Browse the corpus

Travel and the Emergence of Infectious Diseases Mary E. Wilson, M.D. Harvard School of Public Health and Harvard Medical School, Boston, Massachusetts, USA Member: Harvard Working Group on New and Resurgent Infectious Diseases Travel is a potent force in the emergence of disease. Migration of humans has been the pathway for disseminating infectious diseases throughout recorded history and will continue to shape the emergence, frequency, and spread of infections in geographic areas and populations. The current volume, speed, and reach of travel are unprece- dented. The consequences of travel extend beyond the traveler to the population visited and the ecosystem. When they travel, humans carry their genetic makeup, immu- nologic sequelae of past infections, cultural preferences, customs, and behavioral patterns. Microbes, animals, and other biologic life also accompany them. Today's massive movement of humans and materials sets the stage for mixing diverse genetic pools at rates and in combinations previously unknown. Concomitant changes in the environment, climate, technology, land use, human behavior, and demographics converge to favor the emergence of infectious diseases caused by a broad range of organisms in humans, as well as in plants and animals. Many factors contribute to the emergence of in- fectious diseases. Those frequently identified in- clude microbial adaptation and change, human demographics and behavior, environmental changes, technology and economic development, breakdown in public health measures and surveil- lance, and international travel and commerce (1-4). This paper will examine the pivotal role of global travel and movement of biologic life in the emer- gence of infectious diseases. It will also examine the ways in which travel and movement are inextricably tied at multiple levels to other processes that influ- ence the emergence of disease. Travel is a potent force in disease emergence and spread (5). The current volume, speed, and reach of travel are unprecedented. The consequences of mi- gration extend beyond the traveler to the population visited and the ecosystem (6).

ied at multiple levels to other processes that influ- ence the emergence of disease. Travel is a potent force in disease emergence and spread (5). The current volume, speed, and reach of travel are unprecedented. The consequences of mi- gration extend beyond the traveler to the population visited and the ecosystem (6). Travel and trade set the stage for mixing diverse genetic pools at rates and in combinations previously unknown. Massive movement and other concomitant changes in social, political, climatic, environmental, and technologic factors converge to favor the emergence of infectious diseases. Disease emergence is complex. Often several events must occur simultaneously or sequentially for a disease to emerge or reemerge (Table 1) (6). Travel allows a potentially pathogenic microbe to be introduced into a new geographic area; however, to be established and cause disease a microbe must survive, proliferate, and find a way to enter a sus- ceptible host. Any analysis of emergence must look at a dynamic process, a sequence of events, a milieu, or ecosystem. Movement, changing patterns of resistance and vulnerability, and the emergence of infectious dis- eases also affect plants, animals, and insect vectors. Address for correspondence: Mary E. Wilson, Division of Infectious Diseases, Mt. Auburn Hospital, 330 Mt. Auburn Street, Cambridge, MA 02238 USA, fax 617-965-6632; e-mail mewilson@warren.med.harvard.edu. Table 1. Basic concepts in disease emergence* Emergence of infectious diseases is complex. Infectious diseases are dynamic. Most new infections are not caused by genuinely new pathogens. Agents involved in new and reemergent infections cross taxonomic lines to include viruses, bacteria, fungi, protozoa, and helminths. The concept of the microbe as the cause of disease is inadequate and incomplete. Human activities are the most potent factors driving disease emergence. Social, economic, political, climatic, technologic, and environmental factors shape disease patterns and influence emergence. Understanding and responding to disease emergence require a global perspective, conceptually and geographically. The current global situation favors disease emergence. *Adapted from Wilson ME (6). Perspective Vol. 1, No. 2 -- April-June 1995 39 Emerging Infectious Diseases Analysis of these species can hold important lessons about the dynamics of human disease.

rgence require a global perspective, conceptually and geographically. The current global situation favors disease emergence. *Adapted from Wilson ME (6). Perspective Vol. 1, No. 2 -- April-June 1995 39 Emerging Infectious Diseases Analysis of these species can hold important lessons about the dynamics of human disease. To assess the impact of travel on disease emer- gence, it is necessary to consider the receptivity of a geographic area and its population to microbial in- troduction. Most introductions do not lead to dis- ease. Organisms that survive primarily or entirely in the human host and are spread through sexual contact, droplet nuclei, and close physical contact can be readily carried to any part of the world. For example, AIDS, tuberculosis, measles, pertussis, diphtheria, and hepatitis B are easily carried by travelers and can spread in a new geographic area; however, populations protected by vaccines resist introduction. Organisms that have animal hosts, environmental limitations, arthropod vectors, or complicated life cycles become successively more difficult to "transplant" to another geographic area or population. Epidemics of dengue fever and yellow fever cannot appear in a geographic area unless competent mosquito vectors are present. Schis- tosomiasis cannot spread in an environment unless a suitable snail intermediate host exists in that region. Organisms that survive only under carefully tuned local conditions are less likely to be success- fully introduced. Even if an introduced parasite per- sists in a new geographic area, it does not necessarily cause human disease. In the United States, humans infected with Taenia solium, the parasite that causes cysticercosis, infrequently transmit the infection because sanitary disposal of feces, the source of the eggs, is generally available. In short, the likelihood of transmission involves many biological, social, and environmental vari- ables. Historical Perspective Human migration has been the main source of epidemics throughout recorded history. William McNeill (7), in his book Plagues and Peoples, de- scribes the central role of infectious disease in the history of the world. Patterns of disease circulation have influenced the outcome of wars and have shaped the location, nature, and development of human societies. Trade caravans, religious pilgrimages, and mili- tary maneuvers facilitated the spread of many dis- eases, including plague and smallpox.

ous disease in the history of the world. Patterns of disease circulation have influenced the outcome of wars and have shaped the location, nature, and development of human societies. Trade caravans, religious pilgrimages, and mili- tary maneuvers facilitated the spread of many dis- eases, including plague and smallpox. A map in Donald Hopkins'book,Princes and Peasants: Small- pox in History (8), traces the presumed spread of smallpox from Egypt or India, where it was first thought to have become adapted to humans some- time before 1000 B.C. Smallpox spread easily from person to person through close contact with respira- tory discharges and, less commonly, through contact with skin lesions, linens, clothing, and other mate- rial in direct contact with the patient. Because pa- tients remained infectious for about 3 weeks, many opportunities for transmission were available. Even in this century, until the 1970s, smallpox continued to cause epidemics. A pilgrim returning from Mecca was the source of a large outbreak in Yugoslavia in the early 1970s that resulted in 174 Yugoslav cases and 35 deaths (9). The pilgrim apparently con- tracted the infection in Baghdad while visiting a religious site. Because his symptoms were mild, he was never confined to bed and was able to continue his travels and return home. For most of history, human populations were rela- tively isolated. Only in recent centuries has there been extensive contact between the flora and fauna of the Old and New Worlds. Schoolchildren hear the rhyme "Columbus sailed the ocean blue, in fourteen hundred ninety-two," but may learn little about the disaster brought upon the native populations of the Americas by the arriving explorers. By the end of the fifteenth century, measles, influenza, mumps, small- pox, tuberculosis, and other infections had become common in Europe. Explorers from the crowded urban centers of Europe brought infectious diseases to the New World (10), where isolated populations had evolved from a relatively small gene pool and had no previous experience with many infect ions (11). The first epidemics following the arrival of Europeans were often the most severe. By 1518 or 1519, smallpox appeared in Santo Domingo, where it killed one-third to half of the local population and spread to other areas of the Caribbean and the Americas (10). The population of central Mexico is estimated to have dropped by one-third in the single decade following contact with the Europeans.

t severe. By 1518 or 1519, smallpox appeared in Santo Domingo, where it killed one-third to half of the local population and spread to other areas of the Caribbean and the Americas (10). The population of central Mexico is estimated to have dropped by one-third in the single decade following contact with the Europeans. Travel across the Atlantic Ocean transformed the flora and fauna of the New World as well. Some of the transported materials became important sources of food (plants), clothing, and transportation (animals). Other transfers were less welcome: Japa- nese beetles, Dutch elm disease, and chestnut tree fungus. A.W. Crosby, exploring these exchanges be- tween the Old and the New Worlds, sounds a pessi- mistic note: "The Columbian exchange has left us with not a richer but a more impoverished genetic pool" (10). The explorers also paid a price in loss of lives from disease. Philip Curtin (12) provides a quantitative study of "relocation costs," the excess illness and death among European soldiers in the nineteenth century when they lived or worked in the tropics. Until the most recent armed conflicts, infectious diseases claimed more lives than injuries during wars. Plague holds a prominent place in history and remains with us today. A bacterial infection caused by Yersinia pestis, it is primarily an infection of rodents, spread by their fleas. Human infection is incidental to the maintenance of Y. pestis in animal Perspective Emerging Infectious Diseases 40 Vol. 1, No. 2 -- April-June 1995 reservoirs. Yet plague periodically has erupted in human populations, wreaking great devastation, killing millions and causing infection that can be spread directly from person to person by the respi- ratory route. Human population movement has been essential in the spread of plague and the dispersal of rodents and their fleas to new areas. For centuries plague spread along trade routes. It reached Califor- nia by boat around the turn of this century, caused epidemic infection in San Francisco, and then spread to wildlife, where it persists today in a large enzootic focus. Movement of People Travel for business and pleasure constitutes a small fraction of total human movement (5,13). Peo- ple migrating individually or in groups, may be immigrants, refugees, missionaries, merchant ma- rines, students, temporary workers, pilgrims, or Peace Corps workers. Travel may involve short dis- tances or the crossing of international borders. Its volume, however, is huge.

raction of total human movement (5,13). Peo- ple migrating individually or in groups, may be immigrants, refugees, missionaries, merchant ma- rines, students, temporary workers, pilgrims, or Peace Corps workers. Travel may involve short dis- tances or the crossing of international borders. Its volume, however, is huge. In the early 1990s more than 500 million persons annually crossed interna- tional borders on commercial airplane flights (World Tourism Organization, Madrid, unpublished data). An estimated 70 million persons, mostly from devel- oping countries, work either legally or illegally in other countries (14). Movement may be temporary or seasonal, as with nomadic populations and mi- grant workers who follow the crops. Military maneu- vers worldwide employ and move huge populations. The consequences of armed conflict and political unrest displace millions. In the early 1990s, there were an estimated 20 million refugees and 30million displaced persons worldwide (International Organi- zation for Migration, personal communication). Grubler and Nakicenovic estimated and plotted the average kilometers traveled daily for the French population over a 200-year period (1800-2000) and found that spatial mobility has increased more than 1000-fold (15). In the last 40 years, the size of Aus- tralia's population has doubled and the number of persons moving into and out of Australia has in- creased nearly 100-fold (16). Although social, economic, and political factors push people from an area or draw them to another, environmental resources and their impact on food and water supplies are behind many conflicts lead- ing to displacement of populations. Acute disasters, such as flooding, earthquakes, and hurricanes often force populations to seek shelter and sustenance in new lands. Chronic changes, such as drought, deple- tion of soil, and disappearance of fish from streams, lakes, and oceans, draw people to new territories, or, more frequently, to the fringes of large urban cen- ters. Another type of travel relevant to disease emer- gence is the shift of populations to urban areas. It is estimated that by the year 2010, 50% of the world's population will be living in urban areas. It is pro- jected that by the year 2000, the world will comprise 24 "megacities"--sprawling metropolitan areas with populations exceeding 10 million (World Bank, UNDP, World Health Organization, unpublished data).

areas. It is estimated that by the year 2010, 50% of the world's population will be living in urban areas. It is pro- jected that by the year 2000, the world will comprise 24 "megacities"--sprawling metropolitan areas with populations exceeding 10 million (World Bank, UNDP, World Health Organization, unpublished data). These areas will have the population density to support persistence of some infections and con- tribute to the emergence of others. Many of these areas are located in tropical or subtropical regions, where the environment can support a diverse array of pathogens and vectors. Also developing are huge periurban slums, populated with persons from many geographic origins. Poor sanitation allows breeding of arthropod vectors, rodents, and other disease-car- rying animals. Crowded conditions favor the spread of diseases that pass from person to person, includ- ing sexually transmitted infections. Travel between periurban slum areas and rural areas is common, paving the route for the transfer of microbes and disease. Transfer of resistance genes and genetic recombination may also occur in and spread from crowded environments of transients. Acute disturbances, whether climatic or political, lead to interim living arrangements, such as refugee camps and temporary shelters, that provide ideal conditions for the emergence and spread of infec- tions. Temporary living quarters often share simi- larities with periurban slums: crowding, inadequate sanitation, limited access to medical care, lack of clean water and food, dislocation, multiethnic com- position, and inadequate barriers from vectors and animals. An example is the movement of 500,000- 800,000 Rwandan refugees into Zaire in 1994. Al- most 50,000 refugees died during the first month as epidemics of cholera and Shigella dysenteriae type 1 swept through the refugee camps (17). Movement into a rural environment poses differ- ent risks and often places new rural populations in contact with pathogens that are in the soil and water or are carried by animals or arthropods (18). Some of these pathogens such as Guanarito (19) and Sabia viruses (20) in South America, were only recently recognized as capable of infecting humans. Consequences of Movement Human migration favors the emergence of infec- tious diseases through many mechanisms. When people migrate, they carry their genetic makeup, their accumulated immunologic experience, and much more (Table 2).

es (20) in South America, were only recently recognized as capable of infecting humans. Consequences of Movement Human migration favors the emergence of infec- tious diseases through many mechanisms. When people migrate, they carry their genetic makeup, their accumulated immunologic experience, and much more (Table 2). They may carry pathogens in or on their bodies and may also transport disease vectors, such as lice. Their technology (agricultural and industrial), methods for treating disease, cul- tural traditions, and behavioral patterns may influ- Perspective Vol. 1, No. 2 -- April-June 1995 41 Emerging Infectious Diseases ence their risk for infection in a new environment and their capacity to introduce disease into the new region. Their social standing and resources may affect their exposure to local infections and their access to adequate nutrition and treatment. People also change the environment in many ways when they travel or migrate--they plant, clear land, build, and consume. Travel is relevant in the emergence of disease if it changes an ecosystem. The following examples show the many ways in which migration can influence the emergence of disease in anew area. 1. Humans may carry a pathogen in a form that can be transmitted, then or later, directly or indi- rectly to another person. The pathogen may be silent (during the incubation period, chronic carriage, or latent infection) or clinically evident. Examples in- clude hepatitis B virus, human immunodeficiency virus (HIV), Mycobacterium tuberculosis, M. leprae, Salmonella typhi, and other salmonella. Disease may be especially severe when a pathogen is intro- duced into a population that has no previous expo- sure to the infection. How long the consequences of migration persist varies with the specific infection. The two most critical characteristics are the dura- tion of survival of the pathogen in a potentially infective form and its means of transmission. 2. Epidemic cholera in Africa spread along the West African coast and, when the disease moved inland, followed fishing and trading routes. Mar- kets, funerals, refugee camps--events that involved migration of persons and large gatherings with close contact--helped spread the infection. With El Tor cholera, asymptomatic and mild infections can out- number severe disease by 100 to 1 (21), thus permit- ting those infected to continue to move and work. 3. Pilgrims carried an epidemic strain of group A Neisseria meningitidis from southern Asia to Mecca in 1987.

close contact--helped spread the infection. With El Tor cholera, asymptomatic and mild infections can out- number severe disease by 100 to 1 (21), thus permit- ting those infected to continue to move and work. 3. Pilgrims carried an epidemic strain of group A Neisseria meningitidis from southern Asia to Mecca in 1987. Other pilgrims who became colonized with the epidemic strain introduced it into sub-Saharan Africa, where it caused a wave of epidemics in 1988 and 1989 (22). 4. Humans may carry a pathogen that can be transmitted only if conditions are permissive. This permissiveness can pertain to human behavior, the environment, or the presence of appropriate vectors or intermediate hosts. For example, the ease with which HIV spreads in a population depends on sex- ual practices, condom use, the number of sex part- ners, and intravenous drug use, among other factors. Malaria requires specific mosquito vectors (with access to susceptible humans) to spread to new geographic regions. Schistosomiasis can be intro- duced into a new region only if the appropriate snail host is present and if the eggs excreted (in urine or feces, from an infected person), reach the snails in an appropriate environment. 5. Humans may carry a strain of microbe that has an unusual resistance pattern or virulence genes. A multiple-drug-resistant strain of Klebsiella pneumoniae appears to have been transferred by an asymptomatic woman from a hospital in Bahrain to Oxford, where it caused outbreaks in two British hospitals (23). People also carry their background flora, in the intestinal tract, for example, which may contain plasmids and resistance genes that can in- teract with microbes in a new area. It is not just the classic pathogens that may be relevant to the emer- gence of a new disease but the individual traveler's total microbiologic "baggage." 6. Visitors to a region may lack immunity to locally endemic infections, such as hepatitis A and sand-fly fever. Visitors may suffer severe or different manifestations of infection or disease at an age when the local population is immune to it. Resettlement of populations into malaria-endemic regions can lead to a high death rate from falciparum malaria. 7. Kala-azar caused a deadly outbreak in remote villages in southern Sudan in 1994. The origin was thought to be the villagers' exposure to the sand-fly vector during migration to a food distribution center that had been established by a relief organization (24).

s can lead to a high death rate from falciparum malaria. 7. Kala-azar caused a deadly outbreak in remote villages in southern Sudan in 1994. The origin was thought to be the villagers' exposure to the sand-fly vector during migration to a food distribution center that had been established by a relief organization (24). The migration took a malnourished population from a nonendemic zone into the southern part of the kala-azar-endemic zone. Unfamiliarity with the disease and the poor nutritional status of the popu- lation probably contributed to a high death rate (24). 8. Behavioral patterns in a new region may place visitors at risk for infection, while the local popula- tion, possibly because of their knowledge of disease risks, may not be at risk. Behavior patterns may involve food preparation (such as eating some foods raw), clothing (or lack of it), (for example, going barefooted), sleeping arrangements (sleeping on the ground or out of doors in an unscreened area), and contact with animals. 9. Susceptibility of a population may vary be- cause of genetic differences. A microbe introduced into a new region may have a greater or lesser impact, depending on the host population. Genetic factors influence susceptibility to and expression of several infectious diseases. Although these interac- Table 2. What is carried by humans into new regions? Pathogens in or on body Microbiologic flora Vectors on body Immunologic sequelae of past infections Vulnerability to infections Genetic makeup Cultural preferences, customs, behavioral patterns, technology Luggage and whatever it contains Perspective Emerging Infectious Diseases 42 Vol. 1, No. 2 -- April-June 1995 tions are not yet well defined for most infections, genetic factors influence infections caused by differ- ent classes of organisms, including cholera (25,26), parvovirus infection (27), malaria, and Helicobacter pylori infection (28). To determine the consequences of travel both the traveler and the population visited must be consid- ered. Migration may be in only one direction, though travel often involves returning to the point of origin, perhaps after the traveler has made many stops along the way. The changes in the various ecosys- tems as a consequence of the migration guide the emergence of diseases; anystudy thatsimply focuses on the traveler is too narrow. The distance traversed is less important than the differences in biological life in different areas and differences in receptivity and vulnerability.

way. The changes in the various ecosys- tems as a consequence of the migration guide the emergence of diseases; anystudy thatsimply focuses on the traveler is too narrow. The distance traversed is less important than the differences in biological life in different areas and differences in receptivity and vulnerability. In think- ing about disease emergence, what matters is the potential of a disease to appear in a place, popula- tion, or extent not previously reported. What is the long-term impact of migration and travel on human disease? Carriage of pathogens is only part of the influence on disease emergence. Introduced technology, farming methods, treatment and drugs, chemicals, and pesticides may have a far greater and longer impact on disease patterns in a region than the life of a person. Deforestation, build- ing of dams, and opening of roads into previously inaccessible areas have all been associated with population movements and changes in distribution and frequency of a variety of infections in humans (such as malaria, schistosomiasis, Rift Valley fever, and sexually transmitted diseases). Increasingly the vehicle of transportation is the site or even the source of outbreaks. During travel, people from diverse origins are enclosed in close proximity for a hours or days and then discharged to move on to many distant places. These temporary new habitats, jumbo jets or huge ocean liners, can be the sites for dissemination of the microbes (as happens, for example with Legionella pneumophila infections (29), foodborne infections, and cholera) or provide a milieu for person-to-person transmission (influenza, tuberculosis (30,31)). Shipping and Commerce The biomass of humans constitutes only a frac- tion of the matter moved about the earth. Humans carry and send a huge volume of plants, animals and other materials all over the face of the globe. Much of this movement results from the planned transport of goods from one place to another, but some is an unintended consequence of shipping and travel. All has an impact on the juxtaposition of various species in different ecosystems. "Hitchhikers" include all manner of biologic life, both microscopic and macro- scopic. Animals can carry potential human patho- gens and vectors. The globalization of markets brings fresh fruits and vegetables to dinner tables thousands of miles from where they were grown, fertilized, and picked. Tunnels, bridges, and ferries form means to traverse natural barriers to species spread.

c and macro- scopic. Animals can carry potential human patho- gens and vectors. The globalization of markets brings fresh fruits and vegetables to dinner tables thousands of miles from where they were grown, fertilized, and picked. Tunnels, bridges, and ferries form means to traverse natural barriers to species spread. The roads built to transport people often speed the movement of diseases from one area to another. Mass processing and wide distribution net- works allow for the amplification and wide dissemi- nation of potential human microbes. Examples of introduced species include plants and animals--insects, microbes, and marine organ- isms. 1. Ships convey marine organisms on their hulls and in their ballast water. For example, 367 differ- ent species were identified in ballast water of ships traveling between Japan and Coos Bay, Oregon (32). Introductions have had devastating effects in some areas, for example such as the Black and Azov seas, where newly introduced jellyfishlike creatures called ctenophores have ruined local fishing (33). 2. Vibrio cholerae may have been introduced to South America by shipping (34). Researchers iso- lated the organism in samples of ballast, bilge, and sewage from 3 of 14 cargo ships docked at Gulf of Mexico ports. The ships had last ports of call in Brazil, Colombia, and Chile (35). V. cholerae O1, serotype Inaba, biotype El Tor, indistinguishable from the Latin American epidemic strain, was found in oysters and oyster-eating fish from closed oyster beds in Mobile Bay, Alabama (36). V. cholerae O139 has spread along waterways in Asia, although the people carried on the boats doubtless played a role (37,38). 3. Aedes albopictus was introduced into the United States inside used tires shipped from Asia (39,40). The mosquito's introduction causes concern because it is an aggressive biter, survives in both forest and suburban habitats, and appears to be a competent vector for several human pathogens. It has been associated with epidemic dengue fever transmission in Asia and is a competent laboratory vector of La Crosse, yellow fever, and other viruses (41). In Florida, 14 strains of eastern equine en- cephalitis virus have been isolated from A. albopic- tus (42). The mosquito is now established in at least 21 of the contiguous states in United States and in Hawaii. 4. The African anopheles mosquitoes arrived in Brazil in about 1929. This vector could breed under conditions other New World mosquitoes could not.

e en- cephalitis virus have been isolated from A. albopic- tus (42). The mosquito is now established in at least 21 of the contiguous states in United States and in Hawaii. 4. The African anopheles mosquitoes arrived in Brazil in about 1929. This vector could breed under conditions other New World mosquitoes could not. Although the malaria parasite was already found in Brazil, this new vector expanded the range of trans- mission. An estimated 20,000 persons died of ma- laria before the introduced anopheles mosquitoes were eliminated. 5. It has been repeatedly demonstrated that mos- quitoes are present--and survive--on international Perspective Vol. 1, No. 2 -- April-June 1995 43 Emerging Infectious Diseases flights. In random searches of airplanes in London, mosquitoes were found on 12 of 67 airplanes from tropical countries (43). Arthropods can survive even more extreme environments. In one study, mosqui- toes, house flies, and beetles placed in wheel bays of Boeing 747B aircraft survived flights of 6-9 hours with external temperatures of -42 C (43). Airplanes have also carried infective mosquitoes that caused human infection outside malaria-endemic areas (in Europe, for example). 6. Vehicles can transport vectors over land. Glossina palpalis, a vector for African trypanosomi- asis (sleeping sickness), can fly up to 21 km but can be transported much longer distances on animals and in land vehicles. 7. Seven persons in Marburg, Germany, died after handling blood and tissues from African green monkeys from Uganda. The tissues contained an organism later named Marburg virus (44). 8. Exotic animals transported from their usual habitats are clustered in zoos; others are used in research laboratories where they have occasionally caused severe disease in humans. Two examples are B virus from primates (45) and hemorrhagic fever with renal syndrome from rodents (46). 9. The world trade and globalization of organs, tissues, blood, and blood products is growing. Re- searchers are considering animals as sources for tissues and organs for transplantation (47). 10. Plants may not directly cause human dis- ease. But they can alter an ecosystem and facilitate the breeding of a vector for human disease. This can also displace traditional crops that provide essential nutrition. Vertical transmission of plant pathogens (and spread of plant diseases) can result from seed movement (48). Carriage of seeds into new areas can introduce plant pathogens. 11.

ecosystem and facilitate the breeding of a vector for human disease. This can also displace traditional crops that provide essential nutrition. Vertical transmission of plant pathogens (and spread of plant diseases) can result from seed movement (48). Carriage of seeds into new areas can introduce plant pathogens. 11. Migration and altered environments have in- creased the so-called weedy species. These species migrate easily and have high rates of reproduction. If they lack local predators, they can displace other species and often upset local ecology. Introduction of Species into New Areas Introducing species into new geographic areas is not new, but the current volume and frequency of introductions are unprecedented. A pathogen's sur- vival and spread in a new environment are deter- mined by its basic reproductive rate, which is the average number of successful offspring a parasite can produce (49). To invade and establish itself in a host population, a parasitic species must have a basic reproductive rate exceeding one (49). The sim- plicity of this statement belies the complexity of circumstances that influence invasion and persist- ence. These circumstances encompass biological, so- cial, and environmental factors. As noted already, factors that can influence recep- tivity include climate and environmental conditions, sanitation, socioeconomic conditions (50), behavior, nutrition, and genetics. V. cholerae persists in an aquatic reservoir off the Gulf Coast of the United States, yet epidemic cholera has not been a problem in the United States. Where poverty and poor sani- tation prevail, the presence of V. cholerae can be a source of endemic disease and periodic epidemics. Disease emergence is often complex. An outbreak of malaria in San Diego, California, occurred when parasitemic migrant workers were employed in an area where mosquitoes capable of transmitting ma- laria had access to the workers and to a susceptible human population (51). Many conditions had to be met to allow transmission. Migration may introduce parasites into an area where a different intermediate host or vector could change the incidence of disease. Cycling through a different host can lead to different transmission rates, different infectivity, and even different clinical expression. A parasite may be more successful in a new site because of a larger susceptible population or the absence of predators.

rmediate host or vector could change the incidence of disease. Cycling through a different host can lead to different transmission rates, different infectivity, and even different clinical expression. A parasite may be more successful in a new site because of a larger susceptible population or the absence of predators. Confluence of Events Massive global travel is taking place simultane- ously with many other processes that favor the emergence of disease. For example, the human population is more vulnerable because of aging, im- munosuppression from medical treatment and dis- ease (such as AIDS), the presence of prostheses (e.g., artificial heart valves and joints), exposure to chemi- cals and environmental pollutants that may act synergistically with microbes to increase the risk of diseases, increased poverty, crowding and stress, and increased exposure to UV radiation. Technologic changes, while providing many benefits, can also promote disease dissemination. Resistance of mi- crobes and insects to antimicrobial drugs and pesti- cides interferes with the control of infections and allows transmission to continue. Changes in land use can alter the presence and abundance of vectors and intermediate hosts. Microbes are enormously resilient and adaptable. They have short life spans, which allow rapid genetic change. Humans, by comparison, are slow to change genetically but can change their behavior. People move and construct barriers to prevent contact with microparasites, macroparasites, and the extremes of the environment. Technology fosters a perception of human invincibility but actually createsnew vulner- abilities, as it enables us to go deeper, higher, and into more remote and hostile environments. Studies show that no place on earth is devoid of microbes. Their range and resiliency are truly phenomenal. Only a fraction of the existing microbes have been Perspective Emerging Infectious Diseases 44 Vol. 1, No. 2 -- April-June 1995 characterized. Travel and exploration provide a greater opportunity for humans to come into unsam- pled regions with these uncharacterized microbes. Summary and Conclusions Global travel and the evolution of microbes will continue. New infections will continue to emerge, and known infections will change in distribution, severity and frequency. Travel will continue to be a potent factor in disease emergence. The current world circumstances juxtapose people, parasites, plants, animals, and chemicals in a way that pre- cludes timely adaptation.

nue. New infections will continue to emerge, and known infections will change in distribution, severity and frequency. Travel will continue to be a potent factor in disease emergence. The current world circumstances juxtapose people, parasites, plants, animals, and chemicals in a way that pre- cludes timely adaptation. The combination of move- ment at many levels and profound change in the physical environment can lead to unanticipated dis- eases spread by multiple channels. In many in- stances, the use of containment or quarantine is not feasible. Research and surveillance can map the global movement and evolution of microbes and guide interventions. Integration of knowledge and skills from many disciplines--the social, biological, and physical sciences--is needed. The focus should be system analysis and the ecosystem rather than a disease, microbe, or host. Dr. Wilson is Chief of Infectious Diseases at Mount Auburn Hospital in Cambridgeand Assistant Professor of Population and International Health and Epidemiology at the Harvard School of Public Health. An active participant in the Harvard Working Group on New and Reemergent Infectious Diseases since its inception in 1991, she is the senior editor, with Richard Levins and Andrew Spielman, of Disease in Evolution: Global Changes and Emergence of Infectious Diseases (3), a book based on the 1993 Woods Hole workshop on emerging infections. References 1. Lederberg J, Shope RE, Oaks SC, Jr., eds. Emerging infections: microbial threats to health in the United States. Washington, D.C.: National Academy Press, 1992. 2. Centers for Disease Control and Prevention. Address- ing emerging infectious disease threats: a prevention strategy for the United States. Atlanta: U.S. Depart- ment of Health and Human Services, 1994. 3. Wilson ME, Levins R, Spielman A. Disease in evolu- tion: global changes and emergence of infectious dis- eases. New York: New York Academy of Sciences, 1994;740. 4. Levins R, Awerbuch T, Brinkmann U, et al. The emer- gence of new diseases. American Scientist 1994;82:52- 60. 5. Wilson ME. A world guide to infections: diseases, distribution, diagnosis. New York: Oxford University Press, 1991. 6. Wilson ME. Disease in evolution: introduction. In: Wilson ME, Levins R, Spielman A, eds. Disease in evolution: global changes and emergence of infectious diseases. New York: New York Academy of Sciences, 1994;740:1-12. 7. McNeill WH. Plagues and peoples. Garden City, N.Y.: Anchor Press/Doubleday, 1976. 8. Hopkins DR.

. Wilson ME. Disease in evolution: introduction. In: Wilson ME, Levins R, Spielman A, eds. Disease in evolution: global changes and emergence of infectious diseases. New York: New York Academy of Sciences, 1994;740:1-12. 7. McNeill WH. Plagues and peoples. Garden City, N.Y.: Anchor Press/Doubleday, 1976. 8. Hopkins DR. Princes and peasants: smallpox in his- tory. Chicago: University of Chicago Press, 1983. 9. World Health Organization. Smallpox: Yugoslavia. Wkly Epidemiol Rec 1972;47:161-2. 10. Crosby AW, Jr. The Columbian exchange. Westport, Conn. Greenwood Press, 1972:219. 11. Black FL. Why did they die? Science 1992;258:1739- 40. 12. Curtin PD. Death by migration: Europe's encounter with the tropical world in the nineteenth century. Cambridge, U.K.: Cambridge University Press, 1989. 13. Bradley DJ. The scope of travel medicine: an introduc- tion to the conference on international travel medi- cine. In: Steffen R, Lobel HO, Haworth J, Bradley, eds. Travel Medicine. Berlin: Springer-Verlag, 1989:1-9. 14. Siem H, Bollini P, eds. Migration and health in the 1990s. International Migration 1992;30. 15. Grubler A, Nakicenovic N. Evolution of transport systems. Laxenburg, Vienna: ILASA, 1991. 16. Haggett P. Geographical aspects of the emergence of infectious diseases. Geogr Ann 1994;76 B(2):91-104. 17. Goma Epidemiology Group. Public health impact of Rwandan refugee crisis: what happened in Goma, Zaire, in July, 1994? Lancet 1995;345:339-44. 18. Meslin F.-X. Surve illance and control of emerging zoonoses. World Health Stat Q 1992;45:200-7. 19. Tesh RB, Jahrling R, Salas R, Shope RE. Description of Guanarito virus (Arenaviridae: Arenavirus), the etiologic agent of Venezuelan hemorrhagic fever. Am J Trop Med Hyg 1994;50:452-9. 20. Coimbra TLM, Nassar ES, Burattini NM, et al. A new arenavirus isolated from a fatal case of haemorrhagic fever in Brazil. Lancet 1994;343:391-2. 21. Glass RI, Claeson M, Blake PA, Waldman RJ, Pierce NR. Cholera in Africa: lessons on transmission and control for Latin America. Lancet 1991;338:791-5. 22. Moore PS, Reeves MW, Schwartz B, Gellin BG, Broome CV. Intercontinental spread of an epidemic group A Neisseria meningitidis strain. Lancet 1989;2:260-3. 23. Cookson B, Johnson AP, Azadian B, et al. Interna- tional inter- and intrahospital patient spread of a multiple antibiotic-resistant Klebsiella pneumoniae. J Infect Dis 1995;171:511-3. 24. Mercer A, Seaman J, Sondorp E. Kala azar in eastern Upper Nile Province, southern Sudan. Lancet 1995;345:187-8. 25.

989;2:260-3. 23. Cookson B, Johnson AP, Azadian B, et al. Interna- tional inter- and intrahospital patient spread of a multiple antibiotic-resistant Klebsiella pneumoniae. J Infect Dis 1995;171:511-3. 24. Mercer A, Seaman J, Sondorp E. Kala azar in eastern Upper Nile Province, southern Sudan. Lancet 1995;345:187-8. 25. Glass RI, Holmgren I, Haley CE, et al. Predisposition to cholera of individuals with O blood group. Am J Epidemiol 1985;121:791-6. 26. Clemens JD, Sack DA, Harris JR, et al. ABO blood groups and cholera: new observations on specificity of risk and modifications of vaccine efficacy. J Infect Dis 1989;159:770-3. Perspective Vol. 1, No. 2 -- April-June 1995 45 Emerging Infectious Diseases 27. Brown KE, Hibbs JR, Gallinella G, et al. Resistance to parvovirus B19 infection due to lack of virus recep- tor (erythrocyte P antigen). N Engl J Med 1994:330:1192-6. 28. Boren T, Falk P, Roth KA, Larson G, Normark S. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. Science 1993;292:1982-95. 29. Centers for Disease Control and Prevention. Update: outbreak of Legionnaires' disease associated with a cruise ship. MMWR 1994;43:574-5. 30. Driver DR, Valway SE, Morgan M, Onorato IM, Cas- tro KG. Transmission of Mycobacterium tuberculosis associated with air travel. JAMA 1994;272:10311-35. 31. Centers for Disease Control and Prevention. Expo- sure of passengers and flight crew to Mycobacterium tuberculosis on commercial aircraft, 1992-1995. MMWR 1995;44:137-40. 32. Carlton JT, Geller JB. Ecological roulette: the global transport of non-indigenous marine organisms. Sci- ence 1993;261:78-82. 33. Travis J. Invader threatens Black, Azov Seas. Science 1993;262:1366-7. 34. World Health Organization. Cholera in theAmericas. Wkly Epidemiol Rec. 1992;67:33-9. 35. McCarthy SA, McPhearson RM, Guarino AM. Toxi- genic Vibriocholerae O1 and cargo ships entering Gulf of Mexico. Lancet 1992;339:624-5. 36. DePaola A, Capers GM, Moters ML, et al. Isolation of Latin American epidemic strain of Vibrio cholerae O1 from US Gulf Coast. Lancet 1992;339:624. 37. Ramamurthy T, Garg S, Sharma R, et al. Emergence of novel strain of Vibrio cholerae with epidemic poten- tial in southern and eastern India. Lancet 1993;341:703-4. 38. Albert MJ, Siddique AK, Islam MS, et al. Large out- break of clinical cholera due to Vibrio cholerae non-O1 in Bangladesh. Lancet 1993;341:704. 39. Reiter P, Sprenger D.

g S, Sharma R, et al. Emergence of novel strain of Vibrio cholerae with epidemic poten- tial in southern and eastern India. Lancet 1993;341:703-4. 38. Albert MJ, Siddique AK, Islam MS, et al. Large out- break of clinical cholera due to Vibrio cholerae non-O1 in Bangladesh. Lancet 1993;341:704. 39. Reiter P, Sprenger D. The used tire trade: a mecha- nism for the worldwide dispersal of container-breed- ing mosquitoes. J Am Mosq Control Assoc 1987;3:494-501. 40. Craven RB, Eliason DA, Francy P, et al. Importation of Aedes albopictus and other exotic mosquito species into the United States in used tires from Asia. J Am Mosq Control Assoc 1988;4:138-42. 41. Moore CG, Francy DB, Eliason DA, Monath TP. Aedes albopictus in the United States: rapid spread of a potential disease vector. J Am Mosq Control Assoc 1988;4:356-61. 42. Mitchell CJ, Niebylski ML, Smith GC, et al. Isolation of eastern equine encephalitis virus from Aedes al- bopictus in Florida. Science 1992;257:526-7. 43. Russell RC. Survival of insects in the wheel bays of a Boeing 747B aircraft on flights between tropical and temperate airports. Bull WHO 1987;65:659-62. 44. Martini GA, Siegert R, eds. Marburg virus disease. Berlin:Springer-Verlag, 1971. 45. Weigler BJ, Hird DW, Hilliard JK, Lerche NW, Roberts JA, Scott LM. Epidemiology of cercopithecine herpesvirus 1 (B virus) infection and shedding in a large breeding cohort of rhesus macaques. J Infect Dis 1993;167:257-63. 46. Desmyter J, LeDuc JW, Johnson KM, Brasseur F, Deckers C, van Ypersele de Strihou C. Laboratory rat-associated outbreak of haemorrhagic fever with renal syndrome due to Hantaan-likevirus in Belgium. Lancet 1983;ii:1445-8. 47. Fishman JA. Miniature swine as organ donors for man: strategies for prevention of xenotransplant-as- sociated infections. Xenotransplantation 1994;1:47- 57. 48. Anderson PK, Morales FJ. The emergence of new plant diseases: the case of insect-transmitted plant viruses. In: Wilson ME, Levins R, Spielman A, eds. Disease in evolution: global changes and emergence of infectious diseases. New York: New York Academy of Sciences, 1994;740:181-94. 49. Anderson RM, May RM. Infectious diseases of hu- mans: dynamics and control. Oxford, U.K.: Oxford University Press, 1991. 50. Spence DPS, Hotchkiss J, Williams CSD, Davies PDO. Tuberculosis and poverty. Br Med J 1993;307:759-61. 51. Maldonado YA, Nahlen BL, Roberto RR, et al. Trans- mission of Plasmodium vivax malaria in San Diego County, California, 1986. Am J Trop Med Hyg 1990;42:3-9.

ontrol. Oxford, U.K.: Oxford University Press, 1991. 50. Spence DPS, Hotchkiss J, Williams CSD, Davies PDO. Tuberculosis and poverty. Br Med J 1993;307:759-61. 51. Maldonado YA, Nahlen BL, Roberto RR, et al. Trans- mission of Plasmodium vivax malaria in San Diego County, California, 1986. Am J Trop Med Hyg 1990;42:3-9. Perspective Emerging Infectious Diseases 46 Vol. 1, No. 2 -- April-June 1995

Escherichia coli Serotype O157:H7: Novel Vehicles of Infection and Emergence of Phenotypic Variants Peter Feng, Ph.D. U.S. Food and Drug Administration, Washington, D.C., USA Escherichia coli serotype O157:H7 was only recognized as a human pathogen a little more than a decade ago, yet it has become a major foodborne pathogen. In the United States, the severity of serotype O157:H7 infections in the young and the elderly has had a tremendous impact on human health, the food industry, and federal regulations regarding food safety. The implication of acidic foods as vehicles of infection has dispelled the concept that low-pH foods are safe. Further, the association of nonbovine products with outbreaks suggests that other vehicles of transmission may exist for this pathogen. In laboratory diagnosis, most microbiologic assays rely on a single phenotype to selectively isolate this pathogen. However, the increasing evidence that phenotypic variations exist among isolates in this serogroup may eventually necessitate modifications in assay procedures to detect them. Enterohemorrhagic Escherichia coli (EHEC) has emerged in recent years as the predominant cause of hemorrhagic colitis in humans. This illness, with characteristic symptoms of bloody diarrhea and ab- dominal cramps, can progress into a more severe, life-threatening complication known as hemolytic uremic syndrome (HUS). The pathogenicity of EHEC appears to be associated with a number of virulence factors, including the production of several cytotoxins (1,2). These toxins are collectively re- ferred to as verotoxins or Shiga-like toxins (SLTs) because the SLT-I of E. coli closely resembles the Shiga toxin of Shigella dysenteriae type 1 (2). Al- though more than 60 E. coli serotypes produce SLTs (2) and more are being identified as capable of pro- ducing SLT, serotype O157:H7 is the predominant pathogen in the EHEC group and the one associated most frequently with human infections worldwide. Isolates of the serotype O157:H7 were first impli- cated in foodborne illness in 1982; in the subsequent 10 years, approximately 30 outbreaks were recorded in the United States (1).

LT, serotype O157:H7 is the predominant pathogen in the EHEC group and the one associated most frequently with human infections worldwide. Isolates of the serotype O157:H7 were first impli- cated in foodborne illness in 1982; in the subsequent 10 years, approximately 30 outbreaks were recorded in the United States (1). In early 1993, however, serotype O157:H7 received considerable attention after a large foodborne disease outbreak, traced to the consumption of undercooked, contaminated hamburgers served at a regional fast-food restau- rant (3). More than 700 persons in four states were infected; there were 51 cases of HUS and four deaths. Since that outbreak, the reported incidence of serotype O157:H7 infections has risen, partly because better surveillance systems have been im- plemented and awareness has increased amongphy- sicians, clinical microbiologists, and consumers. Fifteen additional outbreaks were recorded in 1993 and 20 in the first half of 1994. Because serotype O157:H7 has only recently been recognized as a foodborne pathogen, our knowledge is limited. However, the notoriety of recent out- breaks and the severity of serotype O157:H7 infec- tions have stimulated research on the organism, its ecology, antibiotic resistance properties, and viru- lence factors. Much has already been learned from the epidemiologic investigations of past outbreaks. For instance, foodborne infections of serotype O157:H7 have most often been associated with the consumption of bovine products; however, several recent outbreaks have implicated other less likely vehicles of infection and showed that the organism may have some unsuspected characteristics. Al- though genotypic studies show serotype O157:H7 to be a unique clone only distantly related to other E. coli serotypes (4,5), phenotypic diversity within the serogroup (6) may complicate existing laboratory diagnosis procedures. The introduction of various molecular diagnostic techniques may facilitate the detection of this serotype and its phenotypic vari- ants. This review examines unexpected and seemingly unlikely vehicles implicated in recent serotype O157:H7 outbreaks and the impact of emerging phe- notypic variants and their effect on diagnostic as- says used to detect this pathogen in clinical specimens or in the food supply. Novel Vehicles of Transmission So far, serotype O157:H7 has caused a total of 60 outbreaks of foodborne illness in the United States.

otype O157:H7 outbreaks and the impact of emerging phe- notypic variants and their effect on diagnostic as- says used to detect this pathogen in clinical specimens or in the food supply. Novel Vehicles of Transmission So far, serotype O157:H7 has caused a total of 60 outbreaks of foodborne illness in the United States. Consumption of contaminated, undercooked Address for correspondence: Peter Feng (HFS-516), U.S. Food and Drug Administration, 200 C Street, S.W., Washington, DC 20204, USA; fax 202-401-7740; e-mail pxf@fdacf.ssw.dhhs.gov. Synopsis Vol. 1, No. 2 -- April-June 1995 47 Emerging Infectious Diseases ground-beef products has accounted for most out- breaks; however, raw milk was also implicated in several outbreaks in the United States and Canada. Improper hygiene with secondary spread from per- son-to-person contact is another well-documented route of infection (1,2). In the last few years, how- ever, several foodborne outbreaks of serotype O157:H7 have implicated unique and seemingly un- likely vehicles of infection: among them are acidic foods, fruits, salad vegetables, yogurt, and water. Acidic foods In the Retail Food Store Sanitation Code of the U.S. Food and Drug Administration, foods with a pH value of less than 4.6 are generally regarded as low risk in terms of food safety. However, several recent disease outbreaks attributable to serotype O157:H7 have shown that this pathogen can persist in foods with low pH. In the fall of 1991, an outbreak of serotype O157:H7 that affected 23 persons was traced to the consumption of fresh-pressed apple cider (7). The implicated cider, made from unwashed "dropped" apples at a farm, had a pH value of 3.7 to 3.9, was not pasteurized, and contained no preservatives. Although apple cider had been implicated in a pre- vious outbreak of Salmonella typhimurium, it is not a common vehicle of enteric infection because of its high acidity. Several laboratory studies have sub- sequently demonstrated that isolates of serotype O157:H7 can tolerate acidic conditions. Some strains persist in media with pH values as low as 2.0 (8), and in cold (8C) apple cider for 10 to 31 days (7,9). Although the source ofserotypeO157:H7 in the cider that caused illness was never fully established, it was suspected that the dropped apples had been contaminated by cow manure.

erate acidic conditions. Some strains persist in media with pH values as low as 2.0 (8), and in cold (8C) apple cider for 10 to 31 days (7,9). Although the source ofserotypeO157:H7 in the cider that caused illness was never fully established, it was suspected that the dropped apples had been contaminated by cow manure. The ability of serotype O157:H7 to tolerate acidity was substantiated in 1993, when another acidic food was implicated in a series of restaurant outbreaks that infected at least 48 persons. Although the source of the outbreaks was not conclusively identi- fied, epidemiologic investigations and other data implicated mayonnaise or mayonnaise-based dress- ing and sauces. Samples of mayonnaise had a pH of 3.6 to 3.9, and the sauces prepared from it were also acidic, with pH levels of 3.6 to 4.4 (10). After this outbreak, several studies confirmed that although isolates of serotype O157:H7 do not multiply under these conditions, they can persist in commercial mayonnaise up to 55 days at 5C (10,11). How the mayonnaise became contaminated with serotype O157:H7 was not determined; however, improper handling of bulk mayonnaise or cross-contamination with meat juices or meat products was suspected. Water Several recent incidents show that both drinking water and recreational water can serve as vehicles for transmitting serotype O157:H7 infections. The first and largest waterborne outbreak asso- ciated with this pathogen occurred in Missouri in 1989 (12). Of the more than 240 people infected, 32 were hospitalized, and four died. The source of the outbreak was not identified, but backflow during a water main break might have contaminated the drinking water supply (12). Like most E. coli, sero- type O157:H7 isolates are susceptible to the effects of chlorine. Hence, adjustments in the chlorination of the drinking water supply during repairs to the water main might have prevented the outbreak (12). An outbreak caused by serotype O157:H7 and S. sonnei in 1991 may have involved recreational lake water in the vicinity of Portland, Oregon. Of the 59 people affected, 21 (all children) were infected by serotype O157:H7 (13). An epidemiologic survey showed that those who became ill had swum in the lake during the previous 3-week period. Transmis- sion probably occurred when the swimmers swal- lowed lake water that was fecally contaminated by other bathers.

Of the 59 people affected, 21 (all children) were infected by serotype O157:H7 (13). An epidemiologic survey showed that those who became ill had swum in the lake during the previous 3-week period. Transmis- sion probably occurred when the swimmers swal- lowed lake water that was fecally contaminated by other bathers. The lengthy period during which peo- ple became infected suggests that these pathogens can remain viable in water for a long time, or that the water was repeatedly recontaminated. Fecal contamination of recreational water by bathers, es- pecially small children, is not uncommon; however, the contaminants are usually diluted quickly by the large volume of water in recreational lakes, bays, or rivers. That swallowing a small amount of lake water can cause illness suggests that the pathogen has a low infectious dose (13). This fact is already well established for Shigella and seems to be consis- tent with recent epidemiologic data from foodborne outbreaks associated with serotype O157:H7. A similar incident, implicating water from a chil- dren's paddling pool, was reported in Scotland in 1992 (14). Although epidemiologic evidence was not conclusive, the available data suggested that a child with diarrhea had played in the pool and fecally contaminated the water with serotype O157. Be- cause the pool water was not changed or disinfected, it became the vehicle of infection for two other neigh- borhood children, who in turn infected others by person-to-person contact. Other vehicles of transmission Recently, several other unique vehicles have been implicated in foodborne outbreaks associated with serotype O157:H7. A 1993 outbreak in an Oregon restaurant was apparently caused by the consump- tion of cantaloupe or other items from the salad bar, which were most likely cross-contaminated by meat products during preparation. One study showed that Synopsis Emerging Infectious Diseases 48 Vol. 1, No. 2 -- April-June 1995 serotype O157:H7 can survive and grow on salad vegetables stored at 12C and 21C for up to 14 days (15). An outbreak in the United Kingdom in 1991 was traced to the consumption of yogurt, which infected 16 persons, 11 of them children (16). Al- though consumption of raw milk has caused past outbreaks, serotype O157:H7 is susceptible to heat treatment and thus does not usually survive the pasteurization process.

days (15). An outbreak in the United Kingdom in 1991 was traced to the consumption of yogurt, which infected 16 persons, 11 of them children (16). Al- though consumption of raw milk has caused past outbreaks, serotype O157:H7 is susceptible to heat treatment and thus does not usually survive the pasteurization process. Even though the implicated yogurt was prepared from pasteurized milk, the milk might have become contaminated with sero- type O157:H7 after pasteurization. A puzzling incident was reported from northern Italy, where 15 cases of HUS, caused by serotype O157 and other EHEC serotypes, was recorded over a 5-month period in 1993 (17). These cases occurred in small towns scattered over a large area with little apparent connection to each other; therefore, com- mon food vehicles and exposure to cattle were elimi- nated as possible sources of infection. However, data from the epidemiologic investigations suggested that contact with live poultry or with chicken coops may have been the source of infection, even though no toxin-producing EHEC strains were isolated from poultry feces. Arecent study showed that inoculating 1-day-old chicks with strains of serotype O157:H7 resulted in rapid colonization of the cecal tissue of the chicks. The chicks then became long-term (up to 11 months) shedders of serotype O157:H7, and this microorganism was subsequently recovered from the shells of their eggs (18). It is conceivable, there- fore, that live poultry were the source of infection in the outbreaks reported from northern Italy. In December 1994, dry cured salami was impli- cated as the source of serotype O157:H7 in a disease outbreak in the state of Washington (19). A prior study showed that although isolates of serotype O157:H7 do not grow in seeded sausage batter, they can tolerate the acidity produced during sausage fermentation and survive the drying and the cold storage associated with the preparation of dry sau- sages (20). Fermented sausages can attain a pH as low as 4.8 (20). The ability of serotype O157:H7 isolates to persist under these conditions is consis- tent with the acid-tolerant properties this organism exhibited in the previously discussed studies with apple cider (7) and mayonnaise (10). Although the consumption of bovine products still accounts for most of the serotype O157:H7 infec- tions, the incidents described above show that other food types can also serve as vehicles in transmitting infections with this serotype.

bited in the previously discussed studies with apple cider (7) and mayonnaise (10). Although the consumption of bovine products still accounts for most of the serotype O157:H7 infec- tions, the incidents described above show that other food types can also serve as vehicles in transmitting infections with this serotype. Emergence of Phenotypic Variants Multilocus enzyme electrophoresis of E. coli strains associated with enteric disease show that serotype O157:H7 is in a well-defined group only distantly related to other SLT-producing serotypes (4,5). Recently, however, several phenotypic variants of this serotype were isolated in Europe. Thus, in addition to causing infections through food vehicles, the problems associated with serotype O157:H7 are compounded by the emergence of phenotypic vari- ants, which may have an impact on diagnostic as- says used to detect this pathogen. The clonal nature of serotype O157:H7 has facili- tated its phenotypic identification. Unlike other E. coli, isolates of serotype O157:H7 do not ferment sorbitol in 24 hours (21) and are negative in the methyl-umbelliferyl glucuronide assay (22), which measures glucuronidase activity (23). These pheno- types, especially the absence of sorbitol fermenta- tion, are used extensively to distinguish isolates of serotype O157:H7 from related bacteria. Isolation of serotype O157:H7 from foods, on selective media, such as hemorrhagic colitis agar (24) and cefixime- tellurite sorbitol-MacConkey agar (25) is based on the sorbitol phenotype. Similarly, sorbitol-Mac- Conkey agar (26) is used in the clinical laboratory as the primary screening medium to analyze patient specimens for the presence of serotype O157:H7. Prompt culturing of bloody stools with this agar has been very effective in isolating serotype O157:H7 from stool specimens (1). Although extremely useful, isolating and identi- fying the pathogen exclusively on the absence of sorbitol fermentation has some limitations. Other enteric bacteria, such as E. hermanii and Hafnia spp., share similar phenotypes and resemble sero- type O157:H7 on sorbitol-containing medium. Like- wise, strains of O157, of non-H7 serotype that are not pathogenic and do not ferment sorbitol have occasionally been isolated from foods (27). Because of the presence of phenotypically similar species sorbitol negative isolates must be serologically con- firmed with O157 and H7 antisera (28).

l-containing medium. Like- wise, strains of O157, of non-H7 serotype that are not pathogenic and do not ferment sorbitol have occasionally been isolated from foods (27). Because of the presence of phenotypically similar species sorbitol negative isolates must be serologically con- firmed with O157 and H7 antisera (28). Though intended solely to select for serotype O157:H7, sorbitol-containing media may also ex- clude the isolation of other pathogenic E. coli sero- types, many of which ferment sorbitol. It appears that serotype O157:H7 is the predominant patho- genic serotype worldwide; however, a large number of other serotypes also produce SLT (1,2). Although many of these have not been implicated in disease or are known to cause only nonbloody diarrhea, some reports indicate that selected SLT-producing, non- O157:H7 serotypes may have caused cases of hem- orrhagic colitis and HUS in Europe (29,30). In the United States, disease caused by non-O157:H7 sero- type is rare; however, a recent outbreak of bloody diarrhea in Montana was suspected to have been caused by a SLT-II-producing E. coli of serotype O104:H21 (31). A more relevant finding, and one that has stronger implications regarding the reliance on sor- Synopsis Vol. 1, No. 2 -- April-June 1995 49 Emerging Infectious Diseases bitol phenotype for identifying pathogens, comes from a recent study which showed that isolates of serotype O157:H7 in sorbitol-containing foods can mutate from a sorbitol-nonfermenting to a sorbitol- fermenting phenotype (32). Moreover, the frequency of isolation of sorbitol-fermenting O157 strains in Europe appears to be increasing. In Germany, for instance, strains of serotype O157:H- that produce SLT-II have been isolated from HUS patients (33). Unlike serotype O157:H7, these strains fermented sorbitol and were positive in the methyl-umbelliferyl glucuronide assay. Initially, these strains were con- sidered atypical. However, other studies confirmed that pathogenic, sorbitol-fermenting, serotype O157:H- strains were fairly prevalent in HUS pa- tients in central Europe (34). In another report, serologic and biochemical characterization of 41 SLT-producing, O157 strains (including H7 and H- serotypes) determined that as many as 25% of the isolates were sorbitol positive. Furthermore, there was considerable variation among pathogenic sero- type O157 isolates not only with respect to sorbitol fermentation, but also with respect to other pheno- typic characteristics (6).

ng, O157 strains (including H7 and H- serotypes) determined that as many as 25% of the isolates were sorbitol positive. Furthermore, there was considerable variation among pathogenic sero- type O157 isolates not only with respect to sorbitol fermentation, but also with respect to other pheno- typic characteristics (6). These variants are not de- tected by sorbitol-containing media and may not be identified by the routine biochemical tests used to characterize serotype O157:H7. The notoriety of recent outbreaks has stimulated the development of many new assays to detect sero- type O157:H7; some of them may also be useful for detecting phenotypic variants. Many of these assays use molecular techniques, and some are commer- cially available. Several new molecular subtyping methods have also been introduced. Although typing methods will not be discussed here, such techniques as ribotyping, pulsed-field gel electrophoresis (35), lambda restriction fragment length polymorphism (36), and others have been extremely useful in study- ing the epidemiology of serotype O157:H7 in food- borne outbreaks. Phenotypic variants of serotype O157:H7 retain the O157 antigen; hence, antibodies to O157 antigen can be used to identify both serotype O157:H7 and its variants. In the clinical laboratory, anti-O157 sera are used effectively in agglutination or latex agglutination tests to rapidly screen or serologically confirm isolates. Some anti-O157 antibodies have also been coupled to magnetic beads and used to selectively isolate this pathogen from foods (37) or have been incorporated into enzyme immunoassays to directly detect serotype O157:H7 in foods and clinical specimens. The latter application of anti- O157 sera, however, has had some drawbacks. Many preparations of anti-O157 sera cross-react with other bacteria, including Citrobacter freundii (38), E. hermanii, and Yersinia enterocolitica O:9 (39). Moreover, the O157 antigen is present on other non-H7 E. coli serotypes (6,40), many of which are not pathogenic. For example, when anti-O157 serum was used in an analysis of various food products, nonpathogenic O157 isolates were found that nei- ther produced SLT nor were of the H7 serotype (27). Therefore, positive test results of food samples tested with assays that use anti-O157 sera should be confirmed by other methods.

enic. For example, when anti-O157 serum was used in an analysis of various food products, nonpathogenic O157 isolates were found that nei- ther produced SLT nor were of the H7 serotype (27). Therefore, positive test results of food samples tested with assays that use anti-O157 sera should be confirmed by other methods. Pre-absorption of diagnostic antisera to remove cross-reacting anti- bodies or the use of antibodies specific for other non-O157 surface antigens of serotype O157:H7 may reduce the frequency of serologic cross-reactions (41). Phenotypic variants also appear to retain the pathogenicity of serotype O157:H7 (6,33); therefore, assays specific for virulence factors are not affected by the phenotypic variations described above. For example, anti-SLT antibodies can be used to screen fecal specimens for toxins, and SLT gene-specific DNA probes and polymerase chain reaction (PCR) can be used to identify all SLT-producing pathogens regardless of phenotype. However, assays specific for SLT or SLT genes do not provide sufficient data for epidemiologic investigations and "trace-back" stud- ies. More than 60 E. coli serotypes have been found to produce SLT (1,2), and even strains from more distantly related genera, such as C. freundii, report- edly produce SLT-II-like cytotoxins (42). Many of these SLT-producing E. coli serotypes have not been implicated in disease; therefore, the mere detection of potential SLT-producing strains in foods or in patients'specimens by these assays is not conclusive evidence that the bacteria caused the illness. Some new assays do not have these limitations. One PCR assay, designed as a mismatch amplifica- tion mutation assay, preferentially amplifies an al- lele in the uidA gene that is unique only to serotype O157:H7, including its phenotypic variants of sero- type O157:H- that are sorbitol and methyl-umbellif- eryl glucuronide positive (43). Coupled with primers specific for SLT genes, this multiplex PCR assay can simultaneously identify isolates of serotype O157:H7 and the type of SLT they encode (44). Analysis of pure culture isolates showed that this assay detected all SLT-producing serotypes and was able to distinguish isolates of serotype O157:H7, including the phenotypic variants. Advantages of these new molecular methods in- clude specificity, sensitivity, and the ability to detect phenotypic variants of serotype O157:H7. However, these assays are far too complex and costly for use in the routine analysis of food or clinical specimens.

tes of serotype O157:H7, including the phenotypic variants. Advantages of these new molecular methods in- clude specificity, sensitivity, and the ability to detect phenotypic variants of serotype O157:H7. However, these assays are far too complex and costly for use in the routine analysis of food or clinical specimens. Furthermore, although the emergence of phenotypic variants is of concern, they have only been observed sporadically and are not prevalent worldwide. Nonetheless, should the frequency of isolation or the incidence of infection caused by these variants in- crease or should other SLT-producing serotypes of E. coli become more firmly established as causative Synopsis Emerging Infectious Diseases 50 Vol. 1, No. 2 -- April-June 1995 agents of illness, other media or assays may need to be incorporated into existing diagnostic methods. In the interim, the continued use of a sorbitol-contain- ing medium such as sorbitol-MacConkey agar to screen bloody stool specimens is a useful and eco- nomical laboratory procedure for the early diagnosis of serotype O157:H7 infections. Conclusions Bovine products have most often been implicated in foodborne infections with E. coli serotype O157:H7. However, recent outbreaks indicate that other food types may also serve as vehicles of trans- mission for this pathogen. Most notably, acidic foods that were once thought to be of low risk can no longer be considered safe because of the acid-tolerant prop- erties of this bacterium. Most microbiologic media and diagnostic assays have been designed specifi- cally to detect serotype O157:H7. However, there is phenotypic diversity within the serogroup. Should these variants become more prevalent in infections, the use of sorbitol medium alone may become inade- quate in detecting the diversity of strains in this pathogenic serotype. Acknowledgments The author thanks G.J. Jackson, FDA, for critical review of this manuscript and L. Tomlinson, FDA, for editorial assistance. Dr. Feng is a research microbiologist with the Food and Drug Administration in Washington, D.C. He was a postdoctoral fellow in molecular biology at Purdue University and a senior scientist at IGEN Inc. His current research centers on developing rapid diagnostic methods for detecting foodborne bacterial pathogens. He is a member of the Microbiology Committee, Association of Official Analytical Chemists International, and serves as science advisor to international health organizations and foreign governments. References 1.

current research centers on developing rapid diagnostic methods for detecting foodborne bacterial pathogens. He is a member of the Microbiology Committee, Association of Official Analytical Chemists International, and serves as science advisor to international health organizations and foreign governments. References 1. Griffin PM, Tauxe RV. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohem- orrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev 1991;13:60-98. 2. Karmali MA. Infection by Verocytotoxin-producing Escherichia coli. Clin Microbiol Rev 1989;2:15-38. 3. Centers for Disease Control and Prevention. Update: multistate outbreak of Escherichia coli O157:H7 in- fections from hamburgers--western United States, 1992-1993. MMWR 1993;42:258-63. 4. Whittam TS, Wachsmuth IK, Wilson RA. Genetic evidence of clonal descent of Escherichia coli O157:H7 associated with hemorrhagic colitis and hemolytic uremic syndrome. J Infect Dis 1988;157:1124-33. 5. Whittam TS, Wolfe ML, Wachsmuth IK, Orskov F, Orskov I, Wilson RA. Clonal relationship among Es- cherichia coli strains that cause hemorrhagic colitis and infantile diarrhea. Infect Immun 1993;61:1619- 29. 6. Aleksic S, Karch H, Bockemuhl J. A biotyping scheme for Shiga-like (Vero) toxin-producing Escherichia coli O157 and a list of serological cross-reactions between O157 and other gram-negative bacteria. Zentralblatt fur Bakteriologie 1992;276:221-30. 7. Besser RE, Lett SM, Weber JT, et al. An outbreak of diarrhea and hemolytic uremic syndrome from Es- cherichia coli O157:H7 in fresh-pressed apple cider. JAMA 1993;269:2217-20. 8. Miller LG, Kaspar CW. Escherichia coli O157:H7 acid tolerance and survival in apple cider. J Food Prot 1994;57:460-4. 9. Zhao T, Doyle MP, Besser RE. Fate of enterohemor- rhagic Escherichia coli O157:H7 in apple cider with and without preservatives. Appl Environ Microbiol 1993;59:2526-30. 10. Weagant SD, Bryant JL, Bark DH. Survival of Es- cherichia coli O157:H7 in mayonnaise and mayon- naise-based sauces at room and refrigerated temperatures. J Food Prot 1994;57:629-31. 11. Zhao T, Doyle MP. Fate of enterohemorrhagic Es- cherichia coli O157:H7 in commercial mayonnaise. J Food Prot 1994;57:780-3. 12. Swerdlow DL, Woodruff BA, Brady RC, et al. Awater- borne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death. Ann Intern Med 1992;117:812-9. 13. Keene WE, McAnulty JM, Hoesly FC, et al.

hemorrhagic Es- cherichia coli O157:H7 in commercial mayonnaise. J Food Prot 1994;57:780-3. 12. Swerdlow DL, Woodruff BA, Brady RC, et al. Awater- borne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death. Ann Intern Med 1992;117:812-9. 13. Keene WE, McAnulty JM, Hoesly FC, et al. A swim- ming-associated outbreak of hemorrhagic colitis caused by Escherichia coli O157:H7 and Shigella son- nei. N Engl J Med 1994;331:579-84. 14. Brewster DH, Browne MI, Robertson D, Houghton GL, Bimson J, Sharp JCM.An outbreak ofEscherichia coli O157 associated with a children's paddling pool. Epidemiol Infect 1994;112:441-7. 15. Abdul-Raouf UM, Beuchat LR, Ammar MS. Survival and growth of Escherichia coli O157:H7 on salad vegetables. Appl Environ Microbiol 1993;59:1999- 2006. 16. Morgan D, Newman CP, Hutchinson DN, Walker AM, Rowe B, Majid F. Verotoxin producing Escherichia coli O157 infections associated with the consumption of yoghurt. Epidemiol Infect 1993;111:181-7. 17. Tozzi AE, Niccolini A, Caprioli A, et al. A community outbreak of haemolytic-uraemic syndromein children occurring in a large area of Northern Italy over a period of several months. Epidemiol Infect 1994;113:209-19. 18. Schoeni JL, Doyle MP. Variable colonization of chick- ens perorally inoculated with Escherichia coli O157:H7 and subsequent contamination of eggs. Appl Environ Microbiol 1994;60:2958-62. 19. Food Chemical News. Unusual E. coli strain causes foodborne illness in Montana. 1994;36:37. 20. Glass KA, Loeffelholz JM, Ford JP, Doyle MP. Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage. Appl Environ Microbiol 1992;58:2513. Synopsis Vol. 1, No. 2 -- April-June 1995 51 Emerging Infectious Diseases 21. Farmer JJ, Davis BR. H7 antiserum-sorbitol fermen- tation medium: a single-tube screening medium for detecting Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol 1985;22:620-5. 22. Doyle MP, Schoeni JL. Survival and growth charac- teristics of Escherichia coli associated with hemor- rhagic colitis. Appl Environ Microbiol 1984;48:855-6. 23. Feng P, Hartman PA. Fluorogenic assays for immedi- ate confirmation of Escherichia coli. Appl Environ Microbiol 1982;43:1320-9. 24. Szabo RA, Todd ECD, Jean A. Method to isolate Es- cherichia coli O157:H7 from food. J Food Prot 1986;49:768-72. 25. Chapman PA, Siddon CA, Zadik PM, Jewes L. An improved selective medium for the isolation of Es- cherichia coli O157.

mmedi- ate confirmation of Escherichia coli. Appl Environ Microbiol 1982;43:1320-9. 24. Szabo RA, Todd ECD, Jean A. Method to isolate Es- cherichia coli O157:H7 from food. J Food Prot 1986;49:768-72. 25. Chapman PA, Siddon CA, Zadik PM, Jewes L. An improved selective medium for the isolation of Es- cherichia coli O157. J Med Microbiol 1991;35:107-10. 26. March SB, Ratnam S. Sorbitol-MacConkey medium for detection of Escherichia coli O157:H7 associated with hemorrhagic colitis. J Clin Microbiol 1986;23:869-72. 27. Willshaw GA, Smith HR, Roberts D, Thirlwell J, Cheasty T, Rowe B. Examination of raw beef products for the presence of Vero cytotoxin producing Es- cherichia coli, particularly those of serogroup O157. J Appl Bacteriol 1993;75:420-6. 28. Padhye NV, Doyle MP. Escherichia coli O157:H7: epidemiology, pathogenesis, and methods for detec- tion in food. J Food Prot 1992;55:555-65. 29. Mariani-Kurkdijian P, Denamur E, Milon A, Picard B, Cave H, Lambert-Zechovsky N, et al. Identification of Escherichia coli O103:H2 as a potential agent of hemolytic-uremic syndrome in France. J Clin Micro- biol 1993;31:296-301. 30. Bockemuhl J, Aleksic S, Karch H. Serological and biochemical properties of Shiga-like toxin (Verocyto- toxin)-producing strains of Escherichia coli, other than O-group 157, from patients in Germany. Zen- tralbl Bakteriol 1992;276:189-195. 31. Food Chemical News. E. coli in salami possibly linked to illness outbreaks. 1994;36:19. 32. Fratamico PM, Buchanan RL, Cooke PH. Virulence of an Escherichia coli O157:H7 sorbitol-positive mutant. Appl Environ Microbiol 1993;59: 4245-52. 33. Gunzer F, Bohm H, Russman H, Bitzan M, Aleksic S, Karch H. Molecular detection of sorbitol-fermenting Escherichia coli O157 in patients with hemolytic-ure- mic syndrome. J Clin Microbiol 1992;30:1807-10. 34. Bitzan M, Ludwig K, Klemt M, Konig H, Buren J, Muller-Wiefel DE. The role of Escherichia coli O157 infections in the classical (enteropathic) haemolytic uraemic syndrome: results of a Central European, multicenter study. Epidemiol Infect 1993;110:183-96. 35. Barrett TJ, Lior H, Green JH, et al. Laboratory inves- tigation of a multistate food-borne outbreak of Es- cherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing. J Clin Microbiol 1994;32:3013-17. 36. Samadpour M, Grimm LM, Desai B, Alfi D, Ongerth JE, Tarr PI.

iol Infect 1993;110:183-96. 35. Barrett TJ, Lior H, Green JH, et al. Laboratory inves- tigation of a multistate food-borne outbreak of Es- cherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing. J Clin Microbiol 1994;32:3013-17. 36. Samadpour M, Grimm LM, Desai B, Alfi D, Ongerth JE, Tarr PI. Molecular epidemiology of Escherichia coli O157:H7 strains by bacteriophage lambda restric- tion fragment length polymorphism analysis: applica- tion toa multistate foodborne outbreak and a day-care center cluster. J Clin Microbiol 1993;31:3179-83. 37. Wright DJ, Chapman PA, Siddon CA. Immunomag- netic separation as a sensitive method for isolating Escherichia coli O157 from food samples. Epidemiol Infect 1994;113:31-9. 38. Bettelheim KA, Evangelidis H, Pearce JL, Sowes E, Strockbine NA. Isolation of Citrobacterfreundii strain which carries the Escherichia coli O157 antigen. J Clin Microbiol 1993;31:760-1. 39. Chart H, Cheasty T, Cope D, Gross RJ, Rowe B. The serological relationship between Yersinia enterocoli- tica O9 and Escherichia coli O157 using sera from patients with yersiniosis and haemolytic uraemicsyn- drome. Epidemiol Infect 1991;107:349-56. 40. Rice EW, Sowers EG, Johnson CH, Dunnigan ME, Strockbine NA, Edberg SC. Serological cross-reaction between Escherichia coliO157 and otherspecies ofthe genus Escherichia. J Clin Microbiol 1992;30:1315-6. 41. Padhye NV, Doyle MP. Rapid procedure for detecting enterohemorrhagic Esherichia coli O157:H7 in foods. Appl Environ Microbiol 1991;57:2693-8. 42. Schmidt H, Montag M, Bockemuhl J, Heesemann J, Karch H. Shiga-like toxin II-related cytotoxins in Citrobacter freundii strains from humans and beef samples. Infect Immun 1993;61:534-43. 43. Feng P. Identification of Escherichia coli O157:H7 by DNA probe specific for an allele of uidA gene. Mol Cell Probes 1993;7:151-4. 44. Cebula TA, Payne WL, Feng P. Simultaneous identi- fication of Escherichia coli of the O157:H7 serotype and their Shiga-like toxin type by MAMA/multiplex PCR. J Clin Microbiol 1995;33:248-50. Synopsis Emerging Infectious Diseases 52 Vol. 1, No. 2 -- April-June 1995

Epidemic-Associated Neisseria meningitidis Detected by Multilocus Enzyme Electrophoresis In Oregon and parts of Washington State, the incidence of serogroup B meningococcal disease in- creased substantially in 1994 (1). Multilocus enzyme electrophoresis (MEE) subtyping of N. meningitidis serogroup B strains collected in these areas during 1993 and 1994 suggested that these increases were due to a group of genetically related strains of the enzyme type-5 (ET-5) complex. ET-5 N. meningitidis serogroup B were first recognized in Norway in 1974 as the cause of a meningococcal disease epidemic that persisted through 1991. Since 1974, serogroup B meningococci of the ET-5 complex have caused epidemics in Europe, Cuba, and South America; these epidemics elevated disease rates for many years in the affected areas (2,3) and led to sustained efforts for vaccine development. This report de- scribes the use of MEE to compare invasive N. men- ingitidis serogroup B meningococcal strains from Oregon and Washington with epidemic serogroup B strains from other countries and with serogroup B strains that have caused endemic disease in other parts of the United States. MEE, first described in 1966 as a molecular ap- proach to the study of genetic variation in eukaryotic systems, has only gradually been adopted by micro- biologists and epidemiologists. The fundamental concept underlying MEE is that differences in the electrophoretic mobility of constitutive enzymes (re- sulting from amino acid substitutions) reflect the chromosomal genotype of strains and thereby allow the calculation of a genetic-relatedness index (Fig- ure 1). As recently as 1984, only one bacterial spe- cies, Escherichia coli, had been studied by MEE. Since then, however, MEE has been used to charac- terize genetic variation among populations of Le- gionella spp., Bordetella spp., Haemophilus influenzae, Streptococcus spp., Listeria monocyto- genes, Neisseria meningitidis, and other bacteria (4). To carry out MEE, crude aqueous extracts of bacteria are electrophoresed in a block of 11% to 12% starch in the presence of a dilute buffer (pH 8.0).

g populations of Le- gionella spp., Bordetella spp., Haemophilus influenzae, Streptococcus spp., Listeria monocyto- genes, Neisseria meningitidis, and other bacteria (4). To carry out MEE, crude aqueous extracts of bacteria are electrophoresed in a block of 11% to 12% starch in the presence of a dilute buffer (pH 8.0). The block is then cut into thin slices, which are stained to detect specific enzymes. The distance traveled by each enzyme is used to create a series of numbers representing the set of enzyme mobilities charac- teristic of individual strains. The number of en- zymes used is somewhat arbitrary and varies between organisms; 15 to 24 enzymes have usually been adequate to characterize genetic diversity among bacterial populations. For this investigation, electrophoretic variations in 24 enzymes were used to describe genetic variability among isolates of N. meningitidis serogroup B. N. meningitidis strains used for this analysis were collected from Oregon (1993-1994, n = 64) and part of Washington State (1993-1994, n = 17; 1992, n = 2; 1990, n = 1; un- known, n = 2); serogroup B meningococcal epidemics outside the United States (1976-1993; Norway n = 1; Cuba n = 1; Brazil n = 1; and Chile n = 2); and active population-based surveillance for meningo- coccal disease in selected areas of the United States (1991-1994, from the San Francisco Bay area, Geor- gia, Maryland, Oklahoma, and Tennessee, n = 57). The epidemic strains tested from Norway and Cuba are the type used for the outer membrane protein vaccines developed and tested in these countries. The MEE data analyzed here (Figure 1) suggest that the increased rates of disease in Oregon and part of Washington are caused by highly genetically related N. meningitidis serogroup B strains of the ET-5 complex. These strains have been relatively rare in the United States. Oregon and Washington strains match a strain isolated in Santiago, Chile, during 1993. The prolonged duration of some ET-5 serogroup B meningococcal epidemics in large re- gions (e.g., Brazil, Argentina, and Chile) demands careful monitoring of this organism in the United States. Efforts to identify potentially modifiable risk factors for the disease and develop a vaccine have been intensified. MEE will continue to be the pri- mary means for epidemiologic tracking and surveil- lance of ET-5 complex N. meningitidis serogroup B in the United States. Michael W. Reeves,* Bradley A. Perkins* Marion Diermayer, and Jay D.

otentially modifiable risk factors for the disease and develop a vaccine have been intensified. MEE will continue to be the pri- mary means for epidemiologic tracking and surveil- lance of ET-5 complex N. meningitidis serogroup B in the United States. Michael W. Reeves,* Bradley A. Perkins* Marion Diermayer, and Jay D. Wenger* *National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA

otentially modifiable risk factors for the disease and develop a vaccine have been intensified. MEE will continue to be the pri- mary means for epidemiologic tracking and surveil- lance of ET-5 complex N. meningitidis serogroup B in the United States. Michael W. Reeves,* Bradley A. Perkins* Marion Diermayer, and Jay D. Wenger* *National Center for Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia, USA Emerging Infections Program, Oregon Dept. of Health, Portland, Oregon, USA, and Epidemiology Program Office, CDC, Atlanta, Georgia, USA References 1. Centers for DiseaseControl and Prevention. Serogroup B meningococcal disease--Oregon, 1994. MMWR 1995;44:121-4. 2. Caugant DA, Froholm LO, Bovre K, Holten E, Frasch CE, Mocca LF, Zollinger WD, Selander RK. Interconti- nental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic dis- ease. Proc Natl Acad Sci USA 1986;83:4927-31. 3. Sacchi CT, Pessoa LL, Ramos SR, Milagres LG, Camargo MCC, Hidalgo NTR, Melles CEA, Caugant DA, Frasch CE. Ongoing group B Neisseria meningi- tidis epidemic in Sao Paulo, Brazil, due to increased prevalence of a single clone of the ET-5 complex. J Clin Microbiol 1992;30:1734-8. 4. Selander RK, Caugant DA, Ochman H, Musser JM, Gil- mour MN, Whittam TS. Methods of multilocus enzyme electrophoresis for bacterial population genetics and sys- tematics. Appl Environ Microbiol 1986;51:873-84. Dispatches Vol. 1, No. 2 -- April-June 1995 53 Emerging Infectious Diseases 0.15 0.10 0.05 0 1 (2%) - - 1 (2%) 1 (12%) 3 (5%) 1 (6%) 1 (2%) 46 (82%) 46 (82%) 14 (88%) 14 (88%) 1 (20%) 1 (20%) 1 (12%) 1 (12%) 2 (4%) 1 (12%) 1 (2%) 4 (80%) 1 (12%) 2 (25%) 1 (12%) 1 (12%) 1 (2%) 1 (6%) Genetic-Relatedness Index - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 1 (2%) 3 (5%) 3 (5%) 1 (4%) 11 (19%) 5 (8%) 18 (32%) 2 (3%) 1 (2%) 9 (16%) 1 (2%) 1 (2%) 8 (14%) 8 (14%) 5 (100%) 5 (100%) 56 (88%) 56 (88%) 16 (73%) 16 (73%) 1 (2%) 1 (2%) 5 (23%) 0.25 0.30 0.35 0.40 0.45 Genetic-Relatedness Index US Endemic Non-US Epidemic Washington Oregon - - - - - - - - - - - - - - - - - - - - - - - - - US Endemic Non-US Epidemic Washington Oregon 0 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 Genetic-Relatedness Index B C C B A Figure 1. Genetic relatedness of serogroup B strains of Neisseria meningitidis from Oregon, Washington, other countries, and endemic-disease cases in the United States. A. Computer-generated dendrogram for all isolates; 68 enzyme types (ETs) were identified with the 24 enzymes used in this study.

edness Index B C C B A Figure 1. Genetic relatedness of serogroup B strains of Neisseria meningitidis from Oregon, Washington, other countries, and endemic-disease cases in the United States. A. Computer-generated dendrogram for all isolates; 68 enzyme types (ETs) were identified with the 24 enzymes used in this study. To determine the relatedness of two ETs, start at the left side of the dendrogram at the line (or leg) representing the ET of interest and follow the leg horizontally to the right angle turn (up or down), allowing a path to the other ET. The point on the x-axis at which a right angle turn (up or down) is made to move horizontally back to the left indicates the genetic relatedness. For example,the ET at the top of the dendrogram is related to the other ETs at slightly more than 0.44; the next two ETs are related to each other at an index of approximately 0.17. B. Expanded view of dendrogram with a genetic-relatedness index of 0.25 to 0.45.The ET-5 complex cluster is shown in bold type. This portion of the dendrogram represents the population structure of group B meningococci in this study; all strains with a genetic-relatedness level of 0.25 or less are shown as single legs or "complexes" of related strains. The distribution of serogroup B meningococcal strains by site and epidemiologic type (Oregon, Washington, non-U.S. epidemic, and U.S. endemic) and by ET group (or complex) is shown in columns to the left of the dendrogram. At a genetic-relatedness level of 0.25, the dendrogram is divided into 11 ET complexes. The ET-5 complex is the ninth leg down (or third from the bottom), shown in bold type. Of strains endemic in the United States, the highest proportion, 18 (32%) of 56, comprise an ET complex located at the fifth leg from the top and are related to the ET-5 complex at a genetic-relatedness index of just over 0.35. In contrast, 56 (88%) of 64 Oregon strains, 16 (73%) of 22 Washington strains, and 5 of 5 non-U.S. epidemic strains are in the ET-5 complex. Only 8 (14%) of 57 strains endemic in the United States are in the ET-5 complex. C. Expanded view of ET-5 portion of the dendrogram with genetic-relatedness index of 0 to 0.16.The distribution of strains by site and epidemiologic type, within the ET-5 complex, is shown to the left of the dendrogram; 16 ETs are represented in the ET-5 complex. The eight strains endemic in the United States in the ET-5 complex are distributed among seven ETs.

drogram with genetic-relatedness index of 0 to 0.16.The distribution of strains by site and epidemiologic type, within the ET-5 complex, is shown to the left of the dendrogram; 16 ETs are represented in the ET-5 complex. The eight strains endemic in the United States in the ET-5 complex are distributed among seven ETs. Forty-six (82%) of 56 Oregon strains and 14 (88%) of 16 Washington strains are clustered at the seventh leg down. One of the five non-U.S. epidemic strains, one of the two strains from Chile, and one (of 56) of strains endemic in the United States match these strains. The other four non-U.S. epidemic strains are located at the tenth leg down, along with one strain from Oregon; these are related to the cluster at the seventh leg at a genetic-relatedness index of approximately 0.06. This slight difference in relatedness results from a difference in the electrophoretic mobility of a single enzyme. Bacteria strains were provided by K. Steingart, Southwest Washington Health Dist., and M. Goldoft, Washington Dept. of Health. Dispatches Emerging Infectious Diseases 54 Vol. 1, No. 2 -- April-June 1995

Dengue/Dengue Hemorrhagic Fever: The Emergence of a Global Health Problem Dengue and dengue hemorrhagic fever (DHF) are caused by one of four closely related, but antigeni- cally distinct, virus serotypes (DEN-1, DEN-2, DEN- 3, and DEN-4), of the genus Flavivirus (1). Infection with one of these serotypes does not provide cross- protective immunity, so persons living in a dengue- endemic area can have four dengue infections during their lifetimes. Dengue is primarily an urban dis- ease of the tropics, and the viruses that cause it are maintained in a cycle that involves humans and Aedes aegypti, a domestic, day-biting mosquito that prefers to feed on humans. Infection with a dengue virus serotype can produce a spectrum of clinical illness, ranging from a nonspecific viral syndrome to severe and fatal hemorrhagic disease. Important risk factors for DHF include the strain and serotype of the virus involved, as well as the age, immune status, and genetic predisposition of the patient. The first reported epidemics of dengue fever oc- curred in1779-1780 in Asia, Africa,and NorthAmer- ica; the near simultaneous occurrence of outbreaks on three continents indicates that these viruses and their mosquito vector have had a worldwide distri- bution in the tropics for more than 200 years. During most of this time, dengue fever was considered a benign, nonfatal disease of visitors to the tropics. Generally, there were long intervals (10-40 years) between major epidemics, mainly because the vi- ruses and their mosquito vector could only be trans- ported between population centers by sailing vessels. A global pandemic of dengue begun in Southeast Asia after World War II and has intensified during the last 15 years. Epidemics caused by multiple serotypes (hyperendemicity) are more frequent, the geographic distribution of dengue viruses has ex- panded, and DHF has emerged in the Pacific region and the Americas (1,2). In Southeast Asia, epidemic DHF first appeared in the 1950s, but by 1975 it had become a leading cause of hospitalization and death among children in many countries.

6-53. 2. Stool SE, Field MJ. The impact of otitis media. Pediatr Infect Dis J 1989;8:S11-S14. 3. Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol Rev 1990;3:171-96. 4. Breiman RF, Butler JC, Tenover FC, Elliott JA, Fack- lam RR. Emergence of drug-resistant pneumococcal infections in the United States. JAMA 1994;271:1831-5. 5. Centers for Disease Control and Prevention. Drug-re- sistant Streptococcus pneumoniae--Kentucky and Ten- nessee, 1993. MMWR 1994;43:23-5,31. 6. Leggiadro RJ, Barrett, FF, Chesney PJ, Davis Y, Tenover FC. Invasive pneumococci with high level penicillin and cephalosporin resistance at a mid-South children's hos- pital. Pediatr Infect Dis J 1994;13:320-2. 7. Centers for Disease Control and Prevention. Preva- lence of penicillin-resistant Streptococcal pneumo- niae--Connecticut 1992-1993. MMWR 1994;43:216-7, 223. 8. Advisory Committee for Immunization Practices. Up- date on adult immunization: recommendations of the Immunization Practices Advisory Committee (ACIP). MMWR 1991;40 (RR-12):42-4. Commentary Vol. 1, No. 2 -- April-June 1995 65 Emerging Infectious Diseases

micity) are more frequent, the geographic distribution of dengue viruses has ex- panded, and DHF has emerged in the Pacific region and the Americas (1,2). In Southeast Asia, epidemic DHF first appeared in the 1950s, but by 1975 it had become a leading cause of hospitalization and death among children in many countries. In the 1980s, DHF began a second expansion into Asia when Sri Lanka, India, and the Maldive Islands had their first major DHF epidemics; Pakistan first reported an epidemic of dengue fever in 1994. The recent epidem- ics in Sri Lanka and India were associated with multiple dengue virus serotypes, but DEN-3 was predominant and was genetically distinct from DEN-3 viruses previously isolated from infected per- sons in those countries (3). After an absence of 35 years, epidemic dengue fever occurred in both Taiwan and the People's Re- public of China in the 1980s. The People's Republic of China had a series of epidemics caused by all four serotypes, and its first major epidemic of DHF, caused by DEN-2, was reported on Hainan Island in 1985 (4). Singapore also had a resurgence of den- gue/DHF from 1990 to 1994 after a successful control program had prevented significant transmission for over 20 years (5). In other countries of Asia where DHF is endemic, the epidemics have become pro- gressively larger in the last 15 years. In the Pacific, dengue viruses were reintroduced in the early 1970s after an absence of more than 25 years. Epidemic activity caused by all four serotypes has intensified in recent years with major epidemics of DHF on several islands (6). Despite poor surveillance for dengue in Africa, we know that epidemic dengue fever caused by all four serotypes has increased dramatically since 1980. Most activity has occurred in East Africa, and major epidemics were reported for the first time in the Seychelles (1977), Kenya (1982, DEN-2), Mozam- bique (1985, DEN-3), Djibouti (1991-92, DEN-2), Somalia (1982, 1993, DEN-2), and Saudi Arabia (1994, DEN-2) (1,6, CDC, unpublished data). Epi- demic DHF has been reported in neither Africa nor the Middle East, but sporadic cases clinically com- patible with DHF have been reported from Mozam- bique, Djibouti, and Saudi Arabia (CDC, unpublished data). The emergence of dengue/DHF as a major public health problem has been most dramatic in the American region. In an effort to prevent urban yel- low fever, which is also transmitted by Ae. aegypti, the Pan American Health Organization organized a campaign that eradicated Ae.

uti, and Saudi Arabia (CDC, unpublished data). The emergence of dengue/DHF as a major public health problem has been most dramatic in the American region. In an effort to prevent urban yel- low fever, which is also transmitted by Ae. aegypti, the Pan American Health Organization organized a campaign that eradicated Ae. aegypti from most Central and South American countries in the 1950s and 1960s. As a result, epidemic dengue occurred only sporadically in some Caribbean islands during this period. The Ae. aegypti eradication program, which was officially discontinued in the United States in 1970, gradually eroded elsewhere, and this species began to reinfest countries from which it had been eradicated. In 1995, the geographic distribu- tion of Ae. aegypti was similar to its distribution before the eradication program (Figure 1). In 1970, only DEN-2 virus was present in the Americas, although DEN-3 may have had a focal distribution in Colombia and Puerto Rico (7). In 1977, DEN-1 was introduced and caused major epi- demics throughout the region over a 16-year period (7). DEN-4 was introduced in 1981 and caused simi- lar widespread epidemics (7). Also in 1981, a new strain of DEN-2 from Southeast Asia caused the first major DHF epidemic in the Americas (Cuba) (7). Dispatches Vol. 1, No. 2 -- April-June 1995 55 Emerging Infectious Diseases This strain has spread rapidly throughout the region and has caused outbreaks of DHF in Venezuela, Colombia, Brazil, French Guiana, Suriname, and Puerto Rico. By 1995, 14 countries in the American region had reported confirmed DHF cases (Figure 2), and DHF is endemic in many of these countries. DEN-3 virus recently reappeared in the Americas after an absence of 16 years. This serotype was first detected in association with a 1994 dengue/DHF epidemic in Nicaragua (8). Almost simultaneously, DEN-3 was confirmed in Panama and, in early 1995, in Costa Rica (8, CDC, unpublished data). In Nica- ragua, considerable numbers of DHF were associ- ated with the epidemic, which was apparently caused by DEN-3. In Panama and Costa Rica, the cases were classic dengue fever. Viral envelope gene sequence data from the DEN- 3 strains isolated from Panama and Nicaraguahave shown that this new American DEN-3 virus strain was likely a recent introduction from Asia since it is genetically distinct from the DEN-3 strain found previously in the Americas, but is identical to the DEN-3 virus serotype that caused major DHF epi- demics in Sri Lanka and India in the 1980s (R.

ama and Nicaraguahave shown that this new American DEN-3 virus strain was likely a recent introduction from Asia since it is genetically distinct from the DEN-3 strain found previously in the Americas, but is identical to the DEN-3 virus serotype that caused major DHF epi- demics in Sri Lanka and India in the 1980s (R. Lanciotti; unpublished data). The new DEN-3 strain, and the susceptibility of the population in the American tropics to it, suggests that DEN-3 will spread rapidly throughout the region and likely will cause major epidemics of dengue/DHF in the near future. In 1995, dengue is the most important mosquito- borne viral disease affecting humans; its global dis- tribution is comparable to that of malaria, and an estimated 2.5 billion people are living in areas at risk for epidemic transmission (Figure 3). Each year, tens of millions of cases of dengue fever occur and, depending on the year, up to hundreds of thousands of cases of DHF. The case-fatality rate of DHF in most countries is about 5%: most fatal cases are among children. There is a small, but significant, risk for dengue outbreaks in the continental United States. Two competent mosquito vectors, Ae. aegypti and Aedes albopictus, are present and, under certain circum- stances, each could transmit dengue viruses. This type of transmission has been detected twice in the last 15 years in south Texas (1980 and 1986) and has been associated with dengue epidemics in northern Mexico (7). Moreover, numerous viruses are intro- duced annually by travelers returning from tropical areas where dengue viruses are endemic. From 1977 to 1994, a total of 2,248 suspected cases of imported dengue were reported in the United States (9, CDC, unpublished data). Although some specimens col- lected were not adequate for laboratory diagnosis, preliminary data indicate that 481 (21%) cases were confirmed as dengue (9, CDC, unpublished data). Many more cases probably go unreported each year because surveillance in the United States is passive and relies on physicians to recognize the disease, inquire about the patient's travel history, obtain proper diagnostic samples, and report the case. These data underscore the fact that southern Texas and the southeastern United States, where Ae. aegypti is found, are at risk for dengue transmission and sporadic outbreaks. The reasons for this dramatic global emergence of dengue/DHF as a major public health problem are complex and not well understood (10).

the case. These data underscore the fact that southern Texas and the southeastern United States, where Ae. aegypti is found, are at risk for dengue transmission and sporadic outbreaks. The reasons for this dramatic global emergence of dengue/DHF as a major public health problem are complex and not well understood (10). However, several important factors can be identified. First, effective mosquito control is virtually nonexistent in most dengue-endemic countries. Considerable em- phasis for the past 20 years has been placed on ultra-low-volume insecticide space sprays for adult mosquito control, a relatively ineffective approach for controlling Ae. aegypti. Second, major global demographic changes have occurred, the most im- portant of which have been uncontrolled urbaniza- tion and concurrent population growth. These demographic changes have resulted in substandard Prior to 1981 1981-1995 Figure 2. American countries with laboratory- confirmed hemorrhagic fever (shaded areas), prior to 1981 and from 1981 to 1995. 1970 1995 Figure 1. Distribution of Aedes aegypti (shaded areas) in the Americas in 1970, at the end of the mosquito eradication program, and in 1995. Dispatches Emerging Infectious Diseases 56 Vol. 1, No. 2 -- April-June 1995 housing and inadequate water, sewer, and waste management systems, all of which increase Ae. aegypti population densities and facilitate transmission of Ae. aegypti-borne disease. Third, increased travel by airplane provides the ideal mechanism for transporting dengue viruses be- tween population centers of the tropics, resulting in a constant exchange of dengue viruses and other pathogens. Lastly, in most countries the public health infrastructure has deteriorated. Limited fi- nancial and human resources and competing priori- ties have resulted in a "crisis mentality" with emphasis on implementing so-called emergency con- trol methods in response to epidemics rather than on developing programs to prevent epidemic trans- mission. This approach has been particularly detri- mental to dengue control because, in most countries, surveillance is very inadequate; the system to detect increased transmission normally relies on reports by local physicians who often do not consider dengue in their diagnoses. As a result, an epidemic has often reached or passed the peak of transmission before it is detected. No dengue vaccine is available. Recently, how- ever, attenuated candidate vaccine viruses have been developed in Thailand.

mally relies on reports by local physicians who often do not consider dengue in their diagnoses. As a result, an epidemic has often reached or passed the peak of transmission before it is detected. No dengue vaccine is available. Recently, how- ever, attenuated candidate vaccine viruses have been developed in Thailand. These vaccines are safe and immunogenic when given in various formula- tions, including a quadrivalent vaccine for all four dengue virus serotypes. Unfortunately, efficacy tri- als in human volunteers have yet to be initiated. Research is also being conducted to develop second- generation recombinant vaccine viruses; the Thai- land attenuated viruses are used as a template. However, an effective dengue vaccine for public use will not be available for 5 to 10 years. Prospects for reversing the recent trend of in- creased epidemic activity and geographic expansion of dengue are not promising. New dengue virus strains and serotypes will likely continue to be intro- duced into many areas where the population densi- ties of Ae. aegypti are at high levels. With no new mosquito control technology available, in recent years public health authorities have emphasized disease prevention and mosquito control through community efforts to reduce larval breeding sources (11). Although this approach will probably be effec- tive in the long run, it is unlikely to impact disease transmission in the near future. We must, therefore, develop improved, proactive, laboratory-based sur- veillance systems that can provide early warning of an impending dengue epidemic. At the very least, surveillance results can alert the public to take action and physicians to diagnose and properly treat dengue/DHF cases. Duane J. Gubler and Gary G. Clark National Center for Infectious Diseases Centers for Disease Control and Prevention Fort Collins, Colorado, and San Juan, Puerto Rico, USA References 1. Gubler DJ. Dengue. In: Epidemiology of arthropod- borne viral disease, Monath TPM, editor. Boca Raton (FL): CRC Press, 1988:223-60. 2. Halstead SB. The XXth century dengue pandemic: need for surveillance and research. Rapp Trimest Sta- tist Sanit Mondo 1992;45:292-8. 3. Lanciotti RS, Lewis JG, Gubler DJ, Trent DW. Molecu- lar evolution and epidemiology of dengue-3 viruses. J Gen Virol 1994;75:65-75. 4. Qiu FX, Gubler DJ, Liu JC, Chen, QQ. Dengue in China: a clinical review. Bull WHO 1993;71:349-59. 5. Anonymous. The dengue situation in Singapore. Epidemiol Bull 1994;20:31-3. 6. Gubler DJ.

iotti RS, Lewis JG, Gubler DJ, Trent DW. Molecu- lar evolution and epidemiology of dengue-3 viruses. J Gen Virol 1994;75:65-75. 4. Qiu FX, Gubler DJ, Liu JC, Chen, QQ. Dengue in China: a clinical review. Bull WHO 1993;71:349-59. 5. Anonymous. The dengue situation in Singapore. Epidemiol Bull 1994;20:31-3. 6. Gubler DJ. Dengue haemorrhagic fever: a global up- date [Editorial]. Virus Information Exchange News. 1991;8:2-3. 7. Gubler DJ. Dengue and dengue haemorrhagic fever in the Americas. In: WHO, Regional Office for SE Asia, New Delhi, Monograph. SEARO 1993;22:9-22. 8. Centers for Disease Control and Prevention. Dengue type 3 infection--Nicaragua and Panama, October- November 1994. MMWR 1995;44:21-4. 9. Rigau-Perez JG, Gubler DJ, Vorndam AV, Clark GG. Dengue surveillance--United States, 1986-1992. MMWR 1994;43:7-19. 10. Gubler DJ, Trent DW. Emergence of epidemic den- gue/dengue hemorrhagic fever as a public health prob- lem in the Americas. Infect Agents Dis 1994;2:383-93. 11. Gubler DJ, Clark GG. Community-based integrated control of Aedes aegypti: a brief overview of current programs. Am J Trop Med Hyg 1994:50:50-60. Figure 3. World distribution of denguevirusesand their mosquito vector, Aedes aegypti, in 1995. Dispatches Vol. 1, No. 2 -- April-June 1995 57 Emerging Infectious Diseases

Progress Toward the Eradication of Dracunculiasis (Guinea Worm Disease): 1994 Dracunculiasis, or guinea worm disease (GWD), is a disabling infection caused by the nematode parasite Dracunculus medinensis. The disease is endemic in India, Africa, and the Middle East. Peo- ple become infected when they drink water contain- ing tiny crustaceans, called copepods or "water fleas," that act as intermediate hosts of the organism and harbor infective larvae. When the ingested co- pepods are killed by the digestive juices in the stom- ach, the larvae are released and move to the small intestine. They penetrate the intestinal wall and migrate to the connective tissues of the thorax, where male and female larvae mature and mate 60 to 90 days after infection. Over the next year, female worms grow to maturity, reach a length of 70 cm or more (2-3 feet), and slowly migrate to the surface of the body. Worms emerge from the lower extremities in about 90% of cases, but they can also appear in the upper extremities, the trunk, buttocks, genita- lia, or other parts of the body. Infected persons remain asymptomatic for ap- proximately a year after infection when the mature female worm approaches the skin and forms a pain- ful papule in the dermis. This papule can become a blister within 24 hours or may enlarge for several days before becoming a blister. Eventually it rup- tures, exposing the worm. Shortly before the skin lesion forms, pronounced systemic symptoms may occur, including erythema and urticarial rash with intense pruritus, nausea, vomiting, diarrhea, and dizziness. On contact with fresh water, a loop of the worm's uterus opens and discharges a swarm of motile larvae. This process may be repeated, if the lesion is resubmerged in water,until the entire brood of larvae is discharged. Larvae ingested by copepods mature in the body cavity of the intermediate host in about 2 weeks. Stagnant sources of drinking water such as ponds, cisterns, pools in dried-up river beds, temporary hand-dug wells, and step-wells commonly harbor populations of freshwater cope- pods and are the usual sites where the infection is transmitted.

opepods mature in the body cavity of the intermediate host in about 2 weeks. Stagnant sources of drinking water such as ponds, cisterns, pools in dried-up river beds, temporary hand-dug wells, and step-wells commonly harbor populations of freshwater cope- pods and are the usual sites where the infection is transmitted. As the worm emerges through the skin lesion, the affected person pulls it out slowly and carefully, usually by winding a few centimeters each day on a stick. This very painful process may last many weeks. Pain and other symptoms may lessen with the rupture of the blister, but at this time pyogenic organisms often invade the superficial lesion and worm tract and aggravate the condition. If the worm breaks during traction, an intense inflammatory reaction occurs, with pain, swelling, and cellulitis along the worm track. Infected persons are often incapacitated for several weeks, or for months, if complications caused by secondary bacterial infec- tions ensue. Infected persons do not develop immunity, and there is no cure for GWD. Pain from emerging worms may be relieved by applying wet compresses to the lesion or by immersing the lesion in a container of water, and then safely disposing of the released larvae. Placing an occlusive bandage on the wound to keep it clean prevents the patient from contami- nating sources of drinking water. Once the worm appears, oral medications to relieve the associated pain and topical antiseptics or antibiotic ointment to minimize the risk for secondary infections allows the worm to be removed by gentle traction over a number of days. The World Health Organization (WHO) has tar- geted GWD for eradication by the end of 1995. A coalition of agencies, institutions, and organizations are providing technical and financial assistance to national eradication programs. An understanding of the factors that contribute to the emergence of dracunculiasis provides the ba- sis for the current elimination program.

GWD for eradication by the end of 1995. A coalition of agencies, institutions, and organizations are providing technical and financial assistance to national eradication programs. An understanding of the factors that contribute to the emergence of dracunculiasis provides the ba- sis for the current elimination program. GWD can be eradicated for several reasons: 1) there is no human carrier beyond the 1-year incubation period; 2) there is no known animal reservoir; 3) detection of patent infections (i.e., worms protruding from skin lesions) is an easy way to assess the presence of the disease in communities, and protrusion of the worm is required for transmission; 4) transmission of the disease is markedly seasonal, facilitating the timing and effectiveness of surveillance and control inter- ventions, including containment of cases; 5) the methods for controlling transmission are simple, and 6) the disease is well recognized by the local population in areas where it is endemic. The overall strategy of national GWD eradication programs includes three operational phases: l) con- ducting baseline surveys to identify villages where the disease is endemic; 2) training village-based health workers to use case registries for monthly surveillance and to implement control interventions in affected communities; and 3) using case-contain- ment, i.e., prompt detection of all remaining cases (before or within 24 hours of worm emergence), treatment of the lesions to prevent transmission, and close supervision of village-based health work- ers to ensure that each case has been properly con- tained (1). The thrust of control interventions is to educate affected persons about the origin of the disease and about the measures they and their communities can take to prevent it. Affected households are provided Dispatches Emerging Infectious Diseases 58 Vol. 1, No. 2 -- April-June 1995 with cloth filters and taught how to safely use them to remove the copepods from water. Household mem- bers are also taught that those with emerging worms should be kept from entering sources of drinking water. Other control interventions include providing safe drinking water to affected villages and selectively using the insecticide temefos to re- duce copepod populations. In 1986, an estimated 3.32 million cases of GWD occurred in Africa (2). That same year, only 22,610 cases of GWD were reported from India, the only affected country with a national eradication cam- paign then underway.

r to affected villages and selectively using the insecticide temefos to re- duce copepod populations. In 1986, an estimated 3.32 million cases of GWD occurred in Africa (2). That same year, only 22,610 cases of GWD were reported from India, the only affected country with a national eradication cam- paign then underway. Worldwide cases reported to WHO have declined from 781,219 in 1988 to 221,055 in 1993 (3). By 1990, 9 of the 19 affected countries had initiated eradication programs and had con- ducted baseline surveys to assess the extent of GWD (Figure 1, Table 1). At the end of 1994, all 19 coun- tries were actively combating the diseases, and the provisional number of cases reported to WHO was 164,750 (Figure 2, Table 2). The number of affected villages was reduced from more than 23,000 in 1993 to fewer than 10,000; 52% of the affected villages were in the case-containment phase. In 1994, Pakistan had no cases (4). (Pakistan is the first country where GWD was endemic during the 1980s to have eliminated indigenous transmis- sion of the disease for 1 year.) Yemen reported small foci of disease transmission for the first time in several years, and Sudan reported 28,899 GWD cases during active village-based searches, and through its passive surveillance system (5). In early March 1995, Sudan revised the number of cases reported to WHO for 1994 to 53,092 cases. Although Figure 1. Status of dracunculiasis eradication in Africa: 1990. Figure 2. Status of dracunculiasis eradication in Africa: 1994. Table 1. Dracunculiasis cases reported, 1990* Country Number of cases Nigeria 394,082 Ghana 117,034 Burkina Faso 42,187 Benin 37,414 Mauritania 8,036 India 4,798 Togo 3,042 Cameroon 742 Pakistan 160 Chad - Cote d'Ivoire - Ethiopia - Kenya - Mali - Niger - Senegal - Sudan - Uganda - Yemen - *Cases reported to the World Health organization from coun- tries completing national case searches or from active vil- lage-based surveillance.

,187 Benin 37,414 Mauritania 8,036 India 4,798 Togo 3,042 Cameroon 742 Pakistan 160 Chad - Cote d'Ivoire - Ethiopia - Kenya - Mali - Niger - Senegal - Sudan - Uganda - Yemen - *Cases reported to the World Health organization from coun- tries completing national case searches or from active vil- lage-based surveillance. No information available at this time. Table 2. Dracunculiasis cases reported, 1994* Country Number of cases Sudan 53,092 Nigeria 39,774 Niger 23,568 Uganda 10,409 Ghana 8,432 Burkina Faso 6,859 Mali 5,396 Togo 5,045 Mauritania 5,029 Cote d'Ivoire 4,700 Benin 3,440 Ethiopia 1,252 Chad 640 India 371 Senegal 186 Yemen 74 Kenya 37 Cameroon 30 Pakistan 0 *Provisional data reported to the World Health Organization from countries completing national case searches or from active village-based surveillance. Sudan's report to WHO includes cases reported by the Passive Surveillance System. Dispatches Vol. 1, No. 2 -- April-June 1995 59 Emerging Infectious Diseases the number of cases reported by Sudan may not be precise, their relative magnitude likely reflects that the incidence of disease there exceeds that reported by any of the other affected countries. The global GWD eradication campaign faces two major challenges in 1995: to complete implementa- tion of the case-containment strategy and other con- trol interventions, such as insecticide use, in villages of all affected countries and to mobilize greater pub- lic support in these countries. The countries that pose the greatest eradication challenge are Sudan, Niger, and Nigeria. However, even in the countries with the fewest cases, markedly tighter control measures will be required to completely interrupt transmission of GWD by the end of 1995. Success of the eradication campaign depends on continued funding and on the ability of national programs and collaborating agencies and organizations to com- plete the needed surveillance and control interven- tions. Ernesto Ruiz-Tiben, Donald R. Hopkins, Trenton K. Ruebush,* and Robert L. Kaiser Global 2000 Project, The Carter Center of Emory University, Atlanta, Georgia, USA *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA References 1. Hopkins DR, Ruiz-Tiben E. Strategies for dracunculi- asis eradication. Bull WHO 1991;69:533-40. 2. Watts S. Dracunculiasis in Africa in 1986: its geo- graphic extent, incidence, and at-risk population. Am J Trop Med Hyg 37:119-25. 3. WHO.

, Centers for Disease Control and Prevention, Atlanta, Georgia, USA References 1. Hopkins DR, Ruiz-Tiben E. Strategies for dracunculi- asis eradication. Bull WHO 1991;69:533-40. 2. Watts S. Dracunculiasis in Africa in 1986: its geo- graphic extent, incidence, and at-risk population. Am J Trop Med Hyg 37:119-25. 3. WHO. Dracunculiasis--global surveillance summary 1993. Wkly Epidemiol Rec 1995:17:121-8. 4. Centers for Disease Control and Prevention. Update: dracunculiasis eradication--Pakistan, 1994. MMWR 1995;44:117-9. 5. WHO. Dracunculiasis update--Sudan. Wkly Epidemiol Rec 1995:7:48-50. Dispatches Emerging Infectious Diseases 60 Vol. 1, No. 2 -- April-June 1995

Heat-Stable Enterotoxin-Producing Escherichia coli O169:H41 in Japan To the Editor: Enterotoxigenic Escherichia coli (ETEC) cause diarrhea by producing either a heat- labile enterotoxin, a heat-stable enterotoxin (ST), or both. Consequently, ETEC can be identified either by detecting the enterotoxin in the culture fluid by immunologic assays or by detecting enterotoxin-cod- ing genes with DNA probes or polymerase chain reaction (PCR) amplification. For many clinical laboratories, however, serologic typing is the most common test used to determine if isolates are mem- bers of known pathogenic groups. Although E. coli serotype O169:H8 has been recognized as one of the ETEC strains (1), serotype O169:H41 is not established worldwide as one of the diarrheagenic E. coli of the ETEC group. Ando et al. first reported that an outbreak at a school for physi- ologically handicapped children in Saitama Prefec- ture was due to ST-producing E. coli serotype O169:H41 (2). We report an outbreak of diarrhea caused by E. coli O169:H41 that predates the out- break reported by Ando et al. and information about additional outbreaks in Japan since 1991. In June 1991, we isolated toxigenic E. coli from the stool of two of three ill members of an eight-mem- ber family during an outbreak of diarrheal illness in Osaka, Japan. An epidemiologic investigation impli- cated pickles (kimchi) purchased during a visit to Korea; only the three members of the family who ate the pickles became ill. The major symptoms were diarrhea (3/3), abdominal pain (2/3), and fever (2/3) of 38 C. The incubation period was estimated at 33 hours. The serotype of these ST-producing E. coli isolateswas not recognized immediately because the cultures were non-typable by the lot of E. coli antis- erum available when the cultures were first isolated. The cultures were identified as E. coli O169:H41 when a new lot of antiserum became available. In another outbreak investigated by the Osaka City Department of Environment and Health and the Osaka Prefecture Department of Environment and Health, food poisoning occurred among 776 of 1,242 guests of wedding receptions held at a wedding facility during September 1993.

1 when a new lot of antiserum became available. In another outbreak investigated by the Osaka City Department of Environment and Health and the Osaka Prefecture Department of Environment and Health, food poisoning occurred among 776 of 1,242 guests of wedding receptions held at a wedding facility during September 1993. The main symptoms were diarrhea (98%) and abdominal cramps (74%), and the mean incubation period was 40.5 hours. E. coli O169:H41 was isolated from the stool speci- mens of 7 of 14 patients. Astrain ofE. coli O169:H41 was isolated from frozen, ready-to-eat seafood recov- ered from a distributor who provided foods to the wedding facility. In addition to the outbreaks mentioned above, Japanese surveillance reports describe foodborne outbreaks in different prefectures between January 1991 and September 1994. In addition to being cul- tured for E. coli, stools were also routinely cultured for Shigella, Salmonella (including typhi and pa- ratyphi), Vibrio, Clostridium, Aeromonas, Plesio- monas, Bacillus cereus, and Staphylococcus aureus. Stools were also examined for rotaviruses and small round viruses by electron microscopy. E. coli sero- type O169:H41 was isolated from patients' stools in 11 of 40 outbreaks; recovery rates were 10%-100%. In 7 of the 11 outbreaks, recovery rates of serotype O169:H41 exceeded 75%. PCR was used to examine the diarrheagenicity of 31 E. coli isolates selected from reported outbreaks that occurred from 1991 to 1994 (3). ST production of the 31 isolates was also examined by COLI ST EIA (Denka Seiken Co., Ltd., Tokyo, Japan), a competitive enzyme-linked immu- nosorbent assay (ELISA) for toxigenic and invasive strains of E. coli. Strains were grown in Casamino Acids-Yeast Extract broth shaken at 37 C for 18 hours. The supernatant obtained after the cen- trifugation of cells was used for the test according to the manufacturer's instructions. Thirty of the 31 E. coli O169:H41 isolates tested demonstrated toxigenicity by both PCR and ELISA. Collaborative studies are in progress to further char- acterize these isolates and to study the relationships between different isolates by molecular epidemiologic methods. Five cultures of E. coli O169:H41 have been ribotyped by a digoxigenin-la- beled (Genius System, Boehringer Mannheim) probe prepared from pKK3535 according to the manufacturer's instructions.

ther char- acterize these isolates and to study the relationships between different isolates by molecular epidemiologic methods. Five cultures of E. coli O169:H41 have been ribotyped by a digoxigenin-la- beled (Genius System, Boehringer Mannheim) probe prepared from pKK3535 according to the manufacturer's instructions. The resulting patterns were indistinguishable when the restriction en- zymes EcoRI, SmaI, BglII, BamHI, SalI, PstI, or HindIII were used to digest chromosomal DNA. We suggest that this comparatively new serotype of ETEC may be spreading across Japan and urge that studies be conducted to determine its distribu- tion and association with gastroenteritis worldwide. References 1. Orskov I, Orskov F, Rowe B. Six new Escherichia coli O groups O165, O166, O167, O168, O169, and O170. Acta Pathol Microbiol Immunol Scand Sect B 1984;92:189-93. 2. Ando K, Itaya T, Aoki A, Saito A, Masaki H, Tokumaru Y. An outbreak of food poisoning caused by enterotoxi- genic Escherichia coli O169:H41. Jpn J Food Microbiol 1993;10:77-81. 3. Itoh F, Ogino T, Itoh K, Watanabe H. Differentiation and detection of pathogenic determinants among diar- rheagenic Escherichia coli by polymerase chain reac- tion using mixed primers. Jpn J Clin Med 1992;50:343-7. Yoshikazu Nishikawa, Masaki Hanaoka, Jun Ogasawara, Nelson P. Moyer,* and Teruo Kimura Osaka City Institute of Public Health and Environmental Sciences, Osaka 543, Japan *Hygienic Laboratory, University of Iowa, Iowa City, Iowa 52242-5002, USA Letters Vol. 1, No. 2 -- April-June 1995 61 Emerging Infectious Diseases The GAP Project in Southeastern Turkey: The Potential for Emergence of Diseases To the Editor: The undersigned, representing interested scientists from both Turkey and the United States, recently visited the water develop- ment projects in southeastern Anatolia, Turkey. This letter describes our observations and projections on the possible health-related consequences of these projects with specific emphasis on infectious dis- eases. When new irrigation schemes are introduced into previously dry areas, disease frequently follows the new water. The Southeastern Anatolia Irrigation Project or GAP (its Turkish acronym) is one of the largest projects ever undertaken in Turkey. This water resources development program includes the construction of 22 dams and 19 hydroelectric plants on the Euphrates and Tigris rivers in southeastern Turkey.

follows the new water. The Southeastern Anatolia Irrigation Project or GAP (its Turkish acronym) is one of the largest projects ever undertaken in Turkey. This water resources development program includes the construction of 22 dams and 19 hydroelectric plants on the Euphrates and Tigris rivers in southeastern Turkey. Upon completion, the project will also in- clude an irrigation network for 1.7 million hectares of land, covering eight provinces corresponding to approximately 10% of Turkey's total population and surface area (1). In its entirety, GAP comprises in- vestments in development projects linked to agricul- ture, energy, transportation, telecommunications, health care, education, and urban and rural infra- structures. To ensure the success of the project, an agency has been established (the Republic of Turkey Prime Ministry Southeastern Anatolia Project Re- gional Development Administration) to oversee and implement all of these projects. The largest of the completed dams on the Euphrates River is the Ataturk Dam. It is the sixth- largest rock-filled dam in the world; its hydroelectric systems have already produced more than seven billion kilowatt hours of power since 1992 (2). Water from the Ataturk Dam reservoir is diverted to the plains of upper Mesopotamia through the Sanliurfa Irrigation Tunnel System. This system consists of two parallel tunnels, each 26.5-km long and 7.62 m in diameter, and numerous other irrigation net- works and canal systems. The first water started to flow to the plains of Harran in November 1994. Additional lands will be incorporated into the irriga- tion scheme as the canals are completed. (The year 2020 is the target date for completion.) When fully operational, GAP is expected to double Turkey's hy- droelectric production, increase irrigated areas by 50%, more than double the per capita income in the region, more than quadruple the gross national product, and create two million new jobs in the coming decade (3). The total surface area affected by the irrigation scheme is about 75,000 km2; of this, 46.2% is cultivated (36% semiarid rain-fed farmland), 33.3% is dry pastures, 20.5% is forest and bush. One of GAP's major goals is to remove the socio- economic disparity between the country's more de- veloped regions and the project area. For GAP to reach its targeted and sustainable economic aims, projects in various other sectors also need to be considered and integrated.

3% is dry pastures, 20.5% is forest and bush. One of GAP's major goals is to remove the socio- economic disparity between the country's more de- veloped regions and the project area. For GAP to reach its targeted and sustainable economic aims, projects in various other sectors also need to be considered and integrated. Inthis context,the public health consequences of emerging diseases in this setting must be anticipated so that appropriate health education and disease prevention measures can be implemented. To anticipate changing patterns in disease asso- ciated with microclimatic and other environmental changes, knowledge of existing diseases in the re- gion is vital. Since arthropods, reservoir animals, and other intermediate hosts are involved in the transmission of many waterborne parasitic dis- eases, a clear understanding of the existing spe- cies--especially of insect vectors--is equally important. Historically, occasional cases of malaria have oc- curred in the region; however, limited records show that this disease is clearly on the rise. Cutaneous leishmaniasis is also endemic and on the rise, but few data are available on the prevalence of the visceral form of the disease. Other common diseases in the region include bacterial and helminthic gas- trointestinal infections as well as trachoma. According to data from the Malaria Division of the Turkish Health Ministry, the reported cases of Plasmodium vivax malaria rose from 8,680 in 1990 to 18,676 in 1992 (4). The province of Sanliurfa (population one million in 1990), which is at the heart of the irrigated plains in GAP, has reported that malaria cases increased from 785 in 1990 to 5,125 in 1993. The numbers of cases in the first 9 months of 1994 alone were already significantly higher than those reported in 1993 (S. Aksoy, unpub- lished data). Although presumably P. vivax malaria is most common, cases of P. falciparum malaria have also been reported in the country. Three cases of P. falciparum malaria were recently documented in Izmir, which is on the Aegean Sea coast of Turkey (4). No cases of drug-resistant malaria have been reported. Another endemic disease on the rise in the south- eastern region is leishmaniasis, transmitted by bit- ing sand flies. In Sanliurfa the number of documented cases of the cutaneous form of this disease has risen from 552 in 1990 to 1,955 in 1993. In the first 9 months of 1994 alone, the number of reported cases was more than 3,000 (S. Aksoy, un- published data).

south- eastern region is leishmaniasis, transmitted by bit- ing sand flies. In Sanliurfa the number of documented cases of the cutaneous form of this disease has risen from 552 in 1990 to 1,955 in 1993. In the first 9 months of 1994 alone, the number of reported cases was more than 3,000 (S. Aksoy, un- published data). At Sanliurfa's Diyarbakir Hospital, in 1991, in addition to cases of the cutaneous forms of the disease, there were 80 potential cases of vis- ceral leishmaniasis (kala-azar) in children ages 2 to 10 (5). Leishmania donovani is often the causative agent of kala-azar, but both L. tropica and L. infan- tummay also be involved (6). As the economic oppor- Letters Emerging Infectious Diseases 62 Vol. 1, No. 2 -- April-June 1995

The GAP Project in Southeastern Turkey: The Potential for Emergence of Diseases To the Editor: The undersigned, representing interested scientists from both Turkey and the United States, recently visited the water develop- ment projects in southeastern Anatolia, Turkey. This letter describes our observations and projections on the possible health-related consequences of these projects with specific emphasis on infectious dis- eases. When new irrigation schemes are introduced into previously dry areas, disease frequently follows the new water. The Southeastern Anatolia Irrigation Project or GAP (its Turkish acronym) is one of the largest projects ever undertaken in Turkey. This water resources development program includes the construction of 22 dams and 19 hydroelectric plants on the Euphrates and Tigris rivers in southeastern Turkey. Upon completion, the project will also in- clude an irrigation network for 1.7 million hectares of land, covering eight provinces corresponding to approximately 10% of Turkey's total population and surface area (1). In its entirety, GAP comprises in- vestments in development projects linked to agricul- ture, energy, transportation, telecommunications, health care, education, and urban and rural infra- structures. To ensure the success of the project, an agency has been established (the Republic of Turkey Prime Ministry Southeastern Anatolia Project Re- gional Development Administration) to oversee and implement all of these projects. The largest of the completed dams on the Euphrates River is the Ataturk Dam. It is the sixth- largest rock-filled dam in the world; its hydroelectric systems have already produced more than seven billion kilowatt hours of power since 1992 (2). Water from the Ataturk Dam reservoir is diverted to the plains of upper Mesopotamia through the Sanliurfa Irrigation Tunnel System. This system consists of two parallel tunnels, each 26.5-km long and 7.62 m in diameter, and numerous other irrigation net- works and canal systems. The first water started to flow to the plains of Harran in November 1994. Additional lands will be incorporated into the irriga- tion scheme as the canals are completed.

m. This system consists of two parallel tunnels, each 26.5-km long and 7.62 m in diameter, and numerous other irrigation net- works and canal systems. The first water started to flow to the plains of Harran in November 1994. Additional lands will be incorporated into the irriga- tion scheme as the canals are completed. (The year 2020 is the target date for completion.) When fully operational, GAP is expected to double Turkey's hy- droelectric production, increase irrigated areas by 50%, more than double the per capita income in the region, more than quadruple the gross national product, and create two million new jobs in the coming decade (3). The total surface area affected by the irrigation scheme is about 75,000 km2; of this, 46.2% is cultivated (36% semiarid rain-fed farmland), 33.3% is dry pastures, 20.5% is forest and bush. One of GAP's major goals is to remove the socio- economic disparity between the country's more de- veloped regions and the project area. For GAP to reach its targeted and sustainable economic aims, projects in various other sectors also need to be considered and integrated. Inthis context,the public health consequences of emerging diseases in this setting must be anticipated so that appropriate health education and disease prevention measures can be implemented. To anticipate changing patterns in disease asso- ciated with microclimatic and other environmental changes, knowledge of existing diseases in the re- gion is vital. Since arthropods, reservoir animals, and other intermediate hosts are involved in the transmission of many waterborne parasitic dis- eases, a clear understanding of the existing spe- cies--especially of insect vectors--is equally important. Historically, occasional cases of malaria have oc- curred in the region; however, limited records show that this disease is clearly on the rise. Cutaneous leishmaniasis is also endemic and on the rise, but few data are available on the prevalence of the visceral form of the disease. Other common diseases in the region include bacterial and helminthic gas- trointestinal infections as well as trachoma. According to data from the Malaria Division of the Turkish Health Ministry, the reported cases of Plasmodium vivax malaria rose from 8,680 in 1990 to 18,676 in 1992 (4). The province of Sanliurfa (population one million in 1990), which is at the heart of the irrigated plains in GAP, has reported that malaria cases increased from 785 in 1990 to 5,125 in 1993.

ion of the Turkish Health Ministry, the reported cases of Plasmodium vivax malaria rose from 8,680 in 1990 to 18,676 in 1992 (4). The province of Sanliurfa (population one million in 1990), which is at the heart of the irrigated plains in GAP, has reported that malaria cases increased from 785 in 1990 to 5,125 in 1993. The numbers of cases in the first 9 months of 1994 alone were already significantly higher than those reported in 1993 (S. Aksoy, unpub- lished data). Although presumably P. vivax malaria is most common, cases of P. falciparum malaria have also been reported in the country. Three cases of P. falciparum malaria were recently documented in Izmir, which is on the Aegean Sea coast of Turkey (4). No cases of drug-resistant malaria have been reported. Another endemic disease on the rise in the south- eastern region is leishmaniasis, transmitted by bit- ing sand flies. In Sanliurfa the number of documented cases of the cutaneous form of this disease has risen from 552 in 1990 to 1,955 in 1993. In the first 9 months of 1994 alone, the number of reported cases was more than 3,000 (S. Aksoy, un- published data). At Sanliurfa's Diyarbakir Hospital, in 1991, in addition to cases of the cutaneous forms of the disease, there were 80 potential cases of vis- ceral leishmaniasis (kala-azar) in children ages 2 to 10 (5). Leishmania donovani is often the causative agent of kala-azar, but both L. tropica and L. infan- tummay also be involved (6). As the economic oppor- Letters Emerging Infectious Diseases 62 Vol. 1, No. 2 -- April-June 1995 tunities in the GAP provinces attract populations to the region, visceral leishmaniasis may become a greater threat. The prevalence of the sand-fly spe- cies in the region, their habitats, and the future implications of the microclimatic changes for these habitats must be studied to anticipate future disease patterns. Other prevalent pathogens in the region include Entamoeba histolytica, Giardia lamblia, and As- caris lumbricoides. Of 22,468 stool samples exam- ined in one study, over 90% carried intestinal parasites; in children from infancy to 5 years of age, 60% contained Giardia intestinalis (7). In a second study in Diyarbakir involving 4,670 patients (ages <1-65 years), the incidence of protozoan and helmin- thic infection was approximately 16% (53%, E. his- tolytica; 31%, G. lamblia; and 10%, A. lumbricoides) (8). In both studies, the incidence of amebiasis was approximately 8% to 9%.

ardia intestinalis (7). In a second study in Diyarbakir involving 4,670 patients (ages <1-65 years), the incidence of protozoan and helmin- thic infection was approximately 16% (53%, E. his- tolytica; 31%, G. lamblia; and 10%, A. lumbricoides) (8). In both studies, the incidence of amebiasis was approximately 8% to 9%. In 1989, a survey con- ducted among 1,001 children in four elementary schools in Sanliurfa found parasites in 88% of the stool samples examined (50%Ascaris, 53% Trichuris trichiura, 22% Giardia, 11% Entamoeba coli) (9). Ancylostomiasis, which occurs in the eastern Medi- terranean, is a potential danger for the region (10). The emergence of schistosomiasis, which can quickly reach epidemic proportions in water-related projects unless measures are taken, should not be ignored. A recent study in Sanliurfa has identified Bulinus truncatus, the snail vector of Schistosoma haematobium in the region (11). Whether other re- gions in GAP also harbor this species is not known, although there have been reports of these snails in the Nusaybin and Mardin regions (12). A few dec- ades ago, sporadic cases of disease were also re- ported from southeastern regions (13). As microclimatic changes occur in the GAP area, the presence of these snails and the potential emergence of schistosomiasis should be closely monitored. The costs of combating epidemic diseases can be very large, whereas the costs of prevention are much lower. Large national projects that anticipate eco- nomic benefits may sometimes overlook the distant prospects of disease. Ideally, health planning should be built into a project from its inception for small funds invested for prevalence studies early on can bring high returns later. Earlier dam projects in Senegal, Lake Volta, and Egypt have shown that unless effective measures are taken early, infections can quickly reach epidemic levels (14). The estab- lishment of good surveillance and recording systems is an important first step. Serap Aksoy,* Sedat Ariturk, Martine Y.K. Armstrong,* K.P. Chang, Zeynep Dortbudak,* Michael Gottlieb, M. Ali Ozcel, Frank F. Richards,* and Karl Western *Yale Univerity School of Medicine, New Haven, Connecticut

mic levels (14). The estab- lishment of good surveillance and recording systems is an important first step. Serap Aksoy,* Sedat Ariturk, Martine Y.K. Armstrong,* K.P. Chang, Zeynep Dortbudak,* Michael Gottlieb, M. Ali Ozcel, Frank F. Richards,* and Karl Western *Yale Univerity School of Medicine, New Haven, Connecticut Dicle University, Diyarbakir, Turkey Chicago Medical School, Illinois National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

mic levels (14). The estab- lishment of good surveillance and recording systems is an important first step. Serap Aksoy,* Sedat Ariturk, Martine Y.K. Armstrong,* K.P. Chang, Zeynep Dortbudak,* Michael Gottlieb, M. Ali Ozcel, Frank F. Richards,* and Karl Western *Yale Univerity School of Medicine, New Haven, Connecticut Dicle University, Diyarbakir, Turkey Chicago Medical School, Illinois National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland Ege University Medical Faculty, Bornova, Izmir, Turkey References 1. Republic of Turkey Prime Ministry. GAP action plan. Ankara, Turkey: GAP Regional Development Admini- stration, 1993 (in Turkish). 2. Republic of Turkey Prime Ministry. GAP status report (1993). Ankara, Turkey: GAP Regional Development Administration, 1993 (in Turkish). 3. Republic of Turkey Prime Ministry. Agricultural com- modities marketing survey and planning of crop pat- tern for GAP. Ankara, Turkey: GAP Regional Development Administration, 1992. 4. Ok UZ, Buke M, Sayiner AA, Ozcel MA. Three Falci- parum malaria cases in Izmir. Acta Parasitologica Tur- cica 1994;18:33. 5. Ozerdem N, Mete O. The interpretation of diagnostic methods in kala-azar (visceral leishmaniasis) infec- tion. Acta Parasitologica Turcica 1993;17:1-6. 6. Qy J-Q, Zhong L, Yasinzai-Mascom, M, et al. Serodiag- nosis of Asian leishmaniasis with a recombinant anti- gen from the repetitive domain of a Leishmania kinesin. Trans R Soc Trop Med Hyg 1994;88:543-5. 7. Duran G, Mete O. The epidemiological evaluation of intestinal parasites in our region. Acta Parasitologica Turcica 1993;17:35. 8. Balikci E, Ozel MF, Mete O. The investigation of intes- tinal parasites on the stool flora in patients who were admitted to microbiology laboratory. Acta Parasi- tologica Turcica 1993;17:27. 9. Unat EK, Akaslan I, Akaslan S, et al. Results of the parasitological examination of stools from students of our elementary schools in Sanliurfa. Acta Parasi- tologica Turcica 1989;13:75. 10. Unat EK, et al. Medical parasitology. Istanbul: Istan- bul University Press;1983:323. 11. Sesem R, Yildirim MZ. Life cycle of Bulinus truncatus (Audouin 1827) (Pulmonata:Gastropoda) under labo- ratory conditions. Acta Parasitologica Turcica 1994;18:337. 12. Paydak F. Systematic analysis of freshwater gastro- pods in Diyarbakir, Urfa and Mardin. Diyarbakir Medical School Proceedings 1967;5:243. 13. Cebeci M. Intestinal diseases in East Mediterranean regions. Istanbul University Med Faculty J. 1959;22:701-5. l4. Stanley NF, Alpers MP. In. Man-made lakes and hu- man health. London: Academic Press, 1975. Letters Vol. 1, No.

o- pods in Diyarbakir, Urfa and Mardin. Diyarbakir Medical School Proceedings 1967;5:243. 13. Cebeci M. Intestinal diseases in East Mediterranean regions. Istanbul University Med Faculty J. 1959;22:701-5. l4. Stanley NF, Alpers MP. In. Man-made lakes and hu- man health. London: Academic Press, 1975. Letters Vol. 1, No. 2 -- April-June 1995 63 Emerging Infectious Diseases

Action Plan for Drug-Resistant Streptococcus pneumoniae Streptococcus pneumoniae is a leading cause of illness and death in the United States. It accounts for an estimated 3,000 cases of meningitis, 50,000 cases of bacteremia, 500,000 cases of pneumonia, and more than seven million cases of otitis media annually (1, 2). S. pneumoniae had been almost uniformly susceptible to penicillin; however, with the development and worldwide spread of drug-resistant S. pneumoniae (DRSP), apublic health challenge has emerged. Studies from Australia, Southeast Asia, Africa, and Europe have reported pneumococcal strains resistant to penicillin and other drugs (3). Surveillance data collected at the Centers for Dis- ease Control and Prevention (CDC) have shown that high-level resistance to penicillin increased more than 60-fold--from 0.02% for 1979-1987 to 1.3% in 1992--for pneumococcal isolates from invasive infec- tions (4). In some communities, at least 30% of iso- lates are nonsusceptible to penicillin (5; CDC, unpublished data). Pneumococcal resistance has been reported for beta-lactams, macrolides, chloramphenicol, and sul- fonamides. As multidrug-resistant strains become increasingly prevalent, treatment options will be- come limited. The clinical impact of antimicrobial resistance on the outcome of invasive and noninva- sive DRSP infections remains largely unknown. Van- comycin has been required to treat patients with pneumococcal meningitis caused by strains resistant to extended-spectrum cephalosporins (e.g., cefotaxime and ceftriaxone) (6). Optimal treatment regimens for DRSP infections remain to be defined; CDC is organiz- ing a working group to develop consensus guidelines for the management of pneumococcal infections. The prevalence of pneumococcal resistance to an- timicrobial drugs is not known for most areas of the United States since DRSP infection has not been a reportable condition. Some studies have suggested great geographic and temporal variation in levels of resistance; prevalence rates are 2% to 30% (5,7). In addition, DRSP can spread rapidly through a popu- lation, and the prevalence of resistant isolates can differ in adults and children.

DRSP infection has not been a reportable condition. Some studies have suggested great geographic and temporal variation in levels of resistance; prevalence rates are 2% to 30% (5,7). In addition, DRSP can spread rapidly through a popu- lation, and the prevalence of resistant isolates can differ in adults and children. To make appropriate empiric antimicrobial choices, clinicians need a reli- able and current assessment of the level of antimi- crobial resistance in the community. To address the growing problem of DRSP, a work- ing group of public health practitioners, health care providers, clinical laboratorians, and repre- sentatives of key professional societies was formed in June 1994. The group identified the development of an electronic, laboratory-based surveillance sys- tem for DRSP as the essential first step to address this concern. The group has issued a comprehensive plan, "A National Strategy for the Surveillance, Ap- plied Research, Control, and Prevention of DRSP," to be published in June 1995. This plan focuses on three public health priorities: 1) to define and monitor the prevalence and geographic distribution of DRSP and recognize the emergence of patterns of resistance, 2) to study further the epidemiology of DRSP, and 3) to minimize the complications of DRSP infections through control and prevention. The working group has begun piloting an elec- tronic, laboratory-based surveillance network to de- tect serious illness due to DRSP. For isolates nonsusceptible to oxacillin (zone size <20 mm), mini- mal inhibitory concentrations should routinely be determined for penicillin, an extended-spectrum cephalosporin, chloramphenicol, vancomycin, and other clinically relevant drugs. These data will be analyzed to determine community-specific levels of resistance and will be made available to clinicians to improveantimicrobialuse.Additionally,aggregatedata will be sent to CDC so that national trends in pneumo- coccal resistance can be identified and reported. Clinical laboratory directors, large commercial laboratory operators, and laboratory softwaremanu- facturers indicate that many laboratory software systems can accommodate a paperless, automated mechanism for reporting communicable disease in- formation directly to public health authorities. DRSP surveillance may thus serve as a model for electronic, laboratory-based reporting for other labo- ratory-reportable conditions.

ers indicate that many laboratory software systems can accommodate a paperless, automated mechanism for reporting communicable disease in- formation directly to public health authorities. DRSP surveillance may thus serve as a model for electronic, laboratory-based reporting for other labo- ratory-reportable conditions. Although improved data flow should increase the number and timeliness of reported cases, a strategy for ensuring quality control of data will be required. In the era of emerging antimicrobial resistance, prevention of pneumococcal infections is paramount; vaccination strategies offer an important approach to controlling DRSP. An existing pneumococcal poly- saccharide vaccine that can prevent a substantial number of pneumococcal infections, including those caused by DRSP, is underutilized. The vaccine is recommended by the Advisory Committee for Immu- nization Practices (ACIP) for use in persons older than 2 years of age who have certain underlying medical conditions and for all persons older than 65 years of age (8). It is not recommended for routine use among children under 2 years of age because it does not provide immunity consistently in this age group. An effective vaccine is needed to prevent pneumococcal infections in this population, which has the highest risk for otitis media and meningitis caused by DRSP. If the prevalence of pneumococcal infection (and therefore antimicrobial use) can be substantially reduced by vaccination, the impact of DRSP may diminish. Novel vaccine demonstration projects supported by federal and state health agen- cies are under way to explore means of increasing Commentary Emerging Infectious Diseases 64 Vol. 1, No. 2 -- April-June 1995 coverage with the effective 23-valent pneumococcal polysaccharide vaccine. Applied research is also needed to address the problem of DRSP. CDC has recently funded popula- tion-based investigations to define risk factors, pat- terns of transmission, costs, and health outcomes associated with DRSP. Public health programs for control and prevention of DRSP are being designed for use at local, state, and federal levels. Because unnecessary use of antimicrobial agents has contrib- uted to the emergence of resistant bacteria (1, 3, 5), educational materials and campaigns are being de- veloped for both health care providers and consum- ers to raise awareness of the link between excessive antimicrobial use and the emergence of drug-resis- tant organisms.

ary use of antimicrobial agents has contrib- uted to the emergence of resistant bacteria (1, 3, 5), educational materials and campaigns are being de- veloped for both health care providers and consum- ers to raise awareness of the link between excessive antimicrobial use and the emergence of drug-resis- tant organisms. Through a multifaceted approach, the growing problem of DRSP can be addressed to minimize the complications and costs of resistant pneumococcal infections. Surveillance for DRSP is an important starting point from which control and prevention solutions can proceed. For more information regarding DRSP activities at CDC, write to Division of Bacterial and Mycotic Dis- eases, NCID, CDC Mailstop C-09, 1600 Clifton Rd., Atlanta, GA 30333 or send an e-mail to DRSP@ciddbd1.em.cdc.gov. Martin S. Cetron, Daniel B. Jernigan, Robert F. Breiman, and the DRSP Working Group* National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA *Guthrie Birkhead, New York State Department of Health and the Council of State and Territorial Epidemiologists (CSTE); Jay C. Butler, CDC; Mathew L. Cartter, Connecticut Depart- ment of Public Health and Addiction Services; Joan P. Ches- ney, American Academy of Pediatrics; William Craig, Infectious Diseases Society of America; Robert P. Gaynes, CDC; Mary J. R. Gilchrist, American Society of Microbiolo- gists; Richard E. Hoffman, Colorado Department of Public Health and Environment and CSTE; James Jorgensen, Na- tional Committee for Clinical Laboratory Standards; David Klein, National Institute of Allergy and Infectious Diseases, National Institutes of Health; ThomasO'Brien, World Health Organization Collaborating Center for Antibiotic Resistance, Boston; Benjamin Schwartz, CDC; Albert Sheldon, Jr., Food and Drug Administration; Kenneth C. Spitalny, New Jersey State Department of Health; Fred C. Tenover, CDC; and Ralph J. Timperi, Association of State and Territorial Public Health Laboratory Directors. References 1. ReichlerMR, Allphin AA, Breiman RF, et al. The spread of multiply resistant Streptococcus pneumoniae at a day care center in Ohio. J Infect Dis 1992;166:1346-53. 2. Stool SE, Field MJ. The impact of otitis media. Pediatr Infect Dis J 1989;8:S11-S14. 3. Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol Rev 1990;3:171-96. 4. Breiman RF, Butler JC, Tenover FC, Elliott JA, Fack- lam RR. Emergence of drug-resistant pneumococcal infections in the United States.

WHONET: An Information System for Monitoring Antimicrobial Resistance WHONET is an information system developed to support The World Health Organization's (WHO) goal of global surveillance of bacterial resistance to antimicrobial agents. Microbiologists, clinicians and infection control workers may use its software to enhance monitoring of drug resistance in their hos- pitals and communities and to merge their files into national, regional, and global networks for surveil- lance of drug resistance. WHONET software can be installed on personal computers and be configured for the locations of the patients a laboratory serves and for the antimicrobial agents it tests. The pro- gram accepts susceptibility test results and allows printing of reports and logbooks and retrieval of data. If the laboratory already has a computerized reporting system, a translation program can be cre- ated to download the laboratory's files into WHONET. Either way, the microbiologists and other infectious disease specialists gain new analytical tools to monitor and manage susceptibility test qual- ity and the spread of drug resistance locally and outside their area. WHONET can also analyze stored data. From a single screen, a WHONET user selects the type of analysis to run, the species of bacteria to analyze, the subsets of isolates to include (e.g., all, isolates from urine only, and isolates resistant to gentamicin and from certain locations), and the antimicrobial agents and period to examine. Types of analyses include percentage of data categorized as resistant, intermediate, or susceptible by standard or other breakpoints; distributions of test measurements (zone diameter, minimal inhibitory concentration) in the form of histograms; scatterplots comparing measurements for different agents or methods for the same isolates; and line listings of isolates grouped by combinations of agents to which theyare resistant (antibiotypes) to trace distinctive strains. Isolates with uncommon antibiotypes can also be flagged on entry so that they may be rechecked while stillavailable,andlocaloutbreakscanbedetectedearly.

methods for the same isolates; and line listings of isolates grouped by combinations of agents to which theyare resistant (antibiotypes) to trace distinctive strains. Isolates with uncommon antibiotypes can also be flagged on entry so that they may be rechecked while stillavailable,andlocaloutbreakscanbedetectedearly. Although test results are entered and monitored locally on software configured for local use, they are filed in a universal file format so thatanycopy of the program can analyze the files of anylaboratory. This feature has enabled groups of users in 10 countries to set up passive surveillance systems by pooling and analyzing their files collaboratively. WHONET as- sists such initiatives by providing file encryption options to ensure confidentiality before data are pooled and analyzed. Ongoing local analysis by local workers is the foundation of the system. It detects local problems in testing, which no laboratory can avoid entirely, and thus improves the overall quality of the files. It delineates local spread of drug-resistant strains, which aids infection control and can explain and correct uncommon prevalence of certain types of drug resistance at certain sites. It allows local work- ers to distinguish their problems from those of other sites and focus on infection control or antimicrobial use that might be related to those problems. Expansion of the system has been recommended by the WHO Scientific Working Group on Monitor- ing and Management of Bacterial Resistance to An- timicrobial Agents. For more information or for participation contact Thomas F. O'Brien and John M. Stelling WHO Collaborating Center for Surveillance of Resistance to Antimicrobial Agents Microbiology, Brigham and Women's Hospital Boston, MA 02115, USA tel (1-617) 732-6803 fax (1-617) 732-4144 Internet: whonet@bustoff.bwh.harvard.edu Recommendations for Preventing the Spread of Vancomycin Resistance CDC's Hospital Infection Control Practices Advi- sory Committee (HICPAC) has published "Recom- mendations for Preventing the Spread of Vancomycin Resistance." The recommendations fo- cus on vancomycin-resistant enterococci (VRE). The reported incidence of infection and coloniza- tion with VRE in U.S. hospitals has increased rap- idly in the last 5 years. This increase has compounded the need for antimicrobial drugs to treat VRE infections. Most VRE are also resistant to multiple other drugs (e.g., aminoglycoside and am- picillin), which have been used for treating VRE infections.

and coloniza- tion with VRE in U.S. hospitals has increased rap- idly in the last 5 years. This increase has compounded the need for antimicrobial drugs to treat VRE infections. Most VRE are also resistant to multiple other drugs (e.g., aminoglycoside and am- picillin), which have been used for treating VRE infections. In addition, the possibility that the van- comycin-resistance genes present in VRE may be transferred to other gram-positive microorganisms, especially Staphylococcus aureus, is a serious public health concern. Although the epidemiology of VRE has not been fully elucidated, and most enterococcal infections have been attributed to the patient's endogenous flora, recent studies have demonstratedthat entero- cocci, including VRE, can be spread directly from patient to patient or indirectly by transient carriage on the hands of personnel or contaminated environ- mental surfaces and patient-care equipment. In its recommendations, HICPAC stresses that the prevention and control of vancomycin resistance will require a coordinated, concerted effort from various departments of a hospital. Because the rec- News and Notes Emerging Infectious Diseases 66 Vol. 1, No. 2 -- April-June 1995 ommendations were developed with limited data and further research is needed to find cost-effective ways to control the spread of vancomycin resistance, HICPAC strongly encourages hospitals to develop their own institution-specific plans, which should stress the following elements: 1) prudent vancomy- cin use by clinicians, 2) education of hospital staff regarding vancomycin resistance, 3) early detection and prompt reporting of vancomycin resistance in enterococci and other gram-positive microorgan- isms by the hospital microbiology laboratory, and 4) immediate implementation of appropriate infection- control measures to prevent person-to-person trans- mission of VRE. The recommendations were developed by HICPAC's Subcommittee on the Prevention and Control of Antimicrobial-Resistant Microorganisms in Hospitals and subject-matter experts and repre- sentatives of the American Hospital Association, American Society for Microbiology, Association for Professionals in Infection Control and Epidemiol- ogy, Infectious Diseases Society of America, Society for Healthcare Epidemiology of America, and Surgi- cal Infection Society.

itals and subject-matter experts and repre- sentatives of the American Hospital Association, American Society for Microbiology, Association for Professionals in Infection Control and Epidemiol- ogy, Infectious Diseases Society of America, Society for Healthcare Epidemiology of America, and Surgi- cal Infection Society. The recommendations were published in February in Infection Control and Hos- pital Epidemiology 1995;16:105-13 and will also be published in the April 1995 issue of the American Journal for Infection Control. Hospital Infection Control Practices Advisory Committee National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA Waterborne Cryptosporidiosis Threat Addressed Cryptosporidium parvum was first recognized as a cause of human illness in 1976. From 1976 to 1982, the disease was reported rarely in the United States, primarily among the immunocompromised. In 1982, the number of reported cases began to increase dramatically along with the number of HIV-infected persons; outbreaks among immunocompetent popu- lations also were reported. Recent municipal water- borne outbreaks of cryptosporidiosis in Texas (1984), Georgia (1987), and Oregon (1992), and a massive outbreak in Wisconsin in 1993 that affected more than 400,000 persons have raised awareness about the waterborne transmission of cryptosporidiosis. Since 1993, several smaller cryptosporidiosis out- breaks were reported in the United States: two were related to drinking water, six were linked to recrea- tional water, and one was foodborne. Cryptosporidiosis is caused by ingestion of the environmentally tough oocysts of the protozoan parasite C. parvum, an intracellular organism that can replicate in the gut epithelial cells of most mam- mals. Its oocyst is extremely resistant to chlorine, which is commonly used to treat municipal water. In healthy persons, the disease lasts 1 to 2 weeks and can have considerable economic impact through absenteeism of those affected. In the immunocom- promised, the disease is often severe, lifelong, and life-threatening. No effective therapy is available. The magnitude of the 1993 Wisconsin outbreak and its association with a municipal water plant operating within existing state and federal regula- tions underlined the need for improved surveillance and coordination among public health agencies and spurred efforts for regulatory standards for Crypto- sporidium in drinking water. During 1995-1996, the U.S.

outbreak and its association with a municipal water plant operating within existing state and federal regula- tions underlined the need for improved surveillance and coordination among public health agencies and spurred efforts for regulatory standards for Crypto- sporidium in drinking water. During 1995-1996, the U.S. Environmental Protection Agency (EPA) in- tends to implement the Information Collection Rule, which requires utilities that serve populations of 100,000 or more and use surface water (lakes, rivers, streams) to test that water routinely for Crypto- sporidium oocysts. If oocysts are found, the utility may also have to test finished water (tap water). Utilities that serve populations of 10,000 to 99,000 will also have to test source water, but for a shorter period. They will not be required to test tap water, even if oocysts are found. Authority to issue boil water advisories if oocysts are found varies from state to state. The health risks from ingesting low levels of Cryptosporidium are unknown. More than 300 representatives from 40 states and more than 25 regulatory, public health, water utility, and advo- cacy groups met at the Centers for Disease Control and Prevention (CDC) in Atlanta in September 1994 to discuss the prevention and control of waterborne cryptosporidiosis. Recommendations from the CDC workshop will be published in the next 2 to 3months. CDC held the first meeting of the Working Group on Waterborne Cryptosporidiosis in November 1994. The working group convenes biweekly by teleconfer- ence. For more information about the group, contact Margaret Hurd (phone: 404-488-7769, fax: 404-488- 7761). The working group has three main purposes: 1) promote a regular exchange of ideas, goals, activi- ties, and proposals among individual scientists, agencies, and organizations interested in water- borne cryptosporidiosis; 2) make decisions on public health issues related to waterborne crypto- sporidiosis; and 3) assemble smaller, more focused, task forces with expertise to develop, implement, and evaluate projects of the working group. The working group has created task forces to assist local, state, and national public health depart- ments, water utilities, and regulatory agencies in preparing for and managing outbreaks. The task forces have the following responsibilities: News and Notes Vol. 1, No. 2 -- April-June 1995 67 Emerging Infectious Diseases

and coloniza- tion with VRE in U.S. hospitals has increased rap- idly in the last 5 years. This increase has compounded the need for antimicrobial drugs to treat VRE infections. Most VRE are also resistant to multiple other drugs (e.g., aminoglycoside and am- picillin), which have been used for treating VRE infections. In addition, the possibility that the van- comycin-resistance genes present in VRE may be transferred to other gram-positive microorganisms, especially Staphylococcus aureus, is a serious public health concern. Although the epidemiology of VRE has not been fully elucidated, and most enterococcal infections have been attributed to the patient's endogenous flora, recent studies have demonstratedthat entero- cocci, including VRE, can be spread directly from patient to patient or indirectly by transient carriage on the hands of personnel or contaminated environ- mental surfaces and patient-care equipment. In its recommendations, HICPAC stresses that the prevention and control of vancomycin resistance will require a coordinated, concerted effort from various departments of a hospital. Because the rec- News and Notes Emerging Infectious Diseases 66 Vol. 1, No. 2 -- April-June 1995

ommendations were developed with limited data and further research is needed to find cost-effective ways to control the spread of vancomycin resistance, HICPAC strongly encourages hospitals to develop their own institution-specific plans, which should stress the following elements: 1) prudent vancomy- cin use by clinicians, 2) education of hospital staff regarding vancomycin resistance, 3) early detection and prompt reporting of vancomycin resistance in enterococci and other gram-positive microorgan- isms by the hospital microbiology laboratory, and 4) immediate implementation of appropriate infection- control measures to prevent person-to-person trans- mission of VRE. The recommendations were developed by HICPAC's Subcommittee on the Prevention and Control of Antimicrobial-Resistant Microorganisms in Hospitals and subject-matter experts and repre- sentatives of the American Hospital Association, American Society for Microbiology, Association for Professionals in Infection Control and Epidemiol- ogy, Infectious Diseases Society of America, Society for Healthcare Epidemiology of America, and Surgi- cal Infection Society. The recommendations were published in February in Infection Control and Hos- pital Epidemiology 1995;16:105-13 and will also be published in the April 1995 issue of the American Journal for Infection Control. Hospital Infection Control Practices Advisory Committee National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA Waterborne Cryptosporidiosis Threat Addressed Cryptosporidium parvum was first recognized as a cause of human illness in 1976. From 1976 to 1982, the disease was reported rarely in the United States, primarily among the immunocompromised. In 1982, the number of reported cases began to increase dramatically along with the number of HIV-infected persons; outbreaks among immunocompetent popu- lations also were reported.

of human illness in 1976. From 1976 to 1982, the disease was reported rarely in the United States, primarily among the immunocompromised. In 1982, the number of reported cases began to increase dramatically along with the number of HIV-infected persons; outbreaks among immunocompetent popu- lations also were reported. Recent municipal water- borne outbreaks of cryptosporidiosis in Texas (1984), Georgia (1987), and Oregon (1992), and a massive outbreak in Wisconsin in 1993 that affected more than 400,000 persons have raised awareness about the waterborne transmission of cryptosporidiosis. Since 1993, several smaller cryptosporidiosis out- breaks were reported in the United States: two were related to drinking water, six were linked to recrea- tional water, and one was foodborne. Cryptosporidiosis is caused by ingestion of the environmentally tough oocysts of the protozoan parasite C. parvum, an intracellular organism that can replicate in the gut epithelial cells of most mam- mals. Its oocyst is extremely resistant to chlorine, which is commonly used to treat municipal water. In healthy persons, the disease lasts 1 to 2 weeks and can have considerable economic impact through absenteeism of those affected. In the immunocom- promised, the disease is often severe, lifelong, and life-threatening. No effective therapy is available. The magnitude of the 1993 Wisconsin outbreak and its association with a municipal water plant operating within existing state and federal regula- tions underlined the need for improved surveillance and coordination among public health agencies and spurred efforts for regulatory standards for Crypto- sporidium in drinking water. During 1995-1996, the U.S. Environmental Protection Agency (EPA) in- tends to implement the Information Collection Rule, which requires utilities that serve populations of 100,000 or more and use surface water (lakes, rivers, streams) to test that water routinely for Crypto- sporidium oocysts. If oocysts are found, the utility may also have to test finished water (tap water). Utilities that serve populations of 10,000 to 99,000 will also have to test source water, but for a shorter period. They will not be required to test tap water, even if oocysts are found. Authority to issue boil water advisories if oocysts are found varies from state to state. The health risks from ingesting low levels of Cryptosporidium are unknown.

of 10,000 to 99,000 will also have to test source water, but for a shorter period. They will not be required to test tap water, even if oocysts are found. Authority to issue boil water advisories if oocysts are found varies from state to state. The health risks from ingesting low levels of Cryptosporidium are unknown. More than 300 representatives from 40 states and more than 25 regulatory, public health, water utility, and advo- cacy groups met at the Centers for Disease Control and Prevention (CDC) in Atlanta in September 1994 to discuss the prevention and control of waterborne cryptosporidiosis. Recommendations from the CDC workshop will be published in the next 2 to 3months. CDC held the first meeting of the Working Group on Waterborne Cryptosporidiosis in November 1994. The working group convenes biweekly by teleconfer- ence. For more information about the group, contact Margaret Hurd (phone: 404-488-7769, fax: 404-488- 7761). The working group has three main purposes: 1) promote a regular exchange of ideas, goals, activi- ties, and proposals among individual scientists, agencies, and organizations interested in water- borne cryptosporidiosis; 2) make decisions on public health issues related to waterborne crypto- sporidiosis; and 3) assemble smaller, more focused, task forces with expertise to develop, implement, and evaluate projects of the working group. The working group has created task forces to assist local, state, and national public health depart- ments, water utilities, and regulatory agencies in preparing for and managing outbreaks. The task forces have the following responsibilities: News and Notes Vol. 1, No. 2 -- April-June 1995 67 Emerging Infectious Diseases * Develop and evaluate informational materials about cryptosporidiosis. * Develop guidelines on when to initiate or endboil water advisories. * Help formulate the language for EPA's Informa- tion Collection Rule. * Identify officials with authority to issue boil water advisories. Examine legal issues associ- ated with boil water advisories and the environ- mental testing, surveillance, and diagnostic requirements for waterborne Cryptosporidium. In addition, technical task forces will collect data to develop guidelines for persons who may want to use bottled water and personal-use water filters and provide updates on the status of environmental sam- pling, water testing, and surrogate indicators of Cryp- tosporidium oocysts.

nts for waterborne Cryptosporidium. In addition, technical task forces will collect data to develop guidelines for persons who may want to use bottled water and personal-use water filters and provide updates on the status of environmental sam- pling, water testing, and surrogate indicators of Cryp- tosporidium oocysts. These task forces will also report on the status of clinical diagnostic and serologic tools and provide local cryptosporidiosis infection rates to use in assessing the risk for water- borne transmission. C. parvum oocysts are present in most surface water supplies; better technological tools and epidemiologic assessments are needed to determine the public health risks from these oocysts. Until the risks are fully known, efforts should be made to inform the public about cryptosporidiosis. Informa- tion on opportunistic infections, including crypto- sporidiosis, for physicians who treat diseases in immunocompromised patients will be published this fall in a supplement to Clinical Infectious Dis- eases by authors from CDC and the Infectious Dis- eases Society of America. Daniel G. Colley National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA News and Notes Emerging Infectious Diseases 68 Vol. 1, No. 2 -- April-June 1995

Emerging Infections: Getting Ahead of the Curve David Satcher, M.D., Ph.D. Centers for Disease Control and Prevention, Atlanta, Georgia, USA The early history of infectious diseases was characterized by sudden, unpredictable outbreaks, frequently of epidemic proportion. Scientific advances in the late 19th and early 20th centuries resulted in the prevention and control of many infectious diseases, particularly in industrialized nations. Despite these improvements in health, out- breaks of infectious disease continue to occur, and new infections emerge. Since 1987, the National Academy of Science's Institute of Medicine (IOM) has published three reports that have identified erosion of the public health infrastructure among the factors contributing to new and reemerging infectious diseases. In partnership with many public and private organizations in the United States and abroad, the Centers for Disease Control and Prevention (CDC) has developed a strategic plan that addresses the priorities set forth in the IOM reports and serves as a guide for CDC and its partners to combat emerging microbial threats to health. Laboratory-based surveillance, better communication networks, and improvements in the public health infrastructure are the cornerstones of the strategy. Emerging Infectious Diseases, a new periodical produced by CDC, will serve as a forum for exchange of information about incipient trends ininfectious diseases, analysis of factors contributing to disease emergence, and development and implementation of prevention measures. "Nothing in the world of living things is permanently fixed." Hans Zinnser--Rats, Lice and History, 1935 Early History of Infectious Diseases Infectious diseases have plagued humans since the dawn of civilization (1-5). The history of these diseases provides a valuable perspective for evalu- ating current trends. Humans are presumed to have originated in tropical climates and to have been affected by the same parasitic diseases as other primates in these areas. As available supplies of game diminished, early hunters migrated into tem- perate zones which were free of tropical parasites.

rspective for evalu- ating current trends. Humans are presumed to have originated in tropical climates and to have been affected by the same parasitic diseases as other primates in these areas. As available supplies of game diminished, early hunters migrated into tem- perate zones which were free of tropical parasites. Historians speculate that humans were relatively safe from infectious diseases during that period. Later, however, as agriculture began to provide a substantial portion of the human diet, populations stabilized and grew. Eventually, populations reached a size that would support persistent person- to-person spread of infectious microorganisms. With this newly established mode of transmission, infec- tious diseases soon became widespread. The exact origins of many infectious agents remain obscure, but with the advent of large populations, humans eventually became the established reservoir ofmany agents. Infected animals and contaminated food and water were additional sources of infectious microor- ganisms. Dissemination of infectious diseases intensified as civilizations progressed. Caravans of traders car- ried new pathogens to unsuspecting and susceptible populations. Explorers and later conquering armies brought infectious microorganisms to new conti- nents. Stowaway rats and other vermin in the holds of ships traveled down the moorings when the ships docked, bringing fleas, lice, and deadly pathogens to a new world. Sporadic epidemics of plague, small- pox, typhus, and measles ravaged cities, decimated armies, and altered the course of history. 19th Century Discoveries Lead to Infectious Disease Prevention and Control Control of many infectious diseases became pos- sible with the pioneering work of Robert Koch and Louis Pasteur and the introduction of the germ theory of disease. With bacteriologic cultivation techniques came the first isolation and identifica- tion of etiologic agents; virus cultivation and identi- fication became available some decades later. Reservoirs of microorganisms and their life cycles were identified; the epidemiology and natural his- tory of many infectious diseases were described, and successful control measures were initiated. Water Address for correspondence: David Satcher, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA, fax 404-639-3039, e-mail eideditor@cidod1.em.cdc.gov. Perspectives Vol. 1, No.

ral his- tory of many infectious diseases were described, and successful control measures were initiated. Water Address for correspondence: David Satcher, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30333, USA, fax 404-639-3039, e-mail eideditor@cidod1.em.cdc.gov. Perspectives Vol. 1, No. 1 -- January-March 1995 1 Emerging Infectious Diseases treatment, vector control, and rodent reduction pro- grams followed. By the beginning of the 20th cen- tury, the principles of vaccination, established empirically by Edward Jenner more than 100 years earlier, began to be realized in earnest. Antibiotics were discovered, and disinfectants were developed. Collectively, these control measures dramatically decreased the incidence and prevalence of many infectious diseases and their fatality rates.The early part of this century is appropriately regarded as a golden age in public health. New and Reemerging Infectious Diseases--A Contemporary Problem Compared with earlier generations, we possess an enormous scientific base, and the rate of acquisi- tion of new information about infectious diseases is at a historic high. Moreover, thanksin large measure to effective childhood immunization programs, in- cluding the President's Childhood Immunization In- itiative, many infectious diseases are under control, particularly in the industrialized world. The elimi- nation of smallpox in 1977 stands as a towering achievement in the fight against infectious diseases. However, many infectious diseases have persisted and have displayed a remarkable ability to re- emerge after lengthy periods of stability. Therefore, we must be ever mindful of the cyclical nature of disease trends. A careful review of infectious disease trends shows a fragile equilibrium between humans and infectious microorganisms. Infectious diseases are still broadly endemic and maintain a large reservoir of agents that have the potential for rapid and wide- spread dissemination. Infectious diseases remain the leading cause of death worldwide, even as the International Code of Diseases places many infec- tious diseases in other categories. For example, men- ingitis and cirrhosis are classified as diseases of the nervous system and liver, respectively, and only 17% of deaths attributable to infections are actually in- cluded in the codes for parasitic and infectious dis- eases (6).

al Code of Diseases places many infec- tious diseases in other categories. For example, men- ingitis and cirrhosis are classified as diseases of the nervous system and liver, respectively, and only 17% of deaths attributable to infections are actually in- cluded in the codes for parasitic and infectious dis- eases (6). In the United States, each year, approximately 25% of physician visits are attribut- able to infectious diseases, with direct and indirect costs, including those for human immunodeficiency virus (HIV) infection and related illnesses, esti- mated at more than $120 billion (7). Persons living in tropical climates are still as vulnerable to infectious diseases as their early an- cestors were. Each year more than one million chil- dren die of malaria in sub-Saharan Africa alone (8); worldwide, approximately 200 million people have schistosomiasis (9), and each year 35-60 million peo- ple contract dengue (10). Moreover, infectious dis- eases and their attendant problems are not confined to tropical climates. For example, an estimated 600,000 cases of pneumonia occur in the United States each year and cause 25,000 to 50,000 deaths (11). More than 10,000 cases of diphtheria have occurred in Russia since 1993 because of inadequate levels of immunization (12). Despite a century of scientific progress, infectious diseases still cause enormous human suffering, deplete scarce re- sources, impede social and economic development, and contribute to global instability. The potential for even greater dissemination looms as a continuous threat. Recent outbreaks underscore the potential for the sudden appearance of infectious diseases in cur- rently unaffected populations. In the United States, contamination of the municipal water supply in Mil- waukee, Wisconsin, in 1993 resulted in an outbreak of cryptosporidiosis that affected an estimated 400,000 people; approximately 4,400 persons re- quired hospitalization (13). In the 1990s, epidemic cholera reappeared in the Americas, after being ab- sent for nearly a century; from 1991 through June of 1994 more than one million cases and nearly 10,000 deaths were reported (14). During the 1980s, tuberculosis reemerged in the United States after decades of decline, and drug-resistant strains have made its control more difficult (15,16). The increas- ing prevalence of antibiotic-resistant strains of gonococci, pneumococci, enterococci, and staphylo- cocci portend of other serious treatment and control failures.

tuberculosis reemerged in the United States after decades of decline, and drug-resistant strains have made its control more difficult (15,16). The increas- ing prevalence of antibiotic-resistant strains of gonococci, pneumococci, enterococci, and staphylo- cocci portend of other serious treatment and control failures. Many other examples of emerging infec- tions could be given (17,18). New infectious diseases, often with unknown long-term public health impact, continue to be iden- tified. Table 1 lists major diseases or etiologic agents identified just within the last 20 years (19-41). New agents are regularly added to the list, particularly with the availability of nucleic acid amplification techniques for detecting and identifying otherwise noncultivable microorganisms (40, 42). In some cases, etiologic agents have been identi- fied as the causes of known diseases or syndromes (e.g., rotavirus, parvovirus, human T-cell lympho- tropic viruses I and II (HTLV I/II), and human her- pesvirus type 6, (HHV-6); in other cases, diseases became better recognized or defined (e.g., Legion- naires' disease, Lyme disease, human ehrlichiosis). Still others are entirely new: with some parallels to medieval times, a previously unknown and deadly disease, acquired immunodeficiency syndrome (AIDS), originated from uncertain sources in one part of the world and became globally disseminated; this time the disease spread at a rate that would have been unthinkable in medieval times. Clearly, the complete history of infectious diseases remains to be written. Perspectives Emerging Infectious Diseases 2 Vol. 1, No. 1 -- January-March 1995 Getting Ahead of the Curve Recent disquieting infectious disease trends have not gone unrecognized, and their similarity to earlier disease trends with immense global consequences has not gone unnoticed. Primary responsibility for addressing new and reemerging infectious diseases rests squarely with the custodians of public health. Indeed, the fundamental maxim of public health must guide current prevention programs: the health of the individual is best ensured by maintaining or improving the health of the entire community.

ry responsibility for addressing new and reemerging infectious diseases rests squarely with the custodians of public health. Indeed, the fundamental maxim of public health must guide current prevention programs: the health of the individual is best ensured by maintaining or improving the health of the entire community. Core functions necessary to ensure the health of the pub- lic were defined in the National Academy of Science's Institute of Medicine (IOM) report on The Future of Public Health (43): * Assessment of health status, risks, and services * Development of health policy * Assurance of quality health services Surveillance (assessment) is the sine qua non of infectious disease prevention programs; however, for surveillance to be effective it must be specific. Con- sider, for example, surveillance of viral hepatitis. Only after the various agents of viral hepatitis were identified and specific laboratory testing became available was it possible to explain trends in disease prevalence and establish the epidemiologic princi- ples underlying the different modes of transmission. Specific laboratory testing is also the basis of screen- ing programs that ensure the safety of the blood supply against hepatitis B and hepatitis C. Agent- specific surveillance is a critical component of many immunization programs. Vaccines to Haemophilus influenzae type b, (Hib), for example, were developed in response to laboratory-based surveillance that identified Hib as a major cause of invasive disease in children. The effectiveness of the Hib vaccination campaign in the United States has been dramatic (Figure 1). Similar approaches will ensure appropri- ate formulation of other developmental vaccines. Monitoring antibiotic resistance is yet another im- portant example of the value of laboratory-based Table 1. Major Etiologic Agents, Infectious Diseases Identified Since 1973* Year Agent Disease Reference 1973 Rotavirus Major cause of infantile diarrhea worldwide 19 1975 Parvovirus B19 Fifth disease; Aplastic crisis in chronic hemolytic anemia 20 1976 Cryptosporidium parvum Acute enterocolitis 21 1977 Ebola virus Ebola hemorrhagic fever 22 1977 Legionella pneumophila Legionnaires' disease 23 1977 Hantaan virus Hemorrhagic fever with renal syndrome (HFRS) 24 1977 Campylobacter sp.

1975 Parvovirus B19 Fifth disease; Aplastic crisis in chronic hemolytic anemia 20 1976 Cryptosporidium parvum Acute enterocolitis 21 1977 Ebola virus Ebola hemorrhagic fever 22 1977 Legionella pneumophila Legionnaires' disease 23 1977 Hantaan virus Hemorrhagic fever with renal syndrome (HFRS) 24 1977 Campylobacter sp. Enteric pathogens distributed globally 25 1980 Human T-cell lymphotropic virus-I (HTLV I) T-cell lymphoma--leukemia 26 1981 Staphylococcus toxin Toxic shock syndrome associated with tampon use 27 1982 Escherichia coli O157:H7 Hemorrhagic colitis; hemolytic uremic syndrome 28 1982 HTLV II Hairy cell leukemia 29 1982 Borrelia burgdorferi Lyme disease 30 1983 Human immunodeficiency virus (HIV) Acquired immunodeficiency syndrome (AIDS) 31 1983 Helicobacter pylori Gastric ulcers 32 1988 Human herpesvirus-6 (HHV-6) Roseola subitum 33 1989 Ehrlichia chaffeensis Human ehrlichiosis 34 1989 Hepatitis C Parenterally transmitted non-A, non-B hepatitis 35 1991 Guanarito virus Venezuelan hemorrhagic fever 36 1992 Vibrio cholerae O139 New strain associated with epidemic cholera 37 1992 Bartonella (= Rochalimaea) henselae Cat-scratch disease; bacillary angiomatosis 38, 39 1993 Hantavirus isolates Hantavirus pulmonary syndrome 40 1994 Sabia virus Brazilian hemorrhagic fever 41 *Compiled by CDC staff. Dates of discovery are assigned on the basis of the year the isolation or identification of etiologic agents was reported. Perspectives Vol. 1, No. 1 -- January-March 1995 3 Emerging Infectious Diseases surveillance. Within this context, current discover- ies of etiologic agents and diseases (Table 1) are reasons for optimism. The potential for improve- ments in assessment and prevention of these and other newly discovered diseases is reminiscent of the watershed years of Koch and Pasteur. We cannot overstate the role of behavioral science in our effort to "get ahead of the curve" with emerg- ing infections. Having the science or laboratory tech- nology to control infectious diseases is not enough, unless we can influence people to behave in ways that minimize the transmission of infections and maximize the efforts of medical interventions. For example, even though HIV/AIDS does not have a vaccine or cure, it is almost entirely preventable. For many people, however, reducing the risk for HIV infection and AIDS requires important changes in lifestyle or behavior. We must use our knowledge of human behavior to help people make lifestyle changes and prevent disease.

ple, even though HIV/AIDS does not have a vaccine or cure, it is almost entirely preventable. For many people, however, reducing the risk for HIV infection and AIDS requires important changes in lifestyle or behavior. We must use our knowledge of human behavior to help people make lifestyle changes and prevent disease. Another illustration of the need to use behavior science is the problem of antibiotic resistance. To a great extent, this problem is related to the behavior of both physicians and patients. Physicians continue to use antibiotics inappropriately, and patients con- tinue to demand antibiotic treatment when it is not indicated, for example, for the common cold. As soci- ety changes and institutions such as day care centers and prisons become more crowded, the spread of infectious diseases is exacerbated. For homeless and drug-dependent populations, completing a 6- to 9- month course of therapy for tuberculosis is difficult, and the failure to complete the therapy increases the risk for drug-resistant tuberculosis in the commu- nity. Microbiologists, infectious disease specialists, and other basic and clinical scientists must collabo- rate with behavioral scientists in an interdiscipli- nary effort to prevent and control emerging infections. The Future of Public Health emphasizes the rela- tionship between a sound public health infrastruc- ture and infectious disease prevention programs. Infrastructure improvements must become a na- tional priority: certainly they are among CDC's top priorities. Improvements in infectious disease sur- veillance are particularly needed (44). Enriching the capacity to respond to urgent threats to health and developing nationwide prevention strategies are also CDC priorities. To combat new and reemerging infectious diseases, CDC, in collaboration with other federal agencies, state and local health depart- ments, academic institutions, professional societies, international organizations, and experts in public health infectious diseases and medical microbiology developed a plan--Addressing Emerging Infectious Disease Threats: A Prevention Strategy for the United States (7).

federal agencies, state and local health depart- ments, academic institutions, professional societies, international organizations, and experts in public health infectious diseases and medical microbiology developed a plan--Addressing Emerging Infectious Disease Threats: A Prevention Strategy for the United States (7). The plan has four major goals: * Surveillance and Response--detect, promptly investigate, and monitor emerging pathogens, the diseases they cause, and the factors influenc- ing their emergence * Applied research--integrate laboratory sci- ence and epidemiology to optimize public health practice * Prevention and control--enhance communi- cation of public health information about emerg- ing diseases and ensure prompt implementation of prevention strategies * Infrastructure--strengthen local, state, and federal public health infrastructures to support surveillance and implement prevention and con- trol programs CDC's plan provides a framework for the agency to work collaboratively with its many partners to identify and reverse worrisome trends in infectious diseases. The need for implementing CDC's plan is urgent, given the extremely dynamic nature of disease trends and the complexity of factors contributing to disease emergence; these were outlined in detail in the 1992 IOM report--Emerging Infections: Micro- bial Threats to Health in the United States (45) and are discussed in a companion article by Stephen S. Morse, Ph.D., in this issue. The IOM report concludes that infectious diseases must be viewed as but one component of a dynamic and complex global ecology, which is shaped and buffeted by technologic, socie- tal, economic, environmental, and demographic Figure 1. Race-adjusted incidence rate* of Haemophilus influenzae type b and non-type b disease detected through laboratory-based surveillance among children aged <5 years -- United States, 1989-1993 *Per 100,000 children aged <5 years.

d and buffeted by technologic, socie- tal, economic, environmental, and demographic Figure 1. Race-adjusted incidence rate* of Haemophilus influenzae type b and non-type b disease detected through laboratory-based surveillance among children aged <5 years -- United States, 1989-1993 *Per 100,000 children aged <5 years. The surveillance area population was 10.4 million in four states (three counties in the San Francisco Bay Area, eight counties in metropolitan Atlanta, four counties in Tennessee, and the state of Oklahoma. Source: CDC. Progress toward elimination of Haemophilus influenzae type b disease among infants and children -- United States, MMWR 1994;43:144-8. Perspectives Emerging Infectious Diseases 4 Vol. 1, No. 1 -- January-March 1995 changes, not to mention microbial change and adap- tation. Clearly, broader coalitions are needed, and com- munication must improve if we are to "get ahead of the curve." This new periodical is part of the overall strategy to draw worldwide attention to emerging infections and improve communication. Given the multiplicity of factors contributing to disease emer- gence, Emerging Infectious Diseases (EID) will pre- sent relevant concepts from professionals in multiple disciplines and disseminate information about emerging infectious diseases in order to de- velop and apply ecologically acceptable interven- tions that will benefit humankind. Prevention and control of new and emerging infectious diseases de- pend on the participation of scientists and other professionals in the public and private sectors. CDC will make EID available by print and elec- tronically to facilitate rapid communication. We hope that in the process EID will promote the ex- change of infectious disease information through global electronic networks and bulletin boards. Although there are many similarities between our vulnerability to infectious diseases and that of our ancestors, there is one distinct difference: we have the benefit of extensive scientific knowledge. Ultimately, our success in combatting infectious dis- eases will depend on how well we use available information. A recent report by the Carnegie Com- mission "Science, Technology, and Government for a Changing World," provides valuable insight in this regard (46).

we have the benefit of extensive scientific knowledge. Ultimately, our success in combatting infectious dis- eases will depend on how well we use available information. A recent report by the Carnegie Com- mission "Science, Technology, and Government for a Changing World," provides valuable insight in this regard (46). Commenting on the Earth Summit in Rio de Janeiro in 1992, the report emphasizes the need to shift from the "manifestations of environ- mental changes in the air, land, water, and plant and animal kingdoms to the causes of those changes." Indeed, the advice of that report challenges us all-- "our capacity to generate, integrate, disseminate, and apply knowledge will determine the human prospect in the 21st century." Dr. Satcher is director of the Centers for Disease Control and Prevention, Atlanta, Georgia. He was president of Meharry Medical College, Nashville, Tennessee, from 1982 to 1993 and is a former faculty member of the King-Drew Medical Center and the UCLA School of Medicine, Los Angeles, California. References 1. Zinsser H. Rats, lice and history. Boston: Little, Brown, and Company, 1935. 2. Hopkins DR. Princes and peasants: smallpox in his- tory. Chicago: University of Chicago Press, 1983. 3. Bollet AJ. Plagues and poxes. New York: Demos Pub- lications, 1987. 4. Burnet M, White DO. Natural history of infectious disease. London: Cambridge University Press, 1972. 5. McNeill WH. Plagues and peoples. Garden City, New York: Anchor Press/Doubleday, 1976. 6. Bennett JV, Holmberg SD, Rogers MF, Solomon SL. Infectious and parasitic diseases. In: Amler RW, Dull HB, editors. Closing the gap: the burden of unneces- sary illness. New York:Oxford University Press, 1987. 7. Centers for Disease Control and Prevention. Address- ing emerging infectious disease threats: a prevention strategy for the United States. Atlanta, Georgia: U.S. Department of Health and Human Services, Public Health Service, 1994. 8. World Health Organization. Severe and complicated malaria. Trans R Soc Trop Med Hyg 1990; 84:Supp 2:1-65. 9. World Health Organization. Tropical disease re- search: progress 1991-92--Eleventh Programme Re- port of the UNDP/World Bank, WHO Special Programme for Research and Training in Tropical Diseases (TDR). Geneva: World Health Organization, 1993. 10. Gubler DJ. Vector-borne diseases. In: Encyclopedia of the environment. New York: Houghton Mifflin Co., 1994. 11. Marston BJ, Plouffe JF, Breiman RF, et al.

Programme Re- port of the UNDP/World Bank, WHO Special Programme for Research and Training in Tropical Diseases (TDR). Geneva: World Health Organization, 1993. 10. Gubler DJ. Vector-borne diseases. In: Encyclopedia of the environment. New York: Houghton Mifflin Co., 1994. 11. Marston BJ, Plouffe JF, Breiman RF, et al. Prelimi- nary findings of a community-based pneumonia inci- dence study. In: Barbaree JM, Breiman RF, DufourAP, editors. Legionella: current status and emerging per- spectives. Washington, D.C.; American Society for Mi- crobiology, 1993. 12. Centers for Disease Control and Prevention. Diphthe- ria outbreak--Russian Federation, 1990-1993. MMWR 1993; 42:840-1, 847. 13. MacKenzie WR, Hoxie NJ, Proctor ME, et al. A mas- sive outbreak in Milwaukee of Cryptosporidium infec- tion transmitted through the public water supply. N Engl J Med 1994; 331:161-7. 14. Organizacion Panamericana de la Salud. El colera en las Americas. Informe No. 10; Junio 1994. 15. Centers for Disease Control and Prevention. Ex- panded tuberculosis surveillance and tuberculosis morbidity--United States, 1993. MMWR 1994; 43:361-6. 16. Centers for Disease Control and Prevention. Multi- drug-resistant tuberculosis in a hospital--Jersey City, New Jersey, 1990-1992. MMWR 1994; 43:417-9. 17. Murphy FA. New, emerging, and reemerging infec- tious diseases. Adv Virus Res 1994; 43: 1-52. 18. Morse SS, editor. Emerging viruses. New York: Oxford University Press, 1993. 19. Bishop RF, Davidson GP, Holmes IH, Ruck BJ. Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet 1973; 2:1281-3. 20. Cossart YE, Field AM, Cant B, Widdows D. Par- vovirus-like particles in human sera. Lancet 1975; 1:72-3. 21. Nime FA, Burek JD, Page DL, Holscher MA, Yardley JH. Acute enterocolitis in a human being infected with the protozoan Cryptosporidium. Gastroenterology 1976; 70: 592-8. 22. Johnson KM, Webb PA, Lange JV, Murphy FA. Isola- tion and partial characterization of a new virus caus- ing acute haemorrhagic fever in Zaire. Lancet 1977; 1:569-71. Perspectives Vol. 1, No. 1 -- January-March 1995 5 Emerging Infectious Diseases 23. McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR, Laboratory Investigation Team. Le- gionnaires' disease. 2: Isolation of a bacterium and demonstration of its role in other respiratory disease. N Engl J Med 1977; 297:1197-1203. 24. Lee HW, Lee PW, Johnson KM. Isolation of the etio- logic agent of Korean hemorrhagic fever.

d CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR, Laboratory Investigation Team. Le- gionnaires' disease. 2: Isolation of a bacterium and demonstration of its role in other respiratory disease. N Engl J Med 1977; 297:1197-1203. 24. Lee HW, Lee PW, Johnson KM. Isolation of the etio- logic agent of Korean hemorrhagic fever. J Infect Dis 1978; 137:298-308. 25. Skirrow MB. Campylobacter enteritis: a "new" dis- ease. Br Med J 1977; 2:9-11. 26. Poiesz BJ, Ruscetti FW, Gazdar AF, Bunn PA, Minna JD, Gallo RC. Detection and isolation of type C retrovirus particles from fresh and cultured lympho- cytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci 1980; 77: 7415-9. 27. Schlievert PM., Shands KN, Gan BB, Schmid GP, Nishimura RD. Identification and characterization of an exotoxin from Staphylococcus aureus associated with toxic shock syndrome. J Infect Dis 1981; 143:509- 16. 28. Riley LW, Remis RS, Helgerson SD, et al. Hemor- rhagic colitis associated with a rare Escherichia coli serotype. N Engl J Med 1983; 308:681-5. 29. Kalyanaraman S, Sarangadharan MG, Poiesz B, Ruscetti FW, Gallo RC. Immunological properties of a type C retrovirus isolated from cultured human T- lymphoma cells and comparison to other mammalian retroviruses, J Virol 1981; 38:906-15. 30. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, Davis JP. Lyme disease-a tick-borne spirochetosis?. Science 1982; 216:1317-9. 31. Barre-Sinoussi F, Chermann JC, Rey F, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome. Science 1983; 220:868-71. 32. Marshall B. Comment in: Lancet 1983; 1:1273-5. 33. Yamanishi K, Okuno T, Shiraki K, Takahashi M, Kondo T, Asano Y, Kurata T. Identification of human herpesvirus-6 as a causal agent for exanthem subi- tum. Lancet 1988; 1:1065-7. 34. Dawson JE, Anderson BE, Fishbein DB, Sanchez JL, Goldsmith CS, Wilson KH, Duntley CW. Isolation and characterization of an Ehrlichia sp. from a patient diagnosed with human ehrlichiosis. J Clin Microbiol 1991; 29:2741-5. 35. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 1989; 244:359-61. 36. Salas R, de Manzione N, Tesh RB, Rico-Hesse R, Shope RE, Betancourt A, Goday O, Bruzual R, Pacheco ME, Ramos B, Taibo ME, Tamayo JG, Jaimes E, Vasquez C, Araoz, F, Querales J. Venezuelan hem- orrhagic fever. Lancet 1991; 338:1033-6. 37. World Health Organization.

-B viral hepatitis genome. Science 1989; 244:359-61. 36. Salas R, de Manzione N, Tesh RB, Rico-Hesse R, Shope RE, Betancourt A, Goday O, Bruzual R, Pacheco ME, Ramos B, Taibo ME, Tamayo JG, Jaimes E, Vasquez C, Araoz, F, Querales J. Venezuelan hem- orrhagic fever. Lancet 1991; 338:1033-6. 37. World Health Organization. Epidemic diarrhea due to Vibrio cholerae non-01. Wkly Epidemiol Rec 1993; 68:141-2. 38. Regnery RL, Anderson BE, Clarridge JE III, Ro- driguez-Barradas MC, Jones DC, Carr JH. Charac- terization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immu- nodeficiency virus-positive patient. J Clin Microbiol 1992; 30:265-74. 39. Welch DF, Pickett DA, Slater LN, Steigerwalt AG, Brenner DJ. Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J Clin Microbiol 1992; 30:275-80. 40. Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, Feldmann H, Sanchez A, Childs J, Zaki S, Peters, CJ. Genetic identification of a hantavirus associated with an outbreak of acute respiratory ill- ness. Science 1993; 262:914-7. 41. Lisieux T, Coimbra M, Nassar ES, Burattini MN, de Souza LT, Ferreira I, Rocco IM, daRosa AP, Vascon- celos PF, Pinheiro FP, et al. New arenavirus isolated in Brazil. Lancet 1994; 343:391-2. 42. Relman DA, Falkow S, LeBoit PE, Perkocha LA, Min K-W, Welch DF, Slater LN. The organism causing bacillary angiomatosis, peliosis hepatitis, and fever and bacteremia in immunocompromised patients. N Engl J Med 1991; 324:1514. 43. Institute of Medicine. The future of public health. Washington, D.C.: National Academy Press, 1988. 44. Berkelman RL, Bryan RT, Osterholm MT, LeDuc JW, Hughes JM. Infectious disease surveillance: a crum- bling foundation. Science 1994; 264: 368-70. 45. Institute of Medicine. Emerging infections: microbial threats to health in the United States. Washington, D.C.: National Academy Press, 1992. 46. Malone TF. The institutions of science and the global prospect: the case of environment. In: Science, tech- nology, and government for a changing world: the concluding report of the Carnegie Commission on Science, Technology, and Government. New York: Carnegie Commission, 1993. Perspectives Emerging Infectious Diseases 6 Vol. 1, No. 1 -- January-March 1995

Unraveling Mysteries Associated with Cat-Scratch Disease, Bacillary Angiomatosis, and Related Syndromes Russell Regnery, Ph.D., and Jordan Tappero, M.D. National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA The search for the infectious agents responsible for cat-scratch disease, bacillary angiomatosis, and related syndromes has a long and often circuitous history. Recog- nition of the etiologic agents and a new understanding of the fundamental features of the epidemiology and natural history of modern dayBartonella (formerly Rochali- maea)-associated diseases culminate a multipartite story that combines clinical medicine, traditional microbiology, and novel technological approaches to solve a long-standing enigma. The quest for the etiologic agent of cat-scratch disease (CSD) has frequently been described as a mystery (1,2). Indeed, the search has many qualities of a mystery novel; the pursuit has spanned several decades and recently taken several unexpected turns. During this period of important discovery, major microbial suspects have undergone name changes, novel microbial culprits have been intro- duced, new groups of affected patients have been recognized, and yet significant questions remain to be answered. Scientific and medical interest has been high; approximately 900 publications have dealt with CSD since the first good clinical descrip- tion of the disease in 1950 (3). Clinical Features of CSD Throughout the life of this evolving mystery, the clinical descriptions of "classical" CSD have re- mained remarkably consistent (Dalton MJ, et al. Rochalimaea antibody; a new era in the diagnosis of cat-scratch disease, submitted for publication; 4-6). CSD is typically a benign and self-limited illness lasting 6 to 12 weeks in the absence of antibiotic therapy. Regional lymphadenopathy (axillary, head and neck, inguinal) is the predominant clinical fea- ture of CSD; affected nodes are often tender and occasionally suppurate (4-7). Between 25% and 60% of patients report a primary cutaneous inoculation lesion (0.5- to 1-cm papule or pustule) at the site of a cat scratch or bite (5,7).

l lymphadenopathy (axillary, head and neck, inguinal) is the predominant clinical fea- ture of CSD; affected nodes are often tender and occasionally suppurate (4-7). Between 25% and 60% of patients report a primary cutaneous inoculation lesion (0.5- to 1-cm papule or pustule) at the site of a cat scratch or bite (5,7). The skin lesions typically develop 3 to 10 days after injury and precede the onset of lymphadenopathy by 1 to 2 weeks. Low- grade fever and malaise accompany lymphade- nopathy in up to 50% of patients; headache, ano- rexia, weight loss, nausea and vomiting, sore throat, and splenomegaly may develop. In addition, short- lived, non-specific maculopapular eruptions, erythema nodosum, figurate erythemas, and throm- bocytopenic purpura have been observed (8). Unusual manifestations of CSD, which occur in up to 14% of patients, include Perinaud's oculoglan- dular syndrome (6%), encephalopathy (2%), hepatic granulomas (0.3%), osteomyelitis(0.3%), and pulmo- nary disease (0.2%) (4,5,8). In general, these compli- cations resolve without sequelae. Perinaud's oculoglandular syndrome is manifested by conjuncti- val granuloma, periauricular lymphadenopathy, and nonsuppurative conjunctivitis. Encephalopathy, manifested as fever and coma that progress to con- vulsions, may last for days to weeks; cerebrospinal fluid is unremarkable. Optic neuritis with transient blindness may also occur. For many years, CSD has been clinically diag- nosed when three of the following four criteria are met in a patient: 1) history of traumatic cat contact; 2) positive skin-test response to CSD skin-test anti- gen; 3) characteristic lymph node lesions; and 4) negative laboratory investigation for unexplained lymphadenopathy (8). Althoughbiopsy confirmation of CSD has been rarely required (especially in lieu of a reliable serologic test--see below), a constant pathologic hallmark of CSD-affected tissues has been granuloma formation. With hematoxylin and eosin stains, the primary inoculation lesion of CSD reveals small areas of frank necrosis surrounded by concentric layers of histiocytes, lymphocytes, and nucleated giant cells (9). Affected lymph nodes are characterized by necrotizing granulomas ringed by lymphocytic infiltrates and multinucleated giant cells. Address for correspondence: Russell Regnery, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G13, Atlanta, GA 30333, USA; fax 404-639-1058; e-mail rur1@ciddvd1.em.cdc.gov. Synopsis Vol. 1, No.

are characterized by necrotizing granulomas ringed by lymphocytic infiltrates and multinucleated giant cells. Address for correspondence: Russell Regnery, Centers for Disease Control and Prevention, 1600 Clifton Road, MS G13, Atlanta, GA 30333, USA; fax 404-639-1058; e-mail rur1@ciddvd1.em.cdc.gov. Synopsis Vol. 1, No. 1 -- January-March 1995 16 Emerging Infectious Diseases Enter Afipia felis During the past 44 years, a variety of microbial agents, including herpes viruses and bacteria of the genera Chlamydia and Pasteurella, have been sus- pected as the causes of CSD (3). A major chapter of the CSD saga unfolded with the 1988 announcement by the Armed Forces Institute of Pathology that a bacterial agent had been visualized within CSD-in- volved lymph nodes by using the Warthin-Starry silver stain (10), and a novel bacterial agent had been isolated from a CSD patient's lymph node (11). By 1992, this agent was characterized fully, given the name Afipia felis (Afipia being a latinized acro- nym for the source of the original isolate, the Armed Forces Institute of Pathology, and felis referring to the presumed vertebrate vector of the human infec- tion), and proclaimed the agent of CSD (12). Although A. felis was declared the putative CSD bacillus, evidence of convincing patient humoral or cellular immune responses to laboratory culturedA. felis antigen was not forthcoming. Despite numerous attempts, other laboratories were unable to recover additional isolates of A. felis from CSD patients. In addition, although the majority of patients with CSD reported exposure to a cat(s), no clear link between cats and A. felis was demonstrated. Enter New Syndromes The story of CSD took a significantly divergent path with the recognition that opportunistic infec- tions were an important consideration for patients infected with human immunodeficiency virus (HIV). Bacillary angiomatosis (BA), a newly recognized dis- ease characterized by cutaneous and subcutaneous vascular lesions containing bacillary organisms visualized by Warthin-Starry silver staining, was described predominantly among HIV-infected pa- tients; however, bacterial isolates were not made or identified (13-15). Over the ensuing decade, the clinical spectrum of BA was expanded to include patients with single or multiple vascular lesions affecting virtually every organ system, including lymph node, bone, brain, liver (peliosis hepatis), and spleen (14-17).

tients; however, bacterial isolates were not made or identified (13-15). Over the ensuing decade, the clinical spectrum of BA was expanded to include patients with single or multiple vascular lesions affecting virtually every organ system, including lymph node, bone, brain, liver (peliosis hepatis), and spleen (14-17). Independently,an unidentified gram- negative pathogen was isolated predominantly from HIV-infected patients with fever and bacteremia, however, these patients lacked cutaneous or parenchymal vascular lesions and were not recog- nized as BA patients (18). Because silver staining and electron microscopy of both BA and CSD tissue sections revealed bacil- lary organisms indistinguishable from one another, several authors suggested that BA might represent disseminated CSD in the immunocompromised host (17,19-21). In addition, several anecdotal reports of BA described a history of cat contact preceding the onset of disease (22). Ultimately, the relationships between possible environmental exposures and BA or CSD were sys- tematically investigated. The first case-control study conducted among patients with BA found traumatic contact with a cat (bite or scratch) to be significantly associated with BA disease (22). BA patients were also more likely than controls to have ahousehold kitten (acat <1 year ofage).Asubsequent case-control study of CSD patients found that these patients were more likely than controls to have trau- maticcontactwith acat, to ownat leastonekitten,and to have kittens with fleas (7). Despite the similarities in histochemical staining properties and epidemiology, serious reservations remained concerning a possible link between the causative agents of CSD and BA. The pathologic features of classical CSD (granuloma) and BA (pro- liferative vascular lesions without granuloma) were distinctly different, and the two diseases seemed to respond differently to antibiotic therapy. Although antimicrobial therapy for BAand CSD have not been systematically evaluated, the majority of BA pa- tients evaluated responded quickly to single-agent therapy with either erythromycin or doxycycline (14,23), whereas the symptoms and signs of patients with CSD failed to show consistent rapid resolution following antibiotic therapy (5). In addition, clini- cians' first choices of antibiotics for treating BA and CSD vary (5,6,14,23).

ed responded quickly to single-agent therapy with either erythromycin or doxycycline (14,23), whereas the symptoms and signs of patients with CSD failed to show consistent rapid resolution following antibiotic therapy (5). In addition, clini- cians' first choices of antibiotics for treating BA and CSD vary (5,6,14,23). Enter Rochalimaea henselae A breakthrough occurred when a novel approach was used to identify possible prokaryotic ribosomal DNAextracted from BAskin lesions. When prokary- otic ribosomal gene DNA extracted from BA lesions and amplified by polymerase chain reaction (PCR) was compared with sequenced ribosomal genes from other organisms, it become apparent that the agent associated with BA in this study was related to, but not necessarily identical to, the agent of trench fever, Rochalimaea quintana (24). At nearly the same time in Oklahoma, Rochali- maea-like organisms were being isolated on blood agar from bacteremic patients (18). Independently in Houston, Texas, fastidious, slow-growingRochali- maea-like isolates were recovered on several occa- sions from the blood of an HIV-infected patient with relapsing fever of unknown origin; like the isolates from the Oklahoma patients, the Houston isolate was recovered from a patient in the absence of BA or CSD lesions (25). The Houston isolate (Houston-1) was identified as the prototype isolate of a novel species of Rochalimaea by using traditional as well as genotypic methods, including ribosomal RNA gene analysis similar to that used to identify the nucleic acid found in BA patients' lesions (25). Al- most simultaneously, the group from Oklahoma had Synopsis Emerging Infectious Diseases 17 Vol. 1, No. 1 -- January-March 1995 come to a similar conclusion by using DNA related- ness data (26); most of their isolates also consisted of the novel species, R. henselae. The new species designation, first officially used to describe the Houston-1 isolate, was coined in recognition of the contribution of Diane Hensel, a microbiologist who had isolated several of the initial organisms in Okla- homa (18,25,26). Subsequently, Koehler et al. iso- lated bacilli directly from cutaneous lesions of persons with BA(27); surprisingly,eitherR.henselae or R. quintana was isolated from BA lesions from different HIV-infected patients. At this juncture, R. henselae infections had been described predominantly among immunocom- promised patients with either BA or fever with bac- teremia.

directly from cutaneous lesions of persons with BA(27); surprisingly,eitherR.henselae or R. quintana was isolated from BA lesions from different HIV-infected patients. At this juncture, R. henselae infections had been described predominantly among immunocom- promised patients with either BA or fever with bac- teremia. The availability of isolates made it possible to develop a test for serologic evidence of Rochali- maea infection and to refine PCR methods for iden- tification of Rochalimaea organisms in tissues and other samples. These methods, together with new techniques for recovering Rochalimaea species iso- lates, were crucial to obtaining a more detailed ac- count not only of BA but also of CSD. The Cat-scratch Connection: A Synthesis A Rochalimaea genus-specific, indirect fluores- cence antibody (IFA) test using irradiated whole cell antigen from the Houston-1 isolate of R. henselae was developed by the Centers for Disease Control and Prevention (CDC) to help identify risk factors forRochalimaea-associated disease. Several blinded serum samples from both HIV-infected BA patients and HIV-infected controls residing in San Francisco were sent to CDC for serologic testing. High-titered antibodies were identified in serum samples from several of the BA patients (28). Similar high-titered antibodies were not detected for any of non-BA con- trol patients with one exception; a serum sample from an HIV-infected patient with CSD also demon- strated strong serologic reactivity to R. henselae antigen. Shortly thereafter, single sera collected from pa- tients with suspected CSD to look for A. felis anti- bodies were evaluated with the new R. henselae serologic test; 36 (88%) of 41 sera were positive (29). None of the sera had significantly elevated titers to A. felis antigen. The same set of sera were coded and resubmitted along with sera taken from other well- characterized bacterial and viral diseases and tested again in a blinded manner. The IFA test accurately identified sera of case-patients with suspected CSD. In addition, 6 (6%) of 107 sera from ostensibly healthy persons, obtained from a commercial vendor, showed antibodyby IFAtesting (29). These serologic datawere the first laboratory evidence suggesting that R. hense- lae was associated with CSD. Data further substantiating the role of R. hense- lae in the etiology of CSD soon followed.

6%) of 107 sera from ostensibly healthy persons, obtained from a commercial vendor, showed antibodyby IFAtesting (29). These serologic datawere the first laboratory evidence suggesting that R. hense- lae was associated with CSD. Data further substantiating the role of R. hense- lae in the etiology of CSD soon followed. The newly developed serologic test was used to help investigate a possible cluster of CSD cases in Connecticut; 38 (84%) of 45 suspected CSD cases had elevated Ro- chalimaea antibody titers, whereas 4 (3.6%) of 112 age-matched controls had detectable antibody titers (7). In another investigation, serum samples ob- tained from 600 prospectively evaluated patients with well-characterized CSD (i.e., persons with his- tory of cat scratch, papule at site of inoculation, and enlarged regional lymph node) had a 95% correla- tion with positive Rochalimaea serology. In 1993, R.henselaewas isolated directly from the lymph nodes of two CSD patients and was identified by genotypic means; both patients had strong sero- logic responses to Rochalimaea antigen (30). Evi- dence of R. henselae-specific nucleic acid sequences were found in 21 (84%) of 25 CSD lymph node tissues submitted to CDC for evaluation (31). Additional supporting evidence for a Rochali- maea as the cause of CSD came from archival sources. Skin-test antigen, used rather extensively in the past to help diagnose examples of CSD (4,8), consisted of pasteurized exudate collected from sup- purative CSD lymph nodes. Among a cohort of CSD patients who were skin-test positive, 52 (93%) of 56 had positive IFA antibody titers to the defined Ro- chalimaea reagents (32). Furthermore, various lots of skin-test antigen were shown by PCR analysis to contain Rochalimaea nucleic acid sequences (33), and R. henselae sequences in particular (34). No A. felis DNA sequences could be detected by PCR. These data strongly indicated that microbiologically undefined skin-test reagents, which had been used for many years for the diagnosis and clinical charac- terization of CSD, were in fact R. henselae reagents. Collectively, these data supported a Rochalimaea species etiology for both CSD and BA. Despite nu- merous attempts, recent efforts to implicate A. felis as a cause of either of these two clinical entities have repeatedly failed. Felis domesticus: A Reservoir for Rochalimaea henselae In addition to epidemiologic data, serologic evi- dence also implicated domestic cats with Rochali- maea-associated disease.

espite nu- merous attempts, recent efforts to implicate A. felis as a cause of either of these two clinical entities have repeatedly failed. Felis domesticus: A Reservoir for Rochalimaea henselae In addition to epidemiologic data, serologic evi- dence also implicated domestic cats with Rochali- maea-associated disease. Rochalimaea-specific IFA antibodies were demonstrated in 6 (46%) of 13 pet cats not associated with human disease and among 39 (81%) of 48 cats living in households reporting human CSD in Connecticut (7). Microbiologic evi- dence for the domestic cat as a reservoir for R. henselae soon followed. R. henselae was isolated over a 3-week period from the blood of a single cat not linked to human illness (35). Investigations by Koe- hler et al. established the cat as a reservoir for Synopsis Vol. 1, No. 1 -- January-March 1995 18 Emerging Infectious Diseases R. henselae infection (36). R. henselae was established as the cause of cutaneous BAin three or four patients with the disease. R. henselae was isolated from the blood of all seven asymptomatic pet cats with which these four BA patients had prolonged contact. The prevalence of infection among cats in the greater San Francisco Bay region was also studied; 25 (41%) of 61 pet or impounded cats had asymptomatic R. henselae bacteremia (36). R. henselae was also de- tected by both direct culture and PCR from several cat fleas combed from these bacteremic cats (36). The human body louse (Pediculus humanus) was established as a vector for human-to-human trench fever R. quintana transmission during the First World War (37). Likewise, B. bacilliformis, a closely related organism (see below) found in the mountains of South America, can be transmitted by another arthropod, the Phlebotomus sand fly (38). The obser- vation that related microbes are vectored between humans by arthropods adds credence to the pro- posed role of arthropod vectors of CSD. Despite sev- eral suggestions that fleas or possibly ticks (7,36,39) are associated with R. henselae disease, no experi- mental data exist to clearly demonstrate that ar- thropods act as direct vectors. Changes in Nomenclature: Rochalimaea becomes Bartonella Genotypic evaluation of members of the genus Rochalimaea has led to the conclusion that members of the genus are closely related to Bartonella bacil- liformis, the agent of Carrion disease, Oroya fever, and verruga peruana (40).

ar- thropods act as direct vectors. Changes in Nomenclature: Rochalimaea becomes Bartonella Genotypic evaluation of members of the genus Rochalimaea has led to the conclusion that members of the genus are closely related to Bartonella bacil- liformis, the agent of Carrion disease, Oroya fever, and verruga peruana (40). Because of historical precedence, the genus designation Bartonella is now applied to all species of the old genus Rochalimaea and replaces the Rochalimaea designation (species names remain unchanged). Physicians and researchers need to exercise care in using the term "bartonellosis." This term has classically been used to describe the frequently fatal syndromes caused by B. bacilliformis. To date, B. bacilliformis and its associated syndrome (bar- tonellosis) have been identified exclusively in South America (38,41). Remaining Questions for Ongoing and Future Research Although B. henselae is now regarded as the etio- logic agent of CSD, as well as a cause of BA, endo- carditis (42), and fever with bacteremia, many questions remain unanswered. For example, why did it take so long to isolate and identifyB. henselae? Part of the answer probably stems from the require- ments necessary for growth in vitro, including en- riched, non-selective blood agar incubated over a prolonged period in a CO2 atmosphere. Most hospi- tal laboratories discard their bacteriological plates before primary isolates of B. henselae would be ex- pected to appear (9-40 days). Extreme sensitivity to a wide variety of antibiotics, at least in vitro, sug- gests that residual levels of antibiotics in patients' blood or other tissues (such as lymph node biopsy) might inhibit Bartonella growth during primary iso- lation attempts in vitro. Selective medium has yet to be developed. Novel genotypic methods were cru- cial for identification of B. henselae; thus, isolates may well have been made in the past but remained unidentified. As mentioned above, it has become apparent that in addition to B. henselae, B. quintana can also be another significant cause of BA disease, at least among immunocompromised patients in San Fran- cisco (27). Another focus of B. quintana infections ("urban trench fever") has been identified among homeless alcoholics in Seattle (43,44). How common are B. quintana infections; are they louseborne and vectored strictly between humans, as was believed during World War I (37)? B. quintana-associated disease has no known link with an alternative ver- tebrate vector (such as cats).

nch fever") has been identified among homeless alcoholics in Seattle (43,44). How common are B. quintana infections; are they louseborne and vectored strictly between humans, as was believed during World War I (37)? B. quintana-associated disease has no known link with an alternative ver- tebrate vector (such as cats). Bartonella elizabethae is known only from a sin- gle isolate from a man surviving endocarditis follow- ing aortic valve-replacement surgery (45). Is there further public health significance to this organism? What additional Bartonella species have yet to be identified and what diseases may they cause? Members of the genus Bartonella are exquisitely sensitive to antibiotics in vitro (30,46). Why then do CSD patients not respond as rapidly and consis- tently to antibiotic therapy as BA patients do? One hypothesis is that immunocompetent patients some- how sequester infectious organisms beyond the reach of antibiotics, whereas immunocompromised patients do not. An alternative hypothesis regarding differential antibiotic responsiveness recognizes that many of the signs of CSD are immune mediated; antibiotics, even if effective in neutralizing or killing bacteria, may not immediately alleviate long-dura- tion immunologic tissue manifestations of antigen stimulation. Conversely, in the absenceof the immu- nologic capability to react to bacterial infection by forming granulomas, as in the case of severely im- munocompromised persons with BA, antibiotics are generally effective in alleviating the symptoms and signs of infection. Does this suggest that possible non-granulomatous manifestations of CSD (for ex- ample, neuroretinitis and encephalopathy) should respond well to the appropriate antibiotic therapy? Although BA has been described in immunocom- petent patients (15), the vast majority of BApatients are immunocompromised (14). What are the factors explaining why B. henselae and B. quintana induce vascular proliferative lesions, such as BA and Synopsis Emerging Infectious Diseases 19 Vol. 1, No. 1 -- January-March 1995 parenchymal bacillary peliosis, almost exclusively in severely immunocompromised patients? What percentage of the relatively large numbers of undiagnosed febrile disease among HIV-infected persons is in fact due to Bartonella species infec- tions? The answer to this important question may help alleviate significant morbidity among HIV-in- fected patients.

t exclusively in severely immunocompromised patients? What percentage of the relatively large numbers of undiagnosed febrile disease among HIV-infected persons is in fact due to Bartonella species infec- tions? The answer to this important question may help alleviate significant morbidity among HIV-in- fected patients. The potential for selection for drug- resistance during long-term antimicrobial therapy deserves scrutiny. Does the 4%-6% of IFAantibody-positive, ostensi- bly healthy "control" study populations suggest a relatively common undercurrent of undiagnosed, subclinical Bartonella-associated disease? Is it possible to immunize cats and thereby inter- rupt B. henselae transmission to humans? Prelimi- nary data suggest that asymptomatic bacteremia in cats can be successfully treated with antimicrobial therapy (36). Once cleared of bacteremia, are these cats routinely susceptible to reinfection? Are the complications occasionally associated with CSD and BA associated with different strains of Bartonella species or are the variations in clinical presentation strictly functions of dose, route of in- oculation, and immune status? And finally, in what role, if any, will A. felis reap- pear as an agent of human disease? IsA. felis respon- sible for the relatively small number of cases of CSD-like lymphadenopathy that have no evidence of antibody to B. henselae? Or is there another expla- nation for the originally proposed association be- tweenA. felisand CSD that has not yet come to light? The new recognition of the importance of Bar- tonella-associated diseases will continue to spawn a host of unanswered related questions. Whereas novel subplots will continue to unfold, the new puz- zles are no longer totally shapeless, and answers to questions of natural history and epidemiology, en- hanced diagnosis and treatment, and methods for disease intervention should now begin to unfold rapidly. References 1. Emmons RW. Cat scratch disease: the mystery finally solved? Ann Intern Med 1984;100:303-4. 2. Goldsmith MF. Has AFIP debugged the cat scratch mystery? JAMA 1983;250:2745-7. 3. Emmons RW, Riggs JL, Schachter J. Continuing the search for the etiology of cat scratch disease. J Clin Micro Biol 1976;4:112-4. 4. Carithers HA. Cat scratch disease: An overview based on a study of 1,200 patients. Am J Dis Child 1985; 139:1124-33. 5. Margileth AM. Cat scratch disease. Adv PediatrInfect Dis 1993;8:1-21. 6. Karim AA, Cockerell CJ, Petri WA.

inuing the search for the etiology of cat scratch disease. J Clin Micro Biol 1976;4:112-4. 4. Carithers HA. Cat scratch disease: An overview based on a study of 1,200 patients. Am J Dis Child 1985; 139:1124-33. 5. Margileth AM. Cat scratch disease. Adv PediatrInfect Dis 1993;8:1-21. 6. Karim AA, Cockerell CJ, Petri WA. Cat scratch dis- ease, bacillary angiomatosis, and other infections due to Rochalimaea. N Engl J Med 1994;330:1509-15. 7. Zangwill KM, Hamilton DH, Perkins BA, et al. Epidemiology, risk factors, and evaluation of a new diagnostic test. N Engl J Med 1993;329:8-13. 8. Warwick WJ. The cat-scratch syndrome, many dis- eases or one disease? Prog Med Virol 1967;9:256-301. 9. Johnson WT, Helwig EB. Cat-scratch disease (his- topathologic changes in the skin). Arch Dermatol 1969;100:148-54. 10. Wear DJ, Margileth AM, Hadfield TL, Fisher GW, Schlagel CJ, King FM. Cat scratch disease: a bacterial infection. Science 1983;221:1403-5. 11. English CK, Wear DJ, Margileth AM, Lissner CR, Walsh GP. Cat scratch disease: isolation and culture of the bacterial agent. JAMA 1988;259:1347-52. 12. Brenner DJ, Hollis DG, Moss CW, English CK, et al. Proposal to Afipia gen. nov., with Afipia felis sp. nov. (Formerly the Cat Scratch Bacillus), Afipia clevelan- denis sp. nov. (Formerly the Cleveland Clinic Strain), Afipia broomeae sp. nov., and three unnamed genospe- cies. J Clin Micro 1991;29:2450-60. 13. Stoler MH, Bonfiglio TA, Steigbigel RT, Pereira M. An atypical subcutaneous infection associated with ac- quired immune deficiency syndrome. Am J Clin Pathol 1983;80:714-8. 14. Koehler JE, Tappero JW. AIDS Commentary: bacil- lary angiomatosis and bacillary peliosis in patients infected with human immunodeficiency virus. Clin Infect Dis 1993;17:612-24. 15. Tappero JW, KoehlerJE, BergerTG, Cockerell CJ, Lee T-H, Busch MP, Stites DP, Mohle-Boetani J, Reingold AL, LeBoit PE. Bacillary angiomatosis and bacillary splenitis in immunocompetent adults. Ann Intern Med 1993;118:363-5. 16. Perkocha LA, Geaghan SM, Yen TS, et al. Clinical and pathological features of bacillary peliosis hepatis in association with human immunodeficiency virus in- fection. N Engl J Med 1990;323:1581-6. 17. Kemper CA, Lombard CM, Dersinski SC, Tompkins LS. Visceral bacillary epithelioid angiomatosis: possi- ble manifestations of disseminated cat scratch disease in the immunocompromised host: a report of two cases. Am J Med 1990;89:216-22. 18. Slater LN, Welch DF, Hensel D, Coody DW.

tion. N Engl J Med 1990;323:1581-6. 17. Kemper CA, Lombard CM, Dersinski SC, Tompkins LS. Visceral bacillary epithelioid angiomatosis: possi- ble manifestations of disseminated cat scratch disease in the immunocompromised host: a report of two cases. Am J Med 1990;89:216-22. 18. Slater LN, Welch DF, Hensel D, Coody DW. A new recognized fastidious gram-negative pathogen as a cause of fever and bacteremia. N Engl J Med 1990;323:1587-93. 19. Black JR, Herrington DA, Hadfield TL, Wear DJ, Margileth AM, Shigekawa B. Life-threatening cat scratch disease in an immunocompromised host. Arch Intern Med 1986;146:394-6. 20. Koehler JE, LeBoit PE, Egbert BM, Berger TG. Cuta- neous vascular lesions and disseminated cat scratch disease in patients with the acquired immunodefi- ciency syndrome (AIDS) and AIDS-related complex. Ann Intern Med 1988;109:449-55. 21. LeBoit PE, Berger TG, Egbert BM, Beckstead JH, Yen TS, Stoler MH. Epithelioid haemangioma-like vascu- lar proliferation in AIDS: manifestation of cat-scratch disease bacillus infection? Lancet 1988;1:960-3. 22. Tappero JW, Mohle-Boetani J, Koehler JE, Swami- nathan B, Berger TG, LeBoit PE, Smith LL, Wenger JD, Pinner RW, Kemper CA, Reingold AL. The epidemiology of bacillary angiomatosis and bacillary peliosis. JAMA 1993;269:770-5. Synopsis Vol. 1, No. 1 -- January-March 1995 20 Emerging Infectious Diseases 23. Tappero JW, Koehler JE. Cat scratch disease and bac- illary angiomatosis [letter]. JAMA 1991;266:1938-39. 24. Relman DA, Loutit JS, Schmidt TM, Falkow S, Tomp- kins LS. The agent of bacillary angiomatosis: an ap- proach to the identification of uncultured pathogens. N Engl J Med 1990;323:1573-80. 25. Regnery RL, Anderson BE, Clarridge III, JE, Ro- driguez-Barradas MC, Jones DC, Carr JH. Charac- terization of a novel Rochalimaea species, R. henselae, sp. nov., isolated from blood of a febrile, human immu- nodeficiency virus-positive patient. J Clin Microbiol 1992;30:265-74. 26. Welch DF, Pickett DA, Slater LN, Steigerwalt AG, Brenner DJ. Rochalimaea henselae, sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchy- mal bacillary peliosis. J Clin Microbiol 1992;30:275- 80. 27. Koehler JE, Quinn FD, Berger TG, LeBoit PE, Tap- pero JW. Isolation of Rochalimaea species from cuta- neous and osseous lesions of bacillary angiomatosis. N Engl J Med 1992;327:1625-31. 28. Tappero J, Regnery R, Koehler J, Olson J.

ngiomatosis, and parenchy- mal bacillary peliosis. J Clin Microbiol 1992;30:275- 80. 27. Koehler JE, Quinn FD, Berger TG, LeBoit PE, Tap- pero JW. Isolation of Rochalimaea species from cuta- neous and osseous lesions of bacillary angiomatosis. N Engl J Med 1992;327:1625-31. 28. Tappero J, Regnery R, Koehler J, Olson J. Detection of serologic response to Rochalimaea henselae in pa- tients with bacillary angiomatosis (BA) by im- munofluorescent antibody (IFA) testing. Program Abstr. 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif. Oct10-14, 1992, Abstr. no. 674. 29. Regnery RL, Olson JG, Perkins BA, Bibb W. Serological response to "Rochalimaea henselae" antigen in sus- pected cat scratch disease. Lancet 1992;339:1443-5. 30. Dolan MJ, Wong MT, Regnery RL, Jorgensen JH, Garcia M, Peters J, Drehner D. Syndrome of Rochali- maea henselae adenitis suggesting cat scratch dis- ease. Ann Intern Med 1993;118:331-6. 31. Anderson B, Sims K, Regnery R, Robinson L, Schmidt MJ, Goral S, Hager C, Edwards K. Detection of Ro- chalimaea henselae DNA in specimens from cat scratch disease patients by PCR. J Clin Microbiol 1994;32:942-8. 32. Szelc Kelly C, Edwards KM, Perez-Perez G, Regnery RL, Perkins BA. A new controversy in the etiology of cat scratch disease: Afipia felis or Rochalimaea hense- lae? Program Abstr. 32nd Interscience Conference on Antimicrobial Agents and Chemotherapy, Anaheim, Calif., Oct 10-14, 1992. Abstr. no. 1565. 33. Perkins BA, Swaminathan B, Jackson LA, Brenner DJ, Wenger JD, Regnery RL. Case 22-1992--patho- genesis of cat scratch disease [letter]. N Engl J Med 1992;327:1599-600. 34. Anderson B, Kelley C, Threlkel R, Edwards K. Detec- tion of Rochalimaea henselae in cat scratch disease skin test antigens. J Infect Dis 1993;168:1034-6. 35. Regnery R, Martin M, Olson J. Naturally occurring "Rochalimaea henselae" infection in domestic cat. Lancet. 1992;340:557-8. 36. Koehler JE, Glaser CA, Tappero JW. Rochalimaea henselae infection: a new zoonosis with the domestic cat as reservoir. JAMA 1994;271:531-5. 37. Strong RP (ed.) Trench fever: Report of Commission, Medical Research Committee, American Red Cross. Oxford: Oxford University Press, 1918:40-60. 38. Schultz MG. A history of bartonellosis (Carrion's dis- ease). Am J Trop Med Hyg 1968;17:503-15. 39. Lucey D, Dolan MJ, Moss CW, Garcia M, Hollis DG, Wegner S, Morgan G, Almeida R, Leong D, Greisen KS, Welch DF, Slater LN.

, Medical Research Committee, American Red Cross. Oxford: Oxford University Press, 1918:40-60. 38. Schultz MG. A history of bartonellosis (Carrion's dis- ease). Am J Trop Med Hyg 1968;17:503-15. 39. Lucey D, Dolan MJ, Moss CW, Garcia M, Hollis DG, Wegner S, Morgan G, Almeida R, Leong D, Greisen KS, Welch DF, Slater LN. Relapsing illness due to Rochalimaea henselae in immunocompetent hosts: Implication for therapy and new epidemiological as- sociations. Clin Infect Dis 1992;14:683-8. 40. Brenner DJ, O'Connor SP, Winkler HH, Steigerwalt AG. Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of Bartonella quin- tana comb. nov., Bartonella vinsonii comb. nov., Bar- tonella henselae comb. nov., and Bartonella elizabethae comb. nov., and to remove the family Bar- tonellaceae from the order Rickettsiales. Int J Syst Bacteriol 1993;43:777-86. 41. Noguchi H, Battistini TS. Etiology of Oroya fever. I. Cultivation of Bartonella bacilliformis. J Exp Med 1926;43:851-64. 42. Hadfield TL, Warren R, Kass M, Brun E, Levy C. Endocarditis caused by Rochalimaea henselae. Hu- man Pathol 1993;24:1140-41. 43. Spach DH, Callis KP, Paauw DS, Houze YB, Schoenk- necht FD, Welch DF, Rosen H, Brenner DJ. Endo- carditis caused by Rochalimaea quintana in a patient infected with human immunodeficiency virus. J Clin Microbiol 1993;31:692-4. 44. Spach DH, Larson AM, Coyle MB, Kanter AS, Welch DF, Stamm AM. Unanticipated Rochalimaea quin- tana bacteremia in patients with chronic alcoholism. [Late Breaker Abstracts]. In: Program Supplement of the 33rd Interscience Conference on Antimicrobial Agents and Chemotherapy. Washington, D.C.: Ameri- can Society for Microbiology, 1993. 45. Daly JS, Worthington MG, Brenner DJ, Moss CW, Hollis DG, Weyant RS, Steigerwalt AG, Weaver RE, Daneshvar MI, O'Connor SP. Rochalimaea elizabe- thae sp. nov. isolated from a patient with endocarditis. J Clin Microbiol 1993;31:872-81. 46. Myers WF, Grossman DM, Wisseman CL. Antibiotic suseptability patterns in Rochalimaea quintana, the agent of trench fever. Antimicrob Agents Chemother. 1984; 25:690-3. Synopsis Emerging Infectious Diseases 21 Vol. 1, No. 1 -- January-March 1995

Emergence of Barmah Forest Virus in Western Australia 1 To the editor: Barmah Forest (BF) virus is a mosquito-borne alphavirus, found only in Australia, which causes outbreaks of polyarthritis in humans. The disease is very similar to epidemic polyarthritis caused by infection with Ross River virus, another Australian alphavirus. BF virus was first isolated from mosquitoes in the State of Western Australia in 1989. After this, small clusters of human cases were diagnosed in the arid northern and central regions of Western Australia in 1992, and the first substantial outbreak of human disease due to infec- tion with BF virus (BF virus disease) occurred in the southwestern region of the state during the spring and summer (September-March) of 1993-94 (2). No evidence of BF virus activity had been found in these regions before these events, which suggests that the virus had only recently been introduced to Western Australia. This report describes the timing and dis- tribution of BF virus disease in humans and the isolation of the virus from mosquitoes in Western Australia, which corroborate the view that BF virus is an emerging virus in this state. The ecology of Australian arboviruses that cause human disease, including BF virus, has recently been reviewed (3). BF virus was first isolated from Culex annulirostris mosquitoes collected at the Bar- mah Forest in northern Victoria (southeastern Aus- tralia) in 1974 (4). It was first shown to infect humans in New South Wales (central-eastern and southeastern Australia) in1986 (5) and wasreported as a cause of clinical disease in humans in 1988 (6). The most common clinical features include polyarthritis, arthralgia, myalgia, fever, rash, and lethargy (7); in some cases, symptoms may persist for more than 6 months (2). Although the symptoms are similar to those caused by infection with Ross River virus, there is little cross-reaction between the two viruses in serologic tests (8), and differentiating between infections caused by either is generally not difficult.

gy (7); in some cases, symptoms may persist for more than 6 months (2). Although the symptoms are similar to those caused by infection with Ross River virus, there is little cross-reaction between the two viruses in serologic tests (8), and differentiating between infections caused by either is generally not difficult. The first true outbreak of BF virus disease occurred concurrently with an outbreak of Ross River virus infection at Nhulunbuy in the Northern Territory in early 1992 (9). The principal vectors of BF are believed to be mosquitoes, and although the vertebrate hosts of BF virus are not known, serologic surveys in eastern Australia have suggested that marsupials are in- volved in the natural cycle. BF virus was first detected in Western Australia in 1989. Since then,73 isolates of thevirus havebeen obtained from mosquitoes trapped in several differ- ent regions of Western Australia (Table 1). The first human cases of BF virus disease in Western Austra- lia were reported in 1992, and 67 serologically con- firmed cases have now been diagnosed. The locations of towns where human cases have occurred or where mosquitoes that yielded BF virus were collected are shown in Figure 1. Eight isolates of the virus were obtained from five different mosquito species (Table 1) collected at Bil- liluna, a small, remote aboriginal community in an arid area in the southeastern Kimberley region in April 1989 (10). The infected mosquitoes were col- lected 3 weeks after heavy local rains. Only moder- ate wet season rains were recorded in the remainder of the Kimberley region, and no cases of BF virus disease were reported from any region in Western Australia that year. There have been no subsequent isolations of BF virus from mosquitoes collected at Billiluna, despite annual collections in the region. No human cases have been reported from Billiluna. The first cases of BF virus disease in Western Australian were reported almost 3 years later, either individually or in small clusters from towns in the arid East Kimberley, Pilbara, Gascoyne, Murchison and Southeast (Goldfields) regions between April and September (Autumn-Spring) 1992. Most activ- ity was reported from the towns of Exmouth (six cases) and Carnarvon (four cases). All of these cases occurred during or just after much larger outbreaks of disease caused by Ross River virus.

, Pilbara, Gascoyne, Murchison and Southeast (Goldfields) regions between April and September (Autumn-Spring) 1992. Most activ- ity was reported from the towns of Exmouth (six cases) and Carnarvon (four cases). All of these cases occurred during or just after much larger outbreaks of disease caused by Ross River virus. This sug- gested that BF and Ross River viruses may have similar mosquito vectors and require similar envi- ronmental conditions for successful transmission. The main environmental factor contributing to the 1992 outbreaks of Ross River virus disease was extremely heavy rain in these normally arid regions during autumn and winter (11). BF virus was isolated from five species of mos- quito in the Fortescue region of the Pilbara and from three species in the West Gascoyne, just prior to, and during, these arid-region outbreaks. In coastal re- gions of the Pilbara, the main vector of BF virus appears to be Aedes vigilax, a salt marsh-breeding species. Large numbers of this species develop after very high tides or heavy rains on salt marshes. It is also the main vector of Ross River virus in these regions (12). Several other temporary freshwater ground pool-breeding species in the subgenus Ochlerotatus, particularly Ae. eidsvoldensis and Ae. EN Marks' species #85, were found to be infected with the virus in inland areas or coastal areas where such pools develop. These preliminary investiga- 1 This report is adapted from and expands on a previous bulletin. (1) Dispatches Vol. 1, No. 1 -- January-March 1995 22 Emerging Infectious Diseases tions also suggested that both BF and Ross River virus can co-circulate. Both viruses were isolated from different mosquitoes of the same species col- lected in the same trap on several occasions. A further six cases of BF virus disease were re- ported after record wet season rains in the Kimber- ley region in early 1993. The casesoccurred justafter mosquitoes in the Kimberley region had been col- lected by personnel from this laboratory. These col- lections yielded 12 isolates of BF virus. Eleven of these were from Ae. vigilax and Cx. annulirostris mosquitoes trapped less than 2 weeks after the first heavy wet season rains near the West Kimberley towns of Broome and Fitzroy Crossing and the East Kimberley town of Halls Creek (see Figure 1 for locations, Table 1 for isolation details). A twelfth isolate was obtained from Ae. normanensis collected 5 weeks after the first rains at Willare in the West Kimberley.

first heavy wet season rains near the West Kimberley towns of Broome and Fitzroy Crossing and the East Kimberley town of Halls Creek (see Figure 1 for locations, Table 1 for isolation details). A twelfth isolate was obtained from Ae. normanensis collected 5 weeks after the first rains at Willare in the West Kimberley. The timing of the collections was such that all three mosquito species could have transmit- ted BF virus to the infected persons in the region. Vector competence studies are required to determine if one or more species were likely to have been the main vectors. A single case was reported from the metropolitan area of Perth, the state's capital, in August 1992. This was the first evidence of BF virus activity in the temperate and populous southwestern region of Western Australia. However, the travel history of the patient was not obtained. Then, in early January Table 1. Mosquito species from which BF virus was isolated in Western Australia by region and date, 1989-1993* Region Locality Species Date Isolates East Kimberley Billiluna Ae. bancroftianus 22 Apr 1989 1 East Kimberley Billiluna Ae. eidsvoldensis 22 Apr 1989 3 East Kimberley Billiluna Ae. pseudonormanensis 22 Apr 1989 1 East Kimberley Billiluna An. amictus 22 Apr 1989 2 East Kimberley Billiluna An. annulipes s.l. 22 Apr 1989 1 East Kimberley Halls Creek Cx. annulirostris 11 Feb 1993 1 West Kimberley Broome Ae. vigilax 10-16 Feb 1993 9 West Kimberley Fitzroy Crossing Cx. annulirostris 13 Feb 1993 1 West Kimberley Willare Ae. normanensis 16 Mar 1993 1 Pilbara (Fortescue) Onslow Ae. EN Marks' sp. #85 13-14 Jun 1992 3 Pilbara (Fortescue) Onslow Cx. annulirostris 13 Jun 1992 1 Pilbara (Fortescue) Onslow An. amictus 13-14 Jun 1992 2 Pilbara (Fortescue) Exmouth Ae. vigilax 16 Jun-11Jul1992 7 Gascoyne (West) Minilya Ae. eidsvoldensis 7 Jul 1992 5 Gascoyne (West) Minilya Ae. eidsvoldensis (bloodfed) 7 Jul 1992 1 Gascoyne (West) Minilya Ae. EN Marks' sp. #85 7 Jul 1992 1 Gascoyne (West) Carnarvon Ae. eidsvoldensis 12 Jul 1992 3 Gascoyne (West) Carnarvon Ae. EN Marks' sp. #85 12 Jul 1992 1 Gascoyne (West) Carnarvon Cx. quinquefasciatus 12 Jul 1992 1 Gascoyne (West) Carnarvon Unidentifiable mosquitoes 12 Jul 1992 1 Central Coastal Karnup Cx. annulirostris 4 Jan 1993 1 Central Coastal Karnup Cq. species near linealis 4 Jan 1993 4 Central Coastal Perth Cx. annulirostris 6 Jan 1993 1 South Coastal Australind Ae. camptorhynchus 6 Jul 1993 1 South Coastal Mandurah (Peel) Ae.

yne (West) Carnarvon Unidentifiable mosquitoes 12 Jul 1992 1 Central Coastal Karnup Cx. annulirostris 4 Jan 1993 1 Central Coastal Karnup Cq. species near linealis 4 Jan 1993 4 Central Coastal Perth Cx. annulirostris 6 Jan 1993 1 South Coastal Australind Ae. camptorhynchus 6 Jul 1993 1 South Coastal Mandurah (Peel) Ae. camptorhynchus 5 Aug-5 Oct 1993 10 South Coastal Busselton Ae. camptorhynchus 1Sep-15Nov 1993 9 South Coastal Busselton Cx. globocoxitus 1 Nov 1993 1 Total 73 *Numbers of mosquitoes trapped and processed and estimated minimum field infection rates for each region will be published in detail elsewhere.

yne (West) Carnarvon Unidentifiable mosquitoes 12 Jul 1992 1 Central Coastal Karnup Cx. annulirostris 4 Jan 1993 1 Central Coastal Karnup Cq. species near linealis 4 Jan 1993 4 Central Coastal Perth Cx. annulirostris 6 Jan 1993 1 South Coastal Australind Ae. camptorhynchus 6 Jul 1993 1 South Coastal Mandurah (Peel) Ae. camptorhynchus 5 Aug-5 Oct 1993 10 South Coastal Busselton Ae. camptorhynchus 1Sep-15Nov 1993 9 South Coastal Busselton Cx. globocoxitus 1 Nov 1993 1 Total 73 *Numbers of mosquitoes trapped and processed and estimated minimum field infection rates for each region will be published in detail elsewhere. Refer to Figure 1 for location of regions and towns from which isolates of BF virus were obtained. Dispatches Emerging Infectious Diseases 23 Vol. 1, No. 1 -- January-March 1995 1993, this laboratory isolated BF virus from Cx. annulirostris and Coquillettidia species near linealis2 mosquitoes collected at Karnup, south of Perth (Table 1). A week later a single human case was reported from an address near the site at which the mosquitoes were trapped. BF virus was also isolated from Cx. annulirostris mosquitoes trapped in the southern suburbs of Perth in early January 1993, providing evidence that the virus may be transmitted to humans in the metropolitan area. Two further cases were reported from the Perth metropolitan area, one in February and one in May 1993. Again, no travel histories were available for these patients, but it appears that the virus re- mained and was actively transmitted in the south- west during the autumn and winter of 1993, as it was isolated from Ae. camptorhyn- chus mosquitoes trapped in July and August (Table 1). A larger outbreak of BF virus disease occurred in the southwest region between September 1993 and March 1994. This has been de- scribed in detail elsewhere (2). Twenty-eight serologically con- firmed cases were reported from the southwest region during that pe- riod. Of these, more than half (17 cases) were in or near the small coastal towns of Mandurah (Peel) and Busselton during spring (Sep- tember-November) of 1993. BF vi- rus was isolated on 20 occasions from pools of Ae. camptorhynchus mosquitoes collected in the Man- durah and Busselton regions before and during the outbreak (Table 1), thereby implicating that species, along with Cx. annulirostris and Cq. species near linealis, as an im- portant vector in the southwest. Ae. camptorhynchus breeds in salt marshes and brackish wetlands during all but the hottest months of the year in the southwest and is the main vector of Ross River virus in the region (3, 11). The ratio of the carriage rate of BF virus in Ae. camptorhynchus during the out- break to the number of human cases was very high compared with the rate observed for Ross River virus in Ae.

s during all but the hottest months of the year in the southwest and is the main vector of Ross River virus in the region (3, 11). The ratio of the carriage rate of BF virus in Ae. camptorhynchus during the out- break to the number of human cases was very high compared with the rate observed for Ross River virus in Ae. camptorhynchus in the same regions during previous Ross River virus outbreaks (M.D. Lindsay, C.A. Johansen, and J.S. Mackenzie, unpublished observations). This suggests that the ratio of subclinical (therefore un- reported) to clinical cases may be much larger with BF virus than with Ross River virus or that fewer humans were infected, possibly because Ae. camp- torhynchus may not transmit BF virus to humans as efficiently as it does Ross River virus. Seven cases were reported between November 1993 and March 1994 in small towns in the inland southwest region in a later cycle of virus activity. Unfortunately, no collections of mosquitoes were carried out in the region during that time. Small clusters of cases or individual cases were also reported from several other regions of Western Australia during this time, including three additional cases from Broome in the West Kimberley region, presumably associated with the 1993-94 wet season. BF virus disease was made a notifiable disease in Western Australia in June 1994 as a direct result of Figure 1. Meteorological regions and towns in Western Australia where human cases of Barmah Forest virus disease were reported and Barmah Forest virus was isolated from mosquitoes, 1989-1994. Meteorologic regions: 1. North Kimberley; 2. East Kimberley; 3. West Kimberley; 4. Pilbara (De Grey); 5. Pilbara (Fortescue); 6. West Gascoyne; 7. East Gascoyne; 7A. Murchison; 8. North Coastal; 9. Central Coastal; 9A. South Coastal; 10. North Central; 10A. South Central; 11. Eucla; 12. Southeast (Goldfields); and 13. Northeast (Interior). 2 This species is similar to, but distinct from Coquillettidia linealis, according to E.N. Marks, the leading Culicid taxono- mist in Australia. Dispatches Vol. 1, No. 1 -- January-March 1995 24 Emerging Infectious Diseases the 1993-94 southwest outbreak (Health [Infectious Diseases] Amendment Order 1994, Government Ga- zette, Western Australia, 24 June 1994). The out- break was also the first report of a substantial number of cases in the absence of Ross River virus activity anywhere in Australia.

rch 1995 24 Emerging Infectious Diseases the 1993-94 southwest outbreak (Health [Infectious Diseases] Amendment Order 1994, Government Ga- zette, Western Australia, 24 June 1994). The out- break was also the first report of a substantial number of cases in the absence of Ross River virus activity anywhere in Australia. Ross River virus is endemic in the Mandurah (Peel) region but only one case of Ross River virus disease was reported from the area during spring-summer (September-Febru- ary) 1993-94. This is the lowest recorded number of cases for that period in the region since record keep- ing began in 1984. Environmental conditions and vector mosquito populations in the southwest were unfavorable for Ross River virus transmission dur- ing the BF outbreak. In particular, populations ofAe. camptorhynchus from October onwards were much smaller than in years when larger numbers of cases of Ross River virus disease were reported (M.D. Lindsay, C.A. Johansen, J.S. Mackenzie, unpub- lished observations). It is not known whether the BF virus outbreak occurred because BF virus can circu- late under conditions that are not suitable for Ross River virus activity or whether extremely low levels of immunity in "virgin" vertebrate host and human populations in the southwest may have enhanced transmission cycles. Surveillance and epidemiologic studies carried out by this laboratory in the north of Western Aus- tralia since 1972 and in the southwest since 1987 have found no convincing evidence of BF virus activ- ity in these regions prior to the events described in this report. No BF virus was isolated from the north of Western Australia before 1989, despite large-scale processing of field-caught mosquitoes over a 17-year period that yielded hundreds of isolates of other arboviruses. Similarly, no BF virus isolate was ob- tained from more than400,000 mosquitoes collected throughout the southwest between 1987 and 1992 and processed for virus isolation. Furthermore, an ongoing serosurvey has found no evidence of infec- tion with BF virus in more than 1,000 individuals of 18 vertebrate species collected in the southwest be- fore 1992 (C.A. Johansen, unpublished results). This suggests that the virus responsible for the recent outbreaks was recently introduced to Western Aus- tralia. The means of introduction, initially to the northwest and more recently to the southwest of Western Australia, is not known.

es collected in the southwest be- fore 1992 (C.A. Johansen, unpublished results). This suggests that the virus responsible for the recent outbreaks was recently introduced to Western Aus- tralia. The means of introduction, initially to the northwest and more recently to the southwest of Western Australia, is not known. In view of the activity at Nhulunbuy in the Northern Territory, before the first Western Australia cases in 1992, it is possible that the virus may have been introduced from that region in a viremic human or in livestock. However, little is known about the duration and height of viremia in infected humans or other ani- mals, and it is not known whether person-to-person vector-mediated transmission of Barmah Forest vi- rus can occur. Our laboratory has begun a study to investigate the molecular epidemiology of BF virus, particularly whether the strain of virus responsible for the Western Australia outbreaks was introduced from Eastern Australia or was a local, hitherto un- detected, strain. Acknowledgments The authors thank Tony Wright, (Health Depart- ment of Western Australia) for assistance with col- lection of mosquitoes, Margaret Wallace, Rosa Egerton-Warburton, Roger Clifton, and Dora Li for assistance with laboratory processing of mosquito and virus samples, and staff in the Virology Section, State Health Laboratory Service, the Health De- partment of Western Australia, for carrying out the serologic testing of human sera. This work was funded by a grant from the Health Department of Western Australia. Michael D.A. Lindsay, Cheryl A. Johansen, Annette K. Broom, David W. Smith,* and John S. Mackenzie Department of Microbiology, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, WA *State Health Laboratory Services, Queen Elizabeth II Medical Centre, Nedlands, WA References 1. Lindsay MD, Smith DW, Johansen C, Mackenzie JS. Barmah Forest virus disease in Western Australia. Western Australian Notifiable Diseases Bulletin 1994; 4: 1-4. 2. Lindsay MD, Johansen C, Smith DW, Wallace MJ, Mackenzie JS. An outbreak of Barmah Forest virus disease in the south-west of Western Australia. Med J Aust. In press. 3. Mackenzie JS, Lindsay MD, Coelen RJ, Hall RA, Broom AK, Smith DW. Arboviruses causing human disease in the Australian zoogeographic region. Arch Virol 1994;136:447-7. 4. Marshall ID, Woodroofe GM, Hirsch S.

e JS. An outbreak of Barmah Forest virus disease in the south-west of Western Australia. Med J Aust. In press. 3. Mackenzie JS, Lindsay MD, Coelen RJ, Hall RA, Broom AK, Smith DW. Arboviruses causing human disease in the Australian zoogeographic region. Arch Virol 1994;136:447-7. 4. Marshall ID, Woodroofe GM, Hirsch S. Viruses recov- ered from mosquitoes and wildlife serum collected in the Murray Valley of south-eastern Australia, Febru- ary 1974, during an outbreak of encephalitis. Aust J Exp Biol Med Sci 1982;60:457-70. 5. Vale TG, Carter IW, McPhie KA, James GS, Cloonan MJ. Human arbovirus infections along the south coast of New South Wales. Aust J Exp Biol Med Sci 1986;64:307-9. 6. Boughton CR, Hawkes RA, Naim HM. Illness caused by a Barmah Forest-like virus in New South Wales. Med J Aust 1988;148:146-7. 7. Phillips DA, Murray JR, Aaskov JG, Weimers MA. Clinical and subclinical Barmah Forest virus infection in Queensland. Med J Aust 1990;152:463-6. 8. Hawkes RA, Boughton CR, Naim HM, Myrick BA, Ramsay LG. Barmah Forest virus infections in hu- mans in New South Wales. Med J Aust 1988;146:569- 73. Dispatches Emerging Infectious Diseases 25 Vol. 1, No. 1 -- January-March 1995 9. Merianos A, Farland AM, Patel M, Currie B, Whelan P, Dentith H, Smith D. A concurrent outbreak of Bar- mah Forest and Ross River disease in Nhulunbuy, Northern Territory. Comm Dis Intell (Aust) 1992;16:110-1. 10. Broom AK, Mackenzie JS, Lindsay MD, Wright AE. Epidemiology of Murray Valley encephalitis and Kun- jin viruses in Western Australia, 1980-89. Arbovirus Research in Australia 1989;5:14-8. CSIRO and Queensland Institute of Medical Research. 11. Lindsay MD, Johansen C, Wright AE, Condon R, D'Er- cole M, Smith D, Mackenzie JS. The epidemiology of outbreaks of Ross River virus infection in Western Australia in 1991-1992. Arbovirus Research in Austra- lia 1992;6:72-6. CSIRO and Queensland Institute of Medical Research. 12. Lindsay MD, Broom AK, Wright AE, Johansen CA, Mackenzie JS. Ross River virus isolations from mos- quitoes in arid regions of Western Australia: implica- tion of vertical transmission as a means of persistence of the virus. Am J Trop Med Hyg 1993;49:686-96. An Outbreak of Shigella sonnei Infection Associated with Consumption of Iceberg Lettuce To the Editor: Shigella sonnei outbreaks in England and Wales are typically associated with primary schools and nurseries. The mode of trans- mission is usually from person to person by the fecal-oral route (1).

Hyg 1993;49:686-96. An Outbreak of Shigella sonnei Infection Associated with Consumption of Iceberg Lettuce To the Editor: Shigella sonnei outbreaks in England and Wales are typically associated with primary schools and nurseries. The mode of trans- mission is usually from person to person by the fecal-oral route (1). In a June 1994 outbreak of Sh. sonnei food poisoning among adults in several coun- tries in North West Europe, the vehicle of infection appeared to be iceberg lettuce (2). In early June, the Communicable Disease Sur- veillance Centre (CDSC), Public Health Laboratory Service, received a report of an increase in domestic cases of Sh. sonnei infections in Sweden from the Salmnet network--a European international labo- ratory-based reporting system for human salmo- nella infections that provides a timely on-line database. In this instance the network was used for shigellosis. Of 100 reported cases ofSh. sonnei infec- tion in Sweden, 52 occurred in two outbreaks in mid- May. Many cases seemed to be due to foodborne infection, and iceberg lettuce and peeled frozen prawns were implicated as vehicles of infection. Sh. sonnei phage types 2 and 3 alpha were associated withthe outbreaks,andphagetypes2and65hadbeen isolated from sporadic cases. A message was sent throughout England and Wales on Epinet (a system for rapid electronic data transfer to all Consultants in Communicable Dis- ease Control [CsCDC] in each District Health Authority, Public Health Laboratories [PHLs] and other agencies involved in infectious disease control) asking for information on possible foodborne Sh. sonnei infection to be sent to CDSC and for isolates to be referred to the Laboratory of Enteric Patho- gens (LEP) for phage typing. Epidemiologic studies Laboratory reports of Sh. sonnei infection re- ceived through the routine reporting system at CDSC were scrutinized to determine the age group and sex distributions during weeks 21 to 24. After the Epinet message, CsCDC and laboratory directors who reported clinical cases for which Sh. sonnei was isolated were asked to administer trawl- ing questionnaires to apparently sporadic cases among adults with no recent history of overseas travel. Personal details and history of illness and exposure to particular foods were sought. Several small outbreaks and clusters were re- ported during June. CsCDC was asked for results of any analytical epidemiologic studies to CDSC. The results of the national laboratory reporting system are shown in Table 1.

rseas travel. Personal details and history of illness and exposure to particular foods were sought. Several small outbreaks and clusters were re- ported during June. CsCDC was asked for results of any analytical epidemiologic studies to CDSC. The results of the national laboratory reporting system are shown in Table 1. Although there were fewer reports in the first 20 weeks of 1994 than in a similar period in 1993, there were more reports in the weeks 21 to 24 and many more reports among adults. The proportion of total reports constituted by those from adults was 66% in weeks 21 to 24 of 1994 compared with 44% with the same period in 1993. The propor- tion in women in the 2 periods was 42% in 1994 compared with 26% in 1993. Forty trawling questionnaires were distributed. Almost all case patients (38/40) had eaten various salad items of which the common food was iceberg lettuce. The lettuce had been consumed in restau- rants, pubs, and in the homes of the case-patients. The lettuce was purchased from supermarkets, greengrocers' shops, and street markets. In one out- break in Northampton, 21 (52%) of guests at a party became ill with diarrhea. Sh. sonnei was isolated from fecal specimens. Illness was significantly asso- ciated with consumption of iceberg lettuce (relative risk 3.68, confidence intervals 1.34 - 10.11, p = 0.0004). The hypothesis that consumption of iceberg let- tuce was associated with apparently sporadic Sh. sonnei infection in adults was tested by a case-con- Dispatches Vol. 1, No. 1 -- January-March 1995 26 Emerging Infectious Diseases

9. Merianos A, Farland AM, Patel M, Currie B, Whelan P, Dentith H, Smith D. A concurrent outbreak of Bar- mah Forest and Ross River disease in Nhulunbuy, Northern Territory. Comm Dis Intell (Aust) 1992;16:110-1. 10. Broom AK, Mackenzie JS, Lindsay MD, Wright AE. Epidemiology of Murray Valley encephalitis and Kun- jin viruses in Western Australia, 1980-89. Arbovirus Research in Australia 1989;5:14-8. CSIRO and Queensland Institute of Medical Research. 11. Lindsay MD, Johansen C, Wright AE, Condon R, D'Er- cole M, Smith D, Mackenzie JS. The epidemiology of outbreaks of Ross River virus infection in Western Australia in 1991-1992. Arbovirus Research in Austra- lia 1992;6:72-6. CSIRO and Queensland Institute of Medical Research. 12. Lindsay MD, Broom AK, Wright AE, Johansen CA, Mackenzie JS. Ross River virus isolations from mos- quitoes in arid regions of Western Australia: implica- tion of vertical transmission as a means of persistence of the virus. Am J Trop Med Hyg 1993;49:686-96. An Outbreak of Shigella sonnei Infection Associated with Consumption of Iceberg Lettuce To the Editor: Shigella sonnei outbreaks in England and Wales are typically associated with primary schools and nurseries. The mode of trans- mission is usually from person to person by the fecal-oral route (1). In a June 1994 outbreak of Sh. sonnei food poisoning among adults in several coun- tries in North West Europe, the vehicle of infection appeared to be iceberg lettuce (2). In early June, the Communicable Disease Sur- veillance Centre (CDSC), Public Health Laboratory Service, received a report of an increase in domestic cases of Sh. sonnei infections in Sweden from the Salmnet network--a European international labo- ratory-based reporting system for human salmo- nella infections that provides a timely on-line database. In this instance the network was used for shigellosis. Of 100 reported cases ofSh. sonnei infec- tion in Sweden, 52 occurred in two outbreaks in mid- May. Many cases seemed to be due to foodborne infection, and iceberg lettuce and peeled frozen prawns were implicated as vehicles of infection. Sh.

mely on-line database. In this instance the network was used for shigellosis. Of 100 reported cases ofSh. sonnei infec- tion in Sweden, 52 occurred in two outbreaks in mid- May. Many cases seemed to be due to foodborne infection, and iceberg lettuce and peeled frozen prawns were implicated as vehicles of infection. Sh. sonnei phage types 2 and 3 alpha were associated withthe outbreaks,andphagetypes2and65hadbeen isolated from sporadic cases. A message was sent throughout England and Wales on Epinet (a system for rapid electronic data transfer to all Consultants in Communicable Dis- ease Control [CsCDC] in each District Health Authority, Public Health Laboratories [PHLs] and other agencies involved in infectious disease control) asking for information on possible foodborne Sh. sonnei infection to be sent to CDSC and for isolates to be referred to the Laboratory of Enteric Patho- gens (LEP) for phage typing. Epidemiologic studies Laboratory reports of Sh. sonnei infection re- ceived through the routine reporting system at CDSC were scrutinized to determine the age group and sex distributions during weeks 21 to 24. After the Epinet message, CsCDC and laboratory directors who reported clinical cases for which Sh. sonnei was isolated were asked to administer trawl- ing questionnaires to apparently sporadic cases among adults with no recent history of overseas travel. Personal details and history of illness and exposure to particular foods were sought. Several small outbreaks and clusters were re- ported during June. CsCDC was asked for results of any analytical epidemiologic studies to CDSC. The results of the national laboratory reporting system are shown in Table 1. Although there were fewer reports in the first 20 weeks of 1994 than in a similar period in 1993, there were more reports in the weeks 21 to 24 and many more reports among adults. The proportion of total reports constituted by those from adults was 66% in weeks 21 to 24 of 1994 compared with 44% with the same period in 1993. The propor- tion in women in the 2 periods was 42% in 1994 compared with 26% in 1993. Forty trawling questionnaires were distributed. Almost all case patients (38/40) had eaten various salad items of which the common food was iceberg lettuce. The lettuce had been consumed in restau- rants, pubs, and in the homes of the case-patients. The lettuce was purchased from supermarkets, greengrocers' shops, and street markets.

questionnaires were distributed. Almost all case patients (38/40) had eaten various salad items of which the common food was iceberg lettuce. The lettuce had been consumed in restau- rants, pubs, and in the homes of the case-patients. The lettuce was purchased from supermarkets, greengrocers' shops, and street markets. In one out- break in Northampton, 21 (52%) of guests at a party became ill with diarrhea. Sh. sonnei was isolated from fecal specimens. Illness was significantly asso- ciated with consumption of iceberg lettuce (relative risk 3.68, confidence intervals 1.34 - 10.11, p = 0.0004). The hypothesis that consumption of iceberg let- tuce was associated with apparently sporadic Sh. sonnei infection in adults was tested by a case-con- Dispatches Vol. 1, No. 1 -- January-March 1995 26 Emerging Infectious Diseases trol study. A case was defined as a person aged 14 or more years who became ill after May 1, 1994, and had microbiologic evidence (fecal isolation) of Sh. sonnei infection, no recent history of overseas travel, and no identifiable contact with other case-patients in the 3 days before onset. Controls were nominated by case-patients and matched by sex, age (within a 10-year age band), and area of residence (within a 10-mile radius of the case). For each case three matched controls were sought. A questionnaire was administered by telephone by three interviewers from CDSC. Clinical and demographic details and details of exposure to food items, including iceberg lettuce, mentioned in trawling questionnaires were sought. Twenty-eight case-patients and 49 matched con- trols were interviewed and, after excluding those who had recently traveled abroad and controls who had been ill, results from 27 cases and 44 controls were analyzed. The median age of case-patients was 47 years, and the range was 19 to 79 years. Eight cases were among men and 19 among women. All case-patients had diarrhea (i.e., three or more loose stools in a 24-hour period), although only four of the 27 reported blood in the stools, 25 of the 27 had abdominal pain, and 11 reported vomiting. The me- dian duration of symptoms was 9 days, and the range was 4 to 25 days. Taking into account the matching inherent in the study design, a matched analysis was performed. In any analysis 27 matched sets were possible. For 13 sets there was one control per case, for 11 sets there were 2 per case, and for 3 sets there were 3 controls per case.

mptoms was 9 days, and the range was 4 to 25 days. Taking into account the matching inherent in the study design, a matched analysis was performed. In any analysis 27 matched sets were possible. For 13 sets there was one control per case, for 11 sets there were 2 per case, and for 3 sets there were 3 controls per case. Single variable analysis of the different foodsconsumed revealed the possible risk factors (p < 0.2) (Table 2). A multivari- able model was fitted with all those variables in- cluded. This procedure was repeated, removing nonsignificant items at each stage. In the third model, the only remaining significant item was ice- berg lettuce (p = 0.0172). The estimated odds ratio for iceberg lettuce was 13.8 (95% confidence interval 1.26 to 150.5). In sporadic cases associated with consumption of lettuce from particular restaurants or public houses, it was possible to compare the date of onset with the date of delivery of iceberg lettuce by the wholesalers. The distribution chain was traced back through im- porters supplying wholesale markets in England. The wholesalers were supplied by packers in Spain. This was consistent with the findings of the investi- gators in the Norwegian outbreak. Iceberg lettuce investigated by the Public Health Laboratory serv- ice during the second week of June 1994 did not grow Sh. sonnei. However, the iceberg lettuce season in Spain, which began in October, ended early in June, and the source of lettuce available for testing could not be traced. Laboratory studies All Sh. sonnei isolates referred to LEP after the Epinet message were phage typed by using the scheme described by Hammerstrom (3) and Kallings and Sjoberg (4), according to a protocol supplied by Dr. R. Wollin, Swedish Institute of Infectious Dis- ease Control, Sweden. Isolates were also tested for resistance to a range of antibiotics by an agar dilu- tion technique (5). A total of 495 isolates were referred to LEP be- tween June 14 and July 31, from 51 laboratories in England and Wales. Most isolates were from spo- radic infections, but in a number of local outbreaks, there was a strong epidemiologic association be- tween illness and consumption of iceberg lettuce. Two phage types predominated among the 19 types identified during this period, PT 2 (42.6% of isolates) and a variant of PT65 provisionally designated PT L (15.9%). In contrast, although a small number of isolates of PT 65 and PT L were identified among strains of Sh.

- tween illness and consumption of iceberg lettuce. Two phage types predominated among the 19 types identified during this period, PT 2 (42.6% of isolates) and a variant of PT65 provisionally designated PT L (15.9%). In contrast, although a small number of isolates of PT 65 and PT L were identified among strains of Sh. sonnei isolated in England and Wales in 1991 and 1992, no isolates of PT 2 were seen before May 1994. Towards the end of the outbreak PT 3 and PT 6 were becoming reestablished in Eng- land and Wales as the predominant types, as they had been in previous years. An exception to the recent pattern was an out- break in North Wales, involving several children and adults, in which infection was associated either with eating ice cream at a particular establishment or having contact with children who had done so. All 73 of the isolates of PT 62 were associated with this outbreak. A total of 357 Sh. sonnei isolated during this period (72.1%) were fully sensitive to all drugs Table 1. Shigella sonnei in England and Wales--Laboratory reports to CDSC Year Number of reports (%) Week 1-20 Weeks 21-24 Weeks 1-24 Total Total Adults Women Total 1993 3190 480 (100) 211 (44) 127 (26) 3670 1994 1557 505 (100) 333 (66) 214 (42) 2062 Dispatches Emerging Infectious Diseases 27 Vol. 1, No. 1 -- January-March 1995 tested. Phage types 2 (87% fully sensitive) and PT L (99%) were predominantly sensitive, as were all isolates of PT 62; usually one would expect more than 70% of Sh. sonnei isolates to be resistant to one or more drugs. The use of the same phage-typing scheme across several European countries has facili- tated cross-referencing between the British, Ger- man, and Swedish outbreaks. Phage types 2 and 65 (or the closely related variant PT L) were identified in several countries. From the epidemiologic studies, it was concluded that the strong statistical evidence (p = 0.0172) that consumption of iceberg lettuce was associated with the risk of becoming ill together with reports from other European countries, including Scotland, Swe- den, and Norway, andthe temporal association of the outbreak with the iceberg lettuce season in Spain implicated iceberg lettuce as the vehicle of infection. This was corroborated by the laboratory studies, which showed a change in predominant phage types during the period of the outbreak. The predomi- nance of the same phage types in lettuce-associated Sh. sonnei infections in a number of countries added further weight to this conclusion.

ttuce as the vehicle of infection. This was corroborated by the laboratory studies, which showed a change in predominant phage types during the period of the outbreak. The predomi- nance of the same phage types in lettuce-associated Sh. sonnei infections in a number of countries added further weight to this conclusion. In England, there were several anecdotal ac- counts of dual infection with salmonellae and vi- ruses as well as Sh. sonnei. This was also true of infections in Norway and Sweden (6). A plausible explanation would be that fecally contaminated water was used to irrigate the lettuce or to cool it after packing. If iceberg lettuce is not washed thor- oughly before consumption, contamination could be retained in the leaves. This study demonstrates both the importance of coordinating laboratory results and epidemiologic investigations and the value of rapid communica- tions and common typing techniques in various European countries. J.A. Frost, M.B. McEvoy,* C.A. Bentley, Y. Andersson,** and B. Rowe Laboratory of Enteric Pathogens, Central Public Health Laboratory, London, England *Communicable Disease Surveillance Centre, London, England **Swedish Institute for Infectious Diseases Control, Stockholm, Sweden References 1. Newman CPS. Surveillance and control of Shigella sonnei infection. Communicable Disease Report 1993; 3: R63-8. 2. Anonymous. A foodborne outbreak of Shigella sonnei infection in Europe. Communicable Disease Report 1994; 4 No. 25. Table 2. Sh. sonnei case control study--Single variable matched analysis Case (n=27) Control (n=44) Food Ate Did not eat % ate Ate Did not eat % ate No.

ion. Communicable Disease Report 1993; 3: R63-8. 2. Anonymous. A foodborne outbreak of Shigella sonnei infection in Europe. Communicable Disease Report 1994; 4 No. 25. Table 2. Sh. sonnei case control study--Single variable matched analysis Case (n=27) Control (n=44) Food Ate Did not eat % ate Ate Did not eat % ate No. of sets p-value Prawns 4 22 15 7 37 15 (26s) 0.9388 Shrimps 0 27 0 1 43 2 (27s) 0.3070 Steak 7 19 27 6 37 14 (26s) 0.0139 Burgers 5 22 19 12 32 27 (27s) 0.2045 Salad 25 2 93 34 10 77 (27s) 0.0081 Cold meats 15 10 60 29 13 69 (23s) 0.7708 Tomatoes 22 4 85 33 11 75 (26s) 0.1039 Spring onions 5 21 19 9 35 21 (26s) 0.7556 Celery 5 22 19 5 39 11 (27s) 0.2956 Cucumber 16 11 59 31 13 71 (27s) 0.3798 Other salads 16 10 62 26 18 59 (26s) 1.0000 Lettuce 25 2 93 31 13 71 (27s) 0.0007 Cos lettuce 2 20 9 3 25 11 (17s) 1.0000 Webb's lettuce 5 18 22 4 34 11 (21s) 0.2632 Lamb's lettuce 2 17 11 6 36 14 (19s) 0.1002 Raddicio lettuce 4 20 17 7 34 17 (23s) 0.8720 Iceberg lettuce 17 8 68 19 23 45 (25s) 0.0023 Frisee lettuce 5 18 22 4 37 10 (22s) 0.2582 Home 15 8 65 27 16 63 (23s) 0.8494 Restaurant 6 16 27 5 33 13 (21s) 0.1546 Pub 4 18 18 2 34 6 (20s) 0.0795 Other outlet 10 14 42 7 31 18 (22s) 0.0012 Dispatches Vol. 1, No. 1 -- January-March 1995 28 Emerging Infectious Diseases 3. Hammerstrom E. Phagetyping ofShigella sonnei.Acta Med Scand 1949. 133 (Suppl 223). 4. Kallings LO, Lindberg AA, Sjoberg L. Phage typing of Shigella sonnei. Arch Immun TherExp 1968; 16: 280-7. 5. Frost JA. Testing for resistance to antimicrobial drugs. In: Chart H, ed. Methods in practical laboratory bacte- riology. Boca Raton Fla.: CRC Press; 1994. 6. Kapperud G, Rorvik LM, Hasseltvedt V, et al. Out- break of Shigella sonnei infection traced to imported iceberg lettuce. J Clin Microbiol, in press. ?Lyme Disease in Australia-- Still To Be Proven! To the Editor: The first case of a syndrome consistent with Lyme disease was reported from the Hunter Valley region of New South Wales (NSW) in southeastern Australia in 1982, but there was no confirming serology. More clinical cases, again with- out serologic confirmation, were reported in 1986, two from the south coast and one from the central coast of NSW. The Queensland State Health Labo- ratories reported that 186 (14.9%) of 1,247 sera taken from patients between 1986-1989 showed antibody response to Borrelia burgdorferi of 64 by indirect fluorescence antibody test (IFAT), but none of these results were confirmed by immunoblotting.

nd one from the central coast of NSW. The Queensland State Health Labo- ratories reported that 186 (14.9%) of 1,247 sera taken from patients between 1986-1989 showed antibody response to Borrelia burgdorferi of 64 by indirect fluorescence antibody test (IFAT), but none of these results were confirmed by immunoblotting. In 1988, a multidisciplinary investigation of pu- tative Lyme disease began, encompassing clinical, serologic, vector, and reservoir host studies, and results from these studies have been published (1). What follows herein is derived from the accumu- lated published and unpublished data of the re- search team, the members of which are credited in the acknowledgments. Over the past 6 years, principally because of local publicity, there has been an increase in serologic testing for Lyme disease in Australia, particularly in southeastern Australia. Testing has often been initiated by patients with undiagnosed health prob- lems. Thus, most Lyme disease patients seen by infectious disease specialists are self selected and are referred for assessment on the basis of tick exposure and reported positive serologic test results for Lyme disease. Patients with positive serologic test results fre- quently have long-standing symptoms for which no other diagnosis has been established. The most com- mon symptoms are musculoskeletal, including my- algias and arthralgias without objective evidence of joint swelling, and syndromes involving fatigue and loss of energy resembling chronic fatigue syndrome. Some patients fulfill diagnostic criteria for fibromy- algia. The next most common symptoms are neuro- logical, and include frequent headaches, inability to concentrate, and memory loss. The most common dermatologic manifestation of chronic Lyme disease, acrodermatitis chronica atrophicans, seen occasion- ally in Europe and rarely in the United States, has not been reported from Australia. A few cases of erythema migrans, the charac- teristic dermatologic manifestation of acute Lyme disease, have been reported from southeastern Aus- tralia, but clinical diagnosis can be confounded by hypersensitivity reactions to tick bite; a spectacular erythematous reaction is often associated with the bite of Ixodes holocyclus, the most common tick biting humans in NSW. Only eight specimens sub- mitted to our laboratory included skin biopsies done to isolate spirochetes. B. burgdorferi s.l.

can be confounded by hypersensitivity reactions to tick bite; a spectacular erythematous reaction is often associated with the bite of Ixodes holocyclus, the most common tick biting humans in NSW. Only eight specimens sub- mitted to our laboratory included skin biopsies done to isolate spirochetes. B. burgdorferi s.l. was iso- lated from one patient returning from Europe, but no spirochetes were isolated from local patients. In our serologic diagnostic service, an enzyme- linked immunosorbent assay (ELISA) for IgG and an IFAT for IgG and IgM have been used with antigens derived from North American B. burgdor- feri strain B31 (2). From 1988 to April 1994, 78 (1.8%) of 4,372 local patients were positive for IgG by both methods. All 78 patients were tested by IgG Western blot for confirmation by using the virulent North American B. burgdorferi strain 297 and a German strain designated B7: with B. burgdorferi strain 297, 46 patient samples showed as many as four indicative bands; with the European strain B7, 22 patient samples showed as many as three indica- tive bands; bands used were 18, 21, 28, 30, 31, 34, 39, 41, 45, 58, 66, 83, and 93 kDa, modified from Dressler et al (3). Twenty-four other patients with various bacterial, viral, or autoimmune syndromes not relating to Lyme disease were tested as controls: with strain 297, 11 control samples showed as many as two indicative bands, and with strain B7, 10 control samples showed as many as two indicative bands. A high degree of cross-reactivity was demon- strated with the controls, particularly with respect to the 31, 41, 58, and 66 kDa bands for both the European and the American antigen. As none of the 78 patients, including putative late-stage patients positive by ELISA and IFAT, showed more than four Dispatches Emerging Infectious Diseases 29 Vol. 1, No. 1 -- January-March 1995

3. Hammerstrom E. Phagetyping ofShigella sonnei.Acta Med Scand 1949. 133 (Suppl 223). 4. Kallings LO, Lindberg AA, Sjoberg L. Phage typing of Shigella sonnei. Arch Immun TherExp 1968; 16: 280-7. 5. Frost JA. Testing for resistance to antimicrobial drugs. In: Chart H, ed. Methods in practical laboratory bacte- riology. Boca Raton Fla.: CRC Press; 1994. 6. Kapperud G, Rorvik LM, Hasseltvedt V, et al. Out- break of Shigella sonnei infection traced to imported iceberg lettuce. J Clin Microbiol, in press. ?Lyme Disease in Australia-- Still To Be Proven! To the Editor: The first case of a syndrome consistent with Lyme disease was reported from the Hunter Valley region of New South Wales (NSW) in southeastern Australia in 1982, but there was no confirming serology. More clinical cases, again with- out serologic confirmation, were reported in 1986, two from the south coast and one from the central coast of NSW. The Queensland State Health Labo- ratories reported that 186 (14.9%) of 1,247 sera taken from patients between 1986-1989 showed antibody response to Borrelia burgdorferi of 64 by indirect fluorescence antibody test (IFAT), but none of these results were confirmed by immunoblotting. In 1988, a multidisciplinary investigation of pu- tative Lyme disease began, encompassing clinical, serologic, vector, and reservoir host studies, and results from these studies have been published (1). What follows herein is derived from the accumu- lated published and unpublished data of the re- search team, the members of which are credited in the acknowledgments. Over the past 6 years, principally because of local publicity, there has been an increase in serologic testing for Lyme disease in Australia, particularly in southeastern Australia. Testing has often been initiated by patients with undiagnosed health prob- lems. Thus, most Lyme disease patients seen by infectious disease specialists are self selected and are referred for assessment on the basis of tick exposure and reported positive serologic test results for Lyme disease. Patients with positive serologic test results fre- quently have long-standing symptoms for which no other diagnosis has been established.

seen by infectious disease specialists are self selected and are referred for assessment on the basis of tick exposure and reported positive serologic test results for Lyme disease. Patients with positive serologic test results fre- quently have long-standing symptoms for which no other diagnosis has been established. The most com- mon symptoms are musculoskeletal, including my- algias and arthralgias without objective evidence of joint swelling, and syndromes involving fatigue and loss of energy resembling chronic fatigue syndrome. Some patients fulfill diagnostic criteria for fibromy- algia. The next most common symptoms are neuro- logical, and include frequent headaches, inability to concentrate, and memory loss. The most common dermatologic manifestation of chronic Lyme disease, acrodermatitis chronica atrophicans, seen occasion- ally in Europe and rarely in the United States, has not been reported from Australia. A few cases of erythema migrans, the charac- teristic dermatologic manifestation of acute Lyme disease, have been reported from southeastern Aus- tralia, but clinical diagnosis can be confounded by hypersensitivity reactions to tick bite; a spectacular erythematous reaction is often associated with the bite of Ixodes holocyclus, the most common tick biting humans in NSW. Only eight specimens sub- mitted to our laboratory included skin biopsies done to isolate spirochetes. B. burgdorferi s.l. was iso- lated from one patient returning from Europe, but no spirochetes were isolated from local patients. In our serologic diagnostic service, an enzyme- linked immunosorbent assay (ELISA) for IgG and an IFAT for IgG and IgM have been used with antigens derived from North American B. burgdor- feri strain B31 (2). From 1988 to April 1994, 78 (1.8%) of 4,372 local patients were positive for IgG by both methods. All 78 patients were tested by IgG Western blot for confirmation by using the virulent North American B. burgdorferi strain 297 and a German strain designated B7: with B. burgdorferi strain 297, 46 patient samples showed as many as four indicative bands; with the European strain B7, 22 patient samples showed as many as three indica- tive bands; bands used were 18, 21, 28, 30, 31, 34, 39, 41, 45, 58, 66, 83, and 93 kDa, modified from Dressler et al (3).

rman strain designated B7: with B. burgdorferi strain 297, 46 patient samples showed as many as four indicative bands; with the European strain B7, 22 patient samples showed as many as three indica- tive bands; bands used were 18, 21, 28, 30, 31, 34, 39, 41, 45, 58, 66, 83, and 93 kDa, modified from Dressler et al (3). Twenty-four other patients with various bacterial, viral, or autoimmune syndromes not relating to Lyme disease were tested as controls: with strain 297, 11 control samples showed as many as two indicative bands, and with strain B7, 10 control samples showed as many as two indicative bands. A high degree of cross-reactivity was demon- strated with the controls, particularly with respect to the 31, 41, 58, and 66 kDa bands for both the European and the American antigen. As none of the 78 patients, including putative late-stage patients positive by ELISA and IFAT, showed more than four Dispatches Emerging Infectious Diseases 29 Vol. 1, No. 1 -- January-March 1995 specific bands to either antigen, they would be con- sidered negative by the criteria of Dressler et al (3). Fewer than 1% of all referred patients conformed with the national surveillance case definition used in the United States by the Centers for Disease Control and Prevention. Problems of specificity and sensitivity associated with serologic testing for Lyme disease are well recognized, particularly in Australia where no local spirochete has been iso- lated for use as a reference antigen. Seroprevalence rates for B. burgdorferi infection in humans have been compared between 200 high (rural residents) and 200 low (urban residents) tick exposure groups in coastal NSW, by using the IgG ELISA. No significant difference was found between the two groups, and the overall seropositivity rate was 2.2% (9/400). A parallel survey of dogs in NSW has shown a similar result with an overall seroposi- tivity rate of 2.5% (6/239). These results contrast with those reported from known endemic-disease areas outside Australia that have rural populations with considerably higher seropositive rates. The low rate found by our surveys is similar to that found by other studies undertaken in areas where Lyme dis- ease is not endemic, and humans have 1%-3% posi- tive serologic results cause by cross-reacting antibodies (4). From January 1990 to December 1992, ticks were collected in areas associated with putative Lyme disease cases and were examined for spirochetes to detect a possible causative agent in potential vec- tors.

is- ease is not endemic, and humans have 1%-3% posi- tive serologic results cause by cross-reacting antibodies (4). From January 1990 to December 1992, ticks were collected in areas associated with putative Lyme disease cases and were examined for spirochetes to detect a possible causative agent in potential vec- tors. Ticks were collected along the east coast of Australia, from southern Queensland through NSW into northern Victoria, by flagging in natural habi- tats, and from domestic and other native animals. Detection of spirochetes was attempted bydark-field microscopy and culturing of gut contents and by direct testing of ticks using polymerase chain reac- tion (PCR) to detect the Borrelia-specific flagellin gene (5). In total, more than 12,000 (>1,000 by PCR) ticks were processed, including 7,922 I. holocyclus (1). No spirochetes were detected by dark-field microscopy or by PCR. Spirochete-like objects (SLOs), were ob- served in 94 cultures from bloodfed ticks and only in cultures with bacterial contaminants, presumably from the bloodmeal. Some SLOs yielded positive fluorescence results when tested with Borrelia-spe- cific polyclonal antibodies, but testswith monoclonal antibodies (anti-flagellin H9724, anti-OspA H5332, anti-OspB H6831) were negative. Electron mi- crographs showed that the SLOs were not typical of Borrelia, were composed of fibers, and probably were not spirochetes. The electron micrographs were similar to micrographs of SLOs recovered from con- taminated cultures from ticks in the United States and Europe and thought to be composed of aggrega- tions of bacterial flagella, probably from the con- taminants. Molecular characterization indicated that the SLOs were not related to B. burgdorferi. A small number of native vertebrate animals (13 native rats Rattus fuscipes, 3 bandicoots Perameles nasuta, and 1 marsupial mouse Antechinus stuartii) trapped on the south coast of NSW were sampled by ear-punch biopsy (6) for culture and PCR investiga- tion, but no evidence of borreliae was found. The animal sample was clearly inadequate, and the PCR primers used for the tick and animal studies may have been inappropriate and unable to identify na- tive Australian spirochetes; however the extensive investigations of tick gut contents by culturing and dark field microscopy were also negative for borre- liae.

und. The animal sample was clearly inadequate, and the PCR primers used for the tick and animal studies may have been inappropriate and unable to identify na- tive Australian spirochetes; however the extensive investigations of tick gut contents by culturing and dark field microscopy were also negative for borre- liae. There are some major differences between Austra- lia and the Lyme-disease-endemic areas of the North- ern Hemisphere with respect to the natural history of borreliosis. No ticks of theI. persulcatus complex, the principal vectors to humans in the northern hemi- sphere, occur in Australia. In eastern Australia, the logical candidate vector would be I. holocyclus, which has a wide host range and is the most common tick biting humans. I. holocyclus cannot transmit a North American strain of B. burgdorferi (7) but the association with any possible Australian spirochetes remains unresolved. Likewise, none of the mammal species identified as reservoir hosts in the Northern Hemisphere are present in Australia. There are reports of spirochetes in Australian native animals, and a local mammal could be a reservoir host for an indigenous spirochete that occasionally infects hu- mans through a tick vector and produces a clinical syndrome similar to Lyme disease; however, no spi- rochete was detected in the ticks or animals studied. The diagnosis of Lyme disease outside known disease-endemic areas should not be based solely on serology because unrelated syndromes, such as autoimmune diseases and cross reactions with other bacteria, can produce false-positive results. Like- wise, a definitive diagnosis on clinical grounds alone in a nonendemic-disease area is difficult to justify without adequate scientific support based on isola- tion of the causative agent from the patient or from another patient or known vector from the region. In Australia, disagreement as to what constitutes a positive serologic result has additionally contrib- uted to overdiagnosis of Lyme disease. Until an organism is isolated from a local patient and is characterized, the presence of Lyme disease in Aus- tralia will remain controversial. Acknowledgments My colleagues Rosemary Munro (clinical microbi- ology), Department of Microbiology, Liverpool Hos- pital, and Stephen Doggett (entomology), David Dickeson (serology), Danielle Avery (molecular biol- Dispatches Vol. 1, No.

racterized, the presence of Lyme disease in Aus- tralia will remain controversial. Acknowledgments My colleagues Rosemary Munro (clinical microbi- ology), Department of Microbiology, Liverpool Hos- pital, and Stephen Doggett (entomology), David Dickeson (serology), Danielle Avery (molecular biol- Dispatches Vol. 1, No. 1 -- January-March 1995 30 Emerging Infectious Diseases ogy), Cheryl Hunt (molecular biology), Joanne Mer- cer (microbiology), and Nicole Trivett (electron mi- croscopy), CIDM at Westmead Hospital, and John Ellis (molecular biology), Department of Microbiol- ogy, University of Technology Sydney, contributed to the studies outlined in this paper. Richard Lawrence, Clinical Superintendent of Medicine, Westmead Hospital, provided valuable discussions on clinical aspects and case presentation. Our inves- tigations were supported by theNational Health and Medical Research Council and the Ramaciotti Foun- dations. Richard C. Russell Department of Medical Entomology, Centre for Infectious Diseases and Microbiology, University of Sydney and Westmead Hospital, Westmead, Australia References 1. Russell RC, Doggett SL, Munro R, Ellis J, Avery D, Hunt C, Dickeson D. Lyme disease: a search for the causative agent in ticks in southeastern Australia. Epidemiol Infect 1994;112:375-84. 2. Russell H, Sampson JS, Schmid GP, Wilkinson HW, Plikaytis B. Enzyme-linked immunosorbent assay and direct immunofluorescence assay for Lyme dis- ease. J Infect Dis 1984;149:465-70. 3. Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392-400. 4. Barbour AG, Fish D. The biological and social phe- nomenon of Lyme disease. Science 1993;260:1610-6. 5. Persing DH, Telford III SR, Rys PN, Dodge DE, White TJ, Malawista SE, Spielman A. Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dam- mini ticks. Science 1990;249:1420-3. 6. Sinsky RJ, Piesman J. Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents. J Clin Microbiol 1989;27:1723-7. 7. Piesman J, Stone BF. Vector competence of the Austra- lian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochaete Borrelia burgdorferi. Int J Parasi- tol 1991;21:109-11.

Piesman J. Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents. J Clin Microbiol 1989;27:1723-7. 7. Piesman J, Stone BF. Vector competence of the Austra- lian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochaete Borrelia burgdorferi. Int J Parasi- tol 1991;21:109-11. A Novel Morbillivirus Pneumonia of Horses and its Transmission to Humans To the editor: On September 22 and 23, 1994, veterinary authorities in Queensland and at the CSIRO Australian Animal Health Laboratory were advised of an outbreak of acute respiratory disease in horses at a stable in the Brisbane suburb of Hendra. The trainer of the horses had been hospi- talized for a respiratory disease and was in critical condition. At that time, the cause of the horses' illness was unclear and any link between equine and human disease was thought improbable. Poisoning, bacterial, viral, and exotic disease causes were in- vestigated. The history of thehorses on this property was considered important (Figure 1). Two weeks before the trainer's illness, on September 7, two horses had been moved to the Hendra stable from a spelling paddock in Cannon Hill (6 km). One of these, a pregnant mare, was sick and died within 2 days. The other horse was subsequently moved on and never became sick. By September 26, 13 horses had died: the mare; 10 other horses in the Hendra stable; one horse, which had very close contact with horses in the Hendra stable, on a neighboring prop- erty; and one which had been transported from the stable to another site (150 km). Four Hendra horses and three others (one in an adjacent stable, one moved to Kenilworth, and one to Samford) were later considered to have been exposed and recovered from the illness. Some of these horses were asymp- tomatic. Nine Hendra horses have remained unaf- fected. The sick horses were anorexic, depressed, usually febrile (temperature up to 41C), showed elevated respiratory rates, and became ataxic. Head pressing was occasionally seen, and commonly, a frothy nasal discharge occurred before death. On September 14, a stablehand at the Hendra stable developed an influenza-like illness charac- terized by fever and myalgia. The next day, the horse trainer also became ill with similar symptoms. Both had close contact with the dying mare, particularly the trainer who was exposed to nasal discharge while trying to feed her; he had abrasions on his hands andarms.

stable developed an influenza-like illness charac- terized by fever and myalgia. The next day, the horse trainer also became ill with similar symptoms. Both had close contact with the dying mare, particularly the trainer who was exposed to nasal discharge while trying to feed her; he had abrasions on his hands andarms. Thestablehand, a40-year-old man, remained ill for 6 weeks and gradually recovered. Besides myalgia, he also had headaches, lethargy, and vertigo. The trainer, a 49-year-old man, was a heavy smoker and showed signs consistent with Legionella infection. He ultimately required venti- lation for respiratory distress and died after 6 days (Selvey L, et al. Anovel morbillivirus infection caus- ing severe respiratory illness in humans and horses, submitted). At the beginning of the diagnostic investigation in horses, African horse sickness, equine influenza, and hyperacute equine herpes virus were excluded as possible causes by antigen trapping enzyme- linked immunosorbent assay (ELISA), polymerase Dispatches Emerging Infectious Diseases 31 Vol. 1, No. 1 -- January-March 1995

ogy), Cheryl Hunt (molecular biology), Joanne Mer- cer (microbiology), and Nicole Trivett (electron mi- croscopy), CIDM at Westmead Hospital, and John Ellis (molecular biology), Department of Microbiol- ogy, University of Technology Sydney, contributed to the studies outlined in this paper. Richard Lawrence, Clinical Superintendent of Medicine, Westmead Hospital, provided valuable discussions on clinical aspects and case presentation. Our inves- tigations were supported by theNational Health and Medical Research Council and the Ramaciotti Foun- dations. Richard C. Russell Department of Medical Entomology, Centre for Infectious Diseases and Microbiology, University of Sydney and Westmead Hospital, Westmead, Australia References 1. Russell RC, Doggett SL, Munro R, Ellis J, Avery D, Hunt C, Dickeson D. Lyme disease: a search for the causative agent in ticks in southeastern Australia. Epidemiol Infect 1994;112:375-84. 2. Russell H, Sampson JS, Schmid GP, Wilkinson HW, Plikaytis B. Enzyme-linked immunosorbent assay and direct immunofluorescence assay for Lyme dis- ease. J Infect Dis 1984;149:465-70. 3. Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392-400. 4. Barbour AG, Fish D. The biological and social phe- nomenon of Lyme disease. Science 1993;260:1610-6. 5. Persing DH, Telford III SR, Rys PN, Dodge DE, White TJ, Malawista SE, Spielman A. Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dam- mini ticks. Science 1990;249:1420-3. 6. Sinsky RJ, Piesman J. Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents. J Clin Microbiol 1989;27:1723-7. 7. Piesman J, Stone BF. Vector competence of the Austra- lian paralysis tick, Ixodes holocyclus, for the Lyme disease spirochaete Borrelia burgdorferi. Int J Parasi- tol 1991;21:109-11.

stable developed an influenza-like illness charac- terized by fever and myalgia. The next day, the horse trainer also became ill with similar symptoms. Both had close contact with the dying mare, particularly the trainer who was exposed to nasal discharge while trying to feed her; he had abrasions on his hands andarms. Thestablehand, a40-year-old man, remained ill for 6 weeks and gradually recovered. Besides myalgia, he also had headaches, lethargy, and vertigo. The trainer, a 49-year-old man, was a heavy smoker and showed signs consistent with Legionella infection. He ultimately required venti- lation for respiratory distress and died after 6 days (Selvey L, et al. Anovel morbillivirus infection caus- ing severe respiratory illness in humans and horses, submitted). At the beginning of the diagnostic investigation in horses, African horse sickness, equine influenza, and hyperacute equine herpes virus were excluded as possible causes by antigen trapping enzyme- linked immunosorbent assay (ELISA), polymerase Dispatches Emerging Infectious Diseases 31 Vol. 1, No. 1 -- January-March 1995 chain reaction (PCR), or electronmicroscopy. Tests for Pasteurella, Bacillus anthracis, Yersinia, Le- gionella, Pseudomonas, and Streptobacillus monili- formis were negative, and poisons consistent with the clinical and gross pathology, such as paraquat, were excluded by specific testing. However, within 3 days, a syncytial forming virus was detected in vero-cell cultures inoculated with diseased horse tissues and shortly thereafter was seen to grow in a wide range of cells. These included MDBK, BHK, and RK13 cells. Subsequently, a syncytial forming virus also was isolated in LLK- MK2 cells that had been inoculated with tissue from the deceased trainer's kidney. The isolation of these viruses and their preliminary characterization by electron microscopy, immunoelectromicroscopy, se- rology, and genetic analyses are described elsewhere (Murray PK, et al. Anew morbillivirus which caused fatal disease in horses and man, submitted). In summary, ultrastructural analysis showed that the virus is a member of the Paramyxoviridae family. It is enveloped, pleomorphic (varies in size from 38 nm to more than 600 nm), and is covered with 10 nm and 18 nm surface projections. It con- tains herringbone nucleocapsids that are 18 nm wide with a 5 nm periodicity. The presence of `dou- ble-fringed' surface projections on this virus is con- sidered unique.

family. It is enveloped, pleomorphic (varies in size from 38 nm to more than 600 nm), and is covered with 10 nm and 18 nm surface projections. It con- tains herringbone nucleocapsids that are 18 nm wide with a 5 nm periodicity. The presence of `dou- ble-fringed' surface projections on this virus is con- sidered unique. Immunoelectronmicroscopy showed that both the horse and the human virus react with convalescent-phase horse sera and with sera from the two human cases. PCR primers were synthesized from consensus Paramyxoviridae matrix protein sequences and tested against the horse virus. Those specific for paramyxoviruses and pneumoviruses did not bind, but one pair of morbillivirus primers gave a 400 bp product. Determination of the sequence of this prod- uct enabled the synthesis of horse virus-specific primers. Phylogenetic analyses of the matrix protein sequence indicates that the virus is unique and distantly related to other known members of the group. A comparison of translated M protein se- quence shows that it has a 50% homology with the morbillivirus group (80% if conservative amino acid substitutions are used). This distant relatedness is emphasized by our observations that neutralizing antisera to measles virus, canine distemper, and rinperest virus failed to neutralize the virus. The viruses isolated from the horses and the trainer are ultrastructurally identical. Serum from the horses and the two human cases specifically cross-neutralize the virus, and the horse virus-spe- cific PCR primers provide a positive reaction with the human virus isolate. Therefore, the horses and the trainer were infected with the same virus. At the beginning of the diagnostic investigation, tissues from thelungs and spleens of diseased horses were injected into two recipient horses. After 6 and 10 days, the recipient horses became ill with high fever and severe respiratory signs, demonstrating that the disease was transmissible. Two days later the horses were destroyed. The equine morbillivirus September 1994 7 9 13 14 15 16 17 19-26 Horses Cannon Hill (Paddock) 2 horses moved Hendra (Stables) Mare died 2 horses moved 10 horses dead 4 recovered Hendra (Neighboring property) 1 horse moved 1 horse dead 1 recovered Kenilworth (150 km distant) 1 horse dead 1 recovered Samford (Paddock) 1 recovered New South Wales Humans Stablehand Becomes ill Slow recovery Trainer Becomes ill Hospitalized Died Figure 1.

Stables) Mare died 2 horses moved 10 horses dead 4 recovered Hendra (Neighboring property) 1 horse moved 1 horse dead 1 recovered Kenilworth (150 km distant) 1 horse dead 1 recovered Samford (Paddock) 1 recovered New South Wales Humans Stablehand Becomes ill Slow recovery Trainer Becomes ill Hospitalized Died Figure 1. A chronology of the development of cases of acute equine respiratory disease and associated illness in humans. Dispatches Vol. 1, No. 1 -- January-March 1995 32 Emerging Infectious Diseases was isolated from tissues from both of these horses. To document that the isolated horse virus was patho- genic, experimental transmission tests were also conducted. Two additional horses received a total of 2x 107 TCID50 of tissue culture virusby intravenous inoculation and by intranasal aerosol. Both horses became seriously ill, and after a short, severe clinical episode, were destroyed 4 and 5 days after exposure. At necropsy, they showed gross and histopathologic lesions that were primarily respiratory and consis- tent with the natural disease. Virus was reisolated from their lungs, liver, spleen, kidney, lymph nodes, and blood. The pathology of this infection is interesting. In horses, the dominant gross pathology is lesions in the lungs. These are congested and edematous with prominent lymphatic dilation in the ventral mar- gins. In natural cases, the airways were usually filled with thick, fine, stable foam which was occa- sionally blood-tinged; thiswas not seen in the experi- mental cases. Histologically, in horses, there is interstitial pneumonia, proteinaceous edema with pneumocyte, and capillary degeneration. Virus can be located in endothelial cells by immunofluores- cence and syncytial cells also could be seen in blood vessel walls, confirming the vascular of trophism of this virus (Murray PK, et al, submitted). The trainer's post-mortem findings showed similarities to those of the horses (Selvey L, et al, submitted). No further clinical cases of disease have been seen in horses or humans since this outbreak. Serologic surveillance of people who had close contact with the sick horses, mostly stable workers, veterinary pa- thologists, animal health field staff, or people who lived in the vicinity of the affected stables, was negative (Selvey L, et al, submitted). Serologic testing of all horses on quarantined properties and within 1 kmof the Hendra stable, and a sample of horses from the rest of Queensland was undertaken (Table 1).

y pa- thologists, animal health field staff, or people who lived in the vicinity of the affected stables, was negative (Selvey L, et al, submitted). Serologic testing of all horses on quarantined properties and within 1 kmof the Hendra stable, and a sample of horses from the rest of Queensland was undertaken (Table 1). A total of 1,964 horses were tested from more than 630 premises. The negative results from this testing also indicate that the infec- tion has not spread. In the entire horse survey, only seven horses, all from the Hendra property and the adjoining stables, were positive. Four of these ani- mals had been clinically affected, but three were asymptomatic. Because of the potential risk and the difficulty in establishing freedom from infection, these seven recovered horses were later destroyed. Although persistent virus excretion or carrier states are not known to occur in other morbillivirus infections, this equine virus is unique and it cannot be presumed to behave similarly. Australian veteri- nary authorities are now satisfied that the incident is over. We have described a newly recorded disease of horses with an obvious zoonotic potential; moreover, the causative agent was previously unrecorded and is significantly different from other members of its genus, morbillivirus. Infection seems to have spread from the mare that first showed the now charac- teristic clinical signs, to other horses in the same stables, to a horse in close contact from an adjacent stable, and also to two human attendants. Clearly, this outbreak was not highly contagious and it rap- idly resolved. However, the virus is highly patho- genic with 65% of naturally infected horses and all four experimental horses dying. Further investigations of the virus and the dis- ease are now warranted since it could reemerge in Australia or elsewhere. Investigations of its origin, its replication, its pathogenesis, and its possible occurrence elsewhere in connection with equine res- piratory disease are merited. Keith Murray, Russell Rogers,* Linda Selvey,** Paul Selleck, Alex Hyatt, Allan Gould, Laurie Gleeson, Peter Hooper, and Harvey Westbury CSIRO Australian Animal Health Laboratory, Ryrie Street, East Geelong, Victoria 3220, Australia *Animal Health Bureau, Queensland Department of Primary Industries, 80 Ann Street, Brisbane, Australia **Communicable Diseases, Queensland Health, 160 Mary Street, Brisbane 4000, Queensland, Australia Table 1.

arvey Westbury CSIRO Australian Animal Health Laboratory, Ryrie Street, East Geelong, Victoria 3220, Australia *Animal Health Bureau, Queensland Department of Primary Industries, 80 Ann Street, Brisbane, Australia **Communicable Diseases, Queensland Health, 160 Mary Street, Brisbane 4000, Queensland, Australia Table 1. The premises and horses surveyed by serologic testing for equine morbillivirus, after the disease outbreak Premises Horses Quarantine Premises* 13 107 1 (within 100 m of Hendra stables) 7 54 2 (100 m to 200 m of Hendra stables) 21 122 3 (200 m to 1 km of Hendra stables) 93 730 4/5 (remainder of Queensland) >500 963 Total >630 1,964 *Quarantine premises included those with clinical cases, hold- ing properties associated with the Hendra stables, and other premises where horses under investigation were kept. Dispatches Emerging Infectious Diseases 33 Vol. 1, No. 1 -- January-March 1995

Electronic Communication and the Future of International Public Health Surveillance Recent developments in electronic communica- tion have enhanced national public health surveil- lance systems and facilitated progress in establishing surveillance that crosses national boundaries. The international foodborne outbreak of Shigella sonnei described by Frost and colleagues was first reported through Salmnet (1), a laboratory- based surveillance system designed to include an on-line network database. Salmnet was established in 1994 to improve the prevention and control of human salmonellosis and other foodborne infections in the countries of the European Union and the European Cooperation in Science and Technology. Epidemiologists who have national surveillance responsibilities and heads of reference laboratories in 13 countries currently collaborate in the Salmnet project under the joint leadership of the Directors of the Laboratory of Enteric Pathogens and the Com- municable Disease Surveillance Centre (CDSC) at the Public Health Laboratory Service campus at Colindale, London. During the course of two work- shops, the collaborators agreed to a) develop and apply standardized phage typing for the most com- mon salmonella serotypes within Europe, b) intro- duce an international quality assurance scheme for laboratory performance of Salmonella phage typing, c) establish a core set of data on each laboratory-con- firmed and typed human salmonella isolate for rapid transfer into a shared nonaggregated dataset, and d) develop statistical analysis programs to facilitate the early recognition of international outbreaks. The collaborators also agreed to rapidly report any clus- ters detected and to exchange information concern- ing other infections, including those caused by Shigella, Listeria, and vero-cytotoxin-producing strains of Escherichia coli. Currently six countries share data through the Internet, while the other seven countries rely on faxing material or sending diskettes through the mail. All collaborating countries plan to join the on-line network by the end of 1995.

caused by Shigella, Listeria, and vero-cytotoxin-producing strains of Escherichia coli. Currently six countries share data through the Internet, while the other seven countries rely on faxing material or sending diskettes through the mail. All collaborating countries plan to join the on-line network by the end of 1995. The opportuni- ties offered by electronic communication have en- couraged a remarkable degree of international cooperation in surveillance, as is evident in the far reaching objectives agreed upon for Salmnet. The Salmnet collaboration was already in place when the S. sonnei outbreak in Sweden was reported to Colindale. CDSC responded by sending an elec- tronic message throughout England and Wales by Epinet, an electronic system for the rapid transfer of vital public health information developed by the CDSC Welsh Unit in Cardiff (2). The message was sent to consultants in communicable disease control in each district health authority, to the 53 public health laboratories, and to other agencies in England and Wales involved in infectious disease control. Further information from Norway, Scotland, and Sweden reinforced and stimulated the ongoing in- vestigation in England and Wales. Since its incep- tion, Salmnet has also contributed information with potential international implications concerning sev- eral Salmonella serotypes ( 3,4). The signing of the Treaty of Maastricht (1992) was an important milestone for international coopera- tion in public health surveillance. The treaty estab- lished a basis for European Community action in the field of public health and enjoined cooperation be- tween member states, third countries, and interna- tional public health organizations to protect human health. Sufficient evidence has accrued on the added value of international surveillance of infectious dis- eases, and it is generally accepted that the potential for major public health hazards is amplified as a consequence of the increasing volume of interna- tional travel and the global extension of food distri- bution networks (5). Surveillance systems such as Salmnet and the European Surveillance of Travel- Associated Legionnaires' Disease (6) will pave the way for international surveillance by providing a communications network that will facilitate the rapid collection and analysis of data using standard case definitions, transmission of information for the prevention of communicable diseases, and the pro- motion of effective public health practice.

ase (6) will pave the way for international surveillance by providing a communications network that will facilitate the rapid collection and analysis of data using standard case definitions, transmission of information for the prevention of communicable diseases, and the pro- motion of effective public health practice. Parallel electronic surveillance systems in the United States offer equal opportunity for interna- tional collaboration. For example, since 1985, data on notifiable diseases have been transmitted elec- tronically each week to the Centers for Disease Con- trol and Prevention (CDC) from state health departments by the National Electronic Telecommu- nications System for Surveillance (NETSS) (7). NETSS was developed by CDC and the Council of State and Territorial Epidemiologists for electroni- cally collecting, transmitting, analyzing, and pub- lishing weekly reports of notifiable diseases and injuries from 50 states, New York City, the District of Columbia, Puerto Rico, the Virgin Islands, Guam, American Samoa, and the Commonwealth of the Northern Mariana Islands (8). The operation of NETSS is based on agreements on reporting condi- tions, standard case definitions, and protocols for formatting and transmitting data, rather than pre- scribed software or systems. A second CDC electronic reporting system, the Public Health Laboratory Information System (PHLIS), is used by public health department labo- ratories in all states, New York City, the District of Columbia, and Guam to report laboratory isolate- based surveillance data to CDC. The PC-based sys- tem was developed jointly by the National Center for Commentary Vol. 1, No. 1 -- January-March 1995 34 Emerging Infectious Diseases Infectious Diseases, CDC, and the Association of State and Territorial Public Health Laboratory Di- rectors to maintain a fast and direct link between public health laboratories in the United States and CDC. PHLIS is used to gather, analyze and transmit data (e.g., laboratory testing results, epidemiologic information, findings from special studies and sur- veys) among multiple sources of public health labo- ratory information (e.g., hospitals, laboratories, or public health departments), and it provides an auto- mated program in its longitudinal databases to de- tect outbreaks (9). European and U.S. surveillance databases and information systems should be linked to share public health information of international concern.

ratory information (e.g., hospitals, laboratories, or public health departments), and it provides an auto- mated program in its longitudinal databases to de- tect outbreaks (9). European and U.S. surveillance databases and information systems should be linked to share public health information of international concern. To that end, CDSC and CDC are developing a cooperative communications information system that will use the Internet to mirror vital public health documents (e.g., CDSC's Communicable Disease Report [CDR], CDC's Morbidity and Mortality Weekly Report [MMWR], and selected surveillance data sets). This network is the beginning of a larger international network that will share data, exchange information, and improve public health. This larger network could link such systems as Salmnet, NETSS, and PHLIS to create a virtual on-line library of international surveillance data and information for public health. With the diffusion of technology, internationally networked electronic public health surveillance sys- tems are gaining in importance. Their existence clearly facilitates the rapid collection, analysis, and dissemination of vital public health information and promotes the establishment of effective interna- tional public health policies. T. Demetri Vacalis Epidemiology Program Office Centers for Disease Control and Prevention Atlanta, Georgia, USA Christopher L. R. Bartlett PHLS Communicable Disease Surveillance Centre London, England Cheryl G. Shapiro National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA References 1. Fisher IST, Rowe B, Bartlett C, Gill O Noel."Salm-Net" laboratory-based surveillance of human salmonella in- fections in Europe. PHLS Microbiology Digest 1994; 11:181-2. 2. Palmer S, Henry R. Epinet in Wales: PHLS Cadwyn Cymru development of a public health information system. PHLS Microbiology Digest 1992;9:107-9. 3. Communicable Disease Surveillance Centre. Salmo- nella mikawasima update. Communicable Disease Re- port 1993;3:215. 4. Communicable Disease Surveillance Centre. Salmo- nella javiana in Europe. Communicable DiseaseReport 1994;4:61. 5. Bartlett C, Gill N. International surveillance of dis- ease--communicable disease control after Maastricht: Germs and subsidiarity. Lancet 1993; 341:997-8. 6. Communicable Disease Surveillance Centre. European surveillance of Legionnaires' disease associated with travel. Communicable Disease Report 1994;4:25. 7. Thacker SB, Stroup DF.

N. International surveillance of dis- ease--communicable disease control after Maastricht: Germs and subsidiarity. Lancet 1993; 341:997-8. 6. Communicable Disease Surveillance Centre. European surveillance of Legionnaires' disease associated with travel. Communicable Disease Report 1994;4:25. 7. Thacker SB, Stroup DF. Future directions for compre- hensive public health surveillance and health informa- tion systems in the United States. Am J Epidemiol 1994;140:383-97. 8. Centers for Disease Control and Prevention. National Electronic Telecommunications System for Surveil- lance--United States, MMWR 1990-1991;40:502-3. 9. Bean NH, Martin SM, Bradford H. PHLIS: An elec- tronic system for reporting public health data from remote sites. Am J Public Health 1992;82:1273-6. Commentary Emerging Infectious Diseases 35 Vol. 1, No. 1 -- January-March 1995

Communicable Diseases Intelligence Communicable Diseases Intelligence (CDI) is a fortnightly publicationof the AustralianDepartment of Human Services and Health and the Communica- ble Diseases Network of Australia and New Zealand. The Network comprises representatives of the Aus- tralian Department of Human Services and Health, the State and Territory health authorities, and other organizations involved in communicable disease sur- veillance and control from throughout the country. In addition, there is a representative from New Zea- land. It has fortnightly teleconferences and other meetings to exchange information on emerging com- municable disease activity and to coordinate surveil- lance and control activities. Each issue of CDI incorporates reports from Aus- tralia's national communicable diseases surveillance systems, including the National Notifiable Diseases Surveillance System, the CDI Laboratory Reporting Schemes, and the Australian Sentinel General Prac- titioner Surveillance Network. Reports from the Na- tional Salmonella Surveillance Scheme, the Australian Gonococcal Surveillance Programme and the National HIV, AIDS, and Tuberculosis Reporting Systems are also regularly included. CDI also publishes timely reports of communica- ble disease outbreaks and other articles dealing with a wide range of subjects relevant to the surveillance and control of communicable diseases in Australia. Recently published items have reported, for exam- ple, the first identification of endemically acquired hepatitis E in the Northern Territory of Australia, an outbreak of influenza in a nursing home, the epidemiology of hepatitis A in South Australia, the epidemiology of Barmah Forest virus disease in Western Australia, and the outbreak of respiratory disease in humans and horses due to a previously unrecognized paramyxovirus.

tis E in the Northern Territory of Australia, an outbreak of influenza in a nursing home, the epidemiology of hepatitis A in South Australia, the epidemiology of Barmah Forest virus disease in Western Australia, and the outbreak of respiratory disease in humans and horses due to a previously unrecognized paramyxovirus. CDI is available from The Editor Communicable Diseases Intelligence AIDS and Communicable Diseases Branch Department of Human Services and Health GPO Box 9848 Canberra ACT 2601 Australia Helen Longbottom Department of Human Services and Health Canberra ACT Australia DxMONITOR: Compiling Veterinary Diagnostic Laboratory Results The DxMONITOR is a collaborative effort be- tween the U.S. Department of Agriculture, Animal and Plant Health Inspection Service's Veterinary Services (USDA:APHIS:VS), the American Associa- tion of Veterinary Laboratory Diagnosticians, and the United States Animal Health Association. This quarterly animal health report presents compiled data from national animal disease control and eradi- cation programs (bovine and porcine brucellosis, bo- vine tuberculosis, porcine pseudorabies and equine infectious anemia); patterns of selected diseases based on veterinary diagnostic laboratory data (bo- vine leukosis; bovine bluetongue; bovine, ovine and caprine paratuberculosis; equine arboviral encepha- litis; equine viral arteritis; porcine reproductive and respiratory syndrome); data on selected etiologic agents associated with specific animal health events such as bovine abortion; global disease distribution (bovine spongiform encephalopathy); and notes from veterinary diagnostic laboratories about unusual laboratory findings or new diagnostic procedures. The DxMONITOR has contributed to a greater awareness of animal diseases in the United States. Global trade agreements, the worldwide information explosion, and increasing public concern over the safety and quality of food have focused attention on animal health. Compilation of veterinary diagnostic laboratory data is one component of the USDA: APHIS efforts to respond to these increased demands for animal health information through an integrated and coordinated monitoring and surveillance sys- tem. Animal-health monitoring and disease surveil- lance concern not only animal health per se, but also interactions with the environment, animal welfare, production practices and product wholesomeness which impact animal health.

health information through an integrated and coordinated monitoring and surveillance sys- tem. Animal-health monitoring and disease surveil- lance concern not only animal health per se, but also interactions with the environment, animal welfare, production practices and product wholesomeness which impact animal health. The DxMONITOR is mailed to all interested parties without charge and is increasingly available through electronic dissemi- nation channels. For more information or subscrip- tion, contact DxMonitor Animal Health Report, c/o Centers for Epidemiology and Animal Health, USDA:APHIS:VS, 555 S. Howes, Suite 200, Ft. Col- lins, CO 80521-2586; telephone 303-490-7800; e-mail DXMONITOR@aphis.ag.gov. William Hueston Centers for Epidemiology and Animal Health Veterinary Services APHIS, U.S. Department of Agriculture Fort Collins, Colorado, USA News and Notes Vol. 1, No. 1 -- January-March 1995 36 Emerging Infectious Diseases

WHO Scientific Working Group on Monitoring and Management of Bacterial Resistance to Antimicrobial Agents Antibacterial resistance is a global clinical and public health problem that has emerged with alarm- ing rapidity in recent years and undoubtedly will increase in the near future. Resistant bacteria do not respect national borders, and developments in the remote locations can have an impact throughout the world. Resistance is a problem in the community as well as in health care settings, where transmission of bacteria is greatly amplified, in both developed and developing countries. Because multiple drug resistance is a growing problem, physicians are now confronted with infections for which there is no effec- tive therapy. The morbidity, mortality, and financial costs of such infections pose an increasing burden for health care systems worldwide, but especially in countries with limited resources. The Division of Communicable Diseases at the World Health Organization, Geneva, Switzerland, recently convened a Scientific Working Group to address the problem of drug-resistant bacterial in- fections. From November 29 to December 2, 1994, participants from 23 countries reviewed and dis- cussed scientific data on the nature and costs of drug resistance; recent national and global trends; ap- proaches to limiting the emergence and spread of resistance in community and institutional settings; and strategies to strengthen local, national, and global surveillance. Participants included repre- sentatives from clinical medicine, public health, the clinical laboratory, and the biomedical research arenas and from the pharmaceutical industry. The Working Group formulated a series of recom- mendations to address these issues at local, national, and international levels.

surveillance. Participants included repre- sentatives from clinical medicine, public health, the clinical laboratory, and the biomedical research arenas and from the pharmaceutical industry. The Working Group formulated a series of recom- mendations to address these issues at local, national, and international levels. The recommendations placed emphasis on enhanced surveillance of drug resistance through usage of WHONET software, in- creased monitoring and improved usage of antimi- crobial drugs in human, veterinary, and animal husbandry settings, improved laboratory diagnostic capacity, standardization and quality control of labo- ratory methodology, professional and public educa- tion, development of new drugs and assessment of alternative therapeutic modalities, assessment of vaccine development and delivery priorities related to antimicrobial resistance, better implementation of infection control measures, and evaluation of pre- vention strategies. The Working Group plans to release its final re- port in the spring. Fred C. Tenover National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA James M. Hughes National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA News and Notes Emerging Infectious Diseases 37 Vol. 1, No. 1 -- January-March 1995

Factors in the Emergence of Infectious Diseases Stephen S. Morse, Ph.D. The Rockefeller University, New York, NewYork, USA "Emerging" infectious diseases can be defined as infections that have newly appeared in a population or have existed but are rapidly increasing in incidence or geographic range. Among recent examples are HIV/AIDS, hantavirus pulmonary syndrome, Lyme disease, and hemolytic uremic syndrome (a foodborne infection caused by certain strains of Escherichia coli). Specific factors precipitating disease emergence can be identified in virtually all cases. These include ecological, environ- mental, or demographic factors that place people at increased contact with a pre- viously unfamiliar microbe or its natural host or promote dissemination. These factors are increasing in prevalence; this increase, together with the ongoing evolution of viral and microbial variants and selection for drug resistance, suggests that infections will continue to emerge and probably increase and emphasizes the urgent need for effective surveillance and control. Dr. David Satcher's article and this overview inaugurate "Perspectives," a regular section in this journal intended to present and develop unifying concepts and strategies for considering emerging infections and their underlying factors. The editors welcome, as contributions to the Perspectives section, overviews, syntheses, and case studies that shed light on how and why infections emerge, and how they may be anticipated and prevented. Infectious diseases emerging throughout history have included some of the most feared plagues of the past. New infections continue to emerge today, while many of the old plagues are with us still. These are global problems (William Foege, former CDC direc- tor now at the Carter Center, terms them "global infectious disease threats"). As demonstrated by in- fluenza epidemics, under suitable circumstances, a new infection first appearing anywhere in the world could traverse entire continents within days or weeks. We can define as "emerging" infections that have newly appeared in the population, or have existed but are rapidly increasing in incidence or geographic range (1,2).

enza epidemics, under suitable circumstances, a new infection first appearing anywhere in the world could traverse entire continents within days or weeks. We can define as "emerging" infections that have newly appeared in the population, or have existed but are rapidly increasing in incidence or geographic range (1,2). Recent examples of emerging diseases in various parts of the world include HIV/AIDS; classic cholera in South America and Africa; cholera due to Vibrio cholerae O139; Rift Valley fever; han- tavirus pulmonary syndrome; Lyme disease; and hemolytic uremic syndrome, a foodborne infection caused by certain strains of Escherichia coli (in the United States, serotype O157:H7). Although these occurrences may appear inexpli- cable, rarely if ever do emerging infections appear without reason. Specific factors responsible for dis- ease emergence can be identified in virtually all cases studied (2-4). Table 1 summarizes the known causes for a number of infections that have emerged recently. I have suggested that infectious disease emergence can be viewed operationally as a two-step process: 1) Introduction of the agent into a new host population (whether the pathogen originated in the environment, possibly in another species, or as a variant of an existing human infection), followed by 2) establishment and further dissemination within the new host population ("adoption") (4). Whatever its origin, the infection "emerges" when it reaches a new population. Factors that promote one or both of these steps will, therefore, tend to precipitate dis- ease emergence. Most emerging infections, and even antibiotic-resistant strains of common bacterial pathogens, usually originate in one geographic loca- tion and then disseminate to new places (5). Regarding the introduction step, the numerous examples of infections originating as zoonoses (7,8) suggest that the "zoonotic pool"--introductions of infections from other species--is an important and potentially rich source of emerging diseases; peri- odic discoveries of "new" zoonoses suggest that the zoonotic pool appears by no means exhausted. Once introduced, an infection might then be disseminated through other factors, although rapid course and high mortality combined with low transmissibility are often limiting.

ntially rich source of emerging diseases; peri- odic discoveries of "new" zoonoses suggest that the zoonotic pool appears by no means exhausted. Once introduced, an infection might then be disseminated through other factors, although rapid course and high mortality combined with low transmissibility are often limiting. However, even if a zoonotic agent is not able to spread readily from person to person and establish itself, other factors (e.g., nosocomial infection) mighttransmit the infection.Additionally, if the reservoir host or vector becomes more widely disseminated, the microbe can appear in new places. Address for correspondence: Stephen S. Morse, The Rockefeller University, 1230 York Avenue, Box 120, New York, NY 10021-6399, USA; fax 212-327-7172; e-mail morse@rockvax.rockefeller.edu. Perspectives Vol. 1, No. 1 -- January-March 1995 7 Emerging Infectious Diseases Table 1.

nated, the microbe can appear in new places. Address for correspondence: Stephen S. Morse, The Rockefeller University, 1230 York Avenue, Box 120, New York, NY 10021-6399, USA; fax 212-327-7172; e-mail morse@rockvax.rockefeller.edu. Perspectives Vol. 1, No. 1 -- January-March 1995 7 Emerging Infectious Diseases Table 1. Recent examples of emerging infections and probable factors in their emergence Infection or Agent Factor(s) contributing to emergence Viral Argentine, Bolivian hemorrhagic fever Changes in agriculture favoring rodent host Bovine spongiform encephalopathy (cattle) Changes in rendering processes Dengue, dengue hemorrhagic fever Transportation, travel, and migration; urbanization Ebola, Marburg Unknown (in Europe and the United States, importation of monkeys) Hantaviruses Ecological or environmental changes increasing contact with rodent hosts Hepatitis B, C Transfusions, organ transplants, contaminated hypodermic apparatus, sexual transmission, vertical spread from infected mother to child HIV Migration to cities and travel; after introduction, sexual transmission, vertical spread from infected mother to child, contaminated hypodermic apparatus (including during intravenous drug use), transfusions, organ transplants HTLV Contaminated hypodermic apparatus, other Influenza (pandemic) Possibly pig-duck agriculture, facilitating reassortment of avian and mammalian influenza viruses* Lassa fever Urbanization favoring rodent host, increasing exposure (usually in homes) Rift Valley fever Dam building, agriculture, irrigation; possibly change in virulence or pathogenicity of virus Yellow fever (in "new" areas) Conditions favoring mosquito vector Bacterial Brazilian purpuric fever (Haemophilus influenzae, biotype aegyptius) Probably new strain Cholera In recent epidemic in South America, probably introduced from Asia by ship, with spread facilitated by reduced water chlorination; a new strain (type O139) from Asia recently disseminated by travel (similarly to past introductions of classic cholera) Helicobacter pylori Probably long widespread, now recognized (associated with gastric ulcers, possibly other gastrointestinal disease) Hemolytic uremic syndrome (Escherichia coli O157:H7) Mass food processing technology allowing contamination of meat Legionella (Legionnaires' disease) Cooling and plumbing systems (organism grows in biofilms that form on water storage tanks and in stagnant plumbing) Lyme borreliosis (Borrelia burgdorferi) Reforestation around homes and other condi

cherichia coli O157:H7) Mass food processing technology allowing contamination of meat Legionella (Legionnaires' disease) Cooling and plumbing systems (organism grows in biofilms that form on water storage tanks and in stagnant plumbing) Lyme borreliosis (Borrelia burgdorferi) Reforestation around homes and other condi tions favoring tick vector and deer (a secondary reservoir host) Streptococcus, group A (invasive; necrotizing) Uncertain Toxic shock syndrome (Staphylococcus aureus) Ultra-absorbency tampons Parasitic Cryptosporidium, other waterborne pathogens Contaminated surface water, faulty water purification Malaria (in "new" areas) Travel or migration Schistosomiasis Dam building *Reappearances of influenza are due to two distinct mechanisms: Annual or biennial epidemics involving new variants due to antigenic drift (point mutations, primarily in the gene for the surface protein, hemagglutinin) and pandemic strains, arising from antigenic shift (genetic reassortment, generally between avian and mammalian influenza strains).

e due to two distinct mechanisms: Annual or biennial epidemics involving new variants due to antigenic drift (point mutations, primarily in the gene for the surface protein, hemagglutinin) and pandemic strains, arising from antigenic shift (genetic reassortment, generally between avian and mammalian influenza strains). Perspectives Emerging Infectious Diseases 8 Vol. 1, No. 1 -- January-March 1995 Bubonic plague transmitted by rodent fleas and ratborne hantavirus infections are examples. Most emerging infections appear to be caused by pathogens already present in the environment, brought out of obscurity or given a selective advan- tage by changing conditions and afforded an oppor- tunity to infect new host populations (on rare occasions, a new variant may also evolve and cause a new disease) (2,4). The process by which infectious agents may transfer from animals to humans or disseminate from isolated groups into new popula- tions can be called "microbial traffic" (3,4). Anumber of activities increase microbial traffic and as a result promote emergence and epidemics. In some cases, including many of the most novel infections, the agents are zoonotic, crossing from their natural hosts into the human population; because of the many similarities, I include here vector-borne dis- eases. In other cases, pathogens already present in geographically isolated populations are given an opportunity to disseminate further. Surprisingly often, disease emergence is caused by human ac- tions, however inadvertently; natural causes, such as changes in climate, can also at times be respon- sible (6). Although this discussion is confined largely to human disease, similar considerations apply to emerging pathogens in other species. Table 2 summarizes the underlying factors re- sponsible for emergence. Any categorization of the factors is, of course, somewhat arbitrary but should be representative of the underlying processes that cause emergence. I have essentially adopted the categories developed in the Institute of Medicine report on emerging infections (12), with additional definitions from the CDC emerging infections plan (13). Responsible factors include ecological changes, such as those due to agricultural or economic devel- opment or to anomalies in climate; human demo- graphic changes and behavior; travel and commerce; technology and industry; microbial adaptation and change; and breakdown of public health measures. Each of these will be considered in turn.

include ecological changes, such as those due to agricultural or economic devel- opment or to anomalies in climate; human demo- graphic changes and behavior; travel and commerce; technology and industry; microbial adaptation and change; and breakdown of public health measures. Each of these will be considered in turn. Ecological interactions can be complex, with sev- eral factors often working together or in sequence. For example, population movement from rural areas to cities can spread a once-localized infection. The strain on infrastructure in the overcrowded and rapidly growing cities may disrupt or slow public health measures, perhaps allowing establishment of the newly introduced infection. Finally, the city may also provide a gateway for further dissemination of the infection. Most successful emerging infections, including HIV, cholera, and dengue, have followed this route. Consider HIV as an example. Although the pre- cise ancestry of HIV-1 is still uncertain, it appears to have had a zoonotic origin (9,10). Ecological fac- tors that would have allowed human exposure to a natural host carrying the virus that was the precur- sor to HIV-1 were, therefore, instrumental in the introduction of the virus into humans. This probably occurred in a rural area. A plausible scenario is suggested by the identification of an HIV-2-infected man in a rural area of Liberia whose virus strain resembled viruses isolated from the sooty mangabey monkey (an animal widely hunted for food in rural areas and the putative source of HIV-2) more closely than it did strains circulating in the city (11). Such findings suggest that zoonotic introductions of this sort may occur on occasion in isolated populations but may well go unnoticed so long as the recipients remain isolated. But with increasing movement from rural areas to cities, such isolation is increas- ingly rare. After its likely first move from a rural area into a city, HIV-1 spread regionally along high- ways, then by long distance routes, including air travel, to more distant places. This last step was critical for HIV and facilitated today's global pan- demic. Social changes that allowed the virus to reach a larger population and to be transmitted despite its relatively low natural transmissibility were instru- mental in the success of the virus in its newfound human host.

to more distant places. This last step was critical for HIV and facilitated today's global pan- demic. Social changes that allowed the virus to reach a larger population and to be transmitted despite its relatively low natural transmissibility were instru- mental in the success of the virus in its newfound human host. For HIV, the long duration of infectivity allowed this normally poorly transmissible virus many opportunities to be transmitted and to take advantage of such factors as human behavior (sex- ual transmission, intravenous drug use) and chang- ing technology (early spread through blood transfusions and blood products) (Table 1). Ecological Changes and Agricultural Development Ecological changes, including those due to agri- cultural or economic development, are among the most frequently identified factors in emergence. They are especially frequent as factors in outbreaks of previously unrecognized diseases with high case- fatality rates, which often turn out to be zoonotic introductions. Ecological factors usually precipitate emergence by placing people in contact with a natu- ral reservoir or host for an infection hitherto unfa- miliar but usually already present (often a zoonotic or arthropod-borne infection), either by increasing proximity or, often, also by changing conditions so as to favor an increased population of the microbe or its natural host (2,4). The emergence of Lyme dis- ease in the United States and Europe was probably due largely to reforestation (14), which increased the population of deer and the deer tick, the vector of Lyme disease. The movement of people into these areas placed a larger population in close proximity to the vector. Agricultural development, one of the most com- mon ways in which people alter and interpose them- selves into the environment, is often a factor Perspectives Vol. 1, No. 1 -- January-March 1995 9 Emerging Infectious Diseases (Table 2). Hantaan virus, the cause of Korean hem- orrhagic fever, causes over 100,000 cases a year in China and has been known in Asia for centuries. The virus is a natural infection of the field mouse Apode- mus agrarius. The rodent flourishes in rice fields; people usually contract the disease during the rice harvest from contact with infected rodents. Junin virus, the cause of Argentine hemorrhagic fever, is an unrelated virus with a history remarkably simi- lar to that of Hantaan virus.

fection of the field mouse Apode- mus agrarius. The rodent flourishes in rice fields; people usually contract the disease during the rice harvest from contact with infected rodents. Junin virus, the cause of Argentine hemorrhagic fever, is an unrelated virus with a history remarkably simi- lar to that of Hantaan virus. Conversion of grass- land to maize cultivation favored a rodent that was the natural host for this virus, and human cases increased in proportion with expansion of maize agriculture (15). Other examples, in addition to those already known (2,15), are likely to appear as new areas are placed under cultivation. Perhaps most surprisingly, pandemic influenza appears to have an agricultural origin, integrated pig-duck farming in China. Strains causing the fre- quent annual or biennial epidemics generally result from mutation ("antigenic drift"), but pandemic in- fluenza viruses do not generally arise by this proc- ess. Instead, gene segments from two influenza strains reassort to produce a new virus that can infect humans (16). Evidence amassed by Webster, Scholtissek, and others, indicates that waterfowl, such as ducks, are major reservoirs of influenza and that pigs can serve as "mixing vessels" for new mammalian influenza strains (16). Pandemic influ- enza viruses have generally come from China. Scholtissek and Naylor suggested that integrated pig-duck agriculture, an extremely efficient food Table 2. Factors in infectious disease emergence* Factor Examples of specific factors Examples of diseases Ecological changes (including those due to economic development and land use) Agriculture; dams, changes in water ecosystems; deforestation/reforestation; flood/drought; famine; climate changes Schistosomiasis (dams); Rift Valley fever (dams, irrigation); Argentine hemorrhagic fever (agriculture); Hantaan (Korean hemorrhagic fever) (agriculture); hantavirus pulmonary syndrome, southwestern US, 1993 (weather anomalies) Human demographics, behavior Societal events: Population growth and migration (movement from rural areas to cities); war or civil conflict; urban decay; sexual behavior; intravenous drug use; use of high-density facilities Introduction of HIV; spread of dengue; spread of HIV and other sexually transmitted diseases International travel and commerce Worldwide movement of goods and people; air travel "Airport" malaria; dissemination of mosquito vectors; ratborne hantaviruses; introduction of cholera into South America; dissemination of O139 V.

roduction of HIV; spread of dengue; spread of HIV and other sexually transmitted diseases International travel and commerce Worldwide movement of goods and people; air travel "Airport" malaria; dissemination of mosquito vectors; ratborne hantaviruses; introduction of cholera into South America; dissemination of O139 V. cholerae Technology and industry Globalization of food supplies; changes in food processing and packaging; organ or tissue transplantation; drugs causing immunosuppression; widespread use of antibiotics Hemolytic uremic syndrome (E. coli contamination of hamburger meat), bovine spongiform encephalopathy; transfusion-associated hepatitis (hepatitis B, C), opportunistic infections in immunosuppressed patients, Creutzfeldt-Jakob disease from contaminated batches of human growth hormone (medical technology) Microbial adaptation and change Microbial evolution, response to selection in environment Antibiotic-resistant bacteria, "antigenic drift" in influenza virus Breakdown in public health measures Curtailment or reduction in prevention programs; inadequate sanitation and vector control measures Resurgence of tuberculosis in the United States; cholera in refugee camps in Africa; resurgence of diphtheria in the former Soviet Union *Categories of factors (column 1) adapted from ref. 12, examples of specific factors (column 2) adapted from ref. 13. Categories are not mutually exclusive; several factors may contribute to emergence of a disease (see Table 1 for additional information). Perspectives Emerging Infectious Diseases 10 Vol. 1, No. 1 -- January-March 1995 production system traditionally practiced in certain parts of China for several centuries, puts these two species in contact and provides a natural laboratory for making new influenza recombinants (17). Web- ster has suggested that, with high-intensity agricul- ture and movement of livestock across borders, suitable conditions may now also be found in Europe (16). Water is also frequently associated with disease emergence. Infections transmitted by mosquitoes or other arthropods, which include some of the most serious and widespread diseases (18,19), are often stimulated by expansion of standing water, simply because many of the mosquito vectors breed in water. There are many cases of diseases transmitted by water-breeding vectors, most involving dams, water for irrigation, or stored drinking water in cities.

ome of the most serious and widespread diseases (18,19), are often stimulated by expansion of standing water, simply because many of the mosquito vectors breed in water. There are many cases of diseases transmitted by water-breeding vectors, most involving dams, water for irrigation, or stored drinking water in cities. (See "Changes in Human Demographics and Behavior" for a discussion of dengue.) The incidence of Japanese encephalitis, another mosquito-borne disease that accounts for almost 30,000 human cases and approximately 7,000 deaths annually in Asia, is closely associated with flooding of fields for rice growing. Outbreaks of Rift Valley fever in some parts of Africa have been associated with dam build- ing as well as with periods of heavy rainfall (19). In the outbreaks of Rift Valley fever in Mauritania in 1987, the human cases occurred in villages near dams on the Senegal River. The same effect has been documented with other infections that have aquatic hosts, such as schistosomiasis. Because humans are important agents of ecologi- cal and environmental change, many ofthesefactors are anthropogenic. Of course, this is not always the case, and natural environmental changes, such as climate or weather anomalies, can have the same effect. The outbreak of hantavirus pulmonary syn- drome in the southwestern United States in 1993 is an example. It is likely that the virus has long been present in mouse populations but an unusually mild and wet winter and spring in that area led to an increased rodent population in the spring and sum- mer and thus to greater opportunities for people to come in contact with infected rodents (and, hence, with the virus); it has been suggested that the weather anomaly was due to large-scale climatic effects (20). The same causes may have been respon- sible for outbreaks of hantaviral disease in Europe at approximately the same time (21,22). With chol- era, it has been suggested that certain organisms in marine environments are natural reservoirs for cholera vibrios, and that large scale effects on ocean currents may cause local increases in the reservoir organism with consequent flare-ups of cholera (23). Changes in Human Demographics and Behavior Human population movements or upheavals, caused by migration or war, are often important factors in disease emergence. In many parts of the world, economic conditions are encouraging the mass movement of workers from rural areas to cit- ies.

nsequent flare-ups of cholera (23). Changes in Human Demographics and Behavior Human population movements or upheavals, caused by migration or war, are often important factors in disease emergence. In many parts of the world, economic conditions are encouraging the mass movement of workers from rural areas to cit- ies. The United Nations has estimated that, largely as a result of continuing migration, by the year 2025, 65% of the world population (also expected to be larger in absolute numbers), including 61% of the population in developing regions, will live in cities (24). As discussed above for HIV, rural urbanization allows infections arising in isolated rural areas, which may once have remained obscure and local- ized, to reach larger populations. Once in a city, the newly introduced infection would have the opportu- nity to spread locally among the population and could also spread further along highways and inter- urban transport routes and by airplane. HIV has been, and in Asia is becoming, the best known bene- ficiary of this dynamic, but many other diseases, such as dengue, stand to benefit. The frequency of the most severe form, dengue hemorrhagic fever, which is thought to occur when a person is sequen- tially infected by two types of dengue virus, is in- creasing as different dengue viruses have extended their range and now overlap (25). Dengue hemor- rhagic fever is now common in some cities in Asia, where the high prevalence of infection is attributed to the proliferation of open containers needed for water storage (which also provide breeding grounds for the mosquito vector) as the population size ex- ceeds the infrastructure (19). In urban environ- ments, rain-filled tires or plastic bottles are often breeding grounds of choice for mosquito vectors. The resulting mosquito population boom is comple- mented by the high human population density in such situations, increasing the chances of stable transmission cycles between infected and suscepti- ble persons. Even in industrialized countries, e.g., the United States, infections such as tuberculosis can spread through high-population density set- tings (e.g., day care centers or prisons) (12,26-28). Human behavior can have important effects on disease dissemination. The best known examples are sexually transmitted diseases, and the ways in which such human behavior as sex or intravenous drug use have contributed to the emergence of HIV are now well known.

set- tings (e.g., day care centers or prisons) (12,26-28). Human behavior can have important effects on disease dissemination. The best known examples are sexually transmitted diseases, and the ways in which such human behavior as sex or intravenous drug use have contributed to the emergence of HIV are now well known. Other factors responsible for disease emergence are influenced by a variety of human actions, so human behavior in the broader sense is also very important. Motivating appropri- ate individual behavior and constructive action, both locally and in a larger scale, will be essential for controlling emerging infections. Ironically, as AIDS prevention efforts have demonstrated, human Perspectives Vol. 1, No. 1 -- January-March 1995 11 Emerging Infectious Diseases behavior remains one of the weakest links in our scientific knowledge. International Travel and Commerce The dissemination of HIV through travel has already been mentioned. In the past, an infection introduced into people in a geographically isolated area might, on occasion, be brought to a new place through travel, commerce, or war (8). Trade between Asia and Europe, perhaps beginning with the silk route and continuing with the Crusades, brought the rat and one of its infections, the bubonic plague, to Europe. Beginning in the 16th and 17th centuries, ships bringing slaves from West Africa to the New World also brought yellow fever and its mosquito vector, Aedes aegypti, to the new territories. Simi- larly, smallpox escaped its Old World origins to wreak new havoc in the New World. In the 19th century, cholera had similar opportunities to spread from its probable origin in the Ganges plain to the Middle East and, from there, to Europe and much of the remaining world. Each of these infections had once been localized and took advantage of opportu- nities to be carried to previously unfamiliar parts of the world. Similar histories are being repeated today, but opportunities in recent years have become far richer and more numerous, reflecting the increasing vol- ume, scope, and speed of traffic in an increasingly mobile world. Rats have carried hantaviruses virtu- ally worldwide (29). Aedes albopictus (the Asian tiger mosquito) was introduced into the United States, Brazil, and parts of Africa in shipments of used tires from Asia (30).

numerous, reflecting the increasing vol- ume, scope, and speed of traffic in an increasingly mobile world. Rats have carried hantaviruses virtu- ally worldwide (29). Aedes albopictus (the Asian tiger mosquito) was introduced into the United States, Brazil, and parts of Africa in shipments of used tires from Asia (30). Since its introduction in 1982, this mosquito has established itself in at least 18 states of the United States and has acquired local viruses including Eastern equine encephalomyelitis (31), a cause of serious disease. Another mosquito- borne disease, malaria, is one of the most frequently imported diseases in non-endemic-disease areas, and cases of "airport malaria" are occasionally iden- tified. A classic bacterial disease, cholera, recently en- tered both South America (for the first time this century) and Africa. Molecular typing shows the South American isolates to be of the current pan- demic strain (32), supporting the suggestion thatthe organism was introduced in contaminated bilge water from an Asian freighter (33). Other evidence indicates that cholera was only one of many organ- isms to travel in ballast water; dozens, perhaps hundreds, of species have been exchanged between distant places through this means of transport alone. New bacterial strains, such as the recently identified Vibrio cholerae O139, or an epidemic strain of Neisseria meningitidis (34,35) (also exam- ples of microbial adaptation and change) have dis- seminated rapidly along routes of trade and travel, as have antibiotic-resistant bacteria (5,36). Technology and Industry High-volume rapid movement characterizes not only travel, but also other industries in modern society. In operations, including food production, that process or use products of biological origin, modern production methods yield increased effi- ciency and reduced costs but can increase the chances of accidental contamination and amplify the effects of such contamination. The problem is fur- ther compounded by globalization, allowing the op- portunity to introduce agents from far away. A pathogen present in some of the raw material may find its way into a large batch of final product, as happened with the contamination of hamburger meat by E. coli strains causing hemolytic uremic syndrome (37). In the United States the implicated E. coli strains are serotype O157:H7; additional serotypes have been identified in other countries.

f the raw material may find its way into a large batch of final product, as happened with the contamination of hamburger meat by E. coli strains causing hemolytic uremic syndrome (37). In the United States the implicated E. coli strains are serotype O157:H7; additional serotypes have been identified in other countries. Bovine spongiform encephalopathy (BSE), which emerged in Britain within the last few years, was likely an interspecies transfer of scrapie from sheep to cattle (38) that occurred when changes in render- ing processes led to incomplete inactivation of scra- pie agent in sheep byproducts fed to cattle (39). The concentrating effects that occur with blood and tissue products have inadvertently dissemi- nated infections unrecognized at the time, such as HIV and hepatitis B and C. Medical settings are also at the front line of exposure to new diseases, and a number of infections, including many emerging in- fections, have spread nosocomially in health care settings (Table 2). Among the numerous examples, in the outbreaks of Ebola fever in Africa many of the secondary cases were hospital acquired, most trans- mitted to other patients through contaminated hy- podermic apparatus, and some to the health care staff by contact. Transmission of Lassa fever to health care workers has also been documented. On the positive side, advances in diagnostic tech- nology can also lead to new recognition of agents that are already widespread. When such agents are newly recognized, they may at first often be labeled, in some cases incorrectly, as emerging infections. Human herpesvirus 6 (HHV-6) was identified only a few years ago, but the virus appears to be extremely widespread (40) and has recently been implicated as the cause of roseola (exanthem subitum), a very common childhood disease (41). Because roseola has been known since at least 1910, HHV-6 is likely to have been common for decades and probably much longer. Another recent example is the bacterium Helicobacter pylori, a probable cause of gastric ul- cers (42) and some cancers (43,44). We have lived with these diseases for a long time without knowing Perspectives Emerging Infectious Diseases 12 Vol. 1, No. 1 -- January-March 1995 their cause. Recognition of the agent is often advan- tageous, offering new promise of controlling a pre- viously intractable disease, such as treating gastric ulcers with specific antimicrobial therapy.

ses for a long time without knowing Perspectives Emerging Infectious Diseases 12 Vol. 1, No. 1 -- January-March 1995 their cause. Recognition of the agent is often advan- tageous, offering new promise of controlling a pre- viously intractable disease, such as treating gastric ulcers with specific antimicrobial therapy. Microbial Adaptation and Change Microbes, like all other living things, are con- stantly evolving. The emergence of antibiotic-resis- tant bacteria as a result of the ubiquity of antimicrobials in the environment is an evolution- ary lesson on microbial adaptation, as well as a demonstration of the power of natural selection. Selection for antibiotic-resistant bacteria (5,36) and drug-resistant parasites has become frequent, driven by the wide and sometimes inappropriate use of antimicrobial drugs in a variety of applications (27,45,46). Pathogens can also acquire new antibi- otic resistance genes from other, often nonpatho- genic, species in the environment (36), selected or perhaps even driven by the selection pressure of antibiotics. Many viruses show a high mutation rate and can rapidly evolve to yield new variants (47). A classic example is influenza (48). Regular annual epidemics are caused by "antigenic drift" in a previously circu- lating influenza strain. Achange in an antigenic site of a surface protein, usually the hemagglutinin (H) protein, allows the new variant to reinfect pre- viously infected persons because the altered antigen is not immediately recognized by the immune system. On rare occasions, perhaps more often with non- viral pathogens than with viruses (49), the evolution of a new variant may result in a new expression of disease. The epidemic of Brazilian purpuric fever in 1990, associated with a newly emerged clonal vari- ant of Hemophilus influenzae, biogroup aegyptius, may fall into this category. It is possible, but not yet clear, that some recently described manifestations of disease by group AStreptococcus, such as rapidly invasive infection or necrotizing fasciitis, may also fall into this category. Breakdown of Public Health Measures and Deficiencies in Public Health Infrastructure Classical public health and sanitation measures have long served to minimize dissemination and human exposure to many pathogens spread by tra- ditional routes such as water or preventable by immunization or vector control.

is category. Breakdown of Public Health Measures and Deficiencies in Public Health Infrastructure Classical public health and sanitation measures have long served to minimize dissemination and human exposure to many pathogens spread by tra- ditional routes such as water or preventable by immunization or vector control. The pathogens themselves often still remain, albeit in reduced numbers, in reservoir hosts or in the environment, or in small pockets of infection and, therefore, are often able to take advantage of the opportunity to reemerge if there are breakdowns in preventive measures. Reemerging diseases are those, like cholera, that were once decreasing but are now rapidly increasing again. These are often conventionally understood and well recognized public health threats for which (in most cases) previously active public health meas- ures had been allowed to lapse, a situation that unfortunately now applies all too often in both de- veloping countries and the inner cities of the indus- trialized world. The appearance of reemerging diseases may, therefore, often be a sign of the break- down of public health measures and should be a warning against complacency in the war against infectious diseases. Cholera, for example, has recently been raging in South America (for the first time in this century) (50) and Africa. The rapid spread of cholera in South America may have been abetted by recent reduc- tions in chlorine levels used to treat water supplies (34). The success of cholera and other enteric dis- eases is often due to the lack of a reliable water supply. These problems are more severe in develop- ing countries, but are not confined to these areas. The U.S. outbreak of waterborne Cryptosporidium infection in Milwaukee, Wisconsin, in the spring of 1993, with over 400,000 estimated cases, was in part due to a nonfunctioning water filtration plant (51); similar deficiencies in water purification have been found in other cities in the United States (52). For our Future In his accompanying article, Dr. David Satcher discusses the history of infectious diseases and the many infections that, from the dawn of history to the present, have traveled with the caravans and fol- lowed the invading armies. The history of infectious diseases has been a history of microbes on the march, often in our wake, and of microbes that have taken advantage of the rich opportunities offered them to thrive, prosper, and spread.

from the dawn of history to the present, have traveled with the caravans and fol- lowed the invading armies. The history of infectious diseases has been a history of microbes on the march, often in our wake, and of microbes that have taken advantage of the rich opportunities offered them to thrive, prosper, and spread. And yet the historical processes that have given rise to the emer- gence of "new" infections throughout history con- tinue today with unabated force; in fact, they are accelerating, because the conditions of modern life ensure that the factors responsible for disease emer- gence are more prevalent than ever before. Speed of travel and global reach are further borne out by studies modeling the spread of influenza epidemics (53) and HIV (54,55). Humans are not powerless, however, against this relentless march of microbes. Knowledge of the fac- tors underlying disease emergence can help focus resources on the key situations and areas worldwide (3,4) and develop more effective prevention strate- gies. If we are to protect ourselves against emerging diseases, the essential first step is effective global Perspectives Vol. 1, No. 1 -- January-March 1995 13 Emerging Infectious Diseases disease surveillance to give early warning of emerg- ing infections (3,12,13,56). This must be tied to in- centives, such as national development, and eventually be backed by a system for an appropriate rapid response. World surveillance capabilities are critically deficient (12,56,57). Efforts, such as the CDC plan (13), now under way in the United States and internationally to remedy this situation are the essential first steps and deserve strong support. Research, both basic and applied, will also be vital. This Journal and the "Perspectives" Section Early warning of emerging and reemerging infec- tions depends on the ability to identify the unusual as early as possible. Information is, therefore, essen- tial. Hence this journal, which is intended as a peer-reviewed forum for the discussion of concepts and examples relevant to emerging infectious dis- eases and their causes, and to provide a channel for field reports and observations on emerging infec- tions. The "Perspectives" section will provide gen- eral overviews dealing with factors in disease emergence, conceptual syntheses of information, ap- proaches for studying or predicting emerging infec- tions, and analyses that shed light on how and why infections emerge, and how they may be anticipated and prevented.

tions. The "Perspectives" section will provide gen- eral overviews dealing with factors in disease emergence, conceptual syntheses of information, ap- proaches for studying or predicting emerging infec- tions, and analyses that shed light on how and why infections emerge, and how they may be anticipated and prevented. Submissions for this section are warmly invited. In coming issues, Perspectives will deal in greater detail with many of the factors dis- cussed in this overview article, and with ways to dissect steps in the emergence process. Discussion of technologies that are broadly applicable to the identification or control of emerging diseases are also appropriate for this section. Case studies are welcome if they are used to develop broader lessons. Acknowledgments I thank Dr. John La Montagne, NIAID, for helpful discussions. Supported by NIH grant RR 03121 (from CMP), US DHHS. Dr. Morse, "Perspectives" section editor of this journal, is assistant professor of virology at The Rockefeller University, New York, N.Y. He chaired the NIH Conference on Emerging Viruses (May 1989) and was a member of the committee on Emerging Microbial Threats to Health (and chaired its Task Force on Viruses), convened by the Institute of Medicine, National Academy of Sciences (ref. 12). References 1. Morse SS, Schluederberg A. Emerging viruses: the evolution of viruses and viral diseases. J Infect Dis 1990;162:1-7. 2. Morse SS. Examining the origins of emerging viruses. In: Morse SS, ed. Emerging viruses. New York: Oxford University Press, 1993:10-28. 3. Morse SS. Regulating viral traffic. Issues Sci Technol 1990;7:81-4. 4. Morse SS. Emerging viruses: defining the rules for viral traffic. Perspect Biol Med 1991;34:387-409. 5. Soares S, Kristinsson KG, Musser JM, Tomasz A. Evidence for the introduction of a multiresistant clone of serotype 6B Streptococcus pneumoniae from Spain to Iceland in the late 1980s. J Infect Dis 1993;168:158- 63. 6. Rogers DJ, Packer MJ. Vector-borne diseases, models, and global change. Lancet 1993;342:1282-4. 7. Fiennes RW. Zoonoses and the origins and ecology of human disease. London: Academic Press, 1978. 8. McNeill WH. Plagues and peoples. New York: Anchor Press/ Doubleday, 1976. 9. Myers G, MacInnes K, Korber B. The emergence of simian/human immunodeficiency viruses. AIDS Res Hum Retroviruses 1992;8:373-86. 10. Allan JS, Short M, Taylor ME, et al. Species-specific diversity among simian immunodeficiency viruses from African green monkeys. J Virol 1991;65:2816-28.

Anchor Press/ Doubleday, 1976. 9. Myers G, MacInnes K, Korber B. The emergence of simian/human immunodeficiency viruses. AIDS Res Hum Retroviruses 1992;8:373-86. 10. Allan JS, Short M, Taylor ME, et al. Species-specific diversity among simian immunodeficiency viruses from African green monkeys. J Virol 1991;65:2816-28. 11. Gao F, Yue L, White AT, et al. Human infection by genetically diverse SIVSM-related HIV-2 in West Af- rica. Nature 1992;358:495-9. 12. Institute of Medicine. Emerging infections: Microbial threats to health in the United States (Lederberg J, Shope RE, Oaks SC Jr, eds). Washington, DC: Na- tional Academy Press, 1992. 13. Centers for Disease Control and Prevention. Address- ing emerging infectious disease threats: a prevention strategy for the United States. Atlanta, Georgia: US Dept of Health and Human Services, Public Health Service, 1994. 14. Barbour AG, Fish D. The biological and social phe- nomenon of Lyme disease. Science 1993;260:1610-6. 15. Johnson KM. Emerging viruses in context: an over- view of viral hemorrhagic fevers. In: Morse SS, ed. Emerging viruses. New York: Oxford University Press, 1993:46-7. 16. Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y. Evolution and ecology of influenza A viruses. Microbiol Rev 1992;56:152-79. 17. Scholtissek C, Naylor E. Fish farming and influenza pandemics. Nature 1988;331:215. 18. World Health Organization. Geographical distribu- tion of arthropod-borne diseases and their principal vectors. Geneva: World Health Organization (WHO/VBC/89.967), 1989:138-48. 19. Monath TP. Arthropod-borne viruses. In: Morse SS, ed. Emerging viruses. New York: Oxford University Press, 1993. 20. Levins R, Epstein PR, Wilson ME, Morse SS, Slooff R, Eckardt I. Hantavirus disease emerging. Lancet 1993;342:1292. 21. Le Guenno B, Camprasse MA, Guilbaut JC, Lanoux P, Hoen B. Hantavirus epidemic in Europe, 1993. Lancet 1994;343:114-5. 22. Rollin PE, Coudrier D, Sureau P. Hantavirus epi- demic in Europe, 1993. Lancet 1994;343:115-6. Perspectives Emerging Infectious Diseases 14 Vol. 1, No. 1 -- January-March 1995 23. Epstein, PR, Ford TE, Colwell RR. Marine ecosys- tems. Lancet 1993;342:1216-9. 24. United Nations. World urbanization prospects, 1990. New York: United Nations, 1991. 25. Gubler DJ, Trent DW. Emergence of epidemic den- gue/dengue hemorrhagic fever as a public health problem in the Americas. Infectious Agents and Dis- ease 1993;26:383-93. 26. Krause RM. The origin of plagues: old and new. Sci- ence 1992;257:1073-8. 27.

rld urbanization prospects, 1990. New York: United Nations, 1991. 25. Gubler DJ, Trent DW. Emergence of epidemic den- gue/dengue hemorrhagic fever as a public health problem in the Americas. Infectious Agents and Dis- ease 1993;26:383-93. 26. Krause RM. The origin of plagues: old and new. Sci- ence 1992;257:1073-8. 27. Bloom BR, Murray CJL. Tuberculosis: commentary on a reemergent killer. Science 1992;257:1055-64. 28. Hoge CW, Reichler MR, Dominguez EA, et al. An epidemic of pneumococcal disease in an overcrowded, inadequately ventilated jail. N Engl J Med 1994;331:643-8. 29. LeDuc JW, Childs JE, Glass GE. The hantaviruses, etiologic agents of hemorrhagic fever with renal syn- drome: a possible cause of hypertension and chronic renal disease in the United States. Annu Rev Public Health 1992;13:79-98. 30. Centers for Disease Control and Prevention. Aedes albopictus introduction into continental Africa, 1991. MMWR 1991;40:836-8. 31. Centers for Disease Control and Prevention. Eastern equine encephalitis virus associated with Aedes al- bopictus--Florida, 1991. MMWR 1992;41:115, 121. 32. Wachsmuth IK, Evins GM, Fields PI, et al. The mo- lecular epidemiology of cholera in Latin America. J Infect Dis 1993;167:621-6. 33. Anderson C. Cholera epidemic traced to risk miscal- culation [News]. Nature 1991;354:255. 34. Moore PS. Meningococcal meningitis in sub-Saharan Africa: a model for the epidemic process. Clin Infect Dis 1992;14:515-25. 35. Moore PS, Broome CV. Cerebrospinal meningitis epi- demics. Sci Am 1994;271(5):38-45. 36. Davies J. Inactivation of antibiotics and the dissemi- nation of resistance genes. Science 1994;264:375-82. 37. Centers for Disease Control and Prevention. Update: multistate outbreak of Escherichia coli O157:H7 in- fections from hamburgers--western United States, 1992-1993. MMWR 1993;42:258-63. 38. Morse SS. Looking for a link. Nature 1990;344:297. 39. Wilesmith JW, Ryan JBM, Atkinson MJ. Bovine spongiform encephalopathy: epidemiological studies on the origin. Vet Rec 1991;128:199-203. 40. Inoue N, Dambaugh TR, Pellett PE. Molecular biology of human herpesviruses 6Aand 6B. Infectious Agents and Disease 1993;26:343-60. 41. Yamanishi K, Okuno T, Shiraki K, et al. Identification of human herpesvirus-6 as a causal agent for exan- them subitum. Lancet 1988;i:1065-7. 42. Peterson WL. Helicobacter pylori and peptic ulcer disease. N Engl J Med 1991;324:1043-8. 43. Nomura A, Stemmermann GN, Chyou P-H, Kato I, Perez-Perez GI, Blaser MJ.

3-60. 41. Yamanishi K, Okuno T, Shiraki K, et al. Identification of human herpesvirus-6 as a causal agent for exan- them subitum. Lancet 1988;i:1065-7. 42. Peterson WL. Helicobacter pylori and peptic ulcer disease. N Engl J Med 1991;324:1043-8. 43. Nomura A, Stemmermann GN, Chyou P-H, Kato I, Perez-Perez GI, Blaser MJ. Helicobacter pylori infec- tion and gastric carcinoma among Japanese Ameri- cans in Hawaii. N Engl J Med 1991;325:1132-6. 44. Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991;325:1127-31. 45. Cohen ML. Epidemiology of drug resistance: implica- tions for a post-antimicrobial era. Science 1992;257:1050-5. 46. Neu HC. The crisis in antibiotic resistance. Science 1992;257:1064-72. 47. Domingo E, Holland JJ. Mutation rates and rapid evolution of RNA viruses. In: Morse SS, ed. The evo- lutionary biology of viruses. New York: Raven Press, 1994:161-84. 48. Kilbourne ED. The molecular epidemiology of influ- enza. J Infect Dis 1978;127:478-87. 49. Morse SS. Toward an evolutionary biology of viruses. In: Morse SS, ed. The evolutionary biology of viruses. New York: Raven Press, 1994:1-28. 50. Glass RI, Libel M, Brandling-Bennett AD. Epidemic cholera in the Americas. Science 1992;265:1524-5. 51. MacKenzie WR, Hoxie NJ, Proctor ME, et al. A mas- sive outbreak in Milwaukee of Cryptosporidium infec- tion transmitted through the water supply. N Engl J Med 1994;331:161-7. 52. Centers for Disease Control and Prevention. Assess- ment of inadequately filtered public drinking water-- Washington, D.C., December 1993. MMWR 1994;43:661-3. 53. Longini IM Jr, Fine PEM, Thacker SB. Predicting the global spread of new infectious agents. Am J Epidemiol 1986;123:383-91. 54. Flahault A, Valleron AJ. HIV and travel, no rationale for restrictions. Lancet 1990;336:1197-8. 55. Flahault A, Valleron AJ. A method for assessing the global spread of HIV-1 infection based on air travel. Mathematical Population Studies 1992;3:161-71. 56. Henderson DA. Surveillance systems and intergov- ernmental cooperation. In: Morse SS, ed. Emerging viruses. New York: Oxford University Press, 1993:283-9. 57. Berkelman RL, Bryan RT, Osterholm MT, LeDuc JW, Hughes JM. Infectious disease surveillance: a crum- bling foundation. Science 1994;264:368-70. Perspectives Vol. 1, No. 1 -- January-March 1995 15 Emerging Infectious Diseases

Conference on "Emerging Infectious Diseases: Meeting the Challenge" Emerging infectious diseases, the leading cause of death worldwide, continue to pose difficult chal- lenges to clinicians, public health professionals, and biomedical researchers in academic settings and industry. Addressing these challenges requires a cohesive effort to develop prevention strategies and to communicate them effectively to the health care community, the public, and policy makers. On June 5-6, 1995, the New York Academy of Medicine and the New York State Department of Health convened to examine the problem of emerg- ing infections. The speakers addressed four themes: 1) emerging infectious diseases: why and why now? 2) transmission of emerging infectious diseases: old modes, new agents; 3) surveillance and sentinel systems for infectious diseases; and 4) emerging infectious diseases: what is to be done? The first three themes were addressed through presentations by 20 experts. The fourth was divided into six segments focusing on diagnosis, the role of the microbiology laboratory in surveillance, other surveillance issues, approaches to epidemic investi- gations, risk perception, and global issues. Speakers consistently alluded to recent compla- cency about infectious diseases in the United States and stressed the need for the clinical, public health, and research communities to work with the biomedi- cal industry in confronting emerging infectious dis- ease challenges in this era of transition to managed health care.

ers consistently alluded to recent compla- cency about infectious diseases in the United States and stressed the need for the clinical, public health, and research communities to work with the biomedi- cal industry in confronting emerging infectious dis- ease challenges in this era of transition to managed health care. In his opening address Joshua Leder- berg from Rockefeller University reminded partici- pants that the struggle between humans and microbes could be characterized as a battle of "wits versus genes." Margaret Hamburg, Commissioner of the New York City Department of Health, empha- sized that plague in India and Ebola virus infection in Zaire were reminders that the world is a global village, that considering domestic and international diseases as separate entities is an outmoded con- cept, and that many conditions that contribute to disease emergence or reemergence in the developing world are also present in the United States, adding to our domestic vulnerability to emerging infections. Other speakers focused on the evolution of viru- lence, the molecular basis of pathogenesis, observa- tions on factors contributing to the plague epidemic in India in 1994 and the Ebola outbreak in Zaire in 1976; foodborne and waterborne diseases; airborne diseases; zoonoses; sexually transmitted and blood- borne diseases and the increasing problem of an- timicrobial resistance in both hospital and community settings. Concerns were expressed about the possibility of a "post-antimicrobial era" in which available drugs are no longer effective against common bacterial infections. Other speakers fo- cused on innovative approaches to surveillance at the local, state, national, and international levels. James LeDuc from the World Health Organization (WHO) provided an update on the emerging infec- tions resolution passed by the World Health Assem- bly in May 1995 and other WHO activities related to detecting and responding to emerging and ree- merging diseases. Among the themes recurring throughout the con- ference were the challenges that microbes will con- tinue to pose; the critical role of the modern microbiology laboratory in detecting and responding to emerging and reemerging infections; and the limi- tations of existing capacity at the local, state, na- tional, and international level to respond to these challenges. Human resource, equipment, diagnostic reagent, and facility needs were addressed, and re- source needs were emphasized.

y in detecting and responding to emerging and reemerging infections; and the limi- tations of existing capacity at the local, state, na- tional, and international level to respond to these challenges. Human resource, equipment, diagnostic reagent, and facility needs were addressed, and re- source needs were emphasized. Training needs of medical students, clinicians, epidemiologists, micro- biologists, entomologists, mammalogists, behav- ioral scientists, and other researchers were also stressed. Additional emphasis was placed on the critical importance of communicating alerts about clusters of illness, data on disease trends, and guide- lines for disease prevention; the need for educating professionals, the public, and policy makers about the critical importance of these issues; the need for strengthening existing partnerships and developing new ones, particularly with health maintenance or- ganizations, the pharmaceutical industry, and non- governmental organizations (including medical missionary organizations); and the need to carefully identify priorities. Conference participants resounded the message of the 1992 Institute of Medicine Report, Emerging Infections: Microbial Threats to Health in the United States, "Pathogenic microbes can be resilient, dan- gerous foes. Although it is impossible to predict their individual emergence in time and place, we can be confident that new microbial diseases will emerge." Particular future concerns included a possible influ- enza pandemic, the emergence of vancomycin resis- tance in Staphylococcus aureus, the occurrence of large dengue hemorrhagic fever epidemics in the Western Hemisphere, and the likelihood that addi- tional chronic diseases will be found to have infec- tious etiologies. Concerns were also expressed about the possibility of a terrorist incident involving an infectious agent and the potential difficulties in de- tecting and responding to such an episode. The New York Academy of Medicine plans to use the discussions during the conference in formulating an agenda for further action. James M. Hughes National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA News and Notes Vol. 1, No. 3 -- July-September 1995 101 Emerging Infectious Diseases USPHS and IDSA Collaborate on Guidelines to Prevent Opportunistic Infections in HIV-Infected Persons U.S.

action. James M. Hughes National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA News and Notes Vol. 1, No. 3 -- July-September 1995 101 Emerging Infectious Diseases USPHS and IDSA Collaborate on Guidelines to Prevent Opportunistic Infections in HIV-Infected Persons U.S. Public Health Service (USPHS)/Infectious Diseases Society of America (IDSA) Guidelines for Preventing Opportunistic Infections in HIV-Infected Persons will be published in an August 1995 supple- ment of Clinical Infectious Diseases. The guidelines, which are intended for health care providers, are the result of collaboration between the Centers for Dis- ease Control and Prevention (CDC), the National Institutes of Health, IDSA, numerous federal and nonfederal organizations, community groups, and HIV-infected persons. The guidelines are endorsed by the American Academy of Pediatrics, the Infec- tious Diseases Society of Obstetrics and Gynecology, and the Society of Healthcare Epidemiologists of America. Jonathan E. Kaplan, M.D. (CDC), Henry Masur, M.D. (NIH), and King Holmes, M.D., Ph.D. (University of Washington), chaired the USPHS/IDSA Prevention of Opportunistic Infec- tions Working Group and are guest editors of the Clinical Infectious Diseases supplement. CDC initiated work on the guidelines in early 1994; meetings were held in Atlanta in June and September to discuss and refine the recommenda- tions. The USPHS/IDSA guidelines address 17 oppor- tunistic infections from three angles: 1) preventing exposure to opportunistic pathogens (e.g., sexual, occupational, and environmental exposure as well as exposure through pets, food, water, and interna- tional travel); 2) preventing opportunistic disease by chemoprophylaxis and vaccination; and 3) prevent- ing disease recurrence. In this document, new rec- ommendations were made and earlier recommenda- tions were updated. For example, new guidelines recommend that in nonemergency situations, cyto- megalovirus (CMV)-seronegative HIV-infected per- sons who require blood transfusions receive only Japanese Encephalitis Acquired in Australia Japanese encephalitis (JE), a mosquito-borne flaviviral disease of humans and animals, is a major public health problem in Asia, where an estimated 50,000 cases occur each year. There has been concern that the range of epidemic JE may be expanding. On April 5, 1995, an outbreak of three cases of JE was recognized in Australia.

encephalitis (JE), a mosquito-borne flaviviral disease of humans and animals, is a major public health problem in Asia, where an estimated 50,000 cases occur each year. There has been concern that the range of epidemic JE may be expanding. On April 5, 1995, an outbreak of three cases of JE was recognized in Australia. Two of the cases were fatal; all were among residents of an island in Aus- tralia's Torres Strait, which lies between mainland Queensland and Papua New Guinea. JE was con- firmed in two of the patients by polymerase chain reaction (Jeffrey Hanna, Queensland Health, pers. comm.). No other cases were reported. This is the first recognized episode of JE acquired in Australia. Control activities on the Australian island began on April 7. The community was informed about the importance of personal mosquito protection meas- ures. In addition, larvicides were applied, and areas were fogged to kill adult mosquitoes. The patients were all male, aged 6 to 44 years. All were hospitalized with symptoms that included fe- ver (up to 40oC), stiff or painful neck, headache, and abdominal pain. Two patients were unconscious at the time of admission. Acute-phase sera showed elevated JE virus im- munoglobulin M (IgM) titers. Two of the patients also had detectable levels of Kunjin and Murray Valley encephalitis virus IgM, but the JE IgM titers were significantly higher in each case. Flaviviruses have also been isolated from the sera of each of two asymptomatic island residents. Preliminary tests suggest that these are both JE virus. Blood taken from 10 horses and 12 domestic pigs living near humans on the island was also tested. All 12 pigs and 9 of the horses had high JE titers by hemagglutination inhibition assay. Neu- tralizing antibody to JE virus was detectable in all the pigs and in four of the horses tested to date. Details of the index case are as follows: The patient, a 16-year-old male, was admitted to Thurs- day Island Hospital on March 22, 1995. He was unconscious and was responsive only to painful stimuli. His neck was stiff, and he showed a prefer- ence for moving his right side. His illness had begun 3 days before. The day before admission he com- plained of abdominal pain. This patient had been mildly mentally retarded since birth and occasionally had generalized seizures but was generally healthy. He was transferred to Cairns Base Hospital, where a cerebral CT scan showed a nonenhancing hypodense lesion in his posterior right basal ganglia.

e admission he com- plained of abdominal pain. This patient had been mildly mentally retarded since birth and occasionally had generalized seizures but was generally healthy. He was transferred to Cairns Base Hospital, where a cerebral CT scan showed a nonenhancing hypodense lesion in his posterior right basal ganglia. He had a leukocytosis of 17.3 x 109L, neutrophils, 15.2 x 109. His cerebrospinal fluid contained 150 leukocytes/l with a differential count of 50% poly- morphs and 50% mononuclear cells. He had a generalized seizure and 2 days after admission, required mechanical ventilation. He never regained consciousness and died on day 17 of hospitalization (April 8). Adapted from Hanna J, Ritchie S, Loewenthal M, et al. Probable Japanese encephalitis acquired in the Torres Strait. Communicable Diseases Intelli- gence 1995;19:206-7. News and Notes Emerging Infectious Diseases 102 Vol. 1, No. 3 -- July-September 1995

USPHS and IDSA Collaborate on Guidelines to Prevent Opportunistic Infections in HIV-Infected Persons U.S. Public Health Service (USPHS)/Infectious Diseases Society of America (IDSA) Guidelines for Preventing Opportunistic Infections in HIV-Infected Persons will be published in an August 1995 supple- ment of Clinical Infectious Diseases. The guidelines, which are intended for health care providers, are the result of collaboration between the Centers for Dis- ease Control and Prevention (CDC), the National Institutes of Health, IDSA, numerous federal and nonfederal organizations, community groups, and HIV-infected persons. The guidelines are endorsed by the American Academy of Pediatrics, the Infec- tious Diseases Society of Obstetrics and Gynecology, and the Society of Healthcare Epidemiologists of America. Jonathan E. Kaplan, M.D. (CDC), Henry Masur, M.D. (NIH), and King Holmes, M.D., Ph.D. (University of Washington), chaired the USPHS/IDSA Prevention of Opportunistic Infec- tions Working Group and are guest editors of the Clinical Infectious Diseases supplement. CDC initiated work on the guidelines in early 1994; meetings were held in Atlanta in June and September to discuss and refine the recommenda- tions. The USPHS/IDSA guidelines address 17 oppor- tunistic infections from three angles: 1) preventing exposure to opportunistic pathogens (e.g., sexual, occupational, and environmental exposure as well as exposure through pets, food, water, and interna- tional travel); 2) preventing opportunistic disease by chemoprophylaxis and vaccination; and 3) prevent- ing disease recurrence. In this document, new rec- ommendations were made and earlier recommenda- tions were updated. For example, new guidelines recommend that in nonemergency situations, cyto- megalovirus (CMV)-seronegative HIV-infected per- sons who require blood transfusions receive only Japanese Encephalitis Acquired in Australia Japanese encephalitis (JE), a mosquito-borne flaviviral disease of humans and animals, is a major public health problem in Asia, where an estimated 50,000 cases occur each year. There has been concern that the range of epidemic JE may be expanding.

d transfusions receive only Japanese Encephalitis Acquired in Australia Japanese encephalitis (JE), a mosquito-borne flaviviral disease of humans and animals, is a major public health problem in Asia, where an estimated 50,000 cases occur each year. There has been concern that the range of epidemic JE may be expanding. On April 5, 1995, an outbreak of three cases of JE was recognized in Australia. Two of the cases were fatal; all were among residents of an island in Aus- tralia's Torres Strait, which lies between mainland Queensland and Papua New Guinea. JE was con- firmed in two of the patients by polymerase chain reaction (Jeffrey Hanna, Queensland Health, pers. comm.). No other cases were reported. This is the first recognized episode of JE acquired in Australia. Control activities on the Australian island began on April 7. The community was informed about the importance of personal mosquito protection meas- ures. In addition, larvicides were applied, and areas were fogged to kill adult mosquitoes. The patients were all male, aged 6 to 44 years. All were hospitalized with symptoms that included fe- ver (up to 40oC), stiff or painful neck, headache, and abdominal pain. Two patients were unconscious at the time of admission. Acute-phase sera showed elevated JE virus im- munoglobulin M (IgM) titers. Two of the patients also had detectable levels of Kunjin and Murray Valley encephalitis virus IgM, but the JE IgM titers were significantly higher in each case. Flaviviruses have also been isolated from the sera of each of two asymptomatic island residents. Preliminary tests suggest that these are both JE virus. Blood taken from 10 horses and 12 domestic pigs living near humans on the island was also tested. All 12 pigs and 9 of the horses had high JE titers by hemagglutination inhibition assay. Neu- tralizing antibody to JE virus was detectable in all the pigs and in four of the horses tested to date. Details of the index case are as follows: The patient, a 16-year-old male, was admitted to Thurs- day Island Hospital on March 22, 1995. He was unconscious and was responsive only to painful stimuli. His neck was stiff, and he showed a prefer- ence for moving his right side. His illness had begun 3 days before. The day before admission he com- plained of abdominal pain. This patient had been mildly mentally retarded since birth and occasionally had generalized seizures but was generally healthy.

to painful stimuli. His neck was stiff, and he showed a prefer- ence for moving his right side. His illness had begun 3 days before. The day before admission he com- plained of abdominal pain. This patient had been mildly mentally retarded since birth and occasionally had generalized seizures but was generally healthy. He was transferred to Cairns Base Hospital, where a cerebral CT scan showed a nonenhancing hypodense lesion in his posterior right basal ganglia. He had a leukocytosis of 17.3 x 109L, neutrophils, 15.2 x 109. His cerebrospinal fluid contained 150 leukocytes/l with a differential count of 50% poly- morphs and 50% mononuclear cells. He had a generalized seizure and 2 days after admission, required mechanical ventilation. He never regained consciousness and died on day 17 of hospitalization (April 8). Adapted from Hanna J, Ritchie S, Loewenthal M, et al. Probable Japanese encephalitis acquired in the Torres Strait. Communicable Diseases Intelli- gence 1995;19:206-7. News and Notes Emerging Infectious Diseases 102 Vol. 1, No. 3 -- July-September 1995 CMV-antibody-negative or leukocyte-reduced cellu- lar blood products. The guidelines also recommend that Toxoplasma-seropositive HIV-infected persons who have a CD4+ lymphocyte count <100 cells/L received chemoprophylaxis against toxoplasmosis (such chemoprophylaxis is generally accomplished with anti-Pneumocystis carinii medication). Earlier recommendations for chemoprophylaxis against Pneumocystis carinii pneumonia and Mycobac- terium avium complex disease have also been up- dated. In addition to disease-specific recommendations, the guidelines include an overview article designed to prioritize the recommendations for health care providers. This article provides an approach to the initial and follow-up evaluations of the HIV-infected patient and also contains sections on HIV-infected pregnant women and HIV-exposed/infected chil- dren. The guidelines are followed by 15 background articles, which provide the information on which the recommendations were based and include research priorities generated by the development of the pre- vention recommendations. The guidelines conclude with quality standards and implementation steps on the most standard-of- care recommendations, such as chemoprophylaxis against Pneumocystis carinii pneumonia.

n which the recommendations were based and include research priorities generated by the development of the pre- vention recommendations. The guidelines conclude with quality standards and implementation steps on the most standard-of- care recommendations, such as chemoprophylaxis against Pneumocystis carinii pneumonia. This final section provides a mechanism by which health care facilities can assess their degree of compliance with the recommendations, so that they can detect and correct compliance-related problems. An abbreviated version of the USPHS/IDSA Guidelines will be published in CDC's Morbidity and Mortality Weekly Report in July. Jonathan E. Kaplan Centers for Disease Control and Prevention Atlanta, Georgia, USA Henry Masur National Institutes of Health Bethesda, Maryland, USA King K. Holmes University of Washington Seattle, Washington, USA Recommendations for a Regional Strategy for the Prevention and Control of Emerging Infectious Diseases in the Americas On June 14-15, 1995, a conference on "Combating Emerging Infectious diseases: Challenges for the Americas" was held at the Pan American Health Organization (PAHO) Headquarters in Washington, D.C. The meeting was designed to shape a regional strategy for preventing and controlling emerging infectious diseases that could pose serious threats to the peoples of the Americas. Participants, convened by PAHO, included top offi- cials and infectious disease experts from that organi- zation as well as the World Health Organization, the U.S. Centers for Disease Control and Prevention, the Canadian Laboratory Center for Disease Control, the U.S. Department of Defense, and several Latin Ameri- can and Caribbean countries. This international group of experts noted that an increasing number of new, emerging, and ree- merging infectious diseases have been identified in both developed and developing nations and that these diseases threaten to increase in the near fu- ture. They include human immunodeficiency vi- rus/acquired immunodeficiency syndrome, which emerged in the l980s and now affects some 16 mil- lion people worldwide; and cholera, which returned to the Western Hemisphere for the first time this century in 1991 and has caused more than 1 million cases and 9,000 deaths in the Americas. PAHO esti- mates that it will take more than a decade and over $200 billion to control the current pandemic of this disease. The experts concluded that both early warnings of, and rapid responses to, infectious disease threats are needed.

n 1991 and has caused more than 1 million cases and 9,000 deaths in the Americas. PAHO esti- mates that it will take more than a decade and over $200 billion to control the current pandemic of this disease. The experts concluded that both early warnings of, and rapid responses to, infectious disease threats are needed. The group made several major recom- mendations to PAHO and its member states to im- prove surveillance, research, and communications in developing countries. They also issued more de- tailed recommendations in the areas of antimicro- bial resistance, outbreak control, and information and communication. In addition, a plan of action is forthcoming. The group made the following recommendations for PAHO and its member countries: General Recommendations * Develop and frequently update prioritized dis- ease-specific guidelines for the prevention and control of diseases that are emerging or ree- merging, both at the public health and individual levels. This should include biologic and behav- ioral change measures and will require groups of experts for each disease as well as communica- tions experts. Diseases of interest include yellow fever, dengue, antimicrobial-resistant organisms (malaria, tuberculosis, and enteric diseases), measles, polio, cholera and other foodborne and waterborne diseases, viral hemorrhagic fevers, plague, rabies and other zoonoses, and try- panosomiasis and other vector-borne diseases. * Identify points of contact in the field to receive and transmit information in countries. These contacts should include organizations and indi- viduals outside the government. News and Notes Vol. 1, No. 3 -- July-September 1995 103 Emerging Infectious Diseases

to painful stimuli. His neck was stiff, and he showed a prefer- ence for moving his right side. His illness had begun 3 days before. The day before admission he com- plained of abdominal pain. This patient had been mildly mentally retarded since birth and occasionally had generalized seizures but was generally healthy. He was transferred to Cairns Base Hospital, where a cerebral CT scan showed a nonenhancing hypodense lesion in his posterior right basal ganglia. He had a leukocytosis of 17.3 x 109L, neutrophils, 15.2 x 109. His cerebrospinal fluid contained 150 leukocytes/l with a differential count of 50% poly- morphs and 50% mononuclear cells. He had a generalized seizure and 2 days after admission, required mechanical ventilation. He never regained consciousness and died on day 17 of hospitalization (April 8). Adapted from Hanna J, Ritchie S, Loewenthal M, et al. Probable Japanese encephalitis acquired in the Torres Strait. Communicable Diseases Intelli- gence 1995;19:206-7. News and Notes Emerging Infectious Diseases 102 Vol. 1, No. 3 -- July-September 1995

CMV-antibody-negative or leukocyte-reduced cellu- lar blood products. The guidelines also recommend that Toxoplasma-seropositive HIV-infected persons who have a CD4+ lymphocyte count <100 cells/L received chemoprophylaxis against toxoplasmosis (such chemoprophylaxis is generally accomplished with anti-Pneumocystis carinii medication). Earlier recommendations for chemoprophylaxis against Pneumocystis carinii pneumonia and Mycobac- terium avium complex disease have also been up- dated. In addition to disease-specific recommendations, the guidelines include an overview article designed to prioritize the recommendations for health care providers. This article provides an approach to the initial and follow-up evaluations of the HIV-infected patient and also contains sections on HIV-infected pregnant women and HIV-exposed/infected chil- dren. The guidelines are followed by 15 background articles, which provide the information on which the recommendations were based and include research priorities generated by the development of the pre- vention recommendations. The guidelines conclude with quality standards and implementation steps on the most standard-of- care recommendations, such as chemoprophylaxis against Pneumocystis carinii pneumonia. This final section provides a mechanism by which health care facilities can assess their degree of compliance with the recommendations, so that they can detect and correct compliance-related problems. An abbreviated version of the USPHS/IDSA Guidelines will be published in CDC's Morbidity and Mortality Weekly Report in July. Jonathan E. Kaplan Centers for Disease Control and Prevention Atlanta, Georgia, USA Henry Masur National Institutes of Health Bethesda, Maryland, USA King K. Holmes University of Washington Seattle, Washington, USA Recommendations for a Regional Strategy for the Prevention and Control of Emerging Infectious Diseases in the Americas On June 14-15, 1995, a conference on "Combating Emerging Infectious diseases: Challenges for the Americas" was held at the Pan American Health Organization (PAHO) Headquarters in Washington, D.C.

, USA Recommendations for a Regional Strategy for the Prevention and Control of Emerging Infectious Diseases in the Americas On June 14-15, 1995, a conference on "Combating Emerging Infectious diseases: Challenges for the Americas" was held at the Pan American Health Organization (PAHO) Headquarters in Washington, D.C. The meeting was designed to shape a regional strategy for preventing and controlling emerging infectious diseases that could pose serious threats to the peoples of the Americas. Participants, convened by PAHO, included top offi- cials and infectious disease experts from that organi- zation as well as the World Health Organization, the U.S. Centers for Disease Control and Prevention, the Canadian Laboratory Center for Disease Control, the U.S. Department of Defense, and several Latin Ameri- can and Caribbean countries. This international group of experts noted that an increasing number of new, emerging, and ree- merging infectious diseases have been identified in both developed and developing nations and that these diseases threaten to increase in the near fu- ture. They include human immunodeficiency vi- rus/acquired immunodeficiency syndrome, which emerged in the l980s and now affects some 16 mil- lion people worldwide; and cholera, which returned to the Western Hemisphere for the first time this century in 1991 and has caused more than 1 million cases and 9,000 deaths in the Americas. PAHO esti- mates that it will take more than a decade and over $200 billion to control the current pandemic of this disease. The experts concluded that both early warnings of, and rapid responses to, infectious disease threats are needed. The group made several major recom- mendations to PAHO and its member states to im- prove surveillance, research, and communications in developing countries. They also issued more de- tailed recommendations in the areas of antimicro- bial resistance, outbreak control, and information and communication. In addition, a plan of action is forthcoming. The group made the following recommendations for PAHO and its member countries: General Recommendations * Develop and frequently update prioritized dis- ease-specific guidelines for the prevention and control of diseases that are emerging or ree- merging, both at the public health and individual levels. This should include biologic and behav- ioral change measures and will require groups of experts for each disease as well as communica- tions experts.

prioritized dis- ease-specific guidelines for the prevention and control of diseases that are emerging or ree- merging, both at the public health and individual levels. This should include biologic and behav- ioral change measures and will require groups of experts for each disease as well as communica- tions experts. Diseases of interest include yellow fever, dengue, antimicrobial-resistant organisms (malaria, tuberculosis, and enteric diseases), measles, polio, cholera and other foodborne and waterborne diseases, viral hemorrhagic fevers, plague, rabies and other zoonoses, and try- panosomiasis and other vector-borne diseases. * Identify points of contact in the field to receive and transmit information in countries. These contacts should include organizations and indi- viduals outside the government. News and Notes Vol. 1, No. 3 -- July-September 1995 103 Emerging Infectious Diseases * Develop plans to distribute accurate and timely information to the general public. * Develop plans to improve and make more effi- cient two-way communication on reporting, con- trol, and modification measures. This may require contracting information management specialists to identify and implement the most efficient means. * Make efficient use of the press, including radio, television and newspapers, fliers, and other methods to educate the public and the medical community, with an eye toward social mobiliza- tion of communities to fight emerging diseases. This will require expertise in communications and support to the countries in developing infor- mation dissemination plans. Countries should define populations at greatest risk and focus the information and control measures in these popu- lations. * Define different approaches for educating the public and the medical community. * Focus efforts on intersectorial action, including education of policy makers outside the health community. Antimicrobial Resistance The expert group recommended that both PAHO and its member countries, where applicable, do the following: * Seek ways to reduce availability of over-the- counter antimicrobial agents, including those used in veterinary medicine; this will require efforts beyond the health care community and involve education and dissemination of informa- tion to all sectors. * Intensify assistance to the countries in develop- ing rational drug policies.

reduce availability of over-the- counter antimicrobial agents, including those used in veterinary medicine; this will require efforts beyond the health care community and involve education and dissemination of informa- tion to all sectors. * Intensify assistance to the countries in develop- ing rational drug policies. * Monitor sensitivity to antibiotics in each country to allow for optimum antibiotic use for individual cases and to eliminate antibiotics with little therapeutic value. Employ mechanisms such as WHONET and PHLIS to centralize, analyze, and distribute antimicrobial sensitivity data. * Develop and distribute specific recommendations to extend the useful life of antimicrobial drugs. * Frequently revise the list of essential antimicro- bials based on sensitivity data. * Initiate educational campaigns on the cost-effec- tiveness of rational drug use in hospitals. * Initiate collaboration with the pharmaceutical industry on rational drug use, standardized la- bels and warnings, and ethical marketing strate- gies. Outbreak Control The expert group endorsed the leadership role of PAHO in developing and disseminating guidelines for outbreak evaluation and control and recom- mended that PAHO * Make timely recommendations to coordinate re- sponse to outbreaks or threats, including issues related to travel advice, quarantine, and com- merce. * Develop policies and standard operating plans for response to outbreaks at the regional and country levels. Assist countries in developing national outbreak response plans and assist in training teams. * Identify and list individuals and groups with dis- ease-specific expertise, laboratories with disease- specific diagnostic capabilities, and products, including diagnostic reagents, drugs, and vac- cines (both licensed and investigational prod- ucts). Frequently update these lists. * Establish a standard system for rapid procure- ment of vaccines, reagents, insecticides and an- timicrobial drugs for prompt response to outbreaks. * Establish information management and dissemi- nation procedures for use during outbreaks, in- cluding accurate and frequent release of information to the press and public. * Conduct formal evaluations of responses to each outbreak and use the lessons learned to improve responses to subsequent outbreaks.

to outbreaks. * Establish information management and dissemi- nation procedures for use during outbreaks, in- cluding accurate and frequent release of information to the press and public. * Conduct formal evaluations of responses to each outbreak and use the lessons learned to improve responses to subsequent outbreaks. Information and Communication The experts recommended communicating with high-level government officials and emphasizing to them the importance of a basic public health infra- structure--including improvements in water, sani- tation, and social and economic conditions--in preventing diseases. The group suggested dissemi- nating more information about public health impli- cations of development (such as deforestation, dam construction, urbanization, and other measures) and seeking effective interaction with other sectors. Other Recommendations PAHO should * Create interagency task forces for emerging dis- eases at regional and country levels. * Inform regional governments, other organiza- tions, and the public about the emerging disease initiative and strive for the highest level of politi- cal support. News and Notes Emerging Infectious Diseases 104 Vol. 1, No. 3 -- July-September 1995 * Solicit and allocate specific resources to deal with the emerging diseases initiative, both at the re- gional and country levels. Aportion of these funds should be immediately available when outbreaks are recognized. For more information on these recommendations, the conference, or its plan of action, contact PAHO. Daniel B. Epstein Office of Information & Public Affairs Pan American Health Organization Washington, D.C., USA Tenth Annual ASTPHLD Conference on Human Retrovirus Testing The Tenth Annual Conference on Human Retrovirus Testing, sponsored by the Association of State and Territorial Public Health Laboratory Di- rectors (ASTPHLD), was held March 6 to 9, 1995, in Reno, Nevada. The conference, which was attended by more than 300 representatives of public and private sector laboratories as well as test kit manu- facturers, emphasized three themes: new human immunodeficiency virus (HIV) variants, interna- tional issues, and HIV testing of newborns. The topics discussed included sequence data for type O isolates, the search for new HIV variants, zi- dovudine (AZT) resistance, decreased maternal-neo- natal transmission due to AZT prophylaxis, results of the national anonymous survey of HIV prevalence in the United States, and the ethical concerns of perinatal screening.

opics discussed included sequence data for type O isolates, the search for new HIV variants, zi- dovudine (AZT) resistance, decreased maternal-neo- natal transmission due to AZT prophylaxis, results of the national anonymous survey of HIV prevalence in the United States, and the ethical concerns of perinatal screening. An international perspective on HIV testing was brought to the conference by presentations that fo- cused on India and Latin America. Results were given of a project, funded by a 12-month study grant from the World AIDS Foundation, to provide train- ing on HIV testing to laboratories in India. Four Indian facilitators were trained in the United States; they provided translation and other assis- tance to eight ASTPHLD faculty, who gave work- shops in four training centers in India. This training, which focused on enzyme immunoassay, linked trainees with staff from Indian reference centers and established training materials and trainers for future workshops to be conducted by Indian staff. Laboratory aspects of HIV testing in Latin Ameri- can and the Caribbean were also discussed by a member of the Pan American Health Organization (PAHO), who described the spectrum of HIV inci- dence rates and testing algorithms. PAHO is asking countries of the region to assess their algorithms in terms of sensitivity, specificity, and cost. PAHO aims to support national laboratories by providing guide- lines and quality assurance. Proficiency testing, which is encouraged, will be provided by the Centers for Disease Control and Prevention. ASTPHLD's 11th Annual Human Retrovirus Conference is set for March 6-8, 1996, in Orlando, Florida. Requests for additional information are available; FAX request to 202-887-5098. James L. Pearson Division of Consolidated Laboratory Services, Commonwealth of Virginia, Richmond, Virginia, USA Emerging Infectious Diseases Laboratory Fellowship Program A partnership has been established between the Association of State and Territorial Public Health Laboratory Directors and the Centers for Disease Control and Prevention (CDC) to develop and initi- ate an emerging infectious diseases laboratory fel- lowship program in January 1996. A goal of this fellowship program is to strengthen local, state, and federal public health infrastructures to support sur- veillance and implement prevention and control pro- grams.

se Control and Prevention (CDC) to develop and initi- ate an emerging infectious diseases laboratory fel- lowship program in January 1996. A goal of this fellowship program is to strengthen local, state, and federal public health infrastructures to support sur- veillance and implement prevention and control pro- grams. The fellowship program will help recruit and train microbiologists for laboratories nationwide and provide opportunities for doctoral level scien- tists to conduct high-priority infectious disease re- search. The emerging infectious diseases fellowship pro- gram will offer a 2-year laboratory research track for doctoral level scientists, with emphasis on ap- plied research or development in infectious diseases and a 1-year advanced laboratory training track for bachelor's and master's level scientists, with em- phasis on the practical application of emerging in- fectious diseases technologies, methods, and practices. Fellow training and research will take place at CDC and state and local public health laboratories. For applications or additional information, con- tact Emerging Infectious Diseases Fellowship Program Association of State and Territorial Public Health Laboratory Directors 1211 Connecticut Avenue, Suite 608 Washington, D.C. 20036 Phone: 202-822-5227, Fax: 202-887-5098 News and Notes Vol. 1, No. 3 -- July-September 1995 105 Emerging Infectious Diseases

* Solicit and allocate specific resources to deal with the emerging diseases initiative, both at the re- gional and country levels. Aportion of these funds should be immediately available when outbreaks are recognized. For more information on these recommendations, the conference, or its plan of action, contact PAHO. Daniel B. Epstein Office of Information & Public Affairs Pan American Health Organization Washington, D.C., USA Tenth Annual ASTPHLD Conference on Human Retrovirus Testing The Tenth Annual Conference on Human Retrovirus Testing, sponsored by the Association of State and Territorial Public Health Laboratory Di- rectors (ASTPHLD), was held March 6 to 9, 1995, in Reno, Nevada. The conference, which was attended by more than 300 representatives of public and private sector laboratories as well as test kit manu- facturers, emphasized three themes: new human immunodeficiency virus (HIV) variants, interna- tional issues, and HIV testing of newborns. The topics discussed included sequence data for type O isolates, the search for new HIV variants, zi- dovudine (AZT) resistance, decreased maternal-neo- natal transmission due to AZT prophylaxis, results of the national anonymous survey of HIV prevalence in the United States, and the ethical concerns of perinatal screening. An international perspective on HIV testing was brought to the conference by presentations that fo- cused on India and Latin America. Results were given of a project, funded by a 12-month study grant from the World AIDS Foundation, to provide train- ing on HIV testing to laboratories in India. Four Indian facilitators were trained in the United States; they provided translation and other assis- tance to eight ASTPHLD faculty, who gave work- shops in four training centers in India. This training, which focused on enzyme immunoassay, linked trainees with staff from Indian reference centers and established training materials and trainers for future workshops to be conducted by Indian staff.

tion and other assis- tance to eight ASTPHLD faculty, who gave work- shops in four training centers in India. This training, which focused on enzyme immunoassay, linked trainees with staff from Indian reference centers and established training materials and trainers for future workshops to be conducted by Indian staff. Laboratory aspects of HIV testing in Latin Ameri- can and the Caribbean were also discussed by a member of the Pan American Health Organization (PAHO), who described the spectrum of HIV inci- dence rates and testing algorithms. PAHO is asking countries of the region to assess their algorithms in terms of sensitivity, specificity, and cost. PAHO aims to support national laboratories by providing guide- lines and quality assurance. Proficiency testing, which is encouraged, will be provided by the Centers for Disease Control and Prevention. ASTPHLD's 11th Annual Human Retrovirus Conference is set for March 6-8, 1996, in Orlando, Florida. Requests for additional information are available; FAX request to 202-887-5098. James L. Pearson Division of Consolidated Laboratory Services, Commonwealth of Virginia, Richmond, Virginia, USA Emerging Infectious Diseases Laboratory Fellowship Program A partnership has been established between the Association of State and Territorial Public Health Laboratory Directors and the Centers for Disease Control and Prevention (CDC) to develop and initi- ate an emerging infectious diseases laboratory fel- lowship program in January 1996. A goal of this fellowship program is to strengthen local, state, and federal public health infrastructures to support sur- veillance and implement prevention and control pro- grams. The fellowship program will help recruit and train microbiologists for laboratories nationwide and provide opportunities for doctoral level scien- tists to conduct high-priority infectious disease re- search. The emerging infectious diseases fellowship pro- gram will offer a 2-year laboratory research track for doctoral level scientists, with emphasis on ap- plied research or development in infectious diseases and a 1-year advanced laboratory training track for bachelor's and master's level scientists, with em- phasis on the practical application of emerging in- fectious diseases technologies, methods, and practices. Fellow training and research will take place at CDC and state and local public health laboratories.

diseases and a 1-year advanced laboratory training track for bachelor's and master's level scientists, with em- phasis on the practical application of emerging in- fectious diseases technologies, methods, and practices. Fellow training and research will take place at CDC and state and local public health laboratories. For applications or additional information, con- tact Emerging Infectious Diseases Fellowship Program Association of State and Territorial Public Health Laboratory Directors 1211 Connecticut Avenue, Suite 608 Washington, D.C. 20036 Phone: 202-822-5227, Fax: 202-887-5098 News and Notes Vol. 1, No. 3 -- July-September 1995 105 Emerging Infectious Diseases

Streptococcal Toxic-Shock Syndrome: Spectrum of Disease, Pathogenesis, and New Concepts in Treatment Dennis L. Stevens, Ph.D., M.D. Professor of Medicine, University of Washington School of Medicine, Seattle, Washington Chief, Infectious Disease Section, Veterans Affairs Medical Center, Boise, Idaho Since the 1980s there has been a marked increase in the recognition and reporting of highly invasive group Astreptococcal infections with or without necrotizing fasciitis associated with shock and organ failure. Such dramatic cases have been defined as streptococcal toxic-shock syndrome. Strains of group A streptococci isolated from patients with invasive disease have been predominantly M types 1 and 3 that produce pyrogenic exotoxin A or B or both. In this paper, the clinical and demographic features of streptococcal bacteremia, myositis, and necrotizing fasciitis are presented and compared to those of streptococcal toxic-shock syndrome. Current concepts in the pathogenesis of invasive streptococcal infection are also presented, with emphasis on the interaction between group A Streptococcus virulence factors and host defense mechanisms. Finally, new concepts in the treatment of streptococcal toxic-shock syndrome are discussed. An emerging pathogen can be one that is totally new (e.g., human immunodeficiency virus), one that was known but has only recently been identified (e.g., Helicobacter pylori), or one that is old but has learned new tricks. The last type is, as Dr. Stanley Falkow contends, merely trying to "make a living" in a changing environment. Regardless of environ- mental pressures, many old pathogens have become major clinical problems because of increased viru- lence or antibiotic resistance (e.g., penicillin- resistant pneumococcus, multidrug resistant Myco- bacterium tuberculosis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium). Arguably, group A Streptococcus (GAS) is the quintessence of an old organism that has become more virulent. In this manuscript, the epidemiology, clinical spectrum, and pathogenesis of GAS infec- tion are discussed in relation to the streptococcal toxic-shock syndrome (TSS).

vancomycin-resistant Enterococcus faecium). Arguably, group A Streptococcus (GAS) is the quintessence of an old organism that has become more virulent. In this manuscript, the epidemiology, clinical spectrum, and pathogenesis of GAS infec- tion are discussed in relation to the streptococcal toxic-shock syndrome (TSS). Current and Historical Perspectives on the Prevalence and Severity of Streptococcal Infections The British tabloids have recently coined the term "flesh-eating bacteria" to describe invasive ne- crotizing infections caused by GAS and have sug- gested that epidemics of streptococcal infection are imminent. Such aggrandizement is unfounded, yet it has served to heighten public awareness of this sporadic, but serious, infectious disease. Strictly speaking, an epidemic is defined as an increase in the prevalence of disease over a baseline endemic rate. In this context, we are, in fact, experiencing an epidemic of severe invasive GAS infections; how- ever, few concrete prospective population-based data support this notion. Estimates suggest that the incidence of these infections is 10 to 20 cases/ 100,000 population. Thus, the stimulus for such public interest has not been the incidence of the syndrome, but more likely, the dramatic nature of these infections. Whether these types of group A streptococcal infections will decline, stay the same, or increase is not known. History is replete with descriptions of epidemics of GAS infections and their nonsuppura- tive sequelae. In the 1600s, epidemics of scarlet fever spread from Italy and Spain to Northern Europe (1), and in 1736, an outbreak occurred in the American colonies, killing 4,000 people (2). Major epidemics of rheumatic fever occurred in World War II in the U.S. military (3). Soon afterward post-strep- tococcal glomerulonephritis struck several regions of the United States (4,5). Many of these epidemics waxed and waned before the advent of antibiotics, suggesting that either changes in socioeconomic conditions or variations in the expression of virulence factors by the pathogen were responsible. This concept is best exemplified by the extraordinary mortality rate of scarlet fever documented in the latter part of the 1880s in New York, Chicago, and Norway; 25% to 30% of children with scarlet fever died during that period (5,6). By 1900, the mortality rate had dropped to under 2% in all three locations.

onsible. This concept is best exemplified by the extraordinary mortality rate of scarlet fever documented in the latter part of the 1880s in New York, Chicago, and Norway; 25% to 30% of children with scarlet fever died during that period (5,6). By 1900, the mortality rate had dropped to under 2% in all three locations. Since socioeconomic conditions Address for correspondence: Infectious Disease Section, Veterans Affairs Medical Center, 500 West Fort Street (Bldg 6), Boise, ID 83702, USA; fax: 208-389-7965. Synopses Vol. 1, No. 3 -- July-September 1995 69 Emerging Infectious Diseases likely did not change markedly during that time and antibiotics were not yet available, the decrease in mortality rates must have been caused by reduced expression of a streptococcal virulence factor or by the slow acquisition of herd immunity to that factor. The epidemiology of GAS infection is complex. More than 80 different M types of S. pyogenes exist, and five separate and distinct scarlatina toxins, streptococcal pyrogenic exotoxins (SPEs) (5) have also been described; some of these can be transmit- ted to different M types by bacteriophage. Minor drifts in the antigenic or virulence properties of GAS could account for the 5- to 6-year cycles of scarlet fever documented by Kohler (9). In the same way as antigenic shifts in influenza virus cause pandemics, major alterations in GAS virulence properties could cause major changes in clinical disease. The recent increases in severe GAS infections, following a 50- to 60-year span of relatively benign clinical disease, support this notion. Acute Life-Threatening Group A Streptococcal Infections Streptococcal TSS Recently, severe invasive GAS infections associ- ated with shock and organ failure have been re- ported with increasing frequency, predominantly from North America and Europe (8-18). These infec- tions have been termed streptococcal toxic-shock syndrome (TSS; Table 1) (19). Persons of all ages are affected; most do not have predisposing underlying diseases (11,20-25). This is in sharp contrast to previous reports of GAS bacteremia, in which pa- tients were either under 10 or over 60 years of age, and most had underlying conditions such as cancer, renal failure, leukemia, or severe burns or were receiving corticosteroids or other immunosuppress- ing drugs (20-22).

iseases (11,20-25). This is in sharp contrast to previous reports of GAS bacteremia, in which pa- tients were either under 10 or over 60 years of age, and most had underlying conditions such as cancer, renal failure, leukemia, or severe burns or were receiving corticosteroids or other immunosuppress- ing drugs (20-22). The complications of current GAS infections are severe; bacteremia associated with aggressive soft tissue infection, shock, adult respi- ratory distress syndrome and renal failure are com- mon; 30% to 70% of patients die in spite of aggressive modern treatments (Table 2) (1,8,24-26). Acquisition of Group AStreptococcus The portal of entry of streptococci cannot be proven in at least half the cases (8) and can only be presumed in many others. Patients with sympto- matic pharyngitis rarely develop streptococcal TSS, though such cases have been reported, especially in the last year. Procedures such as suction lipectomy, hysterectomy, vaginal delivery, bunionectomy and bone pinning have provided a portal of entry in many cases (author's unpublished observations). Most commonly, infection begins at a site of minor local trauma, which frequently does not result in a break in the skin (8). Numerous cases have developed within 24 to 72 hours of minor nonpenetrating trauma, resulting in hematoma, deep bruise to the calf, or even muscle strain. Virus infections, such as varicella and influenza, have provided a portal in other cases. In some cases the use of nonsteroidal antiinflammatory agents may have either masked the early symptoms or predisposed the patient to more severe streptococcal infection and shock (1). For the most part, these infections have occurred sporadically and have not been associated with clus- ters of cases or minor epidemics, though outbreaks of severe GAS infections have occurred in closed environments such as nursing homes (27,28). Clinical Symptoms Pain--the most common initial symptom of strep- tococcal TSS--is abrupt in onset and severe, and usually precedes tenderness or physical findings. The pain usually involves an extremity but may also mimic peritonitis, pelvic inflammatory disease, pneumonia, acute myocardial infarction, or peri- carditis. Twenty percent of patients have an influenza-like syndrome characterized by fever, chills, myalgia, nausea, vomiting, and diarrhea (8). Fever is the most common early sign, although hypothermia may be present in patients with shock.

pelvic inflammatory disease, pneumonia, acute myocardial infarction, or peri- carditis. Twenty percent of patients have an influenza-like syndrome characterized by fever, chills, myalgia, nausea, vomiting, and diarrhea (8). Fever is the most common early sign, although hypothermia may be present in patients with shock. Confusion is present in 55% of patients, and in some, coma or combativeness is manifest (8). Eighty per- cent of patients have clinical signs of soft tissue infection, such as localized swelling and erythema, which in 70% of patients progressed to necrotizing fasciitis or myositis and required surgical debride- ment, fasciotomy or amputation (8). An ominous sign is the progression of soft tissue swelling to the formation of vesicles, then bullae, which appear violaceous or bluish. In such patients, emergent surgical exploration should be performed to estab- lish the diagnosis and distinguish GAS infection from other necrotizing soft tissue infections. Among the 20% of patients without soft tissue findings, clinical symptoms include endophthalmitis, myosi- tis, perihepatitis, peritonitis, myocarditis, and over- whelming sepsis. Adiffuse, scarlatina-like erythema occurs in only 10% of patients. Nearly 50% of pa- tients may have normal blood pressure (systolic pressure >110 mm Hg) on admission but develop hypotension within the subsequent 4 hours (8). Laboratory Evaluation of Patients On admission, renal involvement is indicated by the presence of hemoglobinuria and by serum creat- inine values that are, on average, >2.5 times normal. Renal impairment precedes hypotension in 40% to 50% of patients (8). Hypoalbuminemia is associated with hypocalcemia on admission and throughout the hospital course. The serum creatinine kinase level is useful in detecting deeper soft-tissue infections; Synopses Emerging Infectious Diseases 70 Vol. 1, No. 3 -- July-September 1995 when the level is elevated or rising, there is a good correlation with necrotizing fasciitis or myositis. Though the initial laboratory studies demonstrate only mild leukocytosis, the mean percentage of imma- ture neutrophils (including band forms, metamyelo- cytes, and myelocytes) is striking, reaching 40% to 50%. Blood cultures are positive in 60% of cases (8). Clinical Course Shock is apparent at the time of admission or within 4 to 8 hours in virtually all patients (Table 2).

d leukocytosis, the mean percentage of imma- ture neutrophils (including band forms, metamyelo- cytes, and myelocytes) is striking, reaching 40% to 50%. Blood cultures are positive in 60% of cases (8). Clinical Course Shock is apparent at the time of admission or within 4 to 8 hours in virtually all patients (Table 2). In only 10% of patients does systolic blood pres- sure become normal 4 to 8 hours after administra- tion of antibiotics, albumin, and electrolyte solutions containing salts or dopamine; in all other patients, shock persists. Similarly, renal dysfunc- tion progresses or persists in all patients for 48 to 72 hours in spite of treatment, and many patients may require dialysis (8). In patients who survive, serum creatinine values return to normal within 4 to 6 weeks. Renal dysfunction precedes shock in many patients and is apparent early in the course of shock in all others. Acute respiratory distress syndrome Table 1. Case definition of streptococcal toxic-shock syndrome (streptococcal TSS) and necrotizing fasciitis* I. Streptococcal TSS A. Isolation of group A Streptococcus 1. From a sterile site 2. From a nonsterile body site B. Clinical signs of severity 1. Hypotension 2. Clinical and laboratory abnormalities (requires two or more of the following): a) Renal impairment b) Coagulopathy c) Liver abnormalities d) Acute respiratory distress syndrome e) Extensive tissue necrosis, i.e., necrotizing fasciitis f) Erythematous rash Definite Case = A1 + B(1+2) Probable Case = A2 + B(1+2) II. Necrotizing fasciitis A. Definite case 1. Necrosis of soft tissues with involvement of the fascia PLUS 2. Serious systemic disease, including one or more of the following: a) Death b) Shock (systolic blood pressure <90 mm of Hg). c) Disseminated intravascular coagulopathy d) Failure of organ systems a. respiratory failure b. liver failure c. renal failure 3. Isolation of group A Streptococcus from a normally sterile body site B. Suspected case 1. 1 1 + 2 and serologic confirmation of group A streptococcal infection by a 4-fold rise against: a) streptolysin O b) DNase B 2. 1 + 2 and histologic confirmation: Gram-positive cocci in a necrotic soft tissue infection *Streptococcal toxic-shock syndrome (streptococcal TSS) is defined as any group A streptococcal infection associated with the early onset of shock and organ failure. Definitions describing criteria for shock, organ failure, definite cases, and probable cases are included below. Source: reference 61. Synopses Vol. 1, No.

Streptococcal toxic-shock syndrome (streptococcal TSS) is defined as any group A streptococcal infection associated with the early onset of shock and organ failure. Definitions describing criteria for shock, organ failure, definite cases, and probable cases are included below. Source: reference 61. Synopses Vol. 1, No. 3 -- July-September 1995 71 Emerging Infectious Diseases occurs in 55% of patients and generally develops after the onset of hypotension (8). Supplemental oxygen, intubation, and mechanical ventilation are necessary in 90% of the patients in whom this syn- drome develops. Mortality rates vary from 30% to 70% (1,8,24-26). Morbidity is also high; 13 of 20 patients in one series underwent major surgical procedures, which included fasciotomy, surgical de- bridement, exploratory laparotomy, intraocular as- piration, amputation, or hysterectomy (8). Clinical Isolates M types 1, 3, 12, and 28 have been the most common isolates from patients with shock and mul- tiorgan failure (8,29). Recently, 80% of strains in Sweden from all types of GAS infection have been M type 1 (S. Holm, pers. comm.). Pyrogenic exotoxin A and/or B was found in most cases of severe infection. In the United States, pyrogenic exotoxin A is most frequently associated with these infections (8,23,29- 33), while in Sweden and the United Kingdom, exo- toxin B has been most common (12,25). Recently, streptococcal superantigen (SSA), a novel pyrogenic exotoxin, was isolated from an M 3 strain, albeit in small concentrations (34). In addition, mitogenic factor (MF) has been demonstrated in many differ- ent M types of GAS (35,36). Necrotizing Fasciitis Necrotizing fasciitis, a deep-seated infection of the subcutaneous tissue that progressively destroys fascia and fat but may spare the skin and muscle, can be caused by GAS, Clostridium perfringens, or C. septicum. Necrotizing fasciitis caused by mixed organisms such as aerobic gram-negative bacteria, anaerobes, and microaerophilic streptococci may de- velop in diabetic patients or patients with open wounds contaminated with bowel contents. Though Meleney called infections caused by hemolytic strep- tococci "streptococcal gangrene" (37), the process has been renamed necrotizing fasciitis. His patients' infections began at the site of trivial or inapparent trauma. Within 24 hours of the initial lesion--which frequently was only mild erythema--swelling, heat, erythema, and tenderness rapidly developed.

olytic strep- tococci "streptococcal gangrene" (37), the process has been renamed necrotizing fasciitis. His patients' infections began at the site of trivial or inapparent trauma. Within 24 hours of the initial lesion--which frequently was only mild erythema--swelling, heat, erythema, and tenderness rapidly developed. Dur- ing the next 24 to 48 hours, the erythema changed from red to purple and then to blue, and blisters and bullae, which contained clear yellow fluid, appeared. On days 4 and 5, the purple areas became gangre- nous. From day 7 to day 10, the line of demarcation became sharply defined, and the dead skin began to separate at the margins or breaks in the center, revealing an extensive necrosis of the subcutaneous tissue. In more severe cases, the process advanced rapidly until several large areas of skin became gangrenous, and the intoxication rendered the pa- tient dull, unresponsive, mentally cloudy, or even delirious. Meleney was the first to advocate aggres- sive "bear scratch" fasciotomy and debridement. With this treatment, together with irrigation with Dakains solution, the mortality rate dropped to 20% (37). These older reports of necrotizing fasciitis (6) differ from reports of current necrotizing fasciitis cases associated with streptococcal TSS (8). First, recent cases have mainly occurred in young healthy persons who had no underlying disease but sus- tained minor trauma to an extremity. Earlier series describe older patients with multiple medical prob- lems (6). Meleney's cases (reported from China) were probably among young healthy persons who sus- tained minor trauma, though the major difference between them and present cases is the low mortality rate (20% vs 20% to 60% in streptococcal TSS ) (6,37) before antibiotics were available (37). Analysis of Meleney's reports also suggests that most of his patients did not have shock or organ failure, nor did they require amputation. In contrast, present cases of necrotizing fasciitis caused by GAS are invariably associated with severe manifestations of systemic illness and high morbidity despite the absence of underlying disease and the use of antibiotics, dialy- sis, ventilators, intravenous fluids, and improved surgical techniques. In summary, the high mortality rate among current cases of streptococcal necrotiz- ing fasciitis could be due to the emergence of more virulent streptococci (8). Streptococcal Myositis Streptococcal myositis is an extremely uncom- mon GAS infection. Adams et al.

rs, intravenous fluids, and improved surgical techniques. In summary, the high mortality rate among current cases of streptococcal necrotiz- ing fasciitis could be due to the emergence of more virulent streptococci (8). Streptococcal Myositis Streptococcal myositis is an extremely uncom- mon GAS infection. Adams et al. (38) documented only 21 reported cases from 1900 to 1985, and Svane (39) found only four cases in more 20,000 autopsies. Severe pain may be the only early symptom, and swelling and erythema may be the only early physi- cal findings, though muscle compartment syn- dromes may develop rapidly (8-10,38-41). Distinguishing streptococcal myositis from sponta- neous gas gangrene caused by C. perfringens or C. septicum (42) may be difficult, though crepitus or demonstration of gas in the tissue favors clostridial infection (40). Patients with streptococcal TSS may Table 2. Complications of group A streptococcal soft-tissue infection Complication Percentage of Patients Shock 95 Acute respiratory distress syndrome 55 Renal impairment 80 Irreversible 10 Reversible 70 Bacteremia 60 Death 30 Source: reference 1. Synopses Emerging Infectious Diseases 72 Vol. 1, No. 3 -- July-September 1995 have both necrotizing fasciitis and myositis (8,38). In published series, the case-fatality rate for ne- crotizing fasciitis is 20% to 50%, whereas GAS myositis has a fatality rate of 80% to 100% (6). Aggressive surgical debridement is extremely im- portant for establishing a diagnosis and removing devitalized tissue. Bacteremia Streptococcal bacteremia has occurred most com- monly in the very young and in the elderly (5). Among children, predisposing factors (other than scarlet fever) include burns, varicella, malignant neoplasm, immunosuppression, and age less than 2 years (5). In patients with scarlet fever, the pharynx is the most common source of GAS. Frequently such patients have complications, such as extension of infection into the sinuses, peritonsillar tissue, or mastoids (septic scarlet fever or scarlet fever angi- nose); yet documented bacteremia occurs in only 0.3% of febrile patients (43). Among the children with varicella studied by Bullowa and Wischik (43), GAS bacteremia occurred in only approximately 0.5% of patients. In elderly patients the source of GAS infection is invariably the skin and is associated with cellulitis or erysipelas (5).

nted bacteremia occurs in only 0.3% of febrile patients (43). Among the children with varicella studied by Bullowa and Wischik (43), GAS bacteremia occurred in only approximately 0.5% of patients. In elderly patients the source of GAS infection is invariably the skin and is associated with cellulitis or erysipelas (5). GAS sepsis in the elderly (mean age, 50 to 60 years) has also been associated with diabetes, peripheral vascular disease, malignancy, and corticosteroid use. Not surprising, mortality rates of 35% to 80% have been described in this patient population. In the past, GAS bacteremia was rare among persons 14 to 40 years of age; puerperal sepsis accounted for most bacteremia in this age group. Recently, intravenous drug abuse has emerged as a leading cause of GAS bacteremia in this age group (5). Martin and Hoiby have compre- hensively demonstrated that the prevalence of GAS bacteremia in Norway in the late 1980s increased in all age groups, but the greatest increase (600% to 800%) was in adolescents and young adults (10). Thus, the demographics of invasive streptococcal infections have changed dramatically in the past 4 to 6 years. Current Hypotheses Regarding Mechanisms of Shock and Tissue Destruction Caused by Virulent Group A Streptococci Pyrogenic exotoxins cause fever in humans and animals and also help induce shock by lowering the threshold to exogenous endotoxin (5). Streptococcal pyrogenic exotoxins Aand B induce human mononu- clear cells to synthesize not only tumor necrosis factor- (TNF) (44) but also interleukin-1 (IL-1) (45) and interleukin-6 (IL-6) (45), suggesting that TNF could mediate the fever, shock, and tissue injury observed in patients with streptococcal TSS (8). Pyrogenic exotoxin C has been associated with mild cases of scarlet fever in the United States (author's observations) and in England (46). The roles of two newly described pyrogenic exotoxins, SSA and MF (see section on "Clinical Isolates"), in streptococcal TSS have not been elucidated. M protein contributes to invasiveness through its ability to impede phagocytosis of streptococci by human polymorphonuclear leukocytes (47). Con- versely, type-specific antibody against the M protein enhances phagocytosis (47). After infection with a particular M type, specific antibody confers resis- tance to challenge to viable GAS of that M type (47).

s through its ability to impede phagocytosis of streptococci by human polymorphonuclear leukocytes (47). Con- versely, type-specific antibody against the M protein enhances phagocytosis (47). After infection with a particular M type, specific antibody confers resis- tance to challenge to viable GAS of that M type (47). While M types 1 and 3 strains have accounted for most strains isolated from cases of streptococcal TSS, many other M types, including some nontyp- able strains, have also been isolated from such cases. M types 1 and 3 are also commonly isolated from asymptomatic carriers, patients with pharyngitis, and patients with mild scarlet fever (7,29). Could streptococcal TSS be related to the ability of pyrogenic exotoxin or M proteins type 1 or 3 to act as "super antigens" (48)? Data suggest that this exotoxin and a number of staphylococcal toxins (toxic shock syndrome toxin-1 [TSST-1] and staphy- lococcal enterotoxins A, B, and C) can stimulate T-cell responses through their ability to bind to both the Class II major histocompatibility ability com- plex of antigen-presenting cells and the V region of the T-cell receptor (48). The net effect would be to induce T-cell stimulation with production of cyto- kines capable of mediating shock and tissue injury. Recently, Hackett and Stevens demonstrated that pyrogenic exotoxin A induced both TNF and TNF from mixed cultures of monocytes and lymphocytes (49), supporting the role of lymphokines (TNF) in shock associated with strains producing that exo- toxin. Kotb et al. (50) have shown that a digest of M protein type 6 can also stimulate T-cell responses by this mechanism; however, the role of specific super- antigens in this or any other infectious disease has not been proven. Proof would require demonstration of massive expansion of T-cell subsets bearing a V repertoire specific for the putative superantigen. However, quantitation of such T-cell subsets in pa- tients with acute streptococcal TSS demonstrated deletion rather than expansion, suggesting that per- haps the life span of the expanded subset was short- ened by a process of apoptosis (51). In addition, the subsets deleted were not specific for streptococcal pyrogenic exotoxins A, B, C, or mitogenic factor, suggesting that an as yet undefined superantigen may play a role (51). Cytokine production by less exotic mechanisms likely contributes as well to the genesis of shock and organ failure.

poptosis (51). In addition, the subsets deleted were not specific for streptococcal pyrogenic exotoxins A, B, C, or mitogenic factor, suggesting that an as yet undefined superantigen may play a role (51). Cytokine production by less exotic mechanisms likely contributes as well to the genesis of shock and organ failure. Peptidoglycan, lipoteichoic acid (52), and killed organisms (53,54) are capable of inducing TNF production by mononuclear cells in vitro Synopses Vol. 1, No. 3 -- July-September 1995 73 Emerging Infectious Diseases (6,54,55). Exotoxins such as streptolysin O (SLO) are also potent inducers of TNF and IL-1. Pyro- genic exotoxin B, a proteinase precursor, has the ability to cleave pre-IL-1 to release preformed IL- 1 (56). Finally, SLO and exotoxin A together have additive effects in the induction of IL-1 by human mononuclear cells (49). Whatever the mechanisms, induction of cytokines in vivo is likely the cause of shock, and these two exotoxins, cell wall compo- nents, and the like, are potent inducers of TNF and IL-1. The mere presence of virulence factors, such as M protein or pyrogenic exotoxins, may be less impor- tant in streptococcal TSS than the dynamics of their production in vivo. Recently, Cleary et al. proposed a regulon in GAS that controls the expression of a group of virulence genes coding for known virulence factors such as M protein and C5 peptidase (57). When DNA fingerprinting was used, differences were shown between M1 strains isolated from patients with invasive disease and strains from pa- tients with noninvasive GAS infections (58). Finally, genetic information coding for exotoxins A or C may be introduced to strains of GAS by certain bacterio- phage; after lysogenic conversion, synthesis of exo- toxin A would occur during growth of the streptococcus (31,59,60). Multilocus enzyme electro- phoresis demonstrates two patterns that correspond to the M1 and M3 type organisms that produce pyrogenic exotoxin A, a finding that supports epidemiologic studies implicating these strains in invasive GAS infections (33). The interaction between these microbial viru- lence factors and an immune or nonimmune host determines the epidemiology, clinical syndrome, and outcome. Since horizontal transmission of GAS in general is well documented, the only explanation for the absence of a high attack rate of invasive infection is significant herd immunity against one or more of the virulence factors responsible for streptococcal TSS.

t determines the epidemiology, clinical syndrome, and outcome. Since horizontal transmission of GAS in general is well documented, the only explanation for the absence of a high attack rate of invasive infection is significant herd immunity against one or more of the virulence factors responsible for streptococcal TSS. This hypothetical model explains why epidem- ics have not materialized and why a particular strain of GAS can cause different clinical manifes- tations in the same community (8,61) (Figure 1). Treatment Antibiotic Therapy - Cures and Failures with Penicillin S. pyogenes continues to be exquisitely suscepti- ble to -lactam antibiotics, and numerous studies have demonstrated the clinical efficacy of penicillin preparations for streptococcal pharyngitis. Simi- larly, penicillins and cephalosporins have proven efficacy in treating erysipelas, impetigo, and celluli- tis, all of which are most frequently caused by S. pyogenes. In addition, Wannamaker et al. (6) dem- onstrated that penicillin therapy prevents the devel- opment of rheumatic fever following streptococcal pharyngitis if therapy is begun within 8 to 10 days of the onset of sore throat. Nonetheless, some clini- cal failures of penicillin treatment of streptococcal infection do occur. Penicillin treatment of S. pyo- genes has failed to eradicate bacteria from the phar- ynx of 5% to 20% of patients with documented streptococcal pharyngitis (62-64). In addition, more aggressive GAS infections (such as, necrotizing fas- ciitis, empyema, burn wound sepsis, subcutaneous gangrene, and myositis) respond less well to penicil- lin and continue to be associated with high mortality rates and extensive morbidity (6,8,9,12,15,38,65). For example, in a recent report, 25 cases of strepto- coccal myositis had an overall mortality rate of 85% in spite of penicillin therapy (38). Finally, several studies in experimental infection suggest that peni- cillin fails when large numbers of organisms are present (66,67). The Efficacy of Penicillin, Compared to Clindamycin, In Fulminant Experimental S. pyogenes Infection In a mouse model of myositis caused by S. pyo- genes, penicillin was ineffective when treatment was delayed 2 hours after initiation of infection (67). Survival of erythromycin-treated mice was greater than that of both penicillin-treated mice and un- treated controls, but only if treatment was begun within 2 hours.

on In a mouse model of myositis caused by S. pyo- genes, penicillin was ineffective when treatment was delayed 2 hours after initiation of infection (67). Survival of erythromycin-treated mice was greater than that of both penicillin-treated mice and un- treated controls, but only if treatment was begun within 2 hours. Mice receiving clindamycin, how- ever, had survival rates of 100%, 100%, 80%, and 70%, even if treatment was delayed 0, 2, 6, and 16.5 hours, respectively (67,68). Eagle suggested that penicillin failed in this type of infection because of the "physiologic state of the Figure 1. Pathogenesis of scarlet fever, bacteremia, and toxic shock syndrome. M-1+ SPEA+ = a GAS strain that contains M protein type 1 and streptococcal pyrogenic exotoxin A (SPEA); +anti-M-1 = the presence of antibody to M protein type 1; -anti-M-1 = the absence of antibody to M protein type 1'; anti-SPEA+ = antibody to SPEA; and DIC - disseminated intravascular coagulation. Synopses Emerging Infectious Diseases 74 Vol. 1, No. 3 -- July-September 1995 organism" (66). This phenomenon has recently been attributed to both in vitro and in vivo inoculum effects (69,70). Inoculum Size and the "Physiologic State of the Organism": Differential Expression of Penicillin- Binding Proteins Penicillin and other -lactam antibiotics are most efficacious against rapidly growing bacteria. We hy- pothesized that large inocula reach the stationary phase of growth sooner than smaller inocula both in vitro and in vivo. That high concentrations of S. pyogenes accumulate in deep-seated infection is sup- ported by data from Eagle et al. (66). We compared the penicillin-binding protein patterns from mem- brane proteins of group A streptococci isolated from different stages of growth, i.e., mid-log phase and stationary phase. Binding of radiolabeled penicillin by all penicillin-binding proteins was decreased in stationary cells; however, PBPs 1 and 4 were unde- tectable at 36 hours (69). Thus, the loss of certain penicillin-binding proteins during stationary-phase growth in vitro may be responsible for the inoculum effect observed in vivo and may account for the failure of penicillin in treatment of both experimen- tal and human cases of severe streptococcal infec- tion. The Greater Efficacy of Clindamycin in Experimental S.

in penicillin-binding proteins during stationary-phase growth in vitro may be responsible for the inoculum effect observed in vivo and may account for the failure of penicillin in treatment of both experimen- tal and human cases of severe streptococcal infec- tion. The Greater Efficacy of Clindamycin in Experimental S. pyogenes Infections: Mechanisms of Action The greater efficacy of clindamycin is likely mul- tifactorial: First, its efficacy is not affected by inocu- lum size or stage of growth (69,71); secondly, clindamycin is a potent suppressor of bacterial toxin synthesis (72,73); third, it facilitates phagocytosis of S. pyogenes by inhibiting M-protein synthesis (73); fourth, it suppresses synthesis of penicillin-binding proteins, which, in addition to being targets for penicillin, are also enzymes involved in cell wall synthesis and degradation (71); fifth, clindamycin has a longer postantibiotic effect than -lactams such as penicillin; and lastly, clindamycin causes suppression of LPS-induced monocyte synthesis of TNF (74). Thus, clindamycin's efficacy may also be related to its ability to modulate the immune re- sponse. Other Treatment Measures Though antibiotic selection is critically impor- tant, other measures, such as prompt and aggres- sive exploration and debridement of suspected deep-seated S. pyogenes infection, are mandatory. Frequently, the patient has fever and excruciating pain. Later, systemic toxicity develops, and definite evidence of necrotizing fasciitis and myositis ap- pears. Surgical debridement may be too late at this point. Prompt surgical exploration through a small incision with visualization of muscle and fascia, and timely Gram stain of surgically obtained material may provide an early and definitive etiologic diag- nosis. Surgical colleagues should be involved early in such cases, since later in the course surgical intervention may be impossible because of toxicity or because infection has extended to vital areas impossible to debride (i.e., the head and neck, tho- rax, or abdomen). Anecdotal reports suggest that hyperbaric oxy- gen has been used in a handful of patients, though no controlled studies are under way, nor is it clear that this treatment is useful. Because of intractable hypotension and diffuse capillary leak, massive amounts of intravenous flu- ids (10 to 20 liters/day) are often necessary. Pressors such as dopamine are used frequently, though no controlled trials have been performed in streptococ- cal TSS.

der way, nor is it clear that this treatment is useful. Because of intractable hypotension and diffuse capillary leak, massive amounts of intravenous flu- ids (10 to 20 liters/day) are often necessary. Pressors such as dopamine are used frequently, though no controlled trials have been performed in streptococ- cal TSS. In patients with intractable hypotension, vasoconstrictors such as epinephrine have been used, but symmetrical gangrene of digits seems to result frequently (author's unpublished observa- tions), often with loss of limb. In these cases it is difficult to determine if symmetrical gangrene is due to pressors, infection, or both. Neutralization of circulating toxins would be de- sirable; however, appropriate antibodies are not commercially available in the United States or Europe. Two reports describe the successful use of intravenous gamma globulin in treating streptococ- cal TSS in two patients (75,76). In summary, if a wild "flesh-eating strain" has recently emerged, a major epidemic with a high attack rate would normally be expected. Clearly, epidemics of streptococcal infections, including im- petigo, pharyngitis, scarlet fever, and rheumatic fe- ver have occurred in the past. However, in the last decade, subsequent to early reports of streptococcal TSS, we have observed that the incidence has re- mained relatively low. I hypothesize that large out- breaks have not occurred because 1) most of the population probably has immunity to one or more streptococcal virulence factors (6,25); 2) predispos- ing conditions (e.g., varicella, and use of NSAIDs) are required in a given patient; and 3) only a small percentage of the population may have an inherent predisposition to severe streptococcal infection be- cause of constitutional factors such as HLA Class II antigen type (77,78), B-cell (79), or specific V re- gions on lymphocytes. This last hypothesis is further supported by the observation that secondary cases of streptococcal TSS, though reported (80), have been rare. Dr. Stevens is chief, Infectious Diseases Section, Veterans Affairs Medical Center, Boise, Idaho, and professor of medicine, University of Washington School of Medicine, Seattle. He is a member of CDC's Working Synopses Vol. 1, No. 3 -- July-September 1995 75 Emerging Infectious Diseases Group on Streptococcal Infections and a consultant to the National Institutes of Health and the World Health Organization on Streptococcal Infections.

cine, University of Washington School of Medicine, Seattle. He is a member of CDC's Working Synopses Vol. 1, No. 3 -- July-September 1995 75 Emerging Infectious Diseases Group on Streptococcal Infections and a consultant to the National Institutes of Health and the World Health Organization on Streptococcal Infections. On July 1994, he testified before Congress on Severe Streptococcal Infections and is currently President of the American Lancefield Society. References 1. Stevens DL, Tanner MH, Winship J, Swarts R, Reis KM, Schlievert PM, et al. Reappearance of scarlet fever toxin A among streptococci in the Rocky Moun- tain West: severe group A streptococcal infections associated with a toxic shock-like syndrome. N Engl J Med 1989; 321:1-7. 2. The Working Group on Severe Streptococcal Infec- tions. Defining the group A streptococcal toxic shock syndrome: rationale and consensus definition. JAMA 1993; 269:390-1. 3. Sennert D. De febribus libri quator. Editio novissima. Cui accessit fasciculus medicamentorum contra pestem. Libri IV. De peste, Pestilentibusque ac Mal- ingis Febribus. Venice: Francisum Baba, 1641. 4. Douglass W. The practical history of a new epidemical eruptive miliary fever, with an Angina Ulcusculosa, which prevailed in Boston, New England in the years 1735 and 1736. Boston: T. Fleet, 1736. 5. Dillon HC. Impetigo contagiosa: suppurative and non- suppurative complication. Clinical, bacteriologic and epidemiologic characteristics of impetigo. Am J Dis Child 1968; 115:530-41. 6. Wannamaker LW, Rammelkamp CH, Jr., Denny FW, Brink WR, Houser HB, Hahn EO, et al. Prophylaxis of acute rheumatic fever by treatment of the preceding streptococcal infection with various amounts of depot penicillin. Am J Med 1951; 10:673-95. 7. Weaver GH. Scarlet Fever. In: Abt IA, ed., Pediatrics. Philadelphia: W.B. Saunders Co., 1925:298-362. 8. Stevens DL. Invasive group A streptococcus infec- tions. Clin Infect Dis 1992; 14:2-13. 9. Kohler W, Gerlach D, Knoll H. Streptococcal out- breaks and erythrogenic toxin type A. Zbl Bakt Hyg 1987; 266:104-15. 10. Martin PR, Hoiby EA. Streptococcal serogroup A epi- demic in Norway 1987-1988. Scand J Infect Dis 1990; 22:421-9. 11. Holm S. Fatal group A streptococcal infections. Pre- sented at the 89th Conference of the American Society for Microbiology, New Orleans, LA,1989. 12. Wheeler MC, Roe MH, Kaplan EL, Schlievert PM, Todd JK. Outbreak of group A streptococcus septice- mia in children: clinical, epidemiologic, and microbio- logical correlates.

S. Fatal group A streptococcal infections. Pre- sented at the 89th Conference of the American Society for Microbiology, New Orleans, LA,1989. 12. Wheeler MC, Roe MH, Kaplan EL, Schlievert PM, Todd JK. Outbreak of group A streptococcus septice- mia in children: clinical, epidemiologic, and microbio- logical correlates. JAMA 1991; 266:533-7. 13. Gaworzewska ET, Coleman G. Correspondence: group A streptococcal infections and a toxic shock-like syn- drome. N Engl J Med 1989; 321:1546. 14. Schwartz B, Facklam R, Breiman R. The changing epidemiology of group Astreptococcal infections in the U.S.: association with changes in serotype. Presented at the 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, 1990; Ab- stract 88. 15. Bartter T, Dascal A, Carroll K, Curley FJ. "Toxic strep syndrome": manifestation of group A streptococcal infection. Arch Intern Med 1988; 148:1421-4. 16. Hribalova V. Streptococcus pyogenes and the toxic shock syndrome. Ann Intern Med 1988; 108:772. 17. Greenberg RN, Willoughby BG, Kennedy DJ, Otto TJ, McMillian R, Bloomster TG. Hypocalcemia and "toxic" syndrome associated with streptococcal fascii- tis. South Med J 1983; 76:916-8. 18. Jackson MA, Olson LC, Burry VF. Pediatric group A streptococcal (GAS) disease with multi-organ dys- function. Presented at the 30th Interscience Confer- ence on Antimicrobial Agents and Chemotherapy, Atlanta, GA, 1990; Abstract 195. 19. Thomas JC, Carr SJ, Fujioka K, Waterman SH. Com- munity-acquired group A streptococcal deaths in Los Angeles County. J Infect Dis 1989; 160:1086-7. 20. Francis J, Warren RE. Streptococcus pyogenes bac- teraemia in Cambridge: a review of 67 episodes. Q J Med 1988; 256:603-13. 21. Barnham M. Invasive streptococcal infections in the era before the acquired immune deficiency syndrome: a 10 years' compilation of patients with streptococcal bacteraemia in North Yorkshire. J Infect Dis 1989; 18:231-48. 22. Braunstein H. Characteristics of group A streptococ- cal bacteremia in patients at the San Bernardino County Medical Center. Rev Infect Dis 1991; 13:8-11. 23. Schwartz B, Facklam RR, Brieman RF. Changing epidemiology of group A streptococcal infection in the USA. Lancet 1990; 336:1167-71. 24. Holm SE, Norrby A, Bergholm AM, Norgren M. As- pects of pathogenesis of serious group A streptococcal infections in Sweden, 1988-1989. J Infect Dis 1992; 166:31-7. 25. Stegmayr B, Bjorck S, Holm S, Nisell J, Rydvall A, Settergren B.

idemiology of group A streptococcal infection in the USA. Lancet 1990; 336:1167-71. 24. Holm SE, Norrby A, Bergholm AM, Norgren M. As- pects of pathogenesis of serious group A streptococcal infections in Sweden, 1988-1989. J Infect Dis 1992; 166:31-7. 25. Stegmayr B, Bjorck S, Holm S, Nisell J, Rydvall A, Settergren B. Septic shock induced by group A strep- tococcal infections: clinical and therapeutic aspects. Scand J Infect Dis 1992; 24:589-97. 26. Demers B, Simor AE, Vellend H, Schlievert PM, Byrne S, Jamieson F, et al. Severe invasive group A strepto- coccal infections in Ontario, Canada: 1987-1991. Clin Infect Dis 1993; 16:792-800. 27. Auerbach SB, Schwartz B, Facklam RR, Breiman R, Jarvis WR. Outbreak of invasive group A streptococ- cal (GAS) disease in a nursing home. Presented at the 30th Interscience Conferenceon AntimicrobialAgents and Chemotherapy, Atlanta, GA, 1990; Abstract 171. 28. Hohenboken JJ, Anderson F, Kaplan EL. Invasive group Astreptococcal (GAS) serotype M-1 outbreak in a long-term care facility (LTCF) with mortality. Pre- sented at the 34th Interscience Conference on Antimi- crobial Agents and Chemotherapy, Orlando, FL, 1994; Abstract J189. 29. Johnson DR, Stevens DL, Kaplan EL. Epidemiologic analysis of group A streptococcal serotypes associated with severe systemic infections, rheumatic fever, or uncomplicated pharyngitis. J Infect Dis 1992; 166:374-82. 30. Belani K, Schlievert P, Kaplan E, Ferrieri P. Associa- tion of exotoxin-producing group A streptococci and severe disease in children. Pediatr Infect Dis J 1991; 10:351-4. Synopses Emerging Infectious Diseases 76 Vol. 1, No. 3 -- July-September 1995 31. Hauser AR, Goshorn SC, Kaplan E, Stevens DL, Schlievert PM. Molecular analysis of the streptococcal pyrogenic exotoxins. Presented at the Third Interna- tional American Society for Microbiology Conference on Streptococcal Genetics. Minneapolis, MN, 1990. 32. Hauser AR, Stevens DL, Kaplan EL, Schlievert PM. Molecular analysis of pyrogenic exotoxins from Strep- tococcus pyogenes isolates associated with toxic shock- like syndrome. J Clin Microbiol 1991; 29:1562-7. 33. Musser JM, Hauser AR, Kim MH, Schlievert PM, Nelson K, Selander RK. Streptococcus pyogenes caus- ing toxic-shock-like syndrome and other invasive dis- eases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci USA1991; 88:2668-72. 34. Mollick JA, Miller GG, Musser JM, Cook RG, Gross- man D, Rich RR.

Musser JM, Hauser AR, Kim MH, Schlievert PM, Nelson K, Selander RK. Streptococcus pyogenes caus- ing toxic-shock-like syndrome and other invasive dis- eases: clonal diversity and pyrogenic exotoxin expression. Proc Natl Acad Sci USA1991; 88:2668-72. 34. Mollick JA, Miller GG, Musser JM, Cook RG, Gross- man D, Rich RR. A novel superantigen isolated from pathogenic strains of Streptococcus pyogenes with aminoterminal homology to staphylococcal enterotox- ins B and C. J Clin Invest 1993; 92:710-9. 35. Iwasaki M, Igarashi H, Hinuma Y, Yutsudo T. Cloning, characterization and overexpression of a Streptococ- cus pyogenes gene encoding a new type of mitogenic factor. FEBS Lett 1993; 331:187-92. 36. Norrby-Teglund A, Newton D, Kotb M, Holm SE, Norgren M. Superantigenic properties of the group A streptococcal exotoxin SpeF (MF). Infect Immun 1994; 62:5227-33. 37. Meleney FL. Hemolytic Streptococcus gangrene. Arch Surg 1924; 9:317-64. 38. Adams EM, Gudmundsson S, Yocum DE, Haselby RC, Craig WA, Sundstrom WR. Streptococcal myositis. Arch Intern Med 1985; 145:1020-3. 39. Svane S. Peracute spontaneous streptococcal myosi- tis: a report on 2 fatal cases with review of literature. Acta Chir Scand 1971; 137:155-63. 40. Yoder EL, Mendez J, Khatib R. Spontaneous gangre- nous myositis induced by Streptococcus pyogenes: case report and review of the literature. Rev Infect Dis 1987; 9:382-5. 41. Nather A, Wong FY, Balasubramaniam P, Pang M. Streptococcal necrotizing myositis -- a rare entity: a report of two cases. Clin Orthop 1987; 215:206-11. 42. Stevens DL, Musher DM, Watson DA, Eddy H, Hamill RJ, Gyorkey F, Rosen H, et al. Spontaneous, nontrau- matic gangrene due to Clostridium septicum. Rev Infect Dis 1990; 12:286-96. 43. Bullowa JGM, Wischik S. Complications of varicella. I: their occurrence among 2,534 patients. Am J Dis Child 1935;49: 923-6. 44. Fast DJ, Schlievert PM, Nelson RD. Toxic shock syn- drome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production. Infect Immun 1989; 57:291-4. 45. Hackett SP, Schlievert PM, Stevens DL. Cytokine production by human mononuclear cells in response to streptococcal exotoxins. Clin Res 1991; 39:189A. 46. Hallas G. The production of pyrogenic exotoxins by group A streptococci. J Hyg (Camb) 1985; 95:47-7. 47. Lancefield RC. Current knowledge of type specific M antigens of group A streptococci. J Immunol 1962; 89:307-13. 48. Mollick JA, Rich RR.

ear cells in response to streptococcal exotoxins. Clin Res 1991; 39:189A. 46. Hallas G. The production of pyrogenic exotoxins by group A streptococci. J Hyg (Camb) 1985; 95:47-7. 47. Lancefield RC. Current knowledge of type specific M antigens of group A streptococci. J Immunol 1962; 89:307-13. 48. Mollick JA, Rich RR. Characterization of a superan- tigen from a pathogenic strain of Streptococcus pyo- genes. Clin Res 1991; 39:213A. 49. Hackett SP, Stevens DL. Streptococcal toxic shock syndrome: synthesis of tumor necrosis factor and in- terleukin-1 by monocytes stimulated with pyrogenic exotoxin A and streptolysin O. J Infect Dis 1992; 165:879-85. 50. Kotb M, Tomai M, Majumdar G, Walker J, Beachey EH. Cellular and biochemical responses of human T lymphocytes stimulated with streptococcal M protein. Presented at the 11th Lancefield International Sym- posium on Streptococcal Diseases, Siena, Italy, 1990; Abstract L77. 51. Watanabe-Ohnishi R, Low DE, McGeer A, Stevens DL, Schlievert PM, Newton D, et al. Selective deple- tion of V-bearing T cells in patients with severe invasive group A streptococcal infections and strepto- coccal toxic shock syndrome. J Infect Dis 1995; 171:74- 84. 52. Stevens DL, Bryant AE, Hackett SP. Gram-positive shock. Curr Opin Infect Dis 1992; 5:355-63. 53. Hackett S, Ferretti J, Stevens D. Cytokine induction by viable group A streptococci: suppression by strep- tolysin O. Presented at the 93rd Conference of the American Society for Microbiology, Las Vegas, NV, 1994; Abstract B-249. 54. Muller-Alouf H, Alouf JE, Gerlach D, Ozegowski JH, Fitting C, Cavaillon JM. Comparative study of cytok- ine release by human peripheral blood mononuclear cells stimulated withStreptococcus pyogenes superan- tigenic erythrogenic toxins, heat-killed streptococci and lipopolysaccharide. Infect Immun 1994; 62:4915- 21. 55. Hackett SP, Stevens DL. Superantigens associated with staphylococcal and streptococcal toxic shock syn- dromes are potent inducers of tumor necrosis factor beta synthesis. J Infect Dis 1993; 168:232-5. 56. Kappur V, Majesky MW, Li LL, Black RA, Musser JM. Cleavage of Interleukin 1B (IL-1B) precursor to pro- duce active IL-1B by a conserved extracellular cyste- ine protease from Streptococcus pyogenes. Proc Natl Acad Sci USA 1993; 90:7676-80. 57. Cleary R, Chen C, Lapenta D, Bormann N, Heath D, Haanes E. A virulence regulon in Streptococcus pyo- genes.

ack RA, Musser JM. Cleavage of Interleukin 1B (IL-1B) precursor to pro- duce active IL-1B by a conserved extracellular cyste- ine protease from Streptococcus pyogenes. Proc Natl Acad Sci USA 1993; 90:7676-80. 57. Cleary R, Chen C, Lapenta D, Bormann N, Heath D, Haanes E. A virulence regulon in Streptococcus pyo- genes. Presented at the Third International American Society for Microbiology Conference on Streptococcal Genetics, Minneapolis, MN, 1990; Abstract 19. 58. Cleary PP, Kaplan EL, Handley JP, Wlazlo A, Kim MH, Hauser AR, et al. Clonal basis for resurgence of serious Streptococcus pyogenes disease in the 1980s. Lancet 1992; 339:518-21. 59. Nida SK, Ferretti JJ. Phage influence on the synthe- sis of extracellular toxins in group A streptococci. Infect Immun 1982; 36:745-50. 60. Johnson LP, Tomai MA, Schlievert PM. Bacteriophage involvement in group A streptococcal pyrogenic exo- toxin A production. J Bacteriol 1986; 166:623-7. 61. Stevens DL. Invasive group Astreptococcal infections: the past, present and future. Pediatr Infect Dis J 1994; 13:561-6. 62. Kim KS, Kaplan EL. Association of penicillin toler- ance with failure to eradicate group A streptococci from patients with pharyngitis. J Pediatr 1985; 107:681-4. Synopses Vol. 1, No. 3 -- July-September 1995 77 Emerging Infectious Diseases 63. Gatanaduy AS, Kaplan EL, Huwe BB, McKay C, Wannamaker LW. Failure of penicillin to eradicate group A streptococci during an outbreak of pharyngi- tis. Lancet 1980; 2:498-502. 64. Brook I. Role of beta-lactamase-producing bacteria in the failure of penicillin to eradicate group A strepto- cocci. Pediatr Infect Dis 1985; 4:491-5. 65. Kohler W. Streptococcal toxic shock syndrome. Zbl Bakt 1990; 272:257-64. 66. Eagle H. Experimental approach to the problem of treatment failure with penicillin. I. Group A strepto- coccal infection in mice. Am J Med 1952; 13:389-9. 67. Stevens DL, Gibbons AE, Bergstrom R, Winn V. The Eagle effect revisited: efficacy of clindamycin, eryth- romycin, and penicillin in the treatment of streptococ- cal myositis. J Infect Dis 1988; 158:23-8. 68. Stevens DL, Bryant AE, Yan S. Invasive group A streptococcal infection: new concepts in antibiotic treatment. Int J Antimicrob Agents 1994; 4:297-301. 69. Stevens DL, Yan S, Bryant AE. Penicillin-binding protein expression at different growth stages deter- mines penicillin efficacy in vitro and in vivo: an expla- nation for the inoculum effect. J Infect Dis 1993; 167:1401-5. 70. Yan S, Mendelman PM, Stevens DL.

otic treatment. Int J Antimicrob Agents 1994; 4:297-301. 69. Stevens DL, Yan S, Bryant AE. Penicillin-binding protein expression at different growth stages deter- mines penicillin efficacy in vitro and in vivo: an expla- nation for the inoculum effect. J Infect Dis 1993; 167:1401-5. 70. Yan S, Mendelman PM, Stevens DL. The in vitro antibacterial activity of ceftriaxone against Strepto- coccus pyogenes is unrelated to penicillin-binding pro- tein 4. FEMS Microbiol Lett 1993; 110:313-18. 71. Yan S, Bohach GA, Stevens DL. Persistent acylation of high-molecular weight penicillin-binding proteins by penicillin induces the post-antibiotic effect in Streptococcus pyogenes. J Infect Dis 1994; 170:609-14. 72. Stevens DL, Maier KA, Mitten JE. Effect of antibiotics on toxin production and viability of Clostridium per- fringens. Antimicrob Agents Chemother 1987; 31:213- 8. 73. Gemmell CG, Peterson PK, Schmeling D, Kim Y, Mathews J, Wannamaker L, et al. Potentiation of opsonization and phagocytosis of Streptococcus pyo- genes following growth in the presence of clindamycin. J Clin Invest 1981; 67:1249-56. 74. Stevens DL, Bryant AE, Hackett SP. Antibiotic effects on bacterial viability, toxin production and host re- sponse. Clin Infect Dis 1995;20(Suppl 2):S154-7. 75. Barry W, Hudgins L, Donta ST, Pesanti EL. Intrave- nous immunoglobulin therapy for Toxic shock syn- drome. JAMA 1992; 267(24):3315-6. 76. Yong JM. Letter. Lancet 1994; 343:1427. 77. Greenberg LJ, Gray ED, Yunis E. Association of HL- A5 and immune responsiveness in vitro to streptococ- cal antigens. J Exp Med 1975; 141:934-43. 78. Weinstein L, Barza M. Gas gangrene. N Engl J Med 1972; 289:1129. 79. Zabriskie JB, Lavenchy D, Williams RCJ, et al. Rheu- matic-fever associated B-cell alloantigens as identi- fied by monoclonal antibodies. Arthritis Rheum 1985; 28:1047-51. 80. Schwartz B, Elliot JA, Butler JC, Simon PA, Jameson BL, Welch GE, et al. Clusters of invasive group A streptococcal infections in family, hospital, and nurs- ing home settings. Clin Infect Dis 1992; 15:277-84. Synopses Emerging Infectious Diseases 78 Vol. 1, No. 3 -- July-September 1995

Spiral Bacteria in the Human Stomach: The Gastric Helicobacters Andre Dubois, M.D., Ph.D. Digestive Diseases Division, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA During the past decade, Helicobacter pylori has become recognized as one of the most common human pathogens, colonizing the gastric mucosa of almost all persons exposed to poor hygienic conditions from childhood. It also is often found, albeit with a lower frequency, in groups of high socioeconomic status. H. pylori causes chronic active gastritis and is a major factor in the pathogenesis of duodenal ulcers and, to a lesser extent, gastric ulcers. In addition, the presence of this bacterium is now recognized as a risk factor for gastric adenocarcinoma and lymphoma. Nevertheless, most infections appear without clinical consequences. In this second decade of intensive research, it is important to understand why H. pylori is sometimes a dangerous pathogen, and to determine how it can be eradicated in those at highest risk for severe disease. At the end of the 19th century, several types of spirochetes and spirilla were observed for the first time in the stomach of animals (1,2). Beginning at the turn of the 20th century, similar spiral bacteria were found in gastrectomy specimens from patients with gastric cancer and peptic ulcer disease (3,4). In addition, gastroenterologists and surgeons noted-- but could not explain--the almost universal pres- ence of antral gastritis in patients with duodenal ulcers and the frequent presence of atrophic gastri- tis in patients with gastric ulcer and cancer. Never- theless, the possibility that peptic ulcer disease or gastric cancer might be caused by an infectious agent was generally discounted. The observation made in 1975 that gram-negative bacteria were present in 80% of patients with gastric ulcer (5) was largely ignored by the scientific community which, at the time, was busily developing potent antiulcer agents (6).

isease or gastric cancer might be caused by an infectious agent was generally discounted. The observation made in 1975 that gram-negative bacteria were present in 80% of patients with gastric ulcer (5) was largely ignored by the scientific community which, at the time, was busily developing potent antiulcer agents (6). Skepticism remained the overwhelming reaction to the 1983 reports describing the frequent association between antral gastritis and the pres- ence of Campylobacter-like bacteria (7), as well as of their culture and isolation from patients with gas- tritis (8). A similar reaction followed the subsequent demonstration that these Campylobacter-like bacte- ria were present in almost all patients with gastric and duodenal ulcers, and were generally associated with antral gastritis (9). In the past decade, how- ever, a number of studies have confirmed and ex- tended these early observations. A consensus regarding the major role of this bacterium, now named Helicobacter pylori, in causing gastroduode- nal ulceration was formally presented in 1994 (10). Furthermore, in June 1994, the International Agency for Research on Cancer Working Group stated , "H. pylori plays a causal role in the chain of events leading to cancer," referring to adenocarci- noma and lymphoma of the stomach as well as to the more benign mucosal-associated lymphoid tissues (MALT) (11-13). An important consequence of the considerable interest generated by these clinical observations is that extensive bacteriologic and molecular studies have been performed on this bacterium and similar organisms. 16S rRNA gene sequence analysis has revealed important differences between H. pylori and the closely related Campylobacter, Flexispira, and Wolinella genuses. These differences have ne- cessitated the creation of the genus Helicobacter, which, to date, includes eight gastric, three intesti- nal, and two hepatic species (14). Each of these Helicobacter species colonizes different, or a spec- trum of, mammalian species. This review summarizes our current knowledge of the two Helicobacter species that have been ob- served in the human stomach and reported on ex- tensively in the literature: H. pylori, the type strain, and H. heilmannii, also known as Gastrospirillum hominis (15,16). Characteristics of Gastric Helicobacters Observed in Humans H. pylori, a gram-negative bacterium with a curved, spiral, or gull-wing shape, is 2.5 to 3.5 m long and 0.5 to 1.0 m in diameter and has a peri- odicity of 1 to 2 m.

H. pylori, the type strain, and H. heilmannii, also known as Gastrospirillum hominis (15,16). Characteristics of Gastric Helicobacters Observed in Humans H. pylori, a gram-negative bacterium with a curved, spiral, or gull-wing shape, is 2.5 to 3.5 m long and 0.5 to 1.0 m in diameter and has a peri- odicity of 1 to 2 m. It has smooth surfaces, and one to six polar-sheathed flagellae emerge from one of its rounded ends. Since it is morphologically similar to C. jejuni, it was initially named "pyloric Campy- Address for correspondence: Andre Dubois, Department of Medicine, Uniformed Services University, 4301 Jones Bridge Road, Bethesda, MD 20814-4799, USA; fax: 301-295-3676 or -3557; e-mail dubois@usuhsb.usuhs.mil. Synopses Vol. 1, No. 3 -- July-September 1995 79 Emerging Infectious Diseases lobacter" and subsequently C. pyloridis and C. pylori before finally being named H. pylori. This organism colonizes only the non-acid-secreting mucosa of the stomach and is not found where parietal cells are numerous. Thus, it may be observed in the gastric antrum and the cardia, but also in the corpus, when atrophic gastritis is present, and attached to the gastric epithelial cells found in the duodenum, when gastric metaplasia is present. G. hominis (H. heilmannii) is tightly spiraled, and is 3.5 to 7.5 m in length and 0.9 m in diameter; it has a periodicity of 0.8 to 1 m and up to 12 flagellae at each pole. 16S rRNA indicates that this organism belongs to the genus Helicobacter, and is more closely related to a Helicobacter sp. isolated from the stomach of cats (H. felis) than to H. pylori (17). The name H. heilmannii was proposed in honor of the late German pathologist Heilmann. However, the subsequent examination of the rRNAof different clinical isolates indicates that there is enough het- erogeneity among isolates tentatively identified as H. heilmannii that it is premature to propose an official name (17) . This bacterium colonizes only the parietal cell area of the gastric mucosa and may be found within parietal cells (18,19). Diagnosis H. pylori infection may be diagnosed by harvest- ing gastric biopsy specimens during endoscopy, by culturing and isolating the bacterium under mi- croaerobic conditions (90% N2, 5% O2, and 5% CO2), and by characterizing the enzymes (urease, catalase, and oxidase) it produces.

nd within parietal cells (18,19). Diagnosis H. pylori infection may be diagnosed by harvest- ing gastric biopsy specimens during endoscopy, by culturing and isolating the bacterium under mi- croaerobic conditions (90% N2, 5% O2, and 5% CO2), and by characterizing the enzymes (urease, catalase, and oxidase) it produces. Visualization of the bacterium by light microscopy on slides stained with hematoxylin and eosin, Gram, Giemsa, Genta, or Warthin-Starry stain is also of great benefit since it allows the concurrent diagnosis of the extent of the antral chronic-active gastritis that H. pylori causes. However, because H. pylori colonization is focal, negative biopsy results do not exclude the possibility of infection in areas not sampled. Infec- tion also may be diagnosed by determining plasma and salivary immunoglobulin (Ig) G or IgA levels with enzyme-linked immunosorbent assays (20,21). This latter technique is noninvasive, specific, and sensitive and is believed to reflect the mucosal and systemic immunity induced by H. pylori infection. Two other tests, which rely on the production of urease, also can be used to identify H. pylori. One is the CLO (for Campylobacter-like organisms) test, which is performed by placing a mucosal biopsy specimen in medium containing urea and a pH- sensitive dye that changes color in the presence of OH- ions. The second test is the noninvasive 14C or 13C breath test following the oral administration of 14C- or 13C-urea. Neither of these tests is specific for H. pylori since G. hominis, which generates urease, also gives a positive reaction. Until specific methods based on the polymerase chain reaction (PCR) am- plification of 16S rRNA (17) become widely avail- able, the diagnosis of G. hominis infection must rely on histologic morphologic characteristics; histologic identification must be confirmed by transmission electron microscopy since other spiral organisms, e.g., Flexispira rappini, also may be present in the stomach of humans (22). Epidemiology The seroepidemiology of H. pylori has been exten- sively studied in the United States and in other countries (23). The high frequency of seropositivity (up to 100% in some age groups in Albania) and acquisition of the infection during infancy are char- acteristic of disadvantaged socioeconomic groups living in crowded or poor hygienic conditions and appears to be independent of gender and ethnic origin.

and in other countries (23). The high frequency of seropositivity (up to 100% in some age groups in Albania) and acquisition of the infection during infancy are char- acteristic of disadvantaged socioeconomic groups living in crowded or poor hygienic conditions and appears to be independent of gender and ethnic origin. In adults of higher socioeconomic groups, the rate of seroconversion is estimated at 0.5% per year, although the frequency of seropositivity increases with age and may be as high as 40%. A longitudinal study has indicated that the high frequency of sero- positivity in older adults might be due to a higher rate of H. pylori infection in Western countries in the years between the two world wars than during recent years (cohort effect) (24). Alternatively, the increase in frequency of infection in older adults might be due to years of low but cumulative risk for infection. Although the route of transmission for this infection is not known, the contamination of drink- ing water may play a role in certain developing countries (25). In the United States and in other regions, direct contact and/or consumption of food or water contaminated by saliva (26), gastric contents, or feces (27) may be major factors. The recent obser- vation that H. pylori can be isolated from cats (28) suggests that transmission from pets to humans (or humans to pets) is also possible. The epidemiology and route of transmission of G. hominis are largely unknown. The frequency of this infection appears to range from less than 1% of the population in industrialized countries (29) to 3% to 8% in developing countries (30). Although the detec- tion of spirilla in the stomach of cats and dogs suggests possible transmission from pets, marked morphologic differences exist between these spirilla and the organism found in the stomach of humans. Pathogenicity H. pylori is considered a pathogen because its presence is always associated with chronic active gastritis, and eradication of the bacterium is always followed by resolution of gastritis. In addition, Synopses Emerging Infectious Diseases 80 Vol. 1, No. 3 -- July-September 1995 nearly all patients with duodenal ulcer disease have H. pylori gastritis, and ulcer relapse is exceptional after H. pylori eradication. Thus, the presence of H. pylori seems necessary for the production of duode- nal ulcers, with the exception of ulcers attributed to the use of nonsteroidal antiinflammatory agents or to the Zollinger-Ellison syndrome (10).

r disease have H. pylori gastritis, and ulcer relapse is exceptional after H. pylori eradication. Thus, the presence of H. pylori seems necessary for the production of duode- nal ulcers, with the exception of ulcers attributed to the use of nonsteroidal antiinflammatory agents or to the Zollinger-Ellison syndrome (10). The associa- tion with gastric ulcers is not as strong, although H. pylori infection is present in 80% of patients with gastric ulcers who do not consume nonsteroidal anti- inflammatory agents (10). However, most H. pylori- infected persons do not report any clinical symptoms. This may be because these persons are colonized by less virulent strains or because other host or bacterial cofactors are required for overt disease. In addition, three prospective cohort studies have demonstrated that H. pylori-infected persons have an increased risk of developing intestinal-type, but not undifferentiated, gastric adenocarcinoma (10). In fact, the association of H. pylori with either gas- tric ulcer or gastric cancer may be underestimated in these studies: the atrophic gastritis that follows long-term infection makes the gastric niche less hospitable for the bacterium, which may either eliminate H. pylori or make it difficult to detect. Nevertheless, atrophic gastritis per se is believed to be a precancerous lesion that leads to carcinogenesis without the presence of H. pylori. The pathogenicity of G. hominis is unclear. The organism has been associated with upper gastro- intestinal complaints, and its carriage is generally accompanied by gastritis, although the inflamma- tion and gastric atrophy are less than noted with H. pylori (31,32). In addition, G. hominis was observed in gastric cancer patients (3) as well as in patients with only minimal gastritis (29). In this relatively small number of cases, the frequent concurrent in- fection with H. Pylori makes interpreting the re- spective pathogenic role of either bacterium difficult. It is probable that G. hominis will turn out to be at least somewhat pathogenic, as it makes urease and products of urease action that have been implicated in inflammation. Colonization and Virulence Factors H. pylori multiplies with great efficiency in the hostile environment within the stomach but sur- vives poorly in the gastric lumen; it is mainly found where the pH ranges between 4 and 7, i.e., under the mucous layer and in close proximity, or even at- tached, to gastric superficial epithelial cells.

Virulence Factors H. pylori multiplies with great efficiency in the hostile environment within the stomach but sur- vives poorly in the gastric lumen; it is mainly found where the pH ranges between 4 and 7, i.e., under the mucous layer and in close proximity, or even at- tached, to gastric superficial epithelial cells. The virulence and the ecologic niche of G. hominis are unknown, although its presence within parietal cells of patients with gastrointestinal complaints (18,19) suggests that it is even more resistant to acid than H. pylori. The production of urease was the first putative colonization or virulence factor studied. The produc- tion of this enzyme is shared by the two organisms, and it may explain their extraordinary ability to survive in an environment previously considered sterile because of the presence of proteolytic en- zymes, as well as the low pH of gastric contents. Because the ecologic niches of these bacteria are rich in urea, urease generates OH- ions that neutralize gastric acid. Although the neutralization of gastric acid benefits the two bacteria, the production of hydroxide ions also is toxic to gastric epithelial cells in vivo, as indicated by in vitro experiments (33). Two other important virulence factors shared by H. pylori and G. hominis are their spiral shape and the motility of their flagellae, which render them resistant to peristaltic flushing of the gastric con- tents and enable them to persist in the mucous layer. Because G. hominis appears to infect fewer persons than H. pylori, a more important role might be attributable to characteristics that are unique to H. pylori; these include the production of other en- zymes (catalase, oxidase, protease, and phospholi- pase), as well as the synthesis of specific adhesin proteins that enable them to adhere to mucous and epithelial cells, both in vivo and in vitro (34-36). The putative virulence factor of H. pylori that has commanded the most attention during the past few years has been its vacuolating cytotoxin (vacA gene product). Intragastric administration of the toxin to mice causes some (but not all) of the tissue damage seen in H. pylori-infected persons (37). In addition, cytotoxin production is highly correlated with the production of a high molecular weight (120 to 128 kilodaltons) major protein antigen that is called cytotoxin-associated protein (cagA) and is not the toxin itself (38). Diversity of H. pylori H.

f the tissue damage seen in H. pylori-infected persons (37). In addition, cytotoxin production is highly correlated with the production of a high molecular weight (120 to 128 kilodaltons) major protein antigen that is called cytotoxin-associated protein (cagA) and is not the toxin itself (38). Diversity of H. pylori H. pylori isolates may differ with respect to each of the virulence factors described above; this diver- sity is likely to contribute to variation in coloniza- tion or disease. For example, urease-negative strains have been isolated, and the vacuolating cy- toxin is produced by only a subset ofH. pylori strains (vacA+ or tox+ strains) (39-41). This observation is probably clinically relevant because most or all strains from duodenal ulcer patients, and many strains from gastric cancer patients, produce cyto- toxin, whereas only a fraction of strains from pa- tients with gastritis alone produce the cytotoxin (42,43). This phenotypic diversity is mirrored in great diversity on the DNA level. Thus, only cyto- toxin-producing strains contain the gene for this cytotoxin-associated protein (cagA) (38,42), al- though genetic tests have shown that cagA protein is not needed for toxin production (44). Strains that Synopses Vol. 1, No. 3 -- July-September 1995 81 Emerging Infectious Diseases do not produce the 128-kDa cagA protein generally lack the entire cagA gene and additional neighbor- ing genes. Although the function of the cagA region is unknown, its presence or absence is easily scored by hybridization or PCR and thus serves as an easy marker for probable cytotoxin production and possi- ble virulence of H. pylori strains. Additional viru- lence factors are likely to be present. For example, another recently discovered region constitutes at least 21 kilobases of the H. pylori genome in hybridi- zation experiments, and its presence is highly corre- lated with the presence of cagA: 39 of 40 strains lacking cagA also lacked this region, and 50 of 52 strains containing cagA contained this region. This newly discovered region is being called cagII, and the effort to sequence it is nearly complete (D. E. Berg, pers. comm.). Preliminary searches have iden- tified several open reading frames with strong ho- mologies to virulence functions from other microbes (45). In addition to these extensively studied genes, genetic diversity of various H. pylori strains can be demonstrated by the use of two sensitive, efficient, and reliable PCR-based methods (46,47).

rches have iden- tified several open reading frames with strong ho- mologies to virulence functions from other microbes (45). In addition to these extensively studied genes, genetic diversity of various H. pylori strains can be demonstrated by the use of two sensitive, efficient, and reliable PCR-based methods (46,47). This ap- proach is particularly useful because it allows trac- ing of strains in epidemiologic studies. Infection and Immune Response One of the most puzzling aspects of gastric infec- tion with H. pylori is its persistence despite intense local and systemic immune responses. These im- mune responses are extremely complex and vary among infected humans. The systemic response is characterized by a marked increase in plasma IgG, which remains present for months after the infec- tion has been cured. The local response includes the production of IgA, which binds to the surface anti- gens of H. pylori in vitro and coats the bacterium in vivo. In addition, infection is consistently associated with an intense inflammatory response and the in- filtration of cells into the gastric mucosa. Although polymorphonuclear cells are often present, most cells in such infiltrates are mononuclear cells. Both B and T cells are present, and recent studies have indicated that the natural killer activity of periph- eral blood lymphocytes can be increased byH. pylori, possibly by its stimulating the production of inter- feron and other cytokines (48). Thus, the long-term carriage of the infection may be related to the ability of the bacterium to influence the T-cell response. Fragmentary evidence also suggests that this infec- tion can be abortive and cure spontaneously without the use of antibiotics (A. Dubois and D. E. Berg, unpublished). On the other hand, the mucosal response may promote colonization, as indicated by the observa- tion that patients with acquired immunodeficiency syndrome (AIDS) tend to have a lower rate of infec- tion than aged-matched subjects who are negative for human immunodeficiency virus (49,50). The lat- ter study (50) also demonstrated that AIDS patients had a different pattern of gastritis, characterized by greater mononuclear cell responses, fewer lymphoid follicles, and a greater prevalence of intestinal meta- plasia. The immune response may also prevent the invasiveness of H. pylori, as suggested by the anec- dotal but puzzling observation of invasive H. pylori infection in a patient with AIDS (51). Treatment Although H.

eater mononuclear cell responses, fewer lymphoid follicles, and a greater prevalence of intestinal meta- plasia. The immune response may also prevent the invasiveness of H. pylori, as suggested by the anec- dotal but puzzling observation of invasive H. pylori infection in a patient with AIDS (51). Treatment Although H. pylori is sensitive to many antimi- crobial drugs in vitro, it is difficult to eradicate from the stomach. This may be ascribed to antibiotic breakdown by gastric acid, clearance by gastric emp- tying, and the difficult-to-penetrate mucous layer in which the bacterium resides. Resistance of H. pylori to specific antibiotics, especially metronidazole, is also frequent. Therefore, it is generally accepted that a combination of at least two, and possibly three, antimicrobial agents should be given for a minimum of 1 week. The regimen found to be most effective is the administration of amoxicillin (or tet- racycline) plus metronidazole and bismuth subsali- cylate 2 to 4 times a day for 2 to 3 weeks (52). The use of one antibiotic associated with an antisecre- tory agent, such as a histamine H2 receptor antago- nist, has given disappointing results. In contrast, the combination of a proton pump inhibitor (H+-K+ ATPase antagonist) with amoxicillin or acid-stable macrolides (clarythromycin or roxithromycin) ap- pears more promising; a number of studies are being conducted to determine the optimal dose, duration, concomitant therapy, and cost-effectiveness of these compounds (53,54). Recently, it was shown that at least a 7-day course of any of these regimens is required to obtain a high (90%) cure rate, but that continuing treatment for more than 10 days does not significantly improve its efficacy. Finally, topical therapy for 1 h was recently tried with excellent results, albeit in only one center at this time (55). This treatment involves a 2-day administration of a mucolytic agent to dissolve the mucous layer and of a proton pump inhibitor. On the third day, a balloon is introduced into the second portion of the duode- num under fluoroscopic control, and a solution of pronase, amoxicillin, metronidazole, and bismuth subsalicylate is injected into the stomach, where it is left for 1 h. The presence of the duodenal balloon appears to prevent emptying of the antibiotics and the mucolytic agent, thus ensuring maximum effi- cacy of the therapy. Synopses Emerging Infectious Diseases 82 Vol. 1, No.

amoxicillin, metronidazole, and bismuth subsalicylate is injected into the stomach, where it is left for 1 h. The presence of the duodenal balloon appears to prevent emptying of the antibiotics and the mucolytic agent, thus ensuring maximum effi- cacy of the therapy. Synopses Emerging Infectious Diseases 82 Vol. 1, No. 3 -- July-September 1995 Future Research The past 12 years have seen extensive progress in research on H. pylori as a cause of chronic active gastritis, duodenal ulcer disease, and gastric cancer. This has been largely due to an unusual collabora- tion among gastroenterologists, pathologists, mo- lecular geneticists, bacteriologists, and immuno- logists. However, our understanding of howH. pylori colonizes and causes diseases is far from complete, and it will benefit from studies performed in animal models that can be experimentally infected with H. pylori (56-59). In addition, no easily administered treatment leading to eradication of this bacterium in all patients is yet available, although a better knowledge of its physiology may lead to the develop- ment of such a "silver bullet." Studies in animals that are not naturally infected with H. pylori sug- gest possibilities for vaccines (56,57), and ongoing trials in nonhuman primates are exploring the pos- sibility of immunizing hosts that can be naturally infected with this organism. Although the elimina- tion of peptic ulcer disease and of certain forms of gastric cancer will require extensive and coordi- nated efforts from public health authorities, this goal now appears to be within the reach of the scientific and medical community. Acknowledgments The author thanks Drs. P. Baker and D.E. Berg for their helpful comments and suggestions during the preparation of this review. Dr. Dubois is professor of medicine and surgery (research), assistant director, Digestive Diseases Division, and chief, Laboratory of Gastrointestinal and Liver Studies, F. Edward Hebert School of Medicine of the Uniformed Services University of the Health Sciences, Bethesda, Maryland. He studies the physiology and pathophysiology of gastric secretion and gastric emptying as well as the role of gastric infection with H. pylori in gastroduodenal diseases. References 1. Rappin J. Contribution a l'etude de bacteries de la bouche a l'etat normal. 1881. Quoted by Breed RS, Murray EGD, Hitchens AP, Bergey's manual of deter- minative bacteriology, 6th ed. Baltimore: Williams & Wilkins, 1948;217. 2. Bizzozero G.

e of gastric infection with H. pylori in gastroduodenal diseases. References 1. Rappin J. Contribution a l'etude de bacteries de la bouche a l'etat normal. 1881. Quoted by Breed RS, Murray EGD, Hitchens AP, Bergey's manual of deter- minative bacteriology, 6th ed. Baltimore: Williams & Wilkins, 1948;217. 2. Bizzozero G. Sulle ghiandole tubulari del tube gas- troenterico e sui rapporti del loro coll'epitelio de rives- timento della mucosa. Atti R Accad Sci Torino 1892;28:233-51. 3. Krienitz W. Ueber das Auftreten von Spirochaten verschiedener Form im Mageninhalt bei Carcinoma ventriculi. Dtsch Med Wochenschr 1906:28:872-89. 4. Freedburg AS, Barron LE. The presence of spirochetes in human gastric mucosa. Am J Dig Dis 1940;7:443-5. 5. Steer HW, Colin-Jones DG. Mucosal changes in gas- tric ulceration and their response to carbenoxolone sodium. Gut 1975;16:590-7. 6. Black JW, Duncan WAM, Durant CJ, Ganelin CR, Parson EM. Definition and antagonism of histamine H2 receptors. Nature 1972;236:384-90. 7. Warren JR. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983;i:1273. 8. Marshall BJ. Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet 1983;i:1273-5. 9. Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1984;i:1311-5. 10. NIH Consensus Conference. Helicobacter pylori in peptic ulcer disease. JAMA 1994;272:65-9. 11. Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991;325:1127-31. 12. Nomura A, Stemmerman GN, Chyou PH, Kato I, Perez-Perez GI, Blaser MJ. Helicobacter pylori infec- tion and gastric carcinoma in a population of Jap- anese Americans in Hawaii. N Engl J Med 1991;325: 1132-6. 13. Isaacson PG, Spencer J. Is gastric lymphoma an in- fectious disease? Hum Pathol 1993;24:569-70. 14. Fox JG, Yan LL, Dewhirst FE, et al. Helicobacter bilis sp. nov., a novel Helicobacter species isolated from bile, livers, and intestines of aged, inbred mice. J Clin Microbiol 1995;33:445-54. 15. Heilmann KL, Borchard F. Gastritis due to spiral shaped bacteria other than Helicobacter pylori: clini- cal, histological and ultrastructural findings. Gut 1991;32:137-40. 16. McNulty CAM, Dent JC, Curry A, et al. New spiral bacterium in gastric mucosa. J Clin Pathol 1989;42:585-91. 17. Solnick JV, O'Rourke J, Lee A, Tompkins LS.

, Borchard F. Gastritis due to spiral shaped bacteria other than Helicobacter pylori: clini- cal, histological and ultrastructural findings. Gut 1991;32:137-40. 16. McNulty CAM, Dent JC, Curry A, et al. New spiral bacterium in gastric mucosa. J Clin Pathol 1989;42:585-91. 17. Solnick JV, O'Rourke J, Lee A, Tompkins LS. Molecu- lar analysis of urease genes from a newly identified uncultured species of Helicobacter. J Infect Dis 1993;168:379-83. 18. Rollason TP, Stone J, Rhodes JM. Spiral organisms in endoscopic biopsies of the human stomach. J Clin Pathol 1984;37:23-6. 19. Dye KR, Marshall BJ, Frierson HF, Guerrant RL, McCallum RW. Ultrastructure of another spiral or- ganism associated with human gastritis. Dig Dis Sci 1989;34:1787-91. 20. Perez-Perez GI, Dworkin BM, Chodos JE, Blaser MJ. Campylobacter pylori antibodies in humans. Ann In- tern Med 1988;109:11-7. 21. Drumm B, Perez-Perez GI, Blaser MJ, Sherman PM. Intrafamilial clustering of Helicobacter pylori infec- tion. N Engl J Med 1990;322:359-63. 22. Archer JR, Romero S, Ritchie AE, et al. Charac- terization of an unclassified microaerophilic bacte- rium associated with gastroenteritis. J Clin Microbiol 1988;26:101-5. 23. Taylor DN, Blaser MJ. The epidemiology of Helicobac- ter pylori infection. Epidemiol Rev 1991;13:42-59. 24. Cullen DJE, Collins BJ, Christiansen BJ, et al. When is Helicobacter pylori infection acquired? Gut 1993;34:1681-2. Synopses Vol. 1, No. 3 -- July-September 1995 83 Emerging Infectious Diseases 25. Klein PD, Graham DY, Gaillour A, Opekun AR, Smith EO. Gastrointestinal Physiology Working Group. Water source as risk factor for Helicobacter pylori infection in Peruvian children. Lancet 1991:337:1503- 6. 26. Ferguson DA, Li C, Patel NR, Mayberry WR, Chi DS, Thomas E. Isolation ofHelicobacter pylori from saliva. J Clin Microbiol 1993;31:2802-4. 27. Thomas JE, Gibson CR, Darboe MK, Dale A, Weaver LT. Isolation of H. pylori from human faeces. Lancet 1992;340:1194-5. 28. Handt LK, Fox JO, Dewhirst FE, et al. Helicobacter pylori isolated from the domestic cat: public health implications. Infect Immun 1994;62:2367-74. 29. Mazzuchelli L, Wilder-Smith CH, Ruchti C, Meyer- Wyss B, Merki HS. Gastrospirillum hominis in asymptomatic, healthy individuals. Dig Dis Sci 1993;38:2087-9. 30. Chen Z, Wang B, Xu H, et al. Spiral shaped bacteria in the human gastric biopsy. Hua-Hsi I Ko Ta Hsueh Hsueh Pao 1993;24:392-4. 31. Logan RPH, Karim QN, Polson RJ, Walker MM, Baron JH.

th CH, Ruchti C, Meyer- Wyss B, Merki HS. Gastrospirillum hominis in asymptomatic, healthy individuals. Dig Dis Sci 1993;38:2087-9. 30. Chen Z, Wang B, Xu H, et al. Spiral shaped bacteria in the human gastric biopsy. Hua-Hsi I Ko Ta Hsueh Hsueh Pao 1993;24:392-4. 31. Logan RPH, Karim QN, Polson RJ, Walker MM, Baron JH. Gastrospirillum hominis infection of the stomach. Lancet 1989;ii:672. 32. Morris A, Ali MR, Thomsen L, Hollis B. Tightly spiral shaped bacteria in the human stomach: another cause of active chronic gastritis? Gut 1990;31:134-8. 33. Smoot DT, Mobley HLT, Chippendaele GR, Lewison JF, Resau JH. Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect Immun 1991;59:1992-4. 34. Boren T, Falk P, Roth KA, Larson G, Normark S. Attachment of Helicobacter pylori to human gastric epithelium mediated by blood group antigens. Science 1993;262:1892-5. 35. Fauchere J, Blaser MJ. Adherence of Helicobacter pylori cells and their surface components to HeLa cell membranes. Microb Pathol 1990;9:427-39. 36. Hemalatha SG, Drumm B, Sherman PJ. Adherence of Helicobacter pylori to human gastric epithelial cells in vitro. Med Microbiol Immunol 1991;35:197-202. 37. Telford JL, Ghiara P, Dell'Orco M, et al. Gene struc- ture of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. J Exp Med 1994;179:1653-8. 38. Tummuru MK, Cover TL, Blaser MJ. Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect Immun 1993;61:1799-809. 39. Figura N, Gugliemetti P, Rossolini, et al. Cytotoxin production by Campylobacter pylori strains isolated from patients with peptic ulcers and from patients with chronic gastritis only. J Clin Microbiol 1989;27:225-6. 40. Cover TL, Dooley CP, Blaser MJ. Characterization of and human serologic response to proteins in Helico- bacter pylori broth culture supernatants with vacuolizing cytotoxin activity. Infect Immun 1990;58:603-10. 41. Cover TL, Blaser MJ. Purification and charac- terization of the vacuolating toxin from Helicobacter pylori. J Biol Chem 1992;267:10570-5. 42. Covacci A, Censini S, Bugnoli M, et al. Molecular characterization of the 128-kda immunodominant an- tigen of Helicobacter pylori associated with cytotoxic- ity and duodenal ulcer. Proc Natl Acad Sci USA 1993;90:5791-5. 43. Cover TL, Glupczynski Y, Lage AP, et al. Serologic detection of infection with cagA+ Helicobacter pylori strains.

i S, Bugnoli M, et al. Molecular characterization of the 128-kda immunodominant an- tigen of Helicobacter pylori associated with cytotoxic- ity and duodenal ulcer. Proc Natl Acad Sci USA 1993;90:5791-5. 43. Cover TL, Glupczynski Y, Lage AP, et al. Serologic detection of infection with cagA+ Helicobacter pylori strains. J Clin Microbiol 1995;33:1496-500. 44. Tummuru MK, Cover T, Blaser M. Mutation of the cytotoxin-associated cagA gene does not affect the vacuolating cytotoxin activity of Helicobacter pylori. Infect Immun 1993;62:2609-13. 45. Akopyants NS, Kersulyte D, Berg DE. cagII, a new multigenic locus only present in the most virulent Helicobacter pylori strains. Abstracts of the 95th Gen- eral Meeting of the American Society for Microbiology. Washington, DC, 1995:181, Abstract B-90. 46. Akopyanz N, Bukanov NO, Westblom TU, Kresovich S, Berg DE. DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based rapid fin- gerprinting. Nucleic Acid Res 1992;20:5137-42. 47. Akopyanz N, Bukanov NO, Westblom TU, Berg DE. PCR-based RFLP analysis of DNA sequence diversity in the gastric pathogen Helicobacter pylori. Nucleic Acids Res 1992;20:6221-5. 48. Tarkkanen J, Kosunen TU, Saksela E. Contact of lymphocytes with Helicobacter pylori augments natu- ral killer cell activity and induces production of inter- feron. Infect Immun 1993;61:3012-6. 49. Edwards PD, Carrick J, Turner J, Lee A, Mitchell H, Cooper DA. Helicobacter pylori-associated gastritis is rare in AIDS: antibiotic effect or a consequence of immunodeficiency? Am J Gastroenterol 1991;86: 1761-4. 50. Steephen A, Raijman I, Schwarz P, et al. The spectrum of gastritis in Zambian patients with the acquired immunodeficiency syndrome. Gastroenterology 1995;108:A921. 51. Meiselman MS, Miller-Catchpole R, Christ M, Ran- dall E. Campylobacter pylori gastritis in the acquired immunodeficiency syndrome. Gastroenterology 1988;95:209-12. 52. Chiba N, Rao BV, Rademaker JW, Hunt RH. Meta- analysis of the efficacy of antibiotic therapy in eradi- cating Helicobacter pylori. Am J Gastroenterol 1992;87:1716-27. 53. Logan RPH, Gummett PA, Schaufelberger HD, et al. Eradication of Helicobacter pylori with clarythromy- cin and omeprazole. Gut 1994;35:323-6. 54. Graham DY, Opekun AR, Klein PD. Clarythromycin for the eradication of H. pylori. J Clin Gastroenterol 1993;16:292-4. 55. Kimura K, Ido K, Saifuku K, et al. A 1-h topical therapy for the treatment of Helicobacter pylori infec- tion. Am J Gastroenterol 1995;90:60-3. 56.

ythromy- cin and omeprazole. Gut 1994;35:323-6. 54. Graham DY, Opekun AR, Klein PD. Clarythromycin for the eradication of H. pylori. J Clin Gastroenterol 1993;16:292-4. 55. Kimura K, Ido K, Saifuku K, et al. A 1-h topical therapy for the treatment of Helicobacter pylori infec- tion. Am J Gastroenterol 1995;90:60-3. 56. Michetti P, Corthesy-Theulaz I, Davin C, et al. Immu- nization of BALB/c mice against Helicobacter felis infection with Helicobacter pylori urease. Gastroen- terology 1994;107:1002-11. 57. Marchetti M, Arico B, Burroni D, Figura N, Rappuoli R, Ghiara P. Development of a mouse model of Heli- cobacter pylori infection that mimics human disease. Science 1995;267:1655-8. Synopses Emerging Infectious Diseases 84 Vol. 1, No. 3 -- July-September 1995 58. Krakowka S, Morgan DR, Kraft WG, Leunk RD. Establishment of gastric Campylobacter pylori infec- tion in the neonatal gnotobiotic piglet. Infect Immun 1987;55:2789-96. 59. Dubois A, Fiala N, Heman-Ackah LM, et al. Natural gastric infection with Helicobacter pylori in monkeys. A model for human infection with spiral bacteria. Gastroenterology 1994;106:1405-17. Synopses Vol. 1, No. 3 -- July-September 1995 85 Emerging Infectious Diseases

HIV-1 Patients May Harbor Viruses of Different Phylogenetic Subtypes: Implications for the Evolution of the HIV/AIDS Pandemic The virus variants isolated from HIV-infected persons worldwide share remarkable diversity, es- pecially in the envelope glycoprotein, gp120. Phylo- genetic studies have clustered HIV-1 isolates into eight subtypes (A-H). Nevertheless, even within a single infected person, HIV is present as a "quasi- species," or a swarm of closely related variants. This genetic diversity, which in the case of HIV-1 accumu- lates at a rate of approximately one nucleotide sub- stitution per genome per replication cycle, gives the virus an enormous flexibility to respond to a wide array of in vivo selection pressures. As a conse- quence, drug-resistant and immunologic escape mu- tants are rapidly generated in infected persons through all stages of infection. On a global scale, the HIV pandemic is recognized as consisting of many separate epidemics, each with characteristic geog- raphy, affected populations, and predominant viral strain type. With an estimated 15 million infected persons, the geographic distribution of viral sub- types is becoming more dispersed, and these demar- cations are further confounded by growing evidence of mixed infections. The epidemic emergence of mixed heterotypic infections with HIV-1 and HIV-2 variants has been recognized for some time in the geographic areas where both types of viruses are present. We reported these infections in Cote d'Ivoire and Brazil (1, 2); they have also been reported from India (3). In contrast, homotypic mixed infections of distinct HIV-1 variants have only recently been suggested by the presence of broadly reactive sera and evidence of HIV recombinants from geographic regions in which multiple HIV-1 subtypes are circulating. Dual HIV-1 infection in two patients from Thailand has been demonstrated by viral DNA sequence analysis (4). As the HIV-1 pandemic has grown, the simulta- neous presence of multiple subtypes in a region has become common. As a consequence, an increased frequency of HIV-1 mixed infections could be ex- pected.

rculating. Dual HIV-1 infection in two patients from Thailand has been demonstrated by viral DNA sequence analysis (4). As the HIV-1 pandemic has grown, the simulta- neous presence of multiple subtypes in a region has become common. As a consequence, an increased frequency of HIV-1 mixed infections could be ex- pected. Thus, there is a need to estimate the preva- lence and geographic distribution of this type of infection. Sequence analysis of HIV proviral DNA has been the method of choice to characterize HIV genetic diversity. However, because even relatively limited sequence determinations of small poly- merase chain reaction (PCR) fragments are time consuming and very labor-intensive, this method is not particularly practical for large-scale molecular epidemiologic studies. To address this problem, we have developed a genetic method based on restric- tion site polymorphism to screen for homotypic HIV- 1 mixed infections within infected populations. The concept of this assay is based on the observed corre- lation between the restriction maps of HIV-1 isolates with their phylogenetic classification, which is based on the sequence data. Thus, certain restric- tion enzymes may be used to predict the phylogroup of HIV-1 infected samples. The differences in electro- phoretic mobility of endonuclease digestion prod- ucts result from restriction site polymorphisms in the selected region of the HIV-1 genome and allow for quick recognition of the distinct phylogenetic subtypes. A297 bp pol fragment spanning the entire viral protease gene is used for our analysis. The viral gene is amplified by nested PCR using DNA tem- plates from uncultured peripheral blood mononu- clear cells (PBMC) or virus culture. Preliminary classification of HIV-1 strains to well defined sub- types A, B, C, D, and F is done by sequential endo- nuclease restriction analysis. AluI restriction poly- morphism in a PCR-amplified protease gene segre- gates viral strains into two groups: subtypes B and D belong to one group, and subtypes A, C, and F to another (Figure 1A). Further differentiation of HIV- 1 subtypes within those two groups is accomplished by analysis of HinfI, BclI, MaeI, SpeI, and ScaI restriction enzyme digestion patterns of the pro- tease gene (Pieniazek et al., manuscript in prepara- tion). The electrophoretic migration patterns visualized by ethidium bromide staining or by radi- olabeled probes are then determined on a 10% acry- lamide gel.

plished by analysis of HinfI, BclI, MaeI, SpeI, and ScaI restriction enzyme digestion patterns of the pro- tease gene (Pieniazek et al., manuscript in prepara- tion). The electrophoretic migration patterns visualized by ethidium bromide staining or by radi- olabeled probes are then determined on a 10% acry- lamide gel. In single infections, a single restriction pattern is detected, whereas in multiple infections involving HIV-1 strains of distinct subtypes, com- plex digestion patterns are observed in infected per- sons. As an example, in Figure 1A, we present three distinct AluI restriction patterns of the protease gene that are characteristic for single infections by viruses of subtypes A, C, and F (pattern #1) and by subtypes B and D (patterns #2 and #3). In Figure 1B, we show a typical combination of two distinct AluI restriction patterns (#1 and #2) found in a patient infected with two viral strains of subtypes F and B. Basing our analysis on the conserved protease gene region, we should detect most HIV-1 strains; how- ever, some highly divergent isolates could escape PCR amplification as a result of primer mismatches. Moreover, since a single nucleotide substitution could either generate or destroy a restriction site, sequence analysis remains the ultimate tool in iden- tifying variants of multiple infections. Nevertheless, Dispatches Emerging Infectious Diseases 86 Vol. 1, No. 3 -- July-September 1995 this assay can be conveniently applied to screen a large number of samples. By using this method, we have screened HIV-1 proviral DNA from 208 specimens collected from countries in South America, Africa, and Asia where HIV-1 strains of distinct subtypes are found. We observed the simultaneous presence of two distinct digestion patterns in PCR amplified protease gene (Figure 1B) in 31 samples; our observation suggests superinfection with HIV-1 strains of distinct origin. To eliminate the potential for laboratory cross- contamination, we analyzed the restriction patterns of the protease gene from multiple aliquots of the patient's PBMC. In addition, the analysis was re- peated on DNAfrom a second collected blood sample from each of the patients. The analyses for the first five of 31 patients were completed, and data are summarized here (details are in Janini et al., manu- script in preparation).

tease gene from multiple aliquots of the patient's PBMC. In addition, the analysis was re- peated on DNAfrom a second collected blood sample from each of the patients. The analyses for the first five of 31 patients were completed, and data are summarized here (details are in Janini et al., manu- script in preparation). Sequence and phylogenetic analysis of the viral protease gene (Figure 2) in PBMC from those five patients confirmed dual in- fections caused by HIV-1 strains of subtypes B and F in one person (Br5), subtypes F and D in another patient (Br22), and subtypes C and D in a married couple (Br19 and 20). Moreover, in the child (Br30) of this couple, two distinct AluI digestion patterns were also found; the major HIV-1 strain clustered among subtype C viruses of the parents. The minor strain of this child is likely to represent subtype D, but there was not sufficient material for cloning and further sequencing of this strain. Detection of naturally occurring heterotypic and homotypic multiple infections may have important implications for immunotherapies because infection with one HIV subtype may not fully protect against subsequent superinfections with distinct HIV strains. However, we do not know if the acquisition of viruses in the dually infected adult patients was sequential or simultaneous. Nevertheless, the con- sequences of mixed infections may profoundly affect the ability of the virus to change and may modify the direction of the pandemic through altered patterns of viral pathogenesis, increased genetic variation through recombination, and the generation of Figure 1. A. Three distinct AluI digestion patterns of PCR amplified protease gene representing single HIV-1 infections by viral strains of subtypes A, C, and F (pattern #1), and subtypes B and D (patterns #2 and #3). B. The presence of two distinct AluI digestion patterns (#1 and #2) of the protease gene in PBMC of the patient dually infected by viral strains of subtypes F and B (lane 3). Arrows indicate diagnostic fragments detected by hybridization with the radioactive probe (2). MW represents molecular weight markers--X174 RF DNA, HaeIII digest. Figure 2. Phylogenetic classification of HIV-1 strains in dually infected patients. HIV-1 sequences from dual infections (Br5, 19, 20 and 22 ) are indicated by arrows, and the major strain in the infected child (Br30) is boxed.

e radioactive probe (2). MW represents molecular weight markers--X174 RF DNA, HaeIII digest. Figure 2. Phylogenetic classification of HIV-1 strains in dually infected patients. HIV-1 sequences from dual infections (Br5, 19, 20 and 22 ) are indicated by arrows, and the major strain in the infected child (Br30) is boxed. The tree was constructed on the basis of the DNAsequences of the protease gene by using the maximum likelihood method with the fastDNAml program (6). SIV-cpz protease sequence was used as an outgroup. The distinct HIV-1 subtypes are delineated. The scale bar shows the ratio of nucleotide substitutions for given horizontal branch length. Vertical distances are for clarity only. Dispatches Vol. 1, No. 3 -- July-September 1995 87 Emerging Infectious Diseases pseudotype virions, including phenotypically mixed virus particles. It is to be anticipated that such events would ultimately broaden the cellular tropism for HIV and mandate the designed polyva- lent immunotherapies. Finally, our data together with recently published genetic analysis for HIV-1 and HIV-2 (5) suggest that multiple homotypic infec- tions with divergent HIV strains may be more com- mon than previously thought. The screening assay described here will be useful in estimating inci- dences of such HIV-1 infections. We believe that this information is crucial for both evaluating the pan- demic and developing intervention strategies. Danuta Pieniazek,* Luiz M. Janini,* Artur Ramos,* Amilcar Tanuri, Mauro Schechter, Jose M. Peralta, Anna C.P. Vicente, Norman J. Pieniazek,* Gerald Schochetman,* and Mark A. Rayfield* *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA;

ng intervention strategies. Danuta Pieniazek,* Luiz M. Janini,* Artur Ramos,* Amilcar Tanuri, Mauro Schechter, Jose M. Peralta, Anna C.P. Vicente, Norman J. Pieniazek,* Gerald Schochetman,* and Mark A. Rayfield* *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA; Universidade Federal do Rio de Janeiro, and Hospital Clementino Fraga Filho, and Instituto Oswaldo Cruz, Rio de Janeiro, Brazil References 1. Rayfield M, De Cock K, Heyward W, et al. Mixed human immunodeficiency virus (HIV) infection in an individual: demonstration of both type 1 and type 2 proviral sequences by using polymerase chain reac- tion. J Infect Dis 1988:158:1170-6. 2. Pieniazek D, Peralta JM, Ferreira JA, et al. Identifica- tion of mixed HIV-1/HIV-2 infections in Brazil by po- lymerase chain reaction. AIDS 1991;5:1293-9. 3. Grez M, Dietrich U, Balfe P, et al. Genetic analysis of Human Immunodeficiency Virus type 1 and 2 (HIV-1 and HIV-2) mixed infections in India reveals a recent spread of HIV-1 and HIV-2 from a single ancestor for each of these viruses. J Virol 1994;68:2161-8. 4. Artenstein AW, VanCott TC, Mascola JR, et al. Dual infection with HIV type 1 of distinct envelope subtypes in humans. J Infect Dis 1995;171:805-10. 5. Gao F, Yue L, Robertson DL, et al. Genetic diversity of human immunodeficiency virus type 2: evidence for distinct sequence subtypes with differences in virus biology. J Virol 1994;68:7433-47. 6. Larsen N, Olsen GJ, Maidak BN, el al. The ribosomal database project. Nucleic Acids Res. 1993;21:3021-3. Dispatches Emerging Infectious Diseases 88 Vol. 1, No. 3 -- July-September 1995

Simultaneous Infection of Ixodes ricinus Nymphs by Two Borrelia burgdorferi Sensu Lato Species: Possible Implications for Clinical Manifestations Data from European studies indicate that in hu- mans, particular Borrelia burgdorferi genospecies may be associated with specific clinical manifesta- tions of Lyme disease. Infections by B. burgdorferi sensu stricto tend to lead to arthritic symptoms, whereas infections by B. garinii appear to cause neurologic complications. Late cutaneous manifes- tations (acrodermatitis) appear to be associated with B. afzelii (1). Mixed clinical manifestations have also been described (2). Recently it has been demonstrated, by using polymerase chain reaction (PCR), that DNA from more than one of the three Borrelia species associated with Lyme disease in Europe was present in the biological fluids of Lyme disease patients (3). These data raise questions con- cerning the relative growth of the Borrelia species after a bite by a dually infected tick, the clinical significance of human infection caused by more than one species of Borrelia, and the origin of these mul- tiple infections. This last point evokes the following question: do they result from successive bites by two infected ticks or from a single bite by a tick infected by more than one species? To investigate whether ticks are infected by dif- ferent species of the B. burgdorferi complex at the same time, we carried out a survey of the vector Ixodes ricinus during the spring of 1994, in Ram- bouillet Forest near Paris. A total of 249 unfed nymphs, collected from vegetation, were analyzed by PCR. The ticks were then crushed in phosphate- buffered saline, solubilized in 0.5% Tween 20, and boiled for 10 min. The resulting lysate was used as a template for the amplification reactions by either the universal ospA-based primers SL or the three pairs of genospecific-based primers (3). These last primers distinguish the three Lyme disease-associ- ated B. burgdorferi sensu lato species, i.e., B. burgdorferi sensu stricto, B. garinii, and B. afzelii. In some cases, amplified DNA products were di- gested with specific restriction enzymes to confirm the typing of the Borrelia strain.

c-based primers (3). These last primers distinguish the three Lyme disease-associ- ated B. burgdorferi sensu lato species, i.e., B. burgdorferi sensu stricto, B. garinii, and B. afzelii. In some cases, amplified DNA products were di- gested with specific restriction enzymes to confirm the typing of the Borrelia strain. Thirty of the 249 nymphs were positive for B. burgdorferi when SL universal primers were used. Further testing of 5 of 30 nymphs by PCR, using genospecific primer sets and restriction analysis, did not confirm the preliminary results with the universal primers. This may have been due either to the genotypic variability of B. burgdorferi sensu lato or to the existence of other distinct subgroups or genomic species included in B. burgdorferi sensu lato, as other data appear to indicate (4). Of the 25 other nymphs, 22 were analyzed by both restriction analyses and the specific primers, and three by restriction analysis alone. (The available tick mate- rial was not sufficient to perform PCR with genospe- cific primers.) Nineteen nymphs were infected by a single species of Borrelia (four by B. garinii, 15 by B. afzelii), and six were infected by more than one (two by both B. burgdorferi sensu stricto and B. garinii, three by B. garinii and B. afzelii, one by B. burgdorferi sensu stricto and B. afzelii). From these results, it appears that when nymphs are infected with one species, B. afzelii is the most prevalent. This species may actually be prevalent in this study area or may have a greater tropism for dermal tissue and/or for the peripheral circulatory system of the vertebrate than the other two species. In infected nymphs, the simultaneous presence of more than one genospecies in unfed nymphs of I. ricinus was not exceptional (24%), and all combina- tions of two species were observed. The association of three genospecies has not yet been detected. Si- multaneous infections in unfed nymphs could have different explanations. The first is a larval meal on a host infected by more than one species. Recently, Apodemus speciosus (field mice) infected by two dif- ferent species have been found (5). A second possi- bility is successive infectious interrupted larval meals. Athird possibility is an infectious larval meal by a previously transovarially infected larva. The fourth possibility is a mixed infection acquired transovarially.

us speciosus (field mice) infected by two dif- ferent species have been found (5). A second possi- bility is successive infectious interrupted larval meals. Athird possibility is an infectious larval meal by a previously transovarially infected larva. The fourth possibility is a mixed infection acquired transovarially. Bruno Pichon,* Edmond Godfroid, Bernard Hoyois, Alex Bollen, Francois Rodhain,* and Claudine Perez-Eid* * Unite d'Ecologie des Systemes Vectoriels, Institut Pasteur 75724 Paris, France

us speciosus (field mice) infected by two dif- ferent species have been found (5). A second possi- bility is successive infectious interrupted larval meals. Athird possibility is an infectious larval meal by a previously transovarially infected larva. The fourth possibility is a mixed infection acquired transovarially. Bruno Pichon,* Edmond Godfroid, Bernard Hoyois, Alex Bollen, Francois Rodhain,* and Claudine Perez-Eid* * Unite d'Ecologie des Systemes Vectoriels, Institut Pasteur 75724 Paris, France Laboratoire de Genetique Applique, Universite Libre de Bruxelles, Rue de l'Industrie 24, B-1400 Nivelles, Belgium This work was supported by grants from the Recherche & Partage Association, the Gould Foundation, and the Conseil du Departement du Val d'Oise. E. Godfroid, B. Hoyois, and A. Bollen, were supported by a grant from the Walloon Region of Belgium (Convention UIB, Region Wal- lonne No. 2267). References 1. Assous MV, Postic D, Paul G, Nevot P, Baranton G. Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Borre- lia strain used as antigens. Eur J Clin Infect Dis 1993;12:261-8. Dispatches Vol. 1, No. 3 -- July-September 1995 89 Emerging Infectious Diseases 2. Wienecke R, Neubert U, Volkenandt M. Cross-immu- nity among types of Borrelia burgdorferi. Lancet 1993;341:830-1. 3. Demaerschalck I, Ben Messaoud A, De Kesel M, Hoyois B, Lobet Y, Hoet P, et al. Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients. J Clin Microbiol 1995;33:602-8. 4. Nohlmans LMKE, De Boer R, Van Den Boggard AEJM, Van Boven CPA. Genotypic and phenotypic analysis of Borrelia burgdorferi isolates from the Netherlands. J Clin Microbiol 1995;33:119-25. 5. Nakao M, Miyamoto K. Mixed infection of different Borrelia species among Apodemus speciosus mice in Hokkaido, Japan. J Clin Microbiol 1995;33:490-2. Dispatches Emerging Infectious Diseases 90 Vol. 1, No. 3 -- July-September 1995

Epidemiologic and Evolutionary Relationships between Romanian and Brazilian HIV-1 Subtype F Strains The initial classification of HIV-1 viruses as Western or African strains has been replaced by phylogenetic subtyping that uses nucleotide se- quence data. Eight distinct phylogenetic HIV-1 line- ages or subtypes, A to H, have been defined (1). Considering that the rate of HIV-1 genome evolution is estimated at 0.5% to 1% per year and that the average genetic distance between the HIV-1 sub- types is approximately 20%, it is likely that these subtypes originated before the HIV-1 pandemic (1). The global mosaic of HIV-1 subtypes is consistent with the hypothesis that most regional epidemics started with the introduction of one or a few variants that diversified locally rather than through radiant waves of already diversified HIV-1 subtypes that spread from the place of origin. In this report, we address the evolutionary and epidemiologic rela- tionships between the HIV-1 subtype F viruses re- cently identified in two geographically distinct regions, Romania and Brazil. The HIV-1 epidemic among Romanian children living in orphanages was recognized during 1989- 1990 (2). Epidemiologic studies have shown that most children became infected by horizontal trans- mission of HIV-1 through blood transfusions or through the use of unsterilized medical equipment. We have shown by nucleotide sequence analysis that all HIV-1 isolates from children in southeastern Romania are highly related genetically (3). The av- erage interperson nucleotide distance within the C2-V3 region of the env gene was 0.9% to 3.6%. Phylogenetic analysis of these sequences showed that the Romanian HIV-1 strains clustered together with a single Brazilian HIV-1 strain in a previously unrecognized evolutionary clade later designated as the F subtype. We now have the opportunity to augment this comparison with additional F subtype sequences from the two countries and to further address the relationship between Romanian and Brazilian viruses.

gether with a single Brazilian HIV-1 strain in a previously unrecognized evolutionary clade later designated as the F subtype. We now have the opportunity to augment this comparison with additional F subtype sequences from the two countries and to further address the relationship between Romanian and Brazilian viruses. In the phylogenetic analysis of the envelope C2- V3 nucleotide sequences (Figure 1), we included eight HIV-1 F subtype viruses isolated from children in southeastern Romania (L19570-L119579) (3) as well as two representative sequences of the HIV-1 viruses found in children living in northcentral Ro- mania (R18586 and R18598) (Bandea et al., manu- script in preparation). From Brazil, we included, in addition to the sequence of the first identified F subtype strain (BRA7944)(4), three recently re- ported F sequences (BZ126A, BZ162A, and BZ163A) (5) and four F strains (BR46, BR57, BR58, and BR59) isolated in our laboratory from patients in Rio de Janeiro. We also included two HIV-1 strains from Cameroon, CA4 and CA20, that have been tenta- tively classified as F subtype viruses (1,6) and refer- ence nucleotide sequences representing the other HIV-1 subtypes. The results of this phylogenetic analysis show that the Brazilian and Romanian sequences cluster in two highly related but separate groups. The ge- netic distance between Brazilian subtype F se- quences was 5% to 13.8% , which is within the limits of the established intrasubtype distance values (1). Among Romanian C2-V3 nucleotide sequences this distance was 0.9% to 6.5%, and between the two groups it was 7.5% to 12.9%. These values support the inclusion of the Romanian and Brazilian groups within the same F HIV-1 subtype. The reliability of these phylogenetic results was verified by bootstrap analysis (100 data sets) and by pruning, which con- sists of sequential removing of different strains and rerunning the analysis. The two sequences from Cameroon associated only weakly with Romanian and Brazilian groups, and this association was not stable. The genetic distance between the Romanian and Brazilian se- quences and sequences from Cameroon was 16.5% to 24.1%, which is typical of intersubtype rather than intrasubtype genetic distances.

The two sequences from Cameroon associated only weakly with Romanian and Brazilian groups, and this association was not stable. The genetic distance between the Romanian and Brazilian se- quences and sequences from Cameroon was 16.5% to 24.1%, which is typical of intersubtype rather than intrasubtype genetic distances. Our analysis does not support a strong linkage between the Cameroonian strains and the other F subtype vi- ruses; however, in evolutionary terms, these viruses may be closer to each other than to the other sub- types, or they may have undergone convergent evo- lution within the envelope region that we analyzed. Analysis of additional sequences from other regions of the HIV-1 genome may clarify the relationship between the Cameroonian strains and the other subtypes. The amino acid GPGR motif at the tip of the V3 protein loop has been considered a signature sequence for the Brazilian F subtype viruses (5). This relatively conserved motif was noticeable because it is charac- teristic among B subtype viruses, whereas the GPGQ sequence predominates among all the other HIV-1 strains, including all the initial Romanian F viruses. Two of the newly identified Brazilian strains (BR58 and BR59), however, contain the GPGQ motif, and the Romanian strain R18598 contains the GPGH motif (Figure 2). Phylogenetically, these sequences group with their respective geographic clusters (Figure 1), whichindicatesthatindependentmutationsmayhave occurred at this locus. As indicated earlier, the genetic distance between Romanian HIV-1 nucleotide sequences is very small, which suggests a direct epidemiologic link among Dispatches Vol. 1, No. 3 -- July-September 1995 91 Emerging Infectious Diseases these strains and a short period of evolution. The exclusive presence of highly related viruses in two geographically distinct provinces of Romania strongly suggests that the initial pediatric HIV-1 epidemic in this country started from a single infec- tious source. This suggestion is also supported by the fact that F subtype viruses are uncommon and, therefore, the chances for independent multiple in- troduction of highly related F subtype strains in Romanian children are very low.

hat the initial pediatric HIV-1 epidemic in this country started from a single infec- tious source. This suggestion is also supported by the fact that F subtype viruses are uncommon and, therefore, the chances for independent multiple in- troduction of highly related F subtype strains in Romanian children are very low. Although no nu- cleotide sequence data are available about HIV-1 strains circulating in the adult population in Roma- nia, the epidemiologic and serologic studies indicate that the number of infections is small, and probably most HIV-1-infected adults were infected though sexual contact with foreign visitors or with Romani- ans that traveled outside the country (2). It is ex- pected, therefore, that most of the HIV-1 strains infecting the adults represent internationally prevalent subtypes. It is remotely possible, however, that adults were infected with F subtype viruses and could have served as the original or intermediary carriers for HIV-1 transmission among the groups of children living in distinct geographic regions of Ro- mania. In Brazil, the relatively long genetic distance between F subtype viruses could indicate that a single ancestor was introduced during the early phases of the HIV-1 epidemic and diverged locally, or that multiple different F strains were introduced to this country. The low prevalence of F subtype viruses worldwide makes the latter alternative less likely. However, no information is available about the HIV-1 strains present during the early phases of the epidemic in Brazil. Our ongoing studies and the published data (4,5) indicate that the HIV-1 F sub- type infections represent roughly 10% of the esti- mated number of cases. This relatively large number of F subtype infections and the estimated rate of HIV-1 divergence suggest an early introduc- tion of the F subtype viruses in Brazil. Because of the geographic position and the spar- sity of socioeconomic relations between Romania and Brazil, the potential for an epidemiologic link between HIV-1 F subtype viruses is small. A more compelling argument against a direct epidemiologic link between F subtype viruses from these two coun- tries can be made on the basis of the topology of the phylogenetic branches that set apart the two groups of viruses (Figure 1). If a direct epidemiologic link existed between the two groups, the clustering would be integral with one group branching from within the other.

between F subtype viruses from these two coun- tries can be made on the basis of the topology of the phylogenetic branches that set apart the two groups of viruses (Figure 1). If a direct epidemiologic link existed between the two groups, the clustering would be integral with one group branching from within the other. The tree topology shows the two groups of viruses on separate branches with a rela- tively distant common ancestor. Although limited in scope, our findings support an evolutionary relation- ship between the Romanian and Brazilian F subtype Figure 1. Phylogenetic relationship between Romanian and Brazilian subtype F nucleotide sequences. The tree was constructed by using the neighbor joining method included in the Phylip 3.5c package (7). Three hundred and two aligned nucleotides from the envelope C2-V3 region were used for analysis. The vertical distance between the branches is noninformative and for clarity only. Numbers at the branch nodes indicate bootstrap values. The nucleotide sequence distance among strains can be deduced by using the bar scale included in the figure. Dispatches Emerging Infectious Diseases 92 Vol. 1, No. 3 -- July-September 1995 viruses and indicate that the two regional epidemics arose independently. Claudiu I. Bandea,* Artur Ramos,* Danuta Pieniazek,* Rodica Pascu, Amilcar Tanuri, Gerald Schochetman,* and Mark A. Rayfield* *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA;

tember 1995 viruses and indicate that the two regional epidemics arose independently. Claudiu I. Bandea,* Artur Ramos,* Danuta Pieniazek,* Rodica Pascu, Amilcar Tanuri, Gerald Schochetman,* and Mark A. Rayfield* *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA; Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; University of Medicine and Pharmacy, Tg-Mures, Romania References 1. Myers G, Korber B, Wain-Hobson S, Smith R, Pavlakis G, eds. Human retroviruses and AIDS 1994: a compi- lation and analysis of nucleic acid and amino acid sequences. Los Alamos, NM: Los Alamos National Laboratory, 1993. 2. Hersh BS, Popovici F, Apetrei RC, et al. Acquired immunodeficiency syndrome in Romania. Lancet 1991;338:654-9. 3. Dumitrescu D, Kalish ML, Klicks SC, Bandea CI, Levy JA. Characterization of human immunodeficiency vi- rus type 1 isolates from children in Romania: identifi- cation of a new envelope subtype. J Infect Dis 1994;169:281-8. 4. Potts KE, Kalish ML, Lott T, et al. Genetic heteroge- neity of the V3 region of the HIV-1 envelope glycopro- tein in Brazil. AIDS 1993; 7:1191-7. 5. Louwagie J, Delwart EL, Mullins JI, McCutchan FE, Eddy G, Burke DS. Genetic analysis of HIV-1 isolates from Brazil reveals presence of two distinct genetic subtypes. AIDS Res Hum Retroviruses 1994;10:561-7. 6. Nkengasong JN, Janssens W, Heyndrickx L, et al. Genotypic subtypes of HIV-1 in Cameroon. AIDS 1994;8:1405-12. 7. Felsenstein J. PHYLIP-phylogeny interference pack- age (version 3.2). Cladistics 1989;5:164-6. Figure 2. Alignment of deduced amino acid sequences for envelope C2-V3 region of Brazilian and two representative Romanian HIV-1 F subtype strains and their comparison with Cameroonian sequences and the consensus sequences for some of the other subtypes. F CON represents consensus amino acid sequence (single letter code) for the Romanian and Brazilian F subtype HIV-1 viruses presented in this figure. Consensus sequences for the other subtypes are from Ref. 1. Amino acids identical to the F CON are shown as a dash, and the dots represent gaps introduced to align sequences. The top bar shows the peptide motif at the tip of the V3 protein loop. Dispatches Vol. 1, No. 3 -- July-September 1995 93 Emerging Infectious Diseases

Electronic Communication Facilitates Investigation of a Highly Dispersed Foodborne Outbreak: Salmonella on the Superhighway Widely dispersed foodborne disease outbreaks are an emerging public health problem (1-3). In- creasing population mobility and the wide distribu- tion of centrally produced foods mean that when an outbreak of foodborne disease occurs, the affected persons may be distributed across the country or even the world. Widely dispersed outbreaks chal- lenge limited public health resources; they can be difficult to detect, labor-intensive, and time- con- suming to investigate. We report the rapid, efficient investigation of a widely dispersed interstate out- break through electronic communication between the possible patients and public health workers. On August 4, 1994, a resident of a western state contacted the Centers for Disease Control and Pre- vention (CDC) regarding a possible outbreak of food- borne illness. On July 22, the day after returning from a conference in Baltimore that included atten- dees from all 50 states, he became ill with diarrhea, and Salmonella was isolated from his stool. He con- tacted four other conference attendees who had taken the same flight. One of these also had culture- confirmed Salmonella infection; a second, who was taking antibiotics for other reasons, had a diarrheal illness with a negative stool culture, and two had nonspecific diarrheal illnesses. Because of the pos- sibility of a multistate outbreak involving the airline or the conference and affecting many people, we initiated a survey of conference attendees to deter- mine the rate and correlates of diarrheal illness. Traditionally, surveys of dispersed populations have been conducted either by telephone, requiring many person-hours of interviewing, or by mail, lead- ing to many days' delay while questionnaires are distributed and returned. However, in this case, the organization that sponsored the conference had an internal electronic mail (e-mail) system. Each sec- tion of the organization, although not each person, had a computer that could receive e-mail messages.

y mail, lead- ing to many days' delay while questionnaires are distributed and returned. However, in this case, the organization that sponsored the conference had an internal electronic mail (e-mail) system. Each sec- tion of the organization, although not each person, had a computer that could receive e-mail messages. On August 5, 1994, the organization's central office e-mailed a questionnaire, developed in consultation with CDC, to all computers in the organization with instructions to organization staff to print and dis- tribute the questionnaire to conference attendees. The questionnaire contained items regarding diar- rheal illness, meal and flight exposures, and demo- graphic information. Because the organization's e-mail system was only internal, responses could not be made to CDC by e-mail. Therefore, attendees were instructed to send their completed question- naires to CDC by fax. By August 12 (7 days later), sufficient responses had been received to evaluate the flight and the conference as sources of an out- break of salmonellosis (Figure 1). Of 390 persons registered at the conference, 86 (22%) returned questionnaires by August 12. Aques- tionnaire was returned by the index patient who made the initial call to CDC but not by the four other persons he contacted who were passengers on the same flight. Six (7%) of the 86 respondents reported having diarrhea (three or more loose stools in a 24-hour period) during the period beginning 12 hours after the conference started and ending 5 days after the conference ended (July 20 to 26). Among questionnaire respondents, only the index patient was diagnosed with salmonellosis. Three respon- dents had taken the initially suspect flight. Illness was not associated with taking the same flight as the index patient (p = 0.20, Fisher's Exact Test, 2- tailed). To further investigate the reports of diarrhea, we interviewed the six persons who reported diarrheal illness by questionnaire, as well as the four persons initially contacted by the index patient who had not completed questionnaires. This group included the two persons with known Salmonella infection, of whom one had completed a questionnaire and one had not. Seven of the other eight persons had mild, nonspecific symptoms of less than 2 days' duration; the onset dates of their illnesses spanned a 5-day period, and none sought medical attention.

es. This group included the two persons with known Salmonella infection, of whom one had completed a questionnaire and one had not. Seven of the other eight persons had mild, nonspecific symptoms of less than 2 days' duration; the onset dates of their illnesses spanned a 5-day period, and none sought medical attention. Because few conference attendees or flight passengers be- came ill with symptoms suggestive of salmonellosis during a likely period, we thought that an airplane- or conference-associated outbreak was improbable. Figure 1. Number of days from questionnaire distribution by e-mail to questionnaire return by fax (n=156). Day 0 is Friday, August 5, 1994. Range, 0 to 35 days; median = 6 days. Dispatches Emerging Infectious Diseases 94 Vol. 1, No. 3 -- July-September 1995 By September 9, questionnaires were returned by 156 (40%) of the conference attendees. No additional cases of diarrhea were reported, confirming our in- itial conclusion that the Salmonella infections were not associated with the flight or the conference. The Salmonella isolates were identified at CDC as Salmonella serotype Norwich, of the Salmonella serogroup C1. S. Norwich is rare; in 1993 and 1994, respectively, 63 and 102 isolates of this serotype were reported to the Public Health Laboratory In- formation System (PHLIS), a nationwide electronic laboratory-based surveillance system that collects and summarizes data on isolates from state public health laboratories (4). Because infection with S. Norwich is so uncommon, it still seemed likely that the two infections could have a common source, such as a restaurant. Subsequent investigation focused on meals that the two persons with salmonellosis shared outside the conference and ultimately revealed the source, a restaurant in Baltimore. In late July 1994, the Maryland Department of Health and Mental Hy- giene received reports that Salmonella, serogroup C1, had been isolated from five other persons who visited Baltimore around the time of the conference. Two persons from one family had driven to Balti- more on July 17, eaten only at one restaurant, then returned to their home state of Pennsylvania. Three persons in a second family, from a different part of Pennsylvania, ate at the same Baltimore restaurant on July 21 during a vacation trip. The Salmonella isolates from members of both families were initially misidentified as other serogroup C1 serotypes. They were retested because of this outbreak and were confirmed as S. Norwich.

second family, from a different part of Pennsylvania, ate at the same Baltimore restaurant on July 21 during a vacation trip. The Salmonella isolates from members of both families were initially misidentified as other serogroup C1 serotypes. They were retested because of this outbreak and were confirmed as S. Norwich. The two conference atten- dees with S. Norwich infection also ate at the impli- cated restaurant on July 21. No single menu item had been eaten by all ill persons. In response to a complaint by the first family, the restaurant had been inspected by the local health department; mul- tiple violations of food safety regulations were found. S. Norwich was isolated from a stool speci- men from an employee who reported a diarrheal illness beginning on July 22 and who ate the restau- rant's food. In the month following the inspection of Restaurant A and subsequent corrective action, no further cases of S. Norwich were reported to PHLIS from Maryland or Pennsylvania. E-mail can expedite questionnaire distribution, especially when the population of interest is on one network. The computer system used to send the e-mail message in this outbreak was not linked to individual conference attendees; therefore, we could not evaluate the rates at which individual attendees obtained and responded to the message. If we had been able to reach attendees directly, our response rate may have been higher, and we would have been able to send additional messages to nonresponders. In the future, when outbreaks occur among persons accessible by e-mail, it may be possible to evaluate strategies to improve response rate and to compare the effectiveness of the delivery of questionnaires by e-mail and by more traditional means. This outbreak illustrates the usefulness of rapid electronic communication in a public health setting. Isolation of a rare Salmonella serotype and national electronic reporting to PHLIS assisted in the detec- tion and investigation of a widely dispersed multis- tate outbreak of salmonellosis. Without the national Salmonella serotyping system, the outbreak would not have been recognized. Questionnaires were dis- tributed rapidly by e-mail; the utility of this method is likely to increase as more people become accessi- ble by e-mail. Fax provided a means for respondents to return questionnaires quickly. Continued on-line analysis of surveillance data with PHLIS confirmed that the outbreak was controlled.

uestionnaires were dis- tributed rapidly by e-mail; the utility of this method is likely to increase as more people become accessi- ble by e-mail. Fax provided a means for respondents to return questionnaires quickly. Continued on-line analysis of surveillance data with PHLIS confirmed that the outbreak was controlled. Rapid communi- cation between public health workers in Maryland and Pennsylvania and at CDC was also essential. The usefulness of electronic communication is not limited to outbreak investigation. New technologies will undoubtedly continue to be useful in addressing emerging public health problems. Barbara E. Mahon,* Dale D. Rohn, Sheila R. Pack, Robert V. Tauxe* *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA;

is not limited to outbreak investigation. New technologies will undoubtedly continue to be useful in addressing emerging public health problems. Barbara E. Mahon,* Dale D. Rohn, Sheila R. Pack, Robert V. Tauxe* *National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA; Epidemiology and Disease Control Program, Maryland Department of Health and Mental Hygiene, Baltimore, Maryland, USA; Division of Acute Communicable Diseases (now in Division of HIV Seroepidemiology), Baltimore City Health Department, Baltimore, Maryland, USA References 1. Tauxe RV. Salmonella: a postmodern pathogen. J Food Protection 1991;54:563-8. 2. Hedberg CW, MacDonald KL, Osterholm MT. Chang- ing epidemiology of food-borne disease: a Minnesota perspective. Clin Infect Dis 1994;18:671-82. 3. Hedberg CW, Levine WC, White KE, Carlson RH, Winsor DK, Cameron DN, et al. An international food- borne outbreak of shigellosis associated with a com- mercial airline. JAMA 1992;268:3208-12. 4. Bean NH, Martin SM, Bradford H. PHLIS: an elec- tronic system for reporting public health data from remote sites. Am J Public Health 1992;82:1273-6. Dispatches Vol. 1, No. 3 -- July-September 1995 95 Emerging Infectious Diseases

Reemergence of Ebola Virus in Africa Members of the family Filoviridae, which cur- rently consists of Ebola and Marburg viruses, cause severe and often fatal hemorrhagic fevers in humans and nonhuman primates. The recent isolation and identification of a new Ebola virus from a single nonfatal human case in Cote d'Ivoire (1) and the more recent outbreak of Ebola hemorrhagic fever in and around Kikwit, Zaire (2, 3), have raised concerns about the public health threat of these human patho- gens. Filoviruses are classified as biosafety level 4 agents because of the extreme pathogenicity of cer- tain strains and the lack of a protective vaccine or effective antiviral drug. Moreover, filoviruses are among the most mysterious groups of viruses known because their natural history and reservoirs remain undefined and their pathogenesis is poorly under- stood. Ebola virus infections were first recognized in 1976, when simultaneous but separate outbreaks of human disease caused by two distinct virus subtypes erupted in northern Zaire and southern Sudan (4) and resulted in hundreds of deaths. The Zaire sub- type of Ebola virus had a higher case-fatality, nearly 90%, while the Sudan subtype had a case-fatality rate of approximately 50%. Before 1995, the last identified outbreak of Ebola disease in Africa oc- curred in 1979, when the Sudan subtype of Ebola virus infected 34 persons (5). In late 1989, in Reston, Virginia, a novel Ebola virus infected a colony of cynomolgus macaques that had been imported from the Philippines (6). The new virus, named Reston virus, was shown by researchers at the Centers for disease Control and Prevention (CDC) to be antigeni- cally and genetically distinct from the African Ebola viruses, yet despite its high pathogenicity for nonhu- man primates, it did not appear to cause disease in humans. Several persons who handled the infected animals developed antibody to Ebola virus but showed no signs of disease; one of these persons was infected while performing an autopsy on an animal that had died of a Reston virus infection.

gh pathogenicity for nonhu- man primates, it did not appear to cause disease in humans. Several persons who handled the infected animals developed antibody to Ebola virus but showed no signs of disease; one of these persons was infected while performing an autopsy on an animal that had died of a Reston virus infection. In 1992, a repeat of the 1989 Reston episode occurred in Siena, Italy when macaques were received from the same Philippine exporter; no evidence of a human infec- tion was found (7). The new Ebola virus recently isolated from a patient in the Cote d'Ivoire has been shown to be genetically distinct from previous Ebola isolates (A. Sanchez, unpublished data) and is the first evidence of Ebola virus in West Africa. Investigations of these outbreaks, as well as of those caused by Marburg viruses, have yet to pro- duce any substantial evidence for the natural reser- voir(s) of filoviruses. Filoviruses do not persist in experimentally infected nonhuman primates; there- fore, nonhuman primates are likely not the natural reservoir. Like humans, these species probably are infected when direct or indirect contact is made with the natural host. The recent news of a large Ebola outbreak in Kikwit, Zaire, alarmed a worldwide audience al- ready sensitized by an array of books, magazine articles, television programs, and movies dealing with the danger of Ebola virus disease. The public concern is underscored by the potential for the spread of these viruses to far regions of the world as a result of international commerce and jet travel. The Kikwit outbreak was similar to the original 1976 episode in Zaire, which was centered around the small village of Yambuku some 1000 km to the north (8). As in the 1976 outbreak, secondary transmission of the virus in Kikwit occurred through close per- sonal contact with infectious blood and other body fluids and was facilitated by the lack of modern medical facilities and medical supplies that could protect those giving care to the initially affected patients. The chief difference between the Yambuku episode and this year's outbreak is that Kikwit is a large and densely populated center close to larger cities, such as Kinshasa and Brazzaville, and the potential for communitywide transmission and spread to neighboring areas is greater. Retrospective case surveillance suggests that the index case may have been a charcoal maker that worked in the forest outside Kikwit.

large and densely populated center close to larger cities, such as Kinshasa and Brazzaville, and the potential for communitywide transmission and spread to neighboring areas is greater. Retrospective case surveillance suggests that the index case may have been a charcoal maker that worked in the forest outside Kikwit. Human to human transmission oc- curred without being recognized until the end of April 1995. Ebola hemorrhagic fever was suspected when nosocomial infections in the surgical teams and the nursing staff followed repeated laparotomies on an infected laboratory technician in Kikwit Gen- eral Hospital. Specimens were send to CDC through the Tropical Institute of Antwerpen (Belgium). Teams of experts from CDC, the World Health Or- ganization, Belgium, France, South Africa, and Swe- den traveled to the region to assist in implementing safe patient care, management, and containment of the Ebola virus outbreak. As of July 1, 1995, 233 deaths had been reported among the 293 cases. Rapid diagnosis and characterization of Ebola virus was performed at CDC in Atlanta on blood specimens from 14 patients received on May 9. Nine hours after the specimens had been delivered to CDC, Ebola virus antigen and/or antibody to this virus was confirmed in specimens from 13 of the patients. Four hours later, reverse transcriptase-po- lymerase chain reaction (RT-PCR) assays targeting conserved regions of filovirus polymerase or Ebola virus glycoprotein genes each detected Ebola virus RNAin 12 of the patients. Subsequent analysis of the genetic profile of the virus was especially important to understanding the epidemiology of the Kikwit outbreak. Within 48 hours of receiving the speci- mens, sequence analysis on the PCR DNA (528 bp) amplified from the glycoprotein gene derived from four different patients showed that the Ebola virus Commentary Emerging Infectious Diseases 96 Vol. 1, No. 3 -- July-September 1995 was a Zaire subtype that differed from the original 1976 strain in four bases (<1%). No differences were seen when the polymerase gene PCR products (~350 bp) from those four patients were sequenced, which indicated that they had been infected with the same virus. Three days later, sequence data from ex- panded analysis of the entire glycoprotein gene were compared with those of the original 1976 Yambuku isolate (9) and showed that the overall difference between these Ebola viruses was less than 1.6%.

re sequenced, which indicated that they had been infected with the same virus. Three days later, sequence data from ex- panded analysis of the entire glycoprotein gene were compared with those of the original 1976 Yambuku isolate (9) and showed that the overall difference between these Ebola viruses was less than 1.6%. Such little change in viruses that caused outbreaks of disease at extreme ends of Zaire separated by a span of nearly 19 years, may indicate that the genomes of Ebola viruses (and filoviruses in general) are unusually stable and have evolved to occupy special niches in the wild. The capability to rapidly diagnose and charac- terize filovirus infections is critical to the ability of public health professionals to identify and limit the spread of future outbreaks of filovirus disease. A continued commitment to research and modern dis- ease-surveillance programs is necessary to minimize or preclude filovirus outbreaks similar to that in Kikwit. The possibility of outbreaks is increasingly likely given the continued human incursions into the African forests and the vulnerability of large impov- erished populations to rapid transmission of disease as a result of inadequate public health services. With the current outbreak under control, CDC and col- laborators have begun their efforts to identify the natural reservoir by sending teams of scientists to collect specimens from the area where the putative index patient worked. Attempts to identify the res- ervoir after outbreaks in 1976 and 1979 were handi- capped by the lack of satisfactory diagnostic tools that are critical to detecting small quantities of the virus. However, now that sensitive enzyme immu- noassays and PCR assays have been developed for filoviruses, the chances are much better that, if ap- propriate materials can be collected in the field, the virus can be detected. In conclusion, we want to alert physicians and public health agencies who encounter persons that have clinical signs and symptoms of hemorrhagic fever disease to the reemergence of Ebola virus. Recommendations for the management of viral hem- orrhagic fevers attributable to filoviruses in the United States were recently published in CDC's Mor- bidity and Mortality Weekly Report (1995;44:475-79). Anthony Sanchez, Thomas G. Ksiazek, Pierre E. Rollin, Clarence J. Peters, Stuart T. Nichol, Ali S. Khan, and Brian W. J. Mahy National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA References 1.

ublished in CDC's Mor- bidity and Mortality Weekly Report (1995;44:475-79). Anthony Sanchez, Thomas G. Ksiazek, Pierre E. Rollin, Clarence J. Peters, Stuart T. Nichol, Ali S. Khan, and Brian W. J. Mahy National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA References 1. Le Guenno B, Formenty P, Wyers M, Gounon P, Walker F, Boesch C. Isolation and partial characterisation of a new strain of Ebola virus. Lancet 1995;345:1271-4 2. Centers for Disease Control and Prevention. Outbreak of Ebola viral hemorrhagic fever--Zaire, 1995. MMWR 1995;44:381-2. 3. Centers for Disease Control and Prevention. Update: outbreak of ebola viral hemorrhagic fever--Zaire, 1995. MMWR 1995;44:399. 4. Bowen ETW, Platt GS, Lloyd G, Baskerville A, Harris WJ, Vella EC. Viral haemorrhagic fever in southern Sudan and northern Zaire: preliminary studies on the aetiologic agent. Lancet 1977;1:571-3. 5. Baron RC, McCormick JB, Zubeir OA. Ebola virus disease in southern Sudan: hospital dissemination and intrafamilial spread. Bull WHO 1983;62:997-1003. 6. Jahrling RB, Geisbert TW, Dalgard DW, et al. Prelimi- nary report: isolation of Ebola virus from monkeys imported to USA. Lancet 1990;335:502-5. 7. World Health Organization. Viral haemorrhagic fever in imported monkeys. Wkly Epidemiol Rec 1992;67:142-3. 8. World Health Organization. Ebola haemorrhagic fever in Zaire, 1976. Bull WHO 1978;56:271-93. 9. Sanchez A, Kiley MP, Holloway BP, Auperin DD. Se- quence analysis of the Ebola virus genome: organiza- tion, genetic elements, and comparison with the genome of Marburg virus. Virus Res 1993;29:215-40. Prospects for the Control of Bolivian Hemorrhagic Fever Bolivian hemorrhagic fever (BHF) was first iden- tified in 1959 as a sporadic hemorrhagic illness in rural areas of Beni department, Bolivia. Clusters of BHF patients were noted the same year, and by 1962 BHF was recognized as a new epidemic infectious disease. In 1963, Machupo virus (a member of the family Arenaviridae) was first isolated from patients with acute hemorrhagic fever in San Joaquin, Bo- livia (1). Ecologic investigations established the ro- dent Calomys callosus, which is indigenous to the disease-endemic region of northern Bolivia, as the reservoir for Machupo virus (2,3). Machupo virus infection in C. callosus results in asymptomatic infection with shedding of virus in saliva, urine, and feces; 50% of experimentally in- fected C.

ons established the ro- dent Calomys callosus, which is indigenous to the disease-endemic region of northern Bolivia, as the reservoir for Machupo virus (2,3). Machupo virus infection in C. callosus results in asymptomatic infection with shedding of virus in saliva, urine, and feces; 50% of experimentally in- fected C. callosus are chronically viremic and shed virus in their bodily excretions or secretions (2). Although the infectious dose of Machupo virus in humans is unknown, exposed persons may become infected by inhaling virus shed in aerosolized secre- tions or excretions of infected rodents, by eating food contaminated with rodent excreta, or by direct con- tact of excreta with abraded skin or oropharyngeal mucous membranes (4). Reports of person-to-person Commentary Vol. 1, No. 3 -- July-September 1995 97 Emerging Infectious Diseases

was a Zaire subtype that differed from the original 1976 strain in four bases (<1%). No differences were seen when the polymerase gene PCR products (~350 bp) from those four patients were sequenced, which indicated that they had been infected with the same virus. Three days later, sequence data from ex- panded analysis of the entire glycoprotein gene were compared with those of the original 1976 Yambuku isolate (9) and showed that the overall difference between these Ebola viruses was less than 1.6%. Such little change in viruses that caused outbreaks of disease at extreme ends of Zaire separated by a span of nearly 19 years, may indicate that the genomes of Ebola viruses (and filoviruses in general) are unusually stable and have evolved to occupy special niches in the wild. The capability to rapidly diagnose and charac- terize filovirus infections is critical to the ability of public health professionals to identify and limit the spread of future outbreaks of filovirus disease. A continued commitment to research and modern dis- ease-surveillance programs is necessary to minimize or preclude filovirus outbreaks similar to that in Kikwit. The possibility of outbreaks is increasingly likely given the continued human incursions into the African forests and the vulnerability of large impov- erished populations to rapid transmission of disease as a result of inadequate public health services. With the current outbreak under control, CDC and col- laborators have begun their efforts to identify the natural reservoir by sending teams of scientists to collect specimens from the area where the putative index patient worked. Attempts to identify the res- ervoir after outbreaks in 1976 and 1979 were handi- capped by the lack of satisfactory diagnostic tools that are critical to detecting small quantities of the virus. However, now that sensitive enzyme immu- noassays and PCR assays have been developed for filoviruses, the chances are much better that, if ap- propriate materials can be collected in the field, the virus can be detected.

lack of satisfactory diagnostic tools that are critical to detecting small quantities of the virus. However, now that sensitive enzyme immu- noassays and PCR assays have been developed for filoviruses, the chances are much better that, if ap- propriate materials can be collected in the field, the virus can be detected. In conclusion, we want to alert physicians and public health agencies who encounter persons that have clinical signs and symptoms of hemorrhagic fever disease to the reemergence of Ebola virus. Recommendations for the management of viral hem- orrhagic fevers attributable to filoviruses in the United States were recently published in CDC's Mor- bidity and Mortality Weekly Report (1995;44:475-79). Anthony Sanchez, Thomas G. Ksiazek, Pierre E. Rollin, Clarence J. Peters, Stuart T. Nichol, Ali S. Khan, and Brian W. J. Mahy National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA References 1. Le Guenno B, Formenty P, Wyers M, Gounon P, Walker F, Boesch C. Isolation and partial characterisation of a new strain of Ebola virus. Lancet 1995;345:1271-4 2. Centers for Disease Control and Prevention. Outbreak of Ebola viral hemorrhagic fever--Zaire, 1995. MMWR 1995;44:381-2. 3. Centers for Disease Control and Prevention. Update: outbreak of ebola viral hemorrhagic fever--Zaire, 1995. MMWR 1995;44:399. 4. Bowen ETW, Platt GS, Lloyd G, Baskerville A, Harris WJ, Vella EC. Viral haemorrhagic fever in southern Sudan and northern Zaire: preliminary studies on the aetiologic agent. Lancet 1977;1:571-3. 5. Baron RC, McCormick JB, Zubeir OA. Ebola virus disease in southern Sudan: hospital dissemination and intrafamilial spread. Bull WHO 1983;62:997-1003. 6. Jahrling RB, Geisbert TW, Dalgard DW, et al. Prelimi- nary report: isolation of Ebola virus from monkeys imported to USA. Lancet 1990;335:502-5. 7. World Health Organization. Viral haemorrhagic fever in imported monkeys. Wkly Epidemiol Rec 1992;67:142-3. 8. World Health Organization. Ebola haemorrhagic fever in Zaire, 1976. Bull WHO 1978;56:271-93. 9. Sanchez A, Kiley MP, Holloway BP, Auperin DD. Se- quence analysis of the Ebola virus genome: organiza- tion, genetic elements, and comparison with the genome of Marburg virus. Virus Res 1993;29:215-40. Prospects for the Control of Bolivian Hemorrhagic Fever Bolivian hemorrhagic fever (BHF) was first iden- tified in 1959 as a sporadic hemorrhagic illness in rural areas of Beni department, Bolivia.

virus genome: organiza- tion, genetic elements, and comparison with the genome of Marburg virus. Virus Res 1993;29:215-40. Prospects for the Control of Bolivian Hemorrhagic Fever Bolivian hemorrhagic fever (BHF) was first iden- tified in 1959 as a sporadic hemorrhagic illness in rural areas of Beni department, Bolivia. Clusters of BHF patients were noted the same year, and by 1962 BHF was recognized as a new epidemic infectious disease. In 1963, Machupo virus (a member of the family Arenaviridae) was first isolated from patients with acute hemorrhagic fever in San Joaquin, Bo- livia (1). Ecologic investigations established the ro- dent Calomys callosus, which is indigenous to the disease-endemic region of northern Bolivia, as the reservoir for Machupo virus (2,3). Machupo virus infection in C. callosus results in asymptomatic infection with shedding of virus in saliva, urine, and feces; 50% of experimentally in- fected C. callosus are chronically viremic and shed virus in their bodily excretions or secretions (2). Although the infectious dose of Machupo virus in humans is unknown, exposed persons may become infected by inhaling virus shed in aerosolized secre- tions or excretions of infected rodents, by eating food contaminated with rodent excreta, or by direct con- tact of excreta with abraded skin or oropharyngeal mucous membranes (4). Reports of person-to-person Commentary Vol. 1, No. 3 -- July-September 1995 97 Emerging Infectious Diseases transmission are uncommon; however, hospital con- tact with a patient resulted in person-to-person spread of Machupo virus to nursing and pathology laboratory staff (5). In 1994, the fatal secondary infection of six family members in Magdalena from a single naturally acquired infection further sug- gested the potential for person-to-person transmis- sion (Ksiazek et al., manuscript in preparation). The pathogenesis of BHF, which resembles that of other South American hemorrhagic fevers due to Arenavirus infection (e.g., Argentine hemorrhagic fever), has been described in clinical and pathologic investigations of naturally infected patients (6,7). Experimental infection of rhesus monkeys with Machupo virus demonstrated an incubation period of 7 to 14 days, which is consistent with clinical observations in human infection (8). Early clinical manifestations in humans are characterized by non- specific signs and symptoms including fever, head- ache, fatigue, myalgia, and arthralgia.

tion of rhesus monkeys with Machupo virus demonstrated an incubation period of 7 to 14 days, which is consistent with clinical observations in human infection (8). Early clinical manifestations in humans are characterized by non- specific signs and symptoms including fever, head- ache, fatigue, myalgia, and arthralgia. Later in the course of disease (usually within 7 days of onset), patients may develop hemorrhagic signs, including bleeding from the oral and nasal mucosa and from the bronchopulmonary, gastrointestinal, and geni- tourinary tracts. During the BHF epidemics in the 1960s, rodent control was recognized as the primary method for the prevention of Machupo virus transmission (9). Since C. callosus was frequently found in domestic and peridomestic environments, rodent control meas- ures (e.g., trapping, poisoning) resulted in an imme- diate reduction in the number of C. callosus and control of BHF outbreaks; an epidemic in 1964 ended after 2 weeks of continuous trapping for C. callosus in homes of the affected community (10). Rodent control programs became a new priority for health officials in Bolivia, and active interventional pro- grams were carried out for many years by survivors of past BHF epidemics known to be immune to Machupo virus (11). From 1973 to 1992, no cases of BHF were re- ported, possibly because of effective control of rodent reservoir populations (12). Since the late 1960s, no epidemics of BHF have occurred that involve rural communities, but recent sporadic cases have been identified in the disease-endemic region (13). Al- though patients with BHF have been treated at hospitals outside the disease-endemic region, these patients had a history of exposure to Machupo virus in the disease-endemic region or secondary contact with BHF patients who became infected in the en- demic region. Additionally, no documented cases of BHF have been exported to other countries. Concurrently with the lack of identification of BHF patients during the 1970s and 1980s, the em- phasis on conducting rodent control programs in the BHF-endemic areas also diminished. Moreover, in recent years, Bolivian health officials have been faced with numerous other public health problems, including diarrheal disease, tuberculosis, Chagas' disease, sexually transmitted diseases, and acquired immunodeficiency syndrome.

conducting rodent control programs in the BHF-endemic areas also diminished. Moreover, in recent years, Bolivian health officials have been faced with numerous other public health problems, including diarrheal disease, tuberculosis, Chagas' disease, sexually transmitted diseases, and acquired immunodeficiency syndrome. Thus, local health authorities are confronted with the challenge of allo- cating limited health resources for the control of BHF as the demand for work with other important diseases increases. Agricultural activities dominate the economy of northern Bolivia where many workers are employed in farming and animal husbandry (14). Farm work- ers may reside for prolonged periods in rural areas also inhabited by C. callosus, and farm houses con- structed with partially open walls may allow rodents access to living areas. Thus, human exposure to infected rodents may occur in and around farm work- ers' shelters or during work in the fields and grass- lands of the BHF-endemic region. Given the projected economic growth in Bolivia, it is likely that agricultural workers' risk for exposure to C. callosus will continue and even increase as development modifies the natural habitat of the rodent reservoir leading to increased contact with humans (e.g., fo- cused rodent habitats with increased densities) (15). Future efforts to control BHF may benefit from recent experience in neighboring Argentina where ongoing work has led to the control of Argentine hemorrhagic fever (AHF), caused by Junin virus, an arenavirus genetically related to Machupo virus. Extensive study of AHF by Maiztegui, Enria, and colleagues has provided new insights into the epidemiology, pathogenesis, treatment, and control of this disease (16,17) and has led to an effective Candid #1 vaccine against Junin virus as well as phase 2 clinical trials that suggest ribavirin may be effective in patients with AHF (18,19). The use of an effective vaccine against AHF and evidence for its cross-protection against Machupo virus suggest that vaccination may play a role in the prevention of BHF for persons at highest risk, such as workers who trap rodents for control programs (20). Intravenous ribavirin has shown promise for the treatment of clinically diagnosed BHF cases subsequently con- firmed in the laboratory (Kilgore, manuscript in preparation). Intravenous ribavirin also appeared effective in the treatment of a laboratory-acquired infection with Sabia virus, a related Arenavirus first isolated in Brazil (21).

n has shown promise for the treatment of clinically diagnosed BHF cases subsequently con- firmed in the laboratory (Kilgore, manuscript in preparation). Intravenous ribavirin also appeared effective in the treatment of a laboratory-acquired infection with Sabia virus, a related Arenavirus first isolated in Brazil (21). Ribavirin could be adminis- tered to patients whose symptoms meet a clinical case definition with subsequent laboratory confirma- tion of Machupo virus infection. Local laboratory handling of specimens or testing by effective rapid enzyme-linked immunosorbent assays for antigen and IgM antibodies is ideally performed under biosafety level 4 containment, but use of biological safety cabinets and addition to samples of inexpen- sive reagents such as Triton X-100, which reduce Commentary Emerging Infectious Diseases 98 Vol. 1, No. 3 -- July-September 1995 viral titers, allow the development of capability for real time testing. The family cluster of BHF patients and later sporadic cases in September and October 1994 high- lighted the diagnostic challenge of BHF for clini- cians. Even local physicians may rarely evaluate BHF patients, and other diseases (e.g., malaria, den- gue fever, and yellow fever) that coexist in the BHF- endemic region may resemble BHF in the early phases of illness. Moreover, no readily available di- agnostic tests exist locally to differentiate BHF from other diseases (22). Bolivian health care providers and public health officials recognized the need for education of health care providers and subsequently established a training program aimed at increasing clinicians' recognition of BHF particularly in the disease-endemic region. The cluster of patients in 1994 also focused public attention on BHF because the illnesses had higher case-fatality rate than other diseases in the region where BHF is endemic. The underrecognition of these illnesses as dangerous and potentially fatal in disease-endemic communities suggests the need for increased public health education to reduce virus exposure and transmission. Proven control meas- ures must be reinforced even in towns affected by large epidemics 30 years ago where younger resi- dents have no recollection of the heavy toll exacted by BHF. Prevention of communitywide epidemics through rodent control programs may be combined with the application of barrier precautions (e.g., gloves, masks) in hospitals or clinics to minimize secondary person-to-person transmission of Machupo virus.

younger resi- dents have no recollection of the heavy toll exacted by BHF. Prevention of communitywide epidemics through rodent control programs may be combined with the application of barrier precautions (e.g., gloves, masks) in hospitals or clinics to minimize secondary person-to-person transmission of Machupo virus. After the familial cluster of BHF in 1994, results of rodent trapping confirmed the ab- sence of reinfestation in towns and indicated that the density of rodent reservoirs was not unusually high in areas of probable exposure for the index patient. The absence of communitywide epidemics of BHF suggests that focused rodent control in towns of the disease-endemic region prevented large urban out- breaks. Prevention of sporadic illness in farm work- ers through widespread elimination of reservoirs may not be feasible, but other measures, such as the administration of Candid #1 AHF vaccine to workers at high risk, may offer a more realistic alternative. Finally, agricultural workers in the disease-endemic region should be taught methods to reduce exposure to rodent reservoirs, especially around rural shelters as a means of reducing their risk of exposure to Machupo virus in the environment. Paul E. Kilgore, Clarence J. Peters, James N. Mills, Pierre E. Rollin, Lori Armstrong, Ali S. Khan, and Thomas G. Ksiazek National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA References 1. MacKenzie RB, Beye HK, Valverde L, Garron H. Epi- demic hemorrhagic fever in Bolivia: a preliminary re- port of the epidemiologic and clinical findings in a new epidemic area in South America. J Trop Med Hyg 1964;13:620-5. 2. Johnson KM, MacKenzie RB, Webb PA, Kuns ML. Chronic infection of rodents by Machupo virus. Science 1965;150:1618-9. 3. Johnson KM, Kuns ML, MacKenzie RB, Webb PA, Yunker CE. Isolation of Machupo virus from wild ro- dent, Calomys callosus. Am J Trop Med Hyg 1966;15;103-6. 4. Johnson KM. Epidemiology of Machupo virus infec- tion: III. Significance of virological observations in man and animals. Am J Trop Med Hyg 1965;14:816-8. 5. Peters CJ, Kuehne RW, Mercado RR. Hemorrhagic fever in Cochabamba, Bolivia, 1971. Am J Epidemiol 1974;99:425-33. 6. Stinebaugh BJ, Scholoeder FX, Johnson KM, MacKen- zie RB, Entwisle G, DeAlba E. Bolivian hemorrhagic fever: a report of four cases. Am J Med 1966;40:217-30. 7. Child PL, MacKenzie RB, Valverde LR, Johnson KM. Bolivian hemorrhagic fever: a pathologic description.

habamba, Bolivia, 1971. Am J Epidemiol 1974;99:425-33. 6. Stinebaugh BJ, Scholoeder FX, Johnson KM, MacKen- zie RB, Entwisle G, DeAlba E. Bolivian hemorrhagic fever: a report of four cases. Am J Med 1966;40:217-30. 7. Child PL, MacKenzie RB, Valverde LR, Johnson KM. Bolivian hemorrhagic fever: a pathologic description. Arch Pathol Lab Med 1967;83:434-45. 8. Kastello MD, Eddy GA, Kuehne RW. A rhesus monkey model for the study of Bolivian hemorrhagic fever. J Infect Dis 1976;133:57-62. 9. Kuns ML. Epidemiology of Machupo virus infection: II. Ecological and control studies of hemorrhagic fever. Am J Trop Med Hyg 1965;14:813-6. 10. Mackenzie RB, Kuns ML, Webb PA. Possibilities for control of hemorrhagic fevers in Latin America. Pan American Health Organization; Scientific Publication No.147:260-265. First International Conference on Vac- cines against Viral and Rickettsial Diseases of Man, 1966, Washington, D.C.. 11. Mercado R. Rodent control programmes in areas af- fected by Bolivian hemorrhagic fever. Bull WHO 1975;52:691-6. 12. Pan American Health Organization. Bolivian hemor- rhagic fever. Epidemiolog Bull; 1982;3:15-6. 13. Centers for Disease Control and Prevention. Bolivian hemorrhagic fever--El Beni Department, Bolivia. MMWR 1994;43:943-6. 14. United Nations. Statistical yearbook. 39th issue, de- partment for economic and social information and pol- icy analysis, statistical division. New York: United Nations, 1994;201-356. 15. United Nations. Economic and social indicators for latin american countries, including industrialized/ agricultural production. Statistical yearbook for Latin American countries and the Caribbean, 1993. New York: United Nations, 1994:238-41. 16. Peters CJ, Johnson KM. Arenaviridae: lymphocytic choriomeningitis virus, lassa virus, and other arenaviruses. In: Mandell GLK, Bennett JE, Dolin R, eds. Principles and practice of infectious diseases, 4th ed. New York: Churchill Livingston, Inc., 1995. 17. Enria D, Garcia Franco S, Ambrosio A, Vallejos D, Levis S, Maiztegui J. Current status of the treatment of Argentine hemorrhagic fever. Med Microbiol Immunol 1986;175:173-6. Commentary Vol. 1, No. 3 -- July-September 1995 99 Emerging Infectious Diseases 18. World Health Organization. Vaccination against Ar- gentine hemorrhagic fever. Wkly Epid Rec 1993;68: 233-4. 19. Enria DA, Maiztegui JU. Antiviral treatment of Argen- tine hemorrhagic fever. Antiviral Res 1994;23:23-31. 20. Jahrling PB, Trotter RW, Barrero O, et al.

uly-September 1995 99 Emerging Infectious Diseases 18. World Health Organization. Vaccination against Ar- gentine hemorrhagic fever. Wkly Epid Rec 1993;68: 233-4. 19. Enria DA, Maiztegui JU. Antiviral treatment of Argen- tine hemorrhagic fever. Antiviral Res 1994;23:23-31. 20. Jahrling PB, Trotter RW, Barrero O, et al. Cross- protection against Machupo virus with Candid 1 Junin virus vaccine III. In: Kurstak E, ed. Proceedings of the second international conference on the impact of viral diseases on the development of Latin American coun- tries and the Caribbean Region. Mar del Plata, Argen- tina, 1988. 21. Barry M, Russi M, Armstrong L, et al. Brief report: occupational exposure to a new arenavirus; Sabia virus clinical course, treatment and biosafety management. N Engl J Med (in press). 22. Webb PA, Maiztegui JI. Argentine and Bolivian hemor- rhagic fevers (South American hemorrhagic fevers). In: Gear JHS, ed. Handbook of viral and rickettsial hem- orrhagic fevers. Boca Raton, FL: CRC Press, Inc., 1988. Commentary Emerging Infectious Diseases 100 Vol. 1, No. 3 -- July-September 1995

The Ascension of Wildlife Rabies: A Cause for Public Health Concern or Intervention? Charles E. Rupprecht, V.M.D., Ph.D., Jean S. Smith, M.S. Makonnen Fekadu, D.V.M., Ph.D., and James E. Childs, Sc.D. Centers for Disease Control and Prevention, Atlanta, Georgia, USA The epidemiology of rabies in the United States has changed substantially during the last half century, as the source of the disease has changed from domesticated animals to wildlife, principally raccoons, skunks, foxes, and bats. Moreover, the changes observed among affected wildlife populations have not occurred without human influ- ence. Rather, human attraction to the recreational and economic resources provided by wildlife has contributed to the reemergence of rabies as a major zoonosis. Although human deaths caused by rabies have declined recently to an average of one or two per year, the estimated costs associated with the decrease in deaths amount to hundreds of millions of dollars annually. In future efforts to control rabies harbored by free-ranging animal reservoirs, public health professionals will have to apply imaginative, safe, and cost-effective solutions to this age-old malady in addition to using traditional measures. Rabies virus is the type species (serotype 1) of the Lyssavirus genus, a group of morphologically similar, antigenically and genetically related, negative-stranded RNA viruses, with a near global distribution (1). The lyssaviruses (Table 1) are well adapted to particular mammalian spe- cies (2) and rarely initiate panzootics. The public health threat of rabies as a preeminent zoonosis relates to the acute, incurable encephalitis that results from transmission of the virus by the bite of an infected animal. An estimated 40,000 to 100,000 human deaths are caused by rabies each year worldwide; in addition, millions of persons, primarily in developing countries of the subtropi- cal and tropical regions (3), undergo costlypostex- posure treatment (PET). Although the number of human rabies cases has been significantly re- duced in the United States, the total number of animal rabies cases approached historical limits in 1993.

llions of persons, primarily in developing countries of the subtropi- cal and tropical regions (3), undergo costlypostex- posure treatment (PET). Although the number of human rabies cases has been significantly re- duced in the United States, the total number of animal rabies cases approached historical limits in 1993. To appreciate the public health signifi- cance that lyssaviruses continue to play as per- sistent and emerging infectious agents, one must understand certain human activities, such as re- cent animal translocations (i.e., the natural or purposeful change by humans of the normal home range or geographic distribution of an ani- mal) and animal ecology. Historical Perspectives The history of rabies in the New World reflects the interaction of chance, evolutionary con- straint, ecologic opportunism, and human sur- veillance activities. Rabies may have existed in the United States before European colonization and the introduction of domesticanimals incubat- ing the disease. Various pathogens could have migrated during the exchanges of fauna and hu- man populations over the Bering Strait some 50,000 years ago; folklore of a rabies-like malady among native people throughout the Pacific Northwest supports this notion (4). Records at the time of the Spanish conquest in Middle America associate vampire bats with human illness (5). If chiropteran rabies viruses were present and well established in the New World at the time of con- tinental interchange, terrestrial virus counter- parts also could have been present. Nonetheless, the first indication of terrestrial rabies did not surface until 1703 in what is now California (5). Dog and fox rabies outbreaks, reported commonly in the mid-Atlantic colonies throughout the late 1700s (4), were probably exacerbated by the intro- duction of dogs and red foxes (Vulpes vulpes), imported for British-style fox hunting, through- out New England in the 1800s; fox rabies epizo- otics ensued and spread to the eastern United States by the 1940s to 1950s (5,6). Skunk rabies reports were also frequent throughout the west- ern states by the 19th century, and they were replete with cowboy tales of "phobey cats" (5). Address for correspondence: Charles E. Rupprecht, Centers for Disease Control and Prevention, 1600 Clifton Rd., MS G33, Atlanta, GA 30333, USA; fax 404-639-1058; e-mail cyr5@ciddvd1.em.cdc.gov. Synopses Vol. 1, No.

t the west- ern states by the 19th century, and they were replete with cowboy tales of "phobey cats" (5). Address for correspondence: Charles E. Rupprecht, Centers for Disease Control and Prevention, 1600 Clifton Rd., MS G33, Atlanta, GA 30333, USA; fax 404-639-1058; e-mail cyr5@ciddvd1.em.cdc.gov. Synopses Vol. 1, No. 4 -- October-December 1995 107 Emerging Infectious Diseases Although individual reports document a high incidence of dog rabies at the beginning of the last century, no national surveillance system existed. Human deaths from rabies in the United States were not commonly reported; the highest official record was of 143 cases, from a survey of death certificates in 1890. During 1938, when rabies in humans and other animals became a nationally reportable disease, the total number of rabies cases reported was 9,412 per year (mostly in domesticated species), with 47 human deaths. These numbers are certainly underestimates, since surveillance was limited, and sensitive diagnostic tests for human and animal rabies were not developed until the mid-1950s. An epizootiologic transition began in the United States in the 1920s, when rabies prevention efforts were no longer focused exclu- sively on human vaccination but began to include programs for the control of rabies in dogs. Domes- tic animal cases gradually declined, largely as a result of local dog rabies control programs that included vaccination, stray animal removal, and leash and muzzle ordinances. However, as such cases decreased, surveillance systems designed to track the source of infection for residual domestic animal foci detected increased cases in wild spe- cies. By 1960, rabies was diagnosed more fre- quently among wildlife than among domesticated animals. In 1971, rabies was reported for the first time from all 48 contiguous states and Alaska. Skunks (primarily the striped skunk, Mephitis mephitis) formed the major animal reservoir from 1961 to 1989, until they were unexpectedly sup- planted by the raccoon (Procyon lotor) during the rabies outbreak in the mid-Atlantic and north- eastern states (7). This epizootic is believed to have started during the late 1970s by the Table 1. Recognized members of the genus Lyssavirus, familyRhabdoviridae Lyssavirus Reservoir History Rabies Found worldwide, except for a few island nations, Australia, and Antarctica.

es outbreak in the mid-Atlantic and north- eastern states (7). This epizootic is believed to have started during the late 1970s by the Table 1. Recognized members of the genus Lyssavirus, familyRhabdoviridae Lyssavirus Reservoir History Rabies Found worldwide, except for a few island nations, Australia, and Antarctica. Endemic and sometimes epidemic in a wide variety of mammalian species, including wild and domestic canids, mustelids, viverrids, and insectivorous and hematophagous bats; >25,000 human cases/year, almost all in areas of uncontrolled domestic dog rabies. Descriptions of clinical disease in Greek and Roman documents. In the late 1800s, Pasteur attenuated the virus by serial passage and desiccation to vaccinate humans and animals. Pathognomonic inclusions in nerve cells described by Negri in 1903. An immunofluorescence test for rabies viral antigen developed in the 1950s. Lagosbat Unknown, but probably fruit bats. 10 cases identified to date, including 3 in domestic animals, in Nigeria, South Africa, Zimbabwe, Central African Republic, Senegal, and Ethiopia. No known human deaths. Isolated in 1956 from brain of Nigerian fruit bats (Eidolon helvum) at Lagos Island, Nigeria, but not characterized until 1970; 3 cases in domestic animals initially diagnosed as rabies, but weak immunofluorescence led to suspicion of "rabies-related" virus, later confirmed by typing with monoclonal antibodies or nucleotide sequence analysis. Marginal cross-protection with rabies vaccines. Mokola Unknown, but probably an insectivore or rodent species. Cases identified in Nigeria, South Africa, Cameroon, Zimbabwe, Central African Republic, and Ethiopia; 17 cases known, including 9 domestic animals and 2 human cases. First isolated from Crocidura sp. shrews trapped in Mokola Forest near Ibadan, Nigeria, in 1968. Characterized in 1970. Like Lagos bat virus, evidence of infection with Mokola was recognized only by poor reaction with anti-rabies reagents. 7 domestic animal cases in Zimbabwe in 1981 and 1982 prompted serologic survey and identification of antibodies to Mokola in rodents, especially bushveld gerbils (Tatera leucogaster). No cross-protection with rabies vaccines. Synopses Emerging Infectious Diseases 108 Vol. 1, No. 4 -- October-December 1995 translocation of infected animals from a south- eastern focus of the disease. The epidemiology of human rabies has also changed considerably over the last 50 years (8,9).

bils (Tatera leucogaster). No cross-protection with rabies vaccines. Synopses Emerging Infectious Diseases 108 Vol. 1, No. 4 -- October-December 1995 translocation of infected animals from a south- eastern focus of the disease. The epidemiology of human rabies has also changed considerably over the last 50 years (8,9). From 1946 to 1965, 70% to 80% of human rabies cases occurred after a known exposure (most often a dog bite), and 50% of the cases before 1975 occurred after treatment with suboptimal vac- cines. Over the last decade, 80% of rabies-related human deaths were among persons who had no definitive history of an animal bite (Table 2), and none resulted from postexposure prophylaxis fail- ures. Almost all the recent human cases occurred after an animal exposure that was unrecognized by the patient as carrying a risk for rabies infec- tion. The apparent source of human rabies has also changed: 14 of the 18 cases acquired in the United States since 1980 involved rabies variants associated with insectivorous bats (10). The latest report, in March 1995, typifies re- cent trends. Abat, subsequentlyfound to be rabid, was found in the bedroom of a 4-year-old girl in Washington State. The child denied any contact with the bat, and no postexposure treatment was initiated. A bat-associated rabies virus variant was later identified in biopsy specimens from the Table 1. (continued) Lyssavirus Reservoir History Duvenhage Unknown, but probably insectivorous bats. Cases identified in South Africa, Zimbabwe, and Senegal; 4 cases known, including 1 human death. No cases in domestic animals. First identified in 1970 in rabies-like encephalitis in man bitten by an insectivorous bat near Pretoria, South Africa. Virus named after the victim. Although Negri bodies detected in histologic examination of brain tissue, negative immunofluorescence tests led to suspicion of rabies-related virus, subsequently confirmed by antigenic and genetic typing. Marginal cross-protection with rabies vaccines. European bat Lyssavirus 1 (EBLV1) European insectivorous bats (probably Eptesicus serotinus); >400 cases in bats. 1 confirmed human case in 1985 and a suspect case in 1977. No known domestic animal cases. Although cases in European bats were reported as early as 1954, identification of the virus was not attempted until 1985, when the first of 100 infected bats was reported in Denmark and Germany. Almost all cases are in the common European house bat, E serotinus.

d a suspect case in 1977. No known domestic animal cases. Although cases in European bats were reported as early as 1954, identification of the virus was not attempted until 1985, when the first of 100 infected bats was reported in Denmark and Germany. Almost all cases are in the common European house bat, E serotinus. Marginal cross-protection with rabies vaccines. European bat Lyssavirus 2 (EBLV2) European insectivorous bats (probably Myotis dasycneme); 5 cases identified, including 1 human death. No known domestic animal cases. First identified in isolate from Swiss bat biologist who died of rabies in Finland. Marginal cross-protection with rabies vaccines. Table 2. Human rabies cases in the United States by exposure category, 1946-1995* Exposure source Case Years Domestic Wildlife Other Unknown (%) total 1946-1955 86 8 0 26 (22) 120 1956-1965 21 7 0 10 (26) 38 1966-1975 6 7 1 2 (13) 16 1976-1985 6 1 2 11 (55) 20 1986-1995* 2 2 0 14 (78) 18 * Through Oct. 1995. Synopses Vol. 1, No. 4 -- October-December 1995 109 Emerging Infectious Diseases child and from the bat's carcass (11). Despite the current prominence of raccoons as the largest wildlife reservoir in the United States (12), no documented human rabies cases have been associated with this ubiquitous carnivore. The Cost of Prevention Rabies prevention and control strategies in the United States have succeeded in lowering the number of human rabies deaths to an average of one to two per year. However, the reason for this low mortality level is a prevention program esti- mated to cost $230 million to $1 billion per year (13-15). This cost is shared by the private sector (primarily the vaccination of companion animals) and by the public (through animal control pro- grams, maintenance of rabies laboratories, and subsidizing of rabies PET). Accurate estimates of these expenditures are not available. The number of PETs given an- nually in the United States is unknown, although the total must be substantially greater than the minimum of 20,000 estimated in 1980 to 1981 (16) when vaccine distribution was more tightly regulated. As rabies becomes epizootic or enzootic in a region, the number of PETs increases (17). Although the cost varies, a course of rabies immu- noglobulin and five doses of vaccine given over a 4-week period typically exceeds $1,000. Potential exposure to a single rabid kitten in New Hamp- shire recently led to the treatment of more than 650 persons at an estimated cost of $1.5 million (18).

Ts increases (17). Although the cost varies, a course of rabies immu- noglobulin and five doses of vaccine given over a 4-week period typically exceeds $1,000. Potential exposure to a single rabid kitten in New Hamp- shire recently led to the treatment of more than 650 persons at an estimated cost of $1.5 million (18). Surveillance-related costs also rise as rabies becomes entrenched in wildlife. During 1993, the New York State rabies diagnostic laboratory re- ceived approximately 12,000 suspected animal submissions. This compares with approximately 3,000 submissions in 1989, before raccoon rabies became epizootic. In New Jersey, rabies preven- tion expenditures in two counties increased from $768,488 in 1988, before the raccoon epizootic, to $1,952,014 in 1990, the first full year of the epizootic (15); vaccination of pet animals ac- counted for 82% of this total. Vaccinated domestic animals are normally administered a booster vac- cine dose after a known or suspected rabid animal exposure (19). This further increases costs, as wildlife rabies epizootics escalate. The cost per human life saved from rabies ranges from ap- proximately $10,000 to $100 million, depending on the nature of the exposure and the probability of rabies in a region (20). What's more, most economic analyses do not take into account the psychological trauma caused by human exposure to rabies, the sub- sequent euthanasia of pets, or the loss of wildlife resources during rabies outbreaks. Rabies in wildlife has now reached historically high levels in the United States (12), and the costs of prevent- ing human rabies are mounting. Human Influences and the Role of Translocation The colonization of the New World had a pro- found effect upon native fauna and consequent rabies epizootiology. Large-bodied carnivores, such as bears, cougars, wolves, and wolverines, were perceived as dangerous and killed outright. A few Carnivora have persisted and flourished. For example, the coyote (Canis latrans), a highly adaptable canid and the subject of many unsuc- cessful control programs, has been gradually ex- panding its range northward and eastward. Despite their widespread distribution and abun- dance (even in suburban neighborhoods), rabid coyotes have been reported rarely and sporadically, except for a brief period from 1915 to 1917, when an extensive outbreak occurred in portions of Utah, Nevada, California,and Oregon.

panding its range northward and eastward. Despite their widespread distribution and abun- dance (even in suburban neighborhoods), rabid coyotes have been reported rarely and sporadically, except for a brief period from 1915 to 1917, when an extensive outbreak occurred in portions of Utah, Nevada, California,and Oregon. While dog rabies has been largely controlled, a region of southern Texas that borders Mexico has persisted as a focus of both dog and coyote rabies. The number of cases of coyote rabies has gradu- ally risen in this area since the late 1980s, ac- counting for 46 of the 50 cases of coyote rabies reported in the United States during 1991, 70 of 75 cases in 1992, and 71 of 74 cases in 1993 (12). The outbreak of coyote rabies has spread to the vicinity of San Antonio. One of the dangers of this outbreak is the continued spillover into the do- mestic dog population (21); at least 25 rabid dogs were reported from the area in 1991, 41 in 1992, and 54 in 1993 (12). Human rabies closely paral- lels the disease in domestic animals; at least two human deaths (in 1991 and 1994), probably due to coyote-dog interactions, have been associated with this canid outbreak in Texas (10,22). The translocation of infected coyotes from the south Texas focus is believed to be responsible for the transmission of this rabies variant to dogs in at least two other states: a single hunting dog in Alabama during 1993 (12) and at least seven cases of apparent dog-to-dog rabies transmission in Florida in 1994 (21). Expanded surveillance similar to that done in 1977 with the raccoon Synopses Emerging Infectious Diseases 110 Vol. 1, No. 4 -- October-December 1995 rabies focus in the mid-Atlantic region (7) is war- ranted for this canid virus. In this effort, state health departments should monitor unusual oc- currences (such as the increased submission of canid specimens to the diagnostic facility), track- ing of their time and location, and establishment of suitable public health interventions. These would include restricting further animal move- ments and enforcing mandatory companion ani- mal rabies vaccination. Assessing control efforts is an important component of any intervention. In addition to the problems posed by the emer- gence of the coyote as a reservoir for rabies, the potential translocation of other species should be recognized.

ther animal move- ments and enforcing mandatory companion ani- mal rabies vaccination. Assessing control efforts is an important component of any intervention. In addition to the problems posed by the emer- gence of the coyote as a reservoir for rabies, the potential translocation of other species should be recognized. Since the transmission of rabies by a bat was first reported in 1953, rabid insectivorous bats have caused an average of 700 to 800 cases annu- ally, and have been found throughout the United States, excluding Alaska and Hawaii (12). The discovery of these cases, coincident with the marked reduction of canine rabies cases, has af- forded a certain epidemiologic luxury to enhance surveillance among wildlife. Similar to the Carnivora, the chiropteran families most impor- tant in rabies perpetuation (e.g.,Vespertilionidae, Molossidae) have several species that are highly adaptable, abundant, and widespread. Rabies vi- rus variants maintained by insectivorous bats appear to be exchanged largely independently from those in terrestrial mammalian reservoirs (23), despite documented spillovers. A similar epidemiologic situation exists among European bats, but with Lyssavirus genotypes (24) that can be readily differentiated from New World rabies isolates. The role of bats in Africa (25,26) in Lys- savirus maintenance is less clear (Table 1). Infec- tions with non-rabies lyssaviruses have resulted in rabies vaccine failures (27). Such infections raisethe specter of potentially serious public health consequences if introduced and subsequently es- tablished in susceptible bat populations. How probable is this scenario? The distances between Africa, Eurasia, Pacific Oceania, and the New World mitigate against the dispersal, migration, and introduction of healthy bats without human intervention (28). However, several recent events illustrate the opportunity for the transoceanic transfer of rabies-infected bats. In March 1986, researchers from Canada inadvertently shipped a big brown bat (Eptesicus fuscus) that was incubating rabies virus to colleagues in Tubingen, Germany. When the bat became ill and was euthanized, a diagnosis of rabies was made (29). A similar event occurred when Boston researchers collected a dozen wild big brown bats from Massachusetts during July 1994 and exported them to researchers in Den- mark.

that was incubating rabies virus to colleagues in Tubingen, Germany. When the bat became ill and was euthanized, a diagnosis of rabies was made (29). A similar event occurred when Boston researchers collected a dozen wild big brown bats from Massachusetts during July 1994 and exported them to researchers in Den- mark. By December 1994, six of the importedbats had died and were confirmed as positive for rabies virus by the Danish Veterinary Services, State Institute for Virus Research (L. Miller, pers. comm.). Commercial enterprises also serve as vehicles for the accidental translocation of animals in- fected with rabies virus. The first confirmed non- indigenous case of rabies in Hawaii resulted from the accidental introduction of a big brown bat (30). In March 1991, a bat was captured within a transport container unloaded from a ship in Honolulu harbor. The container held automobiles from Michigan loaded into the container ship in California. The local department of health labo- ratory diagnosed rabies; this was later confirmed, and the virus was characterized antigenically at the Centers for Disease Control and Prevention. The strain was a variant common to E. fuscus in the midwestern and western United States. None of the three instances cited above appear to have resulted in secondary cases or establishment of the virus in foreign animal populations. No unintended importations of non-rabies lys- saviruses to the United States have been docu- mented. The likelihood of accidental introduction, escape, survival, and perpetuation of infected ex- otic bat species into the United States is remote. However, other more recent deliberate transloca- tion activities may significantly enhance the probability of such introductions. During 1994, a number of improperly issued federal permits allowed as many as several thou- sand wild bats to be imported to the United States for sale in the commercial pet trade. These animals were primarily Egyptian tomb bats (Rousettus aegyptiacus), although several other bat species were imported as well. Sales of im- ported bats (and their offspring) to private collec- tors or as pets in the United States are prohibited, according to the Foreign Quarantine Regulations (42 CFR 71.54). Animals that may be vectors of diseases of public health concern are eligible for entrance only for restricted uses at accredited zoos or research institutions, where contact with the general public is limited. Imported bats that Synopses Vol. 1, No.

ited, according to the Foreign Quarantine Regulations (42 CFR 71.54). Animals that may be vectors of diseases of public health concern are eligible for entrance only for restricted uses at accredited zoos or research institutions, where contact with the general public is limited. Imported bats that Synopses Vol. 1, No. 4 -- October-December 1995 111 Emerging Infectious Diseases will be legally displayed normally undergo an extended quarantine period. Although no reports of lyssaviruses isolated from Egyptian fruit bats exist, active surveillance for such viruses has not been conducted. These bats are relatively common and widespread throughout the area that extends from Turkey and Cyprus to Pakistan, the Arabian peninsula, Egypt, and most of sub-Saharan Africa (31). Be- cause they may roost by the thousands in a wide variety of habitats, there is ample opportunity for interaction with other Chiroptera, such as the widely distributed straw-colored (Eidolon helvum) or epauletted (Epomophorus wahlbergi) fruit bats; both of these species have been impli- cated inLyssavirusepizootiology inAfrica (25,26). The adaptability of Egyptian fruit bats should be a cause for concern because of the potential for survival and interaction among indigenous bat fauna, particularly inthe southern UnitedStates. Additionally, beyond the obvious public health risks and foreign animal disease introduction, imported bat species should not be released into the wild because they may cause serious harm to local agriculture and may displace native species. Bats serve many critical ecologic functions worldwide and generally avoid contact with hu- mans. However, they may be infected with many pathogens without demonstrating obvious clini- cal signs of infection. When bats are placed in a private household or pet shop, the hazard of dis- ease transmission to humans is greatlyincreased. Persons currently possessing imported bats should be advised not to display them in settings where human contact can occur. Intervention Widespread, sustained population reduction of mammalian reservoirs to eliminate rabies is not justified (32) for ecologic, economic, and ethical reasons. Given the multispecies complexity and considerable geographic areas affected by wildlife rabies, and the opportunities for translocation, what alternative preventive strategies exist? Re- cent progress in implementing terrestrial wildlife rabies control programs elsewhere in the world has public health relevance for the UnitedStates.

species complexity and considerable geographic areas affected by wildlife rabies, and the opportunities for translocation, what alternative preventive strategies exist? Re- cent progress in implementing terrestrial wildlife rabies control programs elsewhere in the world has public health relevance for the UnitedStates. Oral rabies vaccination of the red fox with vac- cine-laden baits is an integral aspect of rabies control throughout southeastern Canada and Europe, where more than 75 million doses of vaccine have been distributed over 5 million km2 during the past two decades (33). Consequently, rabies incidence among wild and domestic ani- mals has fallen, as have PETS for human rabies. The raccoon rabies epizootic in the eastern United States provided renewed impetus for re- considering oral vaccination technology, first con- ceived at the Center for Disease Control in the 1960s (34). The shift of the vaccination and bait- ing methods from a fox model to the raccoon involved extensive field and laboratory research during the 1980s. The existing attenuated rabies vaccines for foxes were shown to be less effective for raccoons and other carnivores (35,36). Addi- tionally, studies of new candidate vaccines raised safety issues regarding vaccine-induced disease in wildlife (36). In 1983, a vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine was developed (37) that has proven to be an effective oral immunogen in raccoons and various other important reservoir species (38); vaccine advantages include im- proved thermostability and an inability to cause rabies. (Only the gene for the surface glycoprotein of a vaccine strain of rabies virus was included in the recombinant virus.) Whenvaccine-laden baits are offered under natural conditions, contact with them by nontarget wildlife species cannot be to- tally excluded. However, studies of V-RG virus have shown novaccine-associated morbidity, mor- tality, or gross pathologic lesions in more than 40 warm-blooded vertebrate species examined. Moreover, with rare exceptions, there has been no contact-transfer of vaccine between vaccinated and control animals housed together (38); viral recovery has been limited to a few anatomical sites over a 48-h interval (39). While laboratory evaluations of target and nontarget species proceeded during 1987 to 1989, small-scale trials of V-RG were conducted in Bel- gium and France, with promising results (40).

nated and control animals housed together (38); viral recovery has been limited to a few anatomical sites over a 48-h interval (39). While laboratory evaluations of target and nontarget species proceeded during 1987 to 1989, small-scale trials of V-RG were conducted in Bel- gium and France, with promising results (40). The first North American V-RG vaccine field trial began on August 20, 1990, on Parramore Island off the eastern shore of Virginia (41,42). This limited field trial demonstrated vaccine safety. Efficacy was also suggested: more than 80% of field-vaccinated raccoons survived a severe labo- ratory rabies challenge (7 months after V-RG release) to which more than 90% of control rac- coons succumbed (43). A 1991 Pennsylvania study site closely ap- proximated the ecologic communities of the east- ern United States targeted for use of V-RG Synopses Emerging Infectious Diseases 112 Vol. 1, No. 4 -- October-December 1995 vaccine, while still maintaining relative biosecu- rity through its geographic barriers. The study at this site evaluated the rate of vaccine-laden bait contact and potential vaccine-related adverse ef- fects among nonraccoon species, including ro- dents, carnivores, insectivores, and opossum. Raccoons and other furbearers demonstrated no adverse effects associated with vaccine contact. Examination of more than 750 nontarget indi- viduals, representing 35 species, failed to demon- strate gross lesions suggestive of V-RG contact. After these safety trials, the first efficacy field experiments began in New Jersey during 1992 (44). Between spring 1992 and autumn 1994, more than 100,000 vaccine-laden fishmeal poly- merbaits were distributed by hand and helicopter over an area of 56,000 hectares. This trial at- tempted to create a population of immunized rac- coons across the northern Cape May Peninsula to prevent the spread of epizootic raccoon rabies from affected portions of the state. Surveillance demonstrated a significant decrease in the rate of spread and overall rabies incidence in the target and other monitored areas (44), suggesting the potential effectiveness of this strategy. In the United States, oral vaccination of rac- coons is now under way in Massachusetts (45), New York (46), and Florida, and an experimental extension of the program to coyotes is under way in south Texas. However, the future of such vac- cination for wildlife in the United States may be seriously questioned.

strategy. In the United States, oral vaccination of rac- coons is now under way in Massachusetts (45), New York (46), and Florida, and an experimental extension of the program to coyotes is under way in south Texas. However, the future of such vac- cination for wildlife in the United States may be seriously questioned. For oral vaccination to become an adjunct to traditional methods, the following major questions need to be answered: 1) What is the relationship between animal popu- lation density and the minimum density of vac- cine/baits needed?2) What level of herd immunity is necessary to eliminate rabies under various environmental circumstances? 3) What bait dis- tribution techniques are optimal? 4) How can these methods be generalized from foxes and rac- coons to other species, such as skunks, mongooses and dogs? 5) What long-term funding sources are available? 6) What are the various costs of rabies control and prevention methods? Given the prob- lems inherent inwildlife control,the greater issue of extending these methods to the control of dog rabies in the developing world will be a challenge well into the next century. Dr. Rupprecht is chief of the Rabies Section, Ms. Smith is a research microbiologist, Dr. Fekadu is a research veterinary medical officer, and Dr. Childs is chief of the Epidemiology Section, Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia. Acknowledgment The authors gratefully acknowledge the technical expertise of the staff of the Rabies and Epidemiology Sections, Viral and Rickettsial Zoonoses Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, without whose assistance this work would not have been possible. References 1. World Health Organization. Worldsurvey of rabies 28 for the year 1992. Geneva: World Health Organiza- tion, 1994. 2. Wandeler A, Nadin-Davis SA, Tinline RR, Rupprecht CE. Rabies epizootiology: an ecological and evolution- ary perspective. In: Rupprecht CE, Dietzschold B, Koprowski H, editors. Lyssaviruses. New York: Sprin- ger-Verlag, 1994:297-324. 3. Meslin FX, Fishbein DB, Matter HC. Rationale and prospects for rabies elimination in developing coun- tries. In: Rupprecht CE, Dietzschold B, Koprowski H, editors. Lyssaviruses. New York: Springer-Verlag, 1994:1-26. 4. Winkler WG. Fox rabies. In: Baer GM, editor.

saviruses. New York: Sprin- ger-Verlag, 1994:297-324. 3. Meslin FX, Fishbein DB, Matter HC. Rationale and prospects for rabies elimination in developing coun- tries. In: Rupprecht CE, Dietzschold B, Koprowski H, editors. Lyssaviruses. New York: Springer-Verlag, 1994:1-26. 4. Winkler WG. Fox rabies. In: Baer GM, editor. The natural history of rabies. 1st ed. New York: Academic Press, 1975:3-22. 5. Baer GM. Rabies--an historical perspective. Infec- tious Agents and Disease 1994;3:168-80. 6. Carey AB, Giles RH, McLean RG. The landscape epidemiology of rabiesin Virginia. Am J Trop Med Hyg 1978;27:573-80. 7. Rupprecht CE, Smith JS. Raccoon rabies--the re- emergence of an epizootic in a densely populated area. Semin Virol 1994;5:155-64. 8. Held JR, Tierkel ES, Steele JH. Rabies in man and animals in the United States, 1946-65. Public Health Rep 1967;82:1009-18. 9. Anderson LJ, Nicholson KG, Tauxe RV, Winkler WG. Human rabies in the United States, 1960 to 1979: epidemiology, diagnosis, and prevention. Ann Intern Med 1984;100:728-35. 10. Centers for Disease Control and Prevention. Human rabies--Alabama, Tennessee, and Texas, 1994. MMWR 1995;44:269-72. 11. Centers for Disease Control and Prevention. Human rabies--Washington state, 1995. MMWR 1995; 44:625-7. Synopses Vol. 1, No. 4 -- October-December 1995 113 Emerging Infectious Diseases 12. Krebs JW, Strine TW, Smith JS, Rupprecht CE, Childs JE. Rabies surveillance in the United States during 1993. J Am Vet Med Assoc 1994;205:1695-709. 13. Stehr-Green JK, Schantz PM. The impact of zoonotic diseases transmitted by pets on humanhealth and the economy. Vet Clin North Am Small Anim Pract 1987;17:1-15. 14. Fishbein DB, Arcangeli S. Rabies prevention in pri- mary care: a four-step approach. Postgrad Med 1987;82:83-90. 15. Uhaa IJ, Dato VM, Sorhage FE, et al. Benefits and costs of using an orally absorbed vaccine to control rabies in raccoons.J Am Vet MedAssoc1992;201:1873- 82. 16. Helmick CG. The epidemiology of human rabies postexposure prophylaxis, 1980-1981. JAMA 1983;250:1990-6. 17. Centers for Disease Control and Prevention. Raccoon rabies epizootic: United States, 1993. MMWR 1994;43:269-73. 18. Centers for Disease Control and Prevention. Mass treatment of humans exposed to rabies--New Hamp- shire, 1994. MMWR 1995;44:483-6. 19. Centers for Disease Control and Prevention. Compen- dium of animal rabies control, 1995. MMWR 1995;44:(RR-2):1-9. 20. Fishbein DB, Robinson LE. Rabies. N Engl J Med 1993;329:1632-8. 21.

Centers for Disease Control and Prevention. Mass treatment of humans exposed to rabies--New Hamp- shire, 1994. MMWR 1995;44:483-6. 19. Centers for Disease Control and Prevention. Compen- dium of animal rabies control, 1995. MMWR 1995;44:(RR-2):1-9. 20. Fishbein DB, Robinson LE. Rabies. N Engl J Med 1993;329:1632-8. 21. Centers for Disease Control and Prevention. Translo- cation of coyote rabies--Florida, 1994. MMWR 1995;44:580-1, 7. 22. Centers for Disease Control and Prevention. Human rabies--Texas, Arkansas, and Georgia, 1991. MMWR 1991;40:765-9. 23. Smith JS, Orciari LA, Yager PA. Molecular epidemiol- ogy of rabies in the United States. Semin Virol (in press). 24. Bourhy H, Kissi B, Lafon M, Sacramento D, Tordo N. Antigenic and molecular characterization of bat ra- bies virus in Europe. J Clin Microbiol 1992;30:2419- 26. 25. Swanepoel R, Barnard BJH, Meredith CD, et al. Ra- bies in southern Africa. Onderstepoort J Vet Res 1993;60:325-46. 26. King AA, Meredith CD, Thomson GR. The biology of southern Africa lyssavirus variants. In: Rupprecht CE, Dietzschold B, Koprowski H, editors. Lys- saviruses. New York: Springer-Verlag, 1994:267-96. 27. Foggin CM. Mokola virus infection in cats and a dog in Zimbabwe. Vet Rec 1983;113:115. 28. Wiles GJ, Hill JE. Accidental aircraft transport of a bat to Guam. J Mamm (full title) 1986;67:600-1. 29. World Health Organization Collaborating Center for Rabies Surveillance and Research. Bat rabies cases in the Federal Republic of Germany. World Health Or- ganization Rabies Bulletin Europe 1986;10:8-9. 30. Sasaki DM, Middleton CR, Sawa TR, Christensen CC, Kobayashi GY. Rabid bat diagnosed in Hawaii. Ha- waii Med J 1992;51:181-5. 31. Nowak RM. Walkers mammals of the world. 5th ed. Baltimore: Johns Hopkins University Press, 1991:198. 32. Debbie JG. Rabies control of terrestrial wildlife by population reduction. In: Baer GM, editor. The natu- ral history of rabies. 2nd ed. Boca Raton, FL: CRC Press, 1991:477-84. 33. World Health Organization. Oral immunization of foxes in Europe in 1994. Wkly Epidemiol Rec 1995;70:89-91. 34. Baer GM. Oral rabies vaccination: an overview. Rev Infect Dis 1988;10 (Suppl 4):S644-8. 35. Rupprecht CE, Dietzschold B, Cox JH, Schneider L. Oral vaccination of raccoons (Procyon lotor) with an attenuated (SAD-B19) rabies virus vaccine. J Wildl Dis 1989;25:548-54. 36. Rupprecht CE, Charlton KM, Artois M, et al.

1. 34. Baer GM. Oral rabies vaccination: an overview. Rev Infect Dis 1988;10 (Suppl 4):S644-8. 35. Rupprecht CE, Dietzschold B, Cox JH, Schneider L. Oral vaccination of raccoons (Procyon lotor) with an attenuated (SAD-B19) rabies virus vaccine. J Wildl Dis 1989;25:548-54. 36. Rupprecht CE, Charlton KM, Artois M, et al. Ineffec- tiveness and comparative pathogenicity of attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis). J Wildl Dis 1990;26:99-102. 37. Wiktor TJ, Macfarlan RI, Reagan KJ, et al. Protection from rabies by a vaccinia virus recombinant contain- ing the rabies virus glycoprotein gene. Proc Natl Acad Sci USA 1984;81:7194-8. 38. Rupprecht CE, Hanlon CA, Hamir AN, Koprowski H. Oral wildlife rabies vaccination: development of a recombinant virus vaccine. Transactions of the North American Wildlife Natural Resources Conference 1992;57:439-52. 39. Rupprecht CE, Hamir AN, Johnston DH, Koprowski H. Efficacy of a vaccinia-rabies glycoprotein recombi- nant virus vaccine in raccoons (Procyon lotor). Rev Infect Dis 1988;10 (4 Suppl):S803-9. 40. Aubert MFA, Masson E, Artois M, Barrat J. Oral wildlife rabies vaccination field trials in Europe, with recent emphasis on France. In: Rupprecht CE, Dietzschold B, Koprowski H, editors. Lyssaviruses. New York: Springer-Verlag, 1995:219-44. 41. Hanlon CA, Hayes DE, Hamir AN, et al. Proposed field evaluation of a rabies recombinant vaccine for raccoons Procyon lotor: site selection target species characteristics and placebo baiting trials. J Wildl Dis 1989;4:555-67. 42. Hanlon CA, Buchanan JR, Nelson E, et al. Avaccinia- vectored rabies vaccine field trial:ante- and post-mor- tem biomarkers. Rev Sci Tech 1993;99-107. 43. Rupprecht CE, Hanlon CA, Niezgoda M, Buchanan JR, Diehl D, Koprowski H. Recombinant rabies vac- cines: efficacy assessment in free-ranging animals. Onderstepoort J Vet Res 1993;60:463-8. 44. Roscoe DE, Holste W, Niezgoda M, Rupprecht CE. Efficacy of the V-RG oral rabies vaccine in blocking epizootic raccoon rabies. Presented at the 5th Annual International Meeting of Rabies in the Americas, Ni- agara Falls, Ontario, Canada, 1994, Abstract, p.33. 45. Robbins AH, Niezgoda M, Levine S, et al. Oral rabies vaccination of raccoons (Procyon lotor) on the Cape Cod Isthmus, Massachusetts. Presented at the 5th Annual International Meeting of Rabies in the Ameri- cas, Niagara Falls, Ontario, Canada, 1994; Abstract, p. 29. 46. Hanlon CA, Trimarchi C, Harris-Valente K, Debbie JG.

bins AH, Niezgoda M, Levine S, et al. Oral rabies vaccination of raccoons (Procyon lotor) on the Cape Cod Isthmus, Massachusetts. Presented at the 5th Annual International Meeting of Rabies in the Ameri- cas, Niagara Falls, Ontario, Canada, 1994; Abstract, p. 29. 46. Hanlon CA, Trimarchi C, Harris-Valente K, Debbie JG. Raccoon rabies in New York State: epizootiology, economics, and control. Presented at the 5th Annual International Meeting of Rabies in the Americas, Niagara Falls, Ontario, Canada, 1994; Abstract, p.16. Synopses Emerging Infectious Diseases 114 Vol. 1, No. 4 -- October-December 1995

Diagnosis of Tuberculosis in Children: Increased Need for Better Methods Ejaz A. Khan, M.D., and Jeffrey R. Starke, M.D. Baylor College of Medicine, Houston, Texas, USA In the last decade tuberculosis (TB) has reemerged as a major worldwide public health hazard with increasing incidence among adults and children. Although cases among children represent a small percentage of all TB cases, infected children are a reservoir from which many adult cases will arise. TB diagnosis in children usually follows discovery of a case in an adult, and relies on tuberculin skin testing, chest radiograph, and clinical signs and symptoms. However, clinical symptoms are nonspecific, skin testing and chest radiographs can be difficult to interpret, and routine laboratory tests are not helpful. Although more rapid and sensitive laboratory testing, which takes into account recent advances in molecular biology, immunology, and chromatography, is being developed, the results for children have been disappointing. Better techniques would especially benefit children and infants in whom early diagnosis is imperative for preventing progressive TB. Despite the availability of effective preventive measures and chemotherapy, the prevalence of tuberculosis (TB) is increasing in the developing world and in much of the industrialized world as well (1-4). According to World Health Organiza- tion (WHO) estimates, in 1990 there were 8 mil- lion new cases of TB and 3 million deaths due to the disease worldwide; 1.3 million new cases and 450,000 deaths were among children under 15 years of age (5). WHO projects that 90 million new cases and 30 million deaths-including 4.5 million deaths among children-will occur in the 1990s (6,7). In developing countries, the risk for TB infection and disease is relatively uniform in the population; annual rates of infection often exceed 2% (5,6). In industrialized countries, risk is more uneven and depends on the individual's past or present activities and exposure to persons at high risk for the disease (Table 1). From 1987 to 1991, the number of TB cases among children under 5 years of age in the United States increased by 49% from 674 cases to 1006 (8).

,6). In industrialized countries, risk is more uneven and depends on the individual's past or present activities and exposure to persons at high risk for the disease (Table 1). From 1987 to 1991, the number of TB cases among children under 5 years of age in the United States increased by 49% from 674 cases to 1006 (8). Although cases among children represent a small percentage of all TB cases, infected children are a reservoir from which many adult cases will arise. The risk for infection by Mycobacterium tuberculosis among children depends primarily on the level of risk of developing infectious TB for the adults in their immediate environment, especially their household. Because most current diagnostic tests for TB infection and disease have low specificity and therefore low positive predictive values, epidemiologic investigation continues to be im- portant in establishing the diagnosis of TB in children. In industrialized countries, clinicians and public health professionals in TB services must always ask: Has the child been exposed to an adult with infectious pulmonary TB? Natural History of TB in Children The natural history of TB in children follows a continuum; however, it is useful to consider three basic stages: exposure, infection, and disease (1). Exposure implies that the child has had recent and substantial contact with an adult or adoles- cent who has suspected or confirmed contagious pulmonary TB (a source case). Exposed children are usually identified during followup investiga- tions for persons with suspected pulmonary TB by publichealth workers (9); the child's tuberculin skin test (TST) is nonreactive, the results of the chest radiograph are normal, and the child is free of physical signs or symptoms of TB. Some exposed children are infected with M. tubercu- losis. The clinician cannot know immedi- ately which exposed children are infected because the development of delayed-type hypersensitivity to tuberculin may take up to 3 months. Unfortu- nately, in children under 5 years of age, severe TB--especially meningeal and disseminated Address for correspondence: Jeffrey R. Starke, Texas Children's Hospital, MC 3-2371, 1102 Bates Street, Houston, TX 77030, USA; fax: 713-770-4347; e-mail jstarke@msmailpo2.is5.tch.tmc.edu. Synopses Vol. 1, No. 4 -- October-December 1995 115 Emerging Infectious Diseases disease--canoccur infewer than 3 months, before the TST becomes reactive (10).

Jeffrey R. Starke, Texas Children's Hospital, MC 3-2371, 1102 Bates Street, Houston, TX 77030, USA; fax: 713-770-4347; e-mail jstarke@msmailpo2.is5.tch.tmc.edu. Synopses Vol. 1, No. 4 -- October-December 1995 115 Emerging Infectious Diseases disease--canoccur infewer than 3 months, before the TST becomes reactive (10). Young children in the exposure stage should receive chemotherapy, usually isoniazid, until infection can been ex- cluded. TB infection is first signaled by a reactive Mantoux TST. In this stage, there are no signs or symptoms, and the results of the chest radio- graph are either normal or show only fibrotic lesions or calcifications in the lung parenchyma or regional lymph nodes. In developing countries, TB infection is rarely discovered and almost never treated. In most industrialized countries, children with a positive TST receive isoniazid for 6 to 12 months. TB disease occurs when signs and symptoms or radiographic manifestations caused by M. tu- berculosis appear. Radiographic abnormalities and clinical manifestations in infected children probably are influenced by the host inflammatory reaction more than by the number of organisms. Studies show that in 40% to 50% of infants with untreated TB infection disease develops within 1 to 2 years (11). The risk decreases to 15% among older children. In 25% to 35% of children TB is extrapulmonary and more difficult to confirm bacteriologically. In adults, the distinction between TB infection and disease is usually clear because most disease is caused by reactivation of dormant organisms years after infection. Disease in adults is usually accompanied by symptoms, and patients fre- quently are infectious. In children, who most often have primary disease, the interval between infection and disease can be several months to several years, and radiographic abnormalities often are not accompanied by symptoms; more- over, these children are rarely infectious. The major reason for separating infection from dis- ease in children is that the perception affects the approach to treatment: infection is generally treated with a single anti-TB drug, whereas ac- tive disease is treated with two or more drugs. The rationale for the difference in treatment is that the likelihood of emergence of resistance to a drug increases as the bacillary population in- creases (3). This distinction is somewhat artificial in children since infection and primary disease are parts of a continuum.

ve disease is treated with two or more drugs. The rationale for the difference in treatment is that the likelihood of emergence of resistance to a drug increases as the bacillary population in- creases (3). This distinction is somewhat artificial in children since infection and primary disease are parts of a continuum. Because anti-TB medi- cations are well tolerated by children and are relatively inexpensive in industrialized countries, the usual paradigm of infection and disease encourages overtreatment rather than undertreatment. Asymptomatic lymphade- nopathy and mild lung parenchymal changes are labeled and treated as disease. When evaluating new diagnostic tests the ba- sic differences between the pathophysiology of TB in adults and children should be considered. Among children with recent TB infection, active multiplication of mycobacteria occurs with or without the presence of radiographic abnormali- ties or clinical symptoms. For example, gastric aspirate cultures yield M. tuberculosis from a small proportion of recently infected children with normal chest radiographs. One can antici- pate that most diagnostic tests designed to detect M. tuberculosis in adults with TB disease will be positive in some proportion of children who have what is usually called TB infection. It will take careful consideration and investigation to determine if and how the results of these new tests should influence the definitions and treat- ment of TB infection and disease in children. Table 1. Persons at high risk for Mycobacterium tuberculosis infection in industrialized countries Persons likely to be exposed to or become infected with M. tuberculosis * Close contacts of a person with infectious tu- berculosis (TB) * Foreign-born persons from high-incidence ar- eas (e.g., Asia, Africa, Latin America) * The elderly * Residents of long-term care facilities (e.g., correctional facilities and nursing homes) * Persons who inject drugs * Other groups identified locally as having in- creased prevalence of TB (e.g., migrant farm workers or homeless persons) * Persons who may have occupational expo- sure to TB Persons at high risk of developing TB disease once infected * Persons recently infected with M. tuberculo- sis (within the past 2 years) * HIV-infected persons * Persons with immunosuppressing condi- tions or medication use * Persons with a history of inadequately treated TB * Infants Synopses Emerging Infectious Diseases 116 Vol. 1, No.

k of developing TB disease once infected * Persons recently infected with M. tuberculo- sis (within the past 2 years) * HIV-infected persons * Persons with immunosuppressing condi- tions or medication use * Persons with a history of inadequately treated TB * Infants Synopses Emerging Infectious Diseases 116 Vol. 1, No. 4 -- October-December 1995 Established Diagnostic Methods Tuberculin Skin Test (TST) The Mantoux TST, which uses five tuberculin units of purified protein derivative, is the stand- ard method for detecting infection by M. tubercu- losis. The reaction is measured as millimeters of induration after 48 to 72 hours. Since TST is the only way to determine asymptomatic infection by M. tuberculosis, the false-negative rate cannot be calculated. A negative TST does not rule out TB disease in a child. Approximately 10% of other- wise normal children with culture-proven TB do not react to tuberculin initially (12,13). Most of these children have reactive skin tests during treatment, which suggests that TB disease con- tributed to the immunosuppression. In most cases, the anergy occurs to all antigens, but in some cases, reactions to tuberculin are negative but reactions to other antigens remain positive (13). The rate of false-negative TST is higher in those who are tested soon after becoming infected with severe TB; in children with debilitating or immunosuppressive illnesses, malnutrition, or viral and certain bacterial infections; in infants; and if poor technique is used (1,14). The rate of false-negative TST in children with TB who are infected with human immunodeficiency virus (HIV) is unknown, but it is certainly higher than 10%. False-positive reactions to TST are often at- tributed to asymptomatic infection by environ- mental nontuberculous mycobacteria (NTM). Vaccination with M. bovis can cause transient reactivity to a subsequent TST, but the associa- tion is weaker than commonly recognized. Most- 80% to 90% in several studies-children who received BCG as infants have a nonreactive TST at 5 years of age (15-17). Among older children or adolescents who receive BCG, most develop a reactive skin test initially; however, by 10 to 15 years postvaccination, 80% to 90% have lost tu- berculin reactivity (18,19). Skin test reactivity can be boosted, probably by antigenic stimula- tion, by serial testing in many children and adults who received BCG (20). Various factors determine the TST reaction size after receipt of BCG (1).

ally; however, by 10 to 15 years postvaccination, 80% to 90% have lost tu- berculin reactivity (18,19). Skin test reactivity can be boosted, probably by antigenic stimula- tion, by serial testing in many children and adults who received BCG (20). Various factors determine the TST reaction size after receipt of BCG (1). Many recipients of BCG have a reactive TST because they are infected with M. tuberculosis and are at risk for disease, especially if they have had recent contact with an infectious TB patient (18). In general, TST reaction should be interpreted in the same manner for persons who have received BCG (20) and for unvaccinated persons. The relatively low sensitivity and specificity of TST make the test very useful for persons at high risk for TB infection or disease but undesirable for use in persons at low risk (21,22). The predic- tive values of TST can be improved by varying the size of induration considered positive according to epidemiologic risk factors for infection (Table 2). However, most of even 15-mm reactions in chil- dren at low risk are false-positive results, and testing of persons at low risk should be discour- aged. Although the scheme in Table 2 is scientifi- cally and mathematically valid, it assumes that the clinician and family are willing and able to develop an accurate history for TB risk factors for children and adults in their environments. Clinical Signs and Symptoms Two scenarios lead the clinician to suspect that a child has TB disease. The first occurs when TB is considered during the differential diagnosis for an ill child. This is a common scenario in the developing world but is less common in developed countries. Infants are more likely to be sympto- matic than older children with pulmonary TB. The most common symptoms are cough, fever, wheezing, and failure to gain weight (13). Clinical Table 2. Cut-off size of reactive area for a positive Mantoux tuberculin reaction 5 mm 10 mm 15 mm Persons who had Foreign-born persons No risk factors contact with from high-prevalence infectious persons countries Persons with an Residents of prisons, abnormal chest nursing homes, radiograph institutions HIV-infected and Persons who inject drugs other immuno- suppressed persons Persons with other medical risk factors Health-care workers Locally identified populations at high risk Children in contact with adults at high risk Infants Synopses Vol. 1, No.

s of prisons, abnormal chest nursing homes, radiograph institutions HIV-infected and Persons who inject drugs other immuno- suppressed persons Persons with other medical risk factors Health-care workers Locally identified populations at high risk Children in contact with adults at high risk Infants Synopses Vol. 1, No. 4 -- October-December 1995 117 Emerging Infectious Diseases signs are surprisingly meager, but rales and wheezes over the affected lung field are most common. Signs and symptoms of extrapulmonary TB are referable to the involved organ. The sen- sitivity and specificity of signs and symptoms are extremely low and can lead to both overdiagnosis and underdiagnosis when radiographs and other tests are not available. The second scenario occurs when evaluating a child who has had significant contact with an adult with suspected or confirmed TB. Usually the TST is applied first and is reactive. A sub- sequent chest radiograph or physical examina- tion leads to discovery of early disease. The child is usually relatively asymptomatic. In the United States, about 50% of childhood TB cases are dis- covered in this manner (13). Radiologic Studies Evidence of pulmonary TB in chest radio- graphs varies (23,24), but usually radiographs show enlargement of hilar, mediastinal, or sub- carinal lymph nodes and lung parenchymal changes (Figure 1). Most of the radiographic ab- normalities are caused by a combination of lung disease and the mechanical changes induced by partial or complete airway obstruction resulting from enlarging intrathoracic nodes. The most common findings are segmental hyperinflation then atelectasis, alveolar consolidation, intersti- tial densities, pleural effusion, and, rarely, a focal mass. Cavitation is rare in young children but is more common in adolescents, who may develop reactivationdisease similarto that seeninadults. The development of radiographic techniques, such as computed tomography (CT) scanning, illustrates some of the issues that arise when newer and more sensitive diagnostic tests become available (24). A CT scan may show enlarged or prominent mediastinal or hilar lymph nodes in some children with recent TB infection and a normal chest radiograph (25). In the absence of a CT scan, the child's disease stage would be called TB infection, and single drug therapy would be used. Many studies, involving thousands of chil- dren have shown this treatment to be successful.

tinal or hilar lymph nodes in some children with recent TB infection and a normal chest radiograph (25). In the absence of a CT scan, the child's disease stage would be called TB infection, and single drug therapy would be used. Many studies, involving thousands of chil- dren have shown this treatment to be successful. However, when the CT scan shows mild ade- nopathy, the clinician may consider this finding indicative of TB disease and treat with several drugs, although this probably is not necessary in the absence of drug resistance. These findings reinforce the idea that pediatric TB is a continuum, and the distinction between infectionand disease is somewhat artificial.There is no current role for the CT scan in the evaluation of the asymptomatic TB-infected child with a normal chest radiograph. This scan can be helpful in selected cases to demonstrate endobronchial disease, pericardial invasion, early cavitation, and bronchiectasis resulting from pulmonary TB when the chest radiograph is abnormal but the pathologic process is not clear. Mycobacterial Detection and Isolation Despite recent advances, early mycobacteri- ologic diagnosis of TB still relies primarily on examination of acid-fast-stained smears from clinical specimens. It is the easiest, least expen- sive, and most rapid procedure for obtaining pre- liminary information. However, children under 12 years of age with pulmonary TB rarelyproduce sputum and are usually unable to expectorate voluntarily. When sputum samples cannot be ob- tained, gastric aspirate samples are used for detection and isolation of M. tuberculosis. Even though an acid-fast bacilli (AFB) stain of sputum is positive in up to 75% of adults with pulmonary TB, fewer than 20% of children with TB have a positive AFB smear of sputum or gastric aspirate (26,27). The newer fluorochrome stains, such as auramine and rhodamine, are superior to classic carbolfuchsin stains (28). The rates of positive AFB stainfrom bodyfluids andtissues in children with extrapulmonary TB also are low, and false- positive results caused by NTM disease are com- mon, especially in cervical lymph nodes. Figure 1. Chest radiograph of a girl with pulmonary tuberculosis. Note the significant hilar adenopathy in association with atelectasis, the so-called collapse-consolidation lesion. Synopses Emerging Infectious Diseases 118 Vol. 1, No. 4 -- October-December 1995 For most children with pulmonary TB, culture confirmation is not needed.

radiograph of a girl with pulmonary tuberculosis. Note the significant hilar adenopathy in association with atelectasis, the so-called collapse-consolidation lesion. Synopses Emerging Infectious Diseases 118 Vol. 1, No. 4 -- October-December 1995 For most children with pulmonary TB, culture confirmation is not needed. Diagnosis is made on the basis of a positive TST, clinical and radio- graphic findings suggestive of TB, and history of contact with an adult source case. The drug-sus- ceptibility test results from the source case isolate can be used to design the optimal treatment for the child. However, cultures should be obtained from the child if the source patient is unknown or has a drug-resistant organism and if the child is immunocompromised or has extrapulmonary TB. The best specimen for culture from children with suspected pulmonary TB is the early morn- ing gastric aspirate obtained in the hospital by using a nasogastric tube before the child arises and peristalsis empties the stomach of the respi- ratory secretions swallowed overnight (29,30). Three consecutive morning gastric aspirates yield M. tuberculosis in only 30% to 50% of cases, al- though the yield from infants is as high as 70% (14). The culture yield from other body fluids or tissues from children with extrapulmonary TB is usually less than 50% (13). Gastric aspiration is inconvenient, expensive, and uncomfortable. The culture yield from random, outpatient gastric as- pirates has not been determined recently. There- fore, this procedure cannot be recommended but should be studied. Bronchoscopy The role of bronchoscopyin evaluating children for TB is controversial. The culture yield is lower from bronchoscopy specimens than from properly obtained gastric aspirates (29,31). Most children do not need flexible fiberoptic bronchoscopy, but the procedure may be useful in diagnosing endo- bronchial TB and excluding other causes of pulmonary abnormality, particularly in immuno- compromised children, such as those with HIV infection in whom other opportunistic infections may coexist with or mimic TB. In a recent study of 36 children with pulmonary TB, bronchoscopy showed endobronchial involvement in 42%; most (63%) of these children had no clinical or radio- graphic evidence of endobronchial TB (31). This technique may be used to determine if a child might benefit from corticosteroid therapy, but guidelines for making this decision have not been established.

ary TB, bronchoscopy showed endobronchial involvement in 42%; most (63%) of these children had no clinical or radio- graphic evidence of endobronchial TB (31). This technique may be used to determine if a child might benefit from corticosteroid therapy, but guidelines for making this decision have not been established. Clinical Scoring System TB is anenormous problem indevelopingcoun- tries, where about 95% of cases occur (6). Cost, technical difficulties, and lack of resources make TB diagnosis in children very difficult in these countries. Various clinical scoring systems have been proposed on the basis of available informa- tion and tests (32,33) (Table 3). Although helpful, many of these systems have low sensitivity and specificity. However, even in industrialized countries, the triad of a positive tuberculin skin test, an abnormal radiograph, and a history of exposure to an adult with TB remains the most effective method for diagnosing TB in children. New Diagnostic Techniques Polymerase Chain Reaction (PCR) Diagnostic PCR is a technique of DNA ampli- fication that uses specific DNA sequences as markers for microorganisms (34). In theory, this technique can detect a single organism in a speci- men such as sputum, gastric aspirate, pleural fluid, cerebrospinal fluid, or blood. Recent publi- cations show that various PCR techniques, most using the mycobacterial insertion element IS6110 as the DNA marker for M. tuberculosis-complex organisms, have a sensitivity and specificity greater than 90% for detecting pulmonary TB in adults (35,36). However, these tests are not per- formed correctly in all clinical laboratories (36) Table 3. A set of criteria for the diagnosis of pulmonary tuberculosis (TB) in children when culture is not available A. Positive acid-fast stain of sputum or gastric aspirate or B. Two or more of the following: * History of contact with a tuberculous adult * Cough lasting longer than 2 weeks * A reactive tuberculin skin test 10 mm in children without prior BCG vaccination 15 mm in children with prior BCG vaccination * Radiographic findings compatible with TB * Response to anti-TB therapy (increased body weight by 10% after 2 months, decrease in symptoms) Source: ref. 33. Synopses Vol. 1, No. 4 -- October-December 1995 119 Emerging Infectious Diseases and may offer little advantage over high-quality microscopic examination of sputum (34).

graphic findings compatible with TB * Response to anti-TB therapy (increased body weight by 10% after 2 months, decrease in symptoms) Source: ref. 33. Synopses Vol. 1, No. 4 -- October-December 1995 119 Emerging Infectious Diseases and may offer little advantage over high-quality microscopic examination of sputum (34). The cost involved and the need for sophisticated equip- ment and scrupulous technique to avoid cross- contamination of specimens preclude the use of PCR techniques in many developing countries. PCR may have a special role in the diagnosis of extrapulmonary TB and pulmonary TB in chil- dren since sputum smears are usually unreveal- ing in these cases. Use of PCR for detecting M. tuberculosis in children has not been evaluated extensively. Pierre et al. (37) used an IS6110-based PCR to detect M. tuberculosis in gastric aspirate samples from 22 children with pulmonary TB. They found that 15 (25%) of 59 samples were positive; how- ever, testing multiple samples or testing samples at least twice improved the sensitivity. When three samples from the same patient were tested two times each, two or more positive results were obtained from 9 of 15 children with TB, but from 0 of 17 controls. However, 2 of 65 single samples from controls were positive by PCR. Using an IS6110-based PCR assay, Starke et al. (38) tested gastric aspirates from 35 hospitalized children with pulmonary TB and 30 controls to detect M. tuberculosis. When compared with the clinical diagnosis, PCR had a sensitivity of 40% and speci- ficity of 80%. Six controls had false-positive PCR results; one had a recent TB infection, two had NTM disease, and three had conditions unrelated to mycobacterial infection. Delacourt et al. (39) studied 199 specimens from 68 children with sus- pected TB. An IS6110-based PCR identified M. tuberculosis in clinical samples from 83% of chil- dren with disease compared to the low yield from positive AFB smears (21%) and positive cultures (42%) (39). PCR identified 70% of children with clinical pulmonary TB but no other microbiologic proof of the infection. However, 39% of children with infection but no radiographic or clinical dis- ease also had positive PCR results. These results again demonstrate the arbitrariness of the distinction between TB infection and disease in children. It appears that PCR may have a useful but limited place in evaluating children for TB.

tion. However, 39% of children with infection but no radiographic or clinical dis- ease also had positive PCR results. These results again demonstrate the arbitrariness of the distinction between TB infection and disease in children. It appears that PCR may have a useful but limited place in evaluating children for TB. A negative PCR result never eliminates TB as a diagnostic possibility, and a positive result does not confirm it. PCR's major use will be in evalu- ating children with significant pulmonary dis- ease, when the diagnosis is not easily established by clinical or epidemiologic grounds. PCR may be particularly helpful in evaluating immunocom- promised children with pulmonary disease, al- though published reports of PCR performance in such children are lacking. PCR may also aid in establishing the diagnosis of extrapulmonary TB, though only rare case reports have been publish- ed.However, performingPCR ongastric aspirates is not a useful test to distinguish between TB infection and disease and should not be used for children with normal chest radiographs. Serology and Antigen Detection Despite dozens of studies published over the past several decades, serology has found little place in the routine diagnosis of TB in adults or children. Several recent studies have used the enzyme-linked immunosorbent assay (ELISA) to detect antibodies to various purified or complex antigens of M. tuberculosis in children. Rosen (40) used mycobacterial sonicates in an ELISA on samples from 31 children with clinical TB and found a sensitivity of 26% and a specificity of 40%. This ELISA was influenced by recent BCG vacci- nation in children under 5 years of age. Barrera et al. (41) used an ELISA that detects antibodies to purified protein derivative and found a sensi- tivity of 51% for culture-positive pulmonary TB cases in children, but the sensitivitywas only 28% for the clinical cases. Hussey et al. (42) used an autoclaved suspension of M. tuberculosis to detect antibodies in serum from 132 children with clini- cal pulmonary TB; the test was 62% sensitive and 98% specific. Higher sensitivity was obtained among patients with positive culture results (69%, n=35), miliary TB (100%, n =6), tuberculous meningitis (80%, n = 15), and pleural effusion (78%, n =16). No correlation was observed with the tuberculin skin-test result, BCG vaccination, or nutritional status whereas duration of therapy, increasing age and chronicity of infection were positively correlated. Delacourt et al.

miliary TB (100%, n =6), tuberculous meningitis (80%, n = 15), and pleural effusion (78%, n =16). No correlation was observed with the tuberculin skin-test result, BCG vaccination, or nutritional status whereas duration of therapy, increasing age and chronicity of infection were positively correlated. Delacourt et al. (43) used an ELISA to detect IgG and IgM antibodies directed against mycobacterial antigen A60 in children with TB. At a chosen specificity of 98%, IgG was detected in 68% of children with clinical disease when results were highly controlled for age and prior BCG vaccination. IgM detection had only a 19% sensitivity. However, using the same anti- A60 ELISA at a defined specificity of 95%, Turneer et al. (44) found the IgG sensitivity to be 26% for past TB, 6% for asymptomatic primary Synopses Emerging Infectious Diseases 120 Vol. 1, No. 4 -- October-December 1995 TB, 14% for symptomatic TB, and 9% for NTM adenitis. No available serodiagnostic test for TB has adequate sensitivity, specificity, or reproduci- bility under various clinical conditions to be use- ful for diagnosing TB in children. Mycobacterial antigen detection has been evaluated in clinical samples from adults, but rarely from children (45,46). Two recent assays detecting M. tuberculosis-specific antigens yielded high sensitivity and specificity in various clinical specimens from adults with TB (47,48). Measurement of tuberculostearic acid, a myco- bacterial mycolic acid, has been used to detect M. tuberculosis in clinical specimens (49). Brooks et al. (50) demonstrated a sensitivity of 95% and specificity of 91% when chromatographic profile of carboxylic acids and detection of tubercu- lostearic acid were combined and compared with culture results and clinical findings in adults with pulmonary TB;however, these techniques require technically advanced equipment and expertise, which are not available where TB in children is most common. Their sensitivity and specificity in children are unknown. Implications for HIV Infection and Drug Resistance The resurgence of TB over the last decade has coincided with the HIV pandemic. HIV-infected infants and children are in close contact with their caregivers, who may be infected with HIV and M. tuberculosis and are at high risk of devel- oping infectious TB as they become immunocom- promised.

fection and Drug Resistance The resurgence of TB over the last decade has coincided with the HIV pandemic. HIV-infected infants and children are in close contact with their caregivers, who may be infected with HIV and M. tuberculosis and are at high risk of devel- oping infectious TB as they become immunocom- promised. None of the available diagnostic tests for TB infection or disease in children has been evaluated systematically in children with HIV infection and pulmonary disease or suspected TB. In adults with HIV infection and TB, the sensitiv- ity of diagnostic tests that rely on the host im- mune response, such as the TST or serology, is much lower than in nonimmunocompromised TB patients. It is likely that the tests'sensitivity also will be lower in HIV-infected children with TB. Tests that directly detect M. tuberculosis, such as PCR or antigen detection assays, may be particu- larly important for HIV-infected children. The culture yield ofM. tuberculosis from children with HIV infection and TB is unknown but appears to be similar to that from non-HIV-infected children. The most important diagnostic clue for detecting TB in HIV-infected children is a history of contact with an adult who has infectious TB. Since TB may not have yet been diagnosed in this adult, a rapid and aggressive evaluation for TB in adults who care for the child is a critical part of the evaluation of the child. The current prevalence of drug resistance among M. tuberculosis isolates in the United States is 8% to 14% (51, 52). Drug resistance is most common in patients who received treat- ment, are not responding to therapy, do not ad- here to treatment, live in developing countries, are immunocompromised, are prisoners, are homeless, or are children exposed to adults at increased risk for drug resistance. Drug-resistant TB has increased significantly among children (52). Because of low culture yields from children with TB, the clinician must often rely on the antimicrobial susceptibility results for the M. tu- berculosis isolate obtained from the adult source case who presumably infected the child. This again emphasizes the crucial need to identify and evaluate the source case for every child with TB. The rapid identification of drug-resistant organ- isms is necessary for control of drug-resistant TB.

results for the M. tu- berculosis isolate obtained from the adult source case who presumably infected the child. This again emphasizes the crucial need to identify and evaluate the source case for every child with TB. The rapid identification of drug-resistant organ- isms is necessary for control of drug-resistant TB. Various new methods, such as high-performance liquid chromatography or PCR and DNA se- quence analysis, may help to identify and test for antimicrobial susceptibility within a few days of diagnosis, but these techniques remain experi- mental. Summary Most recently developed sensitive and specific diagnostic tests have not found a place in the routine evaluation of children with suspected TB. Clinical criteria, particularly skin-test results, radiographic changes, and documented exposure to an infectious adult remain standard diagnostic methods. In industrialized countries, the local public health entity is a crucial partner to the clinician in establishing the diagnosis in the child and determining if drug resistance is present. As new diagnostic tests are developed, they must be evaluated against clinical criteria. The basic dif- ferences in pathophysiology of TB in adults and children must be considered before new tests are applied inpediatrics. It will be crucial to study the new techniques in children and not simply ex- trapolate from results for adults with TB. Dr. Khan is a postgraduate fellow in pediatric infectious diseases at Baylor College of Medicine, Houston, Texas. Dr. Starke is an associate professor at Baylor College of Medicine and current chairman of Synopses Vol. 1, No. 4 -- October-December 1995 121 Emerging Infectious Diseases CDC's Advisory Committee for the Elimination of Tuberculosis. References 1. Starke JR, Correa AG. Management of mycobacterial infection and disease in children. Pediatr Infect Dis J 1995;14:455-70. 2. Styblo K, Rouillon A. Tuberculosis in developing coun- tries: burden, intervention and cost. Bull Int Union Against Tuber Lung Dis 1990;65:6-24. 3. Starke JR, Jacobs R, Jereb J. Resurgence of tubercu- losis in children. J Pediatr 1992;120:839-55. 4. Cantwell M, Snider D Jr, Cauthen G, Onorato I. Epidemiology of tuberculosis in the United States, 1985 through 1992. JAMA 1994;272:535-9. 5. Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organiza- tion. Tuber Lung Dis 1991;72:1-6. 6. RaviglioneMC, Snider DE, Kochi A.Global epidemiol- ogy of tuberculosis.

n G, Onorato I. Epidemiology of tuberculosis in the United States, 1985 through 1992. JAMA 1994;272:535-9. 5. Kochi A. The global tuberculosis situation and the new control strategy of the World Health Organiza- tion. Tuber Lung Dis 1991;72:1-6. 6. RaviglioneMC, Snider DE, Kochi A.Global epidemiol- ogy of tuberculosis. Morbidity and mortality of a worldwide epidemic. JAMA 1995;273:220-6. 7. Dolin P, Raviglione M, Kochi A. Global tuberculosis incidence and mortality during 1990-2000. Bull World Health Organ 1994;72:213-20. 8. Barnes, PF Borrows, SA. Tuberculosis in the 1990s. Ann Intern Med 1993;119:400-10. 9. Hsu KHK. Contact investigation: a practical ap- proach to tuberculosis eradication. Am J Public Health 1963;53:1761-9. 10. Nolan RJ Jr. Childhood tuberculosis in North Caro- lina: a study of the opportunities for intervention in the transmission of tuberculosis to children. Am J Public Health 1986;76:26-30. 11. Brailey ME. Tuberculosis in white and negro children. II. The epidemiologic aspects of the Harriet Lane study. Cambridge, MA: Harvard University Press, 1958. 12. Steiner P, Rao M, Victoria MS, et al. Persistently negative tuberculin reactions: their presence among children culture positive for M. tuberculosis. Am J Dis Child 1980;134:747-50. 13. Starke JR, Taylor-Watts KT. Tuberculosis in the pedi- atric population of Houston, Texas. Pediatrics 1989;84:28-35. 14. Vallejo J, Ong LT, Starke JR. Clinical features, diag- nosis and treatment of tuberculosis in infants. Pedi- atrics 1994;94:1-7. 15. Lifschitz M. The value of the tuberculin skin test as a screening test for tuberculosis among BCG-vacci- nated children. Pediatrics 1965;36:624-7. 16. Landi S, Ashley MJ, Grzybowski S. Tuberculin sensi- tivity following the intradermal and puncture meth- ods of BCG vaccination. Can Med Assoc J 1967;97:222-5. 17. Joncas JH, Robitaille R, Gauthier T. Interpretation of the PPD skin test in BCG-vaccinated children. Can Med Assoc J 1975;113:127-8. 18. Johnson H, Lee B, Kelly E, McDonnell T. Tuberculin sensitivity and the BCG scar in tuberculosis contacts. Tuber Lung Dis 1995;35:113-7. 19. Menzies R, Vissandjee B. Effect of bacille Calmette- Guerin vaccination on tuberculin reactivity. Am Rev Respir Dis 1992;141:621-5. 20. Sepulveda RL, Burr C, Ferrer X, Sorensen RU. Booster effect of tuberculin testing in healthy 6-year- old school children vaccinated with bacille Calmette- Guerin at birth in Santiago, Chile. Pediatr Infect Dis J 1988;7:578-82. 21. American Thoracic Society.

n tuberculin reactivity. Am Rev Respir Dis 1992;141:621-5. 20. Sepulveda RL, Burr C, Ferrer X, Sorensen RU. Booster effect of tuberculin testing in healthy 6-year- old school children vaccinated with bacille Calmette- Guerin at birth in Santiago, Chile. Pediatr Infect Dis J 1988;7:578-82. 21. American Thoracic Society. Diagnostic standards and classification of tuberculosis. Am Rev Respir Dis 1990;142:725-35. 22. American Academy of Pediatrics Committee on Infec- tious Diseases. Screening for tuberculosis in infants and children. Pediatrics 1994;93:131-4. 23. Schaaf HS, Beyers N, Gie RP, et al. Respiratory tuber- culosis in childhood: the diagnostic value of clinical features and special investigations. Pediatr Infect Dis J 1995;14:189-94. 24. Parisi MT, Jensen MC, Wood BP. Pictorial review of the usual and unusual roentgen manifestations of childhood tuberculosis. Clin Imag 1994;18:149-54. 25. Delacourt C, Mani TM, Bonnerot V, et al. Computed tomography with normal chest radiograph in tuber- culous infection. Arch Dis Child 1993;69:430-2. 26. Strumpf IJ, Tsang AY, Syre JW. Reevaluation of spu- tum staining for the diagnosis of pulmonary tubercu- losis. Am Rev Respir Dis 1979;119:599-602. 27. Lipsky BA, Bates J, Tenover FC, Plorde JJ. Factors affecting the clinical value of microscopy for acid-fast bacilli. Rev Infect Dis 1984;6:214-22. 28. Kent PT, Kubica GP. Public health mycobacteriology - a guide for the level III laboratory. Atlanta, GA; Centers for Disease Control, 1985. 29. Abadco DL, Steiner P. Gastric lavage is better than bronchioalveolar lavage for isolation of Mycobac- terium tuberculosis in childhood tuberculosis. Pediatr Infect Dis J 1992;11:735-8. 30. Carr DT, Karlson AG, Stillwell AA. A comparison of cultures of induced sputum and gastric washings in the diagnosis of tuberculosis. Mayo Clinic Proc 1967;42:23-5. 31. Chan S, Abadco DL, Steiner P. Role of flexible fiberop- tic bronchoscopy in the diagnosis of childhood endo- bronchial tuberculosis. Pediatr Infect Dis J 1994;13:506-9. 32. Glidey Y, Hable D. Tuberculosis in childhood: an analysis of 412 cases. Ethiop Med J 1983;21:161-7. 33. Migliori AB, Borghesi A, Rossanigo P et al. Proposal for an improved score method for the diagnosis of pulmonary tuberculosis in childhood in developing countries. Tuber Lung Dis 1992;73:145-9. 34. Schluger NW, Rom WN. Current approaches to the diagnosis of active pulmonary tuberculosis. Am J Respir Crit Care Med 1994;149:264-7. 35.

orghesi A, Rossanigo P et al. Proposal for an improved score method for the diagnosis of pulmonary tuberculosis in childhood in developing countries. Tuber Lung Dis 1992;73:145-9. 34. Schluger NW, Rom WN. Current approaches to the diagnosis of active pulmonary tuberculosis. Am J Respir Crit Care Med 1994;149:264-7. 35. Eisenach KD, Sifford MD, Cane MD, Bates JH, Craw- ford JT. Detection of Mycobacterium tuberculosis in sputum samples using a polymerase chain reaction. Am Rev Respir Dis 1991;144:1160-3. 36. Noordhock A, Kolk A, Bjune G, et al. Sensitivity and specificity of polymerase chain reaction for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. J Clin Microbiol 1994;32:277-84. Synopses Emerging Infectious Diseases 122 Vol. 1, No. 4 -- October-December 1995 37. Pierre C, Oliver C, Lecossier D, Bousssougant Y, Yemi P, Hance AJ. Diagnosis of primary tuberculosis in children by amplification and detection of mycobacte- rial DNA. Am Rev Respir Dis 1993;147:420-4. 38. Starke JR, Ong LT, Eisenach KD, et al. Detection of M. tuberculosis in gastric aspirate samples from chil- dren using polymerase chain reaction. Am Rev Resp Dis 1993;147(Suppl):A801. 39. Delacourt C, Poveda J-D, Churean C, et al. Use of polymerase chain reaction for improved diagnosis of tuberculosis in children. J Pediatr 1995;126:703-9. 40. Rosen EU. The diagnostic value of an enzyme-linked immunosorbent assay using adsorbed mycobacterial sonicates in children. Tubercle 1990;71:127-30. 41. Barrera L, Miceli I, Ritacco V, et al. Detection of circulating antibodies to purified protein derivative by enzyme-linked immunosorbent assay: its potential for the rapid diagnosis of tuberculosis. Pediatr Infect Dis J 1989;8:763-7. 42. Hussey G, Kibel M, Dempster W. The serodiagnosis of tuberculosis in children: an evaluation of an ELISA test using IgG antibodies to M. tuberculosis, strain H37RV. Ann Trop Paediatr 1991;11:113-8. 43. Delacourt C, Gobin J, Gaillard J-L, de Blic J, Veran M, Scheinmann P. Value of ELISA using antigen 60 for the diagnosis of tuberculosis in children. Chest 1993;104:393-8. 44. Turneer M, Nerom EV, Nyabenda J, Waelbroeck A, Duvivier A, Toppet M. Determination of humoral im- munoglobulins M and G directed against mycobacte- rial antigen 60 failed todiagnoseprimarytuberculosis and mycobacterial adenitis in children. Am J Respir Crit Care Med 1994;150:1508-12. 45. Sada E, Ruiz-Palacios AM, Lopez-Vidal Y, et al.

EV, Nyabenda J, Waelbroeck A, Duvivier A, Toppet M. Determination of humoral im- munoglobulins M and G directed against mycobacte- rial antigen 60 failed todiagnoseprimarytuberculosis and mycobacterial adenitis in children. Am J Respir Crit Care Med 1994;150:1508-12. 45. Sada E, Ruiz-Palacios AM, Lopez-Vidal Y, et al. Detec- tion of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis by enzyme- linked immunosorbent assay. Lancet 1983;2:651-2. 46. Radhakrishnan VV, Sehgal S, Mathai A. Correlation between culture of Mycobacterium tuberculosis and detection of mycobacterial antigens in cerebrospinal fluid of patients with tuberculous meningitis. J Med Microbiol 1990;33:223-6. 47. WadeeAA, BolingL, Reddy SG. Antigen captureassay for detection of a 43-kilodalton Mycobacterium tuber- culosis antigen. J Clin Microbiol 1990;28:2786-91. 48. Sada E, Aguilar D, Torres M, et al. Detection of lipoarabinomannan as a diagnostictest for tuberculo- sis. J Clin Microbiol 1992;30:2415-18. 49. Brooks JB, Daneshvar MI, Fast DM, et al. Selective procedures for detecting femtomole quantities of tu- berculostearic acid in serum and cerebrospinal fluid by frequency-pulsed electron-capture gas-liquid chro- matograph. J Clin Microbiol 1987;25:1201-6. 50. Brooks JB, Daneshvar MI, Harberger RL, et al. Rapid diagnosis of tuberculous meningitis by frequency- pulsed electron-captive gas-liquid chromatography detection of carboxylic acids in cerebrospinal fluid. J Clin Microbiol 1990;28:989-97. 51. Centers for Disease Control and Prevention. National action plan to combat multidrug-resistant tuberculo- sis. MMWR 1992;41:5-50. 52. Bloch AB, Cauthen GM, Onorato IM, et al. Nation- wide survey of drug-resistant tuberculosis in the United States. JAMA 1994;271:665-71. Synopses Vol. 1, No. 4 -- October-December 1995 123 Emerging Infectious Diseases

Data Management Issues for Emerging Diseases and New Tools for Managing Surveillance and Laboratory Data Stanley M. Martin, M.S., Nancy H. Bean, Ph.D. Centers for Disease Control and Prevention, Atlanta, Georgia, USA Data Management Issues for Emerging Diseases Since 1976, when Legionnaires' disease af- fected attendees at the American Legion Conven- tion in Philadelphia (1), the scope of public health has expanded. During the 1976 outbreak investi- gation, public attention was drawn to news ac- counts of the increasing numbers of cases and deaths as well as to speculations about diseases causes and prevention. After the outbreak, public health officials contended with volumes of infor- mation, including clinical data, epidemiologic survey results, and records of specimens collected from patients and the environment. This informa- tion was managed on mainframe computers. In 1980, a cluster of cases of unrecognized illness, primarily affecting young women, created a data management situation similar to that sur- rounding the Legionnaires' disease outbreak. A major epidemiologic investigation, which included examining a multitude of laboratory specimens and analyzing volumes of data, was undertaken by a large team of federal, state, and local public health officials, as well as numerous academic institutions and private industries. The problems with establishing databases and implementing a data management system for toxic shock syn- drome (2) were essentially the same as the data management problems of Legionnaires' disease, except that computer technology had crept for- ward slightly in public health offices. During the spring of 1993, a cluster of cases of another unknown illness, eventually attributed to hantavirus (3), occurred in the southwestern United States. The reaction to this unknown dis- ease by public health officials reflected a startling fact: even though the epidemiologic and labora- tory methods for curtailing the outbreak were in place, a consistent data management strategy had not been established. Ad hoc databases built by outbreak investigators for a multitude of pur- poses began to bog down the investigation.

alth officials reflected a startling fact: even though the epidemiologic and labora- tory methods for curtailing the outbreak were in place, a consistent data management strategy had not been established. Ad hoc databases built by outbreak investigators for a multitude of pur- poses began to bog down the investigation. Cases were recorded in multiple databases that did not recognize duplicate reports of cases. Updates of data about cases were done in some, but not all, databases. Laboratory data about specimens from patients were not linked to other clinical and epidemiologic data about a patient. No single database was available with well-edited, com- plete data about all the cases. Parallel, frag- mented data management efforts evolved in at least 15 locations, with no coordinated mecha- nism to integrate them into one system. Introducing a single system for data manage- ment in the midst of the hantavirus outbreak involved more than the data management issues encountered in the earlier outbreaks. Previously, computer technology was viewed as a solution that, although somewhat cumbersome, enabled officials to move from data management by hand to electronic management. However, during the hantavirus outbreak, computer technology be- came part of the problem; it initially prevented good data management and may have hindered some of the laboratory and epidemiologic efforts to control the outbreak. Data were essentially being locked into various databases and could not be adequately analyzed or merged with data in other databases. In some instances, this peculiar circumstance caused investigators to perform analyses by hand using printouts from electronic databases or entering data again into other sys- tems. In recent years, legal considerations, such as the Privacy Act enacted in 1974 and the Freedom of Information Act enacted in 1966 (4,5), have also complicated data management. These acts, in their efforts to protect individual privacy and ensure availability of data, have in some cases, constrained public health responses to emergency situations and subsequent surveillance efforts by enforcing strict database design and handling requirements.

966 (4,5), have also complicated data management. These acts, in their efforts to protect individual privacy and ensure availability of data, have in some cases, constrained public health responses to emergency situations and subsequent surveillance efforts by enforcing strict database design and handling requirements. Data Management Requirements In epidemiologic investigations, disease prob- lems are generally characterized by person, place, and time, whether the problem concerns the emergence of a new disease, a change in the resistance pattern of a known pathogen, an emer- gency response to an outbreak, or a routine Synopses Emerging Infectious Diseases 124 Vol. 1, No. 4 -- October-December 1995 disease surveillance program. The principles of data gathering, management, and analysis are essentially the same for all these purposes. Com- puter systems developed to manage data associ- ated with these problems should be regarded as tools for the epidemiologic characterization of pathogens, syndromes, cases, and risk factors. Therefore, laboratory data management and re- porting systems must be able to handle data about all of these. The most stringent requirements for data management are imposed by data from labora- tory testing of specimens from patients, human and nonhuman sources, and the environment. A system having a relational data model adequate to properly handle the laboratory data require- ments will almost certainly be adequate to handle the clinical, exposure, and demographic data re- quirements. Two primary data management functions can satisfy the laboratory data demands with multi- ple requirements in each function. The first func- tion, internal laboratory data management, consists of entering test results and tracking specimens. The second, surveillance, includes gathering data and moving data beyond the elec- tronic files of the laboratory to appropriate sites for analysis. A data management system should be able to perform these functions not only during an outbreak but throughout the period of surveil- lance as well.

and tracking specimens. The second, surveillance, includes gathering data and moving data beyond the elec- tronic files of the laboratory to appropriate sites for analysis. A data management system should be able to perform these functions not only during an outbreak but throughout the period of surveil- lance as well. The internal laboratory function, universally similar among most public health laboratories, includes data entry tailored for individual labora- tories at the site; retrieval/query ability; and abil- ity to add or delete tests, manage aliquots, share data input in different laboratories of the site, track the status of every specimen regardless of which laboratory tested it, develop reports for specimen submitters, and in some cases assign costs for laboratory tests performed and prepare invoices for submitters. Requirements for the surveillance function in- clude, in addition to certain critical laboratory data, the following facilities: to record clinical, exposure/risk factor, and demographic data about patients; to include data about multiple speci- mens and aliquots related to the same person, regardless of the interval separating the specimen dates; and to change questions or test results that are recorded for each specimen. Although internal and surveillance functions are clearly separate, they are not independent. Data entered into databases for the internal func- tion should be available without additional effort for the surveillance function. In fact, when the internal function is not electronic or when the internal electronic system is inadequate, the sys- tem performing electronic surveillance should also perform to some extent the internal func- tions. Good laboratory data management does not address the internal function at the exclusion of the surveillance function. If a laboratory data management system is to be useful for emergency situations, it must pro- vide mechanisms for adapting quickly to the emergency situation. For example, it must pro- vide a way to immediately create an electronic data collection instrument and to incorporate this new instrument into the system at all reporting sites electronically. For the surveillance function, these electronic features must include communi- cations facilities to move data electronically from one location to another, mechanisms for sending messages or files, functions for simple analysis, and methods for preparing and printing reports.

stem at all reporting sites electronically. For the surveillance function, these electronic features must include communi- cations facilities to move data electronically from one location to another, mechanisms for sending messages or files, functions for simple analysis, and methods for preparing and printing reports. While some systems perform some of these func- tions, most systems do not provide all of them. With appropriate systems in hand, data man- agement plans for both urgent and routine events can be approached in a sequential fashion. With consensus among all participating investigators, epidemiologists must decide what data (both laboratory and epidemiologic) are needed so that data field characteristics can be defined. Consen- sus should be reached in the early phase of the outbreak investigation; otherwise participants in the investigation will of necessity begin develop- ing ad hoc data management systems. The more thoroughly and carefully this task is performed, the more stable the data will ultimately become. In a well-designed system, the initial defini- tions in an emergency situation can include pro- jections about which data fields will be needed. However, for routine surveillance these can be more thoroughly planned. Thus, the data system should allow fields to be deleted if not needed and to be added if they become important. These modifications should 1) be handled without hav- ing to alterthe system,2) use simple menu-driven functions requiring no computer programmer intervention, 3) accomplish the changes Synopses Vol. 1, No. 4 -- October-December 1995 125 Emerging Infectious Diseases immediately, 4) be distributed to all investigators without disrupting their other functions during the investigation, and 5) be incorporated auto- matically. Next, all known participants in the investiga- tion must be identified. These should include local, state, and federal officials as well as aca- demic or private participants who may provide reports to the central data repository. These par- ticipants must be identified to the system specifi- cally by person and by site for system security. Appropriate state and federal offices should be informed concerning the computer system and the rules for its use well before an emergency occurs; therefore, sites will be on the system in advance of an urgent problem. However, the sys- tem must allow for additional sites to be added quickly.

site for system security. Appropriate state and federal offices should be informed concerning the computer system and the rules for its use well before an emergency occurs; therefore, sites will be on the system in advance of an urgent problem. However, the sys- tem must allow for additional sites to be added quickly. In an emergency, a temporary agreement must be drawn for all participants to cooperate with the demands of the situation, i.e., to use a particular software system and operate under a standard set of rules for collecting and reporting data for the emergency. This agreement may oc- casionally stipulate that participants share data temporarily in a common database for the sake of data integrity. Entering clinical, epidemiologic/risk factor, and laboratory data about the same cases into the same database, rather than merging separate databases after the data are collected, provides such great payoffs in time savings and data integ- rity that the effort to obtain cooperation for a common database during an urgent situation is worthwhile. Although merging multiple data- bases during routine surveillance is feasible, emergency situations do not lend themselves to this type of data management. Therefore, the system to be used for these situations must ac- commodate a common database and provide a means of connecting the reporting sites to the database. When the reporting system is activated and data begin arriving at a central location, the system should facilitate analysis at every report- ing site and provide a mechanism to export data (e.g., ASCII or .dbf files) for external analysis. Emergency situations create unusual de- mands for epidemiologic and laboratory re- sources; therefore, data management should not disrupt or threaten to divert resources devoted to these other purposes. As the system is imple- mented, before emergencies occur, discussions of the resources required should be held with participants. Participants must devote some re- sources to data management, but these should be minimized. This is consistent with implementing a single system in the beginning of the outbreak investigation and continuing with it into the rou- tine surveillance follow-up. Incorporating data into a second system for surveillance could waste resources.

devote some re- sources to data management, but these should be minimized. This is consistent with implementing a single system in the beginning of the outbreak investigation and continuing with it into the rou- tine surveillance follow-up. Incorporating data into a second system for surveillance could waste resources. Although, internal data management does not need to change to accommodate an outbreak, laboratories must implement systems that can directly feed data into the master reporting sys- tem database, either through an import function contained in the master system or by a direct interface between the internal laboratory system and the surveillance reporting system. Data management considerations during out- break investigations and surveillance in the United States include the political concerns of the participants. Political and legal constraints of all participants must be addressed before the need to deal with them arises. On a global scale, this consideration is equally important, especially in countries whose economies may be adversely af- fected by news of a dangerous disease situation. Individual country sovereignty must not be vio- lated by data reporting, and the cooperation of each participating country or political entity (e.g., World Health Organization [WHO], Pan Ameri- can Health Organization [PAHO]) must be ob- tained in an atmosphere of confidentiality. All attempts to obtain, share, or combine data on a regional or global basis must include a well-de- fined set of rules agreed upon by all participants. For example, data forscientific purposes might be received at an office of WHO or PAHO but not sent beyond these organizations. Most often, for the sake of surveillance on a regional or global scale, data management consid- erations must focus first on establishing in-coun- try data management infrastructures. This means that regional or global surveillance will first translate into establishing a master system, or at least compatible systems in individual par- ticipating countries. In most cases, data manage- ment systems available to developing countries do not provide the relational model needed by the laboratory. Therefore, efforts should be initiated to introduce and establish systems that can meet these needs in countries desiring to use them. A plan for regional or global surveillance must include tools to respond to outbreaks and provide Synopses Emerging Infectious Diseases 126 Vol. 1, No.

l model needed by the laboratory. Therefore, efforts should be initiated to introduce and establish systems that can meet these needs in countries desiring to use them. A plan for regional or global surveillance must include tools to respond to outbreaks and provide Synopses Emerging Infectious Diseases 126 Vol. 1, No. 4 -- October-December 1995 for the computing equipment and modems or other means of transmitting the data electroni- cally. Today's environment demands that most data management be done on personal computers located at critical sites where data can be input. However, data volume may ultimately require that the system provide for archiving data onto another medium. This does not preclude the use of personal computers for data management but simply recognizes that current technology limits the volume of data that can practically be man- aged and analyzed on personal computers. The initial data management plan for a coun- try should include a section on reporting proce- dures and the appropriate medium for archiving data. To handle an immediate, urgent situation the system should contain, at a minimum, a per- sonal computer with large hard-disk capacity (at least 1-2 gigabytes at the central level and possi- bly 300-500 megabytes at each reporting site), large memory (at least 4 megabytes of RAM at every reporting site), adequate speed (at least 33 megahertz at every reporting site), and fast mo- dems if appropriate. For sites located in areas with inadequate telephone lines, other provisions for electronic transmissions should be planned (e.g., diskettes). Until security can be assured on the Internet, we do not recommend using this medium for electronic transmission of laboratory clinical data for outbreak investigations and sur- veillance. New Tools for the Management of Surveillance and Laboratory Data The Public Health Laboratory Information System (PHLIS) To address the need for a data management system for outbreak investigations and surveil- lance, the National Center for Infectious Dis- eases, CDC, in cooperation with the Association of State and Territorial Public Health Laboratory Directors in the United States, developed PHLIS. With this system, data entry screens (modules) are created and distributed to all reporting sites electronically, and data are input and reported within hours, without involving computer pro- grammers.

ith the Association of State and Territorial Public Health Laboratory Directors in the United States, developed PHLIS. With this system, data entry screens (modules) are created and distributed to all reporting sites electronically, and data are input and reported within hours, without involving computer pro- grammers. PHLIS provides the capacity for a hierarchical reporting scheme involving reports to multiple, successively higher reporting levels; a database is created at every reporting level so that all data reported to a site or input at the site are included in the database at that site. The most recent version of PHLIS (Version 3.0), is a menu-driven system based on a rela- tional data model sufficient for the needs outlined in the first part of this report. The system allows for a patient record to be input only one time and links multiple specimens for that patient record. This is true even if specimens forthe same patient are entered in different disease modules, or if the patient's name is to be added into a module that contains only epidemiologic data (no laboratory specimens). PHLIS provides a core set of data to be collected on every patient. In addition, each disease module can be customized by adding ad- ditional fields to the core data if needed. The system can accommodate data for epidemiologic, laboratory, survey, and case-control studies, and for other public health needs. Field staff canrapidly add their own data fields to existing disease modules to customize the data entry for special needs at each data reporting site. During an outbreak, a new module can be rapidly developed and electronically transmitted to all participating reporting sites. The system, which includes data communica- tion software, is configured so that data flow in a pyramid reporting structure: that is, data are reported from lower level reporting sites through higher level reporting sites and ultimately to a single central site. As data are passed to each successively higher level, they are automatically assimilated into that site's database. Thus, data- bases are built and updated at successively higher reporting sites. Additional information about a case or specimen may be added at any reporting site; if desired, these additional data are also transmitted to the next higher reporting site.

vel, they are automatically assimilated into that site's database. Thus, data- bases are built and updated at successively higher reporting sites. Additional information about a case or specimen may be added at any reporting site; if desired, these additional data are also transmitted to the next higher reporting site. To meet the need for feedback, PHLIS has a menu-driven option to transmit files or messages up and down the reporting chain, with these files and messages being transmitted automatically when connections are established for each data transmission. This facility is flexible enough to allow any valid user in the reporting chain to transmit files or messages to anyother user in the reporting chain. For example, in the United States, a county health official who is included in the reporting system in one state can send mes- sages or files to a participating county official in Synopses Vol. 1, No. 4 -- October-December 1995 127 Emerging Infectious Diseases another state. The feedback system does not mimic electronic mail because these files and messages are sent along the reporting chain in the same communications configuration as data reporting. Therefore, successful arrival of these messages at their destination(s) depends upon each member of the reporting chain between the sender and the receiver to establish a connection for reporting purposes. However, the system pro- vides an alternative mechanism for sending files and messages directly to any other reporter hav- ing the capacity to receive them without going through the reporting chain. PHLIS is used in all 50 state public health laboratories, as well as the District of Columbia and Guam. Disease modules included are animal rabies, Campylobacter, Escherichia coli O157:H7, Lyme disease, mycobacteria, respiratory and en- teric viruses, humanSalmonella, nonhuman Sal- monella, Shigella, and drug-resistant Strepto- coccus pneumoniae. PHLIS can be implemented independently: or- ganizations can develop their own PHLIS pyra- mid reporting system. For example, PHLIS is currently being implemented at the Caribbean Epidemiology Center (CAREC) in Trinidad and in its member countries for the reporting of HIV/STD infections with the expectation that the reporting system will be expanded to accommo- date other diseases. CAREC can receive reports from the member countries as each country is added to the reporting structure.

Caribbean Epidemiology Center (CAREC) in Trinidad and in its member countries for the reporting of HIV/STD infections with the expectation that the reporting system will be expanded to accommo- date other diseases. CAREC can receive reports from the member countries as each country is added to the reporting structure. Laboratory Information Tracking System (LITS) The second system, LITS, is a PC local area network-based system for tracking laboratory specimens. The system allows specimen informa- tion to be entered at a central specimen receiving site; additional information about the specimen can be entered into the system in any of the laboratories performing tests on that specimen. Although modules are customized for each labo- ratory's needs, laboratorians can add additional tests or delete obsolete ones. Furthermore, users can examine all the data about a specimen, in- cluding data from all laboratories that performed tests on the specimen. Other features in the sys- tem include cost billing, userdefined reports, user defined query, and specimen or patient tracking and security. For emerging diseases, LITS pro- vides a mechanism to standardize laboratory pro- tocol across organizations and a mechanism to share data about specimens within an organiza- tion. Acknowledgments We thank Tim Kuhn for leading the programming team; Bruce Wilson, Dana Crenshaw, Joe Bates,and Neil Jones for programming support; Tim Day for user support; Kathleen Maloney, Joy Goulding, Lori Hutwagner, and Cecile Ivey for evaluating program integrity; Brian Plikaytis for his early involvement with LITS; and Cheryl Shapiro for financial management. References 1. Fraser DW, Tsai TR, Orenstein W, Parkin WE, Beecham HJ, Sharrar RG. Legionnaires' disease: de- scription of an epidemic of pneumonia. N Engl J Med 1977;297:1189-97. 2. Shands KN, Schlech WF III, Hargrett NT, Dan BB, Schmid GP, Bennett JV. Toxic shock syndrome: case- control studies at the Centers for Disease Control. Ann Intern Med 1982;96:895-8. 3. CDC. Outbreak of acute illness--southwestern United States, 1993. MMWR 1993;42:421-4. 4. AdministrativeConferenceof the U.S. PrivacyAct. In: Federal Administrative Procedure Sourcebook, 2nd ed. Office of the Chairman, 1992:863-979. 5. Administrative Conference of the U.S. Freedom of Information Act. In: Federal Administrative Proce- dure Sourcebook, 2nd ed. Office of the Chairman, 1992:633-61. Synopses Emerging Infectious Diseases 128 Vol. 1, No. 4 -- October-December 1995

Helicobacter hepaticus, a Recently Recognized Bacterial Pathogen, Associated with Chronic Hepatitis and Hepatocellular Neoplasia in Laboratory Mice Gastric carcinoma, one of the most prevalent human cancers worldwide, is among the neo- plasms for which epidemiologic evidence of envi- ronmental causes is strongest. The exact nature of these environmental causes was obscure until mounting evidence recently linked chronic infec- tion of the gastric antrum mucosa by Helicobacter pylori (a microaerobic, gram-negative, spiral bac- terium) with elevated cancer risk (1). It is now recognized that gastric B-cell lymphoma of mu- cosa-associated lymphoid tissue is also closely linked to gastric H. pylori infection, and eradica- tion of the infection with antibiotics can result in regression of the lymphoma (2,3). This startling finding has stimulated intense interest in the ge- nus Helicobacter and related organisms; as a re- sult, additional species of Helicobacter are now frequently isolated and characterized from many non-human hosts. Until 1994, however, only H. pylori was known to be associated with tumor development, in humans or in any other animal species. In 1992, at the National Cancer Institute's Frederick Cancer Research and Development Center (FCRDC) in Frederick, Maryland, a high prevalence of liver disease was observed among certain strains of mice; these mice were untreated controls in long-term chemical carcinogenesis ex- periments. Affected strains, notably A/JCr, had been bred at FCRDC under pathogen-free condi- tions and were free of known serologically detect- able murine viruses and parasites; moreover, they had no histologically demonstrable hepatic abnor- malities, except fora very low incidence (1% to 2%) of hepatocellular tumors in mice 15 months of age or older. Over a very short period, the prevalence of a histologically distinctive form of hepatitis increased to virtually 100% in male mice at 1 year of age (Table 1). The earliest demonstrable lesions were small, undistinctive foci of hepatic necrosis seen in young mice aged 2 to 6 months.

s in mice 15 months of age or older. Over a very short period, the prevalence of a histologically distinctive form of hepatitis increased to virtually 100% in male mice at 1 year of age (Table 1). The earliest demonstrable lesions were small, undistinctive foci of hepatic necrosis seen in young mice aged 2 to 6 months. In older mice, aged 6 to 10 months, there was a highly distinctive pericholangitis, consisting of abundant mononuclear cell infiltrates around bile ducts withinportal triads. The biliary epithelium within affected ducts was focallyswollen, and theluminal surfaces of damaged epithelial cells were poorly defined in hematoxylin and eosin-stained sections (4,5). In livers withextensive lesions, bile ductular (oval cell) hyperplasia was also prominent. More- over, mice with hepatitis usually had hepatocellu- lar tumors, often multiple, that included both adenomas and carcinomas (4). Hepatocellular tumors in mice are one of the most common endpoints in bioassays for chemical carcinogens. They were not, at that time, known to be associated with infectious agents. Accord- ingly, initial efforts to identify the cause of the hepatitis/hepatocellular tumor syndrome were di- rected toward possible sources of chemical expo- sure. The possibility of accidental exposure to experimental substances within the research ani- mal facilities was ruled out when liver disease was identified in mice that had never left the breeding areas which are located in separate buildings. Extensive chemical analyses of food, bedding, water, and other possible sources of toxic sub- stances had negative results. Detailed pathologic examination by light mi- croscopy of tissue sections from diseased livers was continued, and many special stains were used. One such stain, Steiner's silver impregna- tion procedure for spirochetes (6), revealed in he- patic tissue uniform bodies that were consistent in size and shape with bacteria. Homogenates of fresh liver tissue from diseased mice proved effec- tive in transmitting hepatitis to A/J mice pur- chased from commercial sources outside FCRDC, when given by intraperitoneal injection (5). In addition, from these homogenates, a motile, spiral bacterium could be cultivated on blood agar plates incubated at 37o C under anaerobic or microaero- bic conditions. This organism was subsequently characterized by ultrastructural morphologic examination, bio- chemical characteristics, and 16S rRNA gene se- quence. Determined to be a new species related to H.

e, spiral bacterium could be cultivated on blood agar plates incubated at 37o C under anaerobic or microaero- bic conditions. This organism was subsequently characterized by ultrastructural morphologic examination, bio- chemical characteristics, and 16S rRNA gene se- quence. Determined to be a new species related to H. pylori, it was given the name H. hepaticus (7). The bacterium is motile and gram negative, 0.2 to 0.3 m in diameter, 1.5 to 5.0 m long, and curved to spiral in shape, with one to several spirals; it has bipolar sheathed flagella (one at each end) but Dispatches Emerging Infectious Diseases 129 Vol. 1, No. 4 -- October-December 1995 lacks the periplasmic fibers that envelope the bac- terial cells in other mouse Helicobacter species. H. hepaticus has strong urease activity, is oxidase and catalase positive, produces H2S, reduces ni- trate to nitrite, and grows microaerobically at 37o C but not at 25o C or 42o C. It is resistant to cephalothin and nalidixic acid but sensitive to metronidazole. Photographs illustrating its mor- phologic structure by light (4,5) and electron (4,5,7) microscopy have been published. The spe- cies-defining characteristic of the organism, the nucleotide sequence of its 16S rRNA gene, has been used to develop a diagnostic assay based on polymerase chain reaction (8). Systematic examination of rodents of all spe- cies and strains produced at FCRDC, especially retired breeders, showed that the characteristic hepatitis and associated bacteria were present in mice of several strains (A/JCr, DBA/2NCr, C3H/HeNCr) and that within these strains, the male mice were more severely affected than the female. Mice with severe combined immunodefi- ciencies were especially vulnerable. The precise location of organisms demonstrable by Steiner stainwithin infected liver parenchyma was shown by transmission electron microscopy to be invari- ably extracellular and characteristically within bile canaliculi (4,5). No liver disease was seen in some strains (e.g., C57BL/6NCr) or in F1 hybrids between sensitive and resistant strains (e.g., B6C3F1). Rodent species other than mice (e.g., rats, Syrian hamsters, and guinea pigs) were not affected. In infected mice with severe combined immu- nodeficiency, cecal inflammation was histologi- cally demonstrable (5), and organisms were isolated from the mucosa of the large intestine (7), which may mean that the usual ecologic niche occupied by H. hepaticus is that of a commensal colonizer of the intestinal tract (8).

In infected mice with severe combined immu- nodeficiency, cecal inflammation was histologi- cally demonstrable (5), and organisms were isolated from the mucosa of the large intestine (7), which may mean that the usual ecologic niche occupied by H. hepaticus is that of a commensal colonizer of the intestinal tract (8). Since mice are coprophagic, it appears highly likely that natural transmission of the organisms is the oral-fecal route. Why and howH. hepaticus invades the liver in mice of certain strains remainto be determined. Hepatitis is also characteristic of certain other enteric pathogenic bacteria, such as Campylobac- ter jejuni (9) that, unlike H. hepaticus, have not been associated with liver tumor development. The tissue damage that accompanies persistent infection by H. hepaticus, H. pylori, and certain other Helicobacter species may be due, at least in part, to a soluble, trypsin-sensitive cytotoxin of high molecular weight produced by these organ- isms (10). There is no precedent for any direct role of such a toxin in carcinogenesis. On the other hand, chronic infections by viruses, bacteria, or certain parasites are recognized risk factors for human cancers at various sites. The hypothesis that chemically reactive, potentially genotoxic, substances of low molecular weight (including ni- tric oxide and active oxygen species) generated by inflammatory cells at the site of chronic infection may initiate or enhance carcinogenesis has been examined (11). The hypothesis is under active investigation in the context of H. hepaticus-asso- ciated liver disease. H. hepaticus is susceptible to a number of anti- biotics; treatment of susceptible, naturally in- fected 8- to 10-week-old strain A/JCr mice with single or combined antimicrobial agents has been evaluated for efficacy in eradicating established infections (12). Amoxicillin, metronidazole, and tetracycline administered singly failed to eradi- cate bacteria from the gastrointestinal tract, but either amoxicillin or tetracycline, in combination with metronidazole and bismuth, was effective in eradicating H. hepaticus from the liver, cecum, and colon when given by oral gavage for a period Table 1.

nidazole, and tetracycline administered singly failed to eradi- cate bacteria from the gastrointestinal tract, but either amoxicillin or tetracycline, in combination with metronidazole and bismuth, was effective in eradicating H. hepaticus from the liver, cecum, and colon when given by oral gavage for a period Table 1. Increasing prevalence of hepatitis and hepatocellular neoplasia in control male A/JCr mice at the National Cancer Insti- tute's Frederick Cancer Research and Development Center, 1989-1992a Number Age, Mice with Mice with Date killed of mice in weeks hepatitis (%) liver tumors (%) Jan-Mar 1989 48 473 0 0 May-Jul 1989 47 7060 0 1 (2) Jan-Apr 1992 6 364 2 (33) 0 Jul 1992 16 54 16 (100) 1 (6) Aug-Oct 1992 6 643 5 (83) 3 (50) Dec 1992 12 77 12 (100) 11 (92) a Adapted from ref. 4. Dispatches Vol. 1, No. 4 -- October-December 1995 130 Emerging Infectious Diseases of 2 weeks (12). The effect of antibiotic therapy on the carcinogenic process, or in older animals, re- mains to be established. The importance of H. hepaticus to humans is not yet completely known. The organism clearly has the potential to confound bioassays for chemi- cal carcinogens, but this potential has no direct effect on humans. Even though most Helicobacter species identified to date are characteristically associated with (and named after) specific mam- malian host species in which they generally in- habit the gastrointestinal tract (with or without causing gastritis or other chronic inflammatory disease), the potential host range for some species is quite broad. H. pylori, originally isolated from humans, has recently been isolated also from the domestic cat; this raises the possibility that Heli- cobacter pylori may be a zoonotic pathogen that can be transmitted from companion animals to humans (13). Exploring the possibility of zoonotic transmission of H. pylori, H. hepaticus, or any other Helicobacter species would require isolation of the organism in question by culture methods. Serologic methods have not yet been refined to the level of species specificity. Humans infected with H. pylori mount a serum antibody response to the bacteria that is readily detected by enzyme-linked immunosorbent assays and is considered evidence of ongoing disease (1); mice infected withH. hepa- ticus similarly produce serum antibodies to that species that have been demonstrated by Western blotting (5). Antisera to H. pylori can be used to visualize H.

nse to the bacteria that is readily detected by enzyme-linked immunosorbent assays and is considered evidence of ongoing disease (1); mice infected withH. hepa- ticus similarly produce serum antibodies to that species that have been demonstrated by Western blotting (5). Antisera to H. pylori can be used to visualize H. hepaticus in mouse liver tissue sec- tions stained by the avidin-biotin complex immu- nohistochemical procedure (5). The cross- reactivitybetweenthesetwo species precludes use of available serologic methods to establish whether H. hepaticus has infected humans. Regardless of whether H. hepaticus is itself capable of infecting humans, it serves to demon- strate that liver tissue can be persistently infected by at least one member of the genus Helicobacter, and that liver cancer can be a long-term conse- quence of such infection. This discovery raises questions about the existence of a comparable relationship between liver cancer in humans and unrecognized bacterial infections. Reviews are un- der way of tissue blocks from pathology archives in search of organisms demonstrable by the Steiner stain in liver sections from human popu- lations at high risk for liver cancer. Jerry M. Rice Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick, Maryland, USA References 1. Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991;325:1127-31. 2. Wotherspoon AC, Doglioni C, DissTC, Pan L, Moschini A, de Boni M, et al. Regression of primary low-grade B-cell gastric lymphoma of mucosa-associated lym- phoid tissue type after eradication of Helicobacter py- lori. Lancet 1993;342:575-7. 3. Bayerdorffer E, Neubauer A, Rudolph B, Thiede C, Lehn N, Eidt S, et al. Regression of primary gastric lymphoma of mucosa-associated lymphoid tissue type after cure of Helicobacter pylori infection. Lancet 1995;345:1591-4. 4. Ward JM, Fox JG, Anver MR, Haines DC, George CV, Collins MJ Jr, et al. Chronic active hepatitis and asso- ciated liver tumors in mice caused by a persistent bacterial infection with a novel Helicobacter species. J Natl Cancer Inst 1994;86:1222-7. 5. Ward JM, Anver MR, Haines DC, Benveniste RE. Chronic active hepatitis in micecaused by Helicobacter hepaticus. Am J Pathol 1994;145:959-68. 6. Garvey W, Fathi A, Bigelow F. Modified Steiner for the demonstration of spirochetes. J Histotechnology 1985;8:15-7. 7.

elicobacter species. J Natl Cancer Inst 1994;86:1222-7. 5. Ward JM, Anver MR, Haines DC, Benveniste RE. Chronic active hepatitis in micecaused by Helicobacter hepaticus. Am J Pathol 1994;145:959-68. 6. Garvey W, Fathi A, Bigelow F. Modified Steiner for the demonstration of spirochetes. J Histotechnology 1985;8:15-7. 7. Fox JG, Dewhirst FE, Tully JG, Paster BJ, Yan L, Taylor NS, et al. Helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and in- testinal mucosal scrapings from mice. J Clin Microbiol 1994;32:1238-45. 8. Battles JK, Williamson JC, Pike KM, Gorelick PL, Ward JM, Gonda MA. Diagnostic assay for Helicobac- ter hepaticus based on nucleotide sequence of its 16S rRNA gene. 1995;33:1344-7. 9. Kita E, Oku D, Hamuro A, Nishikawa F, Emoto M, Yagyu Y, et al. Hepatotoxic activity of Campylobacter jejuni. J Med Microbiol 1990;33:171-82. 10. Taylor NS, Fox JG, Yan L. In-vitro hepatotoxic factor in Helicobacter hepaticus, H. pylori and other Helico- bacter species. J Med Microbiol 1995;42:48-52. 11. Ohshima H, Bartsch H. Chronic infections and inflam- matory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis. Mutat Res 1994;305:253-64. 12. Foltz CJ, Fox JG, Yan L, Shames B. Evaluation of antibiotic therapies for eradication ofHelicobacter hepa- ticus. Antimicrob Agents Chemother 1995;39:1292-4. 13. Handt LK, Fox JG, Dewhirst FE, Fraser GJ, Paster BJ, Yan LL, et al. Helicobacter pylori isolated from the domestic cat: public health implications. Infect Immun 1994;62:2367-74. Dispatches Emerging Infectious Diseases 131 Vol. 1, No. 4 -- October-December 1995

Hemolytic Uremic Syndrome Due to Shiga-like Toxin Producing Escherichia coli O48:H21 in South Australia Enterohemorrhagic Escherichia coli (EHEC) other than serotypes O157:H7 are increasingly recognized in association with hemolytic uremic syndrome (HUS) (1) and have been reported in Australia (2). While detecting strains of O157:H7 has become easier over the years, identifying the expanding number of other serotypes of EHEC also associated with HUS, with other conditions, and with healthy domestic animals is still very difficult. Cases of HUS have been reported in Australia over a number of years. The most common sero- type found was O111:H-, and Australia's recently reported first HUS outbreak (3) was caused by EHEC O111:H-. We wish to report a case of severe HUS due to serotype O48:H21, which, as far as we know, has not been previously reported as a cause of HUS. This case occurred in 1993, before sur- veillance of HUS had been initiated; after this case, between July and December 1994, 10 cases of HUS (from which four isolates were obtained; two were EHEC O111) were reported to the Aus- tralian Paediatric Surveillance Unit (E. Elliott, pers. comm.). The patient in the 1993 case was an 8-year-old girl, living in a rural setting in the outskirts of Adelaide, SouthAustralia.Herhomewas adjacent to a farm on which cows, sheep, and ducks were kept. Akelpie/healer cross puppy was in the house in November 1993. Also kept were a pet galah (Australian cockatoo) and pet fish. She was well until 23 December 1993, when she had diarrhea described as very smelly and watery "like the juice of tinned crab." The diarrhea became bloody on 2 January 1994 and was associated with severe abdominal pains which made the patient draw up her legs. She was having bowel movements six times a day, had become very weak, and was unable to stand. She was admitted to Adelaide Children's Hospital on 3 January 1994, and her condition progressed to anuric renal failure over the next few days. Serum biochemistry on 7 Janu- ary showed a urea level of 23.3 mmol/L and creat- inine level of 539 mol/L. Her hemoglobin level fell from 157 g/L on 3 January to 86 g/L on 10 January.

admitted to Adelaide Children's Hospital on 3 January 1994, and her condition progressed to anuric renal failure over the next few days. Serum biochemistry on 7 Janu- ary showed a urea level of 23.3 mmol/L and creat- inine level of 539 mol/L. Her hemoglobin level fell from 157 g/L on 3 January to 86 g/L on 10 January. Her hematocrit fell from 48% to 24%, and her platelet count fell from 463 x 109 /L to 47 x 109 /L onthese dates,respectively. The blood film showed microangiopathic hemolytic anemia with frag- mented red cells. She required hemodialysis for 3 weeks and was discharged from the hospital on 31 January 1994. Apart from the patient's 5-year-oldbrother, who had loose bowel movements for 1 dayon 28 Decem- ber 1993, no other family members were affected. An adequate dietary history was not obtained; however, no food had been eaten from commercial food outlets. Stool samples werecollected on 4and5 January 1994. The samples were probed for Shiga-like toxin (SLT)-I and SLT-II genes by polymerase chain reaction (PCR), and the results were posi- tive. Approximately 80% of lactose-fermenting colonies on MacConkey agar were also SLT posi- tive. No sorbitol-negative colonies were observed on sorbitol-MacConkey agar. In addition to being cultured for E. coli, the stools were also routinely cultured forShigella, Salmonella, Yersinia, Vibrio, and Clostridium. In addition, stained concen- trates were examined for Giardia lamblia and Entamoeba histolytica with negative results. Four typical E. coli strains were subjected to further tests. They were typical E. coli, positive in the indole and ONPG tests, negative in the Voges- Proskauer, citrate, TDA, malonate, urease, gela- tine, and H2S tests. The strains fermented glucose, lactose, mannitol, xylose, rhamnose, arabinose, sorbitol, sucrose, and melibiose. They did not ferment inositol, adonitol, salicin, raffi- nose, or amylose. They decarboxylated arginine, lysine, and ornithine. All the strains produced enterohaemolysin (4). The strains were O and H serotyped (5, 6) and found to be serotype O48:H21. Supernatant preparations were tested on Vero cells (7) and found to give typical verocytotoxic reactions in titers of 103 to 104 . The supernatants were also tested by enzyme-linked immunosor- bent assay (ELISA) by using monoclonal antibod- ies 13C4 and 11E10 directed against SLT-I and SLT-II, respectively, and strong reactions with both antibodies were noted, confirming the pres- ence of both SLTs. Dispatches Vol. 1, No.

in titers of 103 to 104 . The supernatants were also tested by enzyme-linked immunosor- bent assay (ELISA) by using monoclonal antibod- ies 13C4 and 11E10 directed against SLT-I and SLT-II, respectively, and strong reactions with both antibodies were noted, confirming the pres- ence of both SLTs. Dispatches Vol. 1, No. 4 -- October-December 1995 132 Emerging Infectious Diseases Stool samples taken from the patient on 8 Feb- ruary 1994 were negative for SLT-I and SLT-II genes by PCR and were not cultured further. Stool samples from the patient's brother and local ani- mals were not forthcoming. That all four E. coli isolates tested were of serotype O48:H21 and demonstrated identical toxigenicity by both PCR and ELISA and the fact that SLT-positive organisms were not found in the stools collected during the patient's convalescence strongly suggest that this serotype was the causa- tive organism. The toxicity, virulence, and part of the molecular structure of the SLT-II gene derived from the EHEC O48:H21 strain reported here (and whose novel serotype was discovered by the authors) have recently been described elsewhere (8). Paul N. Goldwater,* Karl A. Bettelheim *Microbiology and Infectious Disease Services, Women's & Children's Hospital, Adelaide, South Australia, 5006; Biomedical Reference Laboratory, Victorian Infectious Diseases Reference Laboratory, Fairfield Hospital, Victoria, North Australia, Australia 3078 References 1. Bokete TN, O'Callahan CM, Clausen CR, Tang NM, et al. Shiga-like toxin producing Escherichia coli in Seattle children: a prospective study. Gastroenterology 1993; 105: 1724-31. 2. Goldwater PN, Bettelheim KA. The role of entero- haemorrhagic Escherichia coli serotypes other than O157:H7 as causes of disease. Communicable Disease Intelligence 1995;19:2-4. 3. Centers for Disease Control and Prevention. Commu- nity outbreak of hemolytic uremic syndrome attribut- able to Escherichia coli 0111:NM--South Australia, 1995. MMWR 1995;44:550-1, 557-8. 4. Beutin L, Montenegro MA, Orskov I, Orskov F, Prada J, Zimmerman S, et al. Close association of verocyto- toxin (Shiga-like toxin) production with entero- hemolysin production in strains of Escherichia coli. J Clin Microbiol 1989;27:2559-64. 5. Bettelheim KA, Thompson CJ. New method of serotyp- ing Escherichia coli: implementation and verification. J Clin Microbiol 1987;25:781-6. 6. Chandler ME, Bettelheim KA. A rapid method of iden- tifying Escherichia coli "H" antigens.

emolysin production in strains of Escherichia coli. J Clin Microbiol 1989;27:2559-64. 5. Bettelheim KA, Thompson CJ. New method of serotyp- ing Escherichia coli: implementation and verification. J Clin Microbiol 1987;25:781-6. 6. Chandler ME, Bettelheim KA. A rapid method of iden- tifying Escherichia coli "H" antigens. Zentralblatt fur Bakteriologie, Mikrobiologie und Hygiene 1. Abteilung Originale. A 1974;129:74-9. 7. Konowalchuk J, Speirs JL, Stavric S. Vero response to a cytotoxin of Escherichia coli. Infect Immun 1977;18:775-9. 8. Paton AW, Bourne AJ, Manning PA, Paton JC. Com- parative toxicity and virulence of Escherichia coli clones expressing variant and chimeric Shiga-like toxin type II operons. Infect Immun 1995;63:2450-8. Dispatches Emerging Infectious Diseases 133 Vol. 1, No. 4 -- October-December 1995

Does Treatment of Bloody Diarrhea due to Shigella dysenteriae Type 1 with Ampicillin Precipitate Hemolytic Uremic Syndrome? Diarrhea-associated hemolytic uremic syn- drome (HUS), the most common cause of acute renal failure in infancy and childhood, is often associated with infection by organisms producing Shiga toxin (ST) or Shiga-like toxin (SLT), mainly verocytotoxin-producing Escherichia coli (VTEC O157:H7) and Shigella dysenteriae type 1 (1,2). Although antibiotics are believed to be essential in treating shigellosis, treatment of S. dysenteriae type 1 patients with antibiotics to which the or- ganism is resistant has been considered a risk factor for HUS (3,4). Until 1993, HUS was rarely reported from Saudi Arabia. Four cases of diarrhea-associated HUS due to S. dysenteriae type 1 were identified in 1989 (J. Hibbs and A. Mishkas, unpublished report), and one case of HUS attributed to plasma transfusion was documented in 1988 (5). In May 1993, four dysentery-associated HUS cases in two families were reported from north- western Saudi Arabia (Tabuk). S. dysenteriae type 1 was isolated from the stool of each HUS patient. The organism was also isolated from 6 of the other 10 members of thetwofamilies whohaddysentery. All isolates were resistant to trimethoprim-sul- famethoxazole, chloramphenicol, tetracycline, and ampicillin but sensitive to nalidixic acid. The two families had just returned from a 1-week visit to relatives in two neighboring villages in Gizan. This densely populated region in southwestern Saudi Arabia has about 1.2 million people living in more than 4,000 villages; the population is relatively poor and uneducated, and environ- mental sanitation is generally inadequate. We defined a case of HUS as any case of bloody diarrhea (BD) that had all of the following: acute renal failure (serum urea nitrogen, 18 mg/dL (or 6.3 mmol/L); or creatinine, 1.3 mg/dL (or 115 mmol/L)); thrombocytopenia (platelet count 130,000/mm3 ); and hemolytic anemia (hemoglo- bin level less than 10 g/dL; or hematocrit less than 30%; or appearance of fragmented red cells on direct microscopy).

following: acute renal failure (serum urea nitrogen, 18 mg/dL (or 6.3 mmol/L); or creatinine, 1.3 mg/dL (or 115 mmol/L)); thrombocytopenia (platelet count 130,000/mm3 ); and hemolytic anemia (hemoglo- bin level less than 10 g/dL; or hematocrit less than 30%; or appearance of fragmented red cells on direct microscopy). We standardized the treat- ment of BD in the Gizan region as follows: No antibiotics were given for treatment of BD at the primary health care centers (PHCCs) before a stool specimen was taken for culture and sensitiv- ity testing. After reviewing the preliminary re- sults, we either recommended use of nalidixic acid for treatment of BD or were guided by the results of the stool culture. This protocol was followed for management of BD in the entire region. Parasitologic, bacteriologic, and biochemical tests and drug treatment regimens were obtained for all patients admitted with BD or HUS to the regional referral hospital or five district hospitals in the outbreak area. BD cases were identified through hospital admission records, visits to PHCCs in the affected villages, interviews with family members of the identified patients, and school visits. We visited the houses of all HUS and BD patients and interviewed family members to ascertain which antibiotic was used to treat the BD patients; mothers were shown bottles and boxes of antibiotics and were asked to identify the antibiotic used for treating the children with BD. We identified 233 cases of BD occurring from February through July 1993 among 79 families scattered over 19 contiguous villages. Affected vil- lages were predominantly in southern Gizan re- gion near the Yemeni border. One hundred ninety patients (81.5%) consulted PHCCs; of those, 97 (51%) were referred to hospitals, and 81 (43%) were admitted. Thirty-four other BD patients were admitted directly to hospitals (a total of 115 admissions). In nine BD cases patients did not seek medical care including seven (3%) who used traditional treatment (the Wicka plant). In 23 (10%) patients, 13 male and 10 female, BD devel- oped into HUS. Four isolates ofS. dysenteriae type 1 that showed the same antibiotic susceptibility described earlier were obtained from four patients with BD in different villages in the middle of the outbreak. We used Cary-Blair transport medium for transporting stool specimens collected before antibiotic treatment from newly recognized pa- tients with BD.

eriae type 1 that showed the same antibiotic susceptibility described earlier were obtained from four patients with BD in different villages in the middle of the outbreak. We used Cary-Blair transport medium for transporting stool specimens collected before antibiotic treatment from newly recognized pa- tients with BD. However, community- and hospi- tal-based interviews showed that the sequence of symptoms was almost identical in all of the 233 BD cases: the condition started with colicky ab- dominal pain and tenesmus (69%), followed by watery diarrhea (60%), which rapidly became only Dispatches Vol. 1, No. 4 -- October-December 1995 134 Emerging Infectious Diseases mucus and blood (83%) or blood-streaked (17%). Seven patients (3.0%) had rectal prolapse. S. dysenteriae type 1 was not isolated from any of the 23 HUS patients; however, all stool specimens were taken during antibiotic treatment. Most BD cases (92.3%) were among Saudis; the remaining 7.7% were among Yemeni patients. No HUS case occurred among patients over 11 years ofage. The male/femaleratiofor bothBD andHUS was 1.3:1. Three boys and three girls with con- firmed HUS died (case-fatality rate = 26.1%); none of the patients with uncomplicated BD cases died. Of the 23 HUS patients, 18 (78%) became ill with the disease 2 to 14 days after hospital admis- sion for uncomplicated BD. This compares with a hospital admission rate of 40 (27%) of 147 for children of the same age with BD from the same villages (odds ratio = 9.6, 95% confidence interval 3.1-35). Five children, aged 8 to 16 months, got HUS either before or on the day of hospital admis- sion; all had received oral ampicillin at home for 5 to 7 days before their illness progressed to HUS. In comparison, two of nine children of the same age, with BD, who were not hospitalized, received ampicillin at home (OR = infinite, p value = 0.02, Fisher's exact test). EighteenHUS cases occurred after the patients were admitted to Samtah and Abu-Arish, two out of five district hospitals. The demographic, clini- cal, and laboratory profiles of BD and HUS cases are shown in Table 1A-C. Six different antibiotics were used in various combinations for treating BD Places of hospitalization of BD and HUS cases Samtah Abu-Arish KFH Bysh Sabia No. of BD cases 43 42 13 9 8 No.

two out of five district hospitals. The demographic, clini- cal, and laboratory profiles of BD and HUS cases are shown in Table 1A-C. Six different antibiotics were used in various combinations for treating BD Places of hospitalization of BD and HUS cases Samtah Abu-Arish KFH Bysh Sabia No. of BD cases 43 42 13 9 8 No. of HUS cases 8 14 - - 1a Percentage of HUS cases 18.8 33.3 0 0 12.5 Median age in years: BD cases 4.0 5.0 7.0 4.0 4.0 Median age in years: HUS cases 1.8 2.8 - - 0.8 Mean (SD) of duration (in days) between onset of symptoms and admission to hospital: BD 3.8 (1.9) 5.5 (1.6) 4.5 (0.4) 3.7 3.9 (2.0) BD complicated with HUS 5.2 (2.6) 4.5 (2.5) - - - HUS diagnosed on admission 13.0 (2.8) 7.5 (0.7) - - 6.0 (0.0) Laboratory test made on the day of admission to the hospitald Non-HUS cases HUS diagnosed 2-14 days after admission to hospital HUS diagnosed on admission to hospital Mean (N)e SD Mean (N) SD Mean (N) SD Serum creatinine 60 (16) 46 63 (3) 79 279 (3) 94 Blood urea nitrogin (BUN) 5.4 (23) 5.8 15.3 (3) 12.0 23.0 (3) 6.1 Serum sodium 129 (33) 9 137 (3) 4.6 127 (3) 14 Serum potassium 3.8 (47) 0.8 3.9 (7) 1.1 4.6 (5) 1.1 (Leukocytes [WBC] count) 14.2 (48) 6.0 34.0 (6) 24.7 41.2 (5) 18.8 Hemoglobin 11.3 (50) 2.3 12.0 (1) 1.5 6.8 (4) 2.5 Hematocrit 36.1 (13) 3.9 NA NA 15 (2) 7.1 Thrombocytes (platelets) 322 (5) 250 154 (1) - NA NA (Body temperature on admis- sion) 37.8o C (75) 0.8 37.9o C (18) 0.8 37.9o C (5) 0.9 Ampicillin 36.6 70.0 15.4 11.1 100.0 Metronidazole 14.6 55.0 7.7 0 100.0 Gentamicin 22.0 22.5 0 55.6 42.9 Nalidixic acid 70.7 57.5 61.5 66.7 0 Claforan 2.4 20.0 0 11.1 0 Amikacin 9.8 0 7.7 22.2 28.6 a Community case of HUS. b Percent of cases receiving the corresponding antibiotics. A patient may receive more than one antibiotic. Totals do not add up to 100%. c This table does not include 5 cases diagnosed as HUS on admission and treated as cases of BD. d Creatinine in mg/dL, blood urea nitrogen in mg/dL, sodium in mmol/L, potassium in mmol/L, WBC (white blood cell count) in thousands/m, hemoglobin level in g/dL, hematocrit, platelets in thousands/mm3 . Values shown are for children under 12 years of age only. e N = number of cases of BD diagnosed in the hospital. NA = not available. Table 1. A. Profiles of children admitted to hospitals with bloody diarrhea (BD) or hemolytic uremic syndrome (HUS) B. Percentage of 110 BD patients treated with antibioticsb,c C.

s/mm3 . Values shown are for children under 12 years of age only. e N = number of cases of BD diagnosed in the hospital. NA = not available. Table 1. A. Profiles of children admitted to hospitals with bloody diarrhea (BD) or hemolytic uremic syndrome (HUS) B. Percentage of 110 BD patients treated with antibioticsb,c C. Laboratory values of children hospitalized with BD or HUS Dispatches Emerging Infectious Diseases 135 Vol. 1, No. 4 -- October-December 1995 patients in these hospitals Table 1B. Treatment with ampicillin prescribed alone or with other antibiotics except nalidixic acid (Table 2), was associated with development of HUS. However, three BD patients who received nalidixic acid de- veloped HUS. Of 12 BD patients (including eight children under 12 years of age) who received no antibiotic therapy, none got HUS. These results support the implication from Bangladesh and from a parallel investigation in Saudi Arabia that inpatient antibiotic treatment of children with dysentery due to S. dysenteriae type 1 may precipitate HUS. We have extended these observations to show the same association for antibiotic treatment at home. Although Abu- ArishandSamtahhospitals received similarnum- bers of BD patients, more HUS cases were reported from Abu-Arish hospital, which used am- picillin to treat patients with BD. The various combinations of antibiotics used to treat BD pa- tients could be explained by the presence of doc- tors from parts of the world that have different prescriptionpractices.Three BDpatients got HUS despite the use of nalidixic acid. Resistance to nalidixic acid among S. dysenteriae isolates was reported from Bangladesh; resistance increased from 2.1% in 1986 to 57.9% in 1990 (6). We recommend a laboratory-based surveillance system to identify and promptly contain emerging outbreaks. Physicians need to be informed con- tinuously about emerging resistant strains of bac- teria and be cautious when using antibiotics to treat patients with dysentery unless the causative organism and the resistance pattern have been identified. Parents of children with BD need to be educated to take their children to the nearest health facility as soon as possible. S. dysenteriae is a delicate bacterium that does not withstand adverse conditions (e.g., heat and dryness); prompt plating, preferably at bedside, is recommended (7). Failure to isolate S.

ntified. Parents of children with BD need to be educated to take their children to the nearest health facility as soon as possible. S. dysenteriae is a delicate bacterium that does not withstand adverse conditions (e.g., heat and dryness); prompt plating, preferably at bedside, is recommended (7). Failure to isolate S. dysenteriae type 1 from dysenteric stool specimens during this outbreak could be attributed to delayed plating of specimens, lack of appropriate transport media, Table 2. Risk of developing hemolytic uremic syndrome (HUS) by antibiotic combination used for treatment of bloody diarrhea (BD), Gizan, Saudi Arabia, 1993 Antibiotic combination group Antibiotic combinations Dysentery patients Developing Total (N = 110) HUS (N = 18) BD cases admitted to Samtah and Abu- Arish Hospitals HUS rate (%) Risk ratio 95% confidence Intervala Nalidixic acid with or without other antibiotics, but no ampicillanb N(3/23), N+G (0/7) G+N+C+A (0/1), M+N (0/1), M+N+C (0/2), N+C (0/4) 41 3 28 7.3 1 0.23-4.34 No antibiotic No antibiotic (0/12) 12 0 6 0.0 NCc p value = 0.629d Antibiotic other than nalidixic acid or ampicillin A (0/1), C (1/0), G (0/1). M+G (0/1) 4 1 4 25.0 1.58 0.22-11.58e Ampicillin with or without other antibiotics but no nalidixic acid 28 10 19 35.7 6.90 0.98-48.68 Ampicillin only P (3/3) 6 3 5 50.0 6.11 1.31-28.54 Ampicillin with other antibiotics but no nalidixic acid P+G (1/2), P+M (2/3), P+M+A (0/2), P+M+C (1/0) P+M+G (3/8) 22 7 14 31.8 10.50 0.54-205.39 Ampicillin and nalidixic acid with or without other antibiotics P+M+G+N (1/1), P+M+N 0/10), P+M+N+C (1/0). P+N (1/8), P+N+A (1/2) 25 4 24 16.0 1.77 0.31-10.21 HUS rate (%) calculated from total number of BD in the five district hospitals using corresponding antibiotic .a Mantel-Haenszel weighted relative risk adjusted to hospital (Epi Info, version 6.02). Analysis restricted for data from Samtah and Abu-Arish hospitals. HUS was not reported from the other three district hospitals. b Reference group. A = amikacin, C = claforan, g = gentamicin, N = nalidixic acid, M = metronidazole, P = ampicillin. Numbers between parentheses in the second column (antibiotic combination) indicate the (number of patients with HUS who took the corresponding antibiotic combination number or patients with BD who took thesameantibioticcombinationbutdidnotdevelopHUS inthefivedistrict hospitals).c NC= notcalculated.d One-tailed Fisher's exact test. e AdjustedMantel-Haenszel relative risk could not be calculated. Dispatches Vol. 1, No.

tients with HUS who took the corresponding antibiotic combination number or patients with BD who took thesameantibioticcombinationbutdidnotdevelopHUS inthefivedistrict hospitals).c NC= notcalculated.d One-tailed Fisher's exact test. e AdjustedMantel-Haenszel relative risk could not be calculated. Dispatches Vol. 1, No. 4 -- October-December 1995 136 Emerging Infectious Diseases and treatment with antibiotics before stool speci- mens were obtained. Even with direct inoculation of stool specimens in pediatric wards, HUS re- sulted in a low yield of S. dysenteriae type 1 (8,9). Treatment of BD due to S. dysenteriae type 1 with ampicillin may precipitate HUS. It would be valuable to retrospectively examine this associa- tion in other countries where both dysentery due to S. dysenteriae type 1 and HUS are reported. Abdulaziz A. A. Bin Saeed, Hassan E. El Bushra, Nasser A. Al-Hamdan Field Epidemiology Training Program, Department of Preventive Medicine, Ministry of Health, Riyadh, Kingdom of Saudi Arabia References 1. Neild GH. Haemolytic uraemic syndrome in practice. Lancet 1994;343:398-401. 2. Robson WLM, Leung AKC, Kaplan BS.Hemolytic ure- mic syndrome. Curr Prob Pediatr 1993;23:16-33. 3. Butler T, Islam MR, Azad MAK, Jones PK. Riskfactors for development of hemolytic uremic syndrome during shigellosis. J Pediatr 1987;110:894-7. 4. Al-Qarawi S, Fontaine RE, Al-Qahtani MS. An out- break of hemolytic uremic syndrome associated with antibiotic treatment of hospital inpatients for dysen- tery. Emerging Infectious Diseases 1995;1:138-40. 5. Kher K, Hussein M. Severe haemolytic syndrome: re- port of a child treated with fresh frozen plasma infu- sions and dialysis. Saudi Med J 1988;9:205-7. 6. Bennish ML, Salam MA, Hussein MA, Myaux J, Khan EH, Chakraborty J, et al. Antimicrobial resistance of Shigella isolates in Bangladesh, 1983-1990: increasing frequency of strains multiply resistant to ampicillin, trimethoprim sulfamethoxazole, and nalidixic acid. Clin Infect Dis 1992;14:1055-60. 7. Keusch GT, Formal SB, Bennish M. Shigellosis. In: Warren KS, Mahmoud AAF, eds. Tropical and geo- graphical medicine 2nd ed. New York: McGraw-Hill, 1990:760. 8. Khin-Maung-U, Myo-Khin, Tin-Aye, Myo-Min-Aung, Soe-Soe-Aye, Thane-Oke-Kyaw-Myint, et al. Clinical features, including hemolytic uremic syndrome, in Shigella dysenteriae type 1 infection in children of Rangoon. J Diarrhoeal Dis Res 1987;3:175-7. 9. Srivastava RN, Moudgil A, Bagga A, Vasudev AS.

McGraw-Hill, 1990:760. 8. Khin-Maung-U, Myo-Khin, Tin-Aye, Myo-Min-Aung, Soe-Soe-Aye, Thane-Oke-Kyaw-Myint, et al. Clinical features, including hemolytic uremic syndrome, in Shigella dysenteriae type 1 infection in children of Rangoon. J Diarrhoeal Dis Res 1987;3:175-7. 9. Srivastava RN, Moudgil A, Bagga A, Vasudev AS. Hemolytic uremic syndrome in children in northern India. Pediatr Nephrol 1991;5:284-8. Dispatches Emerging Infectious Diseases 137 Vol. 1, No. 4 -- October-December 1995

An Outbreak of Hemolytic Uremic Syndrome Associated with Antibiotic Treatment of Hospital Inpatients for Dysentery Shiga toxin (ST) from Shigella dysenteriae type 1 is accepted as a cause of hemolytic uremic syn- drome (HUS); however, the reasons why HUS develops in only some infected patients are not clear (1). The possibility that antibiotic therapy is associated with the development of HUS has been explored for S. dysenteriae type 1 and for Es- cherichia coliO157:H7 (2-4). In May 1993, during an outbreak of S. dysenteriae type 1 in Gizan, Saudi Arabia, an association between antibiotic treatment and HUS was also observed (5). The strain of S. dysenteriae type 1 was resistant to ampicillin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole (TMP-SMX) and sensitive to nalidixic acid. We report here some of our preliminary observations for a concurrent out- break. In response to the Gizan outbreak, a circular was sent to all regions of Saudi Arabia requesting immediate reports of S. dysenteriae type 1. One region reported three cases of S. dysenteriae type 1 with the same antibiotic resistance pattern. The patients were visitors from the Najran region, on the Yemen border. The affected family reported that many other persons in their community of Barshash, home of about 6,000 Yemeni immi- grants, had recently had dysentery. The regional hospital in Najran confirmed that several local children had been treated for HUS, and an inves- tigation was initiated. Beginning in February 1993, the numbers of dysentery patients at the Barshash Primary Health Care Center began to increase from 6 patients per week to 110 in mid May. After control measures were initiated, weekly incidence de- creased to zero by late June. According to policy, the Primary Health Care Center treated dysen- tery patients withoral rehydration, whereas those admitted to the regional hospital received antibi- otics. At the regional hospital, S. dysenteriae type 1 was isolated from four Barshash residents and one resident of another community.

ro by late June. According to policy, the Primary Health Care Center treated dysen- tery patients withoral rehydration, whereas those admitted to the regional hospital received antibi- otics. At the regional hospital, S. dysenteriae type 1 was isolated from four Barshash residents and one resident of another community. Between March and May, the illness of 10 (of 42) children admitted to the regional hospital for dysentery progressed to HUS from 2 to 10 days (median 5) after admission. For all 10 children, physicians noted HUS onset as a sudden change in the clinical condition characterized by pallor, puffy face, peripheral edema, or oliguria. Within 2 days of clinical onset, blood urea nitrogen (BUN) levels of all 10 children were 10 mmol/L. In nine children, the hematocrit fell to below 75% of its value on admission. One child had microcytic hy- pochromic anemia on admission and was trans- fused with one unit of packed red cells; his hematocrit fell from 29.8% after the transfusion to 24.6% the day afterHUS onset; his BUN level rose from 3.1 to 23.5 mmol/L; and his thrombocyte count fell from 529,000/mm3 to 75,000/mm3 . Red cell fragments were reported for seven patients. Thrombocytopenia, developed in seven patients; in two, thrombocyte counts decreased from admis- sion values. A leukemoid reaction ( 30,000 leuko- cytes/mm3 ) developed in seven patients. HUS cases included six children from Bar- shash, one from a contiguous district, one with relatives in Barshash, and two visiting from southern Gizan region where the concurrent out- break was occurring (5). No child was admitted from the community with HUS. Reasons given for admission were either dysentery or bloody diar- rhea with mild or moderate dehydration. Two dysentery patients who developed HUS were ad- mitted because no one was available to care for them at home. Characteristics on hospital admis- sion of dysentery patients who did or did not develop HUS were similar with the exception of serum sodium level and BUN (Table1).Elevations in creatinine (63 to 133 mmol/L) and urea (6.1 to 7.9 mmol/L) levels on admission were mild, and after intravenous rehydration returned to normal. No enteric pathogens were isolated in stool speci- mens from 35 patients. All 42 dysentery patients received a variety of antibiotic combinations composed of ampicillin, TMP-SMX, nalidixic acid, gentamicin, erythromy- cin, and metronidazole.

on admission were mild, and after intravenous rehydration returned to normal. No enteric pathogens were isolated in stool speci- mens from 35 patients. All 42 dysentery patients received a variety of antibiotic combinations composed of ampicillin, TMP-SMX, nalidixic acid, gentamicin, erythromy- cin, and metronidazole. Higher rates of HUS were observed among patients receiving antibiotics to which the locally circulating S. dysenteriae type 1 was resistant (ampicillin or TMP-SMX) or antibiotics that are ineffective against Shigella (metronidazole, erythromycin, gentamicin) than Dispatches Vol. 1, No. 4 -- October-December 1995 138 Emerging Infectious Diseases among patients treated with nalidixic acid (with or without metronidazole) (Table 2). Stratification of this analysis by elevated creatinine level (62 mmol/L) or BUN (6.0 mmol/L) on admission or by weight for age above and below the fifth percentile had no effect on the magnitude or statistical sig- nificance of these associations. However, stratifi- cation by serum sodium on admission above and below 130 mmol/L yielded increased risk ratios of 15 (95% confidence interval, 1.6 to 147) for any ineffective antibiotic and 15 (95% confidence interval, 2.3 to 99) for ampicillin without nalidixic acid (Mantel-Haentzel analysis). These strong associations of HUS with prior antibiotic therapy suggest that the antibiotics may be influencing the progression of S. dysente- riae type 1 to HUS (2,5). The Najran patients had milder disease on admission, and the risk ratios were higher than in the other two reports. The assumption that S. dysenteriae type 1 caused the dysentery is supported by the isolation of S. dysenteriae type 1 from community members during a concomitant dysentery outbreak and by the absence of other ST producing organisms. However, because culture results are lacking, it is possible that some hospitalized dysentery pa- tients were infected with other organisms. This investigation was retrospective, and with- out randomization, the selection of patients for treatment by severity of illness was managed with stratified analysis. All possible indicators of dehy- drationor severity of dysentery on admissionwere not available for stratification. However, these were also not available to the physicians for selection of patients. Moreover, nalidixic acid was considered effective therapy, and physicians did not indicate in the medical records that they were selecting nalidixic acid for less severely ill pa- tients.

admissionwere not available for stratification. However, these were also not available to the physicians for selection of patients. Moreover, nalidixic acid was considered effective therapy, and physicians did not indicate in the medical records that they were selecting nalidixic acid for less severely ill pa- tients. Because of the variety of antibiotics pre- scribed, the effect of antibiotics given to most patients (metronidazole) in combination with other antibiotics could not be evaluated. The effectiveness of those given to a few patients (TMP-SMX) could not be assessed. Table 1. Characteristics on hospital admission of dysentery patients whose illness did or did not develop into hemolytic uremic syndrome (HUS) during hospitalization, Najran, Saudi Arabia, March through May 1993 Dysentery patients Characteristic Developed HUS (10) Did not develop HUS (32) p valuea Age (yr mean) 4.4 4.8 0.71 Male sex 7 (70%) 23 (72%) 1.0 Percentile weight for ageb (median) 15.4 16.3 0.57 Below fifth percentile 6 (60%) 12 (38%) 0.29 Days with dysentery (median) 3 3 0.70 Range 2-8 1-14 Stools on first hospital day (median) 12.5 9.0 0.23 Range 2-26 2-43 Temperature (mean) 38.2o C 38.0o C 0.64 Range 36.8o C-39.4o C 36.5o C-39.4o C Hematocrit (mean) 38.9% 38.8% 0.94 Below 35.5% 4 (40%) 7 (23%) 0.43 Thrombocytes/mm3 555,000 501,000 0.62 Below 150,000 0 0 Leucocytes/mm3 (mean) 11,440 12,300 0.62 Above 12,000 7 (70%) 22 (69%) 1.0 Above 18,000 1 (10%) 4 (13%) 1.0 Serum sodium, mmol/L (mean) 130 134 0.02 Below 130 mmol/L 4 (40%) 7 (22%) 0.41 Serum potassium, mmol/L (mean) 3.4 3.4 0.86 Below 3.5 mmol/L 5 (50%) 16 (50%) 1.0 Serum creatinine, mmol/L (mean) 72 62 0.62 Above 62 mmol/L 5 (50%) 13 (41%) 1.0 BUN, mmol/L (mean) 4.1 3.2 0.06 Above 6.0 mmol/L 2 (20%) 2 (6%) 0.24 a Differences in means by Student's t-test, in medians by the Kruskal-Wallis test, and in proportions by Fisher's exact test (two-tailed). b National Center for Health Statistics. Dispatches Emerging Infectious Diseases 139 Vol. 1, No. 4 -- October-December 1995 Antibiotic therapy may be associated withHUS in various ways. The antibiotics given may have been ineffective, so the infections were allowed to run their natural course to HUS. However, one would have expected at least a few cases to have developed at home among the hundreds of Barshash residents with dysentery. Another pos- sibility is that ineffective antibiotics suppressed competing microbial flora, allowing less re- strained proliferation of S. dysenteriae type 1.

r natural course to HUS. However, one would have expected at least a few cases to have developed at home among the hundreds of Barshash residents with dysentery. Another pos- sibility is that ineffective antibiotics suppressed competing microbial flora, allowing less re- strained proliferation of S. dysenteriae type 1. This does not account for the 12% HUS attack rate in patients treated with nalidixic acid. An in-vitro study of enhanced Shiga-like toxin I (SLT-1) pro- duction by E. coli O157:H7 suggests another ex- planation. Subinhibitory concentrations of antibiotics resulted in up to a 400% increase in SLT-I recovery relative to controls (6). This effect was maximal at maximum subinhibitory concentrations of antibiotics, and a quinoline an- tibiotic, ciprofloxacin, yielded the greatest in- crease of toxin. These findings are most consistent with the epidemiologic findings in this outbreak, including the occurrence of HUS in patients treated with nalidixic acid and the absence of HUS in untreated patients in the community. With these considerations, antibiotic treatment of dysentery from S. dysenteriae type 1 should be approached with caution. Sami Al-Qarawi,* Robert E. Fontaine, and Mohammed-Saeed Al-Qahtani* *Saudi Arabian Field Epidemiology Training Program, Ministry of Health, Saudi Arabia; Epidemiology Program Office, Centers for Disease Control and Prevention, Atlanta, Georgia, USA. References 1. Moake JL. Haemolyticuremic syndrome:basic science. Lancet 1994;343:393-401. 2. Butler T, Islam MR, Azad MAK, Jones PK. Riskfactors for development of hemolytic uremic syndrome during shigellosis. J Pediatr 1987;110:894-7. 3. Ostroff SM, Kobayahi JM, Lewis JH. Infections with Escherichia coli O157:H7 in Washington State: the first year of statewide disease surveillance. JAMA 1989;262:355-9. 4. Pavia AT, Nichols CR, Green DP, et al. Hemolytic-ure- mic syndrome during an outbreak of Escherichia coli O157:H7 infections in institutions for mentally re- tarded persons: clinical and epidemiologic observa- tions. J Pediatr 1990;116:544-51. 5. Bin Saeed AAA, El Bushra HE, Al-Hamdan NA. Does treatment of bloody diarrhea due to Shigella dysente- riae type 1 with ampicillin precipitate hemolytic ure- mic syndrome? Emerging Infectious Diseases 1995;4:134-8. 6. Walterspiel JN, Ashkenazi S, Morrow AL, Cleary TG. Effect of subinhibitory concentrations of antibiotics on extracellular shiga like toxin I. Infection 1992;20:25-9. Table 2.

rrhea due to Shigella dysente- riae type 1 with ampicillin precipitate hemolytic ure- mic syndrome? Emerging Infectious Diseases 1995;4:134-8. 6. Walterspiel JN, Ashkenazi S, Morrow AL, Cleary TG. Effect of subinhibitory concentrations of antibiotics on extracellular shiga like toxin I. Infection 1992;20:25-9. Table 2. Risk of developing hemolytic uremic syndrome (HUS) by antibiotic combinations used for in-hospital treatment of dysentery during a community outbreak of antibiotic-resistant Shigella dysenteriae type 1, Najran, Saudi Arabia, March through May 1993 Dysentery patients 95% Developed Risk confidence Antibiotic combination HUS (10) Total (42) HUS rate/100 ratioa intervalb Ineffectivec antibiotics without nalidixic 6 12 50 4.3 1.3-15 acidd-j Ampicillin combinations without nalidixic 5 7 71 6.2 1.9-20 acidd-f Ineffective antibiotics without nalidixic acid 1 5 20 1.7 0.22-14 or ampicilling-j Ampicillin and nalidixic acid with (2) or without 1 3 33 2.9 0.40-20 (3) metronidazole Trimethoprim-sulfamethoxazole, nalidixic acid and metronidazole 0 1 0 0 Nalidixic acid with (24) or without (2) metronidazole (reference) 3 26 12 1.0 Reference a Relative to the reference antibiotic combination (nalidixic acid with or without metronidazole). b Taylor series approximation standard. c Antibiotics to which the outbreak strain ofS. dysenteriae type 1 was resistant (ampicillin, trimethoprim-sulfamethoxazole) or which are ineffective against shigella (metronidazole, gentamicin, or erythromycin). d Ampicillin and metronidazole (4 patients). e Ampicillin, metronidazole, and gentamicin (2 patients). f Ampicillin only (1 patient). g Trimethoprim-sul- famethoxazole and metronidazole (1 patient). h Trimethoprim-sulfamethoxazole, erythromycin, and metronidazole (1 patient). i Metronidazole only (2 patients). j Erythromycin and metronidazole (1 patient). Dispatches Vol. 1, No. 4 -- October-December 1995 140 Emerging Infectious Diseases

Epidemic Cholera in the New World: Translating Field Epidemiology into New Prevention Strategies Cholera, a devastating diarrheal disease, has swept through the world in recurrent pandemics since 1817. The seventh and ongoing pandemic began in 1961 when the El Tor biotype of Vibrio cholerae O1 emerged in Indonesia. This pandemic spread through Asia and Africa and finally reached Latin America early in 1991 (1). After explosive epidemics in coastal Peru, it spread rap- idlyandcontinues throughout LatinAmerica (Fig- ure 1). Because of underreporting, the more than 1,000,000 cholera cases and 10,000 deaths re- ported from Latin America through 1994 (Table 1) (2) represent only a small fraction of the actual number of infections. Molecular characterization of V. cholerae O1 strains from Peru has shown that they do not match strains from anywhere else in the world; therefore, the source of the Peruvian epidemic strains remains unknown (3). Moreover, other strains have since appeared in Latin Amer- ica. At least one of these, a strain resistant to multiple antimicrobial drugs, was first identified in Mexico and elsewhere in the world in mid-1991 and has since spread widely throughout Central America (4). The introduction of strains into new areas illustrates therapid global transferof patho- gens. V. cholerae O139 Bengal, which emerged as a new cause of epidemic cholera in Asia in 1992, could also appear in Latin America (5). Such introductions are not easy to prevent, because they may follow the arrival of travelers who are not aware of their infection or of ships carrying contaminated ballast water. The key to controlling epidemic cholera lies in limiting its spread by using measures that prevent sustained transmission. One measure might be using an inexpensive and effective vaccine to provide last- ing protection; however, no such vaccine yet exists, although progress in vaccine development is being made (6-8). Another measure is interrupting transmissionsothat thecausative organismnever reaches the human host.

ustained transmission. One measure might be using an inexpensive and effective vaccine to provide last- ing protection; however, no such vaccine yet exists, although progress in vaccine development is being made (6-8). Another measure is interrupting transmissionsothat thecausative organismnever reaches the human host. This approach to preven- tion successfully controlled many epidemic dis- eases in the industrialized world, including cholera, typhoid fever, plague, and malaria, before vaccines or antibiotics were developed. Over the last century, a large engineering infrastructure, built in industrialized nations, has provided safe water and sewage treatment for nearly all people in these nations and has made sustained trans- mission of cholera in those countries extremely unlikely. Despite sporadic cases along the U.S. Gulf Coast and repeated introduction of the epi- demic organisms by travelers, epidemic cholera has not occurred in the United States since the nineteenth century (9,10). To prevent cholera by interrupting transmis- sion of the organism to the host, it is important to understand precisely how the bacteria are trans- mitted. John Snow demonstrated waterborne transmission of cholera during a large epidemic in London in 1856 (11). He and many others since have suspected that other routes of transmission are also important. Epidemiologic investigations during the seventh pandemic have documented a variety of specific food and water pathways by which the bacteria reach the host, some of which werenewandunsuspected(12).TheEl Tor biotype of V. cholerae O1, for example, multiplies rapidly in moist foods of neutral acidity (13). This bacte- rium also persists in the estuarine environment in niches that are poorly understood but may involve the plankton on which shellfish feed. This means Figure 1. Geographic extent of the Latin American cholera epidemic over time. Lines represent the advancing front of the epidemic at different dates. As of mid-1995, all Latin American countries except Uruguay have reported cases; no cases have been reported from the Caribbean. Dispatches Emerging Infectious Diseases 141 Vol. 1, No. 4 -- October-December 1995 that raw seafood can be contaminated naturally before it is harvested. Understanding these path- ways of transmission in detail has been central to devising successful control measures to block them.

ave been reported from the Caribbean. Dispatches Emerging Infectious Diseases 141 Vol. 1, No. 4 -- October-December 1995 that raw seafood can be contaminated naturally before it is harvested. Understanding these path- ways of transmission in detail has been central to devising successful control measures to block them. For example, advice to drink only boiled or bottled water would be of little use in outbreaks where the source was actually contaminated food, such as shellfish or leftover rice. On at least one occasion, such advice actually worsened the situ- ation because thebottledwater wasitself contami- nated (14). When epidemic cholera appeared in Latin America, after an absence of more than 100 years, we conducted a series of eight rapid field investi- gations in collaboration with national public health authorities and the Pan-American Health Organization to define the pathways of disease transmission and the priorities for prevention. Conducted in various settings between February 1991 and August 1993, these investigations guided the initial emergency prevention efforts and the development of sustained prevention measures (Table 2) (15-21). The same case-control method was used for each investigation. We first interviewed a few patients in great detail about what they had ingested in the 3 days before they became ill, probing for known or potential vehicles of cholera. We then constructed a standardized interview questionnaire that asked about possible expo- sures. Using this questionnaire, we interviewed patients recovering from cholera as well as healthy persons of the same age and sex who lived in the same neighborhood. By comparing the fre- quency of positive and negative responses among the ill and well persons, we could identify the exposures most strongly associated with disease. For example, if 25 of 35 patients, but only 5 of 35 matched controls, reported eating sliced mango from street vendors in the 3 days before the illness began, the probability of observing this difference in proportions by chance alone is 0.000045, and the ratio of the odds of exposure is 15, a measure of the strong association between disease and con- suming sliced mangos. When disease is statisti- cally associated with more than one exposure, multivariate analysis can identify truly inde- pendent risk factors. In the above example, "eat- ing food from a street vendor" would not be independent from "eating sliced mango" if one usually got slicedmangofrom a street vendor. This Table 1.

s. When disease is statisti- cally associated with more than one exposure, multivariate analysis can identify truly inde- pendent risk factors. In the above example, "eat- ing food from a street vendor" would not be independent from "eating sliced mango" if one usually got slicedmangofrom a street vendor. This Table 1. Cholera cases by country, as reported to the Pan American Health Organization, 1991 through 1994 Number of reported cases Country Date of first report 1991 1992 1993 1994 Argentina Feb. 5, 1992 0 553 2,080 889 Belize Jan. 9, 1992 0 159 135 6 Bolivia Aug. 26 1991 206 22,260 10,134 2,710 Brazil Apr. 8, 1991 2,101 30,054 56,286 49,455 Chile Apr. 12, 1991 41 73 32 1 Colombia Mar. 10, 1991 11,979 15,129 230 996 Costa Rica Jan. 3, 1992 0 12 14 38 Ecuador Mar. 1, 1991 46,320 31,870 6,833 1,785 El Salvador Aug. 19, 1992 947 8,106 6,573 11,739 French Guiana Dec. 14, 1992 1 16 2 NRa Guatemala July 24, 1991 3,652 15,686 30,605 4,227 Guyana Nov. 5, 1992 0 5 66 0 Honduras Oct. 13, 1991 17 388 2,290 4,965 Mexico June 13,1991 2,690 8,162 10,712 4,059 Nicaragua Nov. 12, 1991 1 3,067 6,631 7,821 Panama Sept.10, 1991 1,178 2,416 42 0 Paraguay Jan. 25, 1993 0 0 3 0 Peru Jan. 23, 1991 322,562 210,836 71,448 23,887 Suriname Mar. 6, 1992 0 2 0 0 United States Apr. 9, 1991 26 103 22 34 Venezuela Nov. 29, 1991 13 2,842 409 0 Total 391,734 352,300 204,547 112,612 a NR = no reports received. Source: ref. 2. Dispatches Vol. 1, No. 4 -- October-December 1995 142 Emerging Infectious Diseases case-control method can be rapidly carried out in the field at low cost. Investigations showed that cholera was being transmitted by several distinct mechanisms. The predominant route of transmission in a given set- ting depends largely on the degree of sanitation already achieved. Therefore, a multifaceted ap- proach to prevention is needed. Emergency meas- ures, such as advice to boil drinking water or to heat all foods from street vendors, are difficult to sustain because of their inconvenience and high cost. Moreover, the cost of building large-scale water treatment and sanitation systems is ex- traordinary, estimated at $200 billion for all of Latin America (22). The challenge of cholera pre- vention lies in devising low-cost alternatives that are both effective and sustainable. Waterborne transmission was identified in seven of the eight investigations.

rge-scale water treatment and sanitation systems is ex- traordinary, estimated at $200 billion for all of Latin America (22). The challenge of cholera pre- vention lies in devising low-cost alternatives that are both effective and sustainable. Waterborne transmission was identified in seven of the eight investigations. In three of these, the implicated water came from municipal sys- tems or from tanker trucks that reportedly ob- tained water from municipal systems. Water delivered through poorly maintained municipal water systems can be contaminated by sewage because of leaky pipes, frequent pressure drops, andthe lack ofresidualchlorine disinfectant inthe water. In developing countries, water is rarely available 24 hours a day so it is usually stored in the home, where further contamination can easily occur. For example, when we measured the in- crease in contamination of the water as it was distributed and stored in Trujillo, Peru, fecal coli- form counts, an index of sewage contamination, were 1/100 ml in watercollected at the source well, 2/100 ml at public taps, and 20/100 ml in water stored in the home (13) In four investigations, the implicated water was collected from rivers or ponds, where direct sewage contamination was likely. Specificprotective practices were also noted in these investigations, including treating water in the home by boiling it or by adding chlorine bleach, using a small-mouthed vessel to store water, pouring water out of the storage vessel rather than scooping it out with a cup, and having hand soap in the home. In one investigation on the Amazon, we found that the local common practice of adding citrus juice to water to improve its taste was protective because the acid in the fruit killed Vibrio bacteria (14). This observation gave local authorities a new, inexpensive, and immediately available emergency control measure. The first stage of prevention, then, is providing safe drinking water. As a result of the above find- ings, we have developed and are testing simple and inexpensive methods of domestic water disin- fection and storage that would also prevent other diseases transmitted by the same route. A pilot trial in a periurban area of Bolivia showed that disinfecting household water with a calcium hypo- chlorite solution and storing it safely in a special narrow-mouthed container was acceptable to a community of Aymara Indians (20). Compliance, Table 2.

d also prevent other diseases transmitted by the same route. A pilot trial in a periurban area of Bolivia showed that disinfecting household water with a calcium hypo- chlorite solution and storing it safely in a special narrow-mouthed container was acceptable to a community of Aymara Indians (20). Compliance, Table 2. Mechanisms of transmission of epidemic cholera in Latin America, as determined in eight epidemiologic investigations, 1991-1993a Peru Ecuador El Bolivia Brazil Guatemala Trujillo Piura Iquitos Guayaquil Salvador Saipina Fortaleza Guatemala City Urban Urban Urban Urban Rural Rural Rural Urban Transmission mechanism 3/91 (15) 3/91 (16) 7/91 (17) 7/91 (18) 3/91 (19) 2/92 (20) 6/93 +b 7/93 (21) Waterborne Municipal water + + + Surface water + + + + Putting hands in + + water vessel Foodborne Street vendors' foods + + Street vendors' beverages + + + Street vendors' ice/ices + + Leftover rice + + + Fruits/vegetables + Seafood Uncooked seafood + Cooked seafood + + a Reference numbers are in parentheses. b CDC Unpublished data. Dispatches Emerging Infectious Diseases 143 Vol. 1, No. 4 -- October-December 1995 as measured by chlorine residuals in stored water, was high among families using the intervention. The concentration of Escherichia coli bacteria in the stored water, a measure of fecal contamina- tion, was significantly lower in households that used the intervention than in neighboring house- holds that used traditional water handling meth- ods (23). A field trial in rural Bolivia showed that villagers could generate their own disinfectant solution by using a simple electrolytic apparatus (24); with the disinfectant and the special water container the villagers provided clean water in their homes. Households using this intervention had 40% fewer diarrheal episodes than randomly selected neighboring familieswho usedtraditional water-handling methods. The combination of point-of-use disinfection and safer water storage containers could have broad effects, including lo- cal empowerment for production of potable water, safer infant foods, and newmicroindustries for the productionofdisinfectant solutions and waterves- sels (25). Cost-benefit analysis indicates that this strategy is costsaving if it preventsmorethan20% of diarrheal illnesses (26). Inexpensive disinfec- tant generators are now being manufactured for this purpose in Ecuador.

r infant foods, and newmicroindustries for the productionofdisinfectant solutions and waterves- sels (25). Cost-benefit analysis indicates that this strategy is costsaving if it preventsmorethan20% of diarrheal illnesses (26). Inexpensive disinfec- tant generators are now being manufactured for this purpose in Ecuador. In Colombia, the number of cholera cases has dropped dramatically since late 1992 when chlorine disinfectant tablets were distributed for treating household water. The second major route of cholera transmission is food contaminated in the market or home. This includes food and beverages sold by street ven- dors, leftover rice, and unwashed fruits and vege- tables. This was an important route in four of the eight investigated areas, including Guatemala City, where there was no evidence of waterborne transmission (21). Foods and beverages sold by street vendors are a fixture of urban life through- out the developing world; they are often prepared in unhygienic ways and then held at ambient temperatures for hours, which permits rapid bac- terial multiplication. Other problems associated with food and beverages from street vendors in- cluded using unsafe ice to chill beverages and selling homemade frozen drinks. Leftover rice is anexcellent growthmedium forV. cholerae O1 and eating leftover rice without reheating it was asso- ciated with illness in three investigations. In one investigation, illness was associated with eating unwashed produce that was probably splashed with river water while being transported to market in small boats. Thus, the second stage of cholera prevention is to improve food handling, particularly for foods and beverages sold by street vendors. Many coun- tries in Latin America have begun educating street vendors in fundamental food safety and linking this education to licensing (27). By itself, however, education may not improve food safety if clean water to prepare foods and beverages is not available and handwashing anddishwashing with soap and water are not routine. We are field test- ing a combined strategy of point-of-use disinfec- tion, handwashing with soap, and use of a special water/beverage container to improve the micro- bial quality of beverages sold by street vendors. Because street vendors are responsive tocustomer demand, teaching consumers to look for street vendors that are visibly practicing better hygiene may reinforce more hygienic conditions.

shing with soap, and use of a special water/beverage container to improve the micro- bial quality of beverages sold by street vendors. Because street vendors are responsive tocustomer demand, teaching consumers to look for street vendors that are visibly practicing better hygiene may reinforce more hygienic conditions. In addi- tion to these efforts to improve food sold on the street, health authorities should advise the public to reheat leftover rice and wash fruits and vegeta- bles before eating. In Santiago, Chile, suspicion that cholera was caused by vegetables irrigated with fresh sewage led to a ban on this practice; the ban not only prevented cholera transmission by this route, but also decreased the incidence of typhoid fever and hepatitis A dramatically (28). Transmission through seafood (identified in two of the eight investigations) is a third major route of cholera transmission, distinct from other foodborne mechanisms, because it requires differ- ent prevention strategies. One investigation im- plicated both uncooked seafood and cooked crab, and another implicated cooked seafood eaten without reheating. Contaminated seafood also caused three outbreaks of travel-associated chol- era in the United States. In the most dramatic, at least 75 persons contracted cholera after taking a flight from Latin America to California (29); ill- ness was associated with eating cold seafood salad that was loaded onto the plane in Lima, Peru. Two other outbreaks followed the informal transport of cooked crabs from Ecuador to the United States in travelers'suitcases (30,31). Marine creatures may harbor V. cholerae O1 before they are harvested or may be contaminated by seawater used in seaside processing plants. In areas where raw and under- cooked seafood are popular, seafood-associated cholera may occur even if the general level of sanitation and hygiene is high (32). Vibrios sur- vive light cooking and can subsequently grow if Dispatches Vol. 1, No. 4 -- October-December 1995 144 Emerging Infectious Diseases the seafood is held for many hours before eating (33). Preventing seafood-associated cholera in the long term will depend on maintaining sewage-free harvest beds and improving sanitation in process- ing plants. In coastal areas where the organism persists in the environment, even in the absence of sewage contamination, education to discourage the consumption of raw or undercooked shellfish is also needed.

lera in the long term will depend on maintaining sewage-free harvest beds and improving sanitation in process- ing plants. In coastal areas where the organism persists in the environment, even in the absence of sewage contamination, education to discourage the consumption of raw or undercooked shellfish is also needed. Thorough cooking provides the greatest security, but it is sometimes resisted by local populations for cultural reasons. "Ceviche," a popular Latin American dish prepared from seafood that is marinated in citrus juice for vari- able lengths of time, is a case in point. Prolonged marination in acidic liquid is likely to inactivate vibrios if the acid can penetrate throughout the flesh and deep organs of the fish or shellfish (34). Further evaluation of this approach is needed, but for the present, encouraging the use of ceviche recipes that provide sufficient marination time may be a practical intervention. In Latin America, as in other parts of the world, epidemiologic field investigations of cholera have defined the local routes of transmission, identified unsuspected and correctable control points, and quantified the effects of emergency measures. The results of investigations also have generated spe- cific control strategies targeted to blocking the predominant routes. While this multistage por- trait of transmission is complex, it is being trans- lated into action and change. The longstanding deficits in basic urban infrastructure and the need for new efforts to correct them have never been more apparent (35,36). Workable prevention strategies include better domestic water storage containers, point-of-use water disinfection, atten- tion to the education and hygiene of street ven- dors, and simple modifications of traditional recipes. Many other diseases are transmitted by these same waterborne and foodborne routes, so these control measures may prevent other infec- tions in addition to cholera. If it becomes a cata- lyst for long overdue improvements in the safety of water and food, epidemic cholera can have a far-reaching impact on the public health of Latin America. Robert V. Tauxe, M.D., M.P.H., Eric D. Mintz, M.D., M.P.H., Robert E. Quick, M.D., M.P.H. National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA References 1. Centers for Disease Control. Cholera--Peru, 1991. MMWR 1991;40:108-9. 2. Pan-American Health Organization. Cholera in the Americas. Epidemiol Bull 1995;16:11-3. 3.

P.H., Robert E. Quick, M.D., M.P.H. National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA References 1. Centers for Disease Control. Cholera--Peru, 1991. MMWR 1991;40:108-9. 2. Pan-American Health Organization. Cholera in the Americas. Epidemiol Bull 1995;16:11-3. 3. Wachsmuth IK, Evins GM, Fields PI, OlsvikO, Popovic T, Bopp CA, et al. The molecular epidemiology of chol- era in Latin America. J Infect Dis 1993;167:621-6. 4. Evins GM, Cameron DN, Wells JG, Greene KD, Pop- ovic T, Giono-Cerezo S, et al. The emerging diversity of the electrophoretic types of Vibrio cholerae in the West- ern Hemisphere. J Infect Dis 1995;172:173-9. 5. Ramamurthy T, Garg S, Sharma R, Bhattacharya SK, Nair GB, Shimada T, et al. Emergence of a novel strain of Vibrio cholerae with epidemic potential in southern and eastern India. Lancet 1993;341:703-4. 6. Mekalanos JJ, Sadoff JC. Cholera vaccines: fighting an ancient scourge. Science 1994;265:1387-9. 7. Sanchez JL, Vasquez B, BegueRE, Meza R, Castellares G, Cabezas C, et al. Protective efficacy of oral whole- cell/recombinant-B-subunit cholera vaccine in Peru- vian military recruits. Lancet 1994;344:1273-6. 8. Levine MM, Tacket CO. Recombinant live oral vac- cines. In: Wachsmuth IK, Blake PA, and Olsvik O, editors. Vibrio cholerae and Cholera. Washington, DC: American Society for Microbiology, 1994:395-413. 9. Rosenberg CE. The cholera years: The United States in 1832, 1849, and 1866. Chicago: University of Chi- cago Press, 1987. 10. Blake PA. Epidemiology of cholera in the Americas. Gastroenterol Clin North Am 1993;22:639-60. 11. Snow J. On the mode of communication of cholera. The Commonwealth Fund. London: Oxford University Press, 1936. 12. Mintz ED, Popovic T, Blake PA. Transmission of Vibrio cholerae O1. In: Wachsmuth IK, Blake PA, and Olsvik O, editors. Vibrio cholerae and cholera. Washington, DC: American Society for Microbiology, 1994:345-56. 13. Kolvin JL, Roberts D. Studies on the growth of Vibrio cholerae biotype El Tor and biotype classical in foods. J Hyg (Cambridge) 1982;89:243-52. 14. Blake PA, Rosenberg ML, Florencia J, Costa JB, Quintino L do P, Gangarosa EJ. Cholera in Portugal, 1974. II. Modes of transmission. Am J Epidemiol 1977;105:344-8. 15. Swerdlow DL, Mintz ED, Rodriguez M, Tejada E, Ocampo C, Espejo L, et al. Waterborne transmission of epidemic cholera in Trujillo, Peru: lessons for a conti- nent at risk. Lancet 1992;340:28-32. 16.

B, Quintino L do P, Gangarosa EJ. Cholera in Portugal, 1974. II. Modes of transmission. Am J Epidemiol 1977;105:344-8. 15. Swerdlow DL, Mintz ED, Rodriguez M, Tejada E, Ocampo C, Espejo L, et al. Waterborne transmission of epidemic cholera in Trujillo, Peru: lessons for a conti- nent at risk. Lancet 1992;340:28-32. 16. Ries AA, Vugia DJ, Beingolea L, Palacios AM, Vasquez E, Wells JG, et al. Cholera in Piura, Peru: a modern urban epidemic. J Infect Dis 1992;166:1429-33. 17. Mujica OJ, Quick RE, Palacios AM, Beingolea L, Var- gas R, Moreno D, et al. Epidemic cholera in the Ama- zon: The role of produce in disease risk and prevention. J Infect Dis 1994;169;1381-4. 18. Weber JT, Mintz ED, Canizares R, Semiglia A, Gomez I, Sempertegui R, et al. Epidemic cholera in Ecuador: multidrug resistance and transmission by water and seafood. Epidemiol Infect 1994;112:1-11. 19. Quick RE, Thompson BL, Zuniga A, Dominguez G, de Brizuela EL, de Palma O, et al. Epidemic cholera in rural El Salvador: risk factors in a region covered by a cholera prevention campaign. Epidemiol Infect 1995;114:249-55. Dispatches Emerging Infectious Diseases 145 Vol. 1, No. 4 -- October-December 1995 20. Gonzales O, Aguilar A, Antunez D, Levine W. An out- break of cholera in rural Bolivia: rapid identification of a major vehicle of transmission. 32nd Interscience Conference on Antimicrobial Agents and Chemother- apy, Anaheim, 1992. Washington, DC: American Soci- ety for Microbiology 1992; Abstract 937. 21. Koo D, Aragon A, Moscoso V, Gudiel M, Bietti L, Car- rillo N, et al. Epidemic cholera in Guatemala, 1993: transmission of a newly introduced epidemic strain by street vendors. Epidemiol Infect 1995; (in press). 22. de Macedo CG. Presentation of the PAHO regional plan. Proceedings of the Conference: Confronting chol- era, the development of a hemispheric response to the epidemic; 1991 Jul 8-9; Miami: University of Miami, 1991;39-44. 23. Quick R, VenczelL, Gonzales O, Damiani E, Highsmith A, Espada A, et al. Impact of narrow-necked water vessels and home chlorination on fecal coliform and E. coli counts in drinking water. 33rd Interscience Con- ference on Antimicrobial Agents and Chemotherapy, New Orleans, 1993. Washington, DC: American Soci- ety for Microbiology, 1993. 24. Quick R, Venczel L, Mintz E, Bopp C, Soleto L, Bean N, et al. Diarrhea prevention in Bolivia through safe water storage vessels and locally-produced mixed oxi- dant disinfectant.

ence Con- ference on Antimicrobial Agents and Chemotherapy, New Orleans, 1993. Washington, DC: American Soci- ety for Microbiology, 1993. 24. Quick R, Venczel L, Mintz E, Bopp C, Soleto L, Bean N, et al. Diarrhea prevention in Bolivia through safe water storage vessels and locally-produced mixed oxi- dant disinfectant. 35th Interscience Conference on An- timicrobial Agents and Chemotherapy, San Francisco, 1995. Washington, DC: American Society for Microbi- ology, 1995; Abstract 1347. 25. Mintz ED, Reiff FM, Tauxe RV. Safe water treatment and storage in the home: a practical new strategy to prevent waterborne disease. JAMA 1995;273:948-53. 26. Miller M, Quick R, Mintz E, Tauxe R, Teutsch S. Solid stools and solvent citizens: an effective solution for preventing diarrhea in developing countries. 34th In- terscience Conference on Antimicrobial Agents and Chemotherapy, Orlando, 1994. Washington, DC: American Society for Microbiology, 1994. Abstract 1244. 27. Arambulo P, Almeida CR, Cuellar J, Bellotto AJ. Street food vending in Latin America. Bull PAHO 1994;28:244-54. 28. Alcayaga S, Alcagaya J, Gassibe P. Changes in the morbidity profile of certain enteric infections after the cholera epidemic. Rev Chil Infect 1993;1:5-10. 29. Centers for Disease Control and Prevention. Cholera associated with an international airline flight, 1992. MMWR 1992;41:134-5. 30. Finelli L, Swerdlow D, Mertz K, Ragazzoni H, Spitalny K. Outbreak of cholera associated with crab brought from an area with epidemic disease. J Infect Dis 1992;166:1433-5. 31. Centers for Disease Control. Cholera--New York, 1991. MMWR 1991;40:516-8. 32. Lowry PW, Pavia AT, McFarland LM, Peltier BH, Bar- rett TJ, Bradford HB, et al. Cholera in Louisiana: widening spectrum of seafood vehicles. Arch Intern Med 1989;149:2079-84. 33. Blake PA, Allegra DT, Snyder JD, Barrett TJ, McFarland L, Caraway CT, et al. Cholera--a possible endemic focus in the United States. N Engl J Med 1980;302:305-9. 34. Mata L. Efecto del jugo y de la pulpa de frutas acidas sobreel Vibriocholerae. In: El Colera: Historia, preven- cion y control. San Jose, Editorial Universidad Estatal a Distancia - Editorial de la Universidad de Costa Rica, 1992, 275. 35. Sepulveda J, Gomez-Dantes H, Bronfman M. Cholera in the Americas: an overview. Infection 1992;20:243-8. 36. Witt VM, Reiff FM. Environmental health conditions and cholera vulnerability in Latin America and the Caribbean. J Public Health Policy 1991;12:450-63. Dispatches Vol. 1, No.

ersidad de Costa Rica, 1992, 275. 35. Sepulveda J, Gomez-Dantes H, Bronfman M. Cholera in the Americas: an overview. Infection 1992;20:243-8. 36. Witt VM, Reiff FM. Environmental health conditions and cholera vulnerability in Latin America and the Caribbean. J Public Health Policy 1991;12:450-63. Dispatches Vol. 1, No. 4 -- October-December 1995 146 Emerging Infectious Diseases

Are North American Bunyamwera Serogroup Viruses Etiologic Agents of Human Congenital Defects of the Central Nervous System? In 1941 Gregg provided the first evidence that rubella virus (family Togaviridae, genus Ru- bivirus) causes human congenital defects (1). Al- though rubella virus infection usually causes a mild disease comprising fever and rash, rubella epidemics have been associated with congenital defects in children of women who became infected during their first trimester of pregnancy (2). The risk of in utero rubella infection was reduced by the introduction of safe and effective vaccines for women of child-bearing age. Congenital abnor- malities in fetal or neonatal ruminants also are related to exposure of pregnant dams to various viruses, including bovine viral diarrhea virus (family Togaviridae, genus Pestivirus), the arthro- pod-borne bluetongue viruses (family Reoviridae, genus Orbivirus), Wesselsbron virus (family Flaviviridae, genus Flavivirus), Rift Valley fever virus (family Bunyaviridae, genus Phlebovirus), Nairobi sheep disease virus (familyBunyaviridae, genus Nairovirus), and Akabane and Aino (family Bunyaviridae, genus Bunyavirus, Simbu sero- group) viruses (3-10). Infections of livestock with these viruses may produce low-titer viremia with no apparent clinical disease, or high-titer viremia and severe clinical illness in the dam. In utero infections may result in malformations of the de- veloping fetus, fetal death with resorption, mum- mification, or miscarriage. Stillborn ruminants may show various musculoskeletal and central nervous system defects, including a syndrome of arthrogryposis with hydranencephaly (AGH). Bunyamwera serogroup viruses (family Bun- yaviridae, genus Bunyavirus) have been isolated from humans, and some, including Cache Valley (CV) and Tensaw (TEN) viruses, have been iso- lated from symptomatic and asymptomatic large mammals (11). Antibodies to CV virus and other viruses of the Bunyamwera serogroup are preva- lent in livestock and large wild mammals and in humans in the Western Hemisphere from Alaska to Argentina (11).

including Cache Valley (CV) and Tensaw (TEN) viruses, have been iso- lated from symptomatic and asymptomatic large mammals (11). Antibodies to CV virus and other viruses of the Bunyamwera serogroup are preva- lent in livestock and large wild mammals and in humans in the Western Hemisphere from Alaska to Argentina (11). Viruses of this serogroup are isolated primarily from mosquitoes of the genera Aedes and Anopheles. These viruses have focal geographicdistributions, although some are found over great expanses. CV virus, a common North American bunyavirus, has been isolated principally from mosquitoes of the genera Cu- liseta, Aedes, and Anopheles. The geographic dis- tribution of this virus includes all North America, except the extreme southeastern states and south- ern Mexico (11). In the southeastern United States, TEN virus, also isolated from mosquitoes of the genera Anopheles and Aedes, is the only known representative of the Bunyamwera sero- group, probably because of the range of the prin- cipal vectors and vertebrate hosts; mutual exclusion of these two viruses likely occurs be- cause of cross-protectivity between them (12). Serologic and temporal associations of infection with CV virus and congenital malformations, in- cluding primarily AGH, were observed in sheep near San Angelo, Texas, between December 1986 and February 1987, suggesting that this virus causes AGH (13). Subsequent outbreaks of similar congenital defects occurred in sheep in Illinois in 1988 (J. Pearson, pers. comm.) and in North Da- kota, Pennsylvania, Maryland, Michigan, and Ne- braskain1986 and 1987(14).Antibody toCVvirus (but not to other viruses) was found to correlate significantly withtheoccurrenceofAGHandother congenital anomalies during the Texas outbreak (15, 16), and IgM antibody to CV virus was de- tected in colostrum-free neonates with AGH (C.H. Calisher, unpublished data). (Neither maternal IgM nor maternal IgG crosses the placenta in sheep; therefore, antibody in fetuses or in neo- nates before they received colostrum indicates fe- tal exposure to an infectious agent [17].) In 1976, antibody to CV virus was detected in serum from cattle that had delivered calves with AGH in Saskatchewan, Canada, in 1975; however, the prevalence of antibodies to CV virus in the bovine population of that area was not investi- gated (R.E. Shope, pers. comm.). In Texas, in 1981, CV virus was isolated from a sick sheep and from a healthy cow in a herd with reproductive prob- lems (18).

vered calves with AGH in Saskatchewan, Canada, in 1975; however, the prevalence of antibodies to CV virus in the bovine population of that area was not investi- gated (R.E. Shope, pers. comm.). In Texas, in 1981, CV virus was isolated from a sick sheep and from a healthy cow in a herd with reproductive prob- lems (18). This virus also was isolated in Texas in 1988 from a sentinel sheep in pasture where an outbreak of congenital defects had occurred in 1986 to 1987. These historical data suggest that Dispatches Emerging Infectious Diseases 147 Vol. 1, No. 4 -- October-December 1995 CV-virus-related congenital malformations may be more widespread than has been recognized. Experimental infections have provided further evidence that CV virus causes embryonic death and multiple congenital malformations of sheep (19) and cattle (J. Edwards, pers. comm.). No association of TEN virus and disease in livestock, wild animals, or humans has been reported. To determine whether infection with selected Bun- yamwera serogroup viruses, CV and TEN, is asso- ciated with certain human congenital defects, a serosurvey was done with serum samples from mothers of children with microcephaly or macro- cephaly, and the results were compared with those from age- and location-matched controls. The re- sults, reported here, provide the first evidence that these Bunyamwera serogroup viruses may be etiologic agents of certain congenital defects of the human central nervous system. Two groups of 500 each human serum samples were selected at random from an archival collec- tion stored at the National Institutes of Health (NIH), Bethesda, Maryland, and tested for neu- tralizing antibody. These samples were part of a collection of serum specimens obtained between 1959 and 1964, at delivery or post partum, (from about 50,000 women enrolled under defined pro- tocols in a prospective study of congenital rubella syndrome. Specimens had been collected from all pregnant women cared for at the particular insti- tution or from women randomly selected by using the last digit of their hospital registration number. For a first series of tests, samples were selected from 200 mothers of children with macrocephaly (head size at least two standard deviations above the mean) and from 50 mothers of children with microcephaly (head size at least two standard deviations below the mean). The initial specimen from mothers with the respective defects was se- lected for testing.

ere selected from 200 mothers of children with macrocephaly (head size at least two standard deviations above the mean) and from 50 mothers of children with microcephaly (head size at least two standard deviations below the mean). The initial specimen from mothers with the respective defects was se- lected for testing. An equal number of mothers of babies without obvious central nervous system defects were selected as controls, which were age- (2 years) and site-matched, were registered for the studyinthe same month, and were ofthe same race as mothers of children with either macro- cephalyormicrocephaly.Serumsamples comprising this first group had been collected from pregnant women in Boston, Massachusetts (104), Provi- dence, Rhode Island (20), New York, New York (72), Philadelphia, Pennsylvania (36), Baltimore, Maryland(38), Buffalo, New York(50),Minneapolis, Minnesota (42), Charlottesville, Virginia (38), Memphis, Tennessee (52), New Orleans, Louisi- ana (36), and Portland, Oregon (12); these were tested for neutralizing antibody to eight bun- yaviruses (Table 1). An additional 500 samples were tested; 250 paired samples, selected as above, from the same archival collection, two each from women in Bos- ton (120), Buffalo (40), New Orleans (8), New York City (46), Baltimore (8), Charlottesville (12), and Minneapolis (16), including controls (selected as above). These were tested for neutralizing anti- body to CV virus only, to follow up on results of tests with the first set of samples. One sample from each of these women had been collected inthe first trimester of pregnancy, and the second had been collected at least 3 months later. Serum speci- mens were stored frozen at -20o C until they were shipped on dry ice (-70o C) to the Centers for Dis- ease Control laboratory at Fort Collins, Colorado. Twenty-nine of the first 500 serum specimens tested contained neutralizing antibody to CV, 29 had antibody to TEN, 29 to Jamestown Canyon, 26 to La Crosse, nine to Lokern, and six to Button- willow viruses. None had antibody to Main Drain or Mermet virus. No significant differences were observed in antibody prevalences to La Crosse, Jamestown Canyon, Lokern, and Buttonwillow viruses between mothers of microcephalic and macrocephalic infants and age- and location- matched controls (Table 1). Cases were not re- viewed for other defects.

body to Main Drain or Mermet virus. No significant differences were observed in antibody prevalences to La Crosse, Jamestown Canyon, Lokern, and Buttonwillow viruses between mothers of microcephalic and macrocephalic infants and age- and location- matched controls (Table 1). Cases were not re- viewed for other defects. The prevalence of antibody to CV virus inmoth- ers of microcephalic infants was not significantly different from the prevalence of such antibody in their matched control, but the presence of anti- body to CV virus in mothers was significantly correlated with macrocephaly in their infants (2 , d.f. = 1, n = 400, 4.797 p <0.05) (Table 1). None of the samples with neutralizing antibody to CV or TEN virus (titers ranged from 10 to 80) had IgM antibody. To determine whether there were statistically significant differences between prevalences of antibody to CV or TEN virus in mothers of macrocephalic infants and in their age- and site-matched controls, McNemar's chi-square was used. No significant difference was found for the presence of antibody to CV virus (p >0.05), but the presence of antibody to TEN virus (p < 0.05 or antibody to either CV or TEN virus (p <0.02) was related to the occurrence of macrocephaly in the infants of these mothers (Table 2). Dispatches Vol. 1, No. 4 -- October-December 1995 148 Emerging Infectious Diseases Table 3 summarizes the presence of antibody to CV and to TEN virus in women by hospital loca- tion and birth outcome. When prevalence of anti- body to CV or TEN virus in these women was analyzed by location and birth outcome, no statis- tically significant differences were determined (comparative data not shown). When the second set of 500 specimens (250 paired early- and late-pregnancy samples) was tested, specimens from eight women had neutral- izing antibody to CV virus. No diagnostically mean- ingful change in titer was detected between six samplepairs,butfourfoldrisesintiterweredetected in two others,(10 to 80, <10 to40), indicating recent infections with CV virus or with a closely related Bunyamwera virus group. Six of theeight women in this group with antibody to CV virus delivered macrocephalic infants, including the two (one in New Orleans and one in New York City) whose specimens showed rises in titer to CV virus.

to 80, <10 to40), indicating recent infections with CV virus or with a closely related Bunyamwera virus group. Six of theeight women in this group with antibody to CV virus delivered macrocephalic infants, including the two (one in New Orleans and one in New York City) whose specimens showed rises in titer to CV virus. These analyses provide the first evidence that Bunyamwera serogroup viruses in North America are associated with congenital defects in humans: the occurrence of macrocephaly in infants was positively correlated with antibody to CV virus. Antibody to TEN virus and to either CV or TEN virus correlated with microcephaly and with macrocephaly. The presence of antibody to CV and to TEN viruses corresponded with the known geo- graphic distributions of these viruses within the United States. Antibody to either of these viruses in a woman living in an area where that virus is not known to occur may reflect the close antigenic relationships and considerable cross-reactivity of these Bunyamwera serogroup viruses (22), differ- ences between local virus strains and prototype viruses, or travel to an area inwhich the virus does occur (23). The two women with diagnostically significant rises in antibody titer to CV virus (one from New Orleans, where TEN virus has been isolated, and one from New York, where CV virus has been isolated) are, as far as we know, the first two persons with documented rises in antibody to a Bunyamwera serogroup virus in North America. Whether either of them had an associated illness could not be determined from the records. That among all the women tested they were the only ones withrises inantibodytiterandthat bothgave birth to macrocephalic infants is, at the least, a fascinating coincidence. IgM antibody in human infections caused by other bunyaviruses may not persist for much more than a few months after Table 1.

ned from the records. That among all the women tested they were the only ones withrises inantibodytiterandthat bothgave birth to macrocephalic infants is, at the least, a fascinating coincidence. IgM antibody in human infections caused by other bunyaviruses may not persist for much more than a few months after Table 1. Antibody to Cache Valley, Tensaw, La Crosse, Jamestown Canyon, Lokern, or Buttonwillow viruses in moth- ers of microcephalic or macrocephalic infants and matched controls Antibody to virus Infant's condition 2 (p) Cache Valley Microcephaly 3.840 (0.05) Macrocephaly 4.797 (<0.05) Either 0.915 (>0.20) Tensaw Microcephaly 4.891 (<0.05) Macrocephaly 4.071 (<0.05) Either 0.329 (>0.20) Cache Valley or Tensaw Microcephaly 5.983 (<0.02) Macrocephaly 4.806 (<0.05) La Crosse Microcephaly 0.211 (>0.20) Macrocephaly 1.481 (>0.20) Jamestown Canyon Microcephaly 0.709 (>0.20) Macrocephaly 0.037 (>0.20) Lokern Microcephaly 1.010 (>0.20) Macrocephaly 2.041 (>0.10) Buttonwillow Microcephaly 2.041 (>0.10) Macrocephaly <0.001 (>0.20) Bunyaviruses used for all tests were prototypes: (Bunyamwera serogroup) CV (strain 6V-633), TEN (A9-171b), Lokern (FMS-4332), Main Drain (BFS-5015), (California serogroup) La Crosse (Original), Jamestown Canyon (61V-2235), (Simbu serogroup) Buttonwillow (A-7956), and Mermet (AV-782). Samples were tested for antibody by serum dilution-plaque reduction neutralization (20). Briefly, diluted and heat- inactivated (56o C/30 min) serum was added to an equal volume of virus containing approximately 200 plaque-forming units (PFU), such that the final virus dilution was 100 PFU. Fresh human serum at a final concentration of 8% was added to all virus suspensions. Serum-virus mixtures were incubated at 4o C for 18 h, and 0.1-ml aliquots were dropped in the center of Vero cultures grown in 6-well plastic plates. After a 45-min period of adsorption, cells were overlaid with medium containing 2% agar and further incubated for 70 to 75 h at 37o C in 5% CO2-95% air. A second overlay, containing medium, 2% agar, and 1:25,000 neutral red was then added, and the plates again were incubated until plaques were readable, usually 12 to 36 h later. A serum specimen was considered positive when it reduced the number of plaques 90% relative to control titrations, which ranged from 80 to 150 plaques.

overlay, containing medium, 2% agar, and 1:25,000 neutral red was then added, and the plates again were incubated until plaques were readable, usually 12 to 36 h later. A serum specimen was considered positive when it reduced the number of plaques 90% relative to control titrations, which ranged from 80 to 150 plaques. Samples that were positive in a screening (1:10) test were titrated for end-point, and the serum titer was taken as the highest twofold dilution of serum that reduced the number of plaques 90%. Although a human positive control for detecting IgM antibody by capture enzyme-linked immunosorbent assays was not available, serum specimens were tested by a modification of a published technique for detecting IgM antibody to California serogroup viruses (21). Mouse or sheep serum samples containing IgM antibody to CV virus or mouse serum with IgM antibody to TEN virus served as positive and negative IgM antibody controls. Statistical analyses were done by chi-square or McNemar's chi-square. Dispatches Emerging Infectious Diseases 149 Vol. 1, No. 4 -- October-December 1995 infection (21); IgM antibody has not been detected in humans with antibody to Bunyamwera sero- group viruses in North America. Therefore, the absence of IgM antibody in specimens with neu- tralizing antibody likelyindicates that these infec- tions were not acute, i.e., they occurred months before the specimens were collected. A fundamental problem in establishing a rela- tionship between infection of mammals with CV or TEN virus and attendant congenital anomalies is the inherent inadequacy of controls. The pres- ence of antibody in humans or other animals with normal offspring does not necessarily argue against the hypothesis that CV virus causes con- genital defects in humans because infection could haveoccurredbeforepregnancy. Additionally,even if this virus can be an etiologic agent of congenital anomalies,preexisting antibodyto this virus could provide immunity for the mother and protect the fetus from viral infection. Thus, it cannot be deter- mined with certainty whether the presence of antibody to a virus is coincidental to, or a cause of, the observed congenital anomalies.

an etiologic agent of congenital anomalies,preexisting antibodyto this virus could provide immunity for the mother and protect the fetus from viral infection. Thus, it cannot be deter- mined with certainty whether the presence of antibody to a virus is coincidental to, or a cause of, the observed congenital anomalies. Review of NIH records for this relatively small set of samples suggested that macrocephaly oc- curred somewhat more often when the first trimester of pregnancy included the months April and May for women living in New Orleans (4/16), Memphis (5/16), and Charlottesville (3/12) and in late summer-early autumn for women living in Boston (6/49), Minneapolis (3/11), Portland (2/6), and New York City (9/33). In each instance, these periods coincide roughly with the appearance of populations of Culiseta, Aedes, or Anopheles mos- quitoes, the vectors of CV and TEN viruses. How- ever, CV virus cannot be implicated in infections in New Orleans because this virus is not known to occur there, although TEN virus does. The relatively small sample sizes in this study allow statistical interpretation but do not provide sufficient support to warrant statements as to the biological significance of the findings; therefore, we consider these data merely preliminary. Deter- mining whether these data have merit awaits the results of additional studies of mothers of children with congenital defects and their offspring. More extensive studies are also needed to investigate the influence of gestational phase and fetal devel- opment on congenital defects; establish relation- ships between peak abundance of arthropod vectors and first trimesters of pregnancies; se- quence the genomes of CV and TEN virus strains from various geographic areas and establish rela- tionships between different gene sequences and virulence in livestock; and develop diagnostic capacity by using monoclonal antibodies, hybridi- zation assays, polymerase chain reaction, and Western blotting techniques. Table 2. Presence of antibody to Cache Valley and Tensaw viruses in mothers of macrocephalic infants and in matched controls Mothers of Antibody-positive Antibody-negative macrocephalic infants No. controls controls Antibody to CV virus 16 3 13 No antibody to CV virus 184 5 179 McNemar's testa Antibody to TEN virus 15 1 14 No antibody to TEN virus 185 5 180 McNemar's testb Antibody to CV or TEN virus (or both) 19 3 16 No antibody to CV or TEN virus 181 5 176 McNemar's testc a (13-5)2 /18 = 3.556 (d.f. = 1), p > 0.05.

rols controls Antibody to CV virus 16 3 13 No antibody to CV virus 184 5 179 McNemar's testa Antibody to TEN virus 15 1 14 No antibody to TEN virus 185 5 180 McNemar's testb Antibody to CV or TEN virus (or both) 19 3 16 No antibody to CV or TEN virus 181 5 176 McNemar's testc a (13-5)2 /18 = 3.556 (d.f. = 1), p > 0.05. b (14-5)2 /19 = 4.263 (d.f. = 1), p <0.05. c (16-5)2 /21= 5.762 (d.f. = 1), p <0.02. Table 3. Antibody to Cache Valley and Tensaw viruses in women, by location and birth outcome Antibody to CV virus Antibody to TEN virus In women with In women with macrocephalic In macrocephalic: In infants:: controls: infants: controls: Location no. (%) no. (%) no. (%) no. (%) Boston 2/49 (4.1) 1/49 (2.0) 1/49 (2.0) 1/49 (2.0) Providence 0/7 1/7 (14.3) 0/7 1/7 (14.3) New York City 2/33 (6.1) 1/33 (3.0) 1/33 (3.0) 1/33 (3.0) Philadelphia 0/15 2/15 (13.3) 1/15 (6.7) 2/15 (13.3) Minneapolis 1/11 (9.1) 0/1 1/11 (9.1) 0/11 Charlottesville 2/12 (16.7) 0/12 2/12 (16.7 0/12 Memphis 6/16 (37.5) 3/16 (18.8) 5/16 (31.3) 1/16 (6.3) New Orleans 1/16 (6.3) 0/1 2/16 (12.5) 0/16 Portland 2/6 (33.3) 0/6 2/6 (33.3) 0/6 Dispatches Vol. 1, No. 4 -- October-December 1995 150 Emerging Infectious Diseases Given that many members of the family Bun- yaviridae cause congenital defects in naturally and experimentallyinfected livestock, or mayhave such a potential (24,25), it would be worthwhile to continueinvestigations withdomestic animals and to develop laboratory models to assess the terato- genic potential for humans of CV, TEN, and other viruses of the family Bunyaviridae. It is also important to determine the roles of CV and TEN viruses in inducing human congenital defects and the relationships between prevalence of antibody to CV and TEN viruses, prevalence of congenital defects, and conception dates, all with respect to local environmental conditions. Charles H. Calisher, Ph.D.,* and John L. Sever, M.D. *Colorado State University, Fort Collins, Colorado, USA;

g human congenital defects and the relationships between prevalence of antibody to CV and TEN viruses, prevalence of congenital defects, and conception dates, all with respect to local environmental conditions. Charles H. Calisher, Ph.D.,* and John L. Sever, M.D. *Colorado State University, Fort Collins, Colorado, USA; George Washington University Medical Center, Children's National Medical Center, Washington, D.C., USA Acknowledgments The authors thank Dr. Maneth Gravell and Ms. Dorothy O'Neill, National Institutes of Health, Bethesda, Mary- land, for selecting, sorting, and shipping serum samples; Mr. Raymond E. Bailey, Centers for Disease Control and Prevention, Fort Collins, Colorado, for statistical advice; Dr. Thomas P.C. Monath, Division of Vector-Borne Infec- tious Diseases, CDC, Fort Collins, for his encouragement and support; and Drs. Barry Beaty, Colorado State Univer- sity, Fort Collins, and John Edwards, Texas A&M Univer- sity, College Station, Texas, for their editorial suggestions. References 1. Gregg N McA. Congenital cataract following German measles in the mother. Trans Ophthal Soc Austral 1941;3:35-46. 2. Best JM, O'Shea S. Rubella virus. In: Schmidt NJ, Emmons RW, editors. Diagnostic procedures for viral, rickettsial and chlamydial infections, 6th ed. Washing- ton, DC: American Public Health Association 1989:731-95. 3. Oberst RD. Viruses as teratogens. Vet Clin North Am; Food Anim Prac 1993:9:23-31. 4. Erasmus BJ. The history of bluetongue. In: Barber TL, Jochim MM, editors. Bluetongue and related or- biviruses. New York: AR Liss, 1985:7-12. 5. Calisher CH, Monath TP. Togaviridae and Flaviviri- dae: the alphaviruses and flaviviruses. In: Lennette EH, Halonen P, Murphy FA, editors. Laboratory diag- nosis of infectious diseases, vol. 2. New York: Springer- Verlag, 1988:414-34. 6. Calisher CH, Shope RE. Bunyaviridae: the bun- yaviruses. In: Lennette EH, Halonen P, Murphy FA, editors. Laboratory diagnosis of infectious diseases, vol. 2. New York: Springer-Verlag, 626-46. 7. Kurogi H, Inaba Y, Takahashi E, Sato K, Omori T, Miura Y, et al. Epizootic congenital arthrogryposis- hydranencephaly syndrome in cattle: isolation of Akabane virus from affected fetuses. Arch Virol 1976;51:67-74. 8. Kurogi H, Inaba Y, Takahashi E, Sato K, Satoda K, Goto Y, et al. Congenital abnormalities in newborn calves after inoculation of pregnant cows with Akabane virus. Infect Immun 1977:17:338-43. 9. Parsonson IM, Della-Porta AJ, Snowdon WA, Murray MD.

ion of Akabane virus from affected fetuses. Arch Virol 1976;51:67-74. 8. Kurogi H, Inaba Y, Takahashi E, Sato K, Satoda K, Goto Y, et al. Congenital abnormalities in newborn calves after inoculation of pregnant cows with Akabane virus. Infect Immun 1977:17:338-43. 9. Parsonson IM, Della-Porta AJ, Snowdon WA, Murray MD. Congenital abnormalities in foetal lambs after inoculation of pregnant ewes with Akabane virus. Aus- tral Vet J 1975:51:585-6. 10. Coverdale OR, Cybinski DH, St. George TD. Congeni- tal abnormalities in calves associated with Akabane and Aino virus. Austral Vet J 1978:54:151-2. 11. Calisher CH, Francy DB, Smith GC, Muth DJ, Lazuick JS, Karabatsos N, et al. Distribution of Bunyamwera serogroup viruses in North America, 1956-1984. Am J Trop Med Hyg 1986;35:429-43. 12. Calisher CH. Evolutionary significance of the taxo- nomic data regarding bunyaviruses of the family Bun- yaviridae. Intervirology 1988;29:268-76. 13. Crandell RA, Livingston CW. Laboratory investigation of a naturally occurring outbreak of arthrogryposis-hy- dranencephaly in Texas sheep. J Vet Diagn Invest 1988;1:62-5. 14. Rook JS, Yamini B, Steficek B. AGH syndrome: coop- eration answers questions. National Wool Grower 1988(April):24-5. 15. Edwards JF, Livingston CW, Chung SI, Collisson EC. Ovine arthrogryposis and central nervous system malformations associated with in utero Cache Valley virus infection: spontaneous disease. Vet Pathol 1989;26:33-9. 16. Edwards JF. Cache Valley virus. Vet Clin North Am; Food Anim Prac 1994;10:515-24. 17. Campbell SG, Siegel MJ, Knowlton BJ. Sheep immu- noglobulins and transmission to the neonatal lamb. N Z Vet J 1977;25:361-5. 18. McConnell S, Livingston C Jr, Calisher CH, Crandell R. Isolations of Cache Valley virus in Texas, 1981. Vet Microbiol 1987;13:11-8. 19. Chung SI, Livingston CW Jr, Edwards JF, Gauer BB, Collisson EW. Congenital malformations in sheep re- sulting from in utero inoculation of Cache Valley virus. Am J Vet Res 1990;51:1645-8. 20. Lindsey HS, Calisher CH, Mathews JH. Serum dilu- tion neutralization test for California group virus iden- tification and serology. J Clin Microbiol 1976;4:503-10. 21. Calisher CH, Pretzman CI, Muth DJ, Parsons MA, Peterson ED. Serodiagnosis of La Crosse virus infec- tions in humans by detection of immunoglobulin M class antibodies. J Clin Microbiol 1986;23:667-71. 22. Calisher CH, Lazuick JS, Lieb S, Monath TP, Castro KG.

tification and serology. J Clin Microbiol 1976;4:503-10. 21. Calisher CH, Pretzman CI, Muth DJ, Parsons MA, Peterson ED. Serodiagnosis of La Crosse virus infec- tions in humans by detection of immunoglobulin M class antibodies. J Clin Microbiol 1986;23:667-71. 22. Calisher CH, Lazuick JS, Lieb S, Monath TP, Castro KG. Human infections with Tensaw virus in south Florida: evidence that Tensaw virus subtypes stimu- late the production of antibodies reactive with closely related Bunyamwera serogroup viruses. Am J Trop Med Hyg 1988;39:117-22. 23. Calisher CH, Sabattini MS, Wolff KL, Monath TP. Cross-neutralization tests of South American isolates of Cache Valley virus revealing the existence of multi- ple subtypes. J Trop Med Hyg 1988;39:202-5. 24. Parsonson IM, Della-Porta AJ, Snowdon WA. Develop- mental disorders of the fetus in some arthropod-borne virus infections. Am J Trop Med Hyg 1981;30:660-73. 25. McPhee DA, Parsonson IM, Della-Porta AJ, Jarrett RG. Teratogenicity of Australian Simbu serogroup and some other Bunyaviridae viruses: the embryonated chicken egg as a model. Infect Immun 1984;43:413-20. Dispatches Emerging Infectious Diseases 151 Vol. 1, No. 4 -- October-December 1995

Lymphocytic Choriomeningitis Virus: An Unrecognized Teratogenic Pathogen Lymphocytic choriomeningitis virus (LCMV), the first member of the arenavirus family to be isolated, is the causative agent of a zoonosis ac- quired from chronically viremic mice or hamsters (1). The clinical spectrum of acquired human LCMV infection ranges from inapparent and asymptomatic to, in rare instances, severely symptomatic, systemic, and fatal central nervous system (CNS) disease. Intrauterine LCMV infec- tion has resulted in fetal or neonatal death, as well as hydrocephalus and chorioretinitis in infants (2-6). We have diagnosed congenital LCMV infec- tion in three infants (7) and have collated pub- lished and unpublished data on three additional affected infants (8, G.R. Istre, pers. comm.). This report briefly summarizes the salient features of the infection in five of these six American infants and outlines the similarities between these and features observed earlier in Europe. We suggest that LCMV is a more frequent cause of CNS dis- ease in newborns than previously recognized. Congenital LCMV infection was first recog- nized in Great Britain in an infant who died at 12 days of age (3). Subsequently, fetal infection with spontaneous abortion (2) and congenital infection in liveborn infants with hydrocephalus and chori- oretinitis were documented in Germany (4), France (6), and Lithuania (5). We have recently documented congenital LCMV infection in three infants from Arizona (7) and have obtained infor- mation regarding three additional neonates from Arizona, Nebraska (8), and Texas (G.R. Istre, pers. comm.). Detailed clinical and laboratory data are available for five of the six infants. All displayed nonobstructive hydrocephalus with periventricu- larcalcifications, chorioretinitis, and psychomotor retardation. One of the five infants had sensor- ineural deafness. None of the infants had cardiac abnormalities.

rs. comm.). Detailed clinical and laboratory data are available for five of the six infants. All displayed nonobstructive hydrocephalus with periventricu- larcalcifications, chorioretinitis, and psychomotor retardation. One of the five infants had sensor- ineural deafness. None of the infants had cardiac abnormalities. Two infants have had follow-up ophthalmologic and audiologic examinations which have shown neither the progression of chorioretinitis nor the development of new audi- tory deficits.Toxoplasma gondii, cytomegalovirus, Herpes simplex virus, rubella, enterovirus, and Treponema pallidum infections were excluded by culture or serology in all infants. The diagnosis of congenital LCMV infection was confirmed in all infants by immunofluorescence antibody (IFA) and enzyme-linked immunosorbent assays (ELISAs). In addition, serum, CSF, urine, and throat wash specimens from two infants were injected into Vero cell monolayers. Neither cytopa- thic effect nor LCMV antigens were detected after incubation. Because virus isolation was only at- tempted after the disease was first diagnosed when the children were 10 months of age, failure to isolate LCMV was not unanticipated. Laboratory diagnosis of LCMV infection is gen- erally made by serologic techniques. IFAis a more sensitive diagnostic method than either comple- ment fixation or neutralizing antibody techniques (9,10). The newer ELISAs are now being used to evaluate congenitally infected infants. Testing the child's serum and CSF and a simultaneously ob- tained serum specimen from the motheryields the maximum information if done as soon after birth as possible. The mothers of four of the five infants in this report had a history of febrile illness during preg- nancy, in contrast to a minority of mothers of affected infants previously reported. Typical LCMV infection in adults is a biphasic disease with fever, malaise, myalgias, anorexia, nausea, vomiting, pharyngitis, cough, and adenopathy fol- lowed by defervescence and a second phase of CNS disease. However, CNS symptoms may appear without any prodrome ormaynever develop. Men- ingitis and meningoencephalitis are the most fre- quent neurologic manifestations of disease, although myelitis, Guillain-Barre syndrome, and sensorineural deafness have been reported (11). Between 1941 and 1958, 8% to 11% of viral CNS syndromes in hospitalized patients in a Washing- ton, D.C., medical center were etiologically associ- ated with LCMV (12).

e most fre- quent neurologic manifestations of disease, although myelitis, Guillain-Barre syndrome, and sensorineural deafness have been reported (11). Between 1941 and 1958, 8% to 11% of viral CNS syndromes in hospitalized patients in a Washing- ton, D.C., medical center were etiologically associ- ated with LCMV (12). Arthritis, parotitis, orchitis, myocarditis, and rash have also been noted (13). Clinical interest in LCMV, however, has not been maintained, and the disease is rarely considered despite improved serodiagnostic methods. Although a history of contact with rodents and their excreta is of diagnostic utility, it is not uni- versally present. A maternal history of rodent exposure was elicited for three of our five infants. Wild mice (Mus musculus) and hamsters infected in utero with LCMV during maternal viremia develop both persistent viremia and viruria. The Dispatches Vol. 1, No. 4 -- October-December 1995 152 Emerging Infectious Diseases virus is transmitted to humans by direct animal contact; by contact with infected rodent saliva, nasal secretions, urine, feces, semen, and milk; and by infectious aerosols (1). Human-to-human transmission has not been documented. The dis- tributionofLCMV is highlyvariable within mouse populations. Seasonal, annual, and cyclical vari- ations in rodent density and infection have been postulated but remain inadequately studied (14). LCMV spreads to humans in rural settings or when human habitats are substandard. Infected laboratory and pet rodents have also been associ- ated with disease inhumans (1). Serologic surveys and clinical studies have documented both epi- demic and endemic human infection in Europe and the Americas. In Baltimore, 9.0% of house mice and 4.7% of residents have had measurable LCMV antibody (15,16). We hypothesize that congenital LCMV infec- tion is generally undiagnosed and may account for unexplained hydrocephalus with microcephaly or macrocephaly, deafness, blindness, and mental re- tardation (three of the five infants in this report were referred for infectious disease consultation by pediatric geneticists, and two were referred by pediatric neurologists). No accurate data are available regarding the prevalence and persist- ence of LCMV antibodies in unselected infants, children, and adults in diverse geographic locales or in children with unexplained visual and/or auditory deficits, microcephaly, and retardation.

icists, and two were referred by pediatric neurologists). No accurate data are available regarding the prevalence and persist- ence of LCMV antibodies in unselected infants, children, and adults in diverse geographic locales or in children with unexplained visual and/or auditory deficits, microcephaly, and retardation. Increased recreational activities in rural environ- ments, rehabilitation of and habitation in older rodent-infected domiciles, and acquisition of un- screened rodents for pets or laboratory use pose as yet undefined risks for LCMV infection to the fetus, child, and adult. The need for further re- search to define the frequency of LCMV infection in human and animal populations is clear. LCMV infection can best be prevented by educating the public and medical professionals on the hazards of contact with infected rodents. Leslie L. Barton, M.D.,* C.J. Peters, M.D., T.G. Ksiazek, D.V.M., Ph.D. *University of Arizona Health Sciences Center, Department of Pediatrics and Steele Memorial Childrens Research Center, Tucson, Arizona, USA; National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA Acknowledgments We thank Drs. Jane Wilson, Gregory Istre, Stephen Chartrand, and Laurie Seaver for patients' information; Dr. Matafija Seinbergas for his enthusiastic support; and Ms. Amy Sites for technical support. References 1. Jahrling PB, Peters CJ. Lymphocytic choriomeningi- tis: a neglected pathogen of man. Arch Pathol Lab Med 1992;116:486-8. 2. Ackermann R, Stammler A, Armbruster B. Isolierung von Virus der lymphozytaren Choriomeningitis aus Abrasionsmaterial nach Kontakt der Schwangeren mit einem Syrischen Goldhamster (Mesocricetus aura- tus). Infection 1975;3:47-9. 3. Komrower GM, Williams BL, Stones PB. Lymphocytic choriomeningitis in the newborn: probable transpla- cental infection. Lancet 1955;1:697-8. 4. Ackermann R, Korver G, Turss R, Wonne R, Hochge- sand P. Prenatal infection with the lymphocytic choriomeningitis virus. Dtsch Med Wochenschr 1974;13:629-32. 5. Seinbergas MM. Hydrocephalus due to prenatal infec- tion with the lymphocytic choriomeningitis virus. In- fection 1976;4:185-91. 6. Chastel C, Bosshard S, Le Goff F, Quillien MC, Gilly R, Aymard M. Infection transplacentaire par le virus de la choriomeningite lymphocytaire: resultats dune enguete serologique retrospective en France. Nouv Press Med 1978;7:1089-92. 7. Barton LL, Budd SC, Morfitt WS, et al.

ingitis virus. In- fection 1976;4:185-91. 6. Chastel C, Bosshard S, Le Goff F, Quillien MC, Gilly R, Aymard M. Infection transplacentaire par le virus de la choriomeningite lymphocytaire: resultats dune enguete serologique retrospective en France. Nouv Press Med 1978;7:1089-92. 7. Barton LL, Budd SC, Morfitt WS, et al. Congenital lymphocytic choriomeningitis virus infection in twins. Pediatr Infect Dis J 1993;12:942-6. 8. Larsen PD, Chartrand SA, Tomashek KY, Hauser LG, Ksiazek TG. Hydrocephalus complicating lymphocytic choriomeningitis virus infection. Pediatr Infect Dis J 1993;12:528-31. 9. Lewis VJ, Walter PD, Thacker WL, Winkler WG. Com- parison of three tests for the serological diagnosis of lymphocytic choriomeningitis virus infection. J Clin Microbiol 1975;2:193-7. 10. Lehmann-Grube F, Kallay M, Ibscher B, Schwartz R. Serologic diagnosis of human infections with lympho- cytic choriomeningitis virus: comparative evaluation of seven methods. J Med Virol 1979;4:125-36. 11. Lehmann-Grube F. Diseases of the nervous system caused by lymphocytic choriomeningitis virus and other arenaviruses. In: Handbook of clinical neurology. New York: Elsevier, 1989;12:355-81. 12. Meyer HM, Johnson RT, Crawford, IP, Dascomb HE, RogersNG.Central nervous system syndromesof "viral" etiology: a study of 713 cases. Am J Med 1960:334-47. 13. Lewis JM, Utz JP. Orchitis, parotitis and menin- goencephalitis due tolymphocytic-choriomeningitis vi- rus. N Engl J Med 1961;265:776-80. 14. Childs JE, Peters CJ. Ecology and epidemiology of arenaviruses and their hosts. In: The Arenaviridae. New York: Plenum, 1993:331-84. 15. Childs JE, Glass GE, Korch GW, Ksiazek TG, Leduc JW. Lymphocytic choriomeningitis virus infection and house mouse (Mus musculus) distribution in urban Baltimore. Am J Trop Med Hyg 1992;47:27-34. 16. Childs JE, Glass GE, Ksiazek TG, Rossi CA, Barrera Oro JG, Leduc JW. Human-rodent contact and infec- tion with lymphocytic choriomeningitis and Seoul vi- ruses in an intercity population. Am J Trop Med Hyg 1991;44:117-21. Dispatches Emerging Infectious Diseases 153 Vol. 1, No. 4 -- October-December 1995

Hemolytic Uremic Syndrome Along with a report of the first outbreak of hemolytic uremic syndrome (HUS) caused by Shiga-like toxin (SLT) producing E. coli inAustra- lia (1), this issue of Emerging Infectious Diseases presents three papers detailing the investigations of pediatric HUS cases linked to Shiga toxin (ST) and SLT producing bacteria. Goldwater and Bet- telheim present a case of pediatric HUS associ- ated with SLT producing Escherichia coli (SLTEC) O48:H21 in South Australia; this strain has not previously been recognized as an SLTEC. Saeed et al. report on the increasingly common identifi- cation of HUS inSaudi Arabia, its association with multiple-antibiotic-resistant Shigella dysenteriae type 1, and the inherent dangers of treating such patients with ampicillin and nalidixic acid. Al- Qawari et al. report on the results of active sur- veillance for dysentery and HUS in Saudi Arabia and discuss a possibly elevated risk for HUS in patients with bloody diarrhea who are hospital- ized and treated with nalidixic acid during an outbreak of S. dysenteriae type 1. The three papers raise a number of important issues regarding HUS. First, it is clear that a large number of SLT producing bacteria have the poten- tial to cause HUS, particularly among children. Current research has focused on E. coli O157:H7, which since the early 1980s has emerged as a major foodborne cause of bloody diarrhea, hemor- rhagic colitis, and HUS since the early 1980s (5-7). However, the large outbreak of pediatric HUS in South Australia in 1995 caused by foodborne E. coli O111:HNM has demonstrated that minor pathogens can emerge as major causes of HUS (1). Goldwater and Bettelheim (5) and other re- searchers have identified a number of E. coli sero- types isolated from patients with HUS (6,7). A-Qawari et al. and Saeed et al. offer a timely reminder that S. dysenteriae type 1, a pathogen with a human-only reservoir, is an equally serious contender inHUS etiology and pathogenesis when conditions facilitate the person-to-person trans- mission of pathogens. The second key issue raised by the latter two papers concerns treatment of bloody diarrhea.

r a timely reminder that S. dysenteriae type 1, a pathogen with a human-only reservoir, is an equally serious contender inHUS etiology and pathogenesis when conditions facilitate the person-to-person trans- mission of pathogens. The second key issue raised by the latter two papers concerns treatment of bloody diarrhea. Both discuss the potential for antibiotic (am- picillin and nalidixic acid)-mediated HUS and conclude that this issue should be carefully evalu- ated before antibiotics are used to manage bloody diarrhea. Saeed et al. note that the wide variety of antibiotics used to treat bloody diarrhea in Saudi Arabia could be explained by the various prescription practices of doctors recruited from different parts of the world. Antibiotic resistance is a worrying component of the mechanisms of emerging infectious diseases. Inappropriate anti- biotic use is a key factor in the development of resistance, and major efforts must be directed towards educating physicians on effective pre- scribing practices. Central to all three papers is the need for sur- veillance of organisms that cause bloody diarrhea, hemorrhagic colitis, and HUS, as well as for knowledge of the local epidemiology of SLTECs, their potential sources, and the optimal way to investigate and manage outbreaks. Goldwater and Bettelheim discuss the characteristic disap- pearance of E. coli from patients' stools after the development of HUS and, therefore, the impor- tance of early detection in cases of bloody diar- rhea. Laboratory testing of bloody diarrheal specimens is clearly critical to understanding the epidemiology of toxin-producing organisms that relate to the development of HUS (8). However, testing can be difficult, time-consuming and costly. Not all laboratories routinely test for SLTECs orhave the capacity to do so. In Australia, for example, using polymerase chain reaction (PCR) technology to detect SLT genes is expen- sive: a negative test result costs approximately $A15, but if the results are positive, the cost rises to around $A250 when the SLTEC is isolated and typed. Human surveillance is essential to the early detection of outbreaks and to the critical assess- ment of the impact on public health of new ap- proaches to food safety (2,8). We conservatively estimate that the South Australian HUS outbreak has cost around $A20 million indirect and indirect costs, with major impacts being felt by industry. This must surely be considered when contemplat- ing the costs of surveillance.

t of the impact on public health of new ap- proaches to food safety (2,8). We conservatively estimate that the South Australian HUS outbreak has cost around $A20 million indirect and indirect costs, with major impacts being felt by industry. This must surely be considered when contemplat- ing the costs of surveillance. One approach may be to use PCR techniques on all samples of bloody diarrhea in children under the age of 16 because if surveillance is to be effective, it must be specific (9). Intermittent surveys, or the use of sentinel laboratories for all cases of diarrhea (8) could be undertaken and mandatory notification of HUS instituted. Compulsory notification of Shigella infection is a requirement in Australia, and including SLTECs on the list of notifiable diseases is being Commentary Emerging Infectious Diseases 154 Vol. 1, No. 4 -- October-December 1995 considered. A national surveillance scheme for HUS was established in 1994, although notifica- tion is not mandatory. Without formal notification requirements, good reporting of HUS has been associated with either clustering of cases or the fact that few hospitals in a region have the capac- ity to manage these cases (10). Although more attention has been focused on E. coli O157:H7, S. dysenteriae is likely the most common cause of HUS in children worldwide and more attention needs to be given to this pathogen in terms of surveillance and control. A strong and healthy public health infrastructure is required to address the infectious disease issues raised by HUS (11). Mary Beers,* Scott Cameron *National Centre for Epidemiology and Population Health, and South Australian Health Commission; Communicable Disease Control Unit, South Australian Health Commission, Adelaide, Australia References: 1. Cameron AS, Beers MY, Walker CC, et al. Community outbreak of hemolytic uremic syndromeattributable to Escherichia coli O111:NM--South Australia, 1995. MMWR 1995;44:550-8. 2. Waters JR, Sharp JC, Dev VJ. Infection caused by Escherichia coli O157:H7 in Alberta, Canada, and in Scotland: a five-year review, 1987-1991. Clin Infect Dis 1994;19:834-43. 3. Bell BP, Goldoft M, Griffin P, Davis MA, Gordon DC, Tarr P, et al. A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers: the Washington experience. JAMA 1994;272:1349-53. 4. Alexander ER, Boase J, Davis M, et al.

1. Clin Infect Dis 1994;19:834-43. 3. Bell BP, Goldoft M, Griffin P, Davis MA, Gordon DC, Tarr P, et al. A multistate outbreak of Escherichia coli O157:H7-associated bloody diarrhea and hemolytic uremic syndrome from hamburgers: the Washington experience. JAMA 1994;272:1349-53. 4. Alexander ER, Boase J, Davis M, et al. Escherichia coli O157:H7 outbreak linked to commercially distributed dry-cured salami--Washington and California, 1994. MMWR 1995;44:157-60. 5. Goldwater PN, Bettelheim KA. The role of enterohaem- orrhagicEscherichia coli serotypes other than O157:H7 as causes of disease in Australia. Communicable Dis- eases Intelligence 1995;19:2-4. 6. Caprioli A, Luzzi I, Rosmini F, Resti C, Edefonti A, Perfumo F, et al. Communitywide outbreak of hemolytic-uremic syndrome associated with non-O157 verocytotoxin-producing Escherichia coli. J Infect Dis 1994;169:208-11. 7. Griffin PM, Tauxe RV. The epidemiology of infections caused by Escherichia coli O157:H7, other enterohaem- orrhagic E. coli, and the associated hemolytic uremic syndrome. Epidemiol Rev 1991;13:60-98. 8. Alexander ER. Editorial response: surveillance of Es- cherichia coli O157:H7--a necessity for the prevention of an emerging infectious disease. Clin Infect Dis 1994;19:844-5. 9. Satcher D. Emerging infections: getting ahead of the curve. Emerging Infectious Diseases 1995;1:1-6. 10. Siegler RL, Pavia AT, Christofferson RD, Milligan MK. A 20-year population-based study of postdiarrheal hemolytic uremic syndrome in Utah. Pediatrics 1994;94:35-40. 11. MacDonald KL, Osterholm MT. The emergence of Es- cherichia coli O157:H7 infection in the United States: the changing epidemiology of foodborne disease. JAMA 1993;269:2264-6. Commentary Vol. 1, No. 4 -- October-December 1995 155 Emerging Infectious Diseases

Guidelines on the Risk for Transmission of Infectious Agents During Xenotransplants An increasingly critical shortage of human do- nors has limited the availability and benefit of organ and tissue transplantation. This chronic shortage, coupled with recent scientific and biotechnological advances, has been a catalyst for new therapeutic approaches directed at using ani- mal tissues in humans. The use of xenogeneic tissues and organs for transplantation or perfu- sion has raised concerns about the potential of both recognized zoonotic pathogens and unknown xenogeneic agents to infect individual human re- cipients and then spread through human popula- tions. Public health guidelines intended to minimize the risk for transmission of known pathogens through human-to-human transplantation exist. Similar guidelines addressing the issue of infec- tious agents that may be associated with xenotransplantation are being jointly developed by Public Health Service working groups at the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration, and the National Institutes of Health. A provisional draft of these guidelines will be published in the Federal Register in late 1995. Public comment on the proposed guidelines is invited. Critical review by members of the transplant community is par- ticularly sought. Publication of a final version of theseguidelines inCDC'sMorbidityand Mortality Weekly Report is planned for the spring of 1996. Louisa E. Chapman National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA Emerging Infectious Diseases Featured at ICAAC/IDSA Meeting Emerging infectious diseases were highlighted recently at a joint meeting of the Interscience Conference on Antimicrobial Agents and Chemo- therapy (ICAAC) and the Infectious Diseases So- ciety of America (IDSA) in San Francisco. In his opening address, IDSA President Vincent Andri- ole stated that the topic of most pressing concern to both organizations was new and reemerging pathogens.

ing of the Interscience Conference on Antimicrobial Agents and Chemo- therapy (ICAAC) and the Infectious Diseases So- ciety of America (IDSA) in San Francisco. In his opening address, IDSA President Vincent Andri- ole stated that the topic of most pressing concern to both organizations was new and reemerging pathogens. Presentations were made on the fol- lowing subjects: arenavirus hemorrhagic fevers; changing virulence of streptococcal infections; cryptosporidia, cyclospora, and microsporidia; dengue/dengue hemorrhagic fever; emerging fun- gal pathogens; epidemic diphtheria in the newly independent states; Escherichia coli O157:H7; Helicobacter pylori; human ehrlichioses; new lym- photropic herpesviruses; and rabies. Common themes emanated from the presenta- tions. The speakers noted that infectious diseases continue to occur throughout the world, both spo- radically and as outbreaks, because of multiple factors. They observed that the incidence and prevalence of infectious diseases are increasing in certain populations, particularly among immuno- compromised persons. Additionally, new infec- tious diseases and etiologic agents continue to be identified with remarkable frequency, and micro- organisms are being identified as causes of chronic diseases, including cancer. Several presenters ex- pressed concern about the migrations of animal reservoirs and arthropod vectors into new popula- tions and geographic areas. The speakers also called for additional support for the public health infrastructure and for basic sciences that provide the foundation for infectious disease prevention and treatment. Building a Geographic Information System (GIS) Public Health Infrastructure for Research and Control of Tropical Diseases A course on using Atlas GIS software and asso- ciated peripherals, such as digitizing tablets and global positioning systems (GPS), to build a GIS public health infrastructure in Latin American countries was taught August 7 to 18, 1995, at the Centers for Disease Control field station in Gua- temala City, which includes the Medical Entomol- ogy Research Training Unit housed at the Universidad del Valle de Guatemala. The course was funded by the Special Programme on Re- search and Training in Tropical Diseases and pre- sented by the Latin American Tropical Disease Research Training Consortium. Course News and Notes Emerging Infectious Diseases 156 Vol. 1, No. 4 -- October-December 1995

ing of the Interscience Conference on Antimicrobial Agents and Chemo- therapy (ICAAC) and the Infectious Diseases So- ciety of America (IDSA) in San Francisco. In his opening address, IDSA President Vincent Andri- ole stated that the topic of most pressing concern to both organizations was new and reemerging pathogens. Presentations were made on the fol- lowing subjects: arenavirus hemorrhagic fevers; changing virulence of streptococcal infections; cryptosporidia, cyclospora, and microsporidia; dengue/dengue hemorrhagic fever; emerging fun- gal pathogens; epidemic diphtheria in the newly independent states; Escherichia coli O157:H7; Helicobacter pylori; human ehrlichioses; new lym- photropic herpesviruses; and rabies. Common themes emanated from the presenta- tions. The speakers noted that infectious diseases continue to occur throughout the world, both spo- radically and as outbreaks, because of multiple factors. They observed that the incidence and prevalence of infectious diseases are increasing in certain populations, particularly among immuno- compromised persons. Additionally, new infec- tious diseases and etiologic agents continue to be identified with remarkable frequency, and micro- organisms are being identified as causes of chronic diseases, including cancer. Several presenters ex- pressed concern about the migrations of animal reservoirs and arthropod vectors into new popula- tions and geographic areas. The speakers also called for additional support for the public health infrastructure and for basic sciences that provide the foundation for infectious disease prevention and treatment. Building a Geographic Information System (GIS) Public Health Infrastructure for Research and Control of Tropical Diseases A course on using Atlas GIS software and asso- ciated peripherals, such as digitizing tablets and global positioning systems (GPS), to build a GIS public health infrastructure in Latin American countries was taught August 7 to 18, 1995, at the Centers for Disease Control field station in Gua- temala City, which includes the Medical Entomol- ogy Research Training Unit housed at the Universidad del Valle de Guatemala. The course was funded by the Special Programme on Re- search and Training in Tropical Diseases and pre- sented by the Latin American Tropical Disease Research Training Consortium. Course News and Notes Emerging Infectious Diseases 156 Vol. 1, No.

Research Training Unit housed at the Universidad del Valle de Guatemala. The course was funded by the Special Programme on Re- search and Training in Tropical Diseases and pre- sented by the Latin American Tropical Disease Research Training Consortium. Course News and Notes Emerging Infectious Diseases 156 Vol. 1, No. 4 -- October-December 1995 instructors included statisticians and epidemiolo- gists from the Division of Parasitic Diseases, Na- tional Center for Infectious Diseases, Centers for Disease Control and Prevention in Atlanta, Geor- gia, and in Guatemala City, Guatemala, and staff from the National Aeronautics and Space Admini- stration, Center for Health Application of Aero- space Related Technologies, Ames Research Center, Sunnyvale, California. Objectives of the training included the follow- ing: mastery of theprinciples andgeneral concepts of all GIS systems; use of Atlas GIS/DOS to asso- ciatemap files withdatabases toproducethematic maps, manipulate various layers (rivers, high- ways, village locations) of the map files to produce customized maps, create buffers around geo- graphicfeatures,anduse them insimpleanalyses; designing georeferenced data files that canberead by the GIS; digitizing paper maps to acquire new data for building a GIS; use of GPS to obtain latitudes, longitudes, and elevations of villages and other major landmarks and to use this infor- mation in the GIS; and mastery of importing/ex- porting databases and map files. The course was designed to enable participants to set up and use a GIS for research, planning, or operational purposes. Participating were institu- tions from Mexico (two teams), Colombia (two), Puerto Rico, Costa Rica, Venezuela, Guatemala (two), Ecuador, and Brazil. Each team came to the course withideas, maps, and data pertaining to an existing project that would be continued at their home institution. Student project areas included onchocerciasis, malaria, water sanitation, leish- maniasis, and public health and natural resource utilization/preservation. The students were taught digitizing and were asked to use Guinea worm surveillance data to create their own GIS. A full day was devoted to geographic analyses. Topics covered included aggregating data from one geographic layerto another, combining geographic features with common database values, and com- bining selected features to form new map layers.

g and were asked to use Guinea worm surveillance data to create their own GIS. A full day was devoted to geographic analyses. Topics covered included aggregating data from one geographic layerto another, combining geographic features with common database values, and com- bining selected features to form new map layers. A workshop on remote sensing, GIS, and image classification explained that satellite imagery and remotely sensed data are obtained by measuring reflectance on seven spectral frequencies and that ground cover can be partially deduced by the amount of reflectance at each band. Field exer- cises to practice GPS use in the Lake Atitlan area followed. Another workshop covered advanced digitizing and gave each team a good start on the digitizing part of theirprojects.Individual instruc- tions were given on how to import map files from other GIS programs into Atlas GIS. Lastly, the Guatemalan onchocerciasis GIS system was pre- sented as a case study. In addition to the 2 weeks of training, each participating institution received a copy of all lec- ture notes, the critical hardware needed to con- tinue the project at home, and the following software, complete with documentation: Atlas GIS/DOS,Import-Export,and Arcview2. An ongo- ing Internet-based discussion group for class or- ganizers and participants is providing a forum for dialogue and monitoring of participants'progress. Allen W. Hightower Robert E. Klein National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA, and Guatemala City, Guatemala APHA Session Features Emerging Infections Emerging and reemerging infections will be the featured topic of a two-part session at the annual meeting of the American Public Health Associa- tion, October 29-November 2, in San Diego, Cali- fornia. The session, titled "Emerging Infections: Solv- ing the Mysteries in the Field and Laboratory," will focus on the worldwide impact of new and reemerging infections from both an epidemiologic and a laboratory perspective.

ing of the American Public Health Associa- tion, October 29-November 2, in San Diego, Cali- fornia. The session, titled "Emerging Infections: Solv- ing the Mysteries in the Field and Laboratory," will focus on the worldwide impact of new and reemerging infections from both an epidemiologic and a laboratory perspective. Eight speakers from national and international health organizations will discuss the following aspects of the public health threat of these dis- eases: public health strategies for controlling in- fectious diseases; social, geographic, ecologic, and environmental factors that have allowed these diseases to spread; the growing threat of antimi- crobial resistance; the increased need for accurate and meaningful disease surveillance; and the challenge toapplythe latest laboratory technology to rapidly detect and characterize new infectious agents. Martin S. Favero National Center for Infectious Diseases Centers for Disease Control and Prevention Atlanta, Georgia, USA News and Notes Vol. 1, No. 4 -- October-December 1995 157 Emerging Infectious Diseases

instructors included statisticians and epidemiolo- gists from the Division of Parasitic Diseases, Na- tional Center for Infectious Diseases, Centers for Disease Control and Prevention in Atlanta, Geor- gia, and in Guatemala City, Guatemala, and staff from the National Aeronautics and Space Admini- stration, Center for Health Application of Aero- space Related Technologies, Ames Research Center, Sunnyvale, California. Objectives of the training included the follow- ing: mastery of theprinciples andgeneral concepts of all GIS systems; use of Atlas GIS/DOS to asso- ciatemap files withdatabases toproducethematic maps, manipulate various layers (rivers, high- ways, village locations) of the map files to produce customized maps, create buffers around geo- graphicfeatures,anduse them insimpleanalyses; designing georeferenced data files that canberead by the GIS; digitizing paper maps to acquire new data for building a GIS; use of GPS to obtain latitudes, longitudes, and elevations of villages and other major landmarks and to use this infor- mation in the GIS; and mastery of importing/ex- porting databases and map files. The course was designed to enable participants to set up and use a GIS for research, planning, or operational purposes. Participating were institu- tions from Mexico (two teams), Colombia (two), Puerto Rico, Costa Rica, Venezuela, Guatemala (two), Ecuador, and Brazil. Each team came to the course withideas, maps, and data pertaining to an existing project that would be continued at their home institution. Student project areas included onchocerciasis, malaria, water sanitation, leish- maniasis, and public health and natural resource utilization/preservation. The students were taught digitizing and were asked to use Guinea worm surveillance data to create their own GIS. A full day was devoted to geographic analyses. Topics covered included aggregating data from one geographic layerto another, combining geographic features with common database values, and com- bining selected features to form new map layers.

Southeast Asia Intercountry Consultative Meeting on Prevention and Control of New, Emerging, and Reemerging Infectious Diseases An intercountry meeting to identify strategies and approaches for tackling the problems of new, emerging, and reemerging infectious diseases in the Southeast Asia region was held in New Delhi, India, August 21 to 25, 1995. Nine countries (Bangladesh, Bhutan, India, In- donesia, Maldives, Myanmar, Nepal, Sri Lanka, and Thailand) participated. Experts from these countries, England, and the United States, as well as representatives from USAID, DANIDA, UNICEF, the World Bank, and the World Health Organization (WHO) also attended. Meeting participants expressed serious con- cern at the global and regional spread of new, emerging, and reemerging infectious diseases, es- peciallyin theSoutheastAsiaregion. Reportsfrom various countries emphasized that these diseases not only have worldwide health implications but also can disrupt commerce and industry and set back important progress achieved in public health during recent years. The spread of these diseases also has major social and political implications. Participants at the meeting underlined the im- portance of surveillance, prompt epidemiologic in- vestigation, and the build-up of adequate laboratory capacities. The need for maintaining ecologic and environmental integrity in various developmental activities was also emphasized. Member countries were called upon to immedi- ately review and strengthen capacities in epi- demic surveillance and response and to formulate country-specific strategies and action plans to an- ticipate, quickly recognize, and rapidly respond to the threat of emerging infections. To ensure program sustainability, participants stressed that strategies to combat emerging dis- eases should be an integral part of existing na- tional infrastructures, particularly infectious disease control programs, and should build on the capacities that already exist.

respond to the threat of emerging infections. To ensure program sustainability, participants stressed that strategies to combat emerging dis- eases should be an integral part of existing na- tional infrastructures, particularly infectious disease control programs, and should build on the capacities that already exist. Four broad areas for tackling the challenge of emerging diseases were identified: strengthening communicable disease surveillance and response, strengthening the ex- isting infrastructure, capacitybuilding forpreven- tion and control, and applied research. A total of 12 actions were recommended. One, for example, was that each country create a rapid response team to react to epidemic situations. A second recommendation was that countries de- velop linkages between their national reference laboratories and WHO collaborating centers. This would be to strengthen diagnostic capacities, fa- cilitate quality assurance, and promote national self-reliance in laboratory diagnosis. Eight recommended actions for WHO to under- take were also enumerated. These included as- sessing and monitoring microbial susceptibility to antibiotics, and vector susceptibility to insecti- cides. For the complete list of conclusions and recom- mendations of this meeting, contact WHO's Re- gional Office for South-East Asia. Samlee Plianbangchang WHO, Regional Office for Southeast Asia World Health House New Delhi-110 002, India News and Notes Emerging Infectious Diseases 158 Vol. 1, No. 4 -- October-December 1995

The Infectious Diseases Impact Statement: A Mechanism for Addressing Emerging Diseases Edward McSweegan, Ph.D. National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA The use of an Infectious Diseases Impact Statement (IDIS) is proposed for predictive assessments of local changes in infectious diseases arising from human-engineered activi- ties. IDIS is intended to be analogous to an Environmental Impact Statement. The drafting of an IDIS for specific activities, particularly in developing nations, would provide a formal mechanism for examining potential changes in local health conditions, including infected and susceptible populations, diseases likely to fluctuate in response to development, existing control measures, and vectors likely to be affected by human activities. The resulting survey data could provide a rational basis and direction for development, surveillance, and preven- tion measures.An IDIS process that balances environmental alterations, local human health, and economic growth could substantially alter the nature of international development efforts and infectious disease outbreaks. A 1995 report by Aksoy et al. (1) describing the GAP (Turkish acronym for the Southeastern Ana- tolia Irrigation Project) irrigation project in Tur- key suggests that anticipating the emergence or expansion of vector-borne and zoonotic diseases in a limited environment is a useful exercise.Accord- ing to the report, a number of diseases (e.g., leish- maniasis, malaria, and schistosomiasis) are likely to increase in direct response to the expansion of irrigation and the increases in under water acre- age and human population in the GAP region.The succinct overview of the disease and vector condi- tions in the GAP area could serve as a starting point for creating what will be referred to in this article as an Infectious Diseases Impact State- ment (IDIS), a document that would be analogous to the Environmental Impact Statement (EIS) routinely used in the United States to assess the likely effects of construction, irrigation, agricul- ture, and similar activities on a local environment or region.

red to in this article as an Infectious Diseases Impact State- ment (IDIS), a document that would be analogous to the Environmental Impact Statement (EIS) routinely used in the United States to assess the likely effects of construction, irrigation, agricul- ture, and similar activities on a local environment or region. An IDIS, however, would not assess the environment directly, but rather would predict changes in local disease patterns resulting from changes to the local environment. Like an EIS, an IDIS would be a predictive and proactive assessment. Drafting an IDIS for a par- ticular region or microenvironment would provide a formal mechanism for asking (and attempting to answer) specific questions about future changes in local health conditions. For example, what are the diseases and vectors in the given area? How are the proposed changes to the environment (e.g., dam-building, forest-clearing) likely to change the incidence and the prevalence of those diseases and vectors? What actions should be taken during the course of a given project and in the future to prevent potential increases in disease and vector populations? If an increase in human disease is likely, is the expense of the proposed project war- ranted? Will the economic benefits of a particular development or agriculture project be offset by increased costs in health care, vaccination, and vector control? The 1969 National Environmental Policy Act was designed to provide a legal mechanism in the United States for evaluating potential impact to the environment from development activities and for permitting the public to participate in the evaluation process at the earliest stages (i.e., "scoping"). A Council of Environmental Quality in the executive branch of the federal government was established as a monitor, and the EIS process was implemented to inform decision makers and the public of potential environmental problems and reasonable alternatives to proposed actions. Environmental Protection Agency (EPA) require- ments for environmental assessments are out- lined in the Code of Federal Regulations (CFR).An EIS is intended to prospectively examine impact "upon the quality of the human environment of the United States and, in appropriate cases, upon the Address for correspondence: Edward McSweegan, Ph.D., National Institute of Allergy and Infectious Diseases, National Institutes of Health, Solar Bldg., Rm. 3A34, Bethesda, MD 20892-7630, USA; fax: 301-402-0659; e-mail: em8p@nih.gov. Perspectives Vol. 2, No.

nment of the United States and, in appropriate cases, upon the Address for correspondence: Edward McSweegan, Ph.D., National Institute of Allergy and Infectious Diseases, National Institutes of Health, Solar Bldg., Rm. 3A34, Bethesda, MD 20892-7630, USA; fax: 301-402-0659; e-mail: em8p@nih.gov. Perspectives Vol. 2, No. 2-- April-June 1996 103 Emerging Infectious Diseases environment of the global commons outside the jurisdiction of any nation"(46 CFR sec.504.7).The requirements of an EIS typically include descrip- tions of human populations in the designated area, current land use patterns, air quality, noise levels, locations of wetlands and coastal zones, sites of historical or cultural value, and non-point source pollution. The protection of human health is im- plied in the EIS process,but this concern is usually assumed to pertain to the location of industrial plants and dumps and to exposure to toxic chemi- cals, heavy metals, ionizing radiation, and pesti- cides. The EIS process contains no explicit reference to infectious diseases or disease vectors affecting human health in response to deliberate environmental changes. In 1995, the only publish- ed EIS references to diseases, infectious or other- wise, were for proposed control measures at two California plant nurseries.Yet past events suggest that attention should be directed toward changes in infectious disease patterns directly attributable to human-engineered events. For example, the construction of the Aswan Dam in Egypt is widely believed to have precipi- tated the appearance of Rift Valley fever (RVF) in Egypt during the 1970s (2). Tens of thousands of RVF cases and hundreds of deaths followed. Simi- larly, completion of the Diama Dam in Senegal, in 1987, led to epidemics of malaria and RVF (3); impoundment of the Volta Lake in Ghana,in 1968, led to an explosive outbreak of schistosomiasis (4). Increased agriculture on the Argentine pampas and along the edges of Bolivian forests has contrib- uted to frequent hemorrhagic fever outbreaks caused by Junin and Machupo viruses, respec- tively (5). Mining operations in the Brazilian jun- gles have led to outbreaks of malaria (6). Road-building projects under way in Papua New Guinea are likely to bring large numbers of sus- ceptible human hosts into contact with rare and yet-to-be-discovered viruses. These epidemics and encounters with new diseases are the unforeseen consequences of critically altering the local envi- ronment.

ks of malaria (6). Road-building projects under way in Papua New Guinea are likely to bring large numbers of sus- ceptible human hosts into contact with rare and yet-to-be-discovered viruses. These epidemics and encounters with new diseases are the unforeseen consequences of critically altering the local envi- ronment. As a consequence, development and ag- riculture projects initiated to improve the lives of local populations can have the opposite effect by increasing disease prevalence and causing new epidemics. Embedding an IDIS requirement into the planning and execution of large-scale projects likely to alter local environments could prevent new epidemics and reduce infectious disease- associated morbidity and mortality. How Would an IDIS Work? An IDIS would first need to be established as an integral component of any activity likely to affect the health of a local population. In tropical and developing regions of the world, that would include a variety of national and international development activities. The area designated for large-scale alteration would be surveyed for cur- rent disease vectors, and the local populations would be examined for diseases likely to be af- fected by the project in question.The quality of the surveillance and the extrapolation of expected changes brought on by a particular activity would vary, depending on the knowledge of diseases, vec- tors, local host immunity, and other factors. Al- though the variables increase the margin of uncertainty in such extrapolations, these esti- mates would be expected to improve as the state of field and laboratory research improves and ex- perience with preparing an IDIS increases. A ret- rospective examination of earlier projects in similar environments would also provide informa- tion for developing an IDIS. The standards of "existing credible scientific evidence" and "reason- ably foreseeable" impact that current environ- mental impact assessments rely on could also be applied to the early stages of the IDIS process. The resulting preproject assessment would pro- vide a snapshot of conditions in a defined area, including the following:diseases likely to fluctuate in response to project activities, numbers of in- fected and susceptible hosts, existing control measures, and vectors likely to be affected by project activities. Such baseline data are fre- quently absent from development and agriculture activities (7).

fined area, including the following:diseases likely to fluctuate in response to project activities, numbers of in- fected and susceptible hosts, existing control measures, and vectors likely to be affected by project activities. Such baseline data are fre- quently absent from development and agriculture activities (7). Knowing what diseases are already present, and how they might be changed, allows one to ask how anticipated changes in disease prevalence and distribution might be prevented or controlled through changes in the proposed pro- ject, improved case finding and treatment, changes in local sanitation and housing, increased vaccination or prophylaxis, or pest management programs. Some or all of the above health mainte- nance measures could then become components of the overall planning, budgeting, and execution of any major development or agricultural activity in the area. Health and health maintenance would become factors in the overall design and cost of the project. In many instances, local disease surveil- lance would become an ongoing part of the project, with supplemental assessments being made to refine the original IDIS. Perspectives Emerging Infectious Diseases 104 Vol. 2, No. 2-- April-June 1996 Who Would Request an IDIS,and Who Would Respond to the Request? Initial candidates would likely be donor organi- zations (the U.S. Agency for International Devel- opment and the World Bank, for example) that provide funding and oversight. In the absence of federal or international statutes, these organiza- tions have the stature and financial capability to make infectious disease control an integral part of their development projects. Indeed, they should have an urgent interest in doing so because in- creases in diseases or new epidemics increase financial demands on them for medicines, vac- cines, and pest control. In the end, more money would be spent beating back the outbreaks and epidemics that foresight might have prevented. National health ministries, state and territorial health departments, and local medical communi- ties in developing countries might also request or initiate an IDIS. The practice of drafting an IDIS and implementing its recommendations might also rejuvenate underfunded areas of interna- tional health, vector biology, parasitology, and medical entomology as professionals in these fields are called on to conduct infectious disease assessments of development activities.

tiate an IDIS. The practice of drafting an IDIS and implementing its recommendations might also rejuvenate underfunded areas of interna- tional health, vector biology, parasitology, and medical entomology as professionals in these fields are called on to conduct infectious disease assessments of development activities. The peer- reviewed literature and electronic services such as ProMED,Outbreak,and the World Health Organi- zation (WHO) and Centers forDisease Control and Prevention World-Wide Web sites could provide the public "scoping"role that posting in theFederal Register and allowing a period for public comment provide in the EIS process in the United States. The first application of an IDIS to a large-scale development or environmental activity could come from western donor organizations working in the developing world.The successful demonstration of an IDIS could encourage other organizations, na- tional health officials, and health activists to push for the routine integration of public health with national development. This could happen in the United States, as well. The United States recently experienced the emergence of Sin Nombre virus in the Southwest and is theoretically open to the introduction of five vector-borne diseases:malaria, Rift Valley fever, yellow fever, dengue, and ar- bovirus encephalitides (15). Public health officials and citizen activists could initiate independent IDIS for activities perceived to threaten the balance between health, the environment, and domestic productivity. What are the Strengths and Limitations of the IDIS Process? A project-embedded IDIS would not be the same as an environmental management program, which seeks to control disease vectors through environmental modification and manipulation and through reduced human contact with vectors (8). An IDIS would, in fact, precede environmental management control measures by first postulat- ing the likely emergence of specific pathogens and vectors.The usefulness of an IDIS lies in its ability to provide a conceptual framework for identifying potential disease problems, and, indeed, prevent- ing them by altering or curtailing the very activi- ties that could lead to disease emergence. In an activist sense, an IDIS could be wielded as a tool of caution or prevention, much as an EIS is wielded in the United States to alter or halt some activity perceived to be a threat to the envi- ronment.

indeed, prevent- ing them by altering or curtailing the very activi- ties that could lead to disease emergence. In an activist sense, an IDIS could be wielded as a tool of caution or prevention, much as an EIS is wielded in the United States to alter or halt some activity perceived to be a threat to the envi- ronment. That ability to influence potential changes and to affect health could be vital; public health concerns connected with agricultural and developmental projects are usually a low priority among foreign ministries, international donor or- ganizations, and engineers (9); neglecting them can leave the full benefits of development unreal- ized. Lest anyone imagines that an IDIS could be used solely as a tool of the political Left, as a kind of "liberation microbiology,"it is important to point out that the same IDIS could be used to justify the use of pesticides and other organized control measures, including the relocation of local popula- tions.Recently,for example,pesticide use has come under attack by various environmental groups, and donor organizations have become increasingly reluctant to fund such activities (10).In the United States, EPA's Endangered Species Act has also tended to thwart the use of pesticides because of potentially adverse impact on some birds and mammals (11). However, an IDIS describing the probable emergence of important disease vectors could be used to justify such use. Thus, a health care issue could be twisted into a health scare by either the political Right or the Left. The recent ratification of the North American Free Trade Agreement (NAFTA) was preceded in the United States by an effort to stall the treaty with an EIS requirement.If an IDIS had predicted new disease outbreaks from increased border trade and traffic, that concern might have had greater impact on the public imagination than more abstract concerns about atmospheric particulates in the border Perspectives Vol. 2, No. 2-- April-June 1996 105 Emerging Infectious Diseases region and could have been effectively used by anti-NAFTA forces. An IDIS should be not a politi- cal tool but rather a valuable information source that helps guide economic development and land use. How Can an IDIS Complement Existing Surveillance Systems? Almost half of the planet's five billion people are at risk for one or more vector-borne diseases (12, 13). Surveillance remains a key tool for moni- toring these diseases and identifying new cases and outbreaks.

at helps guide economic development and land use. How Can an IDIS Complement Existing Surveillance Systems? Almost half of the planet's five billion people are at risk for one or more vector-borne diseases (12, 13). Surveillance remains a key tool for moni- toring these diseases and identifying new cases and outbreaks. Four types of surveillance are used in the control of vector-borne diseases:1) recording human cases, 2) determining vector distribution and infectivity, 3) monitoring vertebrate reser- voirs, and 4) tracking weather patterns to predict vector distribution (14). But throughout the devel- oping world and across tropical boundaries, effec- tive and continuous surveillance is extremely difficult, if not impossible. Cases are missed; out- breaks go unreported.Effective case reporting and continuous field monitoring are best conducted in limited, well-defined areas. Within the microenvi- ronments of human activities, an IDIS could pro- vide valuable baseline surveillance data before changes to that area occur and affect disease and vector distributions. This information could pro- vide a rational basis and direction for ongoing monitoring and corrective measures (e.g., vaccina- tion,relocation,pest control).Focusing on a limited area and a limited number of diseases in that area may also expand the use of promising but under- utilized technologic methods such as remote sens- ing and geographic information systems (GIS). Haines et al. (15) noted the importance of vec- tor-borne disease monitoring and recommended that remote sensing and GIS be used to detect changes in ecosystems and vector populations. To a large extent,however,the advantages of satellite imagery and GIS have not been realized, in part, because of the frequent absence of "ground truth" (data on diseases, vectors, and other factors in the area) and of having to wait to observe natural environmental changes likely to affect disease and disease transmission (16-18).Satellite imagery for much of the planet has been collecting in data- bases since 1972 (16). By 1998, accumulated sat- ellite data will be in the petabyte (1,000 terabyte) range, 1,000 times larger than the contents of the Library of Congress (B. Montgomery, NASA, pers. comm.). High-resolution, multispectral, multiyear images for many potential development and agricultural sites are available.

since 1972 (16). By 1998, accumulated sat- ellite data will be in the petabyte (1,000 terabyte) range, 1,000 times larger than the contents of the Library of Congress (B. Montgomery, NASA, pers. comm.). High-resolution, multispectral, multiyear images for many potential development and agricultural sites are available. Using a preproject IDIS to "ground truth" the project's environment with current satellite imagery, it may be possible to more completely describe local disease and vec- tor conditions and make more accurate predictions about their plasticity during periods of construc- tion, flooding, or farming. The result would be a firmer linkage of ground surveillance and satellite imagery to monitor public health changes within a well-defined and limited environment. In recent years, the sudden emergence of rare or forgotten diseases such as Ebola virus infection, dengue, yellow fever, plague, and hantavirus (Sin Nombre virus) infection has attracted the atten- tion of the public and inspired renewed commit- ments to surveillance and control. WHO recently formed a rapid response unit (the Division of Emerging, Viral and Bacterial Diseases Surveil- lance and Control) to deal with outbreaks of new and reemerging infections (19). Similarly, nine Southeast Asian countries held a meeting on emerging diseases and concluded that each coun- try should also develop rapid response teams for epidemics (20). However, these disease control ef- forts are almost entirely passive, with staff, equip- ment, and budgets idling in anticipation of something eventually happening somewhere. It is difficult to maintain a high degree of public and financial support for such wait-and-see ap- proaches to disease control.The United States has suffered a serious decline in national surveillance and outbreak investigations, in part, because of decreased support for passive monitoring pro- grams (11). Is an IDIS Really Needed When the Existing EIS Statutes Already Cover Human Health and Safety Concerns? In the United States the need is not clear.

ed States has suffered a serious decline in national surveillance and outbreak investigations, in part, because of decreased support for passive monitoring pro- grams (11). Is an IDIS Really Needed When the Existing EIS Statutes Already Cover Human Health and Safety Concerns? In the United States the need is not clear. Infec- tious diseases caused by environmental manipu- lation may be assumed to fall under the general EIS category of human health.However,infectious diseases have not often been considered in the past, and it is easy to imagine that if they were a factor in the EIS process, an environmental/infec- tious disease issue could be smothered under the weight of government regulations and the adver- sarial legal system.EPA operates under 16 federal statutes and 70 congressional committees and subcommittees and is engaged in some 600 law- suits at any given time (21). Moreover, emerging infectious disease issues could bring EPA and the EIS process into conflict with the missions of Perspectives Emerging Infectious Diseases 106 Vol. 2, No. 2-- April-June 1996 federal agencies and state and local health departments. Outside the United States, beyond federal statutes and informed public debate, the need foran IDIS is clearer.Inthe developing world, epidemics and substandard health care are com- mon, and the national goals of healthy environ- ment and healthy economy are usually at odds.An IDIS process that balances environmental altera- tions, local human health, and economic develop- ment could substantially alter the nature of international development efforts and infectious disease outbreaks. To the ancient Greeks, the past appeared in front of them, real and visible; the future was behind them, unseen and unknowable. With that perspective, they were always glancing nervously backward, looking for a future that usually man- aged to creep up and tap them on the shoulder. In a sense, we have the same perspective for disease surveillance and control that the ancient Greeks had for time. Past epidemics and our responses to them are readily apparent; it is that unexpected tap on the shoulder by a hantavirus or an Ebola virus that is always so startling. We cannot know when and where such pathogens will emerge. Their appearance is often a chance event initiated by unpredictable changes in weather or the acci- dental encounter of a single person with a myste- rious vector. These taps on the shoulder are an affront to our sense of control and understanding of disease.

g. We cannot know when and where such pathogens will emerge. Their appearance is often a chance event initiated by unpredictable changes in weather or the acci- dental encounter of a single person with a myste- rious vector. These taps on the shoulder are an affront to our sense of control and understanding of disease. Moreover, it is unsettling to the public's sense of security and its faith in medical research. Although we cannot expect to eliminate the sur- prises of emergent pathogens in the near future, we can take control of situations in which our own actions directly lead to the emergence of diseases. Generating an IDIS in areas where human activi- ties are likely to disrupt endemic-disease patterns would be an important step in controlling future outbreaks. Routine application of a preproject IDIS could improve local surveillance and health care planning by 1) providing baseline data on endemic-disease and vector prevalence and com- petence; 2) embedding projected health mainte- nance costs into the planning and cost of any project or activity likely to influence the environ- ment and public health; and 3) providing a mecha- nism for instituting project alterations and health care measures to offset adverse effects on the health of local populations. Acknowledgments Special thanks to Dr. Michael Gottlieb, National Institute of Allergy and Infectious Diseases, Parasitology and International Programs Branch, and Donald C. Baur, Esq., Perkins-Cole, Washington, D.C. for critical comments and suggestions. Dr. McSweegan is a member of the Parasitology and International Programs Branch at the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, Md. He has a long-standing interest in international science and health, and emerging diseases. He served in the U.S. State Department as an AAAS Fellow before joining NIAID's Tropical Medicine and International Research Office in 1988. References 1. Aksoy S, Ariturk S, Armstrong MYK, Chang KP, Dort- budak Z, Gottlieb M, et al. The GAP project in south- eastern Turkey: the potential for emergence of diseases. Emerging Infectious Diseases 1995; 1:62-3. 2. Meegan JM, Shope RE. Emerging concepts on Rift Valley fever. In: Pollard M, editor. Perspectives in virology. New York: Alan R. Liss, 1981. 3. Lederberg J, Shope RE, Oaks SC, editors. Emerging infections: microbial threats to health in the United States. Washington, DC: Institute of Medicine, Na- tional Academy Press, 1992;71-2. 4.

ope RE. Emerging concepts on Rift Valley fever. In: Pollard M, editor. Perspectives in virology. New York: Alan R. Liss, 1981. 3. Lederberg J, Shope RE, Oaks SC, editors. Emerging infections: microbial threats to health in the United States. Washington, DC: Institute of Medicine, Na- tional Academy Press, 1992;71-2. 4. Scott D, Senker K, England EC. Epidemiology of hu- man Schistosoma haematobium infection around Volta Lake, Ghana, 1973-75. Bull WHO 1982;60:89- 100. 5. Morse SS. Emerging viruses: defining the rules for viral traffic. Perspect Biol Med 1991;34:387-409. 6. de Andrade ALSS, et al. High prevalence of asympto- matic malaria in gold mining areas in Brazil. Clin Infect Dis 1995;20:475. 7. Service MW. Rice, a challenge to health. Parasitol Today 1989;5:162-5. 8. Ault SK. Environmental management: a re-emerging vector control strategy. Am J Trop Med Hyg 1994;50(Suppl):35-49. 9. Silver GA. 1995. International Health Organization Policy Watch. The Federation of American Scientists. (http://www.clark.net/pub/gen/fas/ihm). 10. Arata AA. Difficulties facing vector control in the 1990s. Am J Trop Med Hyg. 1994;50(Suppl):6-10. 11. Longstreth J, Wiseman J. Human health. In: Smith JB, Tirpak DA, editors. The potential effects of global climate change on the United States: Appendix G, Health. Washington, DC: U.S. Environmental Protec- tion Agency, 1989. 12. Beck LR, Rodriguez MH, Dister SW, Rodriguez AD, Rejmankova E, Ulloa, et al. Remote sensing as a landscape epidemiologic tool to identify villages at high risk for malaria transmission. Am J Trop Med Hyg 1994;51:271-80. Perspectives Vol. 2, No. 2-- April-June 1996 107 Emerging Infectious Diseases 13. Knudsen AB, Sloof R. Vector-borne disease problems in rapid urbanization: new approaches to vector con- trol. Bull WHO 1992;70:1-6. 14. Consortium for International Earth Sciences Infor- mation Network (CIESIN) Thematic Guides. Provi- sional Release. 1995. Programs for surveillance, treatment, and control of vector-borne diseases. 15. Haines A, Epstein PR, McMichael AJ. Global health watch: monitoring impacts of environmental change. Lancet 1993;342:1464-9. 16. Washino RK, Wood RL. Application of remote sensing to arthropod vector surveillance and control. Am J Trop Med Hyg. 1994;50Suppl:134-44. 17. Rogers DJ, Williams BG. Monitoring trypanosomiasis in space and time. Parasitology 1993;106:S77-S92. 18. Barinaga M. Satellite data rocket disease control ef- forts into orbit. Science 1993; 261:31-2. 19. World Health Organization.

o arthropod vector surveillance and control. Am J Trop Med Hyg. 1994;50Suppl:134-44. 17. Rogers DJ, Williams BG. Monitoring trypanosomiasis in space and time. Parasitology 1993;106:S77-S92. 18. Barinaga M. Satellite data rocket disease control ef- forts into orbit. Science 1993; 261:31-2. 19. World Health Organization. WHO establishes new rapid-response unit to combat growing world-wide threat of emerging diseases. WHO/75. Press release, 17 October 1995. 20. Plianbangchang S. Southeast Asia intercountry con- sultative meeting. Emerging Infectious Diseases 1995;1:158. 21. Environmental Protection Agency.The common sense initiative. Pub. No. EPA100F94004. Washington, DC: Environmental Protection Agency. Perspectives Emerging Infectious Diseases 108 Vol. 2, No. 2-- April-June 1996

Emerging Disease Issues and Fungal Pathogens Associated with HIV Infection Neil M. Ampel, M.D. University of Arizona College of Medicine, Tucson Veterans Affairs Medical Center, Tucson, Arizona, USA Fungal diseases are increasing among patients infected with human immunodeficiency virus (HIV) type 1. Infections due to Candida and Cryptococcus are the most common. Although mucocutaneous candidiasis can be treated with oral antifungal agents, increasing evidence suggests that prolonged use of these drugs results in both clinical and microbi- ologic resistance. The optimal therapy for cryptococcal meningitis remains unresolved, although initial treatment with amphotericin B, followed by life-long maintenance therapy with fluconazole, appears promising. Most cases of histoplasmosis, coccidioidomycosis, and blastomycosis occur in regions where their causative organisms are endemic, and increasing data suggest that a significant proportion of disease is due to recent infection. Aspergillosis is increasing dramatically as an opportunistic infection in HIV-infected patients, in part because of the increased incidence of neutropenia and corticosteroid use in these patients. Infection due to Penicillium marneffei is a rapidly growing problem among HIV-in- fected patients living in Southeast Asia. Although the advent of oral azole antifungal drugs has made primary prophylaxis against fungal diseases in HIV-infected patients feasible, many questions remain to be answered before the preventive use of antifungal drugs can be advocated. Over the last decade, the incidence of fungal infections has increased dramatically. The human immunodeficiency virus (HIV) type 1 epidemic accounts for a large share of this increase. This article is not a general guide to the diagnosis or treatment of fungal diseases but rather a review of emerging disease issues in regard to these infections among HIV-infected patients. The infec- tions discussed include candidiasis; cryptococ- cosis; the endemic mycoses histoplasmosis, coccidioidomycosis, and blastomycosis; aspergil- losis; and penicilliosis.

nosis or treatment of fungal diseases but rather a review of emerging disease issues in regard to these infections among HIV-infected patients. The infec- tions discussed include candidiasis; cryptococ- cosis; the endemic mycoses histoplasmosis, coccidioidomycosis, and blastomycosis; aspergil- losis; and penicilliosis. In addition, the role of preventive therapy for fungal infections in HIV-in- fected patients is assessed. Fungal pathogens and their most common manifestations in HIV-in- fected patients are listed separately (Table). Candidiasis Mucocutaneous candidiasis is one of the most common manifestations of HIV infection. In one prospective study, 84% of HIV-infected patients had oropharyngeal colonization by Candida spe- cies on at least one occasion, and 55% developed clinical thrush (1). While other yeasts may occasionally cause clinical disease, Candida albi- cans is the organism isolated from most patients (1, 2). Candida species normally colonize the gas- trointestinal tract of healthy adults, and most infections in HIV-infected patients are endo- genously acquired. In some cases, candidal strains can be transmitted from person to person (1). Address for correspondence: Neil M. Ampel, M.D., Medical Service (111), Veterans Affairs Medical Center, 3601 S. Sixth Avenue, Tucson, AZ 85713, USA; fax: 520-629-1861; e-mail: nampl@aol.com. Table. Common fungal pathogens in HIV infection and their most frequent clinical syndromes Organism Clinical syndrome Candida albicans Thrush, vaginal candidiasis, esophageal candidiasis Cryptococcus neoformans Meningitis Histoplasma capsulatum Disseminated infection with fever and weight loss Coccidioides immitis Diffuse and focal pulmonary disease Blastomyces dermatitidis Localized pulmonary disease and disseminated infection, including meningitis Aspergillus fumigatus Pulmonary disease with fever, cough, and hemoptysis Penicillium marneffei Fever alone or with pulmonary infiltrates, lymphadenopathy, or cutaneous lesions Synopsis Vol 2, No. 2--April-June 1996 109 Emerging Infectious Diseases During the course of HIV infection, patients ap- pear to be colonized with one or a few dominant strains, which tend not to change over time. Pow- derly and colleagues isolated the same strain ofC. albicans in 11 of 17 patients with recurrent yeast infection, by DNA probe analysis (2). In another study, using contour-clamped homogeneous elec- tric field electrophoresis, Sangeorzan et al.

with one or a few dominant strains, which tend not to change over time. Pow- derly and colleagues isolated the same strain ofC. albicans in 11 of 17 patients with recurrent yeast infection, by DNA probe analysis (2). In another study, using contour-clamped homogeneous elec- tric field electrophoresis, Sangeorzan et al. found that 60% of patients were colonized with one domi- nant strain of C. albicans. In 74% of these pa- tients, recolonization with the same strain occurred after antifungal therapy (1). Using bio- typing and restriction fragment length polymor- phism analysis of 25S ribosomal DNA, Whelan and colleagues found that strains of C. albicans isolated from 24 patients with AIDS were not significantly different from strains from 23 pa- tients without HIV infection (3). Thus, the candi- dal strains causing disease in patients with HIV infection appear to be the same as those colonizing patients without HIV infection and, in most pa- tients, do not change over time. In HIV-infected patients, candidiasis is virtu- ally always mucocutaneous, involving the oro- pharynx, the esophagus, and the vagina. HIV infection by itself is not associated with the syn- drome of disseminated candidiasis, which is char- acterized by candidemia, endophthalmitis, and multiple organ involvement. The precise immu- nologic processes that control candidal infection in HIV-infected patients are not known. However, mucocutaneous candidiasis is clearly related to the development of clinical cellular immunodefi- ciency. In fact, oropharyngeal candidiasis is an independent predictor of immunodeficiency in pa- tients with AIDS (4). Moreover, a CD4 lymphocyte count < 200/l is a major risk factor for the devel- opment of clinical thrush in HIV-infected persons (1). Although oropharyngeal candidiasis is fre- quent in men, recurrent vaginal candidiasis is a common early manifestation of HIV infection in women. The location and severity of candidiasis in women with HIV infection appear to be closely associated with the degree of cellular immunode- ficiency, based on the peripheral blood CD4 lym- phocyte count. In a study of 66 women, mucocutaneous candidiasis developed in more than half of the women over a median of 14 months of follow-up; vaginal candidiasis, with a mean CD4 lymphocyte count of 506/l, developed only in 10, while oropharyngeal candidiasis, with a mean count of 230/l developed in 16, and esophagitis, with a mean count of 30/l developed in nine (5).

ndidiasis developed in more than half of the women over a median of 14 months of follow-up; vaginal candidiasis, with a mean CD4 lymphocyte count of 506/l, developed only in 10, while oropharyngeal candidiasis, with a mean count of 230/l developed in 16, and esophagitis, with a mean count of 30/l developed in nine (5). Mucocutaneous candidiasis can be treated either topically or with systemic antifungal agents (1, 6), but such therapy does not eradicate coloni- zation (1). Recently, several reports have noted the failure of azole drugs, particularly fluconazole, to treat recurrent cases of oropharyngeal candidiasis (1, 7). While factors such as diminishing cellular immunity, drug interactions, or decreased drug absorption may account for some of these treat- ment failures, increasing evidence suggests that Candida organisms are developing drug resis- tance. In the past, a lack of consensus on the methods for performing antifungal susceptibility tests made it difficult to establish whether clinical fail- ure of antifungal therapy was due to resistance of the organism. However, the National Committee for Clinical Laboratory Standards (NCCLS) has now developed reference methods allowing for uniform testing of yeast isolates (8). Using an NCCLS method, Sangeorzan et al. found that the MIC of fluconazole for Candida isolates increased over time among patients who had received flu- conazole compared with those who received clotri- mazole. Clinical resistance to fluconazole was associated with an increased MIC required by the isolate as well as the patient's low CD4 lymphocyte count (1). These data strongly suggest that contin- ued use of antifungal agents, particularly flucona- zole, leads to both clinical treatment failure and antifungal resistance, especially in highly immu- nodeficient patients. Cryptococcosis A rare disease before the HIV epidemic, crypto- coccosis was identified very early in the epidemic as one of the most common life-threatening infec- tions in AIDS patients (9). However, issues regard- ing its epidemiology and therapy remain unresolved. A single species, Cryptococcus neoformans, is responsible for virtually all clinical cases of cryp- tococcosis. The species exists in two varieties, neo- formans and gattii, which inhabit different ecologic niches. C. neoformans var. neoformans has been isolated in many parts of the world from numerous sites, most frequently from soil contain- ing high amounts of dried bird excreta, particu- larly of pigeons and chickens.

cosis. The species exists in two varieties, neo- formans and gattii, which inhabit different ecologic niches. C. neoformans var. neoformans has been isolated in many parts of the world from numerous sites, most frequently from soil contain- ing high amounts of dried bird excreta, particu- larly of pigeons and chickens. It has long been Synopsis Emerging Infectious Diseases 110 Vol 2, No. 2--April-June 1996 presumed that inhalation of soil contaminated with such excreta is the most likely source of cryptococcal infection. However, few data support this hypothesis. In contrast, the only known environmental source of C. neoformans var. gattii is the river red gum tree (Eucalyptus camaldulen- sis), which grows in rural Australia. Although infection due to var. neoformans is worldwide, cases due to var. gattii have only been identified in tropical and subtropical regions, including ar- eas where E. camaldulensis is not found (10). Virtually all instances of cryptococcosis among HIV-infected persons have been caused by var. neoformans. The ubiquity of var. neoformans in the environment may be making exposure and subsequent infection likely; however, no clear link has ever been established between environmental sources of C. neoformans and the development of cryptococcosis in patients with HIV infection (10). In a recent study, Varma and colleagues, using genomic probe analysis, could not find a direct link between environmental sources of C. neoformans and infection in patients with AIDS or a unique strain of C. neoformans that was infecting these patients (11). Suppression of cellular immunity appears to be a critical factor in the development of cryptococcosis in HIV-infected patients, withthe development of disease relating directly to the risk for AIDS and to the CD4 lymphocyte count (12, 13). The appropriate therapy for cryptococcal men- ingitis in HIV-infected patients is unsettled at this time. The combination of amphotericin B plus flucytosine for 4 to 6 weeks has been considered standard for patients without HIV infection. How- ever, concern about increased toxicity and de- creased efficacy (12) has led to a reconsideration of this regimen in HIV-infected patients. Several studies have examined the use of oral fluconazole in lieu of amphotericin B for initial therapy of cryptococcal meningitis in patients with HIV infection.

V infection. How- ever, concern about increased toxicity and de- creased efficacy (12) has led to a reconsideration of this regimen in HIV-infected patients. Several studies have examined the use of oral fluconazole in lieu of amphotericin B for initial therapy of cryptococcal meningitis in patients with HIV infection. Larsen and colleagues studied 21 patients and found that amphotericin B plus oral flucytosine resulted in fewer clinical failures and faster cryptococcal clearance of the cerebro- spinal fluid than fluconazole alone (14). A large collaborative trial compared results with a rela- tively low dose of intravenous amphotericin B to those with oral fluconazole and found no signifi- cant difference in the overall mortality rate (15). However, in this trial, the early mortality rate, defined as death within the first 2 weeks of ther- apy, was slightly higher, and time to first negative CSF culture was somewhat longer among patients in the fluconazole group. The recurrence of cryptococcosis, even after in- itial therapy has rendered the CSF culture sterile, is extremely common in HIV-infected patients (16); continued antifungal therapy appears to reduce the risk for recurrence (12, 16). Fluconazole in daily doses has been shown to be effective in preventing relapse (16) and is superior to am- photericin B in weekly doses (17). Current information suggests that therapy for cryptococcal meningitis in HIV-infected patients should begin with amphotericin B, with or without flucytosine. Suppressive therapy with fluconazole should be given subsequently to prevent a relapse. When available, the results of a study sponsored by the Mycoses Study Group and AIDS Clinical Treatment Group of the National Institute of Al- lergy and Infectious Diseases, National Institutes of Health, should clarify these issues. Histoplasmosis, Coccidioidomycosis, and Blastomycosis Unlike candidiasis and cryptococcosis, infec- tions caused by Histoplasma capsulatum, Coc- cidioides immitis, and Blastomyces dermatitidis are acquired in specific geographic regions. Infec- tions due to these fungi were not initially associ- ated with HIV infection because the HIV epidemic in the United States began in the large urban areas of the East and West Coasts, outside the areas in which these fungi are endemic.

itis, and Blastomyces dermatitidis are acquired in specific geographic regions. Infec- tions due to these fungi were not initially associ- ated with HIV infection because the HIV epidemic in the United States began in the large urban areas of the East and West Coasts, outside the areas in which these fungi are endemic. As HIV infection spread to the Midwest, where histoplas- mosis and blastomycosis are endemic, and to the Southwest, where coccidioidomycosis occurs, these fungi became recognized as major opportun- istic agents. Histoplasmosis H. capsulatumvar. capsulatumcauses infection worldwide and is the organism most associated with disease in HIV-infected patients. A few cases of histoplasmosis in HIV-infected patients have been due to H. capsulatum var. duboisii (18), which is found in tropical Africa. In the Western Hemisphere, infection is concentrated in the east- ern United States but is also found in the Carib- bean as well as in Central and South America. H. capsulatum var. capsulatum is typically isolated from soil contaminated with avian or bat excreta, Synopsis Vol 2, No. 2--April-June 1996 111 Emerging Infectious Diseases and a number of epidemics among persons with- out HIV infection have been associated with dis- ruption of contaminated soil. Most cases of histoplasmosis in patients with HIV infection have occurred within the recognized area for endemic H. capsulatuminNorthAmerica, the Ohio and Mississippi River valleys. However, within that area, great variability has been seen in the incidence of disease, with most cases being reported from a single city, Indianapolis, Indiana (19, 20). Since 1978, Wheat has recorded several outbreaks of histoplasmosis in that city, mostly centered in areas where active construction led to soil disruption. In the most recent outbreak, pa- tients with AIDS accounted for more than 50% of the culture-proven cases of histoplasmosis (21); these data suggest that many of these cases rep- resent new infection due to recent exposure rather than reactivation of latent disease. Cases of histoplasmosis and HIV-infection have also been reported well outside the recognized histoplasmosis-endemic areas (22, 23). In some instances, these cases represent reactivation of infection acquired during residence in or travel to disease-endemic regions. In other instances, they appear to represent acute infection after disrup- tion of microfoci of H. capsulatum that exist out- side the recognized disease-endemic areas (19).

as (22, 23). In some instances, these cases represent reactivation of infection acquired during residence in or travel to disease-endemic regions. In other instances, they appear to represent acute infection after disrup- tion of microfoci of H. capsulatum that exist out- side the recognized disease-endemic areas (19). Patients with progressive disseminated histo- plasmosis, the most common form of disease among HIV-infected persons, usually have fever, malaise, and weight loss over a period of weeks. Diagnosis is often established by isolating the fungus from respiratory secretions, blood, or bone marrow, but detecting Histoplasma capsulatum var. capsulatum polysaccharide antigen (HPA) in the serum or urine is also helpful (19). Amphotericin B therapy is usually effective for progressive disseminated histoplasmosis in pa- tients with HIV infection. Itraconazole may be useful for less severe cases (24). However, relapses are extremely common when therapy is stopped (19, 25), and maintenance therapy with intermit- tent amphotericin B (25) or with itraconazole (26) is required to prevent this. Fluconazole is less effective but can be used by those who cannot tolerate amphotericin B or itraconazole (27). Urine and serum HPA levels decline with success- ful therapy and can be useful in determining a patient's response to therapy as well as assessing a patient for relapse (26-28). Coccidioidomycosis Within the disease-endemic area (U.S. South- west), coccidioidomycosis is one of the most fre- quent opportunistic infections in persons with AIDS (29). In a prospective study in Tucson, Ari- zona, active coccidioidomycosis developed in 25% of a cohort of HIV-infected patients over a 41- month period (30). The major risk for developing disease was immunosuppression, as manifested by a CD4 lymphocyte count below 250/l, a diag- nosis of AIDS, or anergy indicated on control skin tests. Length of time in the disease-endemic area, a history of prior coccidioidomycosis, and a posi- tive coccidioidal skin test were not associated with the development of active coccidioidomycosis. These data suggest that most coccidioidomycosis cases among HIV-infected persons in a disease-en- demic area are recently acquired and not due to reactivation of latent infection. However, as with histoplasmosis, in a small number of HIV-infected patients, previously acquired infection is reacti- vated, and clinical coccidioidomycosis develops while the patient is residing outside the disease- endemic area (29).

n- demic area are recently acquired and not due to reactivation of latent infection. However, as with histoplasmosis, in a small number of HIV-infected patients, previously acquired infection is reacti- vated, and clinical coccidioidomycosis develops while the patient is residing outside the disease- endemic area (29). Despite recent outbreaks of coccidioidomycosis in the San Joaquin Valley and in Northridge, California (31, 32), most cases of coccidioidomy- cosis among HIV-infected patients have been re- ported from Arizona, particularly the metropolitan areas of Phoenix and Tucson (30, 33). Whether this represents underreporting of dis- ease in California or a true increase in incidence in Arizona is unknown. Most HIV-infected patients with coccidioidomy- cosis seek treatment for pulmonary disease. In many, chest radiographs show a diffuse, reticu- lonodular pattern. Approximately 70% of patients with this pattern die within 1 month despite anti- fungal therapy (30, 33). In fact, this radiographic pattern may mimic that seen with Pneumocystis carinii pneumonia (34). Sites of dissemination fre- quently seen in patients without HIV infection, such as skin, soft tissue, bone, joint, and meninges, appear less common among patients with HIV infection (30, 33). Serologic tests can be useful in diagnosing coccidioidomycosis in HIV-infected pa- tients, although they are more likely to have nega- tive results than patients without HIV infection (35). On the other hand, a positive coccidioidal complement-fixation serologic test result in an HIV-infected patient, even in the absence of Synopsis Emerging Infectious Diseases 112 Vol 2, No. 2--April-June 1996 clinical illness, predicts impending active coc- cidioidomycosis (36). Comparative trials regarding the therapy of coccidioidomycosis in HIV-infected patients have not been carried out. For severe disease, such as for diffuse, reticulonodular pneumonia, am- photericin B is recommended. However, for less fulminant infection, fluconazole has been effec- tive. As with cryptococcosis and histoplasmosis, life-long maintenance therapy with an oral azole is recommended, although relapses have occurred despite this. Blastomycosis Blastomycosis is the least common of the three endemic mycoses in North America among pa- tients with HIV infection; fewer than 25 cases have been reported.

ith cryptococcosis and histoplasmosis, life-long maintenance therapy with an oral azole is recommended, although relapses have occurred despite this. Blastomycosis Blastomycosis is the least common of the three endemic mycoses in North America among pa- tients with HIV infection; fewer than 25 cases have been reported. Only recently has an environ- mental link been established for infection, when the organism was isolated from riverbank soil in association with an outbreak (37). Pappas and colleagues have published the larg- est series of cases associated with HIV infection, consisting of 15 patients from 10 medical centers (38). Ten of the 15 cases were reported from sites within the known disease-endemic area, the mid- western and southeastern United States, and the other five patients had resided within the disease- endemic area at some time before the diagnosis. Most patients were clinically immunodeficient at the time of diagnosis and had either chronic pul- monary infection or disease disseminated beyond the lungs, including meningitis. In all but one case, the diagnosis was established by culture of B. dermatitidis, whereas results of serologic tests, when performed, were uniformly negative. Both amphotericin B and ketoconazole were used suc- cessfully as therapy in the series. Aspergillosis Although disseminated aspergillosis was origi- nally listed as an infection at least moderately predictive of AIDS, it was removed from the list in 1984 because only three cases had been reported among 1,762 patients (39). However, over the last 5 years, the number of cases of invasive aspergil- losis among HIV-infected patients has dramati- cally increased (40-44); more than 75 cases now have been documented. The largest series, con- taining 33 patients, was reported by Lortholary and colleagues from France (43). All patients had AIDS and a median CD4 lymphocyte count of 27/l. About half the patients had the traditional risk factors for invasive aspergillosis (neutropenia or corticosteroid use) at the time of diagnosis. Culture of fluid from bronchoalveolar lavage was positive in all cases in which it was done; A. fumigatus grew in 29 of 31 patients. In this study, isolating the fungus from bronchoalveolar lavage fluid correlated with histologic evidence of inva- sive aspergillosis in 14 of 15 patients. In all series, the death rate has been extremely high despite therapy.

avage was positive in all cases in which it was done; A. fumigatus grew in 29 of 31 patients. In this study, isolating the fungus from bronchoalveolar lavage fluid correlated with histologic evidence of inva- sive aspergillosis in 14 of 15 patients. In all series, the death rate has been extremely high despite therapy. Intravenous amphotericin B has been used most often for treatment, but itra- conazole has been tried in some instances (40, 43). In a recent study of the use of itraconazole for the treatment of invasive aspergillosis in a variety of patients, therapy was unsuccessful in all 16 pa- tients with AIDS and aspergillosis (45). In seven of these, either toxicity to the drug developed or clinical symptoms worsened while they were re- ceiving itraconazole; nine died, two directly of aspergillosis and seven of other causes. If the incidence of aspergillosis is increasing among HIV-infected patients, the factors associ- ated with this increase remain unclear. The iden- tified risk factors for aspergillosis have no doubt increased among HIV-infected patients in the last decade with the use of such drugs as zidovudine and ganciclovir, which are associated with neu- tropenia, and corticosteroids for the treatment of severe Pneumocystis carinii pneumonia and other conditions. Others have postulated that prior pneumonia, which may result in diminished macrophage function (43) and contaminated air, inhaled during marijuana smoking (40), may also play a role. Further studies are needed to clarify this. Penicilliosis Piehl and colleagues reported the first case of infection due to Penicillium marneffei in an AIDS patient in 1988 when they described a patient in Chicago with persistent fever, anorexia, and a papular skin rash. The patient's travel history was not given. Blood, bone marrow, sputum, and skin biopsy specimen cultures all grew P. marneffei. The patient responded to therapy with am- photericin B, but relapsed once the therapy was discontinued (46). Since that report, the number of reported cases among HIV infected patients has risen rapidly, particularly in association with the HIV epidemic Synopsis Vol 2, No. 2--April-June 1996 113 Emerging Infectious Diseases in Thailand. In a series of 80 patients reported by Supparatpinyo and colleagues (47), most patients were men from the Chiang Mai province of north- ern Thailand.

nfected patients has risen rapidly, particularly in association with the HIV epidemic Synopsis Vol 2, No. 2--April-June 1996 113 Emerging Infectious Diseases in Thailand. In a series of 80 patients reported by Supparatpinyo and colleagues (47), most patients were men from the Chiang Mai province of north- ern Thailand. The most common characteristic in these patients was a generalized papular rash; some of the lesions had central ubilication remi- niscent of molluscum contagiosum. Diagnosis was usually established by examining Wright-stained samples of bone marrow aspirates or touch smears of skin biopsy specimens. Treatment with amphotericin B has been suc- cessful in most patients. Itraconazole and flucona- zole may also be useful. The mortality rate appears most related to a delay in diagnosis. Re- lapse is common once therapy is stopped (48); therefore, antifungal therapy should be life-long in the HIV-infected patient with penicilliosis. In northern Thailand, penicilliosis is now the third most common opportunistic infection (after tuberculosis and cryptococcosis) among HIV-in- fected persons (48). Given that P. marneffei ap- pears endemic in Southeast Asia, penicilliosis can be expected to become an even larger problem as the HIV epidemic continues to expand in this part of the world. Prevention of Fungal Infections in HIV-Infected Patients With the rise of fungi as opportunistic patho- gens among patients infected with HIV and with the success of preventive therapy for other oppor- tunists, such as P. carinii, primary prevention of fungal disease in HIV-infected patients should be pursued. The goal of any such prevention would be to increase both the length and the quality of the patient's life. Since the availability of the oral azoles, using antifungal agents to prevent fungal diseases in HIV-infected patients has become a promising ap- proach. However, several questions must be con- sidered before antifungal chemoprophylaxis is used (49). Is the prevalence of disease high enough to make the drug useful in most patients? Is the efficacy of the drug in preventing disease suffi- cient? Does the therapy induce the development of drug resistance? Is the cost of the drug reasonable? Finally, does the drug have an acceptable toxicity profile, and does it interact or interfere with the metabolism of other drugs? Few answers to these questions are available.

of the drug in preventing disease suffi- cient? Does the therapy induce the development of drug resistance? Is the cost of the drug reasonable? Finally, does the drug have an acceptable toxicity profile, and does it interact or interfere with the metabolism of other drugs? Few answers to these questions are available. In a recently published prospective, randomized study comparing fluconazole to topical clotrima- zole (13), fluconazole was significantly better than clotrimazole in preventing oropharyngeal and esophageal candidiasis as well as cryptococcal meningitis. The benefit of fluconazole was great- est among patients whose CD4 lymphocyte count was < 50/l. However, 10% of the patients had at least one episode of candidiasis while taking flu- conazole, which suggests drug resistance. More- over, the overall and fungal-disease-related death rates were no different between the two groups. Given the present data, further studies will be needed before the issue of primary prevention of fungal disease through chemoprophylaxis is set- tled (50). Dr. Ampel is an associate professor of medicine at the University of Arizona College of Medicine and director of HIV Clinical Services at the Tucson Veterans Affairs Medical Center. His research focuses on the immune response in coccidioidomycosis. References 1. Sangeorzan JA, Bradley SF, He X, Zarins LT, et al. Epidemiology of oral candidiasis in HIV-infected pa- tients: colonization, infection, treatment, and emer- gence of fluconazole resistance. Am J Med 1994; 97:339-46. 2. Powderly WG, Robinson K, Keath EJ. Molecular epidemiology of recurrent oral candidiasis in human immunodeficiency virus-positive patients: evidence for two patterns of recurrence. J Infect Dis 1993; 168:463-6. 3. Whelan WL, Kirsch DR, Kwon-Chung KJ, Wahl SM, et al. Candida albicans in patients with the acquired immunodeficiency syndrome: absence of a novel or hypervirulent strain. J Infect Dis 1990; 162:513-8. 4. Klein RS, Harris CA, Butkus Small C, Moll B, et al. Oral candidiasis in high-risk patients as the initial manifestation of the acquired immunodeficiency syn- drome. N Engl J Med 1984; 311:354-8. 5. Imam N, Carpenter CC, Mayer KH, Fisher A, et al. Hierarchical pattern of mucosal Candida infections in HIV-seropositive women. Am J Med 1990; 89:142-6. 6. Stevens DA, Greene SI, Lang OS.

asis in high-risk patients as the initial manifestation of the acquired immunodeficiency syn- drome. N Engl J Med 1984; 311:354-8. 5. Imam N, Carpenter CC, Mayer KH, Fisher A, et al. Hierarchical pattern of mucosal Candida infections in HIV-seropositive women. Am J Med 1990; 89:142-6. 6. Stevens DA, Greene SI, Lang OS. Thrush can be prevented in patients with acquired immunodefi- ciency syndrome and the acquired immunodeficiency syndrome-related complex: randomized, double- blind, placebo-controlled study of 100-mg oral flu- conazole daily. Arch Intern Med 1991; 151:2458-64. 7. Redding S, Smith J, Farinacci G, Rinaldi M, et al. Resistance of Candida albicans to fluconazole during treatment of oropharyngeal candidiasis in a patient with AIDS: documentation by in vitro susceptibility testing and DNA subtype analysis. Clin Infect Dis 1994; 18:240-2 Synopsis Emerging Infectious Diseases 114 Vol 2, No. 2--April-June 1996 8. Rex JH, Pfaller MA, Rinaldi MG, Polack A, et al. Antifungal susceptibility testing. Clin Microbiol Rev 1993; 6:357-81. 9. Dismukes WE. Cryptococcal meningitis in patients with AIDS. J Infect Dis 1988; 157:624-8. 10. Levitz SM. The ecology of Cryptococcus neoformans and the epidemiology of cryptococcosis. Rev Infect Dis 1991; 13:1163-9. 11. Varma A, Swinne D, Staib F, Bennett JE, et al. Diver- sity of DNA fingerprints in Cryptococcus neoformans. J Clin Microbiol 1995; 33:1807-14. 12. Chuck SL, Sande MA. Infections with Cryptococcus neoformans in the acquired immunodeficiency syn- drome. N Engl J Med 1989; 321:793-9. 13. Powderly WG, Finkelstein D, Feinberg J, Frame P, et al. (NIAID AIDS Clinical Trials Group). Arandomized trial comparing fluconazole with clotrimazole troches for the prevention of fungal infections in patients with advanced human immunodeficiency virus infection. N Engl J Med 1995; 332:700-5. 14. Larsen RA, Leal MAE, Chan LS. Fluconazole com- pared with amphotericin B plus flucytosine for cryp- tococcal meningitis in AIDS: a randomized trial. Ann Intern Med 1990; 113:183-7. 15. Saag MS, Powderly WG, Cloud GA, Robinson P, et al. (NIAID Mycoses Study Group and the AIDS Clinical Trials Group). Comparison of amphotericin B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. N Engl J Med 1992; 326:83-9. 16. Bozzette SA, Larsen RA, Chiu J, Leal MAE, et al. A placebo-controlled trial of maintenance therapy with fluconazole after treatment of cryptococcal meningitis in the acquired immunodeficiency syndrome.

B with fluconazole in the treatment of acute AIDS-associated cryptococcal meningitis. N Engl J Med 1992; 326:83-9. 16. Bozzette SA, Larsen RA, Chiu J, Leal MAE, et al. A placebo-controlled trial of maintenance therapy with fluconazole after treatment of cryptococcal meningitis in the acquired immunodeficiency syndrome. N Engl J Med 1991; 324:580-4. 17. Powderly WG, Saag MS, Cloud GA, Robinson P, et al. A controlled trial of fluconazole or amphotericin B to prevent relapse of cryptococcal meningitis in patients with the acquired immunodeficiency syndrome. N Engl J Med 1992; 326:793-9. 18. Chandenier J, Goma D, Moyen G, Samba-Lefebvre MC, et al. [African histoplasmosis due to Histoplasma capsulatum var. duboisii: relationship with AIDS in recent Congolese cases]. Sante 1995; 5:227-34. 19. Wheat LJ, Connolly-Stringfield PA, Baker RL, Curfman MF, et al. Disseminated histoplasmosis in the acquired immune deficiency syndrome: clinical findings, diagnosis and treatment, and review of the literature. Medicine [Baltimore] 1990; 69:361-74. 20. Wheat LJ, Slama TG, Zeckel ML. Histoplasmosis in the acquired immune deficiency syndrome. Am J Med 1985; 78:203-10. 21. Wheat LJ. Histoplasmosis in Indianapolis. Clin Infect Dis 1992; 14 (Suppl 1):S91-9. 22. Huang CT, McGarry T, Cooper S, Saunders R, et al. Disseminated histoplasmosis in the acquired immu- nodeficiency syndrome: report of five cases from a nonendemic area. Arch Intern Med 1987; 147:1181-4. 23. Salzman SH, Smith RL, Aranda CP. Histoplasmosis in patients at risk for the acquired immunodeficiency syndrome in a nonendemic setting. Chest 1988; 93:916-21. 24. Wheat J, Hafner R, Korzun AH, Limjoco MT, et al. (AIDS Clinical Trials Group). Itraconazole treatment of disseminated histoplasmosis in patients with the acquired immunodeficiency syndrome. Am J Med 1995; 98:336-42. 25. McKinsey DS, Gupta MR, Riddler SA, Driks MR, et al. Long-term amphotericin B therapy for dissemi- nated histoplasmosis in patients with the acquired immunodeficiency syndrome (AIDS). Ann Intern Med 1989; 111:655-9. 26. Wheat J, Hafner R, Wulfsohn M, Spencer P, et al. (NIAID Clinical Trials and Mycoses Study Group Collaborators). Prevention of relapse of histoplas- mosis with itraconazole in patients with the acquired immunodeficiency syndrome. Ann Intern Med 1993; 118:610-6. 27. Norris S, Wheat J, McKinsey D, Lancaster D, et al. Prevention of relapse of histoplasmosis with flucona- zole in patients with the acquired immunodeficiency syndrome.

Prevention of relapse of histoplas- mosis with itraconazole in patients with the acquired immunodeficiency syndrome. Ann Intern Med 1993; 118:610-6. 27. Norris S, Wheat J, McKinsey D, Lancaster D, et al. Prevention of relapse of histoplasmosis with flucona- zole in patients with the acquired immunodeficiency syndrome. Am J Med 1994; 96:504-8. 28. Wheat LJ, Connolly-Stringfield P, Blair R, Connolly K, et al. Effect of successful treatment with am- photericin B on Histoplasma capsulatum variety cap- sulatum polysaccharide antigen levels in patients with AIDS and histoplasmosis. Am J Med 1992; 92:153-60. 29. Jones JL, Fleming PL, Ciesielski CA, Hu DJ, et al. Coccidioidomycosis among persons with AIDS in the United States. J Infect Dis 1995; 171:961-6. 30. Ampel NM, Dols CL, Galgiani JN. Coccidioidomycosis during human immunodeficiency virus infection: re- sults of a prospective study in a coccidioidal endemic area. Am J Med 1993; 94:235-40. 31. Einstein HE, Johnson RH. Coccidioidomycosis: new aspects of epidemiology and therapy. Clin Infect Dis 1993; 16:349-56. 32. CDC. Coccidioidomycosis following the Northridge Earthquake--California, 1994. MMWR 1994; 43:194- 5. 33. Fish DG, Ampel NM, Galgiani JN, Dols CL, et al. Coccidiodiomycosis during human immunodeficiency virus infection: a review of 77 patients. Medicine [Baltimore] 1990; 69:384-91. 34. Mahaffey KW, Hippenmeyer CL, Mandel R, Ampel NM. Unrecognized coccidioidomycosis complicating Pneumocystis carinii pneumonia in patients infected with the human immunodeficiency virus and treated with corticosteroids: a report of two cases. Arch Intern Med 1993; 153:1496-8. 35. Antoniskis D, Larsen RA, Akil B, Rarick MU, et al. Seronegative disseminated coccidioidomycosis in pa- tients with HIV infection. AIDS 1990; 4:691-3. 36. Arguinchona HL, Ampel NM, Dols CL, Galgiani JN, et al. Persistent coccidioidal seropositivity without clinical evidence of active coccidioidomycosis in pa- tients infected with human immunodeficiency virus. Clin Infec Dis 1995; 20:1281-5. 37. Klein BS, Vergeront JM, Weeks RJ, Kumar UN, et al. Isolation of Blastomyces dermatitidis in soil associ- ated with a large outbreak of blastomycosis in Wis- consin. N Engl J Med 1986; 314:529-34. 38. Pappas PG, Pottage JC, Powderly WG, Fraser VJ, et al. Blastomycosis in patients with the acquired immu- nodeficiency syndrome. Ann Intern Med 1992; 116:847-53. 39. Jaffe HW, Selik RM.

Blastomyces dermatitidis in soil associ- ated with a large outbreak of blastomycosis in Wis- consin. N Engl J Med 1986; 314:529-34. 38. Pappas PG, Pottage JC, Powderly WG, Fraser VJ, et al. Blastomycosis in patients with the acquired immu- nodeficiency syndrome. Ann Intern Med 1992; 116:847-53. 39. Jaffe HW, Selik RM. Acquired immunodeficiency syn- drome: is disseminated aspergillosis predictive of un- derlying cellular deficiency? [letter]. J Infect Dis 1984; 149:829. Synopsis Vol 2, No. 2--April-June 1996 115 Emerging Infectious Diseases 40. Denning DW, Follansbee SE, Scolaro M, Norris S, et al. Pulmonary aspergillosis in the acquired immu- nodeficiency syndrome. N Engl J Med 1991; 324:654- 62. 41. Singh N, Yu VL, Rihs JD. Invasive aspergillosis in AIDS. South Med J 1991; 84:822-6. 42. Klapholz A, Salomon N, Perlman DC, Talavera W. Aspergillosis in the acquired immunodeficiency syn- drome. Chest 1991; 100:1614-8. 43. Lortholary O, Meyohas MC, Dupont B, Cadranel J, et al. (French Cooperative Study Group on Aspergillosis in AIDS). Invasive aspergillosis in patients with ac- quired immunodeficiency syndrome: report of 33 cases. Am J Med 1993; 95:177-87. 44. Miller WT, Jr., Sais GJ, Frank I, Gefter WB, et al. Pulmonary aspergillosis in patients with AIDS: clini- cal and radiographic correlations. Chest 1994; 105:37- 44. 45. Denning DW, Lee JY, Hostetler JS, Pappas P, et al. NIAID Mycoses Study Group multicenter trial of oral itraconazole therapy for invasive aspergillosis. Am J Med 1994; 97:135-44. 46. Piehl MR, Kaplan RL, Haber MH. Disseminated penicilliosis in a patient with acquired immunodefi- ciency syndrome. Arch Pathol Lab Med 1988; 112:1262-4. 47. Supparatpinyo K, Sirisanthana T. Disseminated Penicillium marneffei infection diagnosed on exami- nation of a peripheral blood smear of a patient with human immunodeficiency virus infection. Clin Infect Dis 1994; 18:246-7. 48. Supparatpinyo K, Khamwan C, Baosoung V, Nelson KE, et al. Disseminated Penicillium marneffei infec- tion in southeast Asia. Lancet 1994; 344:110-3. 49. Perfect JR. Antifungal prophylaxis: to prevent or not? Am J Med 1993; 94:233-4. 50. Powderly WG. Prophylaxis for HIV-related infections: a work in progress. Ann Intern Med 1996; 124:342-4. Synopsis Emerging Infectious Diseases 116 Vol 2, No. 2--April-June 1996

An Outbreak of Ross River Virus Disease in Southwestern Australia More than 540 serologically confirmed cases of Ross River (RR) virus disease have been reported from the southwest region of Western Australia since November 1995 (Figure 1). Most affected by the mosquito-borne disease are communities on the Swan Coastal Plain south of Perth. Cases have also been reported from towns farther south or inland and from Perth itself. These regions were foci of RR virus activity during previous southwest outbreaks in 1988-89 and 1991-92 (1,2); however, the current outbreak differs somewhat in the tim- ing and location of virus activity. This article is a preliminary overview of the incidence of disease, mosquito and virus activity, and environmental conditions before and during the outbreak. Monitoring of the incidence of human disease provided no indication of abnormally high levels of RR virus activity until mid-December 1995 when the number of reported cases began to rise sharply.In contrast,monitoring of mosquito breed- ing sites,adult mosquito populations,and environ- mental conditions in late October and November 1995 showed a potential for high levels of virus transmission. The areas affected most, in terms of numbers of cases and attack rates (not shown), are coastal towns and communities around the Leschenault Inlet (including the city of Bunbury), between 165 and 190 km south of Perth and in the shires of Capel and Busselton, as well as on the coast be- tween 190 and 245 km south of Perth (Table 1). These regions are popular tourist destinations during the summer holidays.It appears that many holiday-makers from elsewhere in the southwest, as well as local residents, were exposed to infected mosquitoes in these regions during the Christ- mas-New Year period. Many of the Perth cases are from semirural, outlying suburbs, but some are from suburbs closer to the city center, often near the Swan and Canning rivers or fresh water wetlands and lakes. Follow-up questionnaires indicate that a consider- able proportion of metropolitan cases were in per- sons exposed in the southwest, particularly in the Leschenault, Capel, and Busselton regions.

t some are from suburbs closer to the city center, often near the Swan and Canning rivers or fresh water wetlands and lakes. Follow-up questionnaires indicate that a consider- able proportion of metropolitan cases were in per- sons exposed in the southwest, particularly in the Leschenault, Capel, and Busselton regions. How- ever, many cases from Perth also appear to have been locally acquired. Locally acquired cases were also present during the two previously reported outbreaks. Many cases have also been reported from the Peel region, 70 km to 130 km south of Perth, surrounding the Peel Inlet and Harvey Estuary. However, considerably more cases had been re- ported by late February in the Peel region during the 1988-89 and 1991-92 outbreaks. Also, during 1988 and 1991 virus activity in the Peel outbreaks commenced earlier than in the Leschenault and Capel-Busselton regions; this is apparently not the case during the current outbreak (Table 1).The reasons for these differences are not yet clear, but extensive control of saltmarsh mosquito breeding has been carried out in the Peel region this season. Large saltmarshes and brackish wetlands in the Peel, Leschenault, Capel, and Busselton re- gions provide an ideal breeding habitat for Ae. camptorhynchus mosquitoes (5,6). This species is the major vector of RR virus in the southwest of Figure 1. Serologically confirmed cases of Ross River virus disease, by month of onset, in the southwest of Western Australia, July 1995 to February 1996, as reported by doctors to the Health Department of Western Australia (when possible, case follow-up questionnaires were administered by environmental health officers from relevant local authorities). Only a small number of cases diagnosed by state and private laboratories, although the patient was not notified, have been included. Consequently, the number of cases shown is almost certainly an underestimate of the true number of serologically confirmed cases. Almost 65% of cases have dates of onset in January 1996. However, further notifications and analysis of follow-up questionnaires that have not yet been carried out for many January/February cases may alter this pattern. Previous southwest outbreaks also peaked in January or February but were considerably less acute. Dispatches Vol. 2, No. 2 --April-June 1996 117 Emerging Infectious Diseases Western Australia.

analysis of follow-up questionnaires that have not yet been carried out for many January/February cases may alter this pattern. Previous southwest outbreaks also peaked in January or February but were considerably less acute. Dispatches Vol. 2, No. 2 --April-June 1996 117 Emerging Infectious Diseases Western Australia. Surveillance during previous outbreaks has clearly shown that the risk for RR virus transmission in coastal regions of the south- west increases markedly if large populations of adult Ae. camptorhynchus persist into late spring and summer (1,2). Adult mosquito populations and RR virus activity are monitored routinely by our laboratory at up to 40 sites between Rocking- ham and Dunsborough (50 km to 260 km south of Perth) each fortnight through spring and summer. In addition,saltmarsh mosquito breeding sites are regularly monitored by local authorities and the health department. Widespread breeding of Ae. camptorhynchus (larvae) was observed in the Capel--Busselton region in late October 1995 and prompted a health department warning of an increased risk for RR virus transmission in the southwest. However, almost no activities to control mosquito larvae were carried out in the worst-affected regions. The adult mosquito monitoring program subsequently showed that extremely large populations of Ae. camptorhynchus survived through November and December. (Figures 2 and 3). The number of mosquitoes collected per trap per night in the Capel-Busselton region during November and December 1995 (up to 10,000 mosquitoes per trap at some sites) is unprecedented in the 5 years of surveillance in the region. Similar results were obtained in the Leschenault region where the number of Ae. camptorhynchus mosquitoes col- lected during December 1995 and January 1996 were similar to those observed during the 1991-92 outbreak. These observations, along with the expected seasonal exodus of city dwellers to these areas during the Christmas holidays, prompted a second warning by the health depart- ment in December 1995. Fourteen isolates of RR virus were obtained from Ae. camptorhynchus mosquitoes collected at a major wet- land west of Busselton on December 7, 1995 (Figure 3). Large popula- tions of potential vertebrate hosts (western gray kangaroos, Macropus fuliginosus) were also observed in close proximity to this site through- out the spring and summer. Case fol- low-up questionnaires indicate thata large percentage of Busselton pa- tients were exposed in this locality.

1995 (Figure 3). Large popula- tions of potential vertebrate hosts (western gray kangaroos, Macropus fuliginosus) were also observed in close proximity to this site through- out the spring and summer. Case fol- low-up questionnaires indicate thata large percentage of Busselton pa- tients were exposed in this locality. The last time RR virus was isolated from this site was during the 1991-92 outbreak in the region. Mosquito populations in the Capel-Busselton region (Figure 3) and most areas of the Les- chenault region dropped rapidly by mid-January 1996. However, further cases with dates of onset in February have been reported (Table 1), which suggests a high infection rate in the remaining adults. The results of processing of adult mosqui- toes collected in February are, therefore, eagerly awaited. Several isolates of RR virus were also obtained from mosquitoes collected in the Peel region (Table 2). The recent isolations from Ae. vigilax are of particular concern. This species is regarded as the major vector of RR virus in coastal areas of north- ern and eastern Australia (3,4), but until now it has had little or no role in transmitting RR virus in the southwest (1,2). Ae. vigilax has become the Table 1. Cases of Ross River virus disease, by month of onset and geographic region, in the southwest of Western Australia, July 1995 to February 1996a Southwest region Jul Aug Sep Oct Nov Dec Jan Feb Totals Metro. area 1 1 6 23 99 17 147 Peel 1 3 1 1 8 34 9 57 Leschenault 2 1 3 9 114 30 159 Capel/Busselton 2 1 26 73 20 122 Inland/south coast 2 1 2 3 30 27 65 North/east of Perth 1 4 2 5 3 15 Totals 3 4 4 9 15 69 355 106 565 a Cases are recorded by region in which exposure most likely occurred, where available (from case follow-up questionnaires), or by region of residence.Data are incomplete.Case follow-up questionnaires are available for many January/February cases but have not yet been analyzed, and most pathology laboratory reports (nonnotified serologically confirmed patients (i.e., figures represent underestimate of true number of confirmed cases) are not included. Figure 2. Mean number of adult mosquitoes (total population and dominant species) and isolations of Ross River virus from mosquitoes at Capel-Busselton region, wetland site, January 1994 to April 1995. Dispatches Emerging Infectious Diseases 118 Vol. 2, No.

restimate of true number of confirmed cases) are not included. Figure 2. Mean number of adult mosquitoes (total population and dominant species) and isolations of Ross River virus from mosquitoes at Capel-Busselton region, wetland site, January 1994 to April 1995. Dispatches Emerging Infectious Diseases 118 Vol. 2, No. 2 --April-June 1996 dominant species in the Peel region between De- cember and March since the opening of the Dawes- ville Channel.This mosquito is a vicious biter,even during the day if weather conditions are suitable, and is known to disperse considerable distances from breeding sites. Thus, the potential for inter- action between infected mosquitoes and humans in the Peel region may be greater and occur over a wider area than originally thought. Analyses of environmental conditions before and during the outbreak are not yet complete. However, record-high daily rainfall was recorded in October at numerous centers in the southwest. Above-average rainfall occurred in November in Perth and Mandurah and in December in Man- durah, Banbury, and Capel-Busselton. These were accompanied by above-average October and No- vember temperatures at many southwest centers. A series of extremely high tides was also recorded along the Peel-Leschenault region coast around December 20. This resulted from unusually early cyclonic activity (three cyclones) along the north and west coasts of Western Australia during December. Clearly, a combination of some or all of these factors enabled widespread breeding and survival of vector mosquito species. Late spring and summer rains, a short-term rise in sea level (accompanied by higher tides), and mild spring and summer temperatures were predisposing fac- tors during previous outbreaks in the southwest (2,4). Preliminary analysis of the location of virus activity (measured as either human cases or iso- lations from mosquitoes) shows that activity is far less likely in regions in which virus activity was detected in the previous season. Thus, length of time since the previous outbreak also appears to be a predisposing factor for higher levels of virus activity in the southwest.The reason for this is not yet known but may be due to higher levels of immunity in recently infected populations of en- zootic or amplifying vertebrate hosts. This may help explain the comparatively reduced numbers of cases in the Peel region this season following moderate levels of virus activity last year, which coincided with the opening of the Dawesville Channel.

e to higher levels of immunity in recently infected populations of en- zootic or amplifying vertebrate hosts. This may help explain the comparatively reduced numbers of cases in the Peel region this season following moderate levels of virus activity last year, which coincided with the opening of the Dawesville Channel. A small number of cases of Barmah Forest virus infection have been diagnosed during the current outbreak. Numerous cases of a RR virus-like dis- ease have also been reported,as in the 1988-89 and 1991-92 outbreaks.Serum from these patients has been tested for IgM antibody to RR and Barmah Forest viruses but is negative for both viruses. Some of these cases may be in persons that had not seroconverted at the time of the first blood sample. However, many have since provided fur- ther samples, all of which have had negative test results. Sera from these patients are being tested against a wide range of other Australian ar- boviruses, and more blood samples will be sought to ensure that the phenomenon is not due to an extremely delayed immunologic response to RR virus. Michael Lindsay,* Nidia Oliveira, Eva Jasinska,* Cheryl Johansen, Sue Harrington, A.E Wright, and David Smith *University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia; University of Queensland, Brisbane, Queensland;

A small number of cases of Barmah Forest virus infection have been diagnosed during the current outbreak. Numerous cases of a RR virus-like dis- ease have also been reported,as in the 1988-89 and 1991-92 outbreaks.Serum from these patients has been tested for IgM antibody to RR and Barmah Forest viruses but is negative for both viruses. Some of these cases may be in persons that had not seroconverted at the time of the first blood sample. However, many have since provided fur- ther samples, all of which have had negative test results. Sera from these patients are being tested against a wide range of other Australian ar- boviruses, and more blood samples will be sought to ensure that the phenomenon is not due to an extremely delayed immunologic response to RR virus. Michael Lindsay,* Nidia Oliveira, Eva Jasinska,* Cheryl Johansen, Sue Harrington, A.E Wright, and David Smith *University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, Western Australia; University of Queensland, Brisbane, Queensland; Health Department of Western Australia, Mt. Claremont, Western Australia; Western Australian Centre for Pathology and Medical Research, Queen Elizabeth II Medical Centre, Nedlands, Western Australia Figure 3. Mean number of adult mosquitoes (total population and dominant species) and isolations of Ross River virus from mosquitoes at Capel-Busselton region, wetland site, January 1995 to January 1996. Table 2. Isolations of Ross River (RR) virus from mosquitoes collected in the Peel region: 1995-96 season Date Species Isolates of RR virus 14-Sep-95 Ae. camptorhynchus 1 24-Oct-95 Ae. camptorhynchus 2 7-Dec-95 Ae.camptorhynchus 1 27-Dec-95 Ae. camptorhynchus 5 15-Jan-96 Ae. camptorhynchus 2 15-Jan-96 Ae. vigilax 3 Dispatches Vol. 2, No. 2 --April-June 1996 119 Emerging Infectious Diseases References 1. Lindsay MD, Condon R, Mackenzie JS, Johansen C, D'Ercole M, Smith D. A major outbreak of Ross River virus infection in the south-west of Western Australia and the Perth metropolitan area. Communicable Dis- ease Intelligence 1992;16:290-4. 2. Lindsay MD,Latchford JA,Wright AE,Mackenzie JS. Studies on the ecology of Ross River virus in the southwest of Western Australia. Arbovirus Research in Australia 1989;5:28-32. 3. Mackenzie JS, Lindsay MD, Coelen RJ, Broom AK, Hall RA, Smith DW. Arboviruses causing human dis- ease in the Australasian zoogeographic region. Arch Virol 1994:136:447-67. 4. Russell RC. Ross River virus: disease trends and vec- tor ecology in Australia. Bull Soc Vector Ecol 1994;19:73-81. 5. Wright AE. Report on the mosquito eradication cam- paign: survey of mosquitoes in the Bunbury region, Western Australia. Health Department of Western Australia, 1988. 6. Wright, AE. Report on the mosquito eradication cam- paign: survey of mosquitoes in the Mandurah region, Western Australia. Health Department of Western Australia, 1988. Dispatches Emerging Infectious Diseases 120 Vol. 2, No. 2 --April-June 1996

Invasive Penicillin-Resistant Pneumococcal Infections: A Prevalence and Historical Cohort Study More than 25 years ago, isolates of Streptococ- cus pneumoniae were uniformly susceptible to penicillin. However, since a penicillin-resistant pneumococcus was first identified in 1967 (1), the incidence of penicillin-resistant S. pneumoniae (PRSP) strains has been gradually increasing. In certain areas of the United States, PRSP strains have become widespread; Alaska has the highest reported prevalence, 26% (2); a recent study con- ducted in Atlanta, Georgia, found a 25% preva- lence of PRSP (3). Outside the United States, an even higher (33%-58%) prevalence of PRSP has been reported (2). Pneumococcal infections are a leading cause of morbidity and mortality in the United States. S. pneumoniae causes more than 500,000 cases of pneumonia, 55,000 cases of bacteremia, and 6,000 cases of meningitis annually, which result in 40,000 deaths (4).The death rate from pneumococ- cal bacteremia approaches 30%, despite the use of appropriate antimicrobial therapy (5). Reports of refractory illness due to resistant pneumococci demonstrate the clinical relevance of these strains (6,7). Identifying risk factors in the development of PRSP infections is important for both the pre- vention and treatment of these infections. The prevalence of invasive infections due to PRSP was previously studied in Denver, Colorado (8,9). We undertook the study described here to determine the prevalence of invasive PRSP infec- tions in the Colorado Front Range and to deter- mine whether invasive PRSP infections have increased in metropolitan Denver since the earlier studies.In addition,we studied a cohort of patients who had invasive pneumococcal disease during 1994 in metropolitan Denver to ascertain risk factors for invasive PRSP infections. Twenty-six hospital microbiology laboratories in the Colorado Front Range, which comprises the 10 largest counties in Colorado and 80% of the state's population (10), submitted to the Colorado Department of Public Health and Environment reports of all blood and cerebrospinal fluid (CSF) isolates of S.

PRSP infections. Twenty-six hospital microbiology laboratories in the Colorado Front Range, which comprises the 10 largest counties in Colorado and 80% of the state's population (10), submitted to the Colorado Department of Public Health and Environment reports of all blood and cerebrospinal fluid (CSF) isolates of S. pneumoniae that were tested for penicillin susceptibility during 1994. (Penicillin susceptibility testing on invasive pneumococcal isolates was standard practice for the laboratories and did not depend on a clinician's request.) For the part of the study that assessed prevalence, penicillin resistance was defined as a minimum inhibitory concentration (MIC) of 0.12 g/ml or an oxacillin zone of inhibition < 20 mm. Isolates that were tested by an MIC method were further classified as intermediately (0.12 g/ml-1.0 g/ml) or highly ( 2.0 g/ml) penicillin resistant. For the part of the study in which the cohort was analyzed, only isolates that were confirmed as penicillin resistant (i.e., MIC 0.12 g/ml) by either broth dilution or the E test (AB Biodisk, North America, Inc., Culver City, California) were included. Data for the cohort study were collected by chart review by the principal investigator, and telephone inter- views of patients were conducted by trained inter- viewers in the Health Statistics Survey Research Unit of the Colorado Department of Public Health and Environment. For patients under 18 years of age, a parent or legal guardian was interviewed. When patients had died, a relative of the patient (if available) was interviewed. Invasive pneumococcal infections were found in 363 patients in the Colorado Front Range; 49 (13%) of the infections were resistant to penicillin. In metropolitan Denver, 29 (14%) of the invasive pneumococcal infections were penicillin-resistant, of which 20 (69%) were intermediately penicillin- resistant (i.e., MIC 0.12-1.0 g/ml), and 9 (31%) were highly penicillin-resistant (i.e., MIC 2.0 g/ml). This prevalence rate of invasive PRSP infections is significantly higher than the pre- viously reported rates in Denver of 1% (8) and 7% (9). Previous surveillance of invasive PRSP iso- lates showed that one region in the United States, which included Colorado, had a significantly higher rate of penicillin resistance among pneu- mococcal isolates than other U.S. regions (11). Half of the PRSP strains in the cohort part of the study that were tested for cephalosporin sus- ceptibility were resistant to an extended-spectrum cephalosporin.

United States, which included Colorado, had a significantly higher rate of penicillin resistance among pneu- mococcal isolates than other U.S. regions (11). Half of the PRSP strains in the cohort part of the study that were tested for cephalosporin sus- ceptibility were resistant to an extended-spectrum cephalosporin. Our results are similar to the re- cent Atlanta study which found that 34% to 54% of PRSP infections were resistant to an extended- spectrum cephalosporin (3). These rates are much higher than other reported rates of cephalosporin resistance among PRSP isolates of 27% in Ken- tucky and 25% in Tennessee, (12). This has impor- tant implications for the management of invasive Dispatches Vol. 2, No. 2 --April-June 1996 121 Emerging Infectious Diseases PRSP infections, especially in meningitis, where MICs of -lactam antibiotics in the cerebrospinal fluid may be less than the MICs of -lactam anti- biotics in the blood (13). The Centers for Disease Control and Prevention recommends that in areas where pneumococcal resistance to cephalosporins is high, empiric therapy with vancomycin plus an extended-spectrum cephalosporin should be con- sidered in all cases of meningitis potentially caused by S. pneumoniae, until the results of cul- ture and susceptibility testing are available (14). A number of studies have addressed the clinical relevance of PRSP infections and have attempted to identify predictive factors for the development of these infections. In our analysis of the demo- graphic and clinical characteristics of the study population (Table), we found that day-care atten- dance by a member of the patient's household in the 3 months before the patient's illness was asso- ciated with invasive PRSP infections. Indeed, 26% of patients with PRSP infections, compared with 7% of patients with penicillin-sensitive infections, had at least one member in their household, excluding the patient, who had been attending a day-care cen- ter before becoming ill. This study is unique in that, to our knowledge, day- care attendance among household members of pa- tients has not been studied. Even though most studies have not specifically con- sidered family members as a potential mode of PRSP transmission, rates of na- sopharyngeal carriage of PRSP are significantly higher in family contacts of children colonized with PRSP who were attending day-care centers (7, 15).

ers of pa- tients has not been studied. Even though most studies have not specifically con- sidered family members as a potential mode of PRSP transmission, rates of na- sopharyngeal carriage of PRSP are significantly higher in family contacts of children colonized with PRSP who were attending day-care centers (7, 15). Our finding that pa- tients with PRSP infec- tions were more likely to have had a child in their household who had at- tended day-care during the months before their illness suggests that day-care set- tings may serve as foci for spreading resistant pneu- mococcal strains. Antibiot- ics are extensively used to treat upper respiratory in- fections that children at- tending day-care centers often have; the practice of administering a prolonged course of prophylactic anti- biotics to children with recurrent otitis media who Table. Demographic and clinical characteristics of patients with invasive penicillin-resistant S. pneumoniae (PRSP) and penicillin-sensitive S. pneumoniae (PSSP) infections PRSP; PSSP; Odds ratio n = 29 (%) n = 180 (%) (95% confidence interval) Demographic characteristics Age, years < 5 11 (38) 39 (22) 1.9 (0.4-9.1) 5-64 8 (28) 93 (52) Reference > 64 10 (34) 48 (27) 1.9 (0.5-7.1) Sex Male 12 (41) 104 (58) Reference Female 17 (59) 76 (42) 1.8 (0.6-5.4) Racea White 16 (55) 100 (56) Reference Nonwhite 11 (38) 68 (38) 1.4 (0.5-3.9) Clinical characteristics Site of infection Blood 26 (90) 171 (95) Reference CSF 3 (10) 9 (5) 0.8 (0.1-5.1) Underlying medical 14 (48) 108 (60) 1.0 (0.3-3.2) condition Antibiotic useb 14 (52) 56 (39) 2.5 (0.9-7.1) No history of 26 (90) 163 (91) 0.7 (0.1-3.4) penicillin allergyc Previous hospitalizationd 5 (19) 33 (23) 0.3 (0.1-1.5) Day-care attendance Patients < 11 yearse 5 (50) 19 (50) 1.1 (0.4-2.7) Household member(s)f 7 (26) 10 (7) 8.1 (2.2-30.7) Residence in a long-term care facility (patients > 64 years)g 2 (20) 13 (27) 0.7 (0.1-3.6) Hospital-acquired 1 (3) 8 (4) 0.4 (0.04-4.9) infection Outcome Survived 25 (86) 156 (87) Reference Died 4 (14) 24 (13) 1.6 (0.4-6.3) a Missing information on 2 PRSP and 12 PSSP patients. b Includes patients who had taken an antibiotic in the 3 months before illness; missing information on 2 PRSP and 36 PSSP patients. c Missing information on 1 PSSP patient. d Patients who were hospitalized in the 3 months before illness; missing information on 2 PRSP and 34 PSSP patients.

n 2 PRSP and 12 PSSP patients. b Includes patients who had taken an antibiotic in the 3 months before illness; missing information on 2 PRSP and 36 PSSP patients. c Missing information on 1 PSSP patient. d Patients who were hospitalized in the 3 months before illness; missing information on 2 PRSP and 34 PSSP patients. e Children < 11 years of age who attended day care in the 3 months before illness; children < 11 years of age, N = 56 (PRSP = 12, PSSP = 44); missing information on 2 PRSP and 6 PSSP patients. f Patients with at least 1 child < 11 years of age (excluding the patient) in the household who attended day care in the 3 months before illness; missing information on 2 PRSP and 39 PSSP patients. g Adults > 64 years of age who resided in a LTC facility in the 3 months before illness; adults > 64 years of age, N = 58 (PRSP = 10, PSSP = 48). Dispatches Emerging Infectious Diseases 122 Vol. 2, No. 2 --April-June 1996 attend day-care centers (16) may promote the se- lection of resistant bacteria in these settings (6, 15). These children may subsequently transmit resistant S. pneumoniae to susceptible persons in their households. Thus, patterns of antibiotic treatment of children who attend day-care centers may explain why day-care attendance might facili- tate PRSP transmission. The likelihood that day- care settings may serve as reservoirs for antibiotic-resistant pneumococci indicates that the efficacy of prophylactic antibiotics for otitis media should be reassessed, especially when PRSP is present in a community. Furthermore, carriage of or infection with PRSP has been associated with recent use of anti- biotics (17). Our study showed that patients with PRSP infections were more likely to have taken an antibiotic in the 3 months before their illness than patients with penicillin-sensitive pneumococcal infections. This finding supports the theory that antibiotic resistance has developed because of the widespread availability and use of antibiotics. Since the beginning of the antibiotic era 50 years ago, it has been well recognized that antibiotics have been and continue to be inappropriately used (18). The emergence of drug-resistantS.pneumoniae emphasizes the importance of following the recom- mendation of the Immunization Practices Advi- sory Committee that all persons 2 years of age and older who are at high risk for pneumococcal dis- ease receive the 23-valent pneumococcal capsular polysaccharide vaccine.

d (18). The emergence of drug-resistantS.pneumoniae emphasizes the importance of following the recom- mendation of the Immunization Practices Advi- sory Committee that all persons 2 years of age and older who are at high risk for pneumococcal dis- ease receive the 23-valent pneumococcal capsular polysaccharide vaccine. Because of its lack of im- munogenicity and efficacy, the pneumococcal vac- cine has not been licensed for children under 2 years of age (14). The high prevalence of PRSP among young children (3, 17), and the potential for these children to transmit PRSP to susceptible persons,underscore the need for an effective pneu- mococcal vaccine for this age group. Antimicrobial resistance contributes to in- creased morbidity, mortality, and health care costs (19). The solution lies in changing antibiotic pre- scribing patterns, changing patient attitudes about the necessity of antibiotics, increasing sur- veillance of drug-resistant organisms, improving techniques for antibiotic susceptibility testing, and investing in research and development of newer antimicrobial agents. Acknowledgments We thank the Health Statistics Survey Research Unit in the Colorado Department of Public Health and Environ- ment for assistance with the telephone interviews, and the supervisors of the microbiology laboratories and the staff in the medical records departments of participating hos- pitals for their cooperation.This publication was supported by Grant No. STC-T2 D33AH18002-09 from the Division of Medicine of the Health Resources and Services Administration. Crystal B. Kronenberger, M.D., M.S.P.H.,* Richard E. Hoffman, M.D., M.P.H., Dennis C. Lezotte, Ph.D.,* and William M. Marine, M.D., M.P.H.* *Department of Preventive Medicine and Biometrics, University of Colorado Health Sciences Center, Denver, Colorado, USA; Colorado Department of Public Health and Environment, Denver, Colorado, USA References 1. Hansman D, Bullen MM. A resistant pneumococcus. Lancet 1967;1:264-5. 2. Appelbaum PC. Antimicrobial resistance in Strepto- coccus pneumoniae: an overview. Clin Infect Dis 1992;15:77-83. 3. Hofmann J, Cetron MS, Farley MM, Baughman WS, Facklam RR, Elliott JA, et al. The prevalence of drug- resistant Streptococcus pneumoniae in Atlanta. N Engl J Med 1995; 333:481-6. 4. Williams WW, Hickson MA, Kane MA, Kendal AP, Spika JS, Hinman AR. Immunization policies and vaccine coverage among adults. Ann Intern Med 1988;108:616-25. 5.

MS, Farley MM, Baughman WS, Facklam RR, Elliott JA, et al. The prevalence of drug- resistant Streptococcus pneumoniae in Atlanta. N Engl J Med 1995; 333:481-6. 4. Williams WW, Hickson MA, Kane MA, Kendal AP, Spika JS, Hinman AR. Immunization policies and vaccine coverage among adults. Ann Intern Med 1988;108:616-25. 5. Butler JC, Breiman RF, Campbell JF, Lipman HB, Broome CV, Facklam RR. Pneumococcal polysaccha- ride vaccine efficacy: an evaluation of current recom- mendations. JAMA 1993;270:1826-31. 6. Ward J. Antibiotic-resistant Streptococcus pneumo- niae:clinical and epidemiologic aspects.Rev Infect Dis 1981;3:254-66. 7. Radetsky MS, Istre GR, Johansen TL, Parmelee SW, Lauer BA, Wiesenthal AM, et al. Multiply resistant pneumococcus causing meningitis: its epidemiology within a day-care centre. Lancet 1981;2:771-3. 8. Lauer BA, Reller LB. Serotypes and penicillin suscep- tibility of pneumococci isolated from blood. J Clin Microbiol 1980;11:242-4. 9. Istre GR, Humphreys JT, Albrecht KD, Thornsberry C, Swenson JM, Hopkins RS. Chloramphenicol and penicillin resistance in pneumococci isolated from blood and cerebrospinal fluid: a prevalence study in metropolitan Denver. J Clin Microbiol 1983;17:472-5. 10. Colorado Vital Statistics, 1990. Health Statistics Sec- tion, Health Statistics and Vital Records Division. Colorado Department of Health, 1990. 11. Spika JS, Facklam RR, Plikaytis BD, Oxtoby MJ, Pneumococcal Surveillance Working Group. Antimi- crobial resistance of Streptococcus pneumoniae in the United States,1979-1987.J Infect Dis 1991;163:1273- 8. 12. Centers for Disease Control and Prevention. Drug-re- sistant Streptococcus pneumoniae--Kentucky and Tennessee, 1993. MMWR 1994;43:23-5,31. Dispatches Vol. 2, No. 2 --April-June 1996 123 Emerging Infectious Diseases 13. Hieber JP, Nelson JD. A pharmacologic evaluation of penicillin in children with purulent meningitis. N Engl J Med 1977;297:410-3. 14. Centers for Disease Control and Prevention. Preva- lence of penicillin-resistant Streptococcus pneumo- niae--Connecticut, 1992-1993. MMWR 1994; 43:216-7,223. 15. Reichler MR, Allphin AA, Breiman RF, Schreiber JR, Arnold JE,McDougal LK,et al.The spread of multiply resistant Streptococcus pneumoniae at a day care center in Ohio. J Infect Dis 1992;166:1346-53. 16. Committee on Infectious Diseases, American Acad- emy of Pediatrics. Antimicrobial prophylaxis. In: Hall PG, Lepow ML, Phillips CF, editors.

AA, Breiman RF, Schreiber JR, Arnold JE,McDougal LK,et al.The spread of multiply resistant Streptococcus pneumoniae at a day care center in Ohio. J Infect Dis 1992;166:1346-53. 16. Committee on Infectious Diseases, American Acad- emy of Pediatrics. Antimicrobial prophylaxis. In: Hall PG, Lepow ML, Phillips CF, editors. Report of the Committee on Infectious Diseases.21st ed.Oak Grove Village, IL: American Academy of Pediatrics, 1988:465-8. 17. Nava JM, Bella F, Garau J, Lite J, Morera MA, Marti C, et al. Predictive factors for invasive disease due to penicillin-resistant Streptococcus pneumoniae: a population-based study. Clin Infect Dis 1994;19:884- 90. 18. Kunin CM. Problems in antibiotic usage. In: Mandel GL, Douglas RG Jr, Bennett JE, editors. Principles and practice in infectious diseases. 3rd ed. New York: Churchill Livingstone;1990:427-34. 19. Holmberg SD, Solomon SL, Blake PA. Health and economic impacts of antimicrobial resistance. Rev In- fect Dis 1987;9:1065-78. Dispatches Emerging Infectious Diseases 124 Vol. 2, No. 2 --April-June 1996

Nosocomial Transmission of Multidrug-Resistant Mycobacterium tuberculosis in Spain Before 1990, outbreaks of multidrug-resistant tuberculosis (MDRTB) were uncommon (1); since then, more than 10 outbreaks have been reported, all in hospitals and prisons in the eastern United States (2-7). Persons traditionally considered at risk for MDRTB (foreign-born TB patients and those inadequately treated for TB) have not been associated with these outbreaks.Instead,the pres- ence of patients with active TB near immunocom- promised patients in HIV-dedicated wards has led to MDRTB-infected HIV patients whose TB cases often go unrecognized. The patients receive inade- quate treatment in facilities without effective pro- cedures for isolating acid-fast bacilli; these circumstances favor nosocomial transmission. Health officials in other geographic areas where HIV and TB are major public health threats have been alerted to this emerging problem, and sur- veillance systems have been designed (8). Spain has the highest reported incidence rate of AIDS in Europe (143.4 cases per million in 1994) (9). Although in Spain TB is not notifiable at the national level, reported rates in the autonomous community of Madrid for 1994 were among the highest in Europe (33.5/100,000) (10). During a 45-month period starting in September 1991, a number of patients and one health care worker became infected with MDRTB in a 120-bed, infec- tious disease reference hospital in urban Madrid. In May 1995, the Field Epidemiology Training Program of the Spanish Ministry of Health was invited to assist the Madrid Department of Health and hospital officials in investigating the out- break. This report describes the findings of the epidemiologic and molecular laboratory investiga- tion and analyzes risk factors associated with the outbreak. The study was designed in three parts: 1) a description of the reported MDRTB cases, including a laboratory investigation of isolates; 2) a case-control study comparing HIV-infected patients who also had MDRTB to HIV-infected patients who did not have MDRTB; and 3) a study of tuberculin conversion among hospital employ- ees.

udy was designed in three parts: 1) a description of the reported MDRTB cases, including a laboratory investigation of isolates; 2) a case-control study comparing HIV-infected patients who also had MDRTB to HIV-infected patients who did not have MDRTB; and 3) a study of tuberculin conversion among hospital employ- ees. We reviewed the medical records and labora- tory specimen testing results of a series of patients with MDRTB in the HIV-dedicated ward of the hospital for January 1991 through June 1995. Cases were defined as patients with culture-con- firmed TB and drug resistance to at least rifampin and isoniazid and with no previous history of inadequate treatment. Demographic and clinical variables were collected to characterize the cluster. Drug susceptibility testing and DNA subtyping analysis were performed with resistant strains available at the time of the study. Drug suscepti- bility testing was performed by the method of proportions in Middlebrook 7H11 medium distrib- uted in petri plate compartments (reference). All 48 reported cases of isoniazid and rifampin resistance were among HIV-infected patients hos- pitalized in the HIV-dedicated ward from Septem- ber 15, 1991, to May 1995. One patient was an HIV-infected nurse who worked on the ward from 1990 to 1994. The mean age of patients was 34.1; 81.3% were male, and 66.6% were intravenous- drug users (Table 1). Of the 47 (97.9%) who died, the mean interval from diagnosis to death was 77.6 days. The epidemiologic curve suggests a propagated transmission pattern of MDRTB among HIV ward patients, starting in 1991 and continuing until June 1995 (Figure 1).By the first 6 months of 1995, 65% of Mycobacterium tuberculosis strains seen among HIV ward patients were multidrug-resis- tant. Table 1. MDRTB patient characteristics Number Variable (n = 48) % Sex Male 39 81.3 Female 9 18.7 Mean age 34.1 (6.8)a HIV status Infected 48 100.0 HIV risk group Intravenous drug users 32 66.6 Homosexual 10 20.8 Others 6 12.6 Outcome Death 47 95.9 Discharge 1 4.1 Mean survival after 77.6 (107.8)a MDRTB diagnosis (in days) a Standard deviation. Dispatches Vol. 2, No. 2 --April-June 1996 125 Emerging Infectious Diseases At the beginning of the outbreak, no consistent antibiogram pattern was observed among isolates.

exual 10 20.8 Others 6 12.6 Outcome Death 47 95.9 Discharge 1 4.1 Mean survival after 77.6 (107.8)a MDRTB diagnosis (in days) a Standard deviation. Dispatches Vol. 2, No. 2 --April-June 1996 125 Emerging Infectious Diseases At the beginning of the outbreak, no consistent antibiogram pattern was observed among isolates. However, beginning in 1993, strains were consis- tently resistant to isoniazid, streptomycin, etham- butol, and rifampin (HSER); of the 26 patients with an MDRTB diagnosis since the last trimester of 1993, 24 (92.3%) had HSER isolates. DNA sub- typing analysis was performed on 12 of these HSER isolates that were available. Eleven of the 12 strains had the same band patterns (Figure 2). Two additional isolates with a different antibi- ogram pattern had different banding patterns. Subtyping of M. tuberculosis strains was per- formed (11) by analyzing DNA located between two copies of repetitive sequence IS6110. A 10-l sample of the extracted DNA was amplified in a reaction mixture containing 0.5 pM of each of the four primers (Ris1, Ris2, Pntb1, and Pntb2), 200 UM DNTPs, 50 mM Tris-HCL, 50 mM KCL (pH 8.8), 2.5 mM MgC12, 0.1% Triton x-100, and 0.5% Taq polymerase. The samples were denatured by incubation at 95o C for 10 min and amplified by 30 cycles of denaturation at 94o C for 1 min, primer alignment at 56o C for 2 min,and primer extension at 72o C for 1 min.The amplification products were analyzed by electrophoresis in 2% agarose gel stained with ethidium bromide and observed with an ultraviolet transilluminator. HIV-infected patients hospitalized on the HIV- dedicated ward between September 15, 1991, and December 31,1994,who had TB diagnosed in 1994 with a known drug-susceptibility pattern were included in a case-control study. Case patients' isolates were resistant to isoniazid, rifampin, streptomycin, and ethambutol; control patients' isolates were sensitive to the same antimicrobial drugs. A time-person line diagram was prepared for each of the case patients and control patients, including all hospitalization dates, ward and room number, potentially infectious days, and possible days exposed to infective patients.Prior admission was defined as all admissions to the HIV-dedicated ward since the MDRTB patient was diagnosed (9/15/91) until the end of the period (12/31/94). TB patients were classified as "potentially infective" from the 2 week-period before sputum or culture results were positive for M.

xposed to infective patients.Prior admission was defined as all admissions to the HIV-dedicated ward since the MDRTB patient was diagnosed (9/15/91) until the end of the period (12/31/94). TB patients were classified as "potentially infective" from the 2 week-period before sputum or culture results were positive for M. tuberculosis until the time sputum results were negative or the patient died. TB-negative patients were classified as "pos- sibly exposed" if they were hospitalized on the HIV-dedicated ward during the time a potentially infective patient was also present, beginning on September 15, 1991, until 2 weeks before TB diag- nosis. Thirty-five patients (18 with cases and 17 con- trols) met the established study case/control defi- nition.Case patients and control patients were not significantly different with respect to age,sex,HIV risk factors, interval between HIV and TB diagno- ses or count of CD4 lymphocytes at the time of TB diagnosis (Table 2). Before the hospitalization during which TB was diagnosed, 76.4% of the case-patients had been hospitalized on the HIV-dedicated ward of the hospital versus 23.5% of control patients (OR = 7.8 [1.4,50.5]) (Table 3). Of the five case patients with no prior hospitalization, three were family mem- bers of previously hospitalized HIV-infected pa- tients who had visited the HIV ward frequently during the outbreak period. 0 1991 Year Number of cases 1992 1993 1994 1995* Sensitive MDRTB Resistant to one drug 10 20 30 40 50 60 on Figure 1. Hospital outbreak, Madrid, Spain, 1995. *1995 data are for first 6 months only. *All strains of resistance pattern HSER (isoniazid, streptomycin, ethambutol, rifampin) had similar DNA subtyping. Figure 2. Evolution of MDRTB strain* over time, hospital outbreak, Madrid, Spain, 1995. MDRTB diagnosis date Hospitalization dates on HIV ward Dispatches Emerging Infectious Diseases 126 Vol. 2, No. 2 --April-June 1996 Case patients and controls were compared with respect to possible exposure to a potentially infec- tive wardmate beginning on 9/15/91 until 2 weeks prior to the diagnosis of TB. Case patients were more likely to have been exposed to potentially infective wardmates (72.2% with a median of 26.4 days) than control patients (41.2% with a median of 7.6 days). When we stratified possible ward exposure by days (0 days vs. 1 to 30 days vs. 30 days), we observed a dose-response effect, and the chi-square for linear trend was statistically sig- nificant (Table 3).

ally infective wardmates (72.2% with a median of 26.4 days) than control patients (41.2% with a median of 7.6 days). When we stratified possible ward exposure by days (0 days vs. 1 to 30 days vs. 30 days), we observed a dose-response effect, and the chi-square for linear trend was statistically sig- nificant (Table 3). Case patients were more likely to die during their initial hospitalization for MDRTB(94.4%) than were control patients (29.4%) during their hospitalization for TB (OR = 40.8 [3.6,1842]) (Table 3). A TB screening clinic visit was offered to all hospital employees after the outbreak was identi- fied. Of the 591 active employees, 565 (95.6%) participated. Of these, 288 (51%) had not partici- pated in previous hospital employee screening programsconductedin1990and1994.Theoverall prevalence of TB infection among participating employees was 450 (80%) of 565;only 115 (20%) of the current employees tested were tuberculin- negative. Employees currently working at the hospital, who had a documented negative (< 6 mm) tuber- culin test between January 1993 and June 1995 were eligible for this skin test conversion study. Many of the employees had received BCG vaccine. For those who had not received BCG vaccine, con- version was defined as an induration of 10 mm or greater with a change of at least 6 mm of indura- tion since the last negative tuberculin test (12).For BCG vaccinees, conversion was defined as a 15- mm induration change since the last negative (< 6 mm) skin test. Employees were defined as occupationally ex- posed if they worked in parts of the hospital where exposure to patients or M. tuberculosis was likely (the HIV-dedicated ward, HIV outpatient clinic, radiology unit, the mycobacteriology laboratory, and the internal medicine ward). Employees were asked to quantify the cumulative number of months spent in these high-risk areas, regardless of their usual place of work, during the 30-month study period. According to the Mantoux tech- nique, 2 tuberculin units of purified protein derivative (PPD) tuberculin RT-23 were administered in the ante- rior forearm of the screened employ- ees, and the results were read within 48 to 72 h. Of the participants, 92 (16.3%) were eligible for the conver- sion study. The incidence of conver- sion during the 30-month period was 24 of 92 (26%).

purified protein derivative (PPD) tuberculin RT-23 were administered in the ante- rior forearm of the screened employ- ees, and the results were read within 48 to 72 h. Of the participants, 92 (16.3%) were eligible for the conver- sion study. The incidence of conver- sion during the 30-month period was 24 of 92 (26%). Employees who had occupational exposure to high-risk areas had higher conversion rates than employees who did not have oc- cupational exposure to high-risk ar- eas (RR = 5.0 [2.7,9.6]) (Table 4). The conversion had a dose-response ef- fect, that is, the more months the per- son is occupationally exposed to high risk-areas,the higher the risk for con- version. This is the first nosocomially trans- mitted MDRTB outbreak reported in Table 2. Population characteristics: case control study Case patients (%) Control patients (%) Variable n = 18 n = 17 OR (95% CI)a Mean age (yr) 34.4 35.2 p = 0.30b Male sex 13 (72.2) 14 (82.3) 1.8 (0.3-12.1) IVDU 11 (61.1) 11 (64.7) 1.2 (0.2-6.7) Interval HIV to TB 1403 1293 p = 0.79b diagnosis in days CD4 lymphocyte 112.9 199.5 p = 0.30b count, median a OR (95% CI) = odds ratio (95% confidence intervals); Fisher's Exact Test. b p value for analysis of variance (ANOVA). IVDU = intravenous drug user. Table 3. Variables associated with MDRTB Case patients (%) Control patients (%) Variable n = 18 n = 17 OR (95% CI)a Prior admission to HIV ward Yes 13 (76.4) 5 (29.4) 6.2 (1.2-37.2) No 5 (23.6) 12 (70.6) Possible ward exposure days None 5 (27.8) 10 (58.8) 1.0 1-30 6 (33.3) 5 (29.4) 2.4 30 7 (38.9) 2 (11.8) 7.0, p = 0.03b Outcome Death 17 (94.4) 5 (29.4) 40.8 (3.6-1842) Discharge 1 (5.6) 12 (70.6) a OR (95% CI) = odds ratio (95% confidence intervals); Fisher's Exact Test. b Chi-square for linear trend. Dispatches Vol. 2, No. 2 --April-June 1996 127 Emerging Infectious Diseases Spain, which, with recent outbreaks in the United Kingdom (13) and Italy, is among the first in Europe. Its characteristics are similar to the other reported outbreaks on that it occurred in an HIV- dedicated ward among non-foreign-born patients who had not been treated for TB; it had a high mortality rate within 3 months of onset during which mycobacteria laboratory surveillance recog- nized similar antibiogram resistant patterns; and identification of MDRTB isolates was followed by DNA subtyping, which confirmed that the same strain was responsible for the outbreak.

not been treated for TB; it had a high mortality rate within 3 months of onset during which mycobacteria laboratory surveillance recog- nized similar antibiogram resistant patterns; and identification of MDRTB isolates was followed by DNA subtyping, which confirmed that the same strain was responsible for the outbreak. Risk fac- tors identified include admission to the HIV-dedi- cated ward and more "possible exposure" days to potentially infective wardmates; additionally, skin test conversion rates among employees were di- rectly related to working in high risk areas of the hospital. The consistency of these findings with those reported in similar outbreaks and the fact that the same isolate was cultured over an exten- sive period among many different patients on a hospital ward without proper room isolation tech- niques support the conclusion that nosocomial transmission was the leading cause of the out- break. CDC's recommended guidelines for hospital TB prevention and control (14) were not fully imple- mented in the hospital. Acid-fast bacilli room iso- lation techniques were not in place; moreover, no ventilation system was available to provide nega- tive pressure to prevent bacilli from passing from the MDRTB patients' rooms to the hallway or to provide the six air interchanges per hour recom- mended for removing bacilli from room air. Surgi- cal masks used in the HIV-dedicated ward during the outbreak as protective masks are not recommended for this purpose be- cause of filtering and facial sealing problems. Observational visits to the HIV-dedicated ward revealed that MDRTB patients without masks were walking, talking, and smoking in the halls and in the lounge next to the unprotected pa- tients, families, and staff. Complete follow-up of employees is an essential component of a hospi- tal control program (14). Because 80% of the employees in this out- break were tuberculin-positive, chest radiographs became an impor- tant component for disease screen- ing; yearly surveillance of the tuberculin-negative employees will help determine if the interventions in place to prevent future outbreaks have worked in this setting. Once the case-control study findings were ana- lyzed, a TB control committee was established. All MDRTB patients were placed in a separate area of the hospital. Hospital staff were informed about the outbreak and alerted about future possible cases and the patient management and treatment schemes.

rked in this setting. Once the case-control study findings were ana- lyzed, a TB control committee was established. All MDRTB patients were placed in a separate area of the hospital. Hospital staff were informed about the outbreak and alerted about future possible cases and the patient management and treatment schemes. The mycobacteria laboratory expanded the antibiogram service to include all second-line antibiotics used by the hospital clinicians. Clini- cians have elaborated a treatment flow chart for MDRTB patients. Masks that fulfilled the sealage and filtering criteria (15) were purchased, and mask use was aggressively implemented. An em- ployee health clinic was also instituted, with a prophylaxis and chest X-ray follow-up program for the 410 infected employees, along with a comput- erized follow-up surveillance system which in- cluded all employees graded by occupational risk category. Plans were made to screen tuberculin- negative employees every 3 months to identify those who had recently seroconverted. Family, community members and wardmates of all pa- tients whose MDRTB had been diagnosed within the previous 6 months were notified of their risk and were scheduled for follow-up evaluations. The HIV-dedicated ward will be transformed into an acid-fast bacilli isolation zone, with an exclusive ventilation system that provides 11 individual rooms, each with 12 air interchanges per hour and negative pressure relative to the hallway. Health officials in Europe need to be updated about this emerging problem, especially in areas Table 4. Employee tuberculin screening conversion study results Convertors (%) Nonconvertors (%) Variable n = 24 n = 68 RR (95% Cl) Occupational exposure to high-risk areas Yes 14 (70.0) 6 (30.0) 5.0 (2.7, 9.6) No 10 (13.9) 62 (86.1) Months exposed in high-risk areas ORb 0 6 (10.7) 50 (89.3) 1.0 1-6 4 (28.6) 10 (71.4) 3.3 7-36 14 (63.6) 8 (36.4) 14.6, p < 0.01 a RR (95% CI) relative risk (95% confidence intervals). b Chi-square for linear trend. Dispatches Emerging Infectious Diseases 128 Vol. 2, No. 2 --April-June 1996 of high HIV and TB prevalence. Basic universal TB control and prevention measures (16,17) should be implemented by all general community hospitals in Spain, especially those with HIV-dedi- cated wards in areas where TB is prevalent. TB surveillance of health care employees is necessary to identify emerging problems as well as to protect employees, patients, and visitors. John V.

prevention measures (16,17) should be implemented by all general community hospitals in Spain, especially those with HIV-dedi- cated wards in areas where TB is prevalent. TB surveillance of health care employees is necessary to identify emerging problems as well as to protect employees, patients, and visitors. John V. Rullan M.D.,* Dionisio Herrera, M.D.,* Rosa Cano, M.D.,* Victoria Moreno, M.D., Pere Godoy, M.D.,* Enrique F. Peiro, M.D.,* Juan Castell, M.D.,* Consuelo Ibanez, M.D.,* Arturo Ortega, M.D., Leopoldo Sanchez Agudo, M.D., and Francisco Pozo, M.D. Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo: *Programa de Epidemiologia Aplicada de Campo-Centro Nacional de Epidemiologia; Centro Nacional de Investigacion Clinica y Medicina Preventiva; Subdireccion General de Salud, Madrid, Spain Drs. Herrera, Peiro, Castell, and Godoy have received a scholarship from the Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III during their 2-year epidemiologic training period in the Field Epidemiology Training Program (Programa de Epidemiologia Aplicada de Campo). Acknowledgments We thank the staff of the Center for Clinical Investiga- tion and Preventive Medicine of the Instituto de Salud Carlos III for their collaboration in this investigation; in particular, staff from the following departments: Admini- stration, Respiratory Diseases, Infectious Diseases, Nurs- ing, and the Mycobacteriology Laboratory. For the employee tuberculin study, we thank Drs. Rafael Rey, and Dura;Leopoldo Sanchez Agudo;Margarita Munoz Arcaniz; Consuelo Yubero Higuera; Concepcion Gonzalez de la Calle; Lucia Martin Gadea; and Pilar Fuertes Rodrigues for their collaboration.We acknowledge Dr. Jack Crawford and staff from the Division of Bacterial and Mycotic Dis- eases, National Center for Infectious Diseases, CDC, for their collaboration as reference laboratory for DNA sub- typing analysis; Dr. Daniel Fishbein for editing the manu- script; and Dr. Rafael Rey Duran for providing us with the 1990 and 1994 data set of the hospital employees tubercu- lin test screening. We thank Luis Dorado for his graphics collaboration. References 1. Kent JH. The epidemiology of multidrug-resistant tuberculosis in the United States. Med Clin North Am 1993;77:1391-1409. 2. Edlin BR, Tokars JI, Grieco MH, et al. An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syn- drome. N Engl J Med 1992;326:1514-21. 3.

Kent JH. The epidemiology of multidrug-resistant tuberculosis in the United States. Med Clin North Am 1993;77:1391-1409. 2. Edlin BR, Tokars JI, Grieco MH, et al. An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syn- drome. N Engl J Med 1992;326:1514-21. 3. Beck-Sague C, Dooley SW, Hutton MD, et al. Hospital outbreak of multidrug-resistant Mycobacterium tu- berculosis infections: factors in transmission to staff and HIV-infected patients. JAMA 1992;268:1280-6. 4. Pearson ML, Jereb JA, Frieden TR, et al. Nosocomial transmission of multidrug-resistant Mycobacterium tuberculosis: a risk to patients and health care work- ers. Ann Intern Med 1992;117:191-6. 5. Fischl MA, Uttamchandani RB, Daikos GL, et al. An outbreak of tuberculosis caused by multiple-drug re- sistant tubercle bacilli among patients with HIV in- fection. Ann Intern Med 1992;117:177-83. 6. Dooley SW, Villarino ME, Lawrence M, et al. Noso- comial transmission of tuberculosis in a hospital unit for HlV-infected patients. JAMA 1992;267:2632-4. 7. Centers for Disease Control.Nosocomial transmission of multidrug-resistant tuberculosis among HIV-in- fected persons--Florida and New York, 1988-1991. MMWR 1991;40:585-91. 8. Ausina V, Riutort N, Vinado B,et al.Prospective study of drug-resistant tuberculosis in a Spanish urban population including patients at risk for HIV infec- tion. Eur J Microbiol Infect Dis 1995;14:105-10. 9. WHO-EC Collaborating Centres on AIDS. AIDS Sur- veillance in Europe-European Centre for the epidemiological monitoring of AIDS.Quarterly Report #45; March 1995. 10. Boletin Epidemiologico de la Comunidad de Madrid 1995. Mayo #5, Vol 4. Informe: Morbilidad por Enfer- medades de Declaracion Obligatoria, 1994. 11. Friedman CR, Stoecle MY, Johnson WD, Riley LW. Double-repetitive-element PCR method for subtyping M. tuberculosis clinical isolates. J Clin Microbiol 1995;33:1383-4. 12. Grupo de Trabajo sobre Tuberculosis. Consenso na- cional para el control de la tuberculosis en Espana. Med Clin (Barc) 1992;98:24-31. 13. Communicable Disease Report. Outbreak of hospital acquired multidrug resistant tuberculosis. United Kingdom PHLS Cornmunicable Disease Surveillance Centre Weekly 1995. 14. Centers for Disease Control and Prevention. Guide- lines for preventing the transmission of Mycobac- terium tuberculosis in health-care facilities, 1994. MMWR 1994;43 (No. RR-13). 15. Lilienfeld, AM, Lilienfeld DE. Foundations of epidemiology. 2nd ed.

gdom PHLS Cornmunicable Disease Surveillance Centre Weekly 1995. 14. Centers for Disease Control and Prevention. Guide- lines for preventing the transmission of Mycobac- terium tuberculosis in health-care facilities, 1994. MMWR 1994;43 (No. RR-13). 15. Lilienfeld, AM, Lilienfeld DE. Foundations of epidemiology. 2nd ed. Oxford, UK: Oxford University Press, 1980;292-5. 16. Maloney SA, Pearson ML, Gordon MT, et al. Efficacy of control measures in preventing nosocomial trans- mission of multidrug-resistant tuberculosis to pa- tients and health care workers. Ann Intern Med 1995;122:90-5. 17. Wenger PN, Otten J, Breeden A, et al. Control of nosocomial transmission of multidrugresistant Myco- bacterium tuberculosis among healthcare workers and HIV-infected patients. Lancet 1995;345:235-40. Dispatches Vol. 2, No. 2 --April-June 1996 129 Emerging Infectious Diseases

Application of Pulsed-Field Gel Electrophoresis to an International Outbreak of Salmonella agona Between 1 December 1994 and 31 January 1995, Salmonella agona infections increased abruptly in England and Wales; isolates of S. agona from 41 patients with diarrheal illness were referred to the Laboratory of Enteric Pathogens of the Public Health Laboratory Service, compared with nine cases in the previous 12 months. Most isolates were from children under 10 years of age. Many of the cases were in Jewish children in London and several other parts of England; two children required hospital admission.S.agona can be subdivided by phage typing and, by using a method developed in the Laboratory of Enteric Pathogens (L. R. Ward, unpublished manuscript), we found that the isolates belonged to S. agona phage type (PT) 15. The outbreak was traced to a kosher savory snack imported into the United Kingdom from Israel, and all isolates from the contaminated product and from patients who had a history of consuming this product were found to belong to S. agona PT 15 (1). (Full details of this outbreak will be published elsewhere.) After notification of the outbreak in the United Kingdom, health authorities in countries where the contaminated snack had also been distributed were warned of the possibility of an upsurge in S. agona infections; these countries included Israel, the United States, and Canada. Countries in the European Union (EU) were also notified through SALM-NET, the European salmonella surveil- lance network (2). As a result of such notification, isolates of S. agona subsequently identified as PT 15 were received from patients in Israel, the United States, and France and from food samples in Israel, the United States, and Canada. For outbreak investigations, the policy of the Laboratory of Enteric Pathogens is to use serotyp- ing, phage typing, and antibiogram analysis (R- typing) for the primary identification and subdivision of isolates,supplemented when appro- priate by a range of DNA-based methods, includ- ing plasmid analysis, ribotyping, insertion sequence (IS) 200 fingerprinting, and pulsed-field gel electrophoresis (PFGE).

serotyp- ing, phage typing, and antibiogram analysis (R- typing) for the primary identification and subdivision of isolates,supplemented when appro- priate by a range of DNA-based methods, includ- ing plasmid analysis, ribotyping, insertion sequence (IS) 200 fingerprinting, and pulsed-field gel electrophoresis (PFGE). The use of these tech- niques has recently been reviewed (3). Of these methods, PFGE has been used for the molecular fingerprinting of S. typhi (4, 5) and for subdivision within epidemic phage types of S. enteritidis (6, 7). Because a small number of patients in England and Wales had been infected with S. agona PT 15 in the 11 months before the kosher snack out- break, we decided to characterize the outbreak isolates by genotypic methods and, if possible, to use such methods for subdivision within the phage type. For S. agona, strains of this serotype do not possess IS200 elements (8), and studies by the Laboratory of Enteric Pathogens have demon- strated that the serotype is unlikely to be subdi- vided by ribotyping (M.D.Hampton,E.J.Threlfall, unpublished observations). PFGE was, therefore, considered the method most likely to provide a genotypic fingerprint suitable for epidemiologic investigations. Isolates from the food product and from all patients infected bothduring the outbreak and in the preceding 11 months were, therefore, examined by PFGE. Similarly, because of the in- ternational distribution of the contaminated food product, S. agona PT 15 organisms isolated in Israel and Canada from the food product and also from specimens from patients in Israel,the United States, and France were examined by PFGE. Analysis by PFGE of the fragments resulting from Xba I digestion of genomic DNA from 78 isolates of S. agona PT 15 made in the United Kingdom, Israel, the United States, Canada, and France between December 1993 and April 1995 showed 11 distinct pulsed-field profiles (PFP) and one variant profile, with 14 to 17 resolvable chro- mosomal fragments, ranging from approximately 25 kb to 680 kb (Figure). These profile types have been designated S. agona PFP (XbaI) 1 through to S. agona PFP (XbaI) 9, and S. agona PFPs (XbaI) 11 and 12; the variant type has been designated S. agona PFP (XbaI) 6a. The pattern designated S. agona PFP (XbaI) 10 had been assigned to an isolate of S. agona of an unrelated phage type, which has been used as a control in PFGE analysis of this serotype. The predominant PFGE profile, S.

) 9, and S. agona PFPs (XbaI) 11 and 12; the variant type has been designated S. agona PFP (XbaI) 6a. The pattern designated S. agona PFP (XbaI) 10 had been assigned to an isolate of S. agona of an unrelated phage type, which has been used as a control in PFGE analysis of this serotype. The predominant PFGE profile, S. agona PFP (XbaI) 6, gave at least 15 resolvable fragments, ranging from 25 kbp to 485 kbp; this profile type was exhibited by 51 of the 78 isolates examined. The PFP 6 variant, designated PFP 6a and iden- tified in one of the Canadian food isolates,could be differentiated from PFP 6 by the presence of an additional fragment of approximately 395 kbp; in Dispatches Emerging Infectious Diseases 130 Vol. 2, No. 2 --April-June 1996 all other respects, S. agona PFP 6a was indistin- guishable from S. agona PFP 6. The distribution ofXbaI-generated PFGE types between source group and country of isolation in isolates of S. agona PT 15 is shown in the Table. Of the 10 PFGE profiles in 46 isolates from per- sons in the United Kingdom, 26 isolates (56.5%) belonged to PFP 6. All isolates with this profile pattern were made between 25 November 1994 and 17 February 1995 and were from patients associated with the kosher snack-related out- break. Two isolates of S. agona PT 15 from the contaminated food product were also examined and both belonged to PFP 6.In contrast,PFP 6 was not identified in any isolates of S. agona PT 15 made in the United Kingdom in the 11 months before the outbreak. In one case, S. agona PT 15 with the PFGE profile PFP 9 was isolated from a patient just after the outbreak began, in Novem- ber 1994. However, the PFGE profile of this isolate was sufficiently unrelated to that of the outbreak isolates (PFP 6) to warrant exclusion of the patient from the kosher snack-related cases. Nine PFGE profiles were identified in the remaining 19 U.K. isolates, with PFPs 5, 2, and 1 predominating (Table). Four of five patients infected with isolates with the PFP 2 pattern lived in northwestern England, and three of five patients with the PFP 5 profile lived in southwestern England or south- ern Wales. None of these nine PFGE profiles were identified in isolates from case-patients associated with the kosher snack outbreak. Nine isolates of S.

infected with isolates with the PFP 2 pattern lived in northwestern England, and three of five patients with the PFP 5 profile lived in southwestern England or south- ern Wales. None of these nine PFGE profiles were identified in isolates from case-patients associated with the kosher snack outbreak. Nine isolates of S. agona PT 15 from patients and two isolates from the contaminated food prod- uct that had been made in Israel were also exam- ined by PFGE and, without exception, all strains belonged to PFP 6.Likewise,all of 10 isolates from outbreak-associated cases in the United States belonged to PFP 6 as did two of three strains isolated in Canada from the contaminated food product; the PFP of the remaining Canadian strain, PFP 6a, was closely related to PFP 6.Three PFPs were identified in the six isolates of S. agona PT 15 from France, of which two (PFP 2 and PFP 5) had been observed in isolates of PT 15 made in the United Kingdom before the outbreak. How- ever, none of the isolates of S. agona PT 15 made in France were of PFP 6 (Table). In conclusion, 11 distinct PFGE profile types and one variant type have been identified in 78 isolates of S. agona PT 15 made in the United Kingdom, Israel, the United States, Canada, and France between December 1993 and April 1995. One PFGE profile type, PFP 6, was specifically associated with isolates from a contaminated sa- vory snack and from persons who consumed this product in the United Kingdom, Israel, and the United States.These results demonstrate that one strain of S. agona PT 15, with a characteristic PFGE profile,was responsible for outbreaks in the United Kingdom, Israel, and the United States in 1994-95. In contrast, the PFGE profile types of isolates of S. agona PT 15 identified in the United Kingdom either in the 11 months before the out- break (or in one case at the start of the outbreak) and of isolates of PT 15 obtained from patients in France were unrelated to PFP 6. We conclude that PFGE fingerprinting provides a genotypic method for subdivision within phage type 15 of S. agona and that this method has provided an invaluable supplement to phage typing for investigation of international outbreaks. It is, however, important to realize that PFGE typing may not be applicable to all salmonella serotypes and phage types; thus, the method's results should be carefully evaluated by using both epidemiologically-related and unrelated isolates before it is used for outbreak investigations. Figure.

international outbreaks. It is, however, important to realize that PFGE typing may not be applicable to all salmonella serotypes and phage types; thus, the method's results should be carefully evaluated by using both epidemiologically-related and unrelated isolates before it is used for outbreak investigations. Figure. PFGE profiles of XbaI-digested genomic DNA from strains of S. agona PT 15. Legend: Tracks 1 - 14 contained: 1 and 14, lambda 48.5-kb ladder (Sigma); 2, S. agona PFP (XbaI) 6; 3, PFP 6a; 4, PFP 4; 5, PFP 10 (= control PFP type for S. agona); 6, PFP 9; 7, PFP 7; 8, PFP 3; 9, PFP 2; 10, PFP 5; 11, PFP 1; 11, PFP 8; 13, PFP 9. Gels were run at 6.0 V cm-1 for 36 h with a 25- to 75-s pulse ramp time. Dispatches Vol. 2, No. 2 --April-June 1996 131 Emerging Infectious Diseases E. John Threlfall, Michael D. Hampton, Linda R. Ward, and Bernard Rowe Laboratory of Enteric Pathogens, Central Public Health Laboratory, London, United Kingdom Acknowledgments We are grateful to the following microbiologists for sending strains of S. agona for typing: Dr. N. Andorn, Ministry of Health, Government Central Laboratories, Jerusalem, Israel; Dr. Mehdi Shayedgani, New York State Department of Health, New York, NY, USA; Dr. Rasik Khakhria, National Laboratory of Enteric Pathogens, Laboratory Centres for Disease Control, Ottawa, Canada; and Professor Patrick Grimont, Unites des Enterobacter- ies, Institut Pasteur, Paris, France. References 1. An outbreak of Salmonella agona due to contami- nated snacks. CDR Weekly 1995; 5: 29-32. 2. Fisher IST, Rowe B, Bartlett CLR, Gill ON. "Salm- Net"--laboratory-based surveillance of human salmo- nella infections in Europe. PHLS Microbiol Digest 1994; 11: 181-2. 3. Threlfall EJ, Powell NG, Rowe B. Differentiation of salmonellas by molecular methods. PHLS Microbiol Digest 1994; 11: 199-202. 4. Thong KL, Cheong YM, Puthucheary S, Koh CL, Pang T. Epidemiologic analysis of sporadic Salmonella ty- phi isolates and those from outbreaks by pulsed-field electrophoresis. J Clin Microbiol 1994; 32: 1135-41. 5. Nair S, Poh CL, Lim YS, Tay L, Yoh KT. Genome fingerprinting of Salmonella typhi by pulsed-field gel electrophoresis for subtyping common phage types. Epidemiol Infect 1994; 113: 391-402. 6. Powell NG,Threlfall EJ,Chart H,Rowe B.Subdivision of Salmonella enteritidis PT 4 by pulsed-field gel electrophoresis: potential for epidemiological surveil- lance. FEMS Microbiol Lett 1994; 119: 193-98. 7. Powell NG, Threlfall EJ, Chart H, Schofield SL, Rowe, B.

mmon phage types. Epidemiol Infect 1994; 113: 391-402. 6. Powell NG,Threlfall EJ,Chart H,Rowe B.Subdivision of Salmonella enteritidis PT 4 by pulsed-field gel electrophoresis: potential for epidemiological surveil- lance. FEMS Microbiol Lett 1994; 119: 193-98. 7. Powell NG, Threlfall EJ, Chart H, Schofield SL, Rowe, B. Correlation of change in phage type with pulsed- field profile and 16S rrn profile in Salmonella enteri- tidis phage types 4, 7 and 9a. Epidemiol Infect 1995; 114: 403-11. 8. Gibert I, Barbe J, Casadesus J. Distribution of inser- tion sequence IS200 in Salmonella and Shigella. J Gen Microbiol 1995; 136: 2555-60. Table. Distribution of Xbal pulsed-field profiles in isolates of Salmonella agona phage type 15 made in the United Kingdom, Israel, USA, Canada, and France, 1993-1995a Number Pulsed-field profiles (PFP) Country Source studied 1 2 3 4 5 6 6a 7 8 9 10 11 12 United Kingdom Human 46 3 5 1 2 5 26b 1 1 1 1 Snack 2 2 Israel Human 9 9 Snack 2 2 United States Human 10 10 Canada Snack 3 2 1 France Human 6 1 4 1 Total 78 3 6 1 2 9 51 1 1 1 1 0 1 1 a Isolates from Israel, the United States, and Canada were made in 1995; of the isolates from France, 3 were obtained in 1994 and 3 in 1995. b Isolated 25 November 1994 to 17 February 1995. Dispatches Emerging Infectious Diseases 132 Vol. 2, No. 2 --April-June 1996

Potential Risk for Dengue Hemorrhagic Fever: The Isolation of Serotype Dengue-3 in Mexico The Americas have a long history of dengue epidemics, which present public health problems because of the potential emergence of dengue hem- orrhagic fever (DHF) (1). Efforts to control Aedes aegypti--the only demonstrated vector of dengue virus in the Americas--were effectively deployed in the 1950s and 1960s when the Pan American Health Organization launched a continental eradication campaign against yellow fever (2). Aedes aegypti was eliminated in Mexico in 1963 (3). However, subsequent social and economic changes in the Americas have permitted the rapid reinfestation of the vector throughout the region. In Mexico, population movement from rural areas to urban centers--brought about by intensive in- dustrialization--were not matched with adequate housing and sufficient water, sewage, and waste management systems. The introduction and pro- liferation of nonrecyclable products provided nu- merous and effective breeding sites for urban mosquitoes. For example, from 1960 to 1990, the annual production of bottles in Mexico increased from 500,000 to 3.5 million, and the annual pro- duction of tires increased from 2 to 17 million (4). Tourism and travel, promoted as essential to the national economy, have also become important mechanisms for transporting dengue viruses. Ad- ditionally, surveillance, prevention, and control programs lack the infrastructure and human re- sources needed to tackle this neglected health problem (1,4).Millions of people living in the tropi- cal and subtropical areas of the region face the reemergence of dengue and DHF (2). In Mexico from 1984 to 1993, DHF cases were sporadically reported; only 26 cases were identi- fied, followed by 30 cases in 1994 (4). During 1995, however, the General Directorate of Epidemiology of the Ministry of Health in Mexico confirmed 358 DHF cases in 18 states with a case-fatality rate of 7.8% (unpublished data). The widespread distri- bution of DHF cases and of the vector and the circulation of different serotypes demonstrate the risk of serious illness throughout the country.

rectorate of Epidemiology of the Ministry of Health in Mexico confirmed 358 DHF cases in 18 states with a case-fatality rate of 7.8% (unpublished data). The widespread distri- bution of DHF cases and of the vector and the circulation of different serotypes demonstrate the risk of serious illness throughout the country. Dengue fever in endemic-disease areas is often not diagnosed properly because of its nonspecific clinical manifestations. Furthermore, only pa- tients with symptoms are treated, and patients rarely demand medical care; thus, the proportion of infected persons in the population is usually underestimated (5). On the other hand, DHF is an acute, life-threatening disease that requires spe- cialized treatment in a medical setting.Identifying dengue serotypes in the continent is one of the most serious problems faced by every surveillance system in the region. The serotype, strain, and sequence of infections by different serotypes are among the most meaningful risk factors for DHF; thus, creating a strong dengue virus surveillance system in every country in the Americas should be a high priority (6, 7). Serologic evidence of dengue in the Americas can be traced back to 1941 in Panama (8). DEN-2 was isolated in Trinidad in 1953 (9). DEN-3 was isolated in the Caribbean and Venezuela in 1963 (2,10), DEN-1 was introduced to the Americas in 1977, and DEN-4 affected the region 4 years later. In 1981, Cuba had a major DHF epidemic caused by a new strain of DEN-2 (11).DEN-3 was detected in Nicaragua and Panama in 1994 and in Costa Rica in 1995 (12), after a long absence from the region; a strain similar to one in Sri Lanka and India in the 1980s caused the DHF epidemics in those countries (12). The identification of DEN-3 in the region increases the probability of DHF cases associated with a newly circulating serotype. In Mexico, this particular situation may have im- portant epidemiologic consequences for several reasons: 1) DEN-3 has not been identified in the country, and the population is totally susceptible to infection by this serotype; 2) infection by DEN-3 would most likely be of the secondary type; 3) population movements through Mexico and to- wards other countries,might disseminate this new serotype to areas where susceptible persons will be exposed to a new serotype; and 4) intensive transmission of dengue would naturally increase the risk for DHF epidemics.

by DEN-3 would most likely be of the secondary type; 3) population movements through Mexico and to- wards other countries,might disseminate this new serotype to areas where susceptible persons will be exposed to a new serotype; and 4) intensive transmission of dengue would naturally increase the risk for DHF epidemics. Surveillance of dengue virus in Mexico began in 1982 when seven isolates of DEN-1 and DEN-2 were identified from outbreaks reported in the south and southeastern regions of the country. From 1982 to 1995, the National Institute of Epidemiological Diagnosis and Reference (INDRE) identified 681 dengue virus isolates.Serotypes were identified by indirect immunofluorescence with spe- cificmonoclonalantibodies donated by the Division Dispatches Vol. 2, No. 2 --April-June 1996 133 Emerging Infectious Diseases of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, Colorado. DEN-1 was the serotype most frequently iso- lated from 1982 to 1995 (47% of all isolates), fol- lowed by DEN-4 (30%) and DEN-2 (21%) (Table). In 1995, DEN-3 was identified in 19 patients with classic dengue fever with no hemorrhagic manifes- tations (Table). Beginning in 1995, active surveil- lance for dengue cases was begun in areas where transmission had been documented. Sentinel sur- veillance centers were implemented to obtain se- rum samples from febrile patients with a clinical picture suggestive of dengue and to isolate and identify the serotype involved. From August to December 1995, 245 isolates of dengue virus were obtained, which represented 36% of isolates ob- tained during the 14-year period. The prevalence of serotypes isolated in 1995 differed from those isolated from 1982 to 1994; DEN-1 was isolated in only 16% of the samples processed, whereas 40% were DEN-2,8% were DEN-3,and 36% were DEN- 4 (Figure 1). It is unclear whether the change in distribution of serotypes is due to more intensive surveillance in certain areas or in a manifestation of herd immunity to serotype 1. This is the first report of DEN-3 in Mexico and reflects the strengthening of the surveillance at INDRE for dengue viruses in areas at risk. The geographic and temporal distribution of DEN-3 isolated in 1995 in Mexico (Figure 2) shows a pattern similar to the one followed by the first dengue epidemics in the early 1980s (2) and may be related to population movements towards the northern border.

the surveillance at INDRE for dengue viruses in areas at risk. The geographic and temporal distribution of DEN-3 isolated in 1995 in Mexico (Figure 2) shows a pattern similar to the one followed by the first dengue epidemics in the early 1980s (2) and may be related to population movements towards the northern border. The role of DEN-3 in increasing DHF cases is still to be determined; to date none of the DHF cases in which dengue virus was iso- lated have been associated with this serotype.Five cases were associated with DEN-1 and 20 cases with DEN-2. Nevertheless, the infection of DEN-3 in persons sensitized by previous infections with other serotypes and the widespread susceptibility of the Mexican population to this serotype must be considered a potential risk factor for future out- breaks. The cost of each DHF case has not been fully determined, but the resources needed to face a DHF epidemic are certainly not available in countries where the health sector has financial constraints due to unstable economic conditions. The development of dengue vaccines is encourag- ing, but the widespread dispersion of mosquito breeding sites exceeds the capabilities of vector control programs. Moreover, the potential role of Table. Number of isolates of dengue virus serotypes in Mexico* Year DEN-1 DEN-2 DEN-3 DEN-4 Total 1982 2 5 0 0 7 1983 5 6 0 2 13 1984 89 2 0 38 129 1985 30 8 0 9 47 1986 65 0 0 24 89 1987 13 0 0 0 13 1988 28 0 0 0 28 1989 21 0 0 0 21 1990 6 0 0 0 6 1991 4 0 0 20 24 1992 1 5 0 19 25 1993 0 10 0 0 10 1994 15 9 0 0 24 1995 40 98 19 88 245 Total 319 143 19 200 681 *SerumsamplesfromsuspectcaseswereaddedtoC6-36cellsgrowninD-MEM with 5% fetal calf serum for 7 days at 28 C and incubated for 1 hour.Cells were washed and further incubated in D-MEM with 0.4% bovine albumin for identifi- cation of cytopathic effect. Figure 2. Geographic and temporal distribution of DEN-3 serotype in Mexico. Figure 1. Frequency of dengue serotypes isolated in 1982 to 1994 and in 1995. Dispatches Emerging Infectious Diseases 134 Vol. 2, No. 2 --April-June 1996 Aedes albopictus in the transmission of dengue virus in Mexico must be evaluated because DHF cases have appeared in areas where A. albopictus has been identified (14). The role of this vector in dengue transmission could increase should its geo- graphic distribution expand and its susceptibility to infection increase (15).

s albopictus in the transmission of dengue virus in Mexico must be evaluated because DHF cases have appeared in areas where A. albopictus has been identified (14). The role of this vector in dengue transmission could increase should its geo- graphic distribution expand and its susceptibility to infection increase (15). The challenge faced by national health services is to improve the early detection of dengue trans- mission, prevent DHF, and decrease the case- fatality rate in DHF patients. This strategy must be supported by a strong surveillance network for viral diseases,which is now being implemented on a regional basis according to the risk of dengue transmission in the country. The detailed knowl- edge of the serotypes involved in future epidemics will provide useful information that will define the role of each serotype in the genesis of DHF cases and target control measures. The threat of a major epidemic requires a control strategy that will pre- vent the emergence of this public health problem. Baltasar Briseno-Garcia,* Hector Gomez-Dantes, Enid Argott-Ramirez,* Raul Montesano, Ana-Laura Vazquez-Martinez,* Sergio Ibanez-Bernal,* Guillermina Madrigal-Ayala,* Cuauhtemoc Ruiz-Matus, Ana Flisser,* and Roberto Tapia-Conyer *Instituto Nacional de Diagnostico y Referencia Epidemiologicos, Direccion General de Epidemiologia, Secretaria de Salud, Mexico D.F., Mexico; Direccion General de Epidemiologia, Secretaria de Salud, Mexico D.F., Mexico; Present address: Centro de Investigacion de Enfermedades Infecciosas, Instituto Nacional de Salud Publica, Secretaria de Salud, Cuernavaca, Mexico References 1. Gubler DJ, Trent DW. Emergence of epidemic den- gue/dengue hemorrhagic fever as a public health problem in the Americas. Infect Agents Dis 1993; 2: 383-393. 2. Pan American Health Organization. Dengue and den- gue hemorrhagic fever in the Americas:Guidelines for prevention and control. Scient Publ 1994;548:3-22. 3. Torres-Munoz A. La fiebre amarilla en Mexico:erradi- cacion de Aedes aegypti. Salud Publica Mex 1995;37:S103-S110. 4. Narro RJ, Gomez-Dantes H. El dengue en Mexico: un problema prioritario de salud publica. Salud Publica Mex 1995; 37:S12-S20. 5. Dietz VJ, Gubler DJ, Rigau-Perez J, Pinheiro F. Epi- demic of dengue 1 in Brazil, 1986: evaluation of a clinically based dengue surveillance system. Am J Epidemiol 1990;131:693-701. 6. Gubler DJ. Vigilancia activa del dengue y de la fiebre hemorragica del dengue. Bol Of Sanit Panam 1989;107:22-30. 7. Rico-Hesse R.

. 5. Dietz VJ, Gubler DJ, Rigau-Perez J, Pinheiro F. Epi- demic of dengue 1 in Brazil, 1986: evaluation of a clinically based dengue surveillance system. Am J Epidemiol 1990;131:693-701. 6. Gubler DJ. Vigilancia activa del dengue y de la fiebre hemorragica del dengue. Bol Of Sanit Panam 1989;107:22-30. 7. Rico-Hesse R. Molecular evolution and distribution of dengue virus type 1 and 2 in nature. Virology 1990;174:479-93. 8. Rosen L. Observations on the epidemiology of dengue in Panama. Am J Trop Med Hyg 1958;68: 45-58. 9. Anderson CR,Downs WG,Hill AE.Isolation of dengue virus from a human being in Trinidad. Science 1956;124:224-5. 10. Pinheiro F. Dengue in the Americas 1980-1987.PAHO Epidemiol Bull 1989;10:1-7. 11. Gubler D. Dengue/dengue hemorrhagic fever in the Americas: prospects for the year 2000. In: Halstead SB, Gomez-Dantes H, editors. Dengue: a worldwide problem, a common strategy. Proceedings of the Inter- national Conference on Dengue and Aedes aegypti community-based control,Merida,Mexico,July 11-16, 1992; Ministry of Health, Mexico,1992:19-27. 12. Gubler JD, Clark GG. Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerging Infectious Diseases 1995;1:55-7. 13. Gubler DJ. Aedes aegypti and Aedes aegypti-borne disease control in the 1990's: top down or bottom up. Am J Trop Med Hyg 1989;40: 571-8. 14. Ibanez-Bernal S, Martinez-Campos C. Aedes albopic- tus in Mexico. J Am Mosq Control Assoc 1994; 10: 231-2. 15. Shope R. Global climate change and infectious dis- eases, Environ Health Perspect 1991;96:171-4. Dispatches Vol. 2, No. 2 --April-June 1996 135 Emerging Infectious Diseases

Improved Serodiagnostic Testing for Lyme Disease: Results of a Multicenter Serologic Evaluation The diverse clinical manifestations of Lyme disease (1-3) have led to frequent confusion in clinical diagnosis, a confusion compounded by problems in the accuracy and precision of diagnos- tic serologic tests (4-11) and the difficulty of isolat- ing the causative organism (12-14), Borrelia burgdorferi. In 1990, more than 20 commercially prepared serologic test kits for Lyme disease were being sold in the United States, but no nationally standardized reference test was available. A col- laborative evaluation of a selected sample of the commercial test kits by the Centers for Disease Control and Prevention (CDC) and the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) demonstrated poor concor- dance of results among these test kits and among a selected group of state health department labo- ratories (11). Because of the lack of a rigorously defined reference serum panel, conclusions could not be drawn about the sensitivity and specificity of the test kits evaluated. An unexpected finding in this study was the low concordance in test results between CDC and two consulting academic reference center laboratories. A number of other studies also have demonstrated low concordance of Lyme disease serologic test results obtained by a variety of laboratories (4-10). As a result of those findings,the study described here was designed to fulfill the following objec- tives: 1) to assemble a serum panel from patients who had clinically well-defined Lyme disease (preferably confirmed by isolation of B. burgdor- feri); healthy controls, and persons residing in non-endemic-disease areas whose potentially cross-reactive specimens had yielded equivocal ELISA results in earlier CDC tests; 2) to test this panel in a blinded fashion by several recognized Lyme disease reference and research laboratories; and 3) to compare the accuracy and precision of tests as a prelude to developing national recom- mendations for standardized serologic testing for antibodies to B. burgdorferi.

esults in earlier CDC tests; 2) to test this panel in a blinded fashion by several recognized Lyme disease reference and research laboratories; and 3) to compare the accuracy and precision of tests as a prelude to developing national recom- mendations for standardized serologic testing for antibodies to B. burgdorferi. Tests were performed by five academic centers active in Lyme disease research (the Marshfield Clinic, Marshfield, Wisconsin; University of Medi- cine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey; State University of New York at Stony Brook, Stony Brook, New York; Tufts/New Eng- land Medical Center, Boston, Massachusetts; and the University of Connecticut Health Center, Farmington, Connecticut) and CDC's Division of Vector-Borne Infectious Diseases,National Center for Infectious Diseases, based in Ft. Collins, Colo- rado. Serum samples from Lyme disease case-pa- tients were obtained from the participating aca- demic investigators (n = 72) and from the CDC Lyme disease reference serum collection (n = 37). All case-patient serum samples (total = 109) were from patients who met the CDC clinical case defi- nition for surveillance of Lyme disease (15). The clinical manifestations in these patients ranged from acute erythema migrans (EM) to late neurologic disease accompanied by Lyme arthritis. B. burgdorferi had been cultured by the method of Berger et al. from 14 of 34 (41%) acute-phase specimens provided by CDC (14). Duplicate speci- mens (n = 85) were randomly selected from the 109 case-patient samples for precision analysis, mak- ing a total of 194 case-patient samples in the panel. Control serum samples were provided by CDC from unpaid healthy blood donors (n = 113) who resided in areas where Lyme disease is not en- demic (Cincinnati, Ohio, and Atlanta, Georgia; travel histories were not available from these do- nors, however. Duplicate specimens (n = 87) also were randomly selected, resulting in 200 noncase samples in the serum panel. Additional control samples were obtained from persons who resided in areas where Lyme disease was not endemic but whose physicians submitted their serum for Lyme disease testing to CDC through their state health department (n = 113). These specimens from pa- tients with suspected cases had borderline (equivocal) seroreactivity in the whole cell soni- cate (WCS) enzyme-linked immunoassay (ELISA) used by CDC before 1992 and are referred to hereafter as "WCS-suspects" (16).

r Lyme disease testing to CDC through their state health department (n = 113). These specimens from pa- tients with suspected cases had borderline (equivocal) seroreactivity in the whole cell soni- cate (WCS) enzyme-linked immunoassay (ELISA) used by CDC before 1992 and are referred to hereafter as "WCS-suspects" (16). The addition of duplicate specimens (n = 87) brought this group to 200 equivocally seroreactive samples. Serum was separated and frozen by the original collectors and shipped frozen to CDC's facilities in Ft. Collins, Colorado. The specimens were divided into aliquots and coded; code labels were applied Dispatches Emerging Infectious Diseases 136 Vol. 2, No. 2 --April-June 1996 by CDC staff not involved in serologic testing of the specimens (n = 594). The panels were then refrozen and shipped on dry ice for blind testing by participating investigators. All specimens were received frozen. To calculate test sensitivity and specificity, only the result of the sample with the lower random code number of each pair was used. Each laboratory employed the testing method that it used routinely at the time this study was undertaken (1992). CDC used an ELISA with a WCS antigen prepared from highly passaged strain B31 (gift of A. Barbour, University of Texas Health Sciences Center, San Antonio, Texas) and an ELISA with a strain B31 flagellar antigen (FLA) then being evaluated (16,17).The other five participating investigators used ELISA tests that employed a WCS antigen of B. burgdorferi. Four used assays developed in their own laboratories, and one used a commercially available test kit (18-22). Three investigators also tested all speci- mens by Western blotting using published meth- ods (19, 20). Two of these three performed immunoblotting for IgM and IgG antibodies sepa- rately. One laboratory tested for IgM and IgG together. Each participating laboratory submitted the raw data of its results, along with a dichotomous interpretation of those results as either positive or negative. By prior agreement, ELISA results that fell into a range ordinarily reported as "equivocal" by that laboratory were treated as negative for this analysis.

gether. Each participating laboratory submitted the raw data of its results, along with a dichotomous interpretation of those results as either positive or negative. By prior agreement, ELISA results that fell into a range ordinarily reported as "equivocal" by that laboratory were treated as negative for this analysis. Statistical analyses undertaken at CDC included calculations of sensitivity (true positives correctly identified), specificity (true negatives correctly identified), precision (frequency of ob- taining the same result on duplicate analysis of a specimen), and a measure of concordance (agree- ment among investigators) of results among the tests using the kappa statistic. The accuracy and precision of the serologic tests as performed in 1992 by all six laboratories is summarized in Table 1. The test methods of inves- tigators 1,2,and 3 produced essentially equivalent results, with moderately high sensitivity (73% to 79%) for the aggregate of all case-patient samples tested and high specificity (98% to 99.5%). Preci- sion was high in these three laboratories for both blood donor samples (97% to 99%) and the WCS- suspects samples submitted from areas where Lyme disease is nonendemic (94% to 98%). Preci- sion was somewhat lower for the case-patient sam- ples (82% to 91%). The performance of the other three laborato- ries, including CDC's, was poor. Both CDC ELISA tests had high sensitivity (92% to 93%), but low specificity (71% to 82%). Precision for case-patient specimens was fairly high (92% to 93%), but low for both non-case-patient (77% to 79%) and WCS- suspects groups (62% to 69%). The method of in- vestigator 4 gave very low sensitivity (49%), moderately high specificity (91%), poor precision with Lyme disease case-patient specimens (79%), but good precision with blood donor and WCS-sus- pects samples (93% to 94%). Investigator 5, who used a commercial test, obtained results with low accuracy and precision. Concordance was high (kappa statistic 0.700) between the results of investigators 1,2,and 3.The CDC FLA test showed moderate concordance (kappa 0.400) with results from investigators 1,2, and 3. The results of investigator 4 showed mod- erate concordance with those of investigators 1 and 2 (kappa 0.400) and low concordance (0.400) with the other results. The results of investigator 5 had low concordance with all other results.

howed moderate concordance (kappa 0.400) with results from investigators 1,2, and 3. The results of investigator 4 showed mod- erate concordance with those of investigators 1 and 2 (kappa 0.400) and low concordance (0.400) with the other results. The results of investigator 5 had low concordance with all other results. The CDC WCS test showed moderate concordance with the FLA test, but low concordance with re- sults of the ELISA tests of the other laboratories. The three investigators with the best results all used WesternblottosupplementtheirELISA.Two of these three investigators submitted their di- chotomous test interpretation with and without using Western blot results. The sensitivity im- proved by 20% for one investigator and by 30% for the otherwhenWesternblotresults were included. The improvement resulted from identifying as Table 1. Accuracy and precision of serologic tests for Lyme disease performed in 1992 Accuracy (%) Precision (%) Case Non-case- WCS Investigator Sensitivity Specificity patients patients suspectsa CDC 93 71 93 77 69 (WCS) CDC 92 82 92 79 62 (FLA) 1 73 99.5 89 99 98 2 76 99 82 99 97 3 79 98 91 97 94 4 49 91 79 94 93 5 40 72 63 74 77 a Specimensfrom patientswith suspectedcases thathadborderline(equivocal) seroreactivity in an enzyme-linked immunosorbent assay with whole-cell soni- cate antigen (WCS). Dispatches Vol. 2, No. 2 --April-June 1996 137 Emerging Infectious Diseases positive by Western blot those case-patient specimens from which an equivocal result was obtained by ELISA and which by study design would have been counted as negative by ELISA results alone. Specificities were not affected by Western blot analysis in this group of three inves- tigators, since the serum panel in this study did not contain cross-reactive sera; and the negative controls and WCS-suspects had negative results by both ELISA and Western blot. Test sensitivity from the three laboratories with the best test specificity (98%) was analyzed according to the clinical manifestations in the case-patients (Table 2). As expected, the sensitivi- ties of the tests were lowest in specimens from patients with early disease, 59% to 66% for erythema migrans and 63% to 75% for early neurologic disease.Sensitivities were much higher for samples of patients with late disease.Sensitivi- ties of 89% to 95% were obtained for Lyme arthri- tis patients and 91% to 100% for persons with late neurologic disease, primarily encephalopathy or polyneuropathy.

to 66% for erythema migrans and 63% to 75% for early neurologic disease.Sensitivities were much higher for samples of patients with late disease.Sensitivi- ties of 89% to 95% were obtained for Lyme arthri- tis patients and 91% to 100% for persons with late neurologic disease, primarily encephalopathy or polyneuropathy. The emergence of a disease can outstrip the development of reliable methods for its laboratory diagnosis. The serodiagnosis of Lyme disease has been fraught with problems of precision and accu- racy. This study provided an opportunity for se- lected academic research centers and CDC to compare the performance of their individual tests by using a serum panel from clinically well-char- acterized patients and controls from non-en- demic-disease areas.The clinical diagnosis of early Lyme disease was supported by the isolation of B. burgdorferi from skin biopsy specimens (14),when possible. The panel, which was coded blind had a sufficiently large number of samples (n = 335) to provide adequate statistical power for the com- parison. Laboratories that supplemented their primary test, an ELISA, with immunoblotting achieved greater test accuracy than those that did not. The use of Western blot as a second test enabled the best performing laboratories to increase test sen- sitivity without a concomitant loss of specificity. This increase in sensitivity occurred as a result of identifying as true positives by Western blot a number of those specimens from patients with clinical cases of Lyme disease that were inter- preted as equivocal by ELISA and would have been otherwise considered in this study as dichoto- mously negative results. Although the investiga- tors employing Western blot tested all panel specimens with this method, they did so at that time to evaluate the potential value of Western blot in Lyme disease serologic diagnosis. The observation that Western blotting could be employed to resolve equivocal ELISA results gave additional impetus for evaluating its potential adjunctive role in Lyme disease serodiagnosis and eventually led to the finally recommended two- test approach (23).The potential utility of Western blotting, however, pointed out the lack of stand- ardized methods for producing blots and stand- ardized interpretive criteria.

tional impetus for evaluating its potential adjunctive role in Lyme disease serodiagnosis and eventually led to the finally recommended two- test approach (23).The potential utility of Western blotting, however, pointed out the lack of stand- ardized methods for producing blots and stand- ardized interpretive criteria. Performance of the CDC WCS and FLA ELISA in this study that did not include known cross-re- active sera suggested that the positive cut-off value for these tests was inappropriately low, thereby increasing sensitivity at the expense of specificity. These results then explained the large number of borderline WCS ELISA results ob- tained by CDC when it tested the sera of pa- tients residing in areas where Lyme disease was not endemic. This group of WCS suspects was nearly uniformly found to be negative on ELISA by the three laboratories with the best performance (Table 1) (23). Table 2. Test sensitivity of laboratories demonstrating a test specificity of 98% Sensitivity, % (positive samples/total) Clinical Manifestations Laboratory 1 Laboratory 2 Laboratory 3 Erythema migrans, all 59 (55/94) 60 (56/94) 66 (62/94) Acute phasea 65 (11/17) 65 (11/17) 76 (13/17) Convalescent phaseb 57 (44/77) 58 (45/77) 64 (49/77) Carditis 100 (2/2) 100 (2/2) 100 (2/2) Lyme arthritisc 89 (58/65) 95 (62/65) 92 (60/65) Neurologic, all 85 (28/33) 88 (29/33) 91 (30/33) Early 63 (5/8) 63 (5/8) 75 (6/8) Late 91 (10/11) 100 (11/11) 91 (10/11) Late and arthritis 93 (13/14) 93 (13/14) 100 (14/14) Total 74 (143/194) 77 (149/194) 79 (154/194) a 30 days from onset of erythema migrans to blood collection. b > 30 days from onset of erythema migrans to blood collection. c Without neurologic signs or symptoms. Dispatches Emerging Infectious Diseases 138 Vol. 2, No. 2 --April-June 1996 Specificity in this study was determined by testing specimens from blood bank donors. With these samples, specificity in the three laboratories that used immunoblotting was very high (98% to 99.5%). The test panel did not, however, contain specimens from patients with conditions known to produce cross-reacting antibodies (e.g.,syphilis) or polyclonal B-cell activation (e.g., Epstein-Barr vi- rus infection or systemic lupus erythematosus). Thus, reported specificities in this study are likely higher than they would have been if cross-reactive specimens were included in the evaluation.

onditions known to produce cross-reacting antibodies (e.g.,syphilis) or polyclonal B-cell activation (e.g., Epstein-Barr vi- rus infection or systemic lupus erythematosus). Thus, reported specificities in this study are likely higher than they would have been if cross-reactive specimens were included in the evaluation. Sub- sequent studies that included cross-reactive sera demonstrated that Western blotting correctly identifies many false-positive ELISA reactions (23, 24). This study confirmed in the reference and re- search laboratory setting the previously docu- mented problems with accuracy and precision of serodiagnostic tests by using WCS antigens of B. burgdorferi (4-11). The study confirmed that a serious disparity existed between the test results obtained by CDC and those obtained by academic reference centers with the best testing perform- ances. These results guided corrective action and led to the adoption by CDC and ASTPHLD of a two-test approach to serodiagnosis (23),whichforms the basis for the future national standardization of Lyme disease serologic testing methods. Robert B. Craven,* Thomas J. Quan,* Raymond E. Bailey,* Raymond Dattwyler, Raymond W. Ryan, Leonard H. Sigal, Allen C. Steere, Bradley Sullivan,# Barbara J.B. Johnson,* David T. Dennis,* and Duane J. Gubler* *Centers for Disease Control and Prevention, Fort Collins, Colorado, USA; State University of New York at Stony Brook, Stony Brook, New York, USA; University of Connecticut Health Center, Farmington, Connecticut, USA; University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA; Tufts/New England Medical Center, Boston, Massachusetts, USA; # Marshfield Clinic, Marshfield, Wisconsin, USA A portion of this information was presented at the VIth International Conference on Lyme Borreliosis, Bologna, Italy, June 1994 and at the Second National Conference on Serologic Diagnosis of Lyme Disease, Dearborn, Michigan, October 1994. References 1. Steere,AC.Lyme disease.N Engl J Med 1989;321:586- 96. 2. Logigian EL, Kaplan RF, Steere AC. Chronic neurologic manifestations of Lyme disease. N Engl J Med 1990;323:1438-44. 3. Steere AC,Bartenhagen NH,Craft JE,et al.The early clinical manifestations of Lyme disease. Ann Intern Med 1989;99:76-82. 4. Bakken LL, Case KL, Callister SM, Bourdeau NJ, Schell RF. Performance of 45 laboratories participat- ing in a proficiency testing program for Lyme disease serology. JAMA 1992;268:891-5. 5.

-44. 3. Steere AC,Bartenhagen NH,Craft JE,et al.The early clinical manifestations of Lyme disease. Ann Intern Med 1989;99:76-82. 4. Bakken LL, Case KL, Callister SM, Bourdeau NJ, Schell RF. Performance of 45 laboratories participat- ing in a proficiency testing program for Lyme disease serology. JAMA 1992;268:891-5. 5. Hedberg CW, Osterholm MT, MacDonald KL, White KE. An interlaboratory study of antibody to Borrelia burgdorferi. J Infect Dis 1987;155:1325-7. 6. Hedberg CW, Osterholm MT. Serologic tests for anti- body to Borrelia burgdorferi--another Pandora's box for medicine? Arch Intern Med 1990;150:732-3. 7. Jones JM. Serodiagnosis of Lyme disease. Ann Intern Med 1991;114:1064. 8. Lane RS, Lennette ET, Madigan JE. Interlaboratory and intralaboratory comparisons of indirect im- munofluorescence assays for serodiagnosis of Lyme disease. J Clin Microbiol 1990;28:1774-9. 9. Luger SW, Krauss E. Serologic tests for Lyme disease: interlaboratory variability. Arch Intern Med 1990;15:761-3. 10. Schwartz BS,Goldstein MD,Ribeiro JMC,SchulzeTL, Shahied SI. Antibody testing in Lyme disease: a com- parison of results in four laboratories. JAMA 1989;262:3431-4. 11. Quan TJ, Wilmoth BA, Carter LG, Bailey RE. A com- parison of some commercially available serodiagnos- tic kits for Lyme disease. In: Proceedings of the First National Conference on Lyme Disease Testing (Dear- born,Michigan).Washington,DC:Association of State and Territorial Public Health Laboratory Directors, 1991:61-73. 12. Steere AC, Grodzicki RL, Kornblatt AN, et al. The spirochetal etiology of Lyme disease. N Engl J Med 1983;308:733-40. 13. Benach JL, Bosler EM, Hanrahan JP, et al. Spiro- chetes isolated from the blood of two patients with Lyme disease. N Engl J Med 1983;308:740-2. 14. Berger BW, Johnson RC, Kodner C, Coleman L. Culti- vation of Borrelia burgdorferi from erythema migrans lesions and perilesional skin. J Clin Microbiol 1992;30:359-61. 15. Centers for Disease Control and Prevention. Case definitions for public health surveillance. MMWR 1990;39(RR-13):19-21. 16. Russell H, Sampson JS, Schmid GP, Wilkinson HW, Plikaytis B. Enzyme-linked immunosorbent assay and indirect immun ofluorescence assay for Lyme disease. J Infect Dis 1984;149:465-70. 17. Hansen K, Asbrink E. Serodiagnosis of erythema mi- grans and acrodermatitis chronica atrophicans by the Borrelia burgdorferi flagellum enzyme-linked immu- nosorbent assay. J Clin Micrbiol 1989;27:545-51. 18. Craft JE, Grodzicki RL, Steere AC.

immun ofluorescence assay for Lyme disease. J Infect Dis 1984;149:465-70. 17. Hansen K, Asbrink E. Serodiagnosis of erythema mi- grans and acrodermatitis chronica atrophicans by the Borrelia burgdorferi flagellum enzyme-linked immu- nosorbent assay. J Clin Micrbiol 1989;27:545-51. 18. Craft JE, Grodzicki RL, Steere AC. Antibody response in Lyme disease: evaluation of tests. J Infect Dis 1984;149:789-95. 19. Grodzicki RL, Steere AC. Comparison of immunoblot- ting and indirect enzyme-linked immunosorbent as- say using different antigen preparations for diagnosing early Lyme disease. J Infect Dis 1988;157:790-97. Dispatches Vol. 2, No. 2 --April-June 1996 139 Emerging Infectious Diseases 20. Dressler F, Whalen JA, Reinhardt BN, Steere AC. Western blotting in the serodiagnosis of Lyme disease. J Infect Dis 1993;167:392-400. 21. Fister RD, Weymouth LA, McLaughlin JC, Ryan RW, Tilton RC. Comparative evaluation of three products for the detection of Borrelia burgdorferi antibody in human serum. J Clin Microbiol 1989;27:2834-37. 22. Dattwyler RJ, Volkman DJ, Luft BJ, Halperin JJ, Thomas J, Golightly MG. Seronegative Lyme disease: dissociation of specific T- and B-lymphocytic response to Borrelia burgdorferi. N Engl J Med 1988;319:1441- 6. 23. Association of State and Territorial Public Health Laboratory Directors and the Centers for Disease Control and Prevention. Recommendations. In: Pro- ceedings of the Second National Conference on Sero- logic Diagnosis of Lyme Disease (Dearborn, Michigan). Washington, DC: Association of State and Territorial Public Health Laboratory Directors 1995:1-5. 24. Johnson BJB, Robbins KE, Bailey RE, et al. Serodiag- nosis of Lyme disease:accuracy of a two-step approach using a flagella-based ELISA and immunoblotting. J Infect Dis 1995; submitted. Dispatches Emerging Infectious Diseases 140 Vol. 2, No. 2 --April-June 1996

Emergence of Bartonella quintana Infection among Homeless Persons Bartonella quintana has episodically emerged as a cause of infection among distinct and diverse populations during the 20th century. The organ- ism was first identified as an important human pathogen during World War I when it caused epi- demics of louse-borne trench fever that affected an estimated 1 million troops in Europe (1, 2). Trench fever was characterized by fever, rash, bone pain, and splenomegaly and ranged in severity from a mild flulike illness to a more severe, relapsing disease. B. quintana infections were rarely recog- nized from the end of World War II until the 1980s when the organism reemerged as an opportunistic pathogen among HIV-infected persons. In this population, B. quintana has been identified as a cause of bacillary angiomatosis, endocarditis, and bacteremia (3-5) and has been isolated from AIDS patients in France (6) and the United States (3-5). In the 1990s, B. quintana has emerged among homeless persons in North America and Europe. In 1993, the organism was isolated from the blood specimens of 10 patients at a single hospital in Seattle, Washington, within a 6-month period (7). These patients had illnesses characterized by fe- ver and persistent bacteremia.Endocarditis devel- oped in two patients,one of whom required a heart valve replacement. All 10 patients had chronic alcoholism, eight were homeless, and the six who were tested for HIV infection were HIV-negative. These six were the first cases of invasive B. quin- tana infection among HIV-negative persons re- ported in the United States. Results of a case-control study indicated that the patients with Bartonella bacteremia were more likely than con- trols (other hospitalized patients from whom blood specimens were obtained at approximately the same time) to be homeless (p = 0.001), to have a history of alcohol abuse (p = 0.001), and to be nonwhite (p = 0.007). The isolates from the 10 patients were identical by polymerase chain reac- tion restriction-fragment-length polymorphism testing,which further suggests that the cases were epidemiologically linked.

the same time) to be homeless (p = 0.001), to have a history of alcohol abuse (p = 0.001), and to be nonwhite (p = 0.007). The isolates from the 10 patients were identical by polymerase chain reac- tion restriction-fragment-length polymorphism testing,which further suggests that the cases were epidemiologically linked. Patients' characteristics were obtained by retrospective medical record re- view, and at the time they sought treatment, three patients reported a recent cat scratch, five had scabies, and one had lice. More complete information, however, on patients' past exposures to animals and ectoparasites was not available. In 1995, Drancourt and co-workers reported three cases of B. quintana endocarditis among HIV-negative, homeless, alcoholic men in France (8). One of the patients had reported contact with a dog, and one had reported contact with dogs and cats; however, a current or past history of infection with lice or scabies was not documented for any of the patients. In 1995, Stein and Raoult also re- ported serologic evidence of B. quintana infection in an HIV-negative, homeless man from Mar- seilles, who had a relapsing febrile illness and a history of louse infestation (9). As a follow-up to the 1993 B. quintana outbreak in Seattle in 1994, we conducted a seroprevalence study of anti-Bartonella antibodies among pa- tients at a community clinic in the "skid row" section of Seattle, which serves a primarily home- less and indigent population (10). The median age of the 192 patients included in the study was 45 years, 156 (81%) of the 192 were male, and 126 (66%) were classified as homeless.B.quintana IgG titers 64 were detected by an indirect fluores- cence antibody assay (11) in 39 (20%) of the 192 clinic patients. In contrast, only 4 (2%) of 199 banked blood specimens from an age-matched and sex-matched comparison group of Seattle volun- teerblood donors had titers 64 (p < 0.001).Among clinic patients, seropositivity (titer 64) was asso- ciated by univariate analysis with older age,home- lessness (relative risk [RR] 2.0; 95% confidence interval [CI] 1.0-4.1), alcohol abuse (RR, 2.5; 95% CI 1.4-4.2), smoking (RR, 2.0; 95% CI 1.2 -3.4), and injection drug use (RR, 2.5; 95% CI 1.3-4.8).

p < 0.001).Among clinic patients, seropositivity (titer 64) was asso- ciated by univariate analysis with older age,home- lessness (relative risk [RR] 2.0; 95% confidence interval [CI] 1.0-4.1), alcohol abuse (RR, 2.5; 95% CI 1.4-4.2), smoking (RR, 2.0; 95% CI 1.2 -3.4), and injection drug use (RR, 2.5; 95% CI 1.3-4.8). By multivariate analysis, only alcohol abuse re- mained independently associated with seroposi- tivity (odds ratio 3.3; 95% CI 1.6-6.9), and of 39 seropositive patients, 21 (54%) had a history of chronic alcoholism.Reliable data on past exposure to animals or ectoparasites were also not available for patients in this study. The study was limited by the well-described cross-reactivity of the assay between Bartonella species (12, 13), and most (62%) clinic patients with B. quintana titers 64 also had titers 64 to B. henselae. It is, therefore, possible that some of the seropositive patients may have been exposed Dispatches Vol. 2, No. 2 --April-June 1996 141 Emerging Infectious Diseases to Bartonella species other than B. quintana. These findings do, however, show that a surpris- ingly high proportion of clinic patients without a history of documented Bartonella infection had detectable anti-Bartonella antibodies and may have been exposed to B. quintana. Multiple factors, including those related to dis- ease transmission, host susceptibility, and ability to detect the organism, have likely contributed to the emergence of B. quintana infection among the homeless. Transmission of B. quintana from hu- man to human by the body louse, Pediculus hu- manus, has been experimentally documented (1) and is believed to have been the predominant mode of transmission of epidemic trench fever in World Wars I and II. Lice reside primarily in the seams of clothing and are easily killed by immer- sion in water 50C or warmer (14), which explains the propensity for louse-borne infections among displaced persons or wartime troops. Although these reports of B. quintana infection among homeless persons lack sufficient information to conclusively determine the disease vector, louse- borne infection remains a plausible hypothesis. Lice, however, have not been associated with bac- illary angiomatosis among AIDS patients, although exposure to cats (and, therefore, possibly to fleas) has been associated with bacillary angio- matosis and bacillary peliosis caused by Bar- tonella species (15) and with cat-scratch disease caused by B. henselae (16). Thus, it is possible that B.

n associated with bac- illary angiomatosis among AIDS patients, although exposure to cats (and, therefore, possibly to fleas) has been associated with bacillary angio- matosis and bacillary peliosis caused by Bar- tonella species (15) and with cat-scratch disease caused by B. henselae (16). Thus, it is possible that B. quintana infection is spread among homeless persons by as yet unidentified vectors or reser- voirs. Homeless persons are also at risk for non-vec- torborne infectious diseases. An increased risk for tuberculosis in this population is well documented (17, 18), and outbreaks of meningococcal disease (19,20),pneumococcal disease (21),and diphtheria (22, 23) have been reported. It is likely that factors such as crowding,altered immunity due to alcohol- ism or other co-existing health problems, and in- adequate or infrequent access to medical care affect the transmission and spread of infectious diseases among the homeless. Previous studies have shown that the clinical response to a stand- ard inoculum of B. quintana varies substantially in experimental study patients (1); this variation indicates that host factors are likely important determinants of the risk for clinical infection fol- lowing exposure to the organism. Although cases of B. quintana bacteremia among homeless persons have thus far been reported only from France and Seattle, Washington, the problem is probably not confined to these locations. B. quintana is a fastidious and slow-growing bacterium that generally requires special culturing techniques for isolation (3-5, 24), and many clinical laboratories do not routinely use blood culturing methods that are sensitive for isolating this organism. Moreover, B. quintana in- fection can result in a broad range of often nonspe- cific clinical manifestations (1, 3-5); therefore, case-patients evaluated for suspected bacteremia may represent only a small proportion of infected persons, as suggested by the results of the Seattle seroprevalence survey. To better define the geo- graphic distribution and prevalence ofB.quintana infection among homeless populations, a height- ened awareness for this infection on the part of clinicians and the use of appropriate culture tech- niques by microbiology laboratories serving this population are needed. In addition, more specific serologic tests would aid in the diagnosis and assessment of the epidemiologic characteristics of B. quintana infections.

ned awareness for this infection on the part of clinicians and the use of appropriate culture tech- niques by microbiology laboratories serving this population are needed. In addition, more specific serologic tests would aid in the diagnosis and assessment of the epidemiologic characteristics of B. quintana infections. The optimal treatment regimen for HIV-nega- tive patients with suspected or confirmed B. quin- tana infection has not been established. Minimal published data exist regarding antimicrobial ther- apy for this infection, and in vitro susceptibility testing has proven unreliable (25).Nonetheless,on the basis of limited data, we believe it is reason- able to treat immunocompetent patients who have uncomplicated B. quintana bacteremia with at least 14 days of oral therapy with erythromycin, azithromycin, doxycycline, or tetracycline. In the 1993 Seattle outbreak, most patients had a satis- factory response to treatment with a beta-lactam agent followed by either erythromycin or az- ithromycin for 14 days (7). Although the number of patients identified with B. quintana endocardi- tis is small, most of these patients have required cardiac valve replacement despite intravenous an- timicrobial therapy (5,8,26).Therefore,we recom- mend that patients with B. quintana endocarditis receive a more prolonged course of at least 4 to 6 months of antimicrobial therapy and cardiac valve replacement if needed. Further study is needed to determine the role of bactericidal agents, such as third generation cephalosporins or quinolones, as monotherapyorincombinationwithabacteriostatic agent for treating invasive B. quintana infections. Dispatches Emerging Infectious Diseases 142 Vol. 2, No. 2 --April-June 1996 Many aspects of the acquisition and pathogene- sis of B. quintana infections, and specifically B. quintana infections among the homeless, are not well defined. Changes in the organism itself that have led to increased virulence may in part ac- count for its reemergence; however, microbiologic data that can support or refute this hypothesis are lacking (27). The absence of recently identified cases in Seattle and in other areas with laborato- ries that use culture techniques appropriate for isolating Bartonella species suggests an episodic pattern of disease, with few or no cases occurring during interepidemic periods.

an support or refute this hypothesis are lacking (27). The absence of recently identified cases in Seattle and in other areas with laborato- ries that use culture techniques appropriate for isolating Bartonella species suggests an episodic pattern of disease, with few or no cases occurring during interepidemic periods. It seems clear, how- ever, that this most recent emergence of an old disease is related, at least in part, to societal factors that have contributed to urban decay and the existence of large homeless populations in our cities. As with other emerging infectious diseases, further efforts to identify, evaluate, and control B. quintana infections among homeless persons are challenges that will require the coordinated effort of clinicians, microbiologists, and public health officials. Lisa A. Jackson, M.D., M.P.H.*, and David H. Spach, M.D. *School of Public Health and Community Medicine, and the Division of Infectious Diseases and the Department of Medicine, University of Washington, Seattle, Washington, USA References 1. Vinson JW, Varela G, Molina-Pasquel C. Trench fever. III. Induction of clinical disease in volunteers inocu- lated with Rickettsia quintana propagated on blood agar. Am J Trop Med Hyg 1969;18:713-22. 2. Slater LN, Welch DF. Rochalimaea species (recently renamed Bartonella). In: Mandell GL, Bennett JE, Dolin R, editors. Principles and practice of infectious diseases, 4th ed. New York: Churchill Livingstone, 1995:1741-7. 3. Welch DF, Pickett DA, Slater LN, Steigerwalt AG, Brenner DJ. Rochalimaea henselae sp. nov., a cause of septicemia, bacillary angiomatosis, and parenchymal bacillary peliosis. J Clin Microbiol 1992;30:275-80. 4. Koehler JE,Quinn FD,Berger TG,LeBoit PE,Tappero JW. Isolation of Rochalimaea species from cutaneous and osseous lesions of bacillary angiomatosis. N Engl J Med 1992;327:1625-31. 5. Spach DH, Callis KP, Paauw DS, et al. Endocarditis caused by Rochalimaea quintana in a patient infected with human immunodeficiency virus.J Clin Microbiol 1993;31:692-4. 6. Maurin M, Roux V, Stein A, Ferrier F, Viraben R, Raoult D. Isolation and characterization by im- munofluorescence, sodium dodecyl sulfate-polyacry- lamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis. J Clin Microbiol 1994;32:1166-71. 7.

zation by im- munofluorescence, sodium dodecyl sulfate-polyacry- lamide gel electrophoresis, western blot, restriction fragment length polymorphism-PCR, 16S rRNA gene sequencing, and pulsed-field gel electrophoresis of Rochalimaea quintana from a patient with bacillary angiomatosis. J Clin Microbiol 1994;32:1166-71. 7. Spach DH,Kanter AS,Dougherty MJ,et al.Bartonella (Rochalimaea) quintana bacteremia in inner-city pa- tients with chronic alcoholism. N Engl J Med 1995;332:424-8. 8. Drancourt M, Mainardi JL, Brouqui P, et al. Bar- tonella (Rochalimaea) quintana endocarditis in three homeless men. N Engl J Med 1995;332:419-23. 9. Stein A, Raoult D. Return of trench fever [letter]. Lancet 1995;345:450-1. 10. Jackson LA, Spach DH, Kippen DA, Sugg NK, Regnery RL, Sayers MH, Stamm WE. Seroprevalence to Bartonella quintana among patients at a commu- nity clinic in downtown Seattle. J Infect Dis 1996;173:1023-6. 11. Regnery RL, Olson JG, Perkins BA, Bibb W. Serologic response to "Rochalimaea henselae" antigen in sus- pected cat scratch disease. Lancet 1992;339:1443-5. 12. Waldvogel K, Regnery RL, Anderson BA, Caduff R, Caduff J, Nadal D. Disseminated cat-scratch disease: detection of Rochalimaea henselae in affected tissues. Eur J Pediatr 1994;153:23-7. 13. Dalton MJ,Robinson LE,Cooper J,Regnery RL,Olson JG,Childs JE.Use of Bartonella antigens for serologic diagnosis of cat-scratch disease at a national referral center. Arch Intern Med 1995;155:1670-6. 14. Elgart ML. Pediculosis. Dermat Clin 1990;8:219-28. 15. Tappero JW, Mohle-Boetani J, Koehler JE, et al. The epidemiology of bacillary angiomatosis and bacillary peliosis. JAMA 1993;269:770-5. 16. Zangwill KM, Hamilton DH, Perkins BA, et al. Cat scratch disease in Connecticut: epidemiology, risk fac- tors, and evaluation of a new diagnostic test. N Engl J Med 1993;329:8-13. 17. Barnes PF, El-Hajj H, Preston-Martin S, et al. Trans- mission of tuberculosis among the urban homeless. JAMA 1996;275:305-7. 18. Nardell E. McInnis B, Thomas B, Weidhaus S. Exoge- nous reinfection with tuberculosis in a shelter for the homeless. N Engl J Med 1986;315:1570-5. 19. Filice GA, Englender SJ, Jacobson JA, et al. Group A meningococcal disease in skid rows: epidemiology and implications for control. Am J Public Health 1984;74:253-4. 20. Counts GW, Gregory DF, Spearman JG, et al. Group A meningococcal disease in the U.S. Pacific Northwest: epidemiology, clinical features, and effect of a vaccina- tion control program. Rev Infect Dis 1984;6:640-8. 21.

disease in skid rows: epidemiology and implications for control. Am J Public Health 1984;74:253-4. 20. Counts GW, Gregory DF, Spearman JG, et al. Group A meningococcal disease in the U.S. Pacific Northwest: epidemiology, clinical features, and effect of a vaccina- tion control program. Rev Infect Dis 1984;6:640-8. 21. DeMaria A Jr, Browne K, Berk SL, et al. An outbreak of type I pneumococcal pneumonia in a men's shelter. JAMA 1980;244:1446-9. Dispatches Vol. 2, No. 2 --April-June 1996 143 Emerging Infectious Diseases 22. Pedersen AHB, Spearman J, Tronca E, et al. Diphthe- ria on skid road, Seattle, Washington, 1972-75. Public Health Rep 1977;92:336-42. 23. Heath CW Jr, Zusman J. An outbreak of diphtheria among skid row men. N Engl J Med 1962;267:809-12. 24. Larson AM, Dougherty MJ, Nowowiejski DJ, Welch DF, Matar GM, Swaminathan B, Coyle MB. Detection of Bartonella (Rochalimaea) quintana by routine ac- ridine orange staining of broth blood cultures. J Clin Microbiol 1994;32:1492-6. 25. Myers WF, Grossman DM, Wisseman CL. Antibiotic susceptibility patterns in Rochalimaea quintana, the agent of trench fever. Antimicrob Agents Chemother 1984;25:690-3. 26. Spach DH, Kanter AS, Daniels NA, et al. Bartonella (Rochalimaea) quintana species as a cause of culture- negative endocarditis. Clin Infect Dis 1995;20:1044-7. 27. Relman DA. Has trench fever returned? N Engl J Med 1995;332:463-4. Dispatches Emerging Infectious Diseases 144 Vol. 2, No. 2 --April-June 1996

The Reemergence of Visceral Leishmaniasis in Brazil Because of a complex array of factors, an in- creasing number of new and reemerging infectious diseases are being recognized in both industrial- ized and developing countries in the Americas (1,2). The expanding population, living in over- crowded conditions with inadequate housing and sanitary facilities, has been exposed to new dis- eases and human pathogens. For example, the appearance of the South American arenaviruses (Junin, Machupo, and Guanarito) illustrates how exploitation of new areas for human settlement and agriculture increases the likelihood that new infectious diseases will emerge. Cholera, plague, AIDS, dengue hemorrhagic fever, and urban/peri- urban visceral leishmaniasis are examples of new and reemerging diseases in the region. In tropical America, zoonotic visceral leishma- niasis caused by Leishmania chagasi, an intracel- lular protozoon,is a long-lasting infectious disease characterized by weight loss, cough, fever, diar- rhea, hepatosplenomegaly, and lethargy. Since not all symptoms appear simultaneously, and many other conditions manifest the same symptoms,the diagnosis is not always opportune. This disease is different from the anthroponotic form of visceral leishmaniasis found in Sudan and India. In the New World this disease is more common among poor, malnourished children (3) under 15 years of age who live in semiarid regions. The case-fatality rate is low if pentavalent antimony therapy is introduced promptly (4).The disease has also been reported in immunodepressed persons, especially those with HIV infection. The domestic dog is the principal animal reser- voir of L. chagasi and, as the constant companion of humans throughout the endemic-disease area, contributes to the dispersal of diseases during human migrations. The protozoon is transmitted from one mammalian host to another, including humans, primarily by the bite of a sand fly who has fed on an infected dog. In the Americas, the principal sand fly vector is Lutzomyia longipalpis (5).

the endemic-disease area, contributes to the dispersal of diseases during human migrations. The protozoon is transmitted from one mammalian host to another, including humans, primarily by the bite of a sand fly who has fed on an infected dog. In the Americas, the principal sand fly vector is Lutzomyia longipalpis (5). This 2- to 3-mm long fly has peridomestic and intradomiciliary habits (6) and avidly bites hu- mans at night,primarily during twilight,while the host is resting. Visceral leishmaniasis is usually diagnosed by identifying the parasite in spleen aspirates;micro- scope examination of bone marrow aspirates offers a satisfactory alternative. Specific antibodies may be detected by immune-enzymatic reactions and immunofluorescence, but these methods lack specificity and are too complex to be carried out in the field. TRALd, a recently developed diagnostic test, consists of a 60-second dipstick, based on a recombinant protein rK39 of a sequence of 298 amino acids and an improved serologic procedure (7). The procedure is undergoing field testing and appears to be a promising tool for control pro- grams. Although visceral leishmaniasis was previously known as a rural disease, large outbreaks and epidemics of visceral leishmaniasis have been re- ported recently in large cities in Brazil because of the favorable epidemiologic conditions associated with the reduction of the natural ecologic space of this zoonosis. Waves of droughts, lack of available farm land, and famine have led to a large migra- tion of the population, to the peripheral suburbs of large cities, creating densely populated settle- ments of shanties (favelas) with minimal infra- structure and sanitation. Most families that migrate are young, less-established farmers of peak child-bearing age; children under 15 years of age account for a large percentage of the entire population.In these communities,the newly intro- duced disease (parasite) encounters a vast number of nonimmune hosts who, because of poor living conditions, are malnourished; malnutrition is one of the prime risk factors for L. chagasi infection and visceral leishmaniasis (3). The habit of keep- ing domestic animals such as dogs, chickens, and horses in the back yard provides an abundance of blood meals for sand fly vectors and raises vector population densities dramatically.

malnourished; malnutrition is one of the prime risk factors for L. chagasi infection and visceral leishmaniasis (3). The habit of keep- ing domestic animals such as dogs, chickens, and horses in the back yard provides an abundance of blood meals for sand fly vectors and raises vector population densities dramatically. Two recent examples of reemerging visceral leishmaniasis in Brazil occurred in the cities of Teresina (population = 678,000), the capital of Piaui State, and Sao Luis (population = 918,000), the capital of Maranhao State. In Teresina, which has the best-documented records, an epidemic of visceral leishmaniasis occurred from 1981 to 1985; it was initially limited to rural settings, but later spread to peripheral areas of the city (9) (Figure 1).Spraying with residual insecticide helped bring the disease under control during the subsequent 3 years. Since 1989, visceral leishmaniasis has gradually reemerged;it reached epidemic levels by the end of 1992 and peaked in 1994. In the State of Maranhao, the disease reemerged in 1993, Dispatches Vol. 2, No. 2 --April-June 1996 145 Emerging Infectious Diseases (Figure 2), 10 years after a previous epidemic (1982 to 1986). In both Teresina and Sao Luis, the epidemics were preceded by prolonged, severe drought. The disease was found predominantly among young people who came from rural areas and lived in periurban, substandard housing and kept domestic animals in their back yards.The two states accounted for approximately 40% to 50% of the number of cases reported in the country during 1993 to 1994 (3,000/year). Emergency control plans based on rapid diag- nosis, complete treatment of human patients, spraying of residual insecticide, ultra-low volume spraying, elimination of infected (seropositive) dogs, and health education were initiated in these two cities in July 1994 (10). The incidence rate dropped markedly from January 1995, as ex- pected, since the incubation period of visceral leishmaniasis is 4 to 6 months (11, 12). The re- maining reported cases include those with longer incubation periods, as well as others, because of the lack of complete coverage of the control inter- ventions and reduced sensitivity of the current diagnostic tools.

as ex- pected, since the incubation period of visceral leishmaniasis is 4 to 6 months (11, 12). The re- maining reported cases include those with longer incubation periods, as well as others, because of the lack of complete coverage of the control inter- ventions and reduced sensitivity of the current diagnostic tools. Technical expertise and effective mechanisms to prevent visceral leishmaniasis epidemics and urban transmission exist, but they require social and political commitment as well as the allocation of funds to provide adequate sanitary and satisfac- tory housing conditions to the population at risk. Visceral leishmaniasis in the Americas must be addressed before it becomes a serious public health problem. Even though it is a zoonosis, control interventions are available that, when properly used, can eliminate urban transmis- sion and keep disease incidence in rural areas at a very low level. Jorge R. Arias, Ph.D.,* Pedro S. Monteiro, and Fabio Zicker, M.D., Ph.D. *Pan American Health Organization, Brasilia, Brazil; National Visceral Leishmaniasis Control Program, Ministry of Health, Brasilia, Brazil; Pan American Health Organization, Washington, D.C. References 1. Pan American Health Organization/World Health Or- ganization. Regional plan for action for combating new, emerging, and reemerging infectious diseases. Resolution CD38/17, August 1995. Pan American Health Organization, Washington, D.C., USA. 2. World Health Organization. Report of the Second WHO Meeting on Emerging Infectious Diseases. Document WHO/CDS/BVI/95.2. Geneva, Switzer- land: World Health Organization, January 1995. 3. Cerf BJ, Jones TC, Badaro R, Sampaio D, Carvalho EM,Rocha H,Texeira R,Johnson Jr WD.Malnutrition as a risk factor for severe visceral leishmaniasis. J Infect Dis 1987;156:1030-2. 4. World Health Organization. Control of the leishmani- ases: report of a WHO expert committee. Technical Report Series 793.Geneva,Switzerland:World Health Organization, 1990. 5. Lainson R, Shaw JJ. Epidemiology and ecology of leishmaniasis in Latin America. Nature 1978;273 (Parasit Suppl):595-600. 6. Young DG, Arias JR. Flebotomos: vectores de leishma- niasis en Las Americas.Pan American Health Organi- zation, 1992: Cuaderno Tecnico No. 33. Pan American Health Organization, Washington, D.C., USA. 7. Reed SG, Sheffler WG, Burns Jr JM, Scott JM, Orge MG, Ghalib HW, et al. An improved serodiagnostic procedure for visceral leishmaniasis. Am J Trop Med Hyg 1990;43:632-9. 8. Wilson ME.

Americas.Pan American Health Organi- zation, 1992: Cuaderno Tecnico No. 33. Pan American Health Organization, Washington, D.C., USA. 7. Reed SG, Sheffler WG, Burns Jr JM, Scott JM, Orge MG, Ghalib HW, et al. An improved serodiagnostic procedure for visceral leishmaniasis. Am J Trop Med Hyg 1990;43:632-9. 8. Wilson ME. Travel and emergence of infectious dis- eases. Emerging Infectious Diseases 1995;1:39-49. 9. Costa CHN, Pereira HF, Araujo MV. Epidemia de leishmaniose visceral no estado do Piaui,Brasil,1980- 1986. Rev Saude Publ S Paulo 1990;24:361-72. 10. Ministerio da Saude. Controle, diagnostico e tratamento da leishmaniose visceral (calazar). nor- mas tecnicas. Normas Tecnicas. 1a. edicao, Brasilia, Brazil: Fundacao Nacional de Saude, Brasil, 1994. 11. Manson-Bahr PEC, Southgate BA, Harvey AEC. De- velopment of kala-azar in man after inoculation with a Leishmania from a Kenya sandfly. Br Med J 1963;I:1208-10. 12. Kirk R. Studies in leishmaniasis in Anglo-Egyptian Sudan. Trans R Soc Trop Med Hyg 1942;35:257-70. Figure 1. Number of cases of visceral leishmaniasis from the state of Piaui, Brazil, 1971 to 1994. Figure 2. Number of cases of visceral leishmaniasis from the state of Maranhao, Brazil, 1979 to 1994. Dispatches Emerging Infectious Diseases 146 Vol. 2, No. 2 --April-June 1996

Molecular Epidemiology of Pneumocystis carinii Pneumonia Pneumocystis carinii pneumonia (PCP) was first recognized as a distinct clinical entity in orphanages in Europe during World War II (1). Today it is the most frequent serious opportunistic infection in AIDS patients. Despite advances in research, numerous questions remain regarding the basic biology and epidemiology of P. carinii. Transmission and Patient Care Although reactivation of latent infections has long been considered the primary explanation for PCP in immunosuppressed patients, over the years a steady flow of reports has described clus- ters of PCP cases (2). In addition, recent studies have suggested that the duration of latency is very limited, i.e., usually less than 1 year (3,4). Still other studies have demonstrated genetic vari- ation in PCR-amplified P. carinii DNA from the lungs of patients during subsequent PCP episodes (5). Together, these observations provide strong circumstantial evidence of person-to-person transmission of P. carinii. Consequently, estab- lishing the role that person-to-person transmis- sion plays in the epidemiology of PCP is urgent. Another important area of PCP epidemiology is determining the predisposing factors for disease. The most frequently discussed predictor of disease is CD4+ cell count, specifically as it relates to care and management of AIDS patients (6); however, it has long been known that malnutrition can be an important contributor (7). The degree to which other factors such as viral infections or pneumoni- tis of other causes, may come into play, is yet to be shown. Much can also be learned regarding the epidemiology of PCP in HIV-infected infants. Re- cent studies report that primary infections in these infants often develop when the child is 3 to 6 months old (8,9). The source of these patients' P. carinii infections (i.e., the hospital setting, their mothers, other children, or an environmental source) is not known. Clinicians working with AIDS patients need a sensitive, reliable, and noninvasive tool for early detection and diagnosis of PCP infections (10,11).

months old (8,9). The source of these patients' P. carinii infections (i.e., the hospital setting, their mothers, other children, or an environmental source) is not known. Clinicians working with AIDS patients need a sensitive, reliable, and noninvasive tool for early detection and diagnosis of PCP infections (10,11). Besides the standard procedures of bronchoalveolar lavage (BAL) and induced spu- tum (IS) sampling, recent studies indicate that it is possible to amplify P. carinii DNA sequences by polymerase chain reaction (PCR) directly from blood or serum samples and from nasopharyngeal aspirates of PCP patients (11,12). Further studies are needed to confirm that a PCR-based diagnostic tool superior to microscopy can be adapted for use in clinical settings. A serologic tool that will dis- tinguish recent PCP infections from those past is also needed. Prophylaxis failures have been reported for both trimethoprim-sulfamethoxazole (TMP-SMX) and pentamidine (13-16). Studies evaluating these cases, however, are frequently complicated by the difficulties in assessing and confirming patient compliance with the prophylaxis regimen. The only factor that has a significant correlation with failure in most cases,however,is the patient's CD4+ T lymphocyte count (14). Although this cor- relation would be expected because of the general increased risk for PCP associated with CD4+ cell depletion (6) and the increase in prophylaxis com- plications in HIV-infected patients (17), these drugs may not eliminate all organisms, and some degree of patient immunity may be required to clear or control the infection. What role, if any, specific antimicrobial resistance mechanisms play in the reported treatment failures has not been shown; however, the emergence of resistance is always a threat. Likewise, long-term TMP-SMX prophylaxis increases the possibility for the selec- tion of antimicrobial resistance in bacterial patho- gens, some of which are important potential causes of pneumonia in HIV-infected patients (18). Identifying potential antimicrobial resistance mechanisms in P. carinii is difficult because of the lack of an established culture system for human P. carinii that would allow traditional antimicro- bial sensitivity testing. At least three separate lines of data suggest that P.

pneumonia in HIV-infected patients (18). Identifying potential antimicrobial resistance mechanisms in P. carinii is difficult because of the lack of an established culture system for human P. carinii that would allow traditional antimicro- bial sensitivity testing. At least three separate lines of data suggest that P. carinii is a commonly encountered organ- ism: the high seroprevalence rates reported in normal populations (19), the rapid rate at which infants acquire primary infections (8) and AIDS patients become reinfected after successful treat- ment (20), and the amplification of P. carinii-spe- cific DNA from ambient air sampled from the environment (e.g., an apple orchard) (21) and from rooms of animals and patients with PCP (22). Airborne transmission has been demonstrated for PCP in rats (23-26) and is by far the most likely mechanism proposed for natural exposure to P. carinii in humans (2,22). Given the similarities Commentary Vol. 2, No. 2 --April-June 1996 147 Emerging Infectious Diseases between P. carinii and various fungal agents and the enigma surrounding the issue of environ- mental sources for P. carinii, it has been suggested that P. carinii may in fact be a dimorphic fungus, ubiquitous in the environment and disseminated by airborne spores (27). Identifying the specific environmental source or sources of P. carinii is critical to understanding the epidemiology of PCP and establishing guidelines for preventing its transmission. It is generally accepted that P. carinii strains from rats do not infect humans and that human strains do not infect rats; however, we do not know the host boundaries for a given P. carinii strain or if all isolates from a given host display the same degree of host restrictions (28,29).In fact,a careful evaluation of the available data concerning P. carinii of numerous hosts suggests that P. carinii may represent a collection of diverse fungal spe- cies (30). Like drug resistance research, studies aimed at strain/species characterization are gen- erally hindered by the difficulties in culturing human P. carinii and the lack of refined molecular biological methods that allow strain identification and characterization.

represent a collection of diverse fungal spe- cies (30). Like drug resistance research, studies aimed at strain/species characterization are gen- erally hindered by the difficulties in culturing human P. carinii and the lack of refined molecular biological methods that allow strain identification and characterization. Molecular Biologic Techniques and Specific Epidemiologic Issues One of the essential reasons for cultivating any particular pathogen is for strain identification and characterization that would elucidate such spe- cific phenotypic characteristics as virulence fac- tors, antimicrobial sensitivity levels, and factors associated with transmissibility.The isolation and cultivation of individual strains, and ultimately of clones, would provide a homogeneous population of organisms from which the desired information can be obtained and a pure source of genetic ma- terial for constructing DNA libraries and identify- ing relevant genes. In the absence of cultivation,investigators have been able to begin addressing some of the basic epidemiologically important issues by applying PCR-based technology. In these studies, the DNA sequence of specific genetic loci from P. carinii is usually amplified from BAL, IS, or serum samples from PCP patients, using highly specific oligonu- cleotide primers. Inherent problems exist in this approach (which are discussed below); however, the approach has allowed the identification of genotypic differences in P. carinii populations sampled from the lungs of different patients and even from the lungs of the same patient during different PCP episodes. Great potential exists in applying this technology to develop molecular pro- files of P. carinii isolates that could ultimately allow the particular genotypes to be linked to specific epidemiologically relevant phenotypes. MolecularTyping Five to ten different genetic loci have been identified as potentially informative for molecular characterization and typing (30-33). Concerning the typing that has actually been performed on human samples, the primary loci evaluated in- clude:1) a 346-bp region of the mitochondrial large subunit rRNA gene (mt lsrRNA) (10) and 2) a 550-bp fragment containing the nuclear ribosomal internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) (34). When these loci are considered collectively,nucleotide variation can be detected at approximately 37 different positions.

46-bp region of the mitochondrial large subunit rRNA gene (mt lsrRNA) (10) and 2) a 550-bp fragment containing the nuclear ribosomal internal transcribed spacer regions 1 and 2 (ITS1 and ITS2) (34). When these loci are considered collectively,nucleotide variation can be detected at approximately 37 different positions. Work is in progress in several laboratories, both to type pa- tient isolates according to the available loci and to identify additional genetic loci to more thoroughly define a given genotype. The primary obstacles to the development of a molecular typing scheme based on PCR-amplified DNA sequence data obtained from PCP patients include the following: 1) multiple strains may in- fect a single patient at a given time; 2) a diploid organism of a single strain may be heterozygous with respect to a particular polymorphic locus; 3) presumed single genes could have multiple copies in a single genome, which could give the appear- ance of genetic polymorphism; 4) amplified DNA sequence data might be confounded because of other fungal agents such as Cryptococcus or Can- dida; and 5) inferences that can be drawn from restricted sequence data (i.e., gene typing versus strain typing) are limited. Although these prob- lems are not insurmountable, they must be con- sidered when evaluating data obtained by this approach. We propose the following recommenda- tions. Recommendations Molecular Epidemiology 1. Recent advances in molecular-based typing should be combined with epidemiologic studies to investigate the transmission of P. carinii and new strategies for control. Commentary Emerging Infectious Diseases 148 Vol. 2, No. 2 --April-June 1996 2. Additional genomic regions must be identi- fied for use in typing, along with the genetic loci that are available. These new loci must be shown to represent single-copy genes. Also, new molecu- lar approaches should be developed that will in- crease the current capacity to resolve genotypic variation among P. carinii strains. 3. Genetic variation should be investigated among P. carinii strains that could be linked to variations in factors such as strain virulence, drug resistance, or transmissibility. 4. The critical issue regarding person-to-person transmission is not so much whether it occurs, as whether it contributes to infection significantly more than airborne sources in the environment.

arinii strains that could be linked to variations in factors such as strain virulence, drug resistance, or transmissibility. 4. The critical issue regarding person-to-person transmission is not so much whether it occurs, as whether it contributes to infection significantly more than airborne sources in the environment. Thus, it must be determined whether there is any benefit to establishing complex protocols that en- sure that patients are carefully protected from each other if they can become infected from other sources in the environment. Consequently, the importance of person-to-person transmission in the epidemiology of PCP should be defined. 5. The role of latent P. carinii infection as a source of PCP in immunocompromised persons should be clarified. Diagnosis,Treatment,and Prevention 1. New tools for noninvasive early diagnosis of PCP, including culture systems, molecular ap- proaches, and serologic tests that can distinguish recent and past PCP infections are needed. 2. In the United States, clinician compliance with recently published U.S. Public Health Serv- ice/Infectious Diseases Society of America guide- lines on the treatment and prophylaxis of PCP should be evaluated. 3. Studies should be initiated to develop addi- tional drugs for PCP treatment and prophylaxis. 4. New approaches for improving patient com- pliance with prescribed PCP prophylaxis must be devised and evaluated. 5.Methods for detecting the possible emergence of drug resistance to P. carinii should be stand- ardized. 6. Standard decontamination procedures for respiratory therapy equipment and pulmonary diagnostic instruments should be evaluated to confirm that they effectively eliminate all viable P. carinii. Environmental Reservoirs and General Biology 1. Environmental sources and the coinciding infective stage(s) of P. carinii should be detected and evaluated. 2. The host range of P. carinii from various sources (i.e., to what degree are humans suscepti- ble to P. carinii from nonhuman sources?) should be determined. Acknowledgments We thank the following colleagues for their contribu- tions: C. William Angus, Marilyn S. Bartlett, Jeanne M. Bertolli, Suzanne Binder, Daniel G. Colley, Frank H. Col- lins, Melanie T. Cushion, Alexandre J. DaSilva, Jeffrey S. Duchin,Brian R.Edlin,Walter T.Hughes,John A.Jernigan, Dennis D. Juranek, Jonathan R. Kaplan, Scott P. Keely, Chao-Hung Lee, Joseph E. McDade, Stephen A. Moser, Norman J. Pieniazek, Frank F. Richards, James W. Smith, Eleanor K. Spicer, James R.

ey, Frank H. Col- lins, Melanie T. Cushion, Alexandre J. DaSilva, Jeffrey S. Duchin,Brian R.Edlin,Walter T.Hughes,John A.Jernigan, Dennis D. Juranek, Jonathan R. Kaplan, Scott P. Keely, Chao-Hung Lee, Joseph E. McDade, Stephen A. Moser, Norman J. Pieniazek, Frank F. Richards, James W. Smith, Eleanor K. Spicer, James R. Stringer, and Craig M. Wilson. Mary E. Bartlett provided editorial assistance. Charles B. Beard, Ph.D., and Thomas R. Navin, M.D. Centers for Disease Control and Prevention Atlanta, Georgia, USA References 1. Walzer PD, editor. Pneumocystis carinii pneumonia. New York: Marcel Dekker, 1994. 2. Cushion MT. Transmission and epidemiology. In: Walzer PD, editor. Pneumocystis carinii pneumonia. New York: Marcel Dekker, 1994:123-40. 3. Chen W, Gigliotti F, Harmsen AG. Latency is not an inevitable outcome of infection with Pneumocystis carinii. Infect Immun 1993;61:5406-9. 4. Vargas SL, Hughes WT, Wakefield AE, Oz HS. Limited persistence in and subsequent elimination of Pneumo- cystis carinii from the lungs after P.cariniipneumonia. J Infect Dis 1995;172:506-10. 5. Keely SP, Stringer JR, Baughman RP, Linka MJ, Walzer PD, Smulian AG. Genetic variation among Pneumocystis carinii hominis isolates in recurrent pneumocystosis. J Infect Dis 1995;172:595-8. 6. Phair J, Munoz A, Detels R, Kaslow R, Rinaldo C, Saah A. The risk of Pneumocystis carinii pneumonia among men infected with human immunodeficiency virus type 1. N Engl J Med 1990;322:161-5. 7. Hughes WT, Price RA, Havron SF, Sisko F, Havron SF, Kafatos AG, Schonland M, et al. Protein-calorie mal- nutrition: a host determinant for Pneumocystis carinii infection. Am J Dis Child 1974;128:44-52. 8. Hughes WT. 1994. Clinical manifestations in children. In:Walzer PD,editor.Pneumocystis cariniipneumonia. New York: Marcel Dekker, 1994:319-29. 9. Simonds RJ, Lindegren ML, Thomas P, Hanson D, Caldwell B, Scott G, et al. Prophylaxis against Pneu- mocystis carinii pneumonia among children with per- inatally acquired human immunodeficiency virus infection in the United States. N Engl J Med 1995;332:786-90. Commentary Vol. 2, No. 2 --April-June 1996 149 Emerging Infectious Diseases 10. Wakefield AE, Pixley FJ, Banerji S, Sinclair K, Miller RF, Moxon ER, et al. Detection of Pneumocystis carinii with DNA amplification. Lancet 1990; 336:451-3. 11. Atzori C, Lu J-J, Jiang B, Bartlett MS, Orlando G, Queener SF, Smith JW, et al.

ommentary Vol. 2, No. 2 --April-June 1996 149 Emerging Infectious Diseases 10. Wakefield AE, Pixley FJ, Banerji S, Sinclair K, Miller RF, Moxon ER, et al. Detection of Pneumocystis carinii with DNA amplification. Lancet 1990; 336:451-3. 11. Atzori C, Lu J-J, Jiang B, Bartlett MS, Orlando G, Queener SF, Smith JW, et al. Diagnosis of Pneumocys- tis carinii pneumonia in AIDS patients by using po- lymerase chain reactions on serum specimens. J Infect Dis 1995;172:1623-6. 12. Richards CGM, Wakefield AE, Mitchell CD. Detection of pneumocystis DNA in nasopharyngeal aspirates of leukaemic infants with pneumonia. Arch Dis Child 1994;71:254-5. 13. Montgomery AB, Feigal DW, Sattler F. Pentamidine aerosol versus trimethoprim-sulfamethoxazole for Pneumocystis carinii in acquired immune deficiency syndrome. Am J Respir Crit Care Med 1995;151:1068- 74. 14. Saah AJ, Hoover DR, Peng Y, Phair JP, Visscher B, Kingsley LA, et al. Predictors for failure of Pneumocys- tis carinii pneumonia prophylaxis. JAMA 1995;273:1197-1202. 15. Lecuit M, Livartowski J, Vons C, Goujard C, Lamaigre G, Delfraissy J-F, et al. Resistance to trimethoprim- sulfamethoxazole and sensitivity to pentamidine ther- apy in an AIDS patient with hepatosplenic pneumocystosis. AIDS 1994;8:1506-7. 16. Torres RA, Barr M, Thorn M, Gregory G, Keily S, Chanin E, et al. Randomized trial of dapsone and aerosolized pentamidine for the prophylaxis of Pneu- mocystis carinii pneumonia and toxoplasmic encepha- litis. Am J Med 1993;95:573-83. 17. Walker RE,Masur H.Current regimens of therapy and prophylaxis. In: Walzer PD, editor. Pneumocystis carinii pneumonia. New York: Marcel Dekker, 1994:439-66. 18. Schwartz RH, Khan WN, Akram S. Penicillin and trimethoprim-sulfamethoxazole-resistant pneumo- cocci isolated from blood cultures of three infants in metropolitan Washington, DC: a harbinger of serious future problems? Pediatr Infect Dis J 1991;10:782-3. 19. Smulian AG, Walzer PD. Serological studies of Pneu- mocystis carinii infection. In: Walzer PD, editor. Pneu- mocystis carinii pneumonia.New York:Marcel Dekker, 1994:141-51. 20. Dohn MN,Frame PT.Clinical manifestations in adults. In:Walzer PD,editor.Pneumocystis cariniipneumonia. New York: Marcel Dekker, 1994:331-59. 21. Wakefield AE. Detection of DNA sequences identical to Pneumocystis carinii in samples of ambient air. J Euk Microbiol 1994;41:116S. 22. Bartlett MS, Lee C-H, Lu J-J, Bauer NL, Betts JF, McLaughlin GL, et al. Pneumocystis carinii detected in air. J Euk Microbiol 1994;41:75S.

ia. New York: Marcel Dekker, 1994:331-59. 21. Wakefield AE. Detection of DNA sequences identical to Pneumocystis carinii in samples of ambient air. J Euk Microbiol 1994;41:116S. 22. Bartlett MS, Lee C-H, Lu J-J, Bauer NL, Betts JF, McLaughlin GL, et al. Pneumocystis carinii detected in air. J Euk Microbiol 1994;41:75S. 23. Hendley JO, Weller TH. Activation and transmission in rats of infection with Pneumocystis. Proc Soc Exp Biol Med 1971;137:1401-4. 24. Walzer PD, Schnelle V, Armstrong D, Rosen PP. Nude mouse: a new experimental model for Pneumocystis carinii infection. Science 1977;197:177-9. 25. Hughes WT, Bartley DL, Smith BM. A natural source of infection due to Pneumocystis carinii. J Infect Dis 1983; 147:595. 26. Hughes WT. Natural habitat and mode of transmis- sion. In: Pneumocystis carinii pneumonitis, vol I. Boca Raton, FL: CRC Press, 1987:97-105. 27. Dei-cas E,Cailliez JC,Palluault F,Aliouat EM,Mazars E, Soulez B, et al. Is Pneumocystis carinii a deep mycosis-like agent? Eur J Epidemiol 1992; 8:460-70. 28. Smith JW, Bartlett MS. Laboratory diagnosis of pneu- mocystosis. Clin Lab Med 1991;11:957-75. 29. Armstrong MYK, Cushion MT. In vitro cultivation. In: Walzer PD, editor. Pneumocystis carinii pneumonia. New York: Marcel Dekker, 1994:3-24. 30. Stringer JR. The identity of Pneumocystis carinii: not a single protozoan, but a diverse group of exotic fungi. Infect Agents Dis 1993;2:109-17. 31. Edman JC, Sogin ML. Molecular phylogeny of Pneu- mocystis carinii. In: Walzer PD, editor, Pneumocystis carinii Pneumonia. New York: Marcel Dekker, Inc., 1994:91-105. 32. The Pneumocystis Workshop. Revised nomenclature for Pneumocystis carinii. J Euk Microbiol 1994;41:121S-22S. 33. Lu J-J, Chen C-H, Bartlett MS, Smith JW, Lee C-H. Comparison of six different PCR methods for detection of Pneumocystis carinii. J Clin Microbiol 1995;33:2785-8. 34. Lu J-J,Bartlett MS,Shaw MM,Queener SF,Smith JW, Ortiz-Rivera M, et al. Typing of Pneumocystis carinii strains that infect humans based on nucleotide se- quence variations of internal transcribed spacers of rRNA genes. J Clin Microbiol 1994;32:2904-12. Commentary Emerging Infectious Diseases 150 Vol. 2, No. 2 --April-June 1996

Needed: Comprehensive Response to the Spread of Infectious Diseases In his article "Globalization,International Law, and Emerging Infectious Diseases," Fidler recog- nizes that biological agents travel by themselves or with their hosts without any recognition of, or regard for,political borders.He notes that with the continued expansion of economic commerce across continents and more rapid transport and travel, persons infected with infectious diseases of very short incubation periods can act as vectors across several nations before they even become sympto- matic. The protective effect of clipper ship travel is long gone. Fidler examines the need for international treaties, agreements, and policies to manage the spread of new or reemerging infections diseases. His concern is that the current international cli- mate requires more enforceable treaties with ade- quate resources to identify, track, interfere with, and contain the spread of infectious diseases per- ceived as an international or global threat. International cooperation within the existing legislative mechanisms has, on occasion, been very successful. International eradication of smallpox was successful because a specific, cost- effective, efficient vaccine was developed; the dis- ease attacked persons regardless of their economic, political, racial, religious, or social affili- ations; the amount of funding was adequate; and all nations recognized the benefits of the eradica- tion program. A similar effort currently in pro- gress to eradicate poliomyelitis will also be successful because of international cooperation. In contrast, international control of other infec- tious diseases, such as malaria and tuberculosis, has been attempted for decades with considerably less success. Notwithstanding the lack of effica- cious vaccines, the reality is that only very limited resources are being committed to prevent and treat all infectious diseases. Outbreaks of Ebola virus infection and plague are routinely reported in the local, national, and international press.

ades with considerably less success. Notwithstanding the lack of effica- cious vaccines, the reality is that only very limited resources are being committed to prevent and treat all infectious diseases. Outbreaks of Ebola virus infection and plague are routinely reported in the local, national, and international press. However, the continued increased incidence and prevalence of tuberculosis, AIDS, and other sexu- ally transmitted diseases are accepted by many as problems of the poor,the immoral,and the expend- able portion of society. Local, national, and inter- national awareness and continued interest are significant problems. International cooperation must extend beyond merely restricting the natural spread of specific diseases. One also has to recognize the need for effective international treaties to prevent the use of biological agents in either tactical or strategic circumstances. Fear of combatants using biologi- cal agents on military and civilian targets inten- sified during and since the Gulf War. The possibility of biological terrorism is no longer lim- ited to the imagination of fiction writers. Fidler does not stress the issue of nonnatural outbreaks of diseases; a global need for an improved non-ad hoc response to emerging infectious disease agents should be completely considered by civilian and military planners. The threat of infectious diseases as weapons provides an additional incen- tive for cooperation among governments. Harold M. Ginzburg, M.D., J.D., M.P.H. Department of Psychiatry and Neurology Tulane University Medical Center New Orleans, Louisiana, USA Commentary Vol. 2, No. 2 --April-June 1996 151 Emerging Infectious Diseases

Transfusion-Associated Malaria To the Editor: A recent article by Zucker (1) described two cases of malaria that were probably transfusion associated. A case of transfusion- associated malaria in which the source was iden- tified was reported in San Francisco in 1991. The case was in an elderly man in whom malaria infection developed after coronary bypass surgery. The patient was born in China and immigrated to the United States in 1940. His only travel outside the United States was a trip to Hong Kong in 1951 for 6 months. The patient's wife was born in China and had malaria in 1941 during World War II. She received no treatment at that time or at any other time. She came to the United States in 1960 and has not left the country since. The patient had six donors, five of whom had no history of malaria, and had negative serologic test results for all four malaria species. Both the pa- tient and his wife had blood smears positive for P. malariae. The patient's wife had positive sero- logic test results for P. vivax and P. ovale (1:64), for P. falciparum (1:258), and for P. malariae (1:1024). Frances Taylor, M.D., M.P.H. Director of Communicable Disease Control, City and County of San Francisco, Department of Public Health, Bureau of Epidemiology, Disease Control, and AIDS, San Francisco, California, USA Reference 1. Zucker J. Changing patterns of autochthonous malaria transmission in the United States: A review of current outbreaks.Emerging Infectious Diseases 1996:2:37-43. Reply to F. Taylor: Dr. Taylor's letter calls attention to the small but important number of induced malaria cases that occur in the United States. From 1957 to 1994, 101 such cases were reported to the Centers for Disease Control and Prevention (CDC); these (including the 1990 case described by Dr. Taylor [1]) are reviewed annually and reported by CDC (2). The occasional occur- rence of induced malaria further emphasizes the importance of including malaria in the differential diagnosis of fevers of unknown origin, even in patients who have not traveled to countries where malaria is endemic.

case described by Dr. Taylor [1]) are reviewed annually and reported by CDC (2). The occasional occur- rence of induced malaria further emphasizes the importance of including malaria in the differential diagnosis of fevers of unknown origin, even in patients who have not traveled to countries where malaria is endemic. Preventing induced malaria requires screening potential blood, tissue, and or- gan donors and deferring those with a history of malaria or travel to malarious areas. Further- more, timely surveillance must be maintained to detect induced cases promptly, identify infected blood donors, and prevent additional cases. The case described by Dr. Taylor was not in- cluded in "Changing patterns of autochthonous malaria transmission in the United States: a re- view of recent outbreaks" (3) because it was a case of induced rather than autochthonous malaria. Each reported malaria case is classified according to standardized terminology (4).Imported malaria (which accounts for most cases in this country) is acquired outside the United States and its territo- ries. Malaria acquired within the United States is rare and occurs by one of three mechanisms: Autochthonous malaria is acquired through the bite of an infective mosquito. Congenital malaria is acquired when a child is infected in utero. In- duced malaria is transmitted by mechanical means such as transfusion of blood or blood prod- ucts, organ transplant, deliberate infection for malariotherapy, or contaminated needles or injec- tion equipment. Congenital and induced cases were not included in this review. When an investigation fails to identify the source of transmission and a case cannot be epidemiologically linked to another case of ma- laria, the case is classified as cryptic. Most cryptic cases are believed to be autochthonous, and there is often evidence to suggest mosquito-borne trans- mission, even when the source of infection re- mains unidentified. For this reason, most cryptic cases were included in this review of autochthonous malaria. The two exceptions noted in the article were excluded because both patients had recent histories of blood transfusion, suggest- ing that their infections were induced. Jane R. Zucker, M.D., M.Sc., and S. Patrick Kachur, M.D., M.P.H. Centers for Disease Control and Prevention, Atlanta, Georgia, USA References 1. Zucker JR, Barber AM, Paxton LA, Schultz LJ, Lobel HO, Roberts JM, et al. Malaria Surveillance--United States,1992.In:CDC Surveillance Summaries,October 20, 1995.

ere induced. Jane R. Zucker, M.D., M.Sc., and S. Patrick Kachur, M.D., M.P.H. Centers for Disease Control and Prevention, Atlanta, Georgia, USA References 1. Zucker JR, Barber AM, Paxton LA, Schultz LJ, Lobel HO, Roberts JM, et al. Malaria Surveillance--United States,1992.In:CDC Surveillance Summaries,October 20, 1995. MMWR 1995:44(SS-5):1-17. 2. Centers for Disease Control. Malaria Surveillance An- nual Summary, 1990. Atlanta: Centers for Disease Con- trol, 1991. 3. Zucker JR. Changing patterns of autochthonous ma- laria transmission in the United States: a review of recent outbreaks. Emerging Infectious Diseases 1996;2:37-43. 4. World Health Organization. Terminology of malaria and malaria eradication.Geneva:World Health Organi- zation, 1963:32. Letters Emerging Infectious Diseases 152 Vol. 2, No. 2-- April-June 1996

An Outbreak of Hemolytic Uremic Syndrome due to Escherichia coli O157:H-: Or Was It? To the Editor: Since the first reported out- breaks of hemolytic uremic syndrome (HUS) and related conditions more than 10 years ago (1), outbreaks of HUS due to Escherichia coli O157 have been reported from many parts of the world, particularly North America and Europe. While most of these reports have incriminated the motile strains of serotype O157:H7, nonmotile serotypes (e.g., O157:H-) have also been associated with HUS; these two serotypes are most commonly associated with both outbreaks and sporadic cases of HUS and related conditions. Over the last dec- ade, a number of techniques for the rapid identifi- cation of these organisms have been developed. Of these, the use of sorbitol-MacConkey agar (2) has perhaps been the most valuable. This technique is based on the fact that these organisms rarely ferment sorbitol on primary isolation, while most other E. coli usually ferment this substrate. We believe that outbreaks due to other enterohemor- rhagic E. coli may have been attributed to sero- group O157 because of the limited technology used in investigating these outbreaks. No outbreaks of HUS due to serogroup O157 have occurred in Australia despite sporadic cases of HUS caused by such strains. Other serogroups (particularly serotype O111:H-) have been associ- ated with most cases of HUS and related condi- tions in Australia (3). No outbreak of HUS had been reported in Australia until January 1995, when an outbreak associated with the consump- tion of contaminated mettwurst (fermented sau- sage) was reported from South Australia (4). Twenty-three children with HUS were hospital- ized. Most required hemodialysis; one died. Vero- cytotoxigenic strains of E. coli O111 producing Shiga-like toxin (SLT) I and II were isolated from 19 patients and from samples of mettwurst. In addition, strains of E. coli O157:H- that produced SLT-I and SLT-II were isolated from three of the patients and the mettwurst. These strains did not ferment sorbitol on the sorbitol-MacConkey agar, which facilitated their isolation.

toxin (SLT) I and II were isolated from 19 patients and from samples of mettwurst. In addition, strains of E. coli O157:H- that produced SLT-I and SLT-II were isolated from three of the patients and the mettwurst. These strains did not ferment sorbitol on the sorbitol-MacConkey agar, which facilitated their isolation. The predominant O111 strains were sorbitol-positive, unlike the O111 strains, recently described as being sorbitol- negative (5).Symptoms of the patients from whom the O157:H- strains were isolated, in addition to E. coli O111:H-, were not significantly different from those of the patients whose specimens yielded only E. coli O111:H-. In addition to O111 (and O157), other serotypes of enterohemorrhagic E. coli, including strains of serogroup O23, O26, and O91, were isolated from the patients. How- ever, antibodies to O111 were detected in nearly all patients, which indicates the serogroup's lead- ing role in the outbreak.The isolation of serogroup O157 is comparatively easy; therefore, it is less likely that these strains would have been missed, than it is that O111 and other serotypes would have been. Even though a negative finding can never be considered conclusive, we consider the inability to isolate serogroup O157 more conclu- sive than the same result for other serotypes. It has frequently been suggested that the O157 se- rogroup is cleared from the patient relatively rap- idly, which makes its isolation difficult or impossible. We found a similar situation with other enterohemorrhagic E. coli serotypes. The fact that most patients elicited an O111 antibody response (and no anti-O157) almost certainly proves this serotype's causal role in this outbreak. The laboratory in South Australia was particu- larly well disposed to deal with such an outbreak because some of its ongoing research programs included studies on aspects of enterohemorrhagic E. coli and related organisms. The most sophisti- cated molecular biologic techniques were immedi- ately available to investigate the outbreak accurately and confirm epidemiologic leads re- garding a common source. Polymerase chain reac- tion (PCR) played a major role not only in identifying SLT-I, SLT-II, and SLT-I and SLT-II producing bacteria in the stool of patients,but also in identifying the suspected source (mettwurst). In addition, PCR, utilizing sequences specific for the O111 serogroup, enabled this serogroup to be rapidly identified in patients' feces samples and suspected source material.