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Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is a devastating form of respiratory failure characterized by intense inflammation and increased permeability in the lungs that usually develops in response to a major insult such as sepsis, trauma, pneumonia, burns, or multiple transfusions [1]. Despite the common occurrence of these risk factors, only a minority of patients who have these injuries develops ALI [2], [3]. ALI/ARDS is now recognized as being more prevalent than initially thought, with an age-adjusted incidence of 86.2/100,000 person-years, with a mortality of 38.5%, and with significant morbidity among the survivors [4], [5].

f these risk factors, only a minority of patients who have these injuries develops ALI [2], [3]. ALI/ARDS is now recognized as being more prevalent than initially thought, with an age-adjusted incidence of 86.2/100,000 person-years, with a mortality of 38.5%, and with significant morbidity among the survivors [4], [5]. Because ALI has such high mortality and morbidity, any intervention that could prevent or treat ALI would have a significant impact on critical care medicine and on public health. Epidemiologic studies can contribute to prevention and treatment by determining the risk factors associated with variable susceptibility and outcomes that could be modified to decrease the risk of developing the disease or of having a poor outcome. The current understanding of why some patients develop and die from ALI and others do not is incomplete. Recently, discoveries about the genetic control and regulation of innate immunity and inflammatory response have raised the question of whether the multiple polymorphic alleles of genes that encode for cytokines and other mediators of inflammation may result in phenotypic differences in host inflammatory response. These differences may account for some of the heterogeneity in individual susceptibility to and prognosis in ARDS.

ve raised the question of whether the multiple polymorphic alleles of genes that encode for cytokines and other mediators of inflammation may result in phenotypic differences in host inflammatory response. These differences may account for some of the heterogeneity in individual susceptibility to and prognosis in ARDS. Since the initial description of ALI, there has been much research on the role of complement, endotoxin, and pro- and anti-inflammatory cytokine response in the pathogenesis and course of ALI/ARDS [6]. Protein biomarkers, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), plasminogen activator inhibitor-1, surfactant protein B (SFTPB), and von Willebrand's factor antigen, may be useful in predicting either development of or outcomes in ALI [7], [8], [9], [10], [11]. Although research on protein biomarkers in ALI/ARDS has contributed greatly to the understanding of the pathogenesis of ALI, it has not yet led to novel interventions.

protein B (SFTPB), and von Willebrand's factor antigen, may be useful in predicting either development of or outcomes in ALI [7], [8], [9], [10], [11]. Although research on protein biomarkers in ALI/ARDS has contributed greatly to the understanding of the pathogenesis of ALI, it has not yet led to novel interventions. Genetic epidemiology is a relatively new discipline that seeks to determine the role of genetic factors and their interactions with the environment in the occurrence of the disease or its outcome within a population [12]. Genetic epidemiology has been applied to the study of ALI. only recently. Genes hold several advantages over protein markers of lung injury, especially for possible prevention. Unlike cytokines, which can vary with the precipitant factor for ALI and with the time course of critical illness, a person's genotype is constant throughout the individual's life, regardless of health status. Thus, there is inherently less variability to the determination of genotypes than protein markers. The variation of many of the protein markers before and during critical illness means that the window of opportunity for assessment must be consistent and is likely to be narrow. Such a window for assessment may be especially impractical in the prevention of ALI/ARDS, because lung injury tends to develop rapidly, within hours to days of the predisposing injury. In addition, in ALI regional differences in the expression and concentrations of some cytokines, such as TNF-α, means that biomarkers may be best measured from alveolar fluid [13]. Measurements from the lungs are invasive, are vulnerable to technical variation, and are not always appropriate for severely hypoxic patients who have ARDS or for the nonintubated patients at risk. DNA for genotype assessment can be obtained easily from peripheral blood samples, and thus genotype assessment can be performed safely for any patient. Another advantage of genes is that any true genetic association with the disease is unlikely to be an epiphenomenon related to lung injury. Any variation in a protein marker may be a product rather than the cause of developing lung injury. The individual's genotype, however, precedes the lung injury and the precipitant to lung injury. Thus, any true genetic association supports the biologic causality of the gene or its product in the development of ALI/ARDS and the targeting of the gene in future preventions.

her than the cause of developing lung injury. The individual's genotype, however, precedes the lung injury and the precipitant to lung injury. Thus, any true genetic association supports the biologic causality of the gene or its product in the development of ALI/ARDS and the targeting of the gene in future preventions. Last, the invariant nature of the genome means that an individual's genetic predisposition to developing lung injury could be determined in advance and noted in the individual's medical records or, conceivably, on an encrypted microchip worn by the individual. This precaution would be especially useful in interventions to prevent ALI/ARDS, because the injury leading to ALI/ARDS is almost always unanticipated, and the window for intervention to prevent lung injury after the insult is narrow. In the last few years, there has been a sudden explosion of studies of the genetic susceptibility of ALI/ARDS. The following sections review the recently published studies in the genetic epidemiology of ALI/ARDS and discuss the relative strengths and limitations of the current approach with a focus on the implications for future prevention and treatment. The possible applications and potential limitations to the translation of genomics and genetic epidemiology to future prevention and treatment of ALI/ARDS are discussed also.

and discuss the relative strengths and limitations of the current approach with a focus on the implications for future prevention and treatment. The possible applications and potential limitations to the translation of genomics and genetic epidemiology to future prevention and treatment of ALI/ARDS are discussed also. Current approach and recent studies in the genetic epidemiology of acute lung injury/acute respiratory distress syndrome Candidate-gene approach Traditionally, the term “pharmacogenomics” referred to the application of whole-genome scanning for the discovery of new drug targets [14]. Genome-wide studies examining anonymous markers spaced throughout the entire genome are not yet practical in ALI/ARDS. Rather, all studies thus far have used the candidate-gene approach, which focuses on specific genes whose products have been well characterized as biologically important in the pathogenesis, progression, or manifestation of ALI/ARDS [15]. The candidate-gene approach is hypothesis driven and founded on current knowledge of the disease process. The validity of the candidate gene rests on the evidence supporting its selection as a candidate in ALI/ARDS. Table 1 details the candidate genes that have been studied in ALI/ARDS and the evidence supporting their selection.Table 1 Candidate genes and polymorphisms examined in acute lung injury/acute respiratory distress syndrome and the evidence supporting their biological plausibility

s selection as a candidate in ALI/ARDS. Table 1 details the candidate genes that have been studied in ALI/ARDS and the evidence supporting their selection.Table 1 Candidate genes and polymorphisms examined in acute lung injury/acute respiratory distress syndrome and the evidence supporting their biological plausibility Polymorphisms studied in ALI/ARDS Evidence supporting importance in ALI/ARDS Candidate gene Polymorphism Functional significance ACE[16] Insertion/deletion polymorphism in intron 16 Yes D allele associated with severity of and mortality in meningococcal disease [17] ACE levels or activity is variable in ARDS patients [18], [19] More recently, ACE linked to ALI in ACE knockout mice [20] CC16[21] −226GA promoter SNP Yes −226GA polymorphism associated with asthma but not critical illnesses Lower Clara cell protein levels correlate with severity of bacterial pneumonia but no reports in ALI/ARDS [22] IL-6[23] −174GC promoter SNP Yes Plasma IL-6 correlate with ARDS mortality [10] IL-6 Haplotypes associated with mortality in systemic inflammatory response [24] Functional genomics indicate altered IL-6 gene expression in ALI [25] IL-10[26] −1082GA promoter SNP Yes In pneumonia,−1082GG genotype is associated with increased mortality [27] −1082GG genotype occurs less frequently in critical illness compared to healthy controls and is associated with lower severity of illness, organ failure, and mortality [28], [29], [30] Low bronchoalveolar lavage IL-10 correlate with ARDS and mortality in ARDS but high plasma IL-10 correlate with ARDS and sepsis mortality [31], [32] MBL-2 codon −221 promoter SNP

−1082GG genotype occurs less frequently in critical illness compared to healthy controls and is associated with lower severity of illness, organ failure, and mortality [28], [29], [30] Low bronchoalveolar lavage IL-10 correlate with ARDS and mortality in ARDS but high plasma IL-10 correlate with ARDS and sepsis mortality [31], [32] MBL-2 codon −221 promoter SNP codon 52 polymorphism codon 54 polymorphism codon 57 polymorphism Yes Variant X, D, B, and C alleles of codon −221, 52, 54, and 57 are associated with low serum MBL deficiency, greater risk of sepsis, greater severity of sepsis, and/or increased mortality in sepsis [33], [34] MLCK Haplotypes examined No Functional genomics indicate altered MLCK gene expression in ALI [35] MLCK involved in ventilator and sepsis associated lung injury in animals [35], [36] PBEF[37] T-1001G promoter SNP C-1543T promoter SNP No Yes Functional genomics indicate altered PBEF gene expression in ALI [35] Increased PBEF protein in animal models of ALI and in humans with ALI [37] SFTPB[38], [39], [40] Insertion/deletion polymorphism in intron 4 +1580CT SNP in codon 131 No Suspected but not known SFTP-B limits lung injury in animals and correlate with respiratory failure in humans [41], [42] Insertion/deletion polymorphism in intron 4 is associated with neonatal respiratory distress syndrome [43] TNF-α and TNF-β [44] −308GA SNP in TNF-α TNF-β1/2 Ncol SNP in TNFB Yes in some but not all studies Increased plasma or bronchoalveolar TNF-α correlate with development of or mortality in ARDS in some but not all studies [9], [10], [32], [45]

Insertion/deletion polymorphism in intron 4 is associated with neonatal respiratory distress syndrome [43] TNF-α and TNF-β [44] −308GA SNP in TNF-α TNF-β1/2 Ncol SNP in TNFB Yes in some but not all studies Increased plasma or bronchoalveolar TNF-α correlate with development of or mortality in ARDS in some but not all studies [9], [10], [32], [45] −308A allele and TNF-β2 homozygotes associated with sepsis in some studies [46], [47], [48] VGEF[49] +936CT SNP Yes Plasma VGEF increases and pulmonary VGEF decreased with ARDS and then normalizes with recovery in ARDS [50] No known association between +936CT polymorphism and critical illnesses Abbreviations: ACE, angiotensin-converting enzyme; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; CC16, Clara cell protein 16; IL-6, interleukin-6; IL-10, interleukin-10; MBL-2, mannose-binding lectin-2; MLCK, myosin light-chain kinase; PBEF, pre-B-cell colony-enhancing factor; SFTPB, surfactant protein B; SNP, single nucleotide polymorphism; TNF-α, tumor necrosis factor-α; TNF-β, tumor necrosis factor-β; VEGF, vascular endothelial growth factor.

6, interleukin-6; IL-10, interleukin-10; MBL-2, mannose-binding lectin-2; MLCK, myosin light-chain kinase; PBEF, pre-B-cell colony-enhancing factor; SFTPB, surfactant protein B; SNP, single nucleotide polymorphism; TNF-α, tumor necrosis factor-α; TNF-β, tumor necrosis factor-β; VEGF, vascular endothelial growth factor. The strongest candidates for investigation are the genes that have been linked to ALI in previous linkage studies, in association studies, or in animal models of the disease ( Fig. 1) [51]. Investigations into the genetic determinants of ALI/ARDS have been undertaken only recently. The selections of many of the candidate genes in recently published studies were supported by previously published reports in other, similar conditions, such as neonatal respiratory distress syndrome for the SFTPB gene and sepsis for the TNF-α, IL-10, mannose binding lectin-2 (MBL-2), and IL-6 genes. Conversely, several candidate genes found to be associated with ARDS (ie, the +1580CT polymorphisms in the SFTPB gene, the T-1001G and C-1543T polymorphisms in the pre–B-cell colony-enhancing factor [PBEF] gene, and the codon 54 polymorphism in the MBL-2 gene) were also found to be associated with increased risk for sepsis or septic shock in the same population [37], [38], [52]. Overall this finding suggests that genes and polymorphisms that have been implicated in sepsis would serve as strong candidate genes in ALI/ARDS.Fig. 1 Criteria for strong candidate genes in ALI. The strongest candidates for investigation in ALI/ARDS are genes in which specific alleles have been linked with ALI/ARDS or related diseases such as sepsis, neonatal respiratory distress syndrome, or other critical illnesses. Alternately, in the absence of such data, there should be evidence supporting the importance of the gene product or function in ALI/ARDS. If a direct candidate-gene approach is used, additional evidence for the functional significance of the allele of interest should exist. (Adapted from Gong MN, Christiani DC. Genetic epidemiology of acute lung injury. In: Mathay MA, editor. Acute respiratory distress syndrome. New York: Marcel Dekker, Inc.; 2003. p. 392; with permission.)

e-gene approach is used, additional evidence for the functional significance of the allele of interest should exist. (Adapted from Gong MN, Christiani DC. Genetic epidemiology of acute lung injury. In: Mathay MA, editor. Acute respiratory distress syndrome. New York: Marcel Dekker, Inc.; 2003. p. 392; with permission.) In the absence of studies of ALI or related conditions, the biologic plausibility of the candidate gene in the pathogenesis of lung injury is important (see Fig. 1). There should be evidence supporting the importance of the gene product or function specifically in ALI.

e-gene approach is used, additional evidence for the functional significance of the allele of interest should exist. (Adapted from Gong MN, Christiani DC. Genetic epidemiology of acute lung injury. In: Mathay MA, editor. Acute respiratory distress syndrome. New York: Marcel Dekker, Inc.; 2003. p. 392; with permission.) In the absence of studies of ALI or related conditions, the biologic plausibility of the candidate gene in the pathogenesis of lung injury is important (see Fig. 1). There should be evidence supporting the importance of the gene product or function specifically in ALI. More recently, novel candidate genes in ALI have come from functional genomic studies that established their biologic plausibility in lung injury. PBEF is a cytokine and adipokine with a variety of functions including the maturation of B-cell precursors, inhibition of neutrophil apoptosis in sepsis, and stimulation of glucose uptake with action similar to insulin [5], [53]. Its role in ALI had not been reported until expression of the PBEF gene was found to be increased in a series of animal models of stretch and liposaccharide-induced lung injury and in vivo studies of patients who had ALI [25]. Other potential candidates in ALI/ARDS that had increased expression included genes previously suspected to be important, such as IL-6, plasminogen activator inhibitor-1, and Myosin light-chain kinase (MLCK). PBEF protein expression also was increased in the lungs, bronchoalveolar lavage fluid, and serum of patients who had ALI. After identifying two common-promoter single-nucleotide polymorphisms (SNPs) in the PBEF gene, Ye and colleagues [37] found that the variant of the T-1001G polymorphism was associated with increased risk of sepsis-induced ALI compared with healthy controls, whereas the variant C-1543T polymorphism was associated with a protective effect in sepsis-induced ALI compared with healthy adults.

(SNPs) in the PBEF gene, Ye and colleagues [37] found that the variant of the T-1001G polymorphism was associated with increased risk of sepsis-induced ALI compared with healthy controls, whereas the variant C-1543T polymorphism was associated with a protective effect in sepsis-induced ALI compared with healthy adults. After the selection of the candidate genes, there are two approaches to investigation. The direct approach focuses on the association between ALI/ARDS and specific polymorphisms, often SNPs, in the candidate gene that are thought to be functional, either because of linkage with other disease processes or because of the known effect on the levels, function, or effectiveness of the gene. Such an approach is effective for hypothesis testing but is limited to previously studied polymorphisms of a gene. This approach is the one most commonly used in ALI/ARDS.

tional, either because of linkage with other disease processes or because of the known effect on the levels, function, or effectiveness of the gene. Such an approach is effective for hypothesis testing but is limited to previously studied polymorphisms of a gene. This approach is the one most commonly used in ALI/ARDS. Alternatively, the indirect approach examines all common SNPs in the gene (> 1% in a sample population), regardless of whether the SNPs have any functional significance. Often these SNPs are examined individually and in combination with other SNPs on the same gene. The term “haplotype” refers to two or more SNPs that are linked and tend to be inherited together en bloc. Multilocus haplotypes can be viewed as signature patterns of allelic variation on a gene that capture and characterize all polymorphisms within the haplotype block. The functional or disease polymorphism may be one of the loci genotyped, or it may reside within the haplotype block and be captured by the haplotype. Thus, the haplotype would serve as a surrogate marker for the functional polymorphism that is truly linked to the disease state. As such, some argue that haplotype analyses could identify functional or disease loci better than a single polymorphism, especially if the penetrance is low, as would be expected in complex diseases like ARDS [54], [55]. Haplotype analyses also can be more efficient in large epidemiology studies, because genotyping can be confined to the minimum number of SNPs that define that haplotype block (haplotype-tagging SNPs) [56]. Haplotype analyses also can capture cis interaction between SNPs. If one polymorphism increases the risk of disease only in the presence of another polymorphism in the same gene, haplotype analysis will be able to discern this relationship, whereas separate analyses of the polymorphisms will not. Last, haplotype analysis can help localize the disease locus to within the haplotype block in the gene and thus may help focus the search for functional variants in subsequent studies. The haplotype approach has become increasing popular in the genetic epidemiology of complex diseases, and this approach was used in the investigation of the PBEF and MLCK genes in ALI.

sease locus to within the haplotype block in the gene and thus may help focus the search for functional variants in subsequent studies. The haplotype approach has become increasing popular in the genetic epidemiology of complex diseases, and this approach was used in the investigation of the PBEF and MLCK genes in ALI. Together, these studies have validated the candidate-gene approach in the search for genetic determinants of ALI/ARDS. Although this approach is hypothesis driven and is well validated in the genetic epidemiology of complex diseases, it is only as strong as the hypothesis supporting the choice of candidates. Thus, the possibility that any candidate gene in ALI/ARDS can serve as a potential target for future preventive and therapeutic measures will rest on the strength of the evidence supporting its role as a candidate gene in ALI/ARDS. This evidence will not depend on any one genetic epidemiology study. Rather, it must be grounded in a series of genetic, molecular, bioinformatics, and clinical studies and confirmatory studies that support the biologic plausibility of the gene in ALI/ARDS.

evidence supporting its role as a candidate gene in ALI/ARDS. This evidence will not depend on any one genetic epidemiology study. Rather, it must be grounded in a series of genetic, molecular, bioinformatics, and clinical studies and confirmatory studies that support the biologic plausibility of the gene in ALI/ARDS. Case-control study design Given the high mortality in ARDS and the generally late age of onset, traditional family-based approaches in genetic epidemiology are either not feasible or impractical. Rather, studies in ALI/ARDS have established the unrelated case-control study as an effective and well-validated design in the investigation of the genetic determinants of ALI/ARDS. Case-control studies require the delineation of a control group and focus on whether the gene of interest occurs at a significantly greater frequency among the patients who have the disease than among the controls. One of the most important advantages of case-control studies in complex disorders such as ALI is the power of the design. Association studies are the most sensitive and powerful of all of the study designs described thus far in detecting common, low-penetrant susceptibility genes in complex disease [12]. In addition, the case-control design is well suited to the study of genetic markers of disease. Genes are stable indicators of disease susceptibility, because they do not change with time or circumstances. The use of genetic markers as the exposure eliminates recall bias that often plagues case-control studies. Case-control studies also are amendable to multivariate modeling, which allows adjustment for important nongenetic factors and interactions.

e susceptibility, because they do not change with time or circumstances. The use of genetic markers as the exposure eliminates recall bias that often plagues case-control studies. Case-control studies also are amendable to multivariate modeling, which allows adjustment for important nongenetic factors and interactions. Because of the power and versatility of association studies, many believe that the future deciphering of the genetics of complex diseases will involve case-control studies [51], [57]. With increasing use of this design, however, comes some misuse as well. The most common and troubling criticisms of association studies are inconsistency and lack of reproducibility. This heterogeneity is caused by a number of factors. The epidemiologic quality of published genetic studies is quite variable [58]. Other factors include the lack of power in some studies (type II errors) and the lack of control for confounders such as population differences or gene–environment interaction. As is true in any case-control design in epidemiology, the strength of the study depends entirely on the proper selection of cases and controls and on the appropriate accounting of the potential confounders, power and type I error [59]. The following section focuses on the features of genetic case-control design as illustrated by studies in ALI. Table 2 details some of these features and the results of recent genetic epidemiology studies in ALI/ARDS.Table 2 Summary of published genetic epidemiology studies in acute lung injury/acute respiratory distress syndrome

ng section focuses on the features of genetic case-control design as illustrated by studies in ALI. Table 2 details some of these features and the results of recent genetic epidemiology studies in ALI/ARDS.Table 2 Summary of published genetic epidemiology studies in acute lung injury/acute respiratory distress syndrome Patient population Major findings Candidate gene Genotype studied Study Case Controls Susceptibility to ALI/ARDS Outcomes in ALI/ARDS ACE Insertion/deletion polymorphism in intron 16 Marshall et al [16] 96 whites with AECC defined ARDS 88 whites with non-ARDS respiratory failure 174 whites after heart surgery 1906 healthy white males D allele and DD genotype associated with increased susceptibility to ARDS compared to all control groups Increasing mortality in ARDS associated with increasing number of D alleles carried Chan et al [60] 17 Chinese patients with AECC defined ARDS from SARS 123 Chinese patients with SARS326 healthy Chinese individuals No association found Not examined CC16 −226GA promoter SNP Frerking et al [21] 117 German with AECC-defined ARDS 373 healthy German newborns No association found Not examined IL-6 −174GC promoter SNP Marshall et al [23] 96 whites with AECC defined ARDS 88 whites with non-ARDS respiratory failure 174 whites after heart surgery 1906 healthy whites males

D allele and DD genotype associated with increased susceptibility to ARDS compared to all control groups Increasing mortality in ARDS associated with increasing number of D alleles carried Chan et al [60] 17 Chinese patients with AECC defined ARDS from SARS 123 Chinese patients with SARS326 healthy Chinese individuals No association found Not examined CC16 −226GA promoter SNP Frerking et al [21] 117 German with AECC-defined ARDS 373 healthy German newborns No association found Not examined IL-6 −174GC promoter SNP Marshall et al [23] 96 whites with AECC defined ARDS 88 whites with non-ARDS respiratory failure 174 whites after heart surgery 1906 healthy whites males No association found −174C allele and −174CC genotype correlated with serum IL-6 levels, and was associated with survival in ARDS and in non-ARDS with respiratory failure IL-10 −1082GA promoter SNP Gong et al [26] 211 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 429 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive who did not develop ARDS −1082GG genotype was associated with ARDS but only in presence of significant interaction between genotype and age −1082GG genotype associated with less organ failure and lower mortality in ARDS MBL-2 codon −221 promoter SNP codon 52 SNP codon 54 SNP codon 57 SNP

No association found −174C allele and −174CC genotype correlated with serum IL-6 levels, and was associated with survival in ARDS and in non-ARDS with respiratory failure IL-10 −1082GA promoter SNP Gong et al [26] 211 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 429 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive who did not develop ARDS −1082GG genotype was associated with ARDS but only in presence of significant interaction between genotype and age −1082GG genotype associated with less organ failure and lower mortality in ARDS MBL-2 codon −221 promoter SNP codon 52 SNP codon 54 SNP codon 57 SNP Gong et al [52] 212 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 442 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive transfusions who did not develop ARDS. Homozygotes for variant codon 54B allele was associated with greater severity of illness and increased susceptibility to ARDS Homozygotes for variant codon 54B allele was associated with greater daily organ failures and increased ARDS mortality MLCK 28 SNPs in whites 25 SNPs in African Americans Gao et al [61] 92 whites with sepsis related AECC defined ALI 114 whites with sepsis 85 healthy whites 51 AA with sepsis 61 healthy African Americans

Gong et al [52] 212 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 442 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive transfusions who did not develop ARDS. Homozygotes for variant codon 54B allele was associated with greater severity of illness and increased susceptibility to ARDS Homozygotes for variant codon 54B allele was associated with greater daily organ failures and increased ARDS mortality MLCK 28 SNPs in whites 25 SNPs in African Americans Gao et al [61] 92 whites with sepsis related AECC defined ALI 114 whites with sepsis 85 healthy whites 51 AA with sepsis 61 healthy African Americans One SNP and one haplotype associated with ALI in whites compared with septic controls Not examined 46 African Americans sepsis-related AECC defined ALI 2 haplotypes associated with ALI in African Americans compared to septic controls PBEF T-1001G promoter SNP C-1543T promoter SNP Ye et al [37] 87 whites with sepsis-related AECC-defined ALI 100 whites with sepsis 84 healthy whites

One SNP and one haplotype associated with ALI in whites compared with septic controls Not examined 46 African Americans sepsis-related AECC defined ALI 2 haplotypes associated with ALI in African Americans compared to septic controls PBEF T-1001G promoter SNP C-1543T promoter SNP Ye et al [37] 87 whites with sepsis-related AECC-defined ALI 100 whites with sepsis 84 healthy whites Compared to healthy controls, variant G1001 allele and 1001G:1543C haplotype were associated with increased susceptibility to ALI while the variant T1543 allele was associated with decreased susceptibility to ALI No association between variant G1001 allele and ARDS mortality No association seen in comparison with septic controls Bajwa et al [62] 375 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 787 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive transfusions who did not develop ARDS Variant G1001 allele and 1001G:1543C haplotype associated with increased susceptibility to ALI in septic and noninfectious risks for ARDS No association between either polymorphism and ARDS mortality Variant T1543 allele not associated with ARDS SFTPB Insertion/deletion polymorphism in intron 4 Max et al [63] 15 Germans with AECC-defined ARDS 21 healthy Americans Variant allele associated with increased susceptibility to ARDS Not examined Gong et al [40] 72 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 117 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive who did not develop ARDS Variant allele associated with increased susceptibility to ARDS and increased susceptibility to severe direct pulmonary injury like pneumonia in women Not examined +1580CT SNP in codon 131 Lin et al [39] 52 German patients with AECC-defined ARDS 46 healthy German adults

aspiration, and massive who did not develop ARDS Variant allele associated with increased susceptibility to ARDS and increased susceptibility to severe direct pulmonary injury like pneumonia in women Not examined +1580CT SNP in codon 131 Lin et al [39] 52 German patients with AECC-defined ARDS 46 healthy German adults 25 whites with trauma, pneumonia, and heart failure +1580C allele and + 1580CC genotype were associated with increased susceptibility to ARDS compared to both control groups Not examined Quasney et al [38] 12 whites and African Americans with ARDS caused by pneumonia 390 whites and African Americans with pneumonia +1580CC genotype were associated with increased susceptibility to respiratory failure, septic shock, and ARDS No association with mortality in pneumonia ARDS mortality not specifically examined TNF-α and TNF-β −308GA SNP in TNF-α TNFβ1/2 Ncol SNP in TNFB Gong et al [44] 237 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 476 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive who did not develop ARDS −308A allele and −308A:TNF-β1 haplotype was associated with increased susceptibility to ARDS in direct pulmonary injury Increasing ARDS mortality with increasing number of −308A alleles with greatest mortality found in younger patients carrying the −308A allele No association with ARDS found for TNF-β1/2 VGEF +936CT SNP Medford et al [49] 117 whites with AECC-defined ARDS 137 healthy whites 103 EA who had respiratory failure

+1580C allele and + 1580CC genotype were associated with increased susceptibility to ARDS compared to both control groups Not examined Quasney et al [38] 12 whites and African Americans with ARDS caused by pneumonia 390 whites and African Americans with pneumonia +1580CC genotype were associated with increased susceptibility to respiratory failure, septic shock, and ARDS No association with mortality in pneumonia ARDS mortality not specifically examined TNF-α and TNF-β −308GA SNP in TNF-α TNFβ1/2 Ncol SNP in TNFB Gong et al [44] 237 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 476 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive who did not develop ARDS −308A allele and −308A:TNF-β1 haplotype was associated with increased susceptibility to ARDS in direct pulmonary injury Increasing ARDS mortality with increasing number of −308A alleles with greatest mortality found in younger patients carrying the −308A allele No association with ARDS found for TNF-β1/2 VGEF +936CT SNP Medford et al [49] 117 whites with AECC-defined ARDS 137 healthy whites 103 EA who had respiratory failure +936CT and +936TT genotype associated with more susceptibility to ARDS compared with both control groups +936CT and +936TT genotype associated with greater severity of illness in ARDS but no association with ARDS mortality was found

+1580C allele and + 1580CC genotype were associated with increased susceptibility to ARDS compared to both control groups Not examined Quasney et al [38] 12 whites and African Americans with ARDS caused by pneumonia 390 whites and African Americans with pneumonia +1580CC genotype were associated with increased susceptibility to respiratory failure, septic shock, and ARDS No association with mortality in pneumonia ARDS mortality not specifically examined TNF-α and TNF-β −308GA SNP in TNF-α TNFβ1/2 Ncol SNP in TNFB Gong et al [44] 237 whites with AECC-defined ARDS from a cohort of ICU patients with sepsis, trauma, aspiration, and massive transfusion 476 whites from same cohort of ICU patients admitted with sepsis, trauma, aspiration, and massive who did not develop ARDS −308A allele and −308A:TNF-β1 haplotype was associated with increased susceptibility to ARDS in direct pulmonary injury Increasing ARDS mortality with increasing number of −308A alleles with greatest mortality found in younger patients carrying the −308A allele No association with ARDS found for TNF-β1/2 VGEF +936CT SNP Medford et al [49] 117 whites with AECC-defined ARDS 137 healthy whites 103 EA who had respiratory failure +936CT and +936TT genotype associated with more susceptibility to ARDS compared with both control groups +936CT and +936TT genotype associated with greater severity of illness in ARDS but no association with ARDS mortality was found Abbreviations: ACE, angiotensin-converting enzyme; EA, European-Americans; IL-6, interleukin-6; IL-10, interleukin-10; PBEF, pre-B-cell colony-enhancing factor; SARS, severe acute respiratory syndrome; SFTPB, surfactant protein B; SNP, single-nucleotide polymorphism; TNF-α, tumor necrosis factor-α; TNF-β, tumor necrosis factor-β; VEGF, vascular endothelial growth factor.

, European-Americans; IL-6, interleukin-6; IL-10, interleukin-10; PBEF, pre-B-cell colony-enhancing factor; SARS, severe acute respiratory syndrome; SFTPB, surfactant protein B; SNP, single-nucleotide polymorphism; TNF-α, tumor necrosis factor-α; TNF-β, tumor necrosis factor-β; VEGF, vascular endothelial growth factor. Case definition As with any case-control study, the choice and phenotype of cases and controls is pivotal to the design, strength, validity, and generalizability of the study. The case definition will differ, depending on the whether the focus is on prevention or treatment. Studies of susceptibility to developing ARDS are more relevant for future prevention, whereas studies on outcomes in ALI/ARDS are more relevant for treatment. In molecular epidemiology studies, factors important in susceptibility studies may not be important in prognostication of outcomes, and vice versa. For example, mutations in the BRCA1 gene, now known to be important in DNA repair, are associated with increased susceptibility to developing early-onset breast or ovarian cancer. However, the BRCA1 gene is not associated with differences in breast cancer recurrence or disease-free survival after therapy, even though BRCA1-associated breast cancer tends to present at a more advanced stage [64].

pair, are associated with increased susceptibility to developing early-onset breast or ovarian cancer. However, the BRCA1 gene is not associated with differences in breast cancer recurrence or disease-free survival after therapy, even though BRCA1-associated breast cancer tends to present at a more advanced stage [64]. When the focus is on prevention and susceptibility rather than on treatment and outcomes in ALI/ARDS, there are inherently more challenges. Genetic epidemiology studies examining outcomes in ALI/ARDS usually use mortality or ventilator-free days as end points. The outcome of ALI/ARDS in genetic susceptibility studies is more heterogeneous and is prone to misclassification, because there is no definitive diagnostic test. The American-European Consensus criteria serve as a uniformly accepted guideline for defining lung injury, but certain criteria, specifically the radiologic criteria, are not always clear and are subject to interobserver variability [65]. In addition, the ratio of partial pressure of arterial carbon dioxide to fraction of inspired oxygen represents a continuum of hypoxemic respiratory failure. The use of a cutoff of 300 mm Hg in the criteria for ALI will result in inevitable random misclassification of cases and controls that tends to bias results toward the null hypothesis. In addition, autopsy studies indicate that the American-European Consensus criteria for ALI/ARDS are sensitive but are not very specific [66]. Nevertheless, the American-European Consensus definition is used uniformly in the studies of ALI/ARDS. Care must be taken to assess carefully the rigor with which the cases adhere to the ALI/ARDS criteria. Similar attention also must be taken to ensure that controls do not actually have ARDS. Reliance on chart review and clinical diagnosis is inadequate, given recent evidence for the underdiagnosis of ALI/ARDS [67]. Thus, the same screening procedures used to determine the cases of ALI/ARDS should be applied to the controls to ensure that they do not also have the condition. Even so, misclassification will occur, and large, well-phenotyped sample sizes will be needed to detect an association.

r the underdiagnosis of ALI/ARDS [67]. Thus, the same screening procedures used to determine the cases of ALI/ARDS should be applied to the controls to ensure that they do not also have the condition. Even so, misclassification will occur, and large, well-phenotyped sample sizes will be needed to detect an association. Choice of controls The choice of controls in case-control studies is equally important, although often neglected. In case-controls studies, controls are not simply people who do not have the disease. Rather, they should represent the population that is at risk for the disease. In other words, they should not have the disease at the time of selection but, under the study design, they would have been included as a case if they did develop the disease [68]. A poor choice of control may result in hidden confounding. In one review, 30% of the genetic epidemiology studies did not delineate adequately the criteria by which the controls were selected, and the controls were improperly chosen in 13.5% of the studies [58].

included as a case if they did develop the disease [68]. A poor choice of control may result in hidden confounding. In one review, 30% of the genetic epidemiology studies did not delineate adequately the criteria by which the controls were selected, and the controls were improperly chosen in 13.5% of the studies [58]. The most common problem is the selection of controls who are not at risk for the disease, making comparisons with the cases difficult. The controls in many of the studies of ALI/ARDS were healthy individuals or hospitalized patients who did not have a clear prior injury placing them at risk for ALI [21], [39], [49], [63]. As discussed previously, genes associated with sepsis are strong candidate genes for ALI, but because sepsis also is the leading precipitant for ALI, one must be careful to avoid confounding from a genetic association with the predisposing injury. When the controls are healthy or have conditions that differ from the precipitating injuries in the ARDS cases, any association found between a candidate gene and ALI/ARDS may actually be caused by an association between the polymorphism and the risk condition for ALI/ARDS, such as sepsis. It is important to use at-risk individuals who have similar conditions as the cases to avoid this confounder. In the investigation of the PBEF gene, both healthy individuals and patients who had sepsis were used as controls. The variant T-1001G and C-1543T alleles were found to be associated with the development of sepsis-associated ALI when compared with healthy controls, but no association was found between patients who had sepsis-associated ALI compared with sepsis patients who did not have ALI [37]. Thus, it is not clear whether the PBEF polymorphisms are associated with ALI or with the severe sepsis that placed the patients at risk for ALI. In a subsequent study in a different cohort of patients who had sepsis, trauma, aspiration, or multiple transfusions, the variant T-1001G, but not the C-1543T allele, was confirmed to be associated with ARDS compared with at-risk individuals [62]. This association was present even among patients who had ARDS of noninfectious origin, extending the generalizability of the genetic association.

rauma, aspiration, or multiple transfusions, the variant T-1001G, but not the C-1543T allele, was confirmed to be associated with ARDS compared with at-risk individuals [62]. This association was present even among patients who had ARDS of noninfectious origin, extending the generalizability of the genetic association. One potential issue with using at-risk controls is that the patients are not drawn randomly from the general population. Rather they are selected as controls on the basis of their critical illness. If the genotype of interest is associated with critical illness, then the genotype frequency may deviate from that predicted by random mating (Hardy Weinberg equilibrium) [69]. Indeed, such was the case with the -1082GA IL-10 and MBL-2 polymorphisms. In such cases, extra effort is needed to ensure that the deviation from Hardy Weinberg equilibrium is not from genotype or clerical error. Such efforts include repeat genotyping, blinding of personnel, or validation of genotyping in a different population.

ch was the case with the -1082GA IL-10 and MBL-2 polymorphisms. In such cases, extra effort is needed to ensure that the deviation from Hardy Weinberg equilibrium is not from genotype or clerical error. Such efforts include repeat genotyping, blinding of personnel, or validation of genotyping in a different population. Race and genetic epidemiology of acute lung injury/acute respiratory distress syndrome Recently the role of race in critical illnesses has been explored. In a retrospective study of decedents, nonwhite race was associated with increased mortality in ARDS, with African Americans, especially young African Americans, having the higher mortality from ARDS than whites and other minorities [70]. It is not clear why African Americans have higher mortality in ARDS than whites. Precipitants for lung injury and other predictors of ARDS were not available for comparison. Because the study focused on deaths from ARDS, it is not clear whether minorities have a higher risk of developing ARDS in the first place.

[70]. It is not clear why African Americans have higher mortality in ARDS than whites. Precipitants for lung injury and other predictors of ARDS were not available for comparison. Because the study focused on deaths from ARDS, it is not clear whether minorities have a higher risk of developing ARDS in the first place. Many have postulated that genetic variability may contribute to the racial disparities in ARDS. Many of the polymorphisms found to be associated with ALI/ARDS susceptibility or outcome, such as the insertion/deletion polymorphism in intron 4 of the SFTP-B gene [71], the -308GA polymorphism in the TNF-α gene [72], and the codon 54 polymorphism in the MBL-2 gene [73], are known to vary in frequency among major racial groups. This variation may be especially important when the haplotype approach is used. The extent of linkage disequilibrium and, hence, of haplotype blocks and frequencies differs between African Americans and non-Africans [74]. Thus, any genotype analysis must be restricted to one racial group or be stratified by race to avoid confounding from differences in ethnic groups (population stratification). In one study, haplotypes in the MLCK gene were found to be associated with variable susceptibility to developing sepsis-induced ALI in both American whites of European heritage and in African Americans [61]. Whites and African Americans differed in the linkage disequilibrium between SNPs and in haplotype block definition, however. Although significant associations between the gene and ALI were found, the disease-associated haplotypes differed between racial groups. The similar location of the race-specific at-risk haplotypes in whites and African Americans suggests that the true disease-associated variant may be located within the 5′ region of the gene.

significant associations between the gene and ALI were found, the disease-associated haplotypes differed between racial groups. The similar location of the race-specific at-risk haplotypes in whites and African Americans suggests that the true disease-associated variant may be located within the 5′ region of the gene. After stratifying by major racial groups, additional methods to adjust for population stratification may not be necessary, especially for studies conducted in the United States. Wacholder and colleagues [75] demonstrated that, among whites, bias from population stratification is small and decreases as the number of ethnic subgroups within the white population increases. This finding may be especially pertinent for whites in the United States, a classification that tends to be composed of many different ethnic subgroups. Similar results were found in the classification of African Americans that contained large numbers of ethnic subgroups [76]. Consistent with these stimulation studies, Gao and colleagues [61] found evidence for ethnic differences within their African Americans subjects, but adjusting for these differences did not significantly change the associations between haplotypes and SNPs in the MLCK gene and ALI except for one SNP, in which the association was actually strengthened.

n studies, Gao and colleagues [61] found evidence for ethnic differences within their African Americans subjects, but adjusting for these differences did not significantly change the associations between haplotypes and SNPs in the MLCK gene and ALI except for one SNP, in which the association was actually strengthened. Currently, most, although not all, studies have restricted their analyses to whites or have stratified their analyses by race. Thus, the findings in genetic epidemiology studies of ALI/ARDS cannot be generalized to nonwhites. Large cohorts of nonwhites will be necessary to confirm previously detected genetic associations in other racial groups. Gene–environment interaction The role of the environment is particularly critical in determining the genetic determinants in a complex disorder in which the gene may have no influence on the risk of disease unless there is concomitant exposure to a particular environmental insult. Such interaction is important in understanding and interpreting the genetic contribution to complex disease such as ALI. Failure to examine the role of environmental exposure can lead to decreased sensitivity in detecting an association between the gene of interest and the disease [77]. Neglect of the gene–environment interaction contributes to the inconsistent findings from genetic association studies of complex disease [78].

ch as ALI. Failure to examine the role of environmental exposure can lead to decreased sensitivity in detecting an association between the gene of interest and the disease [77]. Neglect of the gene–environment interaction contributes to the inconsistent findings from genetic association studies of complex disease [78]. Recently, there is growing evidence to suggest potential gene–environment interaction, from whether the initial precipitant for ARDS is a direct pulmonary injury such as pneumonia or aspiration or an indirect pulmonary injury such as extrapulmonary sepsis or massive transfusion.

ch as ALI. Failure to examine the role of environmental exposure can lead to decreased sensitivity in detecting an association between the gene of interest and the disease [77]. Neglect of the gene–environment interaction contributes to the inconsistent findings from genetic association studies of complex disease [78]. Recently, there is growing evidence to suggest potential gene–environment interaction, from whether the initial precipitant for ARDS is a direct pulmonary injury such as pneumonia or aspiration or an indirect pulmonary injury such as extrapulmonary sepsis or massive transfusion. Two SNPs in the SFTPB gene have been found to be associated with ARDS. SFTPB is essential for the surface tension–lowering properties of pulmonary surfactant, which is known to be dysfunctional in ALI/ARDS. In two small studies, the variant allele in the insertion/deletion polymorphism in intron 4 of the SFTPB gene was found to be associated with susceptibility to ARDS or severe direct pulmonary injury such as pneumonia, especially among women [40], [63]. In another polymorphism in the SFTPB gene, the C allele of +1580CT SNP was found to be associated with ARDS, but this association was confined to patients who had idiopathic insults, mostly direct pulmonary injuries such as pneumonia [39]. No associations were found in the group of patients who had exogenic ARDS, mostly extrapulmonary causes of ARDS [38]. Although healthy controls were used, a subsequent study using ARDS cases and controls who had community-acquired pneumonia confirmed this association between the C allele and ARDS, suggesting that the +1580C allele is associated with ALI/ARDS and not with severe pneumonia. Together, these studies suggest that the SFTPB gene may be important in ARDS susceptibility in direct pulmonary injuries such as pneumonia. This gene also may influence susceptibility to direct pulmonary injury, such as severe pneumonia, that places these patients at risk for ALI/ARDS. The role of the SFTPB gene in lung injury resulting from other causes is not yet clear.

y be important in ARDS susceptibility in direct pulmonary injuries such as pneumonia. This gene also may influence susceptibility to direct pulmonary injury, such as severe pneumonia, that places these patients at risk for ALI/ARDS. The role of the SFTPB gene in lung injury resulting from other causes is not yet clear. A similar gene–environment interaction was found with the −308GA polymorphisms in the TNF-α gene [44]. No association was found between the variant −308A allele and ARDS compared with other critically ill non-ARDS controls who had sepsis, aspiration, massive transfusion, or trauma. After stratification by the site of injury, however, the −308A allele was associated with decreased likelihood of developing ARDS among patients who had direct pulmonary injury (adjusted odds ration [OR], 0.52; 95% confidence interval [CI], 0.30–0.91) but with a nonsignificant increased likelihood of ARDS in indirect pulmonary injury (adjusted OR, 1.7; 95% CI, 0.93–3.2) with evidence for significant effect modification (P = .01).

developing ARDS among patients who had direct pulmonary injury (adjusted odds ration [OR], 0.52; 95% confidence interval [CI], 0.30–0.91) but with a nonsignificant increased likelihood of ARDS in indirect pulmonary injury (adjusted OR, 1.7; 95% CI, 0.93–3.2) with evidence for significant effect modification (P = .01). The reasons for this interaction are unclear. The risk of ARDS is different in direct pulmonary injuries and indirect pulmonary injuries [79]. The cytokine profile and inflammatory markers differ in patients who have ARDS and at-risk patients who do not have ARDS, depending on whether the predisposing injury was sepsis, trauma, acute pancreatitis, or massive transfusion [80]. Certainly, the inflammatory response and the radiologic, histologic, and mechanical properties of the lung differ depending on whether the site of infection or the etiology of ARDS is pulmonary or extrapulmonary [81], [82]. Although these results need to be confirmed in larger studies, these findings overall indicate important gene–environment interactions in the genetic susceptibility to developing ALI/ARDS that depend on the risk factor that predisposes the individual to lung injury.

gy of ARDS is pulmonary or extrapulmonary [81], [82]. Although these results need to be confirmed in larger studies, these findings overall indicate important gene–environment interactions in the genetic susceptibility to developing ALI/ARDS that depend on the risk factor that predisposes the individual to lung injury. Another source of potential gene–environment interaction is age. Older patients have a higher risk than younger individuals of developing and dying from ARDS [4], [83]. In complex diseases, the genetic contribution may be greatest in diseases with an early age of onset rather than in a disease with a late age of onset, in which environmental factors such as comorbidities may figure more prominently. Potential interactions with age have been found in genetic epidemiology studies of ALI/ARDS. Among 212 patients who had ARDS, the −308A allele was associated with more daily organ dysfunction and increased 60-day mortality in ARDS (adjusted OR, 3.5; 95% CI, 1.4–8.6) after adjusting for age, severity of illness, septic shock, transfusion and other potential predictors [44]. Age seemed to be important, with the strongest association found among the 117 ARDS patients younger than median age of 67 years (adjusted OR, 14.9; 95% CI, 3.0–74; P < .001). In the same cohort of critically ill at-risk patients, the −1082GG genotype was associated with increased susceptibility to ARDS in critically ill patients (P < .001), but only in the presence of a statistically significant interaction between age and the −1082GG genotype [26]. When the nature of this interaction was explored further by stratifying the analyses by the median age of 67 years, the −1082GG genotype was protective against ARDS among the older (adjusted OR, 0.63; 95% CI, 0.34–1.2) but not among the younger patients (OR, 1.7; 95% CI, 0.89–3.2), with significant effect modification by age of the association between −1082GG and ARDS (P < .001). Further study with a larger sample size is needed to confirm and define better the age effect on the genetic susceptibility to developing and dying from ARDS. If such interaction does exist, future interventions aimed at preventing and treating ARDS may have variable efficacy, depending on the age of the individual.

). Further study with a larger sample size is needed to confirm and define better the age effect on the genetic susceptibility to developing and dying from ARDS. If such interaction does exist, future interventions aimed at preventing and treating ARDS may have variable efficacy, depending on the age of the individual. Other potential factors that will be worthwhile examining for gene–environment interaction in the future are diabetes and chronic alcohol abuse. A history of diabetes has been found to be protective in ARDS [79], [84]. A growing body of literature is suggesting a role for chronic alcohol abuse in increased susceptibility and poorer outcomes in ALI/ARDS [85]. It is likely that there may be genotypes that are important in ALI/ARDS only in the context of diabetes or chronic alcohol abuse.

been found to be protective in ARDS [79], [84]. A growing body of literature is suggesting a role for chronic alcohol abuse in increased susceptibility and poorer outcomes in ALI/ARDS [85]. It is likely that there may be genotypes that are important in ALI/ARDS only in the context of diabetes or chronic alcohol abuse. Defining these gene–environment interactions is important. Many of the polymorphisms identified in ARDS are common, with frequency greater than 1%. Given their persistence in the genome and the lethality of ARDS, it is unlikely that these polymorphisms are universally detrimental. Rather, it is likely that these variants may be detrimental in some situations and benign or even beneficial in others. Otherwise, there would be selection pressure against their persistence in the population. Thus, any intervention that targets the same causal pathway as the implicated at-risk genes may be beneficial in some circumstances but less helpful in others. Understanding the gene–drug–environment interaction will be important in identifying the patient population that has the most favorable risk/benefit ratio for any particular therapy.

that targets the same causal pathway as the implicated at-risk genes may be beneficial in some circumstances but less helpful in others. Understanding the gene–drug–environment interaction will be important in identifying the patient population that has the most favorable risk/benefit ratio for any particular therapy. Type I errors and power Type I and type II errors also are important in genetic case-control studies. Type I error is the likelihood of a false-positive finding. Although a p-value or a type I error rate of 5% is generally considered acceptable, one may be more likely to find an association by chance alone if multiple comparisons of different genetic loci to the development of disease are performed. Although adjustment for multiple comparisons is ideal, it is not entirely clear what the best strategy is, and current studies of ALI/ARDS may still too small to accommodate statistical correction for multiple comparisons. Ultimately, the likelihood of a cause-and-effect relationship underlying any genetic association will depend on the reproducibility of well-designed studies in different populations and in the strength of the biologic rationale behind the selection of that gene for analysis. Although troublesome to classical geneticists, the need to confirm studies is common in epidemiology. Any population study needs to be validated for different populations and in larger studies.

studies in different populations and in the strength of the biologic rationale behind the selection of that gene for analysis. Although troublesome to classical geneticists, the need to confirm studies is common in epidemiology. Any population study needs to be validated for different populations and in larger studies. Type II errors involve the statistical power of the study. Power to detect an association depends upon the size of the effect, the frequency of the genotype in the population, and the sensitivity of the analysis deployed. Some of the negative studies in genetic epidemiology in ALI are probably caused by the lack of adequate power [21]. The power of the study is especially important when there may be phenotype misclassification and gene–gene or gene–environment interaction. Currently, most ALI/ARDS studies are relatively small for genetic epidemiology studies, and their small size makes examination of interactions difficult. Only the Boston cohort has sufficient sample size to explore for gene–environment interaction. Additional large cohorts will be necessary to confirm previously found associations and interactions [26], [44].

tively small for genetic epidemiology studies, and their small size makes examination of interactions difficult. Only the Boston cohort has sufficient sample size to explore for gene–environment interaction. Additional large cohorts will be necessary to confirm previously found associations and interactions [26], [44]. Genetic epidemiology and its potential application in the prevention and treatment of acute lung injury/acute respiratory distress syndrome With the completion of the HapMap and Human Genome Project, there has been much interest in how genetics may lead to future prevention and treatment of complex diseases. This interest must be tempered to avoid raising false hope. Genetic epidemiology has been applied to the study of ALI/ARDS only recently. Technical and methodological issues in approach and study design are still evolving, and the large cohorts needed for effective genetic epidemiology studies and for the required confirmatory studies in different populations are still being developed. Because the translation of research findings into clinical practice usually takes years, it is likely that genetic epidemiology studies will not lead to any change in clinical practice for years or decades to come. Nevertheless, genetic epidemiology may contribute to future prevention and therapeutic strategies in ALI/ARDS by (1) identifying targets for intervention, (2) enabling risk assessment, and (3) identifying the appropriate patient groups or conditions for interventions (genetic pharmacoepidemiology).

or decades to come. Nevertheless, genetic epidemiology may contribute to future prevention and therapeutic strategies in ALI/ARDS by (1) identifying targets for intervention, (2) enabling risk assessment, and (3) identifying the appropriate patient groups or conditions for interventions (genetic pharmacoepidemiology). Identification of novel targets for intervention Unlike diseases with simple Mendelian inheritance, ALI/ARDS is unlikely to be caused by discrete mutations in a particular gene. Rather, multiple genes with incomplete penetrance and much gene–gene and gene–environment interaction will be important in ALI/ARDS. As such, expecting gene therapy to correct a specific disease-causing mutation or locus is unrealistic. More likely, genetic epidemiology studies may help identify important pathways in the pathogenesis and evolution of lung injury and new therapeutic targets within these pathways for intervention. Hence, the potential of any gene or its product in the future prevention and treatment of ALI/ARDS will depend greatly on the strength of the evidence supporting the biologic role for the candidate gene in ALI/ARDS. In such cases, a multidisciplinary translational approach involving genetic epidemiology, functional genomics, animal models, and bioinformatics will be important. The translational approach may be bidirectional [86]. The “benchside” work may occur before the association study to lend support to its selection as a candidate gene, as was the case with the PBEF and MLCK genes [37], [61]. Alternately, such investigation may occur after the association study to explain better the nature of the genetic association. For example, after an association between the D allele in the angeiotensin-converting enzyme (ACE) gene and ARDS was reported, greater support for the role of ACE in lung injury was established when the loss of ACE activity in ACE-knockout mice was found to protect against lung injury [20]. In contrast, mice deficient in ACE 2, a homologue of ACE, were more susceptible to sepsis and endotoxin-induced lung injury. Inactivation of the ACE gene reduced the injury seen in these ACE 2-knockout mice. These results lend greater strength to the biologic plausibility of ACE in the development of ARDS and, consequently, its potential as a target for intervention.

of ACE, were more susceptible to sepsis and endotoxin-induced lung injury. Inactivation of the ACE gene reduced the injury seen in these ACE 2-knockout mice. These results lend greater strength to the biologic plausibility of ACE in the development of ARDS and, consequently, its potential as a target for intervention. The lack of functional significance of a specific SNP or haplotype found to be associated with disease does not negate the importance of the candidate gene and its pathway in pathogenesis and development of ALI/ARDS. The functional consequence may depend on the stimulus or on activation of other genes. The SNP or haplotype may result in changes that are not easily measured, such as the posttranslational modification or alternative splicing of the gene product. In addition, the disease-associated SNP may not be functional itself but, rather, may be linked to the actual functional susceptibility locus. Nevertheless, if the candidate gene was chosen with sound scientific rationale, a positive association between the candidate gene and the disease supports its importance in ALI, even if the polymorphism studied is not the direct cause of the disease. In such cases, functional studies help support the role of the gene or its product in ALI/ARDS.

date gene was chosen with sound scientific rationale, a positive association between the candidate gene and the disease supports its importance in ALI, even if the polymorphism studied is not the direct cause of the disease. In such cases, functional studies help support the role of the gene or its product in ALI/ARDS. Risk assessment Another way that genetic epidemiology studies can contribute to future prevention and treatment is in risk assessment. In the past, clinical trials of surfactant replacement in ARDS and anti-TNF therapy in sepsis have proved disappointing. It is possible that these therapies may not be beneficial in all patients. For example, anti-TNF therapy may be beneficial only for patients who are genetically predisposed to be exuberant TNF secretors. Anti-TNF therapy may be useless or even detrimental in patients who have the low TNF-secreting genotypes. Such genotype-dependent responses to therapy were demonstrated with recombinant interleukin-1 receptor antagonist (IL1-ra) and the rare IL-1ra +3954 allele in rheumatoid arthritis and with salmeterol therapy and a β-adrenergic receptor genotype in asthma [87], [88]. Better risk assessment of the patient, based on the patient's genetic profile and likelihood of response or adverse reaction to an intervention, will allow better design of future clinical trials. Future trials can target specific patient populations that have genotypes that are more likely to respond. Alternatively, patients can be stratified on the basis of their genotype to allow analyses of drug response by genotype. Given the acuity of the condition, the targeting of individuals who have a certain genotype or the stratification of subjects by genotype before randomization will require rapid and accurate genotyping assays that are not yet available.

fied on the basis of their genotype to allow analyses of drug response by genotype. Given the acuity of the condition, the targeting of individuals who have a certain genotype or the stratification of subjects by genotype before randomization will require rapid and accurate genotyping assays that are not yet available. Understanding genetic risk factors can help with risk assessment in health policy decisions, as well. Young, healthy patients often are considered to have a low risk of serious or complicated influenza infection or pneumonia and are not recommended for routine vaccination or close observation in the hospital [89]. Gene–age interaction for the TNF-α and IL-10 genes in ARDS, however, suggests that certain young individuals have a particularly high risk of developing and dying from ARDS. Knowledge of the genetic predisposition to developing ALI/ARDS might help identify young, healthy patients who would benefit either from early vaccination while still healthy or from closer observation in the event of any insult such as a community-acquired pneumonia.

ularly high risk of developing and dying from ARDS. Knowledge of the genetic predisposition to developing ALI/ARDS might help identify young, healthy patients who would benefit either from early vaccination while still healthy or from closer observation in the event of any insult such as a community-acquired pneumonia. Identification of appropriate patient populations or conditions for intervention In clinical practice, outside the strict inclusion and exclusion criteria and methodology of a randomized, control trial, the patient population is more heterogeneous, and there is a larger variability in the response and the complication rate associated with the intervention [90]. Given potential gene–environment interaction with the site of injury, it is possible that interventions based on surfactant or TNF-α may have varying efficacy depending on whether the initial injury predisposing to ARDS is pulmonary or extrapulmonary. Defining this heterogeneity in response in the context of both environmental and genetic factors in a population falls within the emerging field of genetic pharmacoepidemiology [91]. Obviously, the genes that are directly targeted by the intervention are important. Genes governing drug metabolism, receptor binding to the drug, and other genes in the causal pathways of the disease process probably are important, as well.

opulation falls within the emerging field of genetic pharmacoepidemiology [91]. Obviously, the genes that are directly targeted by the intervention are important. Genes governing drug metabolism, receptor binding to the drug, and other genes in the causal pathways of the disease process probably are important, as well. In essence, this consideration is a special example of gene–environment interaction, in which one of the key environmental factors is the drug or intervention. Identifying these interactions is important in understanding and interpreting the genetic contribution to ALI and in identifying which patient populations and what conditions have the most appropriate risk/benefit ratios warranting a particular intervention. This understanding will be important especially in interventions to prevent ALI/ARDS because of the many different causes that can lead to lung injury. Limitations and barriers to future prevention and treatment in acute lung injury/acute respiratory distress syndrome There is great excitement about how the rapid advances in genomics and genetic epidemiology may lead to individualized medicine in complex diseases such as ALI. This excitement should be tempered to avoid unrealistic expectations. Significant limitations and barriers may limit the application of genomics and genetic epidemiology to future preventive and therapeutic interventions in ALI/ARDS.

etic epidemiology may lead to individualized medicine in complex diseases such as ALI. This excitement should be tempered to avoid unrealistic expectations. Significant limitations and barriers may limit the application of genomics and genetic epidemiology to future preventive and therapeutic interventions in ALI/ARDS. One possible limitation is that any novel intervention based on genetic variation will not be universally beneficial. Rather, its applicability and benefit will be limited to those who have the disease genotype. Thus, the population prevalence of the disease-associated genotypes will determine the size of the patient population that may benefit from this intervention and the generalizability of the intervention. The efficacy of the intervention will be further limited by gene–gene and gene–environment interactions. The pathogenesis of ALI/ARDS consists of interactions and balances between multiple pathways involved in inflammation, coagulation, fibrogenesis, fluid transport, and apoptosis [6]. With such a complex, interdependent process, it is likely that multiple genes are important, and any intervention based on one gene is unlikely to be uniformly and universally beneficial. The presence of gene–environment interactions would further limit the context in which novel therapy will be appropriate.

tosis [6]. With such a complex, interdependent process, it is likely that multiple genes are important, and any intervention based on one gene is unlikely to be uniformly and universally beneficial. The presence of gene–environment interactions would further limit the context in which novel therapy will be appropriate. Another limitation and barrier to the application of genetic epidemiology in ALI/ARDS is the need for large, well-phenotyped cohorts that are sufficiently powered to account adequately for gene–gene and gene–environment interactions. This need is especially pronounced in genetic pharmacoepidemiology. With the need for large DNA databases comes the need for more research on issues surrounding genetic research in the critical care setting, where mortality is high, and consent is obtained from family members or surrogates. There is a need for a better understanding of the concerns of the patients and their surrogates and how best to protect those interests. In addition, the racial difference in ALI means that large cohorts of minority groups will be vital to determine the efficacy of an intervention in different racial groups.

urrogates. There is a need for a better understanding of the concerns of the patients and their surrogates and how best to protect those interests. In addition, the racial difference in ALI means that large cohorts of minority groups will be vital to determine the efficacy of an intervention in different racial groups. Last, the narrow window of opportunity for intervention presents another barrier for any interventions in ALI/ARDS. For example, ALI/ARDS develops rapidly after the initial injury with a median of 1 day after admission to the ICU (interquartile range, 0–5 days) [2], [79]. Such a narrow window for intervention requires rapid identification of appropriate patients for intervention and initiation of intervention as early as possible, possibly in the emergency room. Therapy based on a specific genotype would require either rapid DNA testing or prior genotyping of all patients and storage of this information, either in the medical record or in a secure device on the persons themselves. Neither of these measures is available currently.

ly as possible, possibly in the emergency room. Therapy based on a specific genotype would require either rapid DNA testing or prior genotyping of all patients and storage of this information, either in the medical record or in a secure device on the persons themselves. Neither of these measures is available currently. Summary The application of genetic epidemiology and genomics to the study of ALI/ARDS is in its infancy. Optimal study designs and approaches are still being discussed, and the large, prospective cohorts that will be necessary to examine gene–environment interaction and to confirm prior findings are being developed. There will be technological and analytic challenges to the proper study of genetic determinants of ALI/ARDS that will benefit from a multifaceted approach. There will be significant barriers to the translation of genetic epidemiology studies and genomics to preventive and therapeutic interventions, and any intervention is unlikely to occur in the near future. In oncology, where there is a longer history of genetic and molecular epidemiology studies, commercially available genetic tests now allow individualized risk assessment and tailored therapy for breast cancer. Although significant challenges lie ahead, there is a similar potential for such individualized risk assessment and therapy in critical care medicine. Large, well-phenotyped studies will be crucial to this goal. Dr. Gong is supported by research grant K23 HL67197, R01 HL084060 and HL60710 from the National Heart, Lung, and Blood Institute.

Acute allograft rejection remains a prevalent and serious problem in lung transplantation, with an incidence of 36% in the first year after transplant according to the latest report from the registry of the International Society for Heart and Lung Transplantation (ISHLT).1 Although acute lung rejection in itself is rarely fatal, its indirect consequences have considerable adverse effects on transplant outcomes. Treatment of acute rejection with increased immunosuppression increases the risk for many post-transplant infections. Furthermore, despite treatment, cellular rejection and humoral rejection constitute the major risk factors for bronchiolitis obliterans syndrome (BOS). BOS is a condition of progressive airflow obstruction thought to reflect a manifestation of chronic lung transplant rejection. Most post-transplant deaths beyond the first year occur directly or indirectly as a result of BOS.2

oral rejection constitute the major risk factors for bronchiolitis obliterans syndrome (BOS). BOS is a condition of progressive airflow obstruction thought to reflect a manifestation of chronic lung transplant rejection. Most post-transplant deaths beyond the first year occur directly or indirectly as a result of BOS.2 Compared with other solid organs, the lung appears to be at particularly high risk for rejection. Although the reasons are not entirely clear, increased lung vulnerability to early ischemic injury, recurrent infections, and constant environmental exposures might contribute to the high rates of lung rejection. In this article, the authors present the immunologic basis for acute lung allograft rejection, describing the clinical and pathologic features of acute cellular perivascular (A-grade) rejection and acute cellular airway (B-grade) rejection also known as lymphocytic bronchiolitis (Figs. 1 and 2 ). In addition, the authors discuss the emerging understanding of the importance of humoral rejection in lung transplantation, focusing on the role of anti-HLA antibodies, which can be present before or develop de novo after transplantation (see Figs. 1 and 2). Current strategies will be highlighted for the prevention and treatment of both cellular and humoral allograft rejection.Fig. 1 Venn diagram representing the relationship between acute cellular rejection (grade A and grade B) and humoral rejection manifest by presence of anti-HLA antibodies or histologic findings.

2). Current strategies will be highlighted for the prevention and treatment of both cellular and humoral allograft rejection.Fig. 1 Venn diagram representing the relationship between acute cellular rejection (grade A and grade B) and humoral rejection manifest by presence of anti-HLA antibodies or histologic findings. Fig. 2 Examples of acute lung allograft rejection pathology. (A–D) Grade A acute cellular rejection; arrows indicate vessel lumina. (A) Grade A1 acute rejection with rare perivascular lymphocytes, H&E. (B) Grade A2 acute rejection with a prominent perivascular mononuclear infiltrate, H&E. (C) Grade A3 acute rejection with extensive perivascular infiltrate extending into interstitial spaces, H&E. (D) Grade A4 acute rejection with a diffuse mononuclear infiltrate with lung injury, including fibrinous exudate (arrowhead), H&E. (E) Grade B1R (low-grade) lymphocytic bronchiolitis with small numbers of bronchiolar mononuclear cells, H&E. (F) Grade B2R (high-grade) lymphocytic bronchiolitis with dense bronchiolar mononuclear infiltrate and epithelial involvement, H&E. (G) Neutrophilic capillaritis consistent with humoral rejection (arrowheads indicate neutrophils), H&E, with (H) associated immunofluorescence on frozen lung tissue, demonstrating ring-shaped profiles of C4d staining in alveolar septal capillaries, Immunofluorescent staining. All images are at 200× magnification.

H&E. (G) Neutrophilic capillaritis consistent with humoral rejection (arrowheads indicate neutrophils), H&E, with (H) associated immunofluorescence on frozen lung tissue, demonstrating ring-shaped profiles of C4d staining in alveolar septal capillaries, Immunofluorescent staining. All images are at 200× magnification. Mechanisms of acute rejection In the absence of immunosuppression, the transplant recipient develops a robust response to the allograft, predominantly driven by T-cell recognition of foreign major histocompatibility complex (MHC) proteins, called human leukocyte antigens (HLA) in humans. Foreign MHC, expressed on transplanted tissue cells, is first presented directly to recipient T-cells by donor dendritic cells in the graft (direct pathway). As donor antigen presenting cells (APCs) die out or are destroyed, recipient dendritic cells process and present alloantigens to recipient T-cells (indirect pathway).3

gn MHC, expressed on transplanted tissue cells, is first presented directly to recipient T-cells by donor dendritic cells in the graft (direct pathway). As donor antigen presenting cells (APCs) die out or are destroyed, recipient dendritic cells process and present alloantigens to recipient T-cells (indirect pathway).3 HLA genes are located on the short arm of human chromosome 6 and are traditionally divided into two classes based on historic differentiation. The classical HLA class 1 genes include A, B, and Cw loci, which are expressed on most nucleated cells. The classical HLA class 2 genes include DR, DQ, and DP genes, which are expressed constitutively on B-cells, monocytes, dendritic cells, and other APCs, but can be upregulated on various other cells under inflammatory conditions. The extraordinary diversity of HLA polymorphisms creates a considerable barrier to transplantation, as the donor organ is quickly recognized as nonself on the basis of HLA differences with the recipient.3

ytes, dendritic cells, and other APCs, but can be upregulated on various other cells under inflammatory conditions. The extraordinary diversity of HLA polymorphisms creates a considerable barrier to transplantation, as the donor organ is quickly recognized as nonself on the basis of HLA differences with the recipient.3 The common pathway of acute cellular rejection involves the recruitment and activation of recipient lymphocytes (predominantly effector T-cells) to the lung allograft, which can result in allograft injury and loss of function.3 Consequently, successful outcomes after lung transplantation did not become a possibility until the widespread introduction into clinical practice of the calcineurin inhibitor cyclosporine, which permits a highly effective blockade of T-cell activation and proliferation.4, 5 However, in spite of intensive T-cell suppressive strategies, lung transplant patients continue to experience high rates of rejection. This process of allorecognition is likely augmented by local innate immune activation through endogenous tissue injury and exogenous infection. Innate immune activation can promote alloantigen presentation, costimulation, and T-cell activation.

s, lung transplant patients continue to experience high rates of rejection. This process of allorecognition is likely augmented by local innate immune activation through endogenous tissue injury and exogenous infection. Innate immune activation can promote alloantigen presentation, costimulation, and T-cell activation. Humoral responses following lung transplantation have only recently been appreciated due to the advent of modern highly sensitive solid-phase antibody detection techniques. It is now clear that some patients present for transplantation with preformed anti-HLA antibodies, which are usually acquired through prior pregnancy, transfusions, or transplantation. Immune stimulation by prior infections or autoimmunity might contribute to the development of antibodies to allo-MHC in those patients with no identifiable risk factors. These pre-existing antibodies can react with donor antigens, leading to immediate graft loss (hyperacute rejection) or accelerated humoral rejection and BOS.6 In addition, some lung transplant recipients appear to mount a humoral response to the allograft after transplantation. Most evidence suggests that this humoral response occurs to donor MHC antigens, although other endothelial or epithelial antigens expressed in the lung may become antibody targets as well. T-cells activated through indirect presentation provide help for B-cell memory, antibody class switching, and affinity maturation in the presence of appropriate cytokines and costimulatory factors. Acute and chronic humoral rejection have been well described in renal transplantation.6 Furthermore, histologic features of antibody-mediated rejection can be found on lung biopsy in the absence of measurable anti-HLA antibodies.7

d affinity maturation in the presence of appropriate cytokines and costimulatory factors. Acute and chronic humoral rejection have been well described in renal transplantation.6 Furthermore, histologic features of antibody-mediated rejection can be found on lung biopsy in the absence of measurable anti-HLA antibodies.7 The precise immune mechanisms and their complex interactions leading to stimulation of cellular or humoral immunity and ultimately to lung rejection remain to be fully elucidated. Nevertheless, acute cellular rejection, acute humoral rejection resulting in vascular injury, and the presence of anti-HLA antibodies are processes that overlap clinically and may potentiate each other (see Fig. 1).

mulation of cellular or humoral immunity and ultimately to lung rejection remain to be fully elucidated. Nevertheless, acute cellular rejection, acute humoral rejection resulting in vascular injury, and the presence of anti-HLA antibodies are processes that overlap clinically and may potentiate each other (see Fig. 1). Acute cellular rejection Clinical Presentation and Diagnosis Acute lung allograft rejection can be asymptomatic at the time of pathologic diagnosis. When present, symptoms range from dyspnea, cough, or sputum production to acute respiratory distress, with physical findings that may include fever, hypoxia, and adventitious sounds on lung auscultation.8 Because of the nonspecific nature of symptoms and signs, emphasis should be placed on objective data, mainly pulmonary function testing, in identifying patients at risk for rejection. Spirometry has been found to have a sensitivity of greater than 60% for detecting infection or rejection grade A2 and higher, but it cannot differentiate between the two.9 Radiographic imaging of lung transplant patients is useful in identifying specific causes of symptoms or decreased pulmonary function, such as focal infections or neoplasms. Findings of ground glass opacities, septal thickening, volume loss, and pleural effusions on high-resolution chest computed tomography (CT) scans suggest acute rejection. Although early small studies attempted to demonstrate the usefulness of chest radiographs and chest CT scans in the diagnosis of rejection, more recent data show very low sensitivity for acute rejection (as low as 35%) and no discriminatory value between rejection and other processes.10, 11 Given the poor specificity of pulmonary function tests and radiographic studies, the authors discourage empiric treatment of rejection and recommend histopathologic analysis of lung tissue to diagnose and grade acute lung rejection. The incidence of acute rejection is highest within the first year after transplant, arguing for a high clinical suspicion during this time period.

radiographic studies, the authors discourage empiric treatment of rejection and recommend histopathologic analysis of lung tissue to diagnose and grade acute lung rejection. The incidence of acute rejection is highest within the first year after transplant, arguing for a high clinical suspicion during this time period. Bronchoscopy, with bronchoalveolar lavage (BAL) and transbronchial biopsies, is the most important diagnostic modality for acute allograft rejection and should be considered in any lung transplant recipient with allograft dysfunction. It allows acute rejection to be distinguished from other potential etiologies of allograft dysfunction such as airway stenosis or infection. Most transbronchial biopsies are performed in the lower lobes, a practice that seems reasonable in light of data showing that different lung lobes have similar rejection grades and that if rejection is present, the grade is usually worse in the lower lobes as compared with the upper lobes.12 The Lung Rejection Study Group (LRSG) now recommends 5 pieces of well-expanded alveolated lung parenchyma to provide adequate sensitivity to diagnose rejection.13 Adverse events reported with bronchoscopy in lung transplant recipients are relatively low and include transient hypoxemia (10.5%), bleeding greater than 100 mL (4%), clinically significant pneumothorax (0.6–2.5%), arrhythmia (0.57%–4%), possibly postprocedural pneumonia (8%), and ventilation support (0.32%), but no reported mortality.14, 15, 16, 17

onchoscopy in lung transplant recipients are relatively low and include transient hypoxemia (10.5%), bleeding greater than 100 mL (4%), clinically significant pneumothorax (0.6–2.5%), arrhythmia (0.57%–4%), possibly postprocedural pneumonia (8%), and ventilation support (0.32%), but no reported mortality.14, 15, 16, 17 While there is widespread agreement on the benefit of clinically-directed bronchoscopy in lung transplantation, the role of surveillance bronchoscopy in asymptomatic patients remains disputed. Many centers perform scheduled bronchoscopies at about 1 month, 3 months, 6 months, and annually after transplant, in addition to the clinically-indicated and post-rejection follow-up bronchoscopies.18 The rationale includes the occurrence of clinically silent acute rejection, inadequate surrogate markers for acute rejection, and the relatively low risks of the bronchoscopy procedure. Grade A2 and higher acute rejection has been found in up to 18% to 39% of asymptomatic patients,14, 19, 20 with occasional presence of late-onset acute rejection beyond 1 year after transplant.21 Disputing this approach, one group showed that 3-year outcomes in patients who underwent only clinically-indicated bronchoscopies were comparable to outcomes in patients who underwent surveillance bronchoscopies,15 as well as to the ISHLT database outcomes.22 A randomized trial would be helpful to determine the benefit of surveillance bronchoscopies in lung transplant recipients.

in patients who underwent only clinically-indicated bronchoscopies were comparable to outcomes in patients who underwent surveillance bronchoscopies,15 as well as to the ISHLT database outcomes.22 A randomized trial would be helpful to determine the benefit of surveillance bronchoscopies in lung transplant recipients. In an attempt to obviate the need for surveillance biopsies, many reports have focused on less invasive surrogates of acute lung rejection. Multiple studies have assessed BAL cells and proteins as possible correlates of acute rejection, but many of these studies were small and have not been replicated.23 Acute rejection has been associated with elevated CD8 T-cells, activated CD4 T-cells,24 activated NK cells,25 elevated interleukin (IL)-17,26 IL-15,27 and interferon-gamma in the BAL.28 A pilot study of gene expression in the BAL fluid of lung transplant recipients found that gene expression signatures related to T-lymphocyte function, cytotoxic CD8 activity, and neutrophil degranulation correlate with acute rejection.29 Additional studies are needed to validate these findings and establish whether BAL microarray determinations of acute rejection signature could be cost-effective and provide information that supplements or replaces biopsy results.

n, cytotoxic CD8 activity, and neutrophil degranulation correlate with acute rejection.29 Additional studies are needed to validate these findings and establish whether BAL microarray determinations of acute rejection signature could be cost-effective and provide information that supplements or replaces biopsy results. Even more attractive are studies of noninvasive means of diagnosing acute rejection without bronchoscopy. Although no effective serum biomarkers are currently in use in clinical lung transplant, many have been studied, and some, such as the hepatocyte growth factor, have been shown to correlate with acute rejection in small single-center studies.30 In 2002, the Cylex Immune Cell Function Assay (ImmuKnow; Cylex, Incorporated, Columbia, MA, USA) was approved by the US Food and Drug Administration to measure global immune function in solid organ transplant recipients. This assay measures the in vitro production of adenosine triphosphate (ATP) by the patient’s peripheral blood CD4 T-cells in response to stimulation by phytohemagglutinin-L. Several studies in kidney, liver, heart, and small bowel allograft recipients have demonstrated that low ATP levels (≤225 ng/mL) correlate with infection, while high levels (≥ 525 ng/mL) are associated with rejection.31 Two studies that evaluated this assay in lung transplant recipients demonstrated that low ATP levels correlated with infection,32, 33 but association with acute rejection was not assessed. Preliminary data published in abstract form showed that 87% of lung rejection episodes occurred in the setting of low-to-moderate ATP levels.34 Additionally, exhaled breath analysis studies have shown some promising results. Exhaled nitric oxide (NO) has been correlated with lymphocytic bronchiolitis35 and acute rejection,36 and, in a study of inert gas single-breath washout, the slope of alveolar plateau for Helium (SHe) had a sensitivity of 68% for acute rejection.9 In summary, no surrogate markers have been sufficiently validated as a means to reproducibly identify patients with acute rejection with adequate specificity, and none supplant direct histopathological examination of lung tissue. Nevertheless, further studies in this arena will likely provide valuable information about underlying mechanisms of rejection and better explain clinical heterogeneity of the disease.

identify patients with acute rejection with adequate specificity, and none supplant direct histopathological examination of lung tissue. Nevertheless, further studies in this arena will likely provide valuable information about underlying mechanisms of rejection and better explain clinical heterogeneity of the disease. Histology and Cellular Infiltration of Acute Lung Rejection The histologic appearance of acute lung allograft rejection and the grading rules for acute cellular rejection (A-grade), airway inflammation (B-grade), chronic airway rejection or bronchiolitis obliterans (C-grade), and chronic vascular rejection or accelerated graft vascular sclerosis (D-grade) are outlined in the Working Formulation published by the Lung Rejection Study Group (LRSG) of the ISHLT.13The grading scheme and its key features are summarized in Table 1 , and illustrative images from the authors’ institution are shown in Fig. 2.Table 1 Pathologic grading of lung rejection

ascular sclerosis (D-grade) are outlined in the Working Formulation published by the Lung Rejection Study Group (LRSG) of the ISHLT.13The grading scheme and its key features are summarized in Table 1 , and illustrative images from the authors’ institution are shown in Fig. 2.Table 1 Pathologic grading of lung rejection Category of Rejection Grade Severity Histologic Appearance Grade A: acute rejection 0 None Normal lung 1 Minimal Inconspicuous small mononuclear perivascular infiltrates 2 Mild More frequent, more obvious, perivascular infiltrates; eosinophils may be present 3 Moderate Dense perivascular infiltrates, extension into interstitial space, can involve endothelialitis, eosinophils, and neutrophils 4 Severe Diffuse perivascular, interstitial, & air-space infiltrates with lung injury. Neutrophils may be present. Grade B: airway inflammation 0 None No evidence of bronchiolar inflammation 1R Low grade Infrequent, scattered, or single-layer mononuclear cells in bronchiolar submucosa 2R High grade Larger infiltrates of larger and activated lymphocytes in bronchiolar submucosa; can involve eosinophils and plasmacytoid cells X Ungradable No bronchiolar tissue available Grade C: chronic airway rejection—obliterative bronchiolitis 0 Absent If present describes intraluminal airway obliteration with fibrous connective tissue 1 Present Grade D: chronic vascular rejection—accelerated graft vascular sclerosis No grading Fibrointimal thickening of arteries and poorly cellular hyaline sclerosis of veins; usually requires open lung biopsy for diagnosis Abbreviation: R, revised.

ibes intraluminal airway obliteration with fibrous connective tissue 1 Present Grade D: chronic vascular rejection—accelerated graft vascular sclerosis No grading Fibrointimal thickening of arteries and poorly cellular hyaline sclerosis of veins; usually requires open lung biopsy for diagnosis Abbreviation: R, revised. Data from Stewart S, Fishbein MC, Snell GI, et al. Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant 2007;26(12):1229–42.

ibes intraluminal airway obliteration with fibrous connective tissue 1 Present Grade D: chronic vascular rejection—accelerated graft vascular sclerosis No grading Fibrointimal thickening of arteries and poorly cellular hyaline sclerosis of veins; usually requires open lung biopsy for diagnosis Abbreviation: R, revised. Data from Stewart S, Fishbein MC, Snell GI, et al. Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant 2007;26(12):1229–42. The typical A-grade acute cellular rejection of the lung allograft manifests as perivascular mononuclear inflammatory cell infiltrates with or without interstitial mononuclear cells. Most of these mononuclear cells are T-cells, with a preponderance of CD8 T-cells,37 although a few studies have described increased populations of B-cells or eosinophils.13, 38, 39 Increasing thickness of the mononuclear cell cuff around vessels with increasing mononuclear invasion into the interstitial and alveolar spaces determines the A-grade (see Table 1 and Fig. 2A–D). While the intra-reader agreement for acute rejection has been found to be good (kappa 0.65–0.795),40, 41 the inter-reader reliability of this grading scheme has ranged from good to suboptimal (kappa as high as 0.73 and as low as 0.47).40, 41, 42 Confounding features, such as concurrent infection or alveolar damage early after transplant, may additionally blur the picture and contribute to the inter-reader pathologist discordance.42 In general, the LRSG recommends grading rejection only after the exclusion of infection.

gh as 0.73 and as low as 0.47).40, 41, 42 Confounding features, such as concurrent infection or alveolar damage early after transplant, may additionally blur the picture and contribute to the inter-reader pathologist discordance.42 In general, the LRSG recommends grading rejection only after the exclusion of infection. The B-grade airway mononuclear inflammation is clearly part of the spectrum of acute cellular rejection (see Fig. 2E, F), but grading remains inconsistent due to frequent lack of airway tissue on biopsies, susceptibility to tissue artifacts, and confounding by concurrent infections. Because of low inter-reader reliability of the prior 5-grade (0–4) grading schema for B-grade rejection,40, 41 the LRSG has simplified the B-grading to 3 grades (0–2) (see Table 1).13 This nomenclature is to be used for grading noncartilaginous small airways only after rigorous exclusion of infection.

rrent infections. Because of low inter-reader reliability of the prior 5-grade (0–4) grading schema for B-grade rejection,40, 41 the LRSG has simplified the B-grading to 3 grades (0–2) (see Table 1).13 This nomenclature is to be used for grading noncartilaginous small airways only after rigorous exclusion of infection. Clinical Significance of Acute Rejection Multiple studies have demonstrated that acute rejection is the major risk factor for the development of chronic airflow obstruction: a single episode of acute rejection as well as increased frequency and severity of acute rejection increase the risk for BOS.2, 43 An area of controversy has been the significance of minimal acute rejection (A1) or of a solitary perivascular infiltrate. In the early years of lung transplantation, A1 rejection was usually discounted and not treated. Studies have since found that minimal acute rejection (grade A1) increases the risk of subsequent higher-grade rejections (grade ≥A2)44, 45 and of subsequent BOS46 and that an untreated solitary perivascular monocytic infiltrate may lead to worsening acute rejection.47 Furthermore, based on multiple studies, grade B lymphocytic bronchiolitis is now also known to be an important risk factor for BOS48 and death, independent of acute vascular rejection.2, 48 Although lymphocytic inflammation is frequently seen on endobronchial biopsies of large cartilaginous airways, its clinical and prognostic significance remain unclear, and there is no demonstrated link between lymphocytic inflammation seen on endobronchial biopsies and lymphocytic bronchiolitis or bronchitis seen on transbronchial biopsies.49, 50, 51, 52 Although eosinophils,53 B-cells,38 and mast cells54 have been identified in acute rejection biopsies and have been correlated with worse prognosis, their exact clinical significance remains unclear.

seen on endobronchial biopsies and lymphocytic bronchiolitis or bronchitis seen on transbronchial biopsies.49, 50, 51, 52 Although eosinophils,53 B-cells,38 and mast cells54 have been identified in acute rejection biopsies and have been correlated with worse prognosis, their exact clinical significance remains unclear. Risk Factors for Acute Rejection While many risk factors for acute lung allograft rejection have been studied, this article will focus on those that have been found to be significant and categorize them as allorecognition-related, immunosuppression-related, recipient-related, and infectious.

seen on endobronchial biopsies and lymphocytic bronchiolitis or bronchitis seen on transbronchial biopsies.49, 50, 51, 52 Although eosinophils,53 B-cells,38 and mast cells54 have been identified in acute rejection biopsies and have been correlated with worse prognosis, their exact clinical significance remains unclear. Risk Factors for Acute Rejection While many risk factors for acute lung allograft rejection have been studied, this article will focus on those that have been found to be significant and categorize them as allorecognition-related, immunosuppression-related, recipient-related, and infectious. Allorecognition-related risk factors It is generally thought that the intensity of the host alloimmune response is related to recipient recognition of differences with the donor antigens and that this process drives acute lung allograft rejection. Consistent with this idea, several single-center studies have shown that an increasing degree of HLA mismatch, especially at the HLA-DR, HLA-B, and HLA-A loci, increases the risk of acute rejection.55, 56, 57 Additionally, the ISHLT registry data show a correlation between HLA matching and gender-matching and 5-year survival.1 Although not very well understood, multiorgan transplantation is generally believed to provide an immunologic advantage and lead to lower rates of rejection due to dampening of allo-recognition through a high burden of foreign HLA antigens. Decreased rejection has been shown for grafted kidney, liver, and heart in combined heart–kidney, liver–kidney, and heart–lung transplant recipients,58, 59 although this benefit does not seem to translate into prolonged graft or recipient survival. The data regarding lung rejection in the presence of a second organ remain inconclusive.1, 59, 60, 61

hown for grafted kidney, liver, and heart in combined heart–kidney, liver–kidney, and heart–lung transplant recipients,58, 59 although this benefit does not seem to translate into prolonged graft or recipient survival. The data regarding lung rejection in the presence of a second organ remain inconclusive.1, 59, 60, 61 Immunosuppression-related risk factors While it is clear that adequate immunosuppression is necessary for lung allograft maintenance, the optimal regimen has not been defined. Standard immune suppression includes a calcineurin inhibitor, a cell-cycle inhibitor, and a corticosteroid. Several studies suggest that there may be lower incidence of acute rejection with tacrolimus as opposed to cyclosporine.62, 63 One randomized double-blind trial showed decreased rejection with everolimus as opposed to azathioprine.64 The self-reported ISHLT registry data support the idea of decreased acute rejection episodes with tacrolimus and MMF as compared with cyclosporine and azathioprine.1 Surprisingly very few studies have directly examined the link between levels of immunosuppression and acute rejection. High titers of Epstein-Barr virus (EBV) in peripheral blood, a surrogate marker of a high overall level of immunosuppression, have been found to correlate with lower incidence of acute rejection.65 Furthermore, one episode of early high-grade acute rejection appears predictive of additional acute rejection episodes within the first year after lung transplant, suggesting that more aggressive immunosuppression should be used in these patients.45

ave been found to correlate with lower incidence of acute rejection.65 Furthermore, one episode of early high-grade acute rejection appears predictive of additional acute rejection episodes within the first year after lung transplant, suggesting that more aggressive immunosuppression should be used in these patients.45 Recipient-related risk factors Genetic polymorphisms have also been considered as potential independent risk factors for rejection.66 A genotype leading to increased IL-10 production may protect against acute rejection,67 while a multidrug resistance genotype (MDR1 C3435T) appears to predispose to treatment-resistant acute rejection,68 and a copy number variation in the CCL4L chemokine gene is associated with susceptibility to acute rejection.69 Additionally, the idea has been developed that genetic variation in innate pattern recognition receptors modulates the development of acute rejection after lung transplantation. In this regard, the authors have found reduced acute rejection in association with a variant in toll-like receptor 4 (TLR4) that blunts the innate immune response, and increased rejection with a CD14 variant that augments the innate response.70, 71 Although these studies suggest that polymorphic variants outside of the HLA region also influence the risk for acute rejection, larger multicenter efforts are needed to fully validate these findings and also test for gene–gene and gene–environment interactions among relevant polymorphisms.

ts the innate response.70, 71 Although these studies suggest that polymorphic variants outside of the HLA region also influence the risk for acute rejection, larger multicenter efforts are needed to fully validate these findings and also test for gene–gene and gene–environment interactions among relevant polymorphisms. Collectively these genetic studies provide considerable support for the overall hypothesis that the constant interplay between the environment and pulmonary innate immunity modulates adaptive alloimmunity after lung transplantation. Consistent with this paradigm, gastroesophageal reflux disease also has been associated with increased rates of acute rejection.72 Furthermore, as will be discussed, rejection has been appreciated following a number of respiratory infections. The effect of age on acute rejection appears to be bimodal, with lowest incidence of acute rejection in infancy (younger than age 2)73 and increased risk during childhood as compared with adulthood.74 The incidence of acute rejection in older lung transplant recipients (age 65 or higher) appears to be similar to that of younger adults,75 while their rate of infections appears higher, potentially contributing to increased mortality.76 However, more studies are needed to determine the true effect of age on rejection before a strategy of reduced immunosuppression can be advocated in older recipients.

her) appears to be similar to that of younger adults,75 while their rate of infections appears higher, potentially contributing to increased mortality.76 However, more studies are needed to determine the true effect of age on rejection before a strategy of reduced immunosuppression can be advocated in older recipients. Infectious risk factors Infectious etiologies have been given a lot of attention as potentiators of adaptive immunity in solid organ transplantation. Viral infections have long been thought to modulate the immune system and heighten alloreactivity. Indeed, a high incidence of acute rejection has been found in lung transplant recipients following community-acquired respiratory tract infections with rhinovirus, parainfluenza virus, influenza virus, human metapneumovirus, coronavirus, and respiratory syncytial virus (RSV),77, 78, 79, 80 although respiratory viruses do not appear to be associated with acute rejection during the acute phase of infection.81 Studies on the role of other herpes viruses and polyoma viruses are being conducted, with no evidence of association with acute rejection to date.82, 83, 84 Studies directly linking cytomegalovirus (CMV) infection or CMV prophylaxis strategies with acute rejection have been inconsistent,2 and a recent randomized trial of CMV prophylaxis did not identify a correlation between CMV incidence and acute rejection rates.85 In one study, bacterial infection with Chlamydia pneumoniae was linked to the development of acute rejection and BOS.86

prophylaxis strategies with acute rejection have been inconsistent,2 and a recent randomized trial of CMV prophylaxis did not identify a correlation between CMV incidence and acute rejection rates.85 In one study, bacterial infection with Chlamydia pneumoniae was linked to the development of acute rejection and BOS.86 Treatment of Acute Lung Rejection Treatment of acute lung allograft rejection consists of increased immunosuppression. There has been clear consensus that grade A2 and higher-grade rejection episodes require treatment. However, in light of recent evidence that grade A1 rejection and lymphocytic bronchiolitis are major risk factors for BOS, treatment seems prudent for those entities as well. The mainstays of treatment for acute lung rejection are pulse steroids. Several studies from the 1990s showed successful resolution or improvement of acute rejection after high-dose steroid treatment.38, 87 There are no data to clearly guide dosing of the pulse steroids; a standard dose is 500 mg of methylprednisolone intravenously,4 although centers use doses that range from 125 mg up to 1000 mg per day. Duration of treatment also varies but typically includes at least 3 doses, followed by an oral prednisone taper.

8, 87 There are no data to clearly guide dosing of the pulse steroids; a standard dose is 500 mg of methylprednisolone intravenously,4 although centers use doses that range from 125 mg up to 1000 mg per day. Duration of treatment also varies but typically includes at least 3 doses, followed by an oral prednisone taper. Response to steroids is variable, but early post-transplant rejection seems to respond better than late rejection.88 A major challenge in lung transplantation has been the treatment of persistent or recurrent rejection. A repeat course of corticosteroids is one option. Several studies support switching from cyclosporine to tacrolimus for treatment of persistent acute rejection.89, 90 Many centers use alternative immunosuppressive agents such as polyclonal antithymocyte globulin (ATG), anti-IL-2 receptor (IL2R) antagonists, or muromonab-CD3 (OKT3).91 A recent report demonstrated the utility of alemtuzumab, an anti-CD52 monoclonal antibody, in the treatment of refractory acute rejection in a small cohort of patients who previously failed treatment with ATG.39 Other therapies that have been considered include inhaled cyclosporine,92, 93 extracorporeal photopheresis,94 and total lymphoid irradiation.95 The relationship between acute rejection, its current treatments, and the eventual occurrence of BOS is an area of considerable interest. Although acute rejection appears to be a major risk factor for BOS, it remains unclear how its treatment impacts long-term allograft function and patient survival.

Response to steroids is variable, but early post-transplant rejection seems to respond better than late rejection.88 A major challenge in lung transplantation has been the treatment of persistent or recurrent rejection. A repeat course of corticosteroids is one option. Several studies support switching from cyclosporine to tacrolimus for treatment of persistent acute rejection.89, 90 Many centers use alternative immunosuppressive agents such as polyclonal antithymocyte globulin (ATG), anti-IL-2 receptor (IL2R) antagonists, or muromonab-CD3 (OKT3).91 A recent report demonstrated the utility of alemtuzumab, an anti-CD52 monoclonal antibody, in the treatment of refractory acute rejection in a small cohort of patients who previously failed treatment with ATG.39 Other therapies that have been considered include inhaled cyclosporine,92, 93 extracorporeal photopheresis,94 and total lymphoid irradiation.95 The relationship between acute rejection, its current treatments, and the eventual occurrence of BOS is an area of considerable interest. Although acute rejection appears to be a major risk factor for BOS, it remains unclear how its treatment impacts long-term allograft function and patient survival. Humoral rejection Antibody-mediated allograft rejection is an increasingly recognized entity in lung transplantation. Early observations were based on the phenomenon of hyperacute rejection, where pre-existent donor-specific antibodies led to complement activation and rapid graft loss. With the advent of improved crossmatching before transplant, the incidence of hyperacute rejection in all organs has decreased. However, acute or chronic antibody-mediated lung rejection is an emerging and controversial subject. With the development of improved antibody detection and identification techniques, allograft-specific antibodies have been implicated in both acute and chronic kidney as well as heart rejection, and recent data have expanded the concept to lung transplantation.

d lung rejection is an emerging and controversial subject. With the development of improved antibody detection and identification techniques, allograft-specific antibodies have been implicated in both acute and chronic kidney as well as heart rejection, and recent data have expanded the concept to lung transplantation. The mechanisms by which antibody promotes lung allograft injury remain poorly understood. Antibody binding to allo-MHC or other endothelial or epithelial targets in the lung could lead to activation of the complement cascade with complement deposits leading to endothelial cell injury, production of pro-inflammatory and fibroblast-stimulating molecules, recruitment of inflammatory cells, and increased gene expression and subsequent proliferation,6, 96 potentially contributing to the generation of obliterative airway lesions. This section will discuss emerging issues in humoral lung rejection, including humoral sensitization both before and after lung transplantation, as well as pathologic features of humoral rejection, which can occur with or without the presence of detectable antibodies in the serum. Detection of anti-HLA Antibodies The original methodology for HLA serologic typing, antibody screening and identification, and direct crossmatching was the complement-dependent cytotoxicity (CDC) assay. The assay is based on the specific reactivity between serum antibody and cell surface antigen that activates complement, causing cell death, which can be identified under the microscope using vital dyes for cell staining.

nd identification, and direct crossmatching was the complement-dependent cytotoxicity (CDC) assay. The assay is based on the specific reactivity between serum antibody and cell surface antigen that activates complement, causing cell death, which can be identified under the microscope using vital dyes for cell staining. The CDC assay has now been replaced at most institutions with the more sensitive and specific solid-phase technologies that use a solid matrix coated with purified HLA antigens obtained from either cell lines or recombinant technology. These assays have the ability to detect both complement-fixing antibodies and noncomplement-fixing antibodies. Screening for antibodies is usually achieved by flow cytometry using a panel of 30 populations of beads coated with HLA antigens extracted from 30 individual donors (Fig. 3 ). This assay determines the panel reactive antibody (PRA), which is the percentage of beads or lymphocytes from the given panel that are recognized by patient’s anti-HLA antibodies. Once a patient’s PRA is determined to be positive, the actual HLA specificity of a recipient’s anti-HLA antibodies is determined using a single antigen bead assay with beads coated with recombinant HLA single antigens.97 The most recently developed solid-phase methodology for single-antigen detection is the Luminex single-antigen bead array assay (Luminex Corporation, Austin, TX, USA), which can simultaneously detect a maximum of 100 different colored beads in suspension with a different HLA antigen bound to each colored bead (Fig. 4 ).Fig. 3 Flow cytometric antibody screening for measurement of panel reactive antibody (PRA). FlowPRA beads are coated with purified HLA antigens. After incubation with patient serum and subsequent staining with FITC-labeled antihuman immunoglobulin (Ig)G, FlowPRA beads were analyzed on a flow cytometer. Beads with antibody binding have greater fluorescence intensity as represented by the rightward channel shift compared with the negative control. A percentage value of PRA is calculated based on the area of peak shifted. This patient demonstrated a PRA of 95% for HLA class 1 and a PRA of 94% for HLA class 2. The multiple peaks in the positive flow histogram are due to different bead populations emitting fluorescence of different intensity. The negative control was generated using uncoated beads. FITC, fluorescein isothiocyanate.

ifted. This patient demonstrated a PRA of 95% for HLA class 1 and a PRA of 94% for HLA class 2. The multiple peaks in the positive flow histogram are due to different bead populations emitting fluorescence of different intensity. The negative control was generated using uncoated beads. FITC, fluorescein isothiocyanate. Fig. 4 Standard Luminex single antigen (SA) bead assay results for detection of specific anti-HLA antibodies. SA bead numbers are listed in red on the x-axis. Each SA bead is coated with multiple copies of a single recombinant HLA antigen. The mean fluorescence intensity (MFI), which represents the strength of antibody binding to the beads, is plotted on the y-axis: the color of the bar represents the score of the antibody reactivity strength. The specific HLA antigens tested are listed in the gray chart below the graph. For this patient, positive antibody reactivities were assigned to the 6 beads DQB1*03:01/DQA1*05:03, DQB1*02:01/DQA1*05:01, DQB1*03:01/DQA1*05:05, DQB1*03:01/DQA1*06:01, DQB1*02:01/DQA1*04:01, and DQB1*04:01/DQA1*04:01 based on the cutoff established in the laboratory. Therefore, the patient has specific antibodies against HLA DQA 1 chains encoded by DQA1 alleles DQA1*05:03, DQA1*05:01, DQA1*05:05, DQA1*06:01, and DQA1*04:01. The presence of antibodies against DQB1 chains encoded by alleles DQB1*03:01, DQB1*02:01, and DQB1*04:01 can be excluded based on the negative reactivities with other beads, which also carry the DQB1 chains/antigens encoded by these alleles.

y DQA1 alleles DQA1*05:03, DQA1*05:01, DQA1*05:05, DQA1*06:01, and DQA1*04:01. The presence of antibodies against DQB1 chains encoded by alleles DQB1*03:01, DQB1*02:01, and DQB1*04:01 can be excluded based on the negative reactivities with other beads, which also carry the DQB1 chains/antigens encoded by these alleles. In spite of these technological advances, antibodies may still be present at a level of detection below the sensitivity of the methodology or against antigens not represented by the screening reagents. However, it is believed that antibodies that remain undetected by current methods are mostly weak antibodies and may be clinically irrelevant. Nevertheless, the most definitive compatibility test remains the real-time crossmatch of the recipient serum with the potential donor cells. Flow crossmatch, whereby actual donor cells are incubated with recipient serum and bound antibodies are then tagged with secondary fluorescent anti-immunoglobulin (Ig)G antibodies, has been proven to be up to 10 to 250 times more sensitive than a CDC crossmatch.98

of the recipient serum with the potential donor cells. Flow crossmatch, whereby actual donor cells are incubated with recipient serum and bound antibodies are then tagged with secondary fluorescent anti-immunoglobulin (Ig)G antibodies, has been proven to be up to 10 to 250 times more sensitive than a CDC crossmatch.98 Pre-transplant Considerations for Sensitized Patients One of the major goals in donor selection is to avoid HLA antigens, against which the potential recipient has preformed antibodies. About 10% to 15% of lung transplant recipients are presensitized to HLA antigens.99 Antibody-detection technologies identify unacceptable donor antigens that should be avoided at the time of transplant. When a donor becomes available, information about the donor HLA antigens and the recipient antibodies is compared, constituting a virtual crossmatch and allowing for the real-time prospective crossmatch to be waived. This virtual cross-match approach has significantly shortened the waiting time for presensitized recipients, and correlates highly with cross-match results performed at the time of transplant.100, 101 A high number of anti-HLA antibodies can significantly decrease the donor pool and increase waiting time for a lung transplant candidate. In these instances, interventions to remove or decrease the production of these antibodies may be considered before transplantation.

ults performed at the time of transplant.100, 101 A high number of anti-HLA antibodies can significantly decrease the donor pool and increase waiting time for a lung transplant candidate. In these instances, interventions to remove or decrease the production of these antibodies may be considered before transplantation. Post-transplant Considerations in Sensitized Recipients Even though unacceptable antigens are avoided during the virtual crossmatch, patients with positive pre-transplant PRA (ie, circulating anti-HLA antibodies) are at higher risk for post-transplant complications. Their post-transplant PRA can remain stable or increase via generation of either donor-specific or nondonor-specific anti-HLA antibodies. Similarly, patients who had negative PRA screening tests before transplant can develop de novo nondonor-specific or donor-specific anti-HLA antibodies after transplant. Using modern sensitive antibody detection techniques, recent studies have consistently demonstrated increased incidence of acute rejection,102 persistent rejection, increased BOS,103 and worse overall survival104 in patients with anti-HLA antibodies. This effect is apparent with both pre-transplant HLA sensitization as well as with the development of de novo donor-specific anti-HLA antibodies after transplantation.103

ncreased incidence of acute rejection,102 persistent rejection, increased BOS,103 and worse overall survival104 in patients with anti-HLA antibodies. This effect is apparent with both pre-transplant HLA sensitization as well as with the development of de novo donor-specific anti-HLA antibodies after transplantation.103 The importance of donor specificity and target antigens in humoral rejection is not well understood. The risk of poor outcome may be heightened in the setting of donor-specific antibodies and positive retrospective crossmatches.104 However, patients with positive PRA, with negative crossmatches and without specificity to mismatched donor HLA antigens also have been found to be at increased risk for poor outcome. On the one hand, nondonor-specific antibodies that are present might cross-react with the donor HLA, or antibodies specific to donor HLA might be rapidly absorbed in the lung allograft precluding their detection in the sera. Alternatively, other non-HLA antibodies could contribute to graft injury. For example, de novo autoimmunity after lung transplantation against type V collagen105 and K-alpha1 tubulin expressed on airway epithelial cells have been shown to predispose to BOS.106 Another study demonstrated the presence of anti-endothelial antibody directed against donor antigens in the absence of anti-HLA antibodies.7

mple, de novo autoimmunity after lung transplantation against type V collagen105 and K-alpha1 tubulin expressed on airway epithelial cells have been shown to predispose to BOS.106 Another study demonstrated the presence of anti-endothelial antibody directed against donor antigens in the absence of anti-HLA antibodies.7 It remains unclear exactly how often post-transplant PRAs should be measured and to what extent humoral rejection occurs among lung transplant recipients. Additional research is needed to more precisely define the significance of antibody to donor HLA, to third-party HLA, or to self-antigens after lung transplantation. Pathologic and Clinical Patterns of Humoral Lung Rejection Although uncommon due to the use of cross-match screening, hyperacute rejection, caused by pre-existing recipient antibodies against donor HLA antigens, has been described. Hyperacute rejection usually occurs within hours of transplantation and manifests with acute pulmonary decompensation, profound hypoxemia, diffuse pulmonary edema, and alveolar hemorrhage. Such patients may respond to aggressive antihumoral therapy, but mortality is high.107

inst donor HLA antigens, has been described. Hyperacute rejection usually occurs within hours of transplantation and manifests with acute pulmonary decompensation, profound hypoxemia, diffuse pulmonary edema, and alveolar hemorrhage. Such patients may respond to aggressive antihumoral therapy, but mortality is high.107 More recently, the concept of acute (distinct from hyperacute) humoral rejection, occurring later (weeks to years) in the post-transplant course, has evolved. However, the notion of a specific histopathological syndrome associated with acute humoral rejection remains controversial. Post-transplant vascular injury with pulmonary capillaritis has been described as an atypical form of rejection that may be resistant to steroids but in several cases responsive to plasmapheresis, suggesting that it may represent an antibody-mediated process. The clinical presentation of this form of pulmonary capillaritis typically includes dyspnea, hypoxemia, and pulmonary infiltrates on chest radiograph, mimicking acute cellular rejection.108 Frank hemoptysis, reflecting underlying diffuse alveolar hemorrhage, has been described in a subset of recipients with antibody-mediated capillaritis and should prompt consideration of this entity.108, 109

includes dyspnea, hypoxemia, and pulmonary infiltrates on chest radiograph, mimicking acute cellular rejection.108 Frank hemoptysis, reflecting underlying diffuse alveolar hemorrhage, has been described in a subset of recipients with antibody-mediated capillaritis and should prompt consideration of this entity.108, 109 More recent studies have attempted to evaluate immunoglobulin and complement deposits in the subendothelial space as possible manifestations of antibody-mediated rejection. Septal capillary deposits of immunoglobulins and complement products such as C1q, C3d, C4d (see Fig. 2H), and C5b-9, as well as elevation of C4d in the BAL, have been described in association with circulating anti-HLA antibodies.110, 111 Similar pathologic findings have also been identified in the setting of treatment-resistant cellular rejection,112 decreased pulmonary function tests, or BOS.113, 114 However, other studies have not found evidence of antibody deposits or complement activation in the setting of allograft rejection or vascular injury.115, 116, 117 Others have demonstrated that C3d and C4d staining can occur in lung transplant patients with nonalloimmune lung injury such as infection and primary graft dysfunction with no evidence of anti-HLA antibodies, although this staining does appear to be an independent risk factor for BOS.114 Differences in staining techniques between laboratories and subjective interpretation of results by pathologists may further explain some of the inconsistencies in the published data.

aft dysfunction with no evidence of anti-HLA antibodies, although this staining does appear to be an independent risk factor for BOS.114 Differences in staining techniques between laboratories and subjective interpretation of results by pathologists may further explain some of the inconsistencies in the published data. The LRSG report on the working formulation for the diagnosis of lung rejection remains very cautious in defining the pathologic appearance of humoral rejection. The consensus is that capillary injury can be detected on lung allograft biopsies (see Fig. 2G), although it can be a nonspecific finding. Findings of small vessel injury with intimitis or endothelialitis along with immunohistochemical demonstration of complement deposition should raise the suspicion for acute humoral rejection.13 Although such pathologic findings have been reported without evidence of circulating anti-HLA antibodies and visa versa, the presence in one patient of both circulating anti-HLA antibodies and characteristic pathologic findings should be seen as strong evidence for acute humoral rejection.

or acute humoral rejection.13 Although such pathologic findings have been reported without evidence of circulating anti-HLA antibodies and visa versa, the presence in one patient of both circulating anti-HLA antibodies and characteristic pathologic findings should be seen as strong evidence for acute humoral rejection. Prevention and Therapy for Antibody-Mediated Rejection Plasmapheresis is the mainstay for antibody removal from the circulation and has been shown to lead to clinical improvement in lung transplant recipients with pulmonary capillaritis unresponsive to steroids.108 However, it is usually reserved for severe cases of suspected humoral rejection, given its relatively invasive and cumbersome nature. Intravenous immunoglobulin (IVIG) is one of the most common therapies used to decrease antibody-mediated immunity, with a relatively low adverse effect profile. IVIG causes B-cell apoptosis, reduces B-cell numbers, blocks binding of donor-reactive antibodies, and may inhibit complement activation. The peri-transplant use of IVIG and plasmapheresis at the authors’ institution in presensitized patients led to elimination of antibodies in 6 of 7 patients with class I anti-HLA antibodies and 1 of 3 patients with class II anti-HLA antibodies. As a group, those presensitized patients who received this regimen demonstrated a significant decrease in acute rejection episodes and a trend toward greater freedom from BOS compared with a cohort of presensitized patients who did not receive desensitization therapy.99 Rituximab, an anti-CD20 monoclonal antibody that causes B-cell depletion, has been proven effective in the treatment of presensitized renal transplant recipients in conjunction with IVIG.6, 118 In a recent study of 61 lung transplant recipients with newly acquired post-transplant donor-specific antibodies, a regimen of IVIG combined with rituximab (44 patients) or administered alone (17 patients) led to clearing of antibodies in 62%.119 Notably, freedom from BOS and survival were better in the group of patients who cleared their donor-specific antibodies than those with persistent antibodies. Bortezomib, a selective inhibitor of the 26S proteosome that causes plasma cell apoptosis, is a new therapy that appears useful in the reversal of alloantibody-mediated rejection in renal transplant recipients.120 Its use in lung transplantation has been described in one case report.121

hose with persistent antibodies. Bortezomib, a selective inhibitor of the 26S proteosome that causes plasma cell apoptosis, is a new therapy that appears useful in the reversal of alloantibody-mediated rejection in renal transplant recipients.120 Its use in lung transplantation has been described in one case report.121 Despite new highly sensitive measures to screen for anti-HLA antibodies and evidence that such antibodies are detrimental to the allograft, optimal monitoring, treatment parameters for humoral rejection, and the benefits of pre-emptive strategies to deplete these antibodies remain uncertain. Further studies are needed to determine whether IVIG, plasmapheresis, rituximab, or bortezomib alter the risk for chronic allograft dysfunction in sensitized patients.

optimal monitoring, treatment parameters for humoral rejection, and the benefits of pre-emptive strategies to deplete these antibodies remain uncertain. Further studies are needed to determine whether IVIG, plasmapheresis, rituximab, or bortezomib alter the risk for chronic allograft dysfunction in sensitized patients. Summary Acute cellular rejection affects greater than one-third of lung transplant recipients. Alloreactive T-lymphocytes, responding directly or indirectly to donor antigen, constitute the basis of lung allograft rejection, as diagnosed by well-established histopathological criteria that reflect the severity of perivascular or peribronchial inflammation in the lung allograft. Recent evidence supports a more complex immune response to the allograft with involvement of humoral mechanisms, characterized by circulating antibody to donor HLA and specific patterns of lung injury, occurring in parallel with T-cell-based rejection. Emerging evidence further suggests that the interaction between recipient genetics, immunosuppression therapies, and allograft environmental exposures, including pulmonary infection, contributes to high rejection rates after lung transplantation. A greater understanding of the heterogeneous mechanisms of lung rejection is critical to developing effective therapies that target the precise pathophysiology of the disease and ultimately improve long-term lung transplant outcomes. The authors had financial support from National Institutes of Health (5 KL2 RR024127-03, 1P50-HL084917-01, 1 K24 HL91140-01A2).

Summary Acute cellular rejection affects greater than one-third of lung transplant recipients. Alloreactive T-lymphocytes, responding directly or indirectly to donor antigen, constitute the basis of lung allograft rejection, as diagnosed by well-established histopathological criteria that reflect the severity of perivascular or peribronchial inflammation in the lung allograft. Recent evidence supports a more complex immune response to the allograft with involvement of humoral mechanisms, characterized by circulating antibody to donor HLA and specific patterns of lung injury, occurring in parallel with T-cell-based rejection. Emerging evidence further suggests that the interaction between recipient genetics, immunosuppression therapies, and allograft environmental exposures, including pulmonary infection, contributes to high rejection rates after lung transplantation. A greater understanding of the heterogeneous mechanisms of lung rejection is critical to developing effective therapies that target the precise pathophysiology of the disease and ultimately improve long-term lung transplant outcomes. The authors had financial support from National Institutes of Health (5 KL2 RR024127-03, 1P50-HL084917-01, 1 K24 HL91140-01A2). Financial disclosures/conflicts of interest: The authors have nothing to disclose.

Key points • There is a growing body of literature supporting the role of infectious antigens, in particular mycobacteria and propionibacteria, in sarcoidosis pathogenesis. • Immunologic studies reveal that mycobacterial virulence factors are the targets of the immune response in sarcoidosis diagnostic bronchoalveolar lavage (BAL). • Recently, case reports and clinical trials have emerged reporting the efficacy of antimicrobial therapy on cutaneous and pulmonary sarcoidosis. Although the studies are not conclusive, they demonstrate efficacy on endpoints associated with sarcoidosis morbidity and mortality, such as forced vital capacity (FVC).

• Immunologic studies reveal that mycobacterial virulence factors are the targets of the immune response in sarcoidosis diagnostic bronchoalveolar lavage (BAL). • Recently, case reports and clinical trials have emerged reporting the efficacy of antimicrobial therapy on cutaneous and pulmonary sarcoidosis. Although the studies are not conclusive, they demonstrate efficacy on endpoints associated with sarcoidosis morbidity and mortality, such as forced vital capacity (FVC). Sarcoidosis epidemiology suggests exposure to microbial bioaerosols Sarcoidosis is a granulomatous disease of unknown etiology, most commonly involving the lung, skin, lymph node, and eyes.1 Granulomatous inflammation can be initiated by infectious agents, such as fungi or Mycobacterium tuberculosis (MTB), or by noninfectious agents, such as beryllium (chronic beryllium disease). Analysis of sarcoidosis epidemiology suggests that infectious agents have a role in sarcoidosis pathogenesis. Investigators in A Case Control Etiologic Study of Sarcoidosis observed positive associations between sarcoidosis risk and certain occupations, such as agricultural employment, exposure to insecticides, and moldy environments.2 Another study noted that the hospitalization admissions for African Americans with sarcoidosis in South Carolina increased with proximity to the Atlantic Ocean.3 A unifying factor in environmental and geographic reports is the possibility of exposure to microbial bioaerosols. Natural waters; water distribution systems; biofilm in pipes; peat and potting soil; water droplets; equipment, such as bronchoscopes and catheters; and moldy buildings are natural habitats for environmental opportunistic mycobacteria.4 Aerosolization of environmental opportunistic mycobacteria has been associated with the development of other granulomatous diseases of mycobacterial origin, such as hypersensitivity pneumonitis.5

such as bronchoscopes and catheters; and moldy buildings are natural habitats for environmental opportunistic mycobacteria.4 Aerosolization of environmental opportunistic mycobacteria has been associated with the development of other granulomatous diseases of mycobacterial origin, such as hypersensitivity pneumonitis.5 Molecular and immunologic investigations reveal microbial proteins and DNA The inability to identify microorganisms by histologic staining or to culture microorganisms from pathologic tissues continues to be one of the strongest arguments against a potential role for infectious agents in sarcoidosis pathogenesis. As molecular analysis continues to grow in sensitivity and specificity, current culture and staining methods are known to identify less than 2% of current microbial communities present within the human biological specimens.6, 7 Advanced molecular techniques, such as deep sequencing technologies, also have demonstrated successful identification of novel microorganisms in pathologic tissues not easily identified by traditional methods.8, 9 Molecular analysis of pathologic tissue for microbial nucleic acids and proteins serves as an alternative means of identifying a putative infectious agent. Polymerase chain reaction (PCR) was used to identify the etiologic agents of Whipple disease (Tropheryma whippelii)10 as well as the novel coronavirus as the agent of severe acute respiratory syndrome.11

for microbial nucleic acids and proteins serves as an alternative means of identifying a putative infectious agent. Polymerase chain reaction (PCR) was used to identify the etiologic agents of Whipple disease (Tropheryma whippelii)10 as well as the novel coronavirus as the agent of severe acute respiratory syndrome.11 A growing scientific interest involves defining the microbial community within distinct diseases, that is, microbiome analysis. Microbiome analysis was performed on the upper and lower airway of subjects with interstitial lung diseases, including idiopathic interstitial pneumonia (IIP), non-IIP, and sarcoidosis as well as Pneumocystis jiroveci pneumonia and healthy controls. The microbiota in lower airways of a majority of patients (30; 90%) primarily consisted of Prevotellaceae, Streptococcaceae, and Acidaminococcaceae; α and β diversity measurements revealed no significant differences in airway microbiota composition between the 5 different groups of patients. It was concluded that IIP, non-IIP, and sarcoidosis are not associated with disordered airway microbiota and a pathogenic role of commensals in the disease process is therefore unlikely.12 A more targeted molecular approach for microbial pathogens in sarcoidosis granulomas most strongly supports that propionibacteria and/or mycobacteria have a role in sarcoidosis pathogenesis. Japanese researchers report molecular evidence of Propionibacterium acnes DNA in sarcoidosis specimens, although the DNA could also be isolated from control specimens.13 The distinction lies in the quantitative differences in P acnes DNA between sarcoidosis and controls. The number of genomes of P acnes in BAL cells was correlated with the serum angiotensin-converting enzyme level and the percentage of macrophages in BAL fluid from patients with sarcoidosis. No significant difference was detected between P granulosum and controls.14 A murine model of sarcoidosis pathogenesis was successfully developed using heat-killed propionibacteria by intratracheal challenge.

iotensin-converting enzyme level and the percentage of macrophages in BAL fluid from patients with sarcoidosis. No significant difference was detected between P granulosum and controls.14 A murine model of sarcoidosis pathogenesis was successfully developed using heat-killed propionibacteria by intratracheal challenge. This model demonstrated the contribution of Toll-like receptor (TLR)-1, TLR-2, and TLR-9 to the development of the polarized, TH1 immune response.15 Another study further confirmed the role of TLR-2 in P acnes–specific sarcoidosis immune responses by demonstrating that P acnes–induced granulomatous pulmonary inflammation was markedly attenuated in TLR-2(−/−) mice compared with wild-type C57BL/6 animals.16 A recent meta-analysis involving 9 case-control studies of P acnes associated with sarcoidosis revealed a significantly elevated sarcoidosis risk (odds ratio 19.58; 95% CI, 13.06–29.36).17

–induced granulomatous pulmonary inflammation was markedly attenuated in TLR-2(−/−) mice compared with wild-type C57BL/6 animals.16 A recent meta-analysis involving 9 case-control studies of P acnes associated with sarcoidosis revealed a significantly elevated sarcoidosis risk (odds ratio 19.58; 95% CI, 13.06–29.36).17 Investigations from independent laboratories worldwide have also reported molecular evidence supporting a significant association between mycobacteria and sarcoidosis. One study reported evidence of mycobacterial 16S ribosomal RNA (rRNA) or RNA polymerase B in 60% of the sarcoidosis granulomas and in none of the controls (P<.00002, chi-square).18 Sequence analysis of the 16S rRNA and rpoB amplicons revealed the presence of a novel Mycobacterium, genetically most similar to MTB complex (99% positional identity). Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Song and colleagues19 found MTB katG peptides in 75% of sarcoidosis specimens compared with 14% of control specimens (P = .0006), in situ hybridization localized MTB katG, and 16S rRNA DNA to the inside of sarcoidosis granulomas. Analysis of Polish sarcoidosis lymph nodes revealed MTB complex heat shock protein (HSP) 70, HSP 65, and HSP 16.20 Molecular analysis of American sarcoidosis granulomas also revealed the presence of nucleic acids of the mycobacterial virulence factor, superoxide dismutase A (sodA), in 70% of the sarcoidosis specimens compared with 12% of controls (P = .001). Sequence analysis of the amplicons demonstrated close positional identity with MTB complex, yet genetically distinct.21 DNA of mycobacterial HSPs has been detected in cutaneous lesions of Chinese sarcoidosis patients but absent from control specimens. Sequence analysis was consistent with MTB, M chelonae, and M gordonae.22 Another study reported the ability of real-time PCR analysis to quantitatively differentiate sarcoidosis from tuberculosis using receiver operating characteristic curves.23 Real-time PCR analysis from these independent laboratories demonstrates that if viable mycobacteria are present within the sarcoidosis granulomas, they are present below the sensitivity of the acid-fast bacilli histologic stain.21, 23 Future molecular efforts should delineate if the identified nucleic acids or proteins reflect actively replicating organisms or persistent proteins.

nstrates that if viable mycobacteria are present within the sarcoidosis granulomas, they are present below the sensitivity of the acid-fast bacilli histologic stain.21, 23 Future molecular efforts should delineate if the identified nucleic acids or proteins reflect actively replicating organisms or persistent proteins. Immune responses against mycobacterial virulence factors are present in systemic and active sarcoidosis involvement An equally important modality to delineate if infectious agents have a role in idiopathic disease is to assess for immune responses against microbial proteins. The presence of humoral and cellular responses against microbial antigens is an insightful method for assessing exposure to infectious agents. Increased lymphocyte proliferation induced by P acnes has been reported in patients with active sarcoidosis; however, these responses did not correlate with clinical, roentgenographic, physiologic, and BAL findings in regard to disease severity.24, 25 Sarcoidosis TH1 and TH17 immune responses against viable P acnes that were significantly different from healthy controls were recently reported.26

ients with active sarcoidosis; however, these responses did not correlate with clinical, roentgenographic, physiologic, and BAL findings in regard to disease severity.24, 25 Sarcoidosis TH1 and TH17 immune responses against viable P acnes that were significantly different from healthy controls were recently reported.26 Immune responses against mycobacteria have also been reported. Along with the detection of peptide fragments consistent with katG protein within sarcoidosis granulomas, the existence of humoral immune responses against mycobacterial katG proteins was demonstrated in sarcoidosis patients. Song and colleagues16 noted IgG antibodies to recombinant MTB katG in sera from 48% of sarcoidosis patients compared with 0% in sera from purified protein derivative–negative controls (P = .0059). Sarcoidosis is characterized by polarized CD4+ T cells with a TH1 immunophenotype. The identification of TH1 CD4+ cellular immune responses against mycobacterial ESAT-6 and katG peptides in sarcoidosis peripheral blood mononuclear cells (PBMCs) suggested that the sarcoidosis immune response may be against mycobacterial virulence factors.27 Distinctions in cellular recognition patterns against virulence factors, such as antigen 85A (Ag85A), can differentiate mycobacterial species. For example, patients infected with MTB recognize distinct Ag85A peptides from those infected with M leprae.28 Further investigation of the sarcoidosis immune response pattern against Ag85A confirmed that the pattern detected was distinct from those in patients infected with MTB or M leprae. 29 Another report demonstrated systemic CD4+ TH1 immune responses against multiple mycobacterial virulence factors in sarcoidosis patients. These responses were not only against multiple secreted proteins but also against multiple epitopes within a given protein.30 These findings are more analogous with what is observed in patients with active bacterial infection.

H1 immune responses against multiple mycobacterial virulence factors in sarcoidosis patients. These responses were not only against multiple secreted proteins but also against multiple epitopes within a given protein.30 These findings are more analogous with what is observed in patients with active bacterial infection. A dual molecular and immunologic analysis of sarcoidosis specimens for the mycobacterial virulence factor, sodA, demonstrated nucleic acids sequences closest to MTB, yet distinct. Translation of those sequences into peptides to stimulate sarcoidosis PBMC resulted in reproduction of the sarcoidosis TH1 immunophenotype.21 Mycobacterial proteins, such as soda, are virulence factors that confer pathogenicity to Mycobacterium species.31 It has been demonstrated that the protein secretion system SecA2 is required for the optimal secretion of sodA and katG. Both of these proteins are synthesized without Sec signal sequences and function to detoxify reactive oxygen intermediates generated by the host macrophage. SecA2 is part of a specialized secretion system that contributes to the virulence of pathogenic mycobacteria by countering the oxidative attack of the host and confers their ability to survive within the host macrophage.32, 33

nces and function to detoxify reactive oxygen intermediates generated by the host macrophage. SecA2 is part of a specialized secretion system that contributes to the virulence of pathogenic mycobacteria by countering the oxidative attack of the host and confers their ability to survive within the host macrophage.32, 33 In addition, CD4+ and CD8+ T-cell immune responses against MTB katG have been detected in sarcoidosis BAL. Comparison of immune responses to mycobacterial katG whole protein between American and Swedish sarcoidosis subjects revealed no differences despite distinctions in patient phenotypic, genetic, and prognostic characteristics. It was also demonstrated that although TH1 immune responses were present systemically, katG-reactive CD4+ 1 cells preferentially accumulated in the lung, indicating a compartmentalized response.34 Patients with or without Löfgren syndrome had similar frequencies of katG-specific interferon-gamma–expressing peripheral T cells. This study also demonstrated that circulating katG-reactive T cells were found in chronic active sarcoidosis but not in patients with inactive disease.34 The loss of immune responses to mycobacterial virulence factors after resolution of tuberculosis has also been observed.35 Another report demonstrated that immune responses against these mycobacterial virulence factors are present in sarcoidosis diagnostic BAL and that induction of innate immunity by TLR-2 contributes to the polarized TH1 immune response. Recognition was significantly absent from BAL fluid cells of patients with other lung diseases, including infectious granulomatous diseases.36 The detection of immune responses against ESAT-6, katG, and sodA confirms exposure of sarcoidosis patients to a pathogenic mycobacterial species. These proteins are typically secreted during the stage of active mycobacterial replication, compared with expression of other proteins that are expressed when mycobacteria are in the latent state.37, 38 The immunologic analysis performed to date provides a mechanism for more in-depth analysis of sarcoidosis pathogenesis. These proteins can be used to delineate immunologic pathways that contribute to sarcoidosis resolution or disease progression.

teins that are expressed when mycobacteria are in the latent state.37, 38 The immunologic analysis performed to date provides a mechanism for more in-depth analysis of sarcoidosis pathogenesis. These proteins can be used to delineate immunologic pathways that contribute to sarcoidosis resolution or disease progression. Noninfectious etiologies of sarcoidosis It has been reported that the amyloid precursor protein serum amyloid A (SAA) is strikingly abundant in sarcoidosis tissues, predominantly in a nonfibrillar form, and localized to epithelioid granulomas. SAA has been detected in numerous pulmonary infections, such as tuberculosis, nontuberculous mycobacteria infection, and leprosy.39, 40, 41, 42 By comparison, quantitative immunohistochemistry showed that the extent and distribution of SAA in sarcoidosis is significantly lower in other diseases of granulomatous inflammation.43 Chen and Moller44 elaborate a concept of chronic stimulation of the innate immune system by disaggregated host protein SAA within granulomas after a microbial infection that induces a hyperimmune TH1 immune response to microbial antigens in the absence of ongoing infection. SAA levels are reduced in pulmonary tuberculosis subjects after the initiation of antimicrobial therapy.40 In addition, antibodies against autoantigens, such as zinc finger protein 688 and mitochondrial ribosomal protein L43, have been identified in sarcoidosis BAL and serum. High interindividual heterogeneity was noted.45 Using pulmonary CD4+ T cells from 16 HLA-DRB1∗0301+ patients, HLA-DR molecules were affinity purified and bound peptides acid eluted. The peptidies were separated by reversed-phase high-performance liquid chromatography and analyzed by liquid chromatography–mass spectrometry, resulting in the identification of autoantigens, such as vimentin, and ATP synthase.46 These data support that immune responses against self-antigens are present in local and systemic sites of sarcoidosis subjects.

sed-phase high-performance liquid chromatography and analyzed by liquid chromatography–mass spectrometry, resulting in the identification of autoantigens, such as vimentin, and ATP synthase.46 These data support that immune responses against self-antigens are present in local and systemic sites of sarcoidosis subjects. Microbial induction of sarcoidosis CD4+ T-cell dysfunction Investigation of sarcoidosis immune function on T-cell receptor (TCR) stimulation reveals significant distinctions from healthy controls. The presence of chronic immune stimulation due to persistent microbial antigens has been reported to reduce T-cell function. Sarcoidosis T lymphocytes have also been characterized by reduced cytokine expression and proliferative capacity as well as up-regulation of the inhibitory receptor, programmed death-1 (PD-1), all immunologic phenomena associated with elevated antigenic burdens.

ial antigens has been reported to reduce T-cell function. Sarcoidosis T lymphocytes have also been characterized by reduced cytokine expression and proliferative capacity as well as up-regulation of the inhibitory receptor, programmed death-1 (PD-1), all immunologic phenomena associated with elevated antigenic burdens. As defined by Wherry and colleagues,47 T-cell exhaustion occurs as a result of chronic antigen stimulation that, over the duration of antigen exposure, results in a gradual reduction in the cell’s ability to optimally respond to TCR stimulation. As such, although healthy T cells produce high levels of cytokine and exhibit high levels of proliferation and low levels of apoptosis in response to antigen, exhausted T cells gradually lose these normal functions until they can no longer respond to antigen and instead undergo apoptosis on TCR activation. PD-1 up-regulation on T cells plays a significant role in acquisition of the exhaustion phenotype. As an inhibitory coreceptor, signaling between PD-1 and its ligands, PDL1 and PDL2, functions to modulate tolerance to self antigens and limit the robustness of the adaptive immune response to foreign antigens. Exhausted T cells express high levels of PD-1 that correlates well with the systematic loss of cellular function. Recent findings that PD-1 is up-regulated on dysfunctional sarcoidosis T cells as well as the T cells of other granulomatous diseases characterized by microbial antigens, such as MTB48, 49, 50 and schistosomiasis,51 suggest that this phenotype could result from persistent antigen exposure (Fig. 1 , Table 1 ).Fig. 1 PD-1 inhibits sarcoidosis cellular proliferation. High antigenic loads induce PD-1 up-regulation, which alters cell cycle progression. Cell cycle progression is necessary for normal CD4+ T-cell proliferation to clear microbial or autoantigens, thus leading to clinical resolution. Persistent antigen further PD-1 up-regulation and loss of cellular function.

oliferation. High antigenic loads induce PD-1 up-regulation, which alters cell cycle progression. Cell cycle progression is necessary for normal CD4+ T-cell proliferation to clear microbial or autoantigens, thus leading to clinical resolution. Persistent antigen further PD-1 up-regulation and loss of cellular function. Table 1 Evidence for etiologic agents in sarcoidosis pathogenesis Etiology Evidence Mycobacteria M, I, E18, 19, 20, 21, 22, 27, 29, 30 Propionibacteria M, I13, 14, 15, 24, 25, 26 Fungal antigens M66 Autoantigens M, I45, 46 Abbreviations: E, epidemiologic; I, immunologic; M, molecular.

oliferation. High antigenic loads induce PD-1 up-regulation, which alters cell cycle progression. Cell cycle progression is necessary for normal CD4+ T-cell proliferation to clear microbial or autoantigens, thus leading to clinical resolution. Persistent antigen further PD-1 up-regulation and loss of cellular function. Table 1 Evidence for etiologic agents in sarcoidosis pathogenesis Etiology Evidence Mycobacteria M, I, E18, 19, 20, 21, 22, 27, 29, 30 Propionibacteria M, I13, 14, 15, 24, 25, 26 Fungal antigens M66 Autoantigens M, I45, 46 Abbreviations: E, epidemiologic; I, immunologic; M, molecular. Up-regulation of the PD-1 receptor and reduced proliferative capacity in sarcoidosis BAL and peripheral CD4+ T cells were recently reported.52 Restoration of sarcoidosis CD4+ T-cell proliferative capacity to healthy control levels was apparent after PD-1 pathway blockade.52 Various mechanisms by which PD-1 interferes with T-cell proliferation have been well described. PD-1 has been reported to inhibit CD4+ T-cell proliferation by blocking cell cycle progression through the suppression of Skp2 transcription.53, 54 Skp2 is the substrate recognition component of the ubiquitin ligase complex SCFSkp2 that binds to and degrades p27kip1, a cyclin-dependent kinase (CDK) inhibitor, thereby allowing continuation of the cell cycle. PD-1 cell cycle impediment and, therefore, proliferation hindrance have been shown to be the result of PI3K/Akt and ERK pathway inactivation.53, 54 PD-1 inhibition of T-cell proliferation has been correlated with increased p27 availability and repression of Cdc25A, a CDK-activating phosphatase.53, 54 PD-1 engagement has also been demonstrated to attenuate TCR signaling by preventing ZAP70 and PKCθ activation.55

esult of PI3K/Akt and ERK pathway inactivation.53, 54 PD-1 inhibition of T-cell proliferation has been correlated with increased p27 availability and repression of Cdc25A, a CDK-activating phosphatase.53, 54 PD-1 engagement has also been demonstrated to attenuate TCR signaling by preventing ZAP70 and PKCθ activation.55 The reported up-regulation of PD-1 is particularly important because it has been associated with the emergence of human lymphotropic viruses, such as Epstein-Barr virus and cytomegalovirus.56, 57, 58 These same viruses have been associated with sarcoidosis pathogenesis.59, 60 Clinical trials of antimicrobial therapy in sarcoidosis After the publication of molecular and immunologic support for a role of microorganisms in sarcoidosis pathogenesis, such as fungi, propionibacteria, and mycobacteria, there has been an increasing number of case reports and clinical trials regarding efficacy with antimicrobial therapy. Numerous prior reports of the tetracyclines, in particular, doxycycline and minocycline, have been published in subjects with cutaneous sarcoidosis.61, 62, 63 Although minocycline has antimicrobial effects against P acnes, its mechanism of action is thought immunomodulatory.64 A recent report of the efficacy of clarithromycin, which has efficacy against propionibacteria and mycobacteria, was reported in a Japanese woman with systemic sarcoidosis.65 Conclusive delineation of the mechanism of action is pending.

ial effects against P acnes, its mechanism of action is thought immunomodulatory.64 A recent report of the efficacy of clarithromycin, which has efficacy against propionibacteria and mycobacteria, was reported in a Japanese woman with systemic sarcoidosis.65 Conclusive delineation of the mechanism of action is pending. Fungal antigens are also reported to contribute to sarcoidosis pathogenesis.66 Clinical and radiographic improvement after administration of antifungal therapy, such as posaconazole (300 mg/d) or ketoconazole (200 mg/d) with or without corticosteroids, has been reported in Slovenian sarcoidosis patients. The investigators conducted an open-labelled, patient-preference trial of steroids (methylprednisolone 0.4 mg/kg, antifungal agents (posaconazole 300 mg/d or ketoconazole 200 mg/d), or steroids/antifungal agents. The most significant clinical radiographic improvement was detected in the antifungal group; they also reported a reduction in disease recurrence among those on antifungal therapy. Study limitations include the lack of randomization as well as not being conducted in a double-blind fashion.

r steroids/antifungal agents. The most significant clinical radiographic improvement was detected in the antifungal group; they also reported a reduction in disease recurrence among those on antifungal therapy. Study limitations include the lack of randomization as well as not being conducted in a double-blind fashion. Two clinical trials regarding the efficacy of antimycobacterial therapy in sarcoidosis pathogenesis have been reported. A double blind, placebo-controlled investigation of an antimycobacterial regimen consisting of concomitant levofloxacin, ethambutol, azithromycin and rifampin (CLEAR) compared with placebo was conducted in subjects with cutaneous sarcoidosis. In the intention-to-treat analysis, the CLEAR-treated group had a mean (SD) decrease in lesion diameter of −8.4 (14.0) mm compared with an increase of 0.07 (3.2) mm in the placebo-treated group (P = .05). The CLEAR group had a significant reduction in granuloma burden and experienced a mean (SD) decline of −2.9 (2.5) mm in lesion severity compared with a decline of −0.6 (2.1) mm in the placebo group (P = .02). The observed clinical reductions were present at the 180-day follow-up period. Transcriptome analysis of sarcoidosis CD4+ T cells revealed reversal of pathways associated with disease severity and enhanced T-cell function after TCR stimulation.67

compared with a decline of −0.6 (2.1) mm in the placebo group (P = .02). The observed clinical reductions were present at the 180-day follow-up period. Transcriptome analysis of sarcoidosis CD4+ T cells revealed reversal of pathways associated with disease severity and enhanced T-cell function after TCR stimulation.67 In addition, an open-label investigation of this same regimen was conducted in pulmonary sarcoidosis subjects; 15 chronic, pulmonary sarcoidosis patients with FVCs between 45% and 80% of predicted were enrolled. The primary efficacy endpoint was change in absolute FVC from baseline to completion of therapy. Secondary endpoints were change in functional capacity measured by Six Minute Walk Distance (6MWD) and quality-of-life assessment measured by St. George's Respiratory Questionnaire (SGRQ). Of 15 patients enrolled, 11 completed 4 weeks of therapy, and 8 completed 8 weeks of therapy. The CLEAR regimen was associated with an FVC increase of 0.23 L at 4 weeks and 0.42 L at 8 weeks (P = .0098 and 0.016, respectively). The 6MWD increased by 87 m from baseline to 8 weeks (P = .0078). The mean score of the validated SGRQ was improved at 8 weeks over baseline (P = .023).68 These early trials are promising. Future investigation of the mechanisms by which the antimicrobials work—as antimicrobials, immune modulators, or both—is warranted.

In addition, an open-label investigation of this same regimen was conducted in pulmonary sarcoidosis subjects; 15 chronic, pulmonary sarcoidosis patients with FVCs between 45% and 80% of predicted were enrolled. The primary efficacy endpoint was change in absolute FVC from baseline to completion of therapy. Secondary endpoints were change in functional capacity measured by Six Minute Walk Distance (6MWD) and quality-of-life assessment measured by St. George's Respiratory Questionnaire (SGRQ). Of 15 patients enrolled, 11 completed 4 weeks of therapy, and 8 completed 8 weeks of therapy. The CLEAR regimen was associated with an FVC increase of 0.23 L at 4 weeks and 0.42 L at 8 weeks (P = .0098 and 0.016, respectively). The 6MWD increased by 87 m from baseline to 8 weeks (P = .0078). The mean score of the validated SGRQ was improved at 8 weeks over baseline (P = .023).68 These early trials are promising. Future investigation of the mechanisms by which the antimicrobials work—as antimicrobials, immune modulators, or both—is warranted. Summary Recent molecular, genetic, and immunologic studies from independent laboratories support an association with sarcoidosis and microbial antigens, particularly mycobacteria or propionibacteria. The findings among American sarcoidosis subjects are most strongly associated with mycobacteria and, among Japanese sarcoidosis subjects, propionibacteria. Because epidemiologic studies indicate that both sarcoidosis morbidity and mortality is increasing,69 the impetus on current sarcoidosis researchers is to translate their strong basic research investigations into innovative therapeutics that will have an impact on sarcoidosis pathogenesis and hopefully lead to a cure. The progress to date strongly supports advances toward this goal.

idity and mortality is increasing,69 the impetus on current sarcoidosis researchers is to translate their strong basic research investigations into innovative therapeutics that will have an impact on sarcoidosis pathogenesis and hopefully lead to a cure. The progress to date strongly supports advances toward this goal. This work was supported by National Institutes of Health grants (T32 HL069765 to L.J. Celada; T32 HL094296 to C. Hawkins; and R01 HL117074, U01 112694 to W.P. Drake). Drs L.J. Celada and C. Hawkins have no conflicts of interest to disclose. Dr W.P. Drake serves as a scientific advisor for Celgene.

Bronchiolitis obliterans (BO) in the context of lung transplantation was first described in 1984 at Stanford University in a patient who developed progressive airflow obstruction after heart-lung transplantation.1 Lung biopsies revealed intraluminal polyps of fibromyxoid granulation tissue, which tended to obliterate the lumen of terminal bronchioles, and dense submucosal eosinophilic fibrous scars (Fig. 1 ). Since this early report, BO has been recognized as the major complication and the leading cause of death after lung transplantation.2 Fig. 1 Histological picture of post-transplant BO. The lumen of the bronchiole is almost totally occluded by fibromyxoid granulation tissue.

mucosal eosinophilic fibrous scars (Fig. 1 ). Since this early report, BO has been recognized as the major complication and the leading cause of death after lung transplantation.2 Fig. 1 Histological picture of post-transplant BO. The lumen of the bronchiole is almost totally occluded by fibromyxoid granulation tissue. Because the small airway lesions have a patchy distribution, they can hardly be demonstrated by transbronchial lung biopsies (TBBs), which have a low sensitivity (28%) and specificity (75%).3 As a result, in order to establish the diagnosis of BO without the need for open lung biopsy, the International Society for Heart and Lung Transplantation (ISHLT) proposed in 1993 a clinical definition based on pulmonary function criteria. The term, bronchiolitis obliterans syndrome (BOS), was coined to identify patients with a progressive and irreversible decline in forced expiratory volume in one second (FEV1). In the initial classification, BOS was divided into 4 stages based on the degree of loss in FEV1 compared with the best postoperative value. In the updated classification proposed in 2002, a potential BOS (BOS 0-p) stage—defined by a decline in FEV1 or in midexpiratory flow rates (FEF25-75)—was added to detect early but potentially important changes in pulmonary function (Table 1 ).4 Several conditions needed to be satisfied for a patient to be classified in the staging system: (1) the functional loss had to be present for at least 3 weeks to exclude an acute, reversible process; (2) the loss had to include a decrease in both FEV1 and FEV1/vital capacity ratio (ie, patients with a loss in FEV1 in the context of a restrictive ventilatory defect are not considered as having BOS), and (3) confounding conditions that may produce a decrease in FEV1 (eg, infection, acute rejection, anastomotic complications, disease recurrence, and progression of native lung hyperinflation in patients with single-lung transplantation [SLT] for emphysema) needed to be excluded.Table 1 Bronchiolitis obliterans syndrome classification system

itions that may produce a decrease in FEV1 (eg, infection, acute rejection, anastomotic complications, disease recurrence, and progression of native lung hyperinflation in patients with single-lung transplantation [SLT] for emphysema) needed to be excluded.Table 1 Bronchiolitis obliterans syndrome classification system 1993 Classification 2002 Classification FEV1 80% or more of baseline FEV1 >90% of baseline and FEF25-75 >75% of baseline BOS 0 FEV1 81% to 90% of baseline and/or FEF25-75 = or <75% of baseline BOS 0-p BOS 1 FEV1 66% to 80% of baseline FEV1 66% to 80% of baseline BOS 1 BOS 2 FEV1 51% to 65% of baseline FEV1 51% to 65% of baseline BOS 2 BOS 3 FEV1 50% or less of baseline FEV1 50% or less of baseline BOS 3 Data from Estenne M, Maurer JR, Boehler A, et al. Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. J Heart Lung Transplant 2002;21:297–310.

aseline BOS 1 BOS 2 FEV1 51% to 65% of baseline FEV1 51% to 65% of baseline BOS 2 BOS 3 FEV1 50% or less of baseline FEV1 50% or less of baseline BOS 3 Data from Estenne M, Maurer JR, Boehler A, et al. Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. J Heart Lung Transplant 2002;21:297–310. Transplant centers worldwide have adopted this staging system as a descriptor of chronic lung allograft dysfunction. This proved useful because it provided a common language to classify patients and compare results between programs. Several limitations, however, have become apparent in recent years. First, many patients who have confounding conditions cannot be staged for BOS. Second, as the experience with lung transplantation accrued, an increasing number of patients presented with forms of chronic allograft dysfunction that did not comprise all the characteristic features of BOS. Several types of chronic allograft dysfunction, which differ from BOS, were identified in the past years. These include (1) a reversible phenotype characterized by airway neutrophilia and functional improvement with azithromycin (AZM), (2) a phenotype characterized by a restrictive ventilatory impairment associated with upper lobe fibrosis or persistent parenchymal or pleural abnormalities, (3) exudative or follicular bronchiolitis, and (4) large airway stenosis/malacia. This review deals primarily with classical BOS, which has been more extensively studied, but other recently described presentations of chronic allograft dysfunction also are addressed.

s or persistent parenchymal or pleural abnormalities, (3) exudative or follicular bronchiolitis, and (4) large airway stenosis/malacia. This review deals primarily with classical BOS, which has been more extensively studied, but other recently described presentations of chronic allograft dysfunction also are addressed. The clinical spectrum of chronic allograft dysfunction Classical BOS In the registry report of the ISHLT published in 2010,2 freedom from BOS in a cohort of 12,058 patients followed between April 1994 and June 2009 was 89.7% at 1 year, 67.4% at 3 years, 51.2% at 5 years, and 24.8% at 10 years after surgery. These percentages represent a clear decrease in the prevalence of the complication compared with earlier series. Yet BOS remains by far the most significant long-term complication and the leading cause of late death after lung transplantation, accounting for 20% to 30% of all deaths after the third postoperative year.2

These percentages represent a clear decrease in the prevalence of the complication compared with earlier series. Yet BOS remains by far the most significant long-term complication and the leading cause of late death after lung transplantation, accounting for 20% to 30% of all deaths after the third postoperative year.2 BOS may affect all lung transplant recipients irrespective of donor and recipient characteristics, type of transplantation, and pretransplant disease. The clinical presentation of BOS is heterogeneous.5 The type of presentation, the time from transplantation to onset, and the rate of progression are all variable between patients (Fig. 2 ). BOS may present as an acute illness and imitate a respiratory infection,5 but in most patients it starts as an asymptomatic process that produces an insidious decline in lung function. In approximately 20% of patients, BOS develops within 2 years of transplantation (early-onset BOS), but the vast majority of patients develop the complication at a later point in time.2, 6 Some patients present with a substantial loss of lung function and are already in BOS stage 2 or 3 (high-grade onset) at presentation whereas others show a slow decline over time.6 In a study by Jackson and colleagues5 56% of 204 patients who developed BOS showed a sudden drop in FEV1, whereas 18% presented with a smooth linear decline; time to BOS onset was longer in the latter group. Acute rejection during the first 6 months was significantly associated with acute onset of BOS. Auscultation of the lungs is often normal, but squeaks and coarse crackles may be heard. High-resolution CT may reveal air trapping (Fig. 3 ) and bronchiectasis,7, 8 without significant parenchymal infiltrate. As the disease progresses, permanent airway colonization with pathogens, such as Pseudomonas aeruginosa and Aspergillus fumigatus, frequently develops. Survival at 5 years after diagnosis ranges from 26% to 43%,6, 9, 10, 11 and survival at 5 years after transplantation is 20% to 40% lower in patients with, compared to patients without, BOS.11 There is also evidence that the number of respiratory infections and the aggressiveness with which they are treated have an impact on BOS progression.9 In addition to representing a major obstacle to long-term survival, BOS causes significant morbidity and loss of health-related quality of life.12 Fig. 2 Changes in FEV1 over time elapsed since transplantation in 3 patients with BOS.

and the aggressiveness with which they are treated have an impact on BOS progression.9 In addition to representing a major obstacle to long-term survival, BOS causes significant morbidity and loss of health-related quality of life.12 Fig. 2 Changes in FEV1 over time elapsed since transplantation in 3 patients with BOS. Stages refer to the BOS classification, and horizontal lines indicate transitions between stages 0 and 1, stages 1 and 2, and stages 2 and 3. The figure illustrates the highly variable pattern of functional evolution between patients affected by BOS. (From Estenne M, Maurer JR, Boehler A, et al. Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. J Heart Lung Transplant 2002;21:297–310; with permission.)Fig. 3 Expiratory CT scan in a transplant recipient with BOS. The arrow indicates lobules with low attenuation, a sign of the presence of air trapping. (From Bankier AA, Muylem AV, Knoop C, et al. BOS in heart-lung transplant recipients: diagnosis with expiratory CT. Radiology 2001;218:533–9; with permission.)

(From Estenne M, Maurer JR, Boehler A, et al. Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. J Heart Lung Transplant 2002;21:297–310; with permission.)Fig. 3 Expiratory CT scan in a transplant recipient with BOS. The arrow indicates lobules with low attenuation, a sign of the presence of air trapping. (From Bankier AA, Muylem AV, Knoop C, et al. BOS in heart-lung transplant recipients: diagnosis with expiratory CT. Radiology 2001;218:533–9; with permission.) BOS is used as a surrogate marker of BO but does not equal BO. Therefore, as expected for any functional marker, a drop in FEV1 is likely to have a low specificity for the diagnosis of BO (this is why exclusionary criteria were added to the definition of BOS). This lack of specificity is difficult to assess because a gold standard is rarely available. Yet in a study of lungs explanted at the time of retransplantation for BOS,13 pathology examination always showed at least some degree of BO, but a wide range of other pathologic processes of potential clinical significance was also evident in half of the specimens. Other Forms of Chronic Allograft Dysfunction In contrast to classical BOS, which is characterized by a progressive, irreversible airflow obstruction and few, if any, parenchymal or pleural abnormalities, recently described new phenotypes of chronic allograft dysfunction may include one or more of the following features: partial reversibility of airway obstruction, restrictive ventilatory impairment, parenchymal/pleural abnormalities, and large airway stenosis/malacia.

few, if any, parenchymal or pleural abnormalities, recently described new phenotypes of chronic allograft dysfunction may include one or more of the following features: partial reversibility of airway obstruction, restrictive ventilatory impairment, parenchymal/pleural abnormalities, and large airway stenosis/malacia. Neutrophilic reversible allograft/airways dysfunction Because it is well known that macrolide antibiotics are effective in treating airway diseases which, like BOS, are associated with neutrophilic inflammation (eg, panbronchiolitis and cystic fibrosis), Gerhardt and colleagues14 performed an open trial with AZM in lung transplant recipients. In this study, AZM (250 mg 3 times a week) was added to the current immunosuppressive treatment in 6 patients with BOS; 5 patients responded with a mean improvement in the FEV1 of 21.6% after 14 weeks. One patient even had a complete restoration of FEV1 to peak post-transplant values. This landmark study was followed by at least 6 studies,15, 16, 17, 18, 19, 20 of which 4 confirmed the results published by Gerhardt and colleagues.14 One study by Benden and colleagues21 also reported a positive effect of clarithromycin on FEV1. Taking these publications together, approximately 35% of all patients in different BOS stages responded to macrolide treatment by a mean increase in FEV1 of approximately 14%. Furthermore, a higher bronchoalveolar lavage (BAL) fluid neutrophilia was associated with a greater likelihood of functional response.18 Based on these observations, Verleden and colleagues22 suggested that BOS might be dichotomized into an AZM-responsive phenotype characterized by airway neutrophilia and functional improvement with AZM (the so-called neutrophilic reversible allograft/airways dysfunction [NRAD]), and an AZM-unresponsive phenotype, which corresponds to the classical, fibroproliferative form of BO. These two phenotypes might have different pathophysiology, clinical presentation, and prognosis; for example, NRAD might start earlier after transplantation and progress slower than fibroproliferative BOS; and crackles, increased sputum production, bronchiectasis, and mucus plugging might be more prominent in the former than in the latter.

might have different pathophysiology, clinical presentation, and prognosis; for example, NRAD might start earlier after transplantation and progress slower than fibroproliferative BOS; and crackles, increased sputum production, bronchiectasis, and mucus plugging might be more prominent in the former than in the latter. Upper lobe fibrosis In 2005, a joint retrospective study by the Toronto General Hospital and the Duke University Hospital identified 13 of 686 lung transplant recipients who developed upper lobe fibrosis.23 Radiographic changes started initially as nonspecific interstitial markings in the upper lobes and slowly progressed to honeycombing, traction bronchiectasis, and volume loss (Fig. 4 ). Most patients had a restrictive ventilatory defect, with some eventually developing concomitant airflow obstruction. Open lung biopsy specimens revealed dense interstitial fibrosis, with occasional features of BO, acute fibrinous and organizing pneumonia, bronchiolitis obliterans organizing pneumonia, and aspiration. The rate of progression of clinical symptoms ranged from slow to rapid but, overall, the condition had a poor prognosis. The prevalence and cause of this form of chronic allograft dysfunction are still unclear.Fig. 4 CT scans obtained at two different levels (A and B) in a lung transplant recipient showing the peculiar pattern of upper lobe fibrosis. Culture for infectious agents of BAL specimens were repeatedly negative in these zones and transbronchial biopsies showed nonspecific inflammation and fibrotic changes.

unclear.Fig. 4 CT scans obtained at two different levels (A and B) in a lung transplant recipient showing the peculiar pattern of upper lobe fibrosis. Culture for infectious agents of BAL specimens were repeatedly negative in these zones and transbronchial biopsies showed nonspecific inflammation and fibrotic changes. Recently, Woodrow and colleagues24 reported on lung transplant recipients who had a decline in lung function associated with persistent parenchymal (alveolar, nodular, ground-glass, or interstitial) abnormalities on chest CT—not specifically involving the upper lobes (Fig. 5 ). No precise cause was found for the parenchymal infiltrates and the patients showed a functional deterioration over time that paralleled the course of patients with classical BOS. A similar proportion of patients (approximately 50%) had a restrictive ventilatory defect in the group with, and in the group without, parenchymal infiltrates.Fig. 5 This patient with CF had been transplanted for 10 years when she developed an acute, rapidly progressive, and completely therapy-resistant drop in lung function. CT scan of the lungs repeatedly showed peripheral infiltrates in the upper (A) but also lower (B) lung zones. BAL showed prominent neutrophilia but also—even if to a lesser extent—eosinophilia. No infectious agents could be incriminated; in particular, there were no diagnostic criteria for invasive pulmonary aspergillosis. She, eventually, could undergo redo-double lung transplantation. The explanted lungs showed zones of nonspecific interstitial fibrosis with pneumocyte hyperplasia and fibroblast proliferation in the areas of the parenchymal abnormalities, and advanced BO lesions in other lung regions.

invasive pulmonary aspergillosis. She, eventually, could undergo redo-double lung transplantation. The explanted lungs showed zones of nonspecific interstitial fibrosis with pneumocyte hyperplasia and fibroblast proliferation in the areas of the parenchymal abnormalities, and advanced BO lesions in other lung regions. Chronic pleural inflammation In a study by Woodrow and colleagues,24 36% of the radiographic abnormalities were pleural, and another study showed that at 1 year after transplantation, 50 of 58 patients (86%) had pleural abnormalities (most frequently pleural thickening) on chest CT.25 Such abnormalities may obviously restrict lung volumes, but their relationship with a process of chronic rejection and their long-term impact remain to be clarified. Exudative/follicular bronchiolitis In 2008, McManus and colleagues26 reported on 13 of 99 transplant recipients who presented with exudative bronchiolitis, which appeared on high-resolution CT as a tree-in-bud pattern (centrilobular nodules and branching lines). This condition was associated with early infection post-transplant and a history of Aspergillus infection. Neutrophil count in bronchial washing was increased and most patients improved clinically and radiologically with AZM. Yet, exudative bronchiolitis increased markedly the likelihood of developing BOS. Recently, Vos and colleagues27 described a patient who developed follicular bronchiolitis characterized by abundant peribronchiolar lymphoid follicules; this condition was also associated with the development of BOS.

iologically with AZM. Yet, exudative bronchiolitis increased markedly the likelihood of developing BOS. Recently, Vos and colleagues27 described a patient who developed follicular bronchiolitis characterized by abundant peribronchiolar lymphoid follicules; this condition was also associated with the development of BOS. Large airway stenosis/malacia A recent article by Akindipe and colleagues28 reported on 5 patients who had to be retransplanted for severe recurrent airway narrowing. In all patients, allograft lung pathology revealed evidence of BO. This observation suggests a possible link between airway ischemia/hypoxia, large airway stenosis/malacia, and the development of BO.

by Akindipe and colleagues28 reported on 5 patients who had to be retransplanted for severe recurrent airway narrowing. In all patients, allograft lung pathology revealed evidence of BO. This observation suggests a possible link between airway ischemia/hypoxia, large airway stenosis/malacia, and the development of BO. Pathogenesis and risk factors Pathogenesis In the earlier days of lung transplantation, BOS/BO was believed to be equivalent to chronic allograft rejection (ie, a process caused by an alloimmune reaction). The lung is uniquely exposed to the environment, however, and thus to recurrent nonalloimmune insults, such as infections, inhalation of toxic fumes, or gastroesophageal reflux. Furthermore, recent studies suggest a possible role of autoantibodies developed against specific epithelial proteins and of airway hypoxia in the pathogenesis of BOS/BO. The current view is that these insults, acting alone or in combination, up-regulate dendritric cells in the airway epithelium, leading to epithelial damage and inflammation with production of chemokines and cytokines by airway epithelium and smooth muscle cells, macrophages, and neutrophils. Activated neutrophils further increase epithelial damage via the production of reactive oxygen species and metalloproteinases. After the initial inflammatory phase, a fibroproliferative phase occurs, driven by growth factors and leading to proliferation of smooth muscle cells and myofibroblasts. This process eventually results in aberrant collagen deposition, excessive fibroproliferation, and small airway obliteration. BO would thus represent a final common pathway lesion secondary to multiple, repetitive insults to the airway epithelium.29, 30

eading to proliferation of smooth muscle cells and myofibroblasts. This process eventually results in aberrant collagen deposition, excessive fibroproliferation, and small airway obliteration. BO would thus represent a final common pathway lesion secondary to multiple, repetitive insults to the airway epithelium.29, 30 Alloimmune Risk Factors Ninety-five percent of patients receive grafts with 3 or more HLA mismatches. Using the Collaborative Transplant Study database, 5-year graft outcome according to HLA mismatch was examined in 8020 lung transplants performed during 1989 through 2009. Graft survival rates showed a stepwise decrease as the combined number of HLA-A+B+DR mismatches increased from 1 to 6, with a high number of HLA mismatches having an unfavorable impact on survival.31 Because of the average high number of mismatches, studies to examine the effect of HLA mismatching on the incidence of acute rejection have proved difficult and their results have been inconsistent. Most of them have, however, identified some negative impact of HLA mismatching.32, 33 Multivariate logistic regression analyses of data on 3549 adult lung transplant recipients retrieved from the United Network for Organ Sharing/ISHLT registry demonstrated an association between mismatching at the HLA-A locus (but not at the HLA-B or HLA-DR loci) and acute rejection episodes requiring hospital admission.34 Several studies have confirmed that the development of anti-HLA class I and class II antibodies after surgery is associated with a risk for acute rejection and BOS.35, 36, 37, 38, 39 Binding of these antibodies to airway epithelial cells may induce epithelial injury and proliferation.40

requiring hospital admission.34 Several studies have confirmed that the development of anti-HLA class I and class II antibodies after surgery is associated with a risk for acute rejection and BOS.35, 36, 37, 38, 39 Binding of these antibodies to airway epithelial cells may induce epithelial injury and proliferation.40 Acute vascular rejection histology graded greater than or equal to A2 has been identified in many studies as a statistical risk factor for BOS. Yousem41 reported in 1996 that untreated acute vascular rejection grade A2 leads to the development of BOS in 50% of patients. Several studies have shown that the risk of BOS increases when acute vascular rejection is histologically severe or persistent or recurs after treatment (studies reviewed by Knoop and Estenne42). The impact of minimal acute rejection (grade A1) on the development of BOS, however, has been long neglected. Recently, in a study by Hopkins and colleagues43 less than10% of grade A1 rejections were associated with clinical symptoms but 34% of the asymptomatic patients progressed to higher-grade acute rejection or lymphocytic bronchiolitis (LB) within 3 months. In this report also, patients with multiple A1 episodes during the first 12 months post-transplant had a significantly higher risk of developing BOS, and this occurred earlier than in patients with 1 or less grade A1 episode.43 Khalifa and colleagues44 retrospectively examined data from 228 lung transplant patients followed over a 7-year period and confirmed that grade A1 rejection is a distinct risk factor for BOS. Hachem and colleagues45 from the same group determined that even a single episode of A1 rejection, without recurrence or subsequent progression to a higher rejection grade, was a significant risk factor for the development of BOS. Treatment of grade A1 rejection with diverse approaches in order to augment the net immunosuppression decreased the risk for subsequent progression to BOS stage 1.44

isode of A1 rejection, without recurrence or subsequent progression to a higher rejection grade, was a significant risk factor for the development of BOS. Treatment of grade A1 rejection with diverse approaches in order to augment the net immunosuppression decreased the risk for subsequent progression to BOS stage 1.44 LB in the absence of acute vascular rejection may also predate BOS.46, 47 Glanville and colleagues48 retrospectively assessed data from 1770 TBB specimens obtained from 341 patients over a period of 10 years and showed that the cumulative incidence of BOS was significantly associated with the severity of LB. Another retrospective analysis of 2697 TBB specimens obtained from nearly 300 consecutive patients followed during the first 2 postoperative years at the University of Copenhagen showed that the cumulative incidences of LB (≥ B2) were 33%, 53%, 62%, and 68% at 1 month, 3 months, 6 months, and 12 months, respectively. Approximately 25% and 50% of patients had a second episode graded B2 or higher within 3 months and 2 years of transplantation, respectively. In this study, LB during the first 2 years was independently associated with the frequency and/or severity of acute rejection, and LB grade B2 or higher was associated with an increased risk of BO.49

d 50% of patients had a second episode graded B2 or higher within 3 months and 2 years of transplantation, respectively. In this study, LB during the first 2 years was independently associated with the frequency and/or severity of acute rejection, and LB grade B2 or higher was associated with an increased risk of BO.49 The concept of acute or chronic antibody-mediated rejection is still controversial after lung transplantation.50 There are now well documented reports that this type of rejection exists,51 however, and data on its possible contribution to chronic allograft dysfunction are beginning to appear.52 The observation that patients developing HLA antibodies fare less well than those who do not and the novel data on self-antibodies directed against epithelial antigens in patients with BOS/BO (discussed later) lend some credibility to this hypothesis.

ribution to chronic allograft dysfunction are beginning to appear.52 The observation that patients developing HLA antibodies fare less well than those who do not and the novel data on self-antibodies directed against epithelial antigens in patients with BOS/BO (discussed later) lend some credibility to this hypothesis. Autoimmune Risk Factors Recently, the development of autoimmune processes directed against epithelial-specific proteins has been incriminated in the development of BOS/BO. In one study, collagen type V-reactive CD4+ T cells were associated with a nearly 10-fold increase in the risk of BOS in clinical lung transplantation.53 In another study, anti-K–α1 tubulin antibodies were present in a significant number of patients with BOS.54 Anti-K–α1 tubulin circulating antibodies may induce profibrotic growth factors from airway epithelial cell lines, thus providing evidence that autoimmunity—like alloimmunity—may induce fibrosis.55 Conversely, it has also been shown that alloimmune responses in the lung can promote the development of collagen type V and K-α1-tubulin autoimmunity.56 Thus, the picture has become more complex: alloimmunity, autoimmunity, and the innate immune system (discussed later) may all trigger allograft airway fibrosis, and these processes are moreover likely intertwined.57

ne responses in the lung can promote the development of collagen type V and K-α1-tubulin autoimmunity.56 Thus, the picture has become more complex: alloimmunity, autoimmunity, and the innate immune system (discussed later) may all trigger allograft airway fibrosis, and these processes are moreover likely intertwined.57 Other Risk Factors As discussed previously, the association between acute rejection and BOS has been reported for both early and late rejection episodes. Yet many patients with acute rejection do not develop BOS, and some patients with BOS have never experienced acute rejection. One possible explanation is that the use of intense induction and maintenance immunosuppression and of aggressive treatment of rejection might uncouple the association between acute rejection and BOS. Another potential explanation, however, is that BOS/BO might be triggered by nonalloimmune–dependent factors. These may directly injure the airways—as is the case for gastric aspiration—and/or augment the alloimmune response via activation of the innate immune system, as is the case for respiratory bacterial and viral infections.58

tial explanation, however, is that BOS/BO might be triggered by nonalloimmune–dependent factors. These may directly injure the airways—as is the case for gastric aspiration—and/or augment the alloimmune response via activation of the innate immune system, as is the case for respiratory bacterial and viral infections.58 Bacterial colonization of the graft was formerly believed to be the consequence of BOS/BO. It has been recently reported, however, that bacterial colonization, notably with Pseudomonas aeruginosa, might be one of the possible alloimmune-independent risk factors for BOS/OB. In a study by Botha and colleagues,59 including 155 lung transplant recipients, the development of allograft colonization with Pseudomonas was strongly associated with the development of BOS within 2 years of transplant (23.4% and 7.7% in those colonized and not colonized, respectively). The isolation of Pseudomonas predated the diagnosis of BOS in more than 75% of affected patients by a median exceeding 200 days. Similar findings have been reported for Aspergillus colonization.60 Valentine and colleagues61 analyzed the role of bacterial and fungal respiratory tract infections in the development of BOS in a single-center study comprising 161 lung recipients who had survived at least 180 days. Multivariate analysis indicated that gram-negative, gram-positive, and fungal pneumonias were associated with the development of BOS. Gram-positive pneumonia and fungal pneumonia in the first 100 days conferred hazard ratios of 3.8 and 2.1, respectively. They concluded that early recognition and treatment of these pathogens might improve long-term outcomes.

am-negative, gram-positive, and fungal pneumonias were associated with the development of BOS. Gram-positive pneumonia and fungal pneumonia in the first 100 days conferred hazard ratios of 3.8 and 2.1, respectively. They concluded that early recognition and treatment of these pathogens might improve long-term outcomes. Kumar and colleagues62 screened serial surveillance and diagnostic BAL specimens obtained from 93 lung transplant recipients over 3 years for community-acquired respiratory viral infections (CARVIs) using sensitive molecular methods that simultaneously detected 19 respiratory viral types/subtypes. Respiratory viruses—rhinovirus, parainfluenza virus 1 to 4, coronavirus, influenza, metapneumovirus, and respiratory syncytial virus—were isolated in 48 of 93 (51.6%) patients in at least one BAL sample. Biopsy-proven acute rejection (≥ A2) or decline in FEV1 greater than or equal to 20% occurred in 33.3% of CARVI-positive patients (within 3 months of CARVI) compared with only 6.7% of CARVI-negative patients. No significant difference was seen in the incidence of acute rejection between symptomatic and asymptomatic patients. Biopsy-proved BO was diagnosed in 10 of 16 (62.5%) patients within 1 year of infection, indicating that symptomatic or asymptomatic viral infection may trigger acute rejection and/or BOS/BO. In contrast, Gottlieb and colleagues63 found that only symptomatic CARVI increases the risk of BOS. Chlamydia pneumoniae 64, 65 and human herpesvirus 6 respiratory infections66 are also known to increase the risk of BOS.

icating that symptomatic or asymptomatic viral infection may trigger acute rejection and/or BOS/BO. In contrast, Gottlieb and colleagues63 found that only symptomatic CARVI increases the risk of BOS. Chlamydia pneumoniae 64, 65 and human herpesvirus 6 respiratory infections66 are also known to increase the risk of BOS. CMV mismatching (ie, seronegative recipients receiving organs from seropositive donors) and CMV pneumonitis have been associated with BOS in several series, but others found only a marginal or no relationship at all. These differences might be accounted for, at least in part, by the different strategies used to match recipients with regard to CMV status and to prevent and treat CMV illness over the decades. Valentine and colleagues61 reported that CMV pneumonitis within the first 100 days conferred a hazard ratio of 3.1 to develop BOS. The same group reported on their experience with ganciclovir (GCV) prophylaxis in 130 patients surviving at least 100 days. CMV pneumonitis occurred in 16%, 8%, 17%, and 19% of patients in the D+R+, D−R+, D+R− and D−R− groups, respectively. Ninety patients received indefinite GCV prophylaxis whereas 40 patients discontinued the prophylaxis (STOP). Cumulative incidences of CMV pneumonitis in the indefinite GCV prophylaxis and STOP groups at 5 years were 2% and 57%, respectively. In the STOP cohort, 15 of 40 patients developed CMV pneumonitis after GCV was stopped, and 10 of these developed BOS. The risk of CMV pneumonitis in the STOP cohort was significantly higher when GCV prophylaxis was discontinued within the first year. BOS-free survival and survival were, however, similar across groups.67 On the contrary, Tamm and colleagues68 showed in their series that CMV pneumonitis, when treated with GCV, is not a risk factor for BOS and does not affect survival.

s significantly higher when GCV prophylaxis was discontinued within the first year. BOS-free survival and survival were, however, similar across groups.67 On the contrary, Tamm and colleagues68 showed in their series that CMV pneumonitis, when treated with GCV, is not a risk factor for BOS and does not affect survival. Gastroesophageal reflux disease (GERD) is thought to be a risk factor for the development or progression of BOS/OB. GERD is observed in approximately half of all lung transplant patients.69 The prevalence of delayed gastric emptying is also high.69 In addition, these patients have an impaired cough reflex because of lung denervation and have altered mucociliary clearance. Taken together, these factors increase the likelihood of aspiration and subsequent airway injury. Pepsin70 and bile acids71 can be readily found in the BAL fluid of many lung transplant recipients, which confirms the frequent occurrence of gastric aspiration. The finding of increased bile acids in BAL fluid (a marker of nonacidic reflux) correlates with the presence of BOS/BO.70, 71 Exposure of the airway epithelium to bile acids may predispose to colonization with Pseudomonas aeruginosa and airway neutrophilia. Because of concern that GERD increases the risk of BOS, the general trend has been to propose a surgical solution, namely gastric fundoplication, to all recipients presenting with significant GERD. Supportive evidence for this strategy is derived from retrospective studies in which gastric fundoplication within 3 months after transplant was associated with greater freedom from BOS and increased survival.72 Another study from the same center showed that fundoplication improved lung function in many patients with established BOS.73 Before adopting this radical approach for every single lung transplant recipient, however, it is important to be aware that (1) the best way to diagnose GERD in lung transplant recipients—pH monitoring versus impedance monitoring—is at present controversial; (2) GERD—as well as the cough reflex—may improve over time; (3) fundoplication may have serious side effects (eg, significant weight loss, which can be of importance in recipients with significant malnutrition); (4) the protective function of the surgical sleeve may wane over time; (5) the precise indications, timing, and choice of fundoplication technique are yet to be defined; and (6) the overall impact on lung function and survival are unknown because there are no controlled studies to date.74

ith significant malnutrition); (4) the protective function of the surgical sleeve may wane over time; (5) the precise indications, timing, and choice of fundoplication technique are yet to be defined; and (6) the overall impact on lung function and survival are unknown because there are no controlled studies to date.74 Allograft ischemia may arise during the period of warm ischemia during explantation, because of the absence of bronchial arterial reanastomosis at implantation, or through small airway microvascular damage at later time points.75 In a study of 334 lung transplant recipients of whom 65 developed primary graft dysfunction (which is a severe form of ischemia/reperfusion lung injury), this complication was an independent risk factor for BOS.76 Allograft ischemia might result in hypoxic inflammatory conditions leading to vascular remodeling and angiogenesis, which may in turn be a potent stimulus for airway fibrosis.77, 78 By gaining a better understanding of the complex interaction between airway ischemia, vascular remodeling and angiogenesis-mediated airway fibroproliferation, it might become increasingly possible to rationally design therapies that can halt conditions of maladaptive fibrosis79 and, possibly, decrease the risk of BOS/BO. The role of other risk factors for BOS, such as graft ischemic time, donor-recipient gender or size mismatch, and type of surgical procedure, is currently controversial.

Allograft ischemia may arise during the period of warm ischemia during explantation, because of the absence of bronchial arterial reanastomosis at implantation, or through small airway microvascular damage at later time points.75 In a study of 334 lung transplant recipients of whom 65 developed primary graft dysfunction (which is a severe form of ischemia/reperfusion lung injury), this complication was an independent risk factor for BOS.76 Allograft ischemia might result in hypoxic inflammatory conditions leading to vascular remodeling and angiogenesis, which may in turn be a potent stimulus for airway fibrosis.77, 78 By gaining a better understanding of the complex interaction between airway ischemia, vascular remodeling and angiogenesis-mediated airway fibroproliferation, it might become increasingly possible to rationally design therapies that can halt conditions of maladaptive fibrosis79 and, possibly, decrease the risk of BOS/BO. The role of other risk factors for BOS, such as graft ischemic time, donor-recipient gender or size mismatch, and type of surgical procedure, is currently controversial. The Role of Neutrophils It is widely accepted that BOS/BO involves a neutrophilic airway inflammation, although this feature is lacking in a substantial proportion of patients. Recent studies (summarized by Verleden and colleagues22) have shown that interleukin (IL)-17 may have an important role in the development of BOS/BO. IL-17 is a potent indirect neutrophil-attracting chemokine through its ability to induce IL-8 secretion from different cell types in the airways. IL-17 is increased in the airways of patients with BOS/BO and induces production of IL-8 by airway smooth muscle and epithelial cells, which, in turn, promotes airway neutrophilia. The IL-17/IL-8 axis may be triggered by both alloimmune and autoimmune mechanisms, airway bacterial colonization, and GERD,22 but the reasons why BOS is accompanied by airway neutrophilia in some patients and not in others remain unclear. The effect of AZM on this inflammatory process is likely primarily accounted for by its ability to inhibit the IL-17/IL-8 pathway; other potential mechanisms include a positive effect of AZM on GERD (AZM is a known agonist of motilin) as well as its inhibitory effect on bacterial growth.

d not in others remain unclear. The effect of AZM on this inflammatory process is likely primarily accounted for by its ability to inhibit the IL-17/IL-8 pathway; other potential mechanisms include a positive effect of AZM on GERD (AZM is a known agonist of motilin) as well as its inhibitory effect on bacterial growth. Open Questions and Controversial Issues It is important to stress that BOS and the newly described phenotypes of chronic allograft dysfunction are syndromes defined by clinical criteria, changes in pulmonary function, radiographic features, and analysis of BAL cellularity, alone or in combination. These entities are merely descriptive and do not sort by specific pathogenic pathways, risk factors, pathology, or prognosis. Patients may have more than one phenotype at a time or over time, and different pathogenic pathways and pathology may coexist (discussed previously). More work is required to understand the clinical relevance and pathogenesis of each entity as well as the mechanisms by which the different risk factors produce one phenotype or another.

e more than one phenotype at a time or over time, and different pathogenic pathways and pathology may coexist (discussed previously). More work is required to understand the clinical relevance and pathogenesis of each entity as well as the mechanisms by which the different risk factors produce one phenotype or another. For the time being, we still have to work with the current definition of BOS (ie, a progressive and irreversible airflow obstruction due to a loss of small airway function attributed to BO) although the difficulties associated with a staging system based on a retrospective diagnosis and exclusionary criteria are acknowledged. BOS should probably no longer be presumed to reflect specifically a process of chronic rejection because nonalloimmune insults likely often contribute to the development of the small airway lesions. Finally, whether or not NRAD should be considered a subtype of BOS or a distinct entity is currently debated in the transplant community (discussed later). Diagnosis To the extent that current therapies work to stop or slow down the progression of BOS, they do so mostly by an anti-inflammatory, not an antifibrotic, effect. Therefore, they are more likely to be effective in the early stage of the disease. For this reason, various parameters have been evaluated as early biomarkers of BOS.

e extent that current therapies work to stop or slow down the progression of BOS, they do so mostly by an anti-inflammatory, not an antifibrotic, effect. Therefore, they are more likely to be effective in the early stage of the disease. For this reason, various parameters have been evaluated as early biomarkers of BOS. Lung Function Spirometry is appealing as an early marker because it is widely available, noninvasive, reproducible, and relatively inexpensive. Two studies have assessed the predictive value of BOS stage 0-p for the diagnosis of BOS stage 1. In the study by Hachem and colleagues,80 which included 203 adult bilateral lung transplant (BLT) recipients, the FEV1 criterion had a sensitivity, specificity, positive predictive value, and negative predictive value of 74%, 86%, 79%, and 82%, respectively; corresponding values for the modified FEF25-75 criterion (computed using baseline values obtained at the time of the two highest FEV1 measurements) were 66%, 88%, 81%, and 76%, respectively. In the 197 SLT recipients studied by Lama and colleagues,81 the FEV1 criterion was also predictive of BOS 1; its predictive value was superior to that of the FEF25-75 criterion and was superior in patients with underlying restrictive as opposed to obstructive physiology.

ents) were 66%, 88%, 81%, and 76%, respectively. In the 197 SLT recipients studied by Lama and colleagues,81 the FEV1 criterion was also predictive of BOS 1; its predictive value was superior to that of the FEF25-75 criterion and was superior in patients with underlying restrictive as opposed to obstructive physiology. Exhaled Biomarkers Distribution of ventilation The slope of the alveolar plateau of the single-breath washout test reflects the homogeneity of ventilation distribution and increases as ventilation becomes more heterogeneous. The single-breath test can be performed using an inspiration of pure oxygen and measuring the concentration of nitrogen during expiration; a gas mixture containing inert gases (for example helium) can also be used during inspiration, and the concentration of these gases be measured during expiration. Two prospective studies have assessed the usefulness of the single-breath test for the early detection of BOS in BLT recipients. In these studies, nitrogen slope became abnormal 17882 and 15183 days before the criterion for BOS 1 was met. The positive predictive value of the test was 70% to 80% and the negative predictive value approximately 100%. Furthermore, 2 studies by Estenne and colleagues82 and Van Muylem and colleagues84 showed that helium slope is an even earlier marker than nitrogen slope (Fig. 6 ).Fig. 6 In patients presenting BOS, the slope of the alveolar plateau for helium increases as ventilation becomes more heterogeneous and this early marker of BOS becomes abnormal before spirometric criteria for BOS 1 are fulfilled. This figure shows changes in FEV1 and in the slope of the alveolar plateau for helium obtained during a single-breath washout test (She) in one heart-lung transplant recipient. In the upper panel, the dashed line corresponds to 100% of the two best postoperative values and the continuous line indicates a 20% decrease (BOS stage 1). In the lower panel, the dashed line corresponds to the average of the two lowest postoperative values, and the continuous line is the upper limit of the 97.5% CI computed from data obtained in 10 stable transplant patients. Note that a significant change in She is observed 631 days before the 20% drop in FEV1.

tage 1). In the lower panel, the dashed line corresponds to the average of the two lowest postoperative values, and the continuous line is the upper limit of the 97.5% CI computed from data obtained in 10 stable transplant patients. Note that a significant change in She is observed 631 days before the 20% drop in FEV1. (From Van Muylem A, Knoop C, Estenne M. Early detection of chronic pulmonary allograft dysfunction by exhaled biomarkers. Am J Respir Crit Care Med 2007;175:731–6; with permission.) Two recent studies in recipients of SLT for emphysema or fibrosis suggested that when performed in lateral decubitus, the single-breath test may also provide information on ventilation distribution in the graft in this patient population.85, 86 Exhaled gases Exhaled nitric oxide (eNO) is a well-recognized biomarker of airway inflammation. In stable lung transplant recipients and patients with BOS, eNO reflects the expression of bronchial epithelial inducible NO synthase and positively correlates with airway neutrophilia.87, 88, 89, 90 Carbon monoxide (CO) is produced endogenously by the stress protein heme oxygenase, which is increased in a variety of oxidant/inflammatory-mediated injuries. In BO lesions, heme oxygenase staining correlates with myeloperoxidase expression (reflecting oxidant load) and with neutrophilic infiltration of the bronchial wall. Heme oxygenase degrades heme with the production of iron, biliverdin, and CO. Therefore, both eNO87, 88, 89 and exhaled CO (eCO)90 may reflect airway neutrophilia and, hence, be used as surrogate markers of BOS.

eloperoxidase expression (reflecting oxidant load) and with neutrophilic infiltration of the bronchial wall. Heme oxygenase degrades heme with the production of iron, biliverdin, and CO. Therefore, both eNO87, 88, 89 and exhaled CO (eCO)90 may reflect airway neutrophilia and, hence, be used as surrogate markers of BOS. Four studies have shown that eNO is increased in patients with BOS compared with patients without BOS,89, 91, 92, 93 independent of the type of surgical procedure. The potential contribution of serial eNO measurements to the early detection of BOS, however, was difficult to assess from these studies. In a recent study of 65 recipients of bilateral grafts who were followed for approximately 1250 days, Van Muylem and colleagues84 found that eNO and eCO only transiently increased in BOS 0-p and then returned to baseline as BOS progressed (Fig. 7 ). The sensitivity of exhaled gases for the diagnosis of BOS 0-p was only 50% to 60%, but it increased to approximately 80% when values of eNO and eCO were combined; yet, on average, the increase in exhaled gases did not precede the diagnosis of BOS 0-p. This may be due, at least in part, to a significant proportion of patients with early BOS having no increase in airway neutrophilia.Fig. 7 Changes in eNO, in eCO, and in the slope of the alveolar plateau for helium (She) over time in one transplant recipient. The continuous lines indicate the confidence interval. Note that values of She become abnormal before BOS 0-p, and then increase progressively with the BOS stage; in contrast, values of eNO and eCO are much more variable between successive measurements and do not show a consistent trend as BOS progresses.

nt recipient. The continuous lines indicate the confidence interval. Note that values of She become abnormal before BOS 0-p, and then increase progressively with the BOS stage; in contrast, values of eNO and eCO are much more variable between successive measurements and do not show a consistent trend as BOS progresses. In summary, eNO and eCO have a fair sensitivity and nitrogen or helium slope has a good sensitivity for the detection of BOS. All biomarkers also have a high negative predictive value, but their specificity and positive predictive value are much lower. The low specificity reflects that these markers—like the FEV1—may be affected by complications other than BOS (eg, acute rejection, lymphocytic bronchiolitis, and infection). From a clinical point of view, the high negative predictive value should help detect conditions that may confound the diagnosis of BOS, because in the absence of a significant rise in exhaled biomarkers, BOS is an unlikely explanation for a decline in spirometry. Conversely, a persistent rise in slope or in eNO/eCO should be interpreted as a warning signal and prompt close monitoring of a patient’s lung function and clinical condition.

iagnosis of BOS, because in the absence of a significant rise in exhaled biomarkers, BOS is an unlikely explanation for a decline in spirometry. Conversely, a persistent rise in slope or in eNO/eCO should be interpreted as a warning signal and prompt close monitoring of a patient’s lung function and clinical condition. Other Markers Several other surrogate markers of BOS have been proposed, but their clinical utility is limited by one or more of the following factors: they are invasive or potentially toxic, they are expensive, they are not widely available, or their predictive value has not been appropriately tested or is controversial. These surrogate markers include exhaled breath condensate,94 induced sputum,95 analysis of cellular composition and inflammatory markers in BAL fluid, and imaging techniques, such as CT—in particular, quantification of air trapping at full expiration (see Fig. 3),7, 8, 96 and hyperpolarized 3He MRI.97

is controversial. These surrogate markers include exhaled breath condensate,94 induced sputum,95 analysis of cellular composition and inflammatory markers in BAL fluid, and imaging techniques, such as CT—in particular, quantification of air trapping at full expiration (see Fig. 3),7, 8, 96 and hyperpolarized 3He MRI.97 Treatment Optimization and/or Change in Immunosuppressive Regimen All interventions that target risk factors and may prevent the development of BOS are valuable because therapy is often ineffective when BOS is established. In this context, optimization of the immunosuppressive regimen to prevent the occurrence of acute rejection is a critical issue (discussed previously). Several studies have looked at the effects of increasing the net level of immunosuppression (eg, by using high-dose methylprednisolone, cytolytic therapy, or methotrexate) and/or changing the maintenance regimen (eg, by shifting from cyclosporine A to tacrolimus or from azathioprine to mycophenolate mofetil, or by adding inhaled cyclosporine A) in patients with established BOS (reviewed by Knoop and Estenne42). In some patients, these modalities have been shown to stabilize lung function or decrease the rate of decline of FEV1 for short periods of time. The small number of patients studied, the mostly retrospective design of these studies, the lack of adequate control group, and the relatively short follow-up time, however, make it difficult to assess the effectiveness of these treatments. No single strategy has proved more successful than another. In addition, augmented immunosuppression increases the risk of toxicity and predisposes to intercurrent bronchopulmonary infections, which must be factored into the risk-benefit analysis as they may promote the progression of BOS (discussed previously).

ments. No single strategy has proved more successful than another. In addition, augmented immunosuppression increases the risk of toxicity and predisposes to intercurrent bronchopulmonary infections, which must be factored into the risk-benefit analysis as they may promote the progression of BOS (discussed previously). Macrolides As discussed previously, several studies with a follow-up of 12 to 40 weeks have shown that macrolide treatment improves FEV1 in approximately one-third of patients in different BOS stages. Two studies20, 98 have assessed the long-term effect of AZM. The study by Gottlieb and colleagues included 81 patients with a median follow-up of 1.3 years20 and the study by Vos and colleagues98 included 108 patients treated for a median time of 612 days. An initial response (defined as a 10% or more increase in FEV1) was observed in 30% to 40% of the patients, but 30% to 40% of these subsequently relapsed. By multivariate analysis, initial response to AZM and earlier post-transplant time of initiation of treatment were protective factors for disease progression or relapse; in contrast, the level of BAL neutrophilia had no predictive value. These longitudinal data thus show that AZM provides a sustained functional improvement in the long-term in only a small minority of patients with BOS (approximately 10%–15%). This observation suggests, therefore, that in most patients (even those with the NRAD phenotype), BOS is a condition that worsens with time.

lue. These longitudinal data thus show that AZM provides a sustained functional improvement in the long-term in only a small minority of patients with BOS (approximately 10%–15%). This observation suggests, therefore, that in most patients (even those with the NRAD phenotype), BOS is a condition that worsens with time. Despite this relatively modest effect of AZM on lung function, 3 studies in patients with BOS have shown that this treatment is associated with a significant reduction in the risk of death.20, 98, 99 In studies by Gottlieb and colleagues20 and by Vos and colleagues,98 responders had significantly better overall survival compared with nonresponders; as expected, the difference between groups was more pronounced when only responders with a sustained response were taken into account. Using AZM for the prevention of BOS (ie, when patients are still in BOS stage 0) may have an even greater clinical impact than using it as a treatment. In a recent prospective randomized trial of AZM (40 patients) versus placebo (43 patients), Vos and colleagues100 initiated treatment at discharge and followed the patients for 2 years. BOS occurred less in patients receiving AZM (12.5%) than placebo (44.2%), and BOS-free survival was better with AZM. Patients receiving AZM demonstrated better FEV1, lower BAL neutrophilia, and less systemic inflammation. There was no difference in survival between groups, but this may be due to the short follow-up time.

S occurred less in patients receiving AZM (12.5%) than placebo (44.2%), and BOS-free survival was better with AZM. Patients receiving AZM demonstrated better FEV1, lower BAL neutrophilia, and less systemic inflammation. There was no difference in survival between groups, but this may be due to the short follow-up time. Statins In a large retrospective study published in 2003, Johnson and colleagues101 found that patients who received statins during the first year after transplantation for treatment of hypercholesterolemia were at less risk of developing BOS than patients who were not treated. In addition, patients receiving statins were less likely to develop severe BOS and had better survival. Unfortunately, there have been no subsequent reports to confirm these observations nor have there been controlled studies. In vitro, it has been demonstrated that simvastatin attenuates the release of airway neutrophilic and remodeling mediators from primary bronchial epithelial cells from stable lung transplant patients and inhibits their up-regulation by transforming growth factor β and IL-17.102 In practice, many lung transplant programs systematically prescribe a statin in order to exploit this immunomodulatory effect even if the true clinical benefit is still hypothetical.

hial epithelial cells from stable lung transplant patients and inhibits their up-regulation by transforming growth factor β and IL-17.102 In practice, many lung transplant programs systematically prescribe a statin in order to exploit this immunomodulatory effect even if the true clinical benefit is still hypothetical. Total Lymphoid Irradiation Fisher and colleagues103 summarized their experience with total lymphoid irradiation in 37 patients treated for progressive BOS. In the 27 recipients who completed more than 80% of the treatment, the rate of decline in FEV1 decreased from 122.7 mL/mo pre–total lymphoid irradiation to 25.1 mL/mo post–total lymphoid irradiation. Patients with a greater rate of functional decline before treatment were more likely to respond. Results of these studies are promising, but in the absence of adequately powered randomized control trials, they should be regarded as providing suggestive, rather than convincing, evidence.

post–total lymphoid irradiation. Patients with a greater rate of functional decline before treatment were more likely to respond. Results of these studies are promising, but in the absence of adequately powered randomized control trials, they should be regarded as providing suggestive, rather than convincing, evidence. Photospheresis Two recent single-center reports document experience with extracorporeal photopheresis in the treatment of BOS. Benden and colleagues104 reported on a series of 12 patients with various stages of BOS; rate of decline in FEV1 was 112 mL/mo before photopheresis and 12 mL/mo after completion of 12 cycles. No complications related to therapy were recorded in this study. In a larger study of 60 patients with BOS, Morrell and colleagues105 documented a similarly dramatic reduction in rate of decline in FEV1 from 116 mL/mo prior to treatment to 28.9 mL/mo during the 6 months after initiation of photopheresis. Eight patients experienced catheter-related bacteremias, one patient had a catheter-associated thrombus, and one patient experienced transient hypotension during a treatment. The mechanisms by which photopheresis exerts immunomodulatory and anti-inflammatory effects remain poorly understood. In the absence of randomized trials, it is premature to endorse photopheresis as a definitive therapy for BOS.

-associated thrombus, and one patient experienced transient hypotension during a treatment. The mechanisms by which photopheresis exerts immunomodulatory and anti-inflammatory effects remain poorly understood. In the absence of randomized trials, it is premature to endorse photopheresis as a definitive therapy for BOS. Retransplantation In 1998, Novick and colleagues106 reported results of 230 retransplants performed at 47 centers worldwide, 63% of which were performed for BOS. The report indicated that early survival after retransplantation was reduced compared with first transplants, but results of retransplants performed for BOS were not different than those done for other indications. In addition, recurrent BOS was observed in a frequency similar to that seen after first transplants. Subsequently, three single-center reports have confirmed this observation. Brugière and colleagues107 reported on long-term outcome in 15 single-lung retransplantations for BOS. The median time between primary lung transplantation and retransplantation was 31 months (range, 12 to 39 months). Actuarial survival rates at 1 year, 2 years, and 5 years after retransplantation were 60%, 53%, and 45%, respectively. Ten patients died during long-term follow-up, 6 of them from infection (60%). The retained graft was the initial site of the fatal infection in 4 of these patients. Two other patients experienced disabling chronic purulent expectoration arising from the old graft. Lung retransplantation thus offered a viable therapeutic option for selected SLT recipients with BOS, but given the morbidity and mortality related to the retained graft, the team now favors replacement of the primary graft when retransplantation is considered.107 Strueber and colleagues108 reported on 54 redo-transplants among 614 lung transplantation procedures performed at their institution. Retransplantation for BOS achieved 1-year and 5-year survival rates of 78% and 62%, respectively, which were not different from those observed after first-time lung transplantations. Recipients had a similar incidence of BOS after retransplantation for BOS versus after a first procedure. The same group published similar results for 7 retransplantations performed in children.109 At present, 1% to 2% of lung transplantations performed yearly worldwide are retransplantations.2 In assessing these procedures, medical issues and the issue of equitable use of scarce resources need be addressed.

rst procedure. The same group published similar results for 7 retransplantations performed in children.109 At present, 1% to 2% of lung transplantations performed yearly worldwide are retransplantations.2 In assessing these procedures, medical issues and the issue of equitable use of scarce resources need be addressed. Summary Chronic allograft dysfunction, especially BOS/BO, remains the major obstacle to long-term survival after lung transplantation. Major advances in understanding the risk factors and pathogenic mechanisms leading to irreversible small airway lesions have been made and new options for the prevention and treatment of BOS/BO are available. There is no doubt that the coming years will further improve our ability to cope with this devastating complication of lung transplantation.

Key points • Culture-independent molecular assays detect viral pathogens with great sensitivity and can be used to define the virome in the upper and lower respiratory tract. • The respiratory tract virome is defined by very common pathogens (rhinoviruses, paramyxoviruses) as well as viruses that occur less frequently and those with unknown pathogenicity. • Viruses with the potential for pathogenicity are detected in both symptomatic and asymptomatic people. • Monitoring emerging respiratory pathogens is important, and high-throughput sequencing can be used as a tool to complement epidemiologic studies and to design diagnostics. • In the future, comprehensive pathogen detection and host response may be coupled to create better assays for research studies and diagnostics. Introduction Viral infections of the respiratory tract are very common. In a recent study of 26 households in Utah that were followed weekly over 1 year, modern molecular methods were used to detect respiratory viruses in the anterior nares.1 This study found that children less than 5 years old had about 12 viral episodes in the respiratory tract each year, whereas adults averaged about 6 per year. These numbers are higher than previous studies,2 which is likely explained by the use of molecular assays instead of culture- and serology-based tests and the discovery of new respiratory viruses in intervening years that would not have been assessed in older studies.

ach year, whereas adults averaged about 6 per year. These numbers are higher than previous studies,2 which is likely explained by the use of molecular assays instead of culture- and serology-based tests and the discovery of new respiratory viruses in intervening years that would not have been assessed in older studies. Modern molecular methods for virus detection are highly sensitive and specific. Polymerase chain reaction (PCR) assays are also rapid and generally inexpensive. High-throughput nucleic acid sequencing (HTS) methods are slower but have the potential to be more comprehensive because there is no need to select specific targets beforehand, and the method can detect genomes with substantial sequence variation compared with known reference genomes (Fig. 1 ). With these tools in hand, we can begin to think about characterizing the virome of the respiratory tract, herein defined as all of the viruses in the respiratory tract that can infect and replicate in human cells, which includes known pathogens and viruses with unknown pathogenicity. We have begun to learn about the virome of the respiratory tract through studies of patients with acute infections, chronic lung diseases, and undergoing lung transplantation, among others. The author reviews some of these studies in addition to recent technological developments, which will improve characterization of the respiratory virome and diagnostics in coming years.Fig. 1 Methods for characterizing viruses in the respiratory tract. Current molecular methods, such as PCR and HTS, have clear advantages over older methods (culture and serology) in terms of cost, speed, and sensitivity. Future assays for research and diagnostics will be aimed at capturing and improving on the best features of the current methods.

ing viruses in the respiratory tract. Current molecular methods, such as PCR and HTS, have clear advantages over older methods (culture and serology) in terms of cost, speed, and sensitivity. Future assays for research and diagnostics will be aimed at capturing and improving on the best features of the current methods. Overview of the virome in the respiratory tract Defining the virome in the respiratory tract and understanding the implications of the viruses detected are significant challenges. The work is complicated by several factors. First, the lower airway is not easily accessible and sometimes requires invasive sampling. For instance, bronchoalveolar lavage samples are often only available from symptomatic individuals who are having lavage performed for diagnostic testing and not from asymptomatic controls. Second, a study of the viruses in the lungs of patients with cystic fibrosis (CF) showed that the viral populations were distinct in different regions of the lung.3 This variation within the respiratory tract and lung means it can be difficult to get a clear, or complete, view of the virome. Third, only recently have relatively unbiased approaches to identifying viruses become available in the form of HTS assays. With that said, a great deal of progress has been made in defining the human virome in the respiratory tract (summarized in Table 1 ).Table 1 Common viruses detected in the respiratory tract virome

hird, only recently have relatively unbiased approaches to identifying viruses become available in the form of HTS assays. With that said, a great deal of progress has been made in defining the human virome in the respiratory tract (summarized in Table 1 ).Table 1 Common viruses detected in the respiratory tract virome Virus Groups Species or Types References from this Review Picornaviruses Rhinoviruses A, B, and/or C Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Colvin et al,8 2012; Wylie et al,9 2012; Flight et al,13 2014; Goffard et al,14 2014; Wat et al,15 2008; Graf et al,24 2016; Thorburn et al,25 2015; Zoll et al,26 2015 Enteroviruses Colvin et al,8 2012; Wylie et al,9 2012; Wylie et al,22 2015; Wylie et al,23 2015; Graf et al,24 2016; Thorburn et al,25 2015; Zoll et al,26 2015 Parechovirus Wylie et al,9 2012 Paramyxoviruses Respiratory syncytial virus Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Flight et al,13 2014; Wat et al,15 2008; Graf et al,24 2016; Thorburn et al,25 2015; Zoll et al,26 2015 Parainfluenzaviruses 1–4 Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Colvin et al,8 2012; Wylie et al,9 2012; Flight et al,13 2014; Goffard et al,14 2014; Wat et al,15 2008; Graf et al,24 2016; Thorburn et al,25 2015 Metapneumovirus Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Colvin et al,8 2012; Graf et al,24 2016; Thorburn et al,25 2015; Zoll et al,26 2015 Measles virus Lysholm et al,4 2012; Wang et al,5 2016; Wylie et al,9 2012; Flight et al,13 2014; Graf et al,24 2016 Pneumovirus Wylie et al,9 2012 Orthomyxoviruses Influenzavirus A, B, and/or C Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Colvin et al,8 2012; Wylie et al,9 2012; Flight et al,13 2014; Goffard et al,14 2014; Wat et al,15 2008; Graf et al,24 2016; Thorburn et al,25 2015 Coronaviruses HKU1, OC43, 229E, and/or NL63 Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Colvin et al,8 2012; Wylie et al,9 2012; Goffard et al,14 2014; Wat et al,15 2008; Graf et al,24 2016; Thorburn et al,25 2015 Adenoviruses Adenovirus C or untyped Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Colvin et al,8 2012; Wylie et al,9 2012; Flight et al,13 2014; Graf et al,24 2016; Thorburn et al,25 2015 Parvoviruses Bocavirus or unclassified Lysholm et al,4 2012; Wang et al,5 2016; Colvin et al,8 2012; Wylie et

Adenovirus C or untyped Lysholm et al,4 2012; Wang et al,5 2016; Jain et al,6 2015; Jain et al,7 2015; Colvin et al,8 2012; Wylie et al,9 2012; Flight et al,13 2014; Graf et al,24 2016; Thorburn et al,25 2015 Parvoviruses Bocavirus or unclassified Lysholm et al,4 2012; Wang et al,5 2016; Colvin et al,8 2012; Wylie et  al,9 2012; Willner et al,16 2009; Young et al,17 2015; Graf et al,24 2016; Zoll et al,26 2015 Herpesviruses Cytomegalovirus, Epstein-Barr virus, Roseolovirus, and/or Kaposi sarcomavirus Wang et al,5 2016; Wylie et al,9 2012; Willner et al,16 2009; Young et al,17 2015; Graf et al,24 2016 Anelloviruses Torque teno virus, torque teno midi virus, and/or torque teno mini virus or untyped Lysholm et al,4 2012; Wang et al,5 2016; Wylie et al,9 2012 Papillomaviruses Various Wang et al,5 2016; Willner et al,16 2009; Young et al,17 2015 Polyomaviruses KI and/or WU Lysholm et al,4 2012; Colvin et al,8 2012; Wylie et al,9 2012 The Virome in Patients with Respiratory Tract Infections and Controls One cost-effective approach to broadly identify viruses associated with the respiratory tract is to pool samples and screen for a comprehensive set of viruses. The downside to this approach is that one cannot determine the frequency at which any individual virus occurs among patients. However, as characterization of the respiratory tract virome using molecular methods is a relatively new area of exploration, these studies can be useful in order to determine if viruses beyond the common, known respiratory pathogens are detected.

rmine the frequency at which any individual virus occurs among patients. However, as characterization of the respiratory tract virome using molecular methods is a relatively new area of exploration, these studies can be useful in order to determine if viruses beyond the common, known respiratory pathogens are detected. In one study, 210 adults and children with severe lower respiratory tract infections were sampled.4 Nasopharyngeal aspirates were collected and samples were combined, creating 13 pools of 8 to 24 samples per pool. Virus particles were enriched, and DNA and RNA viruses were assessed using HTS. Thirty-nine viral species were observed in these samples, giving a broad view of the scope of the respiratory tract virome during infection. Based on read counts, the most abundant viruses in the data set were the paramyxoviruses (including human respiratory syncytial virus, human metapneumovirus). Picornaviruses were the next most abundant (primarily rhinoviruses A and C). Orthomyxoviruses were the third most abundant (influenza viruses A, B, and C). There were several rare and/or unexpected viruses represented at low abundance. These viruses included bocavirus, KI polyomavirus, picobirnavirus, measles virus, and anelloviruses. In a study from China, a similar sequencing-based approach was taken to characterize the virome in children less than 6 years old with severe acute respiratory illness and 15 controls without respiratory illness.5 Nasopharyngeal swabs were pooled into 9 pools of 15 samples each; virus particles were enriched; and HTS data were generated to assess both RNA and DNA viruses. The most highly represented viruses included the paramyxoviruses (primarily human respiratory syncytial virus), and the other common viruses detected in the study described earlier were detected. This study also detected human coronaviruses, bocaviruses, picornaviruses, influenza viruses, adenoviruses, and anelloviruses. Rare sequences included those from metapneumovirus, measles, hepatitis B and C, papillomavirus, and others. These two studies demonstrate the power of HTS compared with targeted PCR assays for defining the virome. In both studies, common and expected respiratory pathogens were detected. However, other viruses, some with unknown pathogenicity in the respiratory tract, were also detected. Detection of these viruses is valuable as we aim to fully understand the biology of the respiratory tract.

d PCR assays for defining the virome. In both studies, common and expected respiratory pathogens were detected. However, other viruses, some with unknown pathogenicity in the respiratory tract, were also detected. Detection of these viruses is valuable as we aim to fully understand the biology of the respiratory tract. Although these studies were exploratory, they raise questions about whether some of the viruses with unclear pathogenicity could be contributing to the presentation of symptoms, complicating the course of infection with other pathogens, or are biomarkers for infection or host response. These questions remain to be addressed in future studies.

ere exploratory, they raise questions about whether some of the viruses with unclear pathogenicity could be contributing to the presentation of symptoms, complicating the course of infection with other pathogens, or are biomarkers for infection or host response. These questions remain to be addressed in future studies. The Virome in Children and Adults with Pneumonia Viruses are clearly an important cause of pneumonia, particularly in the postpneumococcal vaccine era. A prospective multicenter study sponsored by the Centers for Disease Control and Prevention focused on the Etiology of Pneumonia in the Community (EPIC).6, 7 This study used extensive diagnostic testing (culture, serology, molecular testing) to understand the causes of pneumonia in more than 2000 adults in the United States, in the time after the implementation of the pneumococcal vaccine. In this study, a pathogen was detected in 38% of the samples. At least 1 virus was detected in 23% of the samples available for testing, and 3% had both bacterial and viral pathogens detected. Rhinoviruses and influenza viruses were the most common pathogens detected in 9% and 6% of patients, respectively. The EPIC study took a similar approach to study the cause of community-acquired pneumonia in children. Again, more than 2000 subjects were enrolled. The children were less than 18 years old, with a median age of 2 years. A microbial pathogen was detected in 81% of samples. At least one virus was detected in 66% of the samples, and both bacterial and viral pathogens were detected in 7% of the children. In children, respiratory syncytial virus, rhinovirus, metapneumovirus, and adenovirus were the most common pathogens. These studies identify viruses as key pathogens in pneumonia since the implementation of the pneumococcal vaccine. Furthermore, it is important to note that no pathogen was detected in 62% of adults and 19% of children, suggesting the possibility that pneumonia was caused by viruses not included in the set of targeted assays used in this study. Identification of the etiologic agent of pneumonia may benefit from unbiased HTS assays to detect unexpected or rare pathogens that are not be included in standard clinical testing.

f children, suggesting the possibility that pneumonia was caused by viruses not included in the set of targeted assays used in this study. Identification of the etiologic agent of pneumonia may benefit from unbiased HTS assays to detect unexpected or rare pathogens that are not be included in standard clinical testing. The Virome in Children with Unexplained Fever and Asymptomatic Controls In a study of children with unexplained fever, both targeted PCR assays8 and HTS9 were used to characterize the respiratory tract virome in individual samples from subjects. Children with fever were compared with afebrile children who were in the hospital for same-day surgery. Nasopharyngeal swabs from 75 febrile children and 116 afebrile children were tested with a panel of PCR assays that targeted common respiratory pathogens and viruses of interest, including influenza A, parainfluenza virus, metapneumovirus, rhinovirus, enterovirus, coronavirus, adenovirus, bocavirus, and the recently discovered KI and WU polyomaviruses. HTS was performed on 50 samples from febrile children and 81 samples from afebrile controls. Using sequencing, 17 viral genera were detected overall. These genera included viruses listed earlier; cytomegalovirus, parechovirus and others were detected in febrile children, whereas Roseolovirus was detected in nasopharyngeal swabs from both febrile and afebrile children. Although febrile children were more likely to have a virus present in the sample compared with controls, afebrile children still carried viruses asymptomatically. Rhinoviruses/enteroviruses and anelloviruses were particularly common in asymptomatic children in this study. However, anelloviruses, particularly torque teno virus, were associated with fever.10 This study demonstrates 2 points very well. First, even with a panel of respiratory virus PCR assays that extends far beyond targets that would be used for clinical testing, additional viruses were detected in the respiratory tract only by sequencing. This finding emphasizes the potential to improve viral diagnostic testing by broadening the set of viruses evaluated. Second, viruses are commonly found in the respiratory tracts of asymptomatic children and many of these viruses have potential to cause symptomatic infection. This point means that pathogen detection may not always be enough to make a clear diagnosis. Other information may be needed, as discussed in more detail later in this review.

s are commonly found in the respiratory tracts of asymptomatic children and many of these viruses have potential to cause symptomatic infection. This point means that pathogen detection may not always be enough to make a clear diagnosis. Other information may be needed, as discussed in more detail later in this review. The Respiratory Tract Virome in Patients with Cystic Fibrosis Although many viral infections are mild or resolve without complication in generally healthy individuals, viruses in the respiratory tract are associated with exacerbations of chronic lung diseases, including CF, chronic obstructive pulmonary disease, and asthma (reviewed in the following11, 12). Exploration of the virome in the respiratory tracts of patients with chronic lung diseases has emphasized the prevalence of common pathogens and also further defines the scope of the respiratory tract virome.

ng diseases, including CF, chronic obstructive pulmonary disease, and asthma (reviewed in the following11, 12). Exploration of the virome in the respiratory tracts of patients with chronic lung diseases has emphasized the prevalence of common pathogens and also further defines the scope of the respiratory tract virome. To illustrate this point, the author discusses a few studies of the virome in patients with CF. Molecular methods of detection, specifically PCR assays, have increased the association of viral infection with pulmonary exacerbation. Incidence of viral infections in adult patients with CF was estimated at 1 to 2 viral infections per year based on a study of 100 patients who were sampled every 2 months for a year and on exacerbation.13 Samples from the respiratory tract (sputum, nose and throat swabs) were tested using PCR assays for common respiratory pathogens, including respiratory syncytial virus, rhinovirus, influenza virus, and others. Rhinovirus and metapneumovirus were the most commonly detected viruses, accounting for 72.5% and 13.2% of virus-positive samples, respectively. Viral infection was associated with pulmonary exacerbation, with virus detected in 40% of exacerbation samples compared with 24% of samples collected on regular visits. In another study, sputum was collected from 46 adult patients and viruses were screened using PCR assays.14 In this study, rhinoviruses and coronaviruses were the most common viruses, and rhinoviruses were associated with exacerbation. In children with CF, exacerbation has also been associated with viral infection. In a study of 71 patients, viruses were assessed in nasal swabs using a panel of targeted PCR assays for common respiratory pathogens.15 Viruses were detected in 46% of samples collected during exacerbation but only 17% of samples collected when asymptomatic. Influenza A, influenza B, and rhinovirus were all associated with exacerbation.

tients, viruses were assessed in nasal swabs using a panel of targeted PCR assays for common respiratory pathogens.15 Viruses were detected in 46% of samples collected during exacerbation but only 17% of samples collected when asymptomatic. Influenza A, influenza B, and rhinovirus were all associated with exacerbation. The virome of patients with CF has also been characterized using HTS. One study aimed to study the virome in sputum from 5 patients with CF and 5 healthy controls.16 Virus particles were enriched, and DNA viruses were sequenced. This study was particularly interesting because the use of HTS instead of targeted PCR assays resulted in the detection of viruses that would not have been included in typical PCR panels evaluating respiratory viruses. In the patients with CF, reticuloendotheliosis virus, Epstein-Barr virus, human herpesvirus 6B, and human herpesvirus 8 were detected. In control patients, human papillomaviruses were detected in 2 samples. Several single-stranded DNA viruses were also detected in patients and/or controls, including geminiviruses and circoviruses. In a second study, the virome was assessed in explanted lungs from patients with CF undergoing lung transplant and lungs obtained post mortem.3 Anelloviruses, papillomaviruses, and herpesviruses were detected; viruses were detected in distinct regions of the lung rather than diffusely throughout. These studies were valuable because many of the viruses detected were not those that would have been included in PCR screens for common respiratory pathogens, yet they may impact disease progression in the lung by creating or promoting inflammation in the respiratory tract.

of the lung rather than diffusely throughout. These studies were valuable because many of the viruses detected were not those that would have been included in PCR screens for common respiratory pathogens, yet they may impact disease progression in the lung by creating or promoting inflammation in the respiratory tract. The Respiratory Tract Virome after Lung Transplantation In one study, the virome was studied in bronchoalveolar lavage and oral washes from lung transplant patients within the first year of transplant, human immunodeficiency virus (HIV) positive subjects without respiratory symptoms, and healthy volunteers.17 Anelloviruses, papillomaviruses, and herpesviruses were detected. The striking observation in this study was that the diversity and abundance of anelloviruses was highly increased in the transplant patients compared with patients with HIV and healthy controls. This finding was true in both the lower airway lavage samples and the upper airway oral washes. Although anelloviruses are not known to be pathogenic, they seem to be a marker of immunosuppression.18 The effects of their dysregulation on engraftment or outcome, if any, are not known at this time.

and healthy controls. This finding was true in both the lower airway lavage samples and the upper airway oral washes. Although anelloviruses are not known to be pathogenic, they seem to be a marker of immunosuppression.18 The effects of their dysregulation on engraftment or outcome, if any, are not known at this time. Emerging Respiratory Viruses Since the discovery of severe acute respiratory syndrome coronavirus in 2003,19 other novel respiratory pathogens have emerged, including coronaviruses NL63, HKU1, and MERS (reviewed in20) and influenza viruses H1N1 pandemic strain and H7N9.21 In each case, the molecular tools to adequately survey the spread of the virus had to be developed. Once assays become available, the transmission of these viruses can be tracked and recommendations for protecting public health can be made. HTS assays can be useful to survey outbreaks of emerging viruses. A recent example of this occurred in the fall of 2014, when the United States experienced a widespread outbreak of enterovirus D68 in 49 states and the District of Columbia. This virus had previously been observed rarely. In multiplex PCR panels, the virus was either undetected or broadly typed only as an enterovirus/rhinovirus, which limited the study of the outbreak. Typing of the virus initially involved a labor-intensive and slow method in which an amplicon was generated and sequenced, and the sequence was compared with reference strains for typing. The genomes of the outbreak strains were sequenced22(and also by the Centers for Disease Control and Prevention), and subsequently a highly specific molecular assay was developed in order to aid in detection and typing of the outbreak strain.23 In this case, sequencing provided specific viral typing and allowed for genomic characterization and design of a specific laboratory developed test. This model could be useful for future outbreaks.

equently a highly specific molecular assay was developed in order to aid in detection and typing of the outbreak strain.23 In this case, sequencing provided specific viral typing and allowed for genomic characterization and design of a specific laboratory developed test. This model could be useful for future outbreaks. High-throughput sequencing in the clinic Sequencing Assays Compared with Standard Clinical Tests The workhorses for clinical testing of respiratory viruses are multiplex PCR panels that target the most common respiratory viruses. These assays are highly sensitive and yield rapid results. However, PCR-based assays can have limitations. In most multiplex panels, the assays cannot be used to subtype viruses, identify drug-resistant alleles, or identify viruses not targeted by the panel. Evolving viruses may also mutate in the region targeted by the PCR primers and be missed by the assay. For these reasons, HTS-based assays could have a role in the clinic.

most multiplex panels, the assays cannot be used to subtype viruses, identify drug-resistant alleles, or identify viruses not targeted by the panel. Evolving viruses may also mutate in the region targeted by the PCR primers and be missed by the assay. For these reasons, HTS-based assays could have a role in the clinic. Recently, several groups have compared the results from HTS with standard clinical assays for detection of viruses in the respiratory tract. One study found that RNA sequencing and the GenMark eSensor Respiratory Virus Panel (RVP) had an 86% correlation rate on one set of 42 known positive nasopharyngeal swab samples and 93% correlation on a second set of 67 samples.24 High-throughput RNA sequencing detected 12 viruses that were either not included in the RVP panel or whose sequence was divergent from the RVP target and, thus, not detected. Furthermore, viral subtypes were determined for influenza A (as well as identification of the oseltamivir resistance mutation), respiratory syncytial viruses, and rhinoviruses. Another study tested 89 nasopharyngeal swabs from adults with upper respiratory tract infections using reverse transcription (RT)-PCR assays for a series of common viruses, including human rhinoviruses, coronaviruses, influenza viruses and others, and by RNA sequencing.25 The HTS assay had a sensitivity of 77% compared with the PCR assays. The viruses that were not detected by HTS had higher cycle threshold (Ct) values in the real-time RT-PCR assays, indicating there were lower levels of viral nucleic acid present in those samples. Again, HTS had the advantage of providing additional subtyping information, in this case for human enteroviruses, rhinoviruses, metapneumovirus, and respiratory syncytial virus. A third study demonstrated that HTS could be used to detect a pathogen (rhinovirus C) in a sample from a child with respiratory symptoms in which no virus had been detected by PCR.26 Taken together, these data show that in some cases HTS could be advantageous compared with PCR assays, but sensitivity of sequencing can be a limitation.

monstrated that HTS could be used to detect a pathogen (rhinovirus C) in a sample from a child with respiratory symptoms in which no virus had been detected by PCR.26 Taken together, these data show that in some cases HTS could be advantageous compared with PCR assays, but sensitivity of sequencing can be a limitation. Improving Sensitivity of High-Throughput Nucleic Acid Sequencing for Virus Detection The studies comparing clinical tests with HTS demonstrate that sequencing can add information to clinical assays in terms of typing viruses and detecting resistance mutations. Importantly, they illustrate that viruses not included in the PCR panels are sometimes present. In each case, despite the slightly different method used for sample collection and preparation and the different patient cohorts used, sensitivity for detection was an issue. Recently 2 groups developed an approach to enrich viral nucleic acids from a comprehensive set of viruses before sequencing.27, 28 This approach uses targeted sequence capture, a hybridization-based approach for selecting targets of interest. Probes or baits are made using target sequences, and these are hybridized to the nucleic acid in the sample of interest. The baits are then captured along with the sequences that hybridized to them and washed; the result is an enrichment of the target nucleic acids in the sequencing assays. These newly developed target-based enrichment strategies do not target specific viruses or viral families that are expected to be associated with a disease, but rather they include targets for all viruses that are known to infect vertebrates, allowing for a comprehensive screen of both expected and unexpected viruses in the same assay. This kind of approach greatly improves sequencing sensitivity, with the percentage of viral reads increasing from approximately 10 to approximately 10,000 fold using targeted sequence capture compared with standard HTS.27, 28 As a result, virus targeted sequence capture may be particularly useful in helping to diagnose infections where no virus has been detected by routine methods. Failures of routine tests occur because the virus has divergent sequence from the target in the PCR panel, the virus is an emerging infectious disease, or the virus is not one of the prominent causes of respiratory infection.

useful in helping to diagnose infections where no virus has been detected by routine methods. Failures of routine tests occur because the virus has divergent sequence from the target in the PCR panel, the virus is an emerging infectious disease, or the virus is not one of the prominent causes of respiratory infection. Host Response to Infection Interestingly, sensitive molecular methods currently used for diagnostics and many research studies demonstrate that viruses can frequently be detected in asymptomatic individuals. In the Utah study of 26 households mentioned earlier, bocaviruses and rhinoviruses could frequently be detected in asymptomatic children and adults.1 A Missouri study that used both PCR assays8 and HTS9 demonstrated that viral nucleic acid could be detected in nasopharyngeal swabs from asymptomatic children, in particular enteroviruses/rhinoviruses. It is necessary to appreciate that the detection of viral nucleic acid with molecular methods does not necessarily indicate the virus had infected the cell and/or was successfully replicating or that symptoms are necessarily resultant from the particular virus that was detected.

n particular enteroviruses/rhinoviruses. It is necessary to appreciate that the detection of viral nucleic acid with molecular methods does not necessarily indicate the virus had infected the cell and/or was successfully replicating or that symptoms are necessarily resultant from the particular virus that was detected. Additional information regarding the host response can be used to determine whether symptoms are caused by viral or bacterial pathogens (Fig. 2 ). Using a set of 30 samples from febrile children and 22 samples from afebrile controls, Hu and colleagues29 demonstrated that there were distinct host gene expression patterns in the blood that distinguished viral and bacterial infections; furthermore, symptomatic and asymptomatic infections could be clearly delineated. Similarly, in 118 adults with lower respiratory tract infections and 40 healthy controls, host gene expression in the blood could distinguish viral and bacterial infections with 95% sensitivity and 92% specificity.30 Bacterial-viral coinfections could also be distinguished. Tsalik and colleagues used publicly available microarray data to develop a host gene expression classifier that could distinguish bacterial, viral, and noninfectious illnesses with 87% accuracy.31 Both the Suarez and colleagues30 and Tsalik and colleagues studies showed that gene expression profiling performed better than procalcitonin. In the future, one might imagine that diagnostics may couple pathogen detection with host response to provide clinicians clear results that indicate whether there is a need for antibiotics in each case. In fact, approaches that couple pathogen detection and host response are being put forth as highly effective diagnostic approach; software tools are being developed to rapidly provide reports that may in the very near future be used by clinicians for diagnostic purposes.32 Fig. 2 Future diagnostics. In the future, respiratory tract infections may be diagnosed by merging pathogen detection (the current method for diagnostics) with host response measures that further define the cause of the symptoms (viral, bacterial, coinfections, not pathogenic). This merger will help clarify diagnoses and define appropriate treatment measures.

respiratory tract infections may be diagnosed by merging pathogen detection (the current method for diagnostics) with host response measures that further define the cause of the symptoms (viral, bacterial, coinfections, not pathogenic). This merger will help clarify diagnoses and define appropriate treatment measures. Summary We are beginning to define the scope of the human respiratory tract virome. The prevalence of individual viruses vary from study to study (see Table 1), likely due to differences in seasonality of sample collection, variation in local virus circulation, and methodological choices (eg, sample type, sample preparation). The studies reviewed here demonstrate that HTS and expanded panels of PCR assays can identify rare viral pathogens that might not be included in multiplex diagnostic panels or PCR panels in which only the most common pathogens or viruses of interest are selected. The relatively unbiased sequencing approach can also reveal viruses that may not directly cause respiratory illness but whose presence may impact the trajectory of illness through mechanisms we do not yet understand. Methodological improvements to virus detection will help us better define the respiratory tract virome and monitor outbreaks. In the future, clinical tests may include both pathogen detection and an assessment of host response in order to more clearly distinguish viral and bacterial infections. Disclosure: The author has nothing to disclose.

Key points • Infection and chronic obstructive pulmonary disease (COPD) can be regarded as comorbid conditions, because infections contribute the progression of COPD, and COPD alters the susceptibility and manifestations of lung infections. • The underlying mechanism of acute exacerbations of COPD is acquisition of new strains of bacteria and viruses. A complex host-pathogen interaction then determines the clinical manifestations and outcomes of such acquisition. • COPD predisposes to community-acquired pneumonia and alters its cause, treatment, and outcomes. • Several lines of evidence now suggest that chronic airway infection by bacteria is prevalent in COPD, and by triggering a chronic inflammatory response contributes to progression of disease. • Lung innate immune defenses are impaired in COPD, making these patients more susceptible to infection. Respiratory pathogens prevalent in COPD use various mechanisms to evade host responses and thereby cause acute and persistent infections.

• Several lines of evidence now suggest that chronic airway infection by bacteria is prevalent in COPD, and by triggering a chronic inflammatory response contributes to progression of disease. • Lung innate immune defenses are impaired in COPD, making these patients more susceptible to infection. Respiratory pathogens prevalent in COPD use various mechanisms to evade host responses and thereby cause acute and persistent infections. Introduction The role of infection in chronic obstructive pulmonary disease (COPD) was first postulated in 1953 by Stuart-Harris and colleagues1 in what is now known as the British hypothesis. They speculated that the decline in the lung function in COPD was the result of mucus hypersecretion and recurrent bacterial infections. In the next 2 decades, several studies were performed to confirm the hypothesis. In some of these studies, sputum microbiology was used to compare the rate of bacterial infection in patients with chronic bronchitis at baseline and during exacerbations, as well as in comparison with individuals without COPD.2, 3, 4, 5, 6, 7 Some differences in bacterial infection related to disease state were found; for example, Smith and colleagues2, 3, 8 found increased colonization with Haemophilus influenzae in patients with severe COPD compared with mild COPD. However, for the most part, differences in the rate of bacterial isolation from sputum at stable state (ie, colonization) versus at acute exacerbation (ie, infection) were not seen in these studies. Advanced molecular biology techniques to differentiate bacterial strains within species had not been developed and were therefore not available to these investigators. Other investigators examined this hypothesis by using serologic studies to determine levels of antibacterial antibodies in patients with chronic bronchitis. These results were also confusing and contradictory and were confounded by the use of laboratory strains as an antigen (discussed in Ref.9). In 1977, Fletcher and colleagues10 published a landmark study that showed that frequency of exacerbations and mucus hypersecretion did not result in faster decline of lung function in patients with COPD. By the early 1980s, because of these observations and the appreciation of the importance of tobacco smoke in COPD pathogenesis, the British hypothesis was rejected, and bacterial infection was relegated to an epiphenomenon in this disease.7

retion did not result in faster decline of lung function in patients with COPD. By the early 1980s, because of these observations and the appreciation of the importance of tobacco smoke in COPD pathogenesis, the British hypothesis was rejected, and bacterial infection was relegated to an epiphenomenon in this disease.7 The role of viral infection in COPD exacerbations was also extensively investigated in the 1960s and 1970s with viral cultures and serology at exacerbation.3, 5, 8 Because of the lack of confounding by chronic colonization and serologic cross reactivity, about 30% of exacerbations were confirmed to be of viral origin. Following 20 to –30 years of scant investigation, the role of infection has been revisited in the last 2 decades with new molecular biology, immunology, and microbiology techniques.11 Understanding of infection in COPD, both in the acute and chronic settings, has consequently developed substantially, as discussed later (Fig. 1 ).Fig. 1 Acute and chronic infection cycles in the pathogenesis of COPD.

revisited in the last 2 decades with new molecular biology, immunology, and microbiology techniques.11 Understanding of infection in COPD, both in the acute and chronic settings, has consequently developed substantially, as discussed later (Fig. 1 ).Fig. 1 Acute and chronic infection cycles in the pathogenesis of COPD. Acute infection Acute infections in COPD are clinically recognized either as exacerbations or as episodes of pneumonia. The differentiation between the two presentations is based on the presence (pneumonia) or absence (exacerbation) of lung parenchymal involvement, which presents as an infiltrate on chest radiology. Although pneumonia has been always considered to be a more significant acute infection, exacerbations occur with much greater frequency and also have serious consequences in COPD. As the British hypothesis was being largely discredited, the importance of exacerbations in COPD was also minimized. They came to be regarded as self-resolving viral illness of little consequence (chest colds) for which no specific therapy was available and that were part of the natural course of the disease. The last 2 decades have seen considerable revision in this point of view, because data have emerged that exacerbations do contribute to the loss of quality of life and lung function in COPD and account for as much as half the cost of care of COPD. Furthermore, bacterial infection contributes to exacerbations, specific therapies are of benefit, and prevention of exacerbations is possible and is an important therapeutic goal in COPD.

ons do contribute to the loss of quality of life and lung function in COPD and account for as much as half the cost of care of COPD. Furthermore, bacterial infection contributes to exacerbations, specific therapies are of benefit, and prevention of exacerbations is possible and is an important therapeutic goal in COPD. Causes of Exacerbations Exacerbations of COPD are airway inflammatory events that are induced by infection in most instances. The aggravating infection can be viral, bacterial, or a combination of viral and bacterial infections. Although there are episodes that are induced by poorly understood noninfectious factors, infections likely account for about 80% of exacerbations (Table 1 ).Table 1 Microbial pathogens in COPD

ction in most instances. The aggravating infection can be viral, bacterial, or a combination of viral and bacterial infections. Although there are episodes that are induced by poorly understood noninfectious factors, infections likely account for about 80% of exacerbations (Table 1 ).Table 1 Microbial pathogens in COPD Microbe Role in Exacerbations Role in Stable Disease Bacteria H influenzae 20%–30% of exacerbations Major pathogen Streptococcus pneumoniae 10%–15% of exacerbations Minor role Moraxella catarrhalis 10%–15% of exacerbations Minor role Pseudomonas aeruginosa 5%–10% of exacerbations, prevalent in advanced disease Likely important in advanced disease Enterobacteriaceae Isolated in advanced disease, pathogenic significance undefined Undefined Haemophilus haemolyticus Isolated frequently, unlikely cause Unlikely Haemophilus parainfluenzae Isolated frequently, unlikely cause Unlikely Staphylococcus aureus Isolated infrequently, unlikely cause Unlikely Viruses Rhinovirus 20%–25% of exacerbations Unlikely Parainfluenza 5%–10% of exacerbations Unlikely Influenza 5%–10% of exacerbations Unlikely Respiratory syncytial virus 5%–10% of exacerbations Controversial Coronavirus 5%–10% of exacerbations Unlikely Adenovirus 3%–5% of exacerbations Latent infection seen, pathogenic significance undefined Human metapneumovirus 3%–5% of exacerbations Unlikely Atypical Bacteria Chlamydophila pneumoniae 3%–5% of exacerbations Commonly detected, pathogenic significance undefined Mycoplasma pneumoniae 1%–2% Unlikely Fungi Pneumocystis jiroveci Undefined Commonly detected, pathogenic significance undefined From Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2356; with permission.

cted, pathogenic significance undefined Mycoplasma pneumoniae 1%–2% Unlikely Fungi Pneumocystis jiroveci Undefined Commonly detected, pathogenic significance undefined From Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2356; with permission. Virus The role of viruses in exacerbation was established in older studies (as discussed earlier) by viral culture and serology. Understanding of viral exacerbations has recently been expanded by the use of molecular diagnostic techniques and with the development of a human experimental model of rhinoviral exacerbations. The most common viruses detected in airway secretions at exacerbation are rhinovirus, influenza, respiratory syncytial virus (RSV), parainfluenza, and adenovirus. A recent systematic review found that viruses were detected in 34.1% of exacerbations.12 More recent studies using molecular detection of virus by polymerase chain reaction (PCR) techniques have found viruses in up to half of all exacerbations.13 The human experimental model of rhinoviral exacerbations was described in a study in which 13 subjects with COPD and 13 control subjects were nasally inoculated with a low dose of rhinovirus.14 An increased neutrophilic inflammatory response in the lower airway, and more prominent lower respiratory symptoms and airway obstruction, were found in COPD compared with controls. An impaired interferon response to the infection was seen in patients with COPD. This work confirms the viral causation of exacerbations and has provided insights into susceptibility and pathogenic mechanisms involved in viral exacerbations.

ry symptoms and airway obstruction, were found in COPD compared with controls. An impaired interferon response to the infection was seen in patients with COPD. This work confirms the viral causation of exacerbations and has provided insights into susceptibility and pathogenic mechanisms involved in viral exacerbations. Bacteria In contrast with the role of viruses, the role of bacteria as a cause of exacerbations has been controversial and was not fully appreciated until recently. At present, pathogens clearly implicated in COPD exacerbations are nontypeable H influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Pseudomonas aeruginosa. Whether Staphylococcus aureus and gram-negative enteric bacteria (Enterobacteriaceae), which are frequently isolated from sputum in COPD, are causative for exacerbations or are only capable of airway colonization is unclear at present.

uenzae, Streptococcus pneumoniae, Moraxella catarrhalis, and Pseudomonas aeruginosa. Whether Staphylococcus aureus and gram-negative enteric bacteria (Enterobacteriaceae), which are frequently isolated from sputum in COPD, are causative for exacerbations or are only capable of airway colonization is unclear at present. Previous studies that defined bacterial pathogens isolated from sputum only at a species level were unable to fully appreciate the dynamic nature of bacterial infection in COPD. In a longitudinal prospective cohort study in patients with COPD, when bacterial strains in sputum were characterized by molecular techniques, combined with a careful analysis of host immune and immunologic responses, an important mechanism that likely underlies exacerbations caused by the 4 major pathogens listed earlier was found (Fig. 2 ).15 The risk of having an exacerbation was increased by more than 2-fold with respiratory tract acquisition of strains of these bacterial pathogens that were new to the patient. In this initial study, 33% of the visits within a month of new strain acquisition were associated with an exacerbation compared with 15.4% without a new strain.16 Subsequent analyses from this study have now shown that the incidence of exacerbations at a visit with a new strain isolated from sputum is 40% to 50%, and that this holds true for each of the 4 major pathogens (nontypeable H influenzae, S pneumoniae, M catarrhalis, and P aeruginosa).17, 18, 19 Additional support for this mechanism for exacerbations comes from various observations. Exacerbation-associated strains of H influenzae are more inflammatory in in vitro and animal models than strains associated with colonization, showing that clinical implications of bacterial acquisition correlate with strain virulence.20 Strain-specific host immune response and a vigorous neutrophilic inflammatory response distinguish new strain exacerbations from those without new strains.21 Fig. 2 Proposed mechanism of bacterial exacerbations in COPD.

tion, showing that clinical implications of bacterial acquisition correlate with strain virulence.20 Strain-specific host immune response and a vigorous neutrophilic inflammatory response distinguish new strain exacerbations from those without new strains.21 Fig. 2 Proposed mechanism of bacterial exacerbations in COPD. (From Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2357; with permission.)

tion, showing that clinical implications of bacterial acquisition correlate with strain virulence.20 Strain-specific host immune response and a vigorous neutrophilic inflammatory response distinguish new strain exacerbations from those without new strains.21 Fig. 2 Proposed mechanism of bacterial exacerbations in COPD. (From Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2357; with permission.) Whether an increase in bacterial concentration (load) in the airway of a preexisting (colonizing) strain can be an additional independent mechanism of exacerbations is controversial. When bacterial sputum concentrations from our longitudinal cohort study were analyzed, either no differences or small differences were found between stable disease and exacerbation, and the small differences were no longer seen once new strain acquisition was taken into account.21 In contrast, Rosell and colleagues22 showed in pooled analysis of their data from bronchoscopic protected brush specimens that 54% of the patients with COPD exacerbation had pathogenic bacteria present in their airway secretions at significant concentrations compared with 29% of the patients with stable COPD. Intracellular H influenzae was found in bronchial mucosal biopsies in 87% of intubated patients with COPD exacerbation, compared with 33% of the patients with stable COPD.23 Garcha and colleagues,24 using quantitative PCR, found higher sputum bacterial loads at exacerbation than at stable state. However, these studies that have shown higher bacterial loads in sputum at exacerbation have not taken into account bacterial strain variation in their specimens.

f the patients with stable COPD.23 Garcha and colleagues,24 using quantitative PCR, found higher sputum bacterial loads at exacerbation than at stable state. However, these studies that have shown higher bacterial loads in sputum at exacerbation have not taken into account bacterial strain variation in their specimens. Coinfection with virus and bacteria A few recent studies have examined the impact of simultaneous or sequential bacterial and viral infection at exacerbation. Papi and colleagues25 examined 64 patients with COPD exacerbation requiring hospital admission; 25% had combined bacterial and viral infections, and these patients had more severe symptoms and longer hospitalization. Presence of cold symptoms and H influenzae in sputum has also been associated with more symptoms and a larger decrease in lung function than when either is present alone.26 In the rhinoviral human experimental model discussed earlier, as many as 60% of patients with COPD developed a secondary bacterial infection with a greater inflammatory response and duration of symptoms.27 However, the severity of the exacerbations was mild and none of the patients required steroids or antibiotics.

.26 In the rhinoviral human experimental model discussed earlier, as many as 60% of patients with COPD developed a secondary bacterial infection with a greater inflammatory response and duration of symptoms.27 However, the severity of the exacerbations was mild and none of the patients required steroids or antibiotics. It is likely that viral infection predisposes the susceptible host to bacterial coinfection and vice versa.28, 29 In cultured airway epithelial cells, Sajjan and colleagues29 found that infection with H influenzae increased expression of intercellular adhesion molecule (ICAM)-1 and Toll-like receptor (TLR)-3, receptors for rhinovirus and its double-stranded RNA. In contrast, in another experimental model, Avadhanula and colleagues28 found increased bacterial adhesion to respiratory epithelial cells after viral infection. In the human rhinovirus experimental model, degradation of antimicrobial peptides such as elafin by neutrophil elastase could explain the occurrence of secondary bacterial infection.27 In summary, bacterial and viral infections play a critical role in COPD exacerbations. Application of molecular diagnostic techniques to exacerbations is likely to further enhance understanding of infectious episodes. The role of opportunistic bacterial pathogens in causing exacerbations still needs to be defined.

It is likely that viral infection predisposes the susceptible host to bacterial coinfection and vice versa.28, 29 In cultured airway epithelial cells, Sajjan and colleagues29 found that infection with H influenzae increased expression of intercellular adhesion molecule (ICAM)-1 and Toll-like receptor (TLR)-3, receptors for rhinovirus and its double-stranded RNA. In contrast, in another experimental model, Avadhanula and colleagues28 found increased bacterial adhesion to respiratory epithelial cells after viral infection. In the human rhinovirus experimental model, degradation of antimicrobial peptides such as elafin by neutrophil elastase could explain the occurrence of secondary bacterial infection.27 In summary, bacterial and viral infections play a critical role in COPD exacerbations. Application of molecular diagnostic techniques to exacerbations is likely to further enhance understanding of infectious episodes. The role of opportunistic bacterial pathogens in causing exacerbations still needs to be defined. Community-acquired Pneumonia Epidemiology Community-acquired pneumonia (CAP) is a major cause of morbidity and mortality worldwide, with incidence of 2.6 to 11 per 1000 adults.30, 31 Mortality can reach 20%, with 14.9% of the mortality risk attributed to smoking, second only to age.32 In a multivariate analysis, COPD was an independent risk factor for developing severe CAP, with an odds ratio (OR) of 1.91.33 Evaluation of COPD subgroups revealed that severe COPD on home oxygen and severe COPD exacerbations requiring hospitalization were independent risk factors for developing CAP.34 Merino-Sanchez and colleagues35 observed a 12.6% incidence of pneumonia in 596 patients with COPD over 3 years, with 55% of the cases with Pneumonia Severity Index (PSI) of 4 and 5. The mortality for a PSI of 5 was 35.7%. In 2 European studies, hospitalized patients with CAP with and without COPD were compared. Although mortality differences between the groups were not seen, patients with COPD experienced more severe pneumonia, higher rates of readmission, and recurrent pneumonia. Lower serum tumor necrosis factor alpha (TNF-α) and interleukin-6 levels were seen in the COPD group, suggesting an impaired inflammatory response in these patients.36, 37 Higher mortality with CAP in COPD has been observed in other studies, reiterating the importance of early recognition and appropriate management in this high-risk population.38, 39, 40

pha (TNF-α) and interleukin-6 levels were seen in the COPD group, suggesting an impaired inflammatory response in these patients.36, 37 Higher mortality with CAP in COPD has been observed in other studies, reiterating the importance of early recognition and appropriate management in this high-risk population.38, 39, 40 Causes of CAP in COPD S pneumoniae remains the most common cause of CAP in COPD. However, because of alterations in the lung microbiome in COPD, pathogens such as H influenzae, M catarrhalis, and P aeruginosa may play a larger role in the development of CAP in these patients. Moreover, patients with COPD are exposed to frequent antibiotic courses and they are more likely to be infected with antibiotic-resistant pathogens, making empiric antibiotic choices challenging.41 In a study of hospitalized patients with COPD with CAP, more infections attributable to P aeruginosa were observed.42 However, the use of respiratory specimens to determine the microbiological cause of CAP in COPD is challenging, because chronic colonization with CAP-associated pathogens is common in COPD.

allenging.41 In a study of hospitalized patients with COPD with CAP, more infections attributable to P aeruginosa were observed.42 However, the use of respiratory specimens to determine the microbiological cause of CAP in COPD is challenging, because chronic colonization with CAP-associated pathogens is common in COPD. Role of inhaled corticosteroids Inhaled corticosteroids (ICS), in combination with long acting beta agonists (LABA), are widely used in COPD, and reduce the frequency of exacerbations and daily symptoms in these patients.43 However, the benefits come at a cost of increased risk of pneumonia. This increased risk was originally observed in the TORCH (Toward a Revolution in COPD Health) study, in which the ICS/LABA group had a higher probability (19.6%) of developing pneumonia over the course of 3 years.43 A recent meta-analysis of 24 randomized controlled trials of ICS in COPD confirmed these results with a calculated relative risk of developing pneumonia at 1.56, and a number needed to harm of 60.44, 45, 46 However, mortality was no different from the use of a LABA alone. The association between ICS use and pneumonia should be interpreted with caution. None of these trials were specifically designed to assess the risk of pneumonia, most episodes lack radiological confirmation, and COPD exacerbations may have been misdiagnosed as pneumonia. Mechanisms underlying this association have not been examined, but corticosteroid-induced impairment of local immune response to microbial pathogens is likely responsible.

igned to assess the risk of pneumonia, most episodes lack radiological confirmation, and COPD exacerbations may have been misdiagnosed as pneumonia. Mechanisms underlying this association have not been examined, but corticosteroid-induced impairment of local immune response to microbial pathogens is likely responsible. Antimicrobial therapy in COPD and CAP In outpatients with CAP, the presence of COPD as a comorbid condition places them in a high-risk group, and treatment with a respiratory fluoroquinolone or a β-lactam plus a macrolide is recommended.47 Monotherapy with a macrolide or doxycycline is not appropriate in these patients. Because antibiotic use is common in these patients, a review of antibiotic use in the previous 3 months should guide empiric choice, and antibiotic classes used in the previous 3 months should be avoided. Among inpatients with CAP and COPD, the same choices are applicable. However, in patients requiring intensive care admission, combination therapy is always recommended, with a β-lactam and a respiratory fluoroquinolone or a macrolide. If Pseudomonas is suspected (previous Pseudomonas isolation, bronchiectasis, malnutrition, recent broad-spectrum antibiotic exposure), an antipseudomonal regimen is recommended.

quiring intensive care admission, combination therapy is always recommended, with a β-lactam and a respiratory fluoroquinolone or a macrolide. If Pseudomonas is suspected (previous Pseudomonas isolation, bronchiectasis, malnutrition, recent broad-spectrum antibiotic exposure), an antipseudomonal regimen is recommended. Chronic infection In contrast with the (almost) sterile airways of a healthy lung, the lower airway of patients with COPD is frequently colonized with bacteria.48, 49 Although a wide variety of pathogens can be isolated, the two most common are H influenzae and P aeruginosa (see Table 1). Until recently, the presence of these bacteria was regarded as colonization, implying an innocuous process in the airway without sequelae. A growing body of evidence now suggests that this colonization in stable COPD, via complex interactions with the host immune-inflammatory system, could contribute to COPD pathogenesis and progression. Vicious-circle Hypothesis Similar to bronchiectasis and cystic fibrosis, the host-pathogen interaction in stable COPD is well described by the vicious-circle hypothesis (Fig. 3 ). Repeated insults to the lung, such as smoking and environmental exposures, lead to impairment of the host immune defenses, thus allowing bacterial colonization. The bacteria cause subclinical inflammatory response in the airway, resulting in further damage to the innate lung defense and persistence of chronic bacterial infection. This process accelerates during acute exacerbations.Fig. 3 The vicious-circle hypothesis of infection and inflammation in COPD.

cterial colonization. The bacteria cause subclinical inflammatory response in the airway, resulting in further damage to the innate lung defense and persistence of chronic bacterial infection. This process accelerates during acute exacerbations.Fig. 3 The vicious-circle hypothesis of infection and inflammation in COPD. (From Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2361; with permission.)

cterial colonization. The bacteria cause subclinical inflammatory response in the airway, resulting in further damage to the innate lung defense and persistence of chronic bacterial infection. This process accelerates during acute exacerbations.Fig. 3 The vicious-circle hypothesis of infection and inflammation in COPD. (From Sethi S, Murphy TF. Infection in the pathogenesis and course of chronic obstructive pulmonary disease. N Engl J Med 2008;359:2361; with permission.) Evidence to Support Chronic Infection Colonization is defined by the absence of damaging effects to the host related to the presence of a pathogen and the absence of a specific immune response. There are several parts of the body where such colonization is seen (eg, skin, colon) and is essential for health. The microbial pathogens that colonize these surfaces constitute a microbiome. Recent advances in research technologies, especially high-throughput genomic characterization, have made it possible to characterize the microbiome. The healthy lung is sterile by standard culture techniques. Even with molecular techniques, the microbiome of the healthy lung is sparse and transient, composed primarily of oral flora that are microaspirated and cleared.50 In contrast, in a third of patients with COPD, potential respiratory pathogens can be retrieved by culture of lower airway samples.49, 51 An abundant microbiome can be found by molecular techniques in COPD lungs.52 Unlike other body surfaces, like the skin and gut mucosa, the lung is not well equipped to handle a microbiome. Therefore, microbial presence in the lower airways in COPD is harmful.

s can be retrieved by culture of lower airway samples.49, 51 An abundant microbiome can be found by molecular techniques in COPD lungs.52 Unlike other body surfaces, like the skin and gut mucosa, the lung is not well equipped to handle a microbiome. Therefore, microbial presence in the lower airways in COPD is harmful. Several studies have described excess inflammation in stable patients with COPD when colonized with bacterial pathogens.51, 53, 54, 55, 56 The airway inflammation associated with bacterial colonization is predominantly neutrophilic. Studies comparing sputum samples from colonized and noncolonized patients have found higher levels of TNF-α, interleukin-8, interleukin-6, leukotriene B4, neutrophil myeloperoxidase, and elastase, and lower levels of the antiprotease secretory leukocyte protease inhibitor.53, 54, 56, 57 Bronchoscopic sampling of the lower airway with bronchoalveolar lavage showed increased levels of neutrophils, TNF-α, interleukin-8, matrix metalloproteinase 9, and endotoxin in association with bacterial colonization.49, 51, 58 Although several mediators contribute to COPD pathogenesis, interleukin-8 in particular has been associated with increased exacerbation frequency, longer recovery periods, worsening airway obstruction, and development of bronchiectasis.58, 59 Bacterial (M catarrhalis) acquisition, even without an increase in symptoms of an exacerbation, has been associated with increases in proteolytic activity and a reduction in antiproteolytic defense, resulting in worsening of the protease/antiprotease imbalance that is thought to cause progressive lung damage in COPD.56 The inflammatory profile seen with bacterial colonization is similar to that seen with bacterial exacerbations, implying that colonization is a low-grade infection.

ction in antiproteolytic defense, resulting in worsening of the protease/antiprotease imbalance that is thought to cause progressive lung damage in COPD.56 The inflammatory profile seen with bacterial colonization is similar to that seen with bacterial exacerbations, implying that colonization is a low-grade infection. Following exacerbations of COPD, specific immune responses, both systemic and mucosal, to the infecting strain are often observed. Similar observations have now been described following colonization.18, 19 This active immune response supports the presence of chronic infection in COPD. Furthermore, with M catarrhalis, a differential immune response is seen with colonization, which is accompanied by a stronger mucosal immune response, compared with a stronger systemic immune response accompanying exacerbations.18 Whether the nature of the immune response dictates the clinical expression of infection or vice versa is not clear.

halis, a differential immune response is seen with colonization, which is accompanied by a stronger mucosal immune response, compared with a stronger systemic immune response accompanying exacerbations.18 Whether the nature of the immune response dictates the clinical expression of infection or vice versa is not clear. The potential contribution of viral and atypical pathogens to chronic infection in COPD has been controversial. Latent adenoviral infection of the lungs, in the form of integration of portions of adenoviral DNA into cellular DNA, was found to enhance the inflammatory response to tobacco smoke, and thereby was thought to contribute to COPD pathogenesis.60 Initial studies showed that such adenoviral integration was more common in COPD than in controls; however, subsequent studies have not supported this observation. Latent RSV infection has been described in COPD by one group of investigators, but has not been found by others.61, 62 The presence and contribution of chronic chlamydial lung infection in COPD remains similarly controversial, with inconsistent observations from various investigators.

ve not supported this observation. Latent RSV infection has been described in COPD by one group of investigators, but has not been found by others.61, 62 The presence and contribution of chronic chlamydial lung infection in COPD remains similarly controversial, with inconsistent observations from various investigators. Besides the direct microbiological evidence of infection and its consequences, other indirect lines of evidence of chronic infection in COPD have emerged from radiological and pathologic studies. In a pathologic study of small airways of patients with COPD, the extent of formation of lymphoid aggregates predominantly composed of B cells had the best correlation with the degree of airflow obstruction.63 It is likely that these aggregates represent a local host immune response to chronic microbial infection. Furthermore, this pathologic finding was replicated in a mouse model of chronic inflammation in the lungs induced by repeated instillation of nontypeable H influenzae lysate.64 Widespread use of high resolution computed tomography scans has revealed that bronchiectasis develops in a substantial proportion of patients with COPD. In a comprehensive study of 92 patients with stable moderate or severe disease, 57.5% had bronchiectasis, and its presence was related to worse lung function, hospital admission in the past year, and chronic bronchitic symptoms.65 Repeated sputum cultures in these patients linked chronic colonization with potential bacterial pathogens (predominantly nontypeable H influenzae and P aeruginosa) with the presence of bronchiectasis.65

presence was related to worse lung function, hospital admission in the past year, and chronic bronchitic symptoms.65 Repeated sputum cultures in these patients linked chronic colonization with potential bacterial pathogens (predominantly nontypeable H influenzae and P aeruginosa) with the presence of bronchiectasis.65 In summary, these various lines of investigation support the paradigm of a vicious circle of infection and inflammation in COPD. However, COPD is a heterogeneous disease and it is likely that in 30% to 50% of these patients chronic infection plays a prominent role, these being the ones with chronic colonization, bronchiectasis, and/or chronic bronchitis. Future longitudinal natural history studies or studies with interventions that decrease bacterial colonization and measure disease progression are needed to prove the vicious-circle hypothesis. Mechanism of Increased Susceptibility to Infection in COPD Although infection is a comorbid condition in COPD and much has been learned about the incidence and consequences of infection in this disease, understanding of the mechanisms underlying increased susceptibility to infection seen in COPD is still in its early stages. Alterations in both the host and pathogen can contribute to establishment of acute and chronic infections, and both play a role in COPD. Disruptions in the host that increase susceptibility to infection can be categorized into changes in innate or adaptive lung defense. Pathogen alterations include host defense evasion mechanisms.

in both the host and pathogen can contribute to establishment of acute and chronic infections, and both play a role in COPD. Disruptions in the host that increase susceptibility to infection can be categorized into changes in innate or adaptive lung defense. Pathogen alterations include host defense evasion mechanisms. Host Defects: Innate Immunity The healthy lung possesses a multilayered, redundant, and highly efficient innate defense system that allows it to maintain an almost pathogen-free environment in spite of being constantly exposed to a variety of microbes through inhalation and microaspiration. This innate immune system of the lung has 3 components: mechanical barrier, humoral, and cellular response systems. This nonspecific immunity is the first line of defense against viruses, bacteria, and other particulates. It recognizes antigenic ligands entering the airway via pattern-recognition receptors and triggers series of responses resulting in complement activation and phagocytosis. The end result is elimination of the antigen or its presentation on the surface of the macrophages and activation of the adaptive immunity. In patients with COPD, several innate responses are impaired, leading to increased susceptibility to infection.

es of responses resulting in complement activation and phagocytosis. The end result is elimination of the antigen or its presentation on the surface of the macrophages and activation of the adaptive immunity. In patients with COPD, several innate responses are impaired, leading to increased susceptibility to infection. Mucociliary clearance The mucociliary clearance is the first barrier to noxious agents, by effectively trapping and clearing inhaled and microaspirated microbial pathogens. Both normal mucus and a normal ciliary apparatus are required for effective mucociliary clearance. Abnormal mucus (such as in cystic fibrosis) and a dysfunctional ciliary apparatus (such as in ciliary dyskinesia) are associated with acute and chronic bronchial infection. Augmented mucus production can be regarded as a defensive response to particulate or microbial exposure. However, when the exposure is chronic, and inflammation and ciliary dysfunction are also present, it could worsen mucociliary clearance.

ciliary dyskinesia) are associated with acute and chronic bronchial infection. Augmented mucus production can be regarded as a defensive response to particulate or microbial exposure. However, when the exposure is chronic, and inflammation and ciliary dysfunction are also present, it could worsen mucociliary clearance. Smoking disrupts mucociliary clearance, not only by augmenting mucus production but also by inducing structural abnormalities in the ciliary apparatus.66 Studies in moderate to heavy smokers have shown longer lung clearance times, although the degree of impairment is variable.67, 68 Further deterioration in mucociliary clearance is seen with development of chronic bronchitis and airway obstruction in smokers.69, 70, 71 Patients with COPD have hypertrophy and hyperplasia of their airway goblet cells and increased mucus stores.72, 73 In tissue and animal models, exposure to S pneumoniae and H influenzae results in further upregulation of mucin production.74, 75 Neutrophilic inflammation also worsens mucociliary function, mediated by increased mucus production, reduced ciliary beating, and altered viscoelastic properties of mucus.

ucus stores.72, 73 In tissue and animal models, exposure to S pneumoniae and H influenzae results in further upregulation of mucin production.74, 75 Neutrophilic inflammation also worsens mucociliary function, mediated by increased mucus production, reduced ciliary beating, and altered viscoelastic properties of mucus. Immunoglobulin A Immunoglobulin (Ig) A, especially polymeric secretory IgA, plays an important role in innate defense by coating the bacterial pathogen, thereby interfering with its ability to interact with the mucosal surface (immune exclusion). IgA can also neutralize infectious agents and could act as an opsonin assisting in pathogen elimination. Localized areas of IgA deficiency in the large and small airways are seen in COPD that were associated with squamous metaplasia. Polymeric IgG receptor expression, a receptor required for transcytosis of the IgA molecule from the basolateral to the apical surface of the epithelial cell, was reduced in these areas.76 These changes in IgA could be an important mechanism of infection susceptibility in COPD.

were associated with squamous metaplasia. Polymeric IgG receptor expression, a receptor required for transcytosis of the IgA molecule from the basolateral to the apical surface of the epithelial cell, was reduced in these areas.76 These changes in IgA could be an important mechanism of infection susceptibility in COPD. Antimicrobial peptides Antimicrobial polypeptides abundant in the airway surface lining fluid have antimicrobial and immunoregulatory functions. One major group, the cationic polypeptides, includes lysozyme, lactoferrin, defensins, the cathelicidins (LL-37), and secretory leukocyte protease inhibitor (SLPI).77, 78, 79, 80, 81, 82 Another important group, the collectins, include surfactant protein-A (SP-A), surfactant protein-D (SP-D), and mannose-binding lectin.83 Complex and dynamic alterations in various antimicrobial polypeptides have been described in COPD, both in the stable state and during exacerbations.

tor (SLPI).77, 78, 79, 80, 81, 82 Another important group, the collectins, include surfactant protein-A (SP-A), surfactant protein-D (SP-D), and mannose-binding lectin.83 Complex and dynamic alterations in various antimicrobial polypeptides have been described in COPD, both in the stable state and during exacerbations. Deficiencies of SLPI and lysozyme in the stable state have been associated with more frequent exacerbations.84, 85 Decreased serum mannose-binding lectin has been linked with exacerbation frequency in COPD, but this has not been a consistent observation.86 Lower airway concentrations of SP-A and SP-D are seen in smokers, with further decreases in association with emphysema.87, 88 Lower levels of beta-defensin 2 and Clara Cell Protein 16 (CC16) and increased levels of elafin and SLPI in sputum supernatants in stable COPD have been observed.89 Decreased levels of beta-defensin 2 in the central airways, but not in the distal airways, of smokers with COPD were found in a study of resected lung specimens.90 Dynamic changes in antimicrobial peptides have also been described with exacerbations of COPD. SLPI levels decrease significantly at the time of such exacerbations, which return to baseline after resolution.78 Lysozyme and lactoferrin levels decrease and LL-37 levels increase with both colonization and infective exacerbations with H influenzae and M catarrhalis.78 In a human model of rhinoviral infection, impaired elafin and SLPI responses following rhinoviral infection were associated with secondary bacterial infection.27

8 Lysozyme and lactoferrin levels decrease and LL-37 levels increase with both colonization and infective exacerbations with H influenzae and M catarrhalis.78 In a human model of rhinoviral infection, impaired elafin and SLPI responses following rhinoviral infection were associated with secondary bacterial infection.27 Macrophage function Key cellular components on innate lung defense are alveolar macrophages and airway epithelial cells. Phagocytic and cytokine responses of alveolar macrophages to bacterial pathogens are crucial for dealing with small pathogen inocula, without invoking potentially damaging inflammatory and adaptive immune responses. Alveolar macrophages from patients with COPD show impaired ability to phagocytose H influenzae and M catarrhalis, but not S pneumoniae or inert microspheres. This impairment is correlated with worsening lung function (Fig. 4 ).91 Alveolar macrophages from patients with COPD also have a less robust cytokine response to bacterial proteins, specifically outer membrane protein P6 and lipo-oligosaccharide (endotoxin) of H influenzae.92, 93, 94 Following exposure to rhinovirus, alveolar macrophages showed decreased cytokine responses to bacterial lipopolysaccharide and lipoteichoic acid,95 which could explain the increased susceptibility to bacterial infection after viral infection in COPD.Fig. 4 Comparison of phagocytosis of nontypeable H influenzae (A), Moraxella catarrhalis (B), Streptococcus pneumoniae (C), and latex microspheres (D) by human alveolar macrophages from healthy controls, current smokers with COPD, and ex-smokers with COPD.

eptibility to bacterial infection after viral infection in COPD.Fig. 4 Comparison of phagocytosis of nontypeable H influenzae (A), Moraxella catarrhalis (B), Streptococcus pneumoniae (C), and latex microspheres (D) by human alveolar macrophages from healthy controls, current smokers with COPD, and ex-smokers with COPD. (Modified from Berenson CS, Kruzel RL, Eberhardt E, et al. Phagocytic dysfunction of human alveolar macrophages and severity of chronic obstructive pulmonary disease. J Infect Dis 2013; with permission.) These decrements in macrophage function are likely secondary to several mechanisms, including reduction in pattern-recognition receptors such as Toll-like receptors TLR2 and TLR4, reduction in scavenger receptors such as macrophage receptor with collagenous structure (MARCO), or alteration in subpopulations of macrophages in the airway.96, 97, 98 TLR2 has been found to be downregulated in smokers, patients with COPD, and farmers exposed to organic dust.96, 99, 100 TLR4 downregulation is associated with the development of emphysema and worse airflow limitation in smokers.101 Polymorphism T399I of the TLR4 gene has been associated with development of COPD phenotype in smokers.102 Pathogen Mechanisms Tissue invasion

These decrements in macrophage function are likely secondary to several mechanisms, including reduction in pattern-recognition receptors such as Toll-like receptors TLR2 and TLR4, reduction in scavenger receptors such as macrophage receptor with collagenous structure (MARCO), or alteration in subpopulations of macrophages in the airway.96, 97, 98 TLR2 has been found to be downregulated in smokers, patients with COPD, and farmers exposed to organic dust.96, 99, 100 TLR4 downregulation is associated with the development of emphysema and worse airflow limitation in smokers.101 Polymorphism T399I of the TLR4 gene has been associated with development of COPD phenotype in smokers.102 Pathogen Mechanisms Tissue invasion H influenzae was traditionally regarded as an extracellular pathogen. However, molecular detection techniques have shown this pathogen in the bronchial epithelium and inside subepithelial macrophages in COPD.23, 48 These tissue bacteria could be shielded from the actions of antibiotics and antibodies, and therefore could be more resistant to eradication. Molecular detection often detects H influenzae in airway secretions and lung samples when cultures are negative, which could be explained by such tissue invasion.

in COPD.23, 48 These tissue bacteria could be shielded from the actions of antibiotics and antibodies, and therefore could be more resistant to eradication. Molecular detection often detects H influenzae in airway secretions and lung samples when cultures are negative, which could be explained by such tissue invasion. Biofilm formation Biofilms are bacteria encased with an extracellular matrix, which is usually composed of polysaccharides produced and secreted by bacteria. Bacteria in the core of the film, which is predominantly anaerobic, are in a low metabolic state. Antibiotic penetration into biofilms is limited, requiring up to 1000 times higher concentrations to achieve eradication.103 Parts of the biofilm can detach and cause distant infection. Pathogens common in COPD, including H influenzae, M catarrhalis, and P aeruginosa, are capable of biofilm formation. Furthermore, smoke exposure has been shown to increase biofilm formation.104 P aeruginosa in cystic fibrosis is the prototypical example of biofilm formation as a mechanism of persistence in the lung. Whether bacterial biofilms are present in COPD airways is not yet known.105 Mucoid P aeruginosa and some strains of H influenzae persist clinically for long periods in spite of repeated antibiotic exposure, which is reminiscent of cystic fibrosis.

example of biofilm formation as a mechanism of persistence in the lung. Whether bacterial biofilms are present in COPD airways is not yet known.105 Mucoid P aeruginosa and some strains of H influenzae persist clinically for long periods in spite of repeated antibiotic exposure, which is reminiscent of cystic fibrosis. Antigenic alteration Pathogens can evade the host immune response by alteration of their surface proteins, which are targets of the host immune system. The P2 outer membrane protein of H influenzae, which is a major target of bactericidal antibodies in COPD, shows extensive antigenic variation among strains of this pathogen.106 Serial persistent isolates of H influenzae in COPD show diminution of high-molecular-weight adhesin expression, which could represent another immune evasion mechanism.107 Future directions The role of infection in COPD is an evolving topic with extensive ongoing research trying to better understand host-pathogen interactions and find suitable targets for intervention. Although exacerbation pathogenesis is better understood now, much still needs to be learned about pathogen virulence and causal overlap. The vicious-circle hypothesis exposes the complex interactions between smoking, innate immunity, and respiratory pathogens. Augmentation and modulation of the innate and adaptive host immunity as well as formulation of novel antibacterial agents and vaccines are paramount in future research and development in COPD. Funding Sources: S. Sethi, supported by VA Merit Review and NHLBI. K. Rangelov, Nil.

Future directions The role of infection in COPD is an evolving topic with extensive ongoing research trying to better understand host-pathogen interactions and find suitable targets for intervention. Although exacerbation pathogenesis is better understood now, much still needs to be learned about pathogen virulence and causal overlap. The vicious-circle hypothesis exposes the complex interactions between smoking, innate immunity, and respiratory pathogens. Augmentation and modulation of the innate and adaptive host immunity as well as formulation of novel antibacterial agents and vaccines are paramount in future research and development in COPD. Funding Sources: S. Sethi, supported by VA Merit Review and NHLBI. K. Rangelov, Nil. Conflict of Interest: The authors declare no conflict of interest.

Key points • Viral pneumonia is a common pulmonary complication in patients with HCT/HM and is associated with high morbidity and mortality. • Because of nonspecific imaging findings, and high rates of coinfection with other viral, bacterial, and fungal pathogens, microbiologic diagnosis generally requires bronchoalveolar lavage with samples sent for culture, direct fluorescent antibody and nucleic acid testing as available. • CMV remains the most common cause of viral pneumonia in HCT/HM, but adoption of preemptive therapy strategies and changes in transplant techniques over the last few decades have resulted in significant improvement in the incidence and mortality associated with CMV pneumonia. • Community respiratory virus (CRV) infections, such as influenza, parainfluenza, and respiratory syncytial virus, are common. Fewer patients develop lower tract disease; however, once established, mortality rates are high. • Infection prevention practices in the community and health care setting are critical in limiting the acquisition and spread of CRVs in this highly susceptible patient population.

• Community respiratory virus (CRV) infections, such as influenza, parainfluenza, and respiratory syncytial virus, are common. Fewer patients develop lower tract disease; however, once established, mortality rates are high. • Infection prevention practices in the community and health care setting are critical in limiting the acquisition and spread of CRVs in this highly susceptible patient population. Introduction Patients undergoing hematopoietic cell transplantation (HCT) or treatment of hematologic malignancy (HM) have profound impairment of cell-mediated and humoral immunity. As such they are at risk of lower respiratory tract infection from reactivation of latent infections, such as cytomegalovirus (CMV), and progression of community-acquired upper respiratory tract infections, such as respiratory syncytial virus (RSV) influenza A and B. The clinical presentation of these infections is varied, and diagnosis is often complicated by high rates of coinfection with bacterial, fungal, and other viral pathogens (Table 1 ).1, 2, 3, 4 This article reviews the epidemiology of the major viral pathogens for pneumonia in patients with HCT/HM with discussion of evidence-based prevention strategies and treatments.Table 1 Incidence of infection, incidence of pneumonia, associated mortality rates, and treatments of most common causes of viral pneumonia in patients with HCT or HM

he epidemiology of the major viral pathogens for pneumonia in patients with HCT/HM with discussion of evidence-based prevention strategies and treatments.Table 1 Incidence of infection, incidence of pneumonia, associated mortality rates, and treatments of most common causes of viral pneumonia in patients with HCT or HM Virus Incidence of Infection Progression to Pneumonia Mortality Treatment Cytomegalovirus 50%–90% seroprevalence 1%–8% after allogeneic HCT with pre-emptive therapy 1%–5% other populations with no surveillance 60%–80% Ganciclovir or foscarnet Influenza A and B (FluA and FluB) 33% of symptomatic patients 14%–30% 15%–28% Oseltamivir or other neuraminidase inhibitors Respiratory syncytial virus 14%–30% of symptomatic patients 40%–75% 28%–55% No direct-acting therapy; inhaled ribavirin most studied Parainfluenza virus 1%–10% of all patients 30% 17%–46% None currently licensed; DAS-181 in phase III trials Adenovirus 8%–17% after allogeneic HCT, 6% after autologous ∼8% N/A Cidofovir Diagnosis of viral pneumonia Clinical Presentation Presenting signs and symptoms of viral pneumonia are variable. Most patients have fever and cough, with hypoxia and increased work of breathing of varying degrees depending on the extent of the infection. Upper respiratory tract infection symptoms, such as nasal congestion, rhinorrhea, sinusitis, myalgias, and fatigue, may be present for infections caused by community respiratory viruses (CRVs).

ave fever and cough, with hypoxia and increased work of breathing of varying degrees depending on the extent of the infection. Upper respiratory tract infection symptoms, such as nasal congestion, rhinorrhea, sinusitis, myalgias, and fatigue, may be present for infections caused by community respiratory viruses (CRVs). Imaging Computed tomography (CT) is helpful in distinguishing between infectious and noninfectious causes of lung disease in this patient population and also between viral and fungal or bacterial infections.5, 6 Viral pneumonias are similar in appearance on CT often demonstrating small centrilobular nodules, patchy bilateral areas of ground glass opacities and consolidation, bronchial wall thickening, and tree-in-bud opacities (Fig. 1 ). If there is significant bronchiolitis, air trapping can also be evident.5, 6, 7, 8, 9, 10 Fig. 1 (A) Transverse and coronal views of CT imaging for a patient with HCT with adenovirus and rhinovirus pneumonia, demonstrating patchy bilateral infiltrates with ground glass opacities and tree-in-bud pattern. (B) Transverse and coronal view of CT imaging for a patient with HCT with pneumonia caused by parainfluenza virus type 3. Note scattered nodular infiltrates with ground glass opacities.

h adenovirus and rhinovirus pneumonia, demonstrating patchy bilateral infiltrates with ground glass opacities and tree-in-bud pattern. (B) Transverse and coronal view of CT imaging for a patient with HCT with pneumonia caused by parainfluenza virus type 3. Note scattered nodular infiltrates with ground glass opacities. Diagnostic Sampling Fiberoptic bronchoscopy with bronchoalveolar lavage is the predominant sampling method to confirm a diagnosis of viral pneumonia in the HCT/HM population. Although sampling of the upper respiratory tract with nasopharyngeal aspirate/wash may provide an early identification of the involved virus or viruses, sampling of the lower tract is usually recommended to confirm the diagnosis and to exclude other copathogens. Because of the risk of hemorrhage in these patients who often have thrombocytopenia, endobronchial biopsy is usually avoided. Surgical lung biopsy, which was once the principle method by which lung abnormalities were evaluated after HCT, is now rarely performed.11

confirm the diagnosis and to exclude other copathogens. Because of the risk of hemorrhage in these patients who often have thrombocytopenia, endobronchial biopsy is usually avoided. Surgical lung biopsy, which was once the principle method by which lung abnormalities were evaluated after HCT, is now rarely performed.11 Virologic Diagnosis Standard viral cultures are of waning utility in the diagnosis of viral pneumonias because it can take up to 2 weeks to become positive and several more recently identified respiratory viruses, such as human metapneumovirus, coronaviruses, and bocavirus, are notoriously difficult to isolate in culture. For most viral pathogens, molecular methods of viral detection, such as direct or indirect fluorescent antibody tests or nucleic acid tests, can be used to give reliable results with a rapid turnaround time. Multiplex polymerase chain reaction (PCR) panels have the advantage of being able to test for multiple viruses at the same time and are more sensitive than fluorescent antibody tests.12, 13, 14, 15, 16, 17

uorescent antibody tests or nucleic acid tests, can be used to give reliable results with a rapid turnaround time. Multiplex polymerase chain reaction (PCR) panels have the advantage of being able to test for multiple viruses at the same time and are more sensitive than fluorescent antibody tests.12, 13, 14, 15, 16, 17 The diagnosis of CMV pneumonia, however, continues to rely on the use of standard viral or shell vial culture, histopathology, or immunohistochemical testing.18 The assumption has been that the CMV PCR tests would be too sensitive and have a low positive predictive value for CMV pneumonitis.19, 20, 21, 22 However, because of the operational advantages of PCR testing with much faster turnaround time, efforts are underway to estimate a quantitative CMV viral load threshold that would be more predictive of CMV pneumonitis rather than asymptomatic shedding.

a low positive predictive value for CMV pneumonitis.19, 20, 21, 22 However, because of the operational advantages of PCR testing with much faster turnaround time, efforts are underway to estimate a quantitative CMV viral load threshold that would be more predictive of CMV pneumonitis rather than asymptomatic shedding. Cytomegalovirus Epidemiology CMV is the most common cause of viral pneumonia after allogeneic HCT. Early reports indicated an incidence of 20% to 70% with an associated mortality of 85% to 90%.23, 24, 25 The development of ganciclovir resulted in significant improvements in the mortality associated with CMV pneumonia but with mortality rates still 60% to 80% focus shifted to prevention of disease.26, 27, 28, 29 The use of ganciclovir for prophylaxis decreased the incidence of pneumonitis and other CMV end-organ disease, but was associated with increased rates of neutropenia and late-occurring disease (ie, after Day 100 posttransplant).25, 30, 31, 32, 33 In the current era of preemptive therapy, where patients are monitored for CMV replication with either pp65 antigen or CMV DNA PCR in the blood or plasma and antiviral treatment is initiated before the development of CMV pneumonia, the incidence of CMV pneumonia is now only 1% to 3% in the early posttransplant period (100 days posttransplant).34, 35, 36 An additional 1% to 8% of patients develop CMV pneumonia within the first year after transplant.36, 37, 38

d or plasma and antiviral treatment is initiated before the development of CMV pneumonia, the incidence of CMV pneumonia is now only 1% to 3% in the early posttransplant period (100 days posttransplant).34, 35, 36 An additional 1% to 8% of patients develop CMV pneumonia within the first year after transplant.36, 37, 38 Risk factors for development of CMV pneumonia after allogeneic HCT are CMV seropositivity, recipient of a cord blood graft, HLA-mismatched donors, myeloablative conditioning regimens, acute and chronic graft-versus-host disease (GVHD), and use of T-cell-depleted stem cells.34, 36, 39, 40, 41, 42, 43 CMV pneumonia is much less common in patients who have received an autologous transplant, or in patients receiving treatment of HM with incidence of 1% to 5% reported in the absence of surveillance and preemptive therapy.44, 45, 46, 47, 48 Treatment Ganciclovir (5 mg/kg intravenous [IV] every 12 hours) remains the first-line treatment for CMV pneumonitis.49 Based on the results of three nonrandomized studies,50, 51, 52 CMV immunoglobulin was often recommended as adjunctive treatment. However, more recent analyses have called into question the additional benefit of this therapy.29, 47, 53, 54 Duration of treatment is generally induction therapy for 21 to 28 days, followed by 21 to 28 days of maintenance therapy (ganciclovir, 5 mg/kg IV every 24 hours). Foscarnet (90 mg/kg IV every 12 hours) may be used in the setting of neutropenia because it is associated with less bone marrow suppression than ganciclovir, but commonly causes significant nephrotoxicity.

rapy for 21 to 28 days, followed by 21 to 28 days of maintenance therapy (ganciclovir, 5 mg/kg IV every 24 hours). Foscarnet (90 mg/kg IV every 12 hours) may be used in the setting of neutropenia because it is associated with less bone marrow suppression than ganciclovir, but commonly causes significant nephrotoxicity. Antiviral resistance mutations have been identified in the viral encoded UL97 kinase, required only by ganciclovir, and the viral DNA polymerase, the target of ganciclovir, foscarnet, and cidofovir.55 Fortunately, antiviral resistance is a rare occurrence in patients with HCT/HM occurring in 0% to 4% of patients with CMV reactivation.56, 57, 58 Foscarnet, cidofovir, and brincidofovir (an oral nucleotide analogue and prodrug of cidofovir) could be used for treatment of resistant CMV caused by UL97 mutations. Maribavir and letermovir, two agents currently undergoing clinical trials, have distinct mechanisms of action that may make them useful for treatment of resistant CMV disease; however, little is known of their genetic barrier to resistance.59, 60, 61, 62 Other Herpesviruses Reactivation of latent varicella zoster virus and herpes simplex virus in immunocompromised patients can result in disseminated disease with pneumonia and was a significant clinical problem for patients with HCT/HM.63, 64 Long-term prophylaxis with acyclovir or valacyclovir has been the standard of care for more than a decade.65, 66, 67, 68 Cases still rarely occur in patients who have discontinued acyclovir prophylaxis, but generally respond well to high-dose parenteral acyclovir.69, 70, 71

nical problem for patients with HCT/HM.63, 64 Long-term prophylaxis with acyclovir or valacyclovir has been the standard of care for more than a decade.65, 66, 67, 68 Cases still rarely occur in patients who have discontinued acyclovir prophylaxis, but generally respond well to high-dose parenteral acyclovir.69, 70, 71 Community respiratory viruses CRVs are a common cause of infection in patients with HCT/HM; however, the risk of pneumonia varies by virus type and patient risk factors. Several of the viruses, such as influenza and RSV, have significant seasonal variation of incidence, whereas others, such as parainfluenza virus (PIV) and adenovirus, tend to cause disease year round. Outbreaks on oncology and HCT hospital wards and ambulatory clinics have been described for many of these viruses,72, 73, 74, 75, 76 emphasizing the importance of infection-prevention policies and procedures that can prevent the transmission of viruses among highly susceptible patients. This is particularly challenging with this patient population because of prolonged viral shedding, which often lasts weeks or months.77, 78, 79

es,72, 73, 74, 75, 76 emphasizing the importance of infection-prevention policies and procedures that can prevent the transmission of viruses among highly susceptible patients. This is particularly challenging with this patient population because of prolonged viral shedding, which often lasts weeks or months.77, 78, 79 Influenza Influenza is diagnosed in approximately 1% of patients with HCT/HM during treatment, and in 33% of patients presenting with respiratory virus symptoms.80, 81, 82 Progression to pneumonia occurs in 14% to 30% of patients and is associated with mortality rates of 15% to 28%.80, 81, 82, 83 During the 2009 H1N1 influenza pandemic, rates of pneumonia were much higher (>50%), but mortality was similar.84 Risk factors for development of pneumonia include lymphopenia (<100 cells/μL), neutropenia (<500 cells/μL), steroid use at time of diagnosis, and absence of antiviral treatment.80, 81, 83, 84 Neuraminidase inhibitors, primarily oseltamivir, are currently the standard of care for influenza treatment and postexposure prophylaxis. Oseltamivir resistance has been described but remains uncommon.85, 86, 87, 88 In the setting of documented or suspected oseltamivir resistance, or in patients who have impaired enteric absorption, inhaled zanamivir or the newly licensed parenteral peramivir have been used.89, 90, 91

atment and postexposure prophylaxis. Oseltamivir resistance has been described but remains uncommon.85, 86, 87, 88 In the setting of documented or suspected oseltamivir resistance, or in patients who have impaired enteric absorption, inhaled zanamivir or the newly licensed parenteral peramivir have been used.89, 90, 91 Seasonal vaccination with the trivalent inactivated vaccine is recommended for all health care workers caring for patients with HCT/HM, family members, and household contacts. Additionally, it is recommended that patients undergoing treatment of leukemia and lymphoma are vaccinated because it may reduce the risk of hospitalization for respiratory illness.92, 93, 94, 95 Ideally, vaccine should be administered at least 2 weeks before any cytotoxic therapy. For HCT recipients, vaccination of patients less than 6 months after transplant is ineffective and is generally not recommended.66, 95, 96 Chemoprophylaxis after exposure is recommended for patients with HCT within 1 year of transplant or for patients with HM during chemotherapy.66, 97

eks before any cytotoxic therapy. For HCT recipients, vaccination of patients less than 6 months after transplant is ineffective and is generally not recommended.66, 95, 96 Chemoprophylaxis after exposure is recommended for patients with HCT within 1 year of transplant or for patients with HM during chemotherapy.66, 97 Respiratory Syncytial Virus Infection with RSV is more common than influenza, occurring in 7% to 10% of patients undergoing allogeneic HCT.98, 99 Among patients with HCT/HM presenting with viral respiratory symptoms, RSV is diagnosed in 14% to 30%.81, 99 Involvement of the lower respiratory tract occurs in 40% to 75% of infected patients.81, 98, 99 Risk factors for progression to pneumonia include patient age, allogeneic HCT, mismatched or unrelated donor, GVHD, myeloablative conditioning regimens, infection less than 30 days posttransplant, prolonged lymphopenia, and lack of ribavirin-based therapy.81, 98, 99, 100, 101 RSV pneumonia is associated with mortality rates of 28% to 55%.100, 102, 103 Treatment with inhaled ribavirin has been shown in several retrospective studies to be associated with decreased rates of progression and a 67% to 83% reduction in the risk of mortality after RSV infection.100, 102 Based on these data, many centers use inhaled ribavirin (2 g for 2 hours every 8 hours for 10 days) in select, high-risk patient populations.66, 104 However, because of recent increases in the cost of this formulation, the use of systemic (oral or parenteral) ribavirin is increasing despite a paucity of evidence to support this practice.102, 105, 106 There are a few noteworthy agents with novel mechanisms of action that are currently in trial: GS5806, an oral RSV entry inhibitor; and ALS8176, a nucleoside RSV polymerase inhibitor.107, 108 Finally, pneumonia caused by RSV has been associated with a significant airflow decline by 1 year posttransplant, an important long-term sequela of this common infection.109

mechanisms of action that are currently in trial: GS5806, an oral RSV entry inhibitor; and ALS8176, a nucleoside RSV polymerase inhibitor.107, 108 Finally, pneumonia caused by RSV has been associated with a significant airflow decline by 1 year posttransplant, an important long-term sequela of this common infection.109 Parainfluenza Virus Unlike influenza and RSV, PIV infections occur without much seasonal variation. The incidence of PIV infection in patients with HCT/HM is 1% to 10%, and 30% of infected patients develop pneumonia; most cases are caused by PIV type 3.110, 111, 112, 113 Death occurs in 17% to 46% of patients who develop pneumonia.110, 112, 113 Risk factors for development of pneumonia include high-dose corticosteroid use, lymphopenia, neutropenia, infection occurring early posttransplantation, the presence of copathogens, and a higher Acute Physiology and Chronic Health Evaluation II score.110, 113, 114 There are currently no licensed treatments for PIV pneumonia. Ribavirin has been used with little noted improvement in mortality or clinical response.110, 114 DAS181, an investigational sialidase fusion protein that works by removing sialic acid–containing receptors from respiratory epithelial cells, preventing PIV from binding, has been successfully used in several cases of adult and pediatric PIV pneumonia in HCT/HM and is currently in phase III clinical trials.115, 116

14 DAS181, an investigational sialidase fusion protein that works by removing sialic acid–containing receptors from respiratory epithelial cells, preventing PIV from binding, has been successfully used in several cases of adult and pediatric PIV pneumonia in HCT/HM and is currently in phase III clinical trials.115, 116 Adenovirus Human adenovirus (HAdV) infections can cause significant disseminated disease in patients with HCT/HM, including severe pneumonia. The most severe disease occurs after HCT, especially in children, and in patients with HM treated with alemtuzumab.117, 118, 119 Adenovirus infection occurs in 8% to 17% of patients undergoing allogeneic HCT, with most cases occurring in children.120, 121 A total of 10% of patients develop HAdV end-organ disease; the lungs are involved in 75% of these cases.120, 121 Infection is less common after autologous HCT, occurring in only 6% of patients, and end-organ disease including pneumonia is rare. In addition to T-cell depletion, risk factors for HAdV disease are lymphopenia, receipt of cord blood grafts, GVHD requiring increased or prolonged immunosuppression, and absence of HAdV-specific T-cell responses.122 Patients with adenoviral pneumonia often have involvement at other sites, such as the gut and liver, and mortality rates with disseminated disease are high. First-line treatment of HAdV disease including pneumonitis is cidofovir (5 mg/kg once weekly, or 1 mg/kg three times weekly) and reduction in immunosuppression whenever possible.66, 122, 123, 124

ia often have involvement at other sites, such as the gut and liver, and mortality rates with disseminated disease are high. First-line treatment of HAdV disease including pneumonitis is cidofovir (5 mg/kg once weekly, or 1 mg/kg three times weekly) and reduction in immunosuppression whenever possible.66, 122, 123, 124 Other Community Respiratory Viruses Other common CRVs, such as human metapneumovirus, novel coronaviruses (eg, SARS-CoV, MERS-CoV), and even human rhinovirus, cause lower respiratory tract infection in patients with HCT/HM.125, 126, 127, 128 Although each of these viruses have their own specific biology and epidemiology, the risk factors for pneumonia that have been identified for other CRVs, such as lymphopenia and infection occurring early after HCT, are shared. Because there are yet no direct-acting treatments for these viruses, efforts to prevent infection in these highly immunosuppressed patients remains paramount. Current guidelines recommend preventing contact from symptomatic health care workers and family members, daily screening of health care workers and visitors to inpatient units for symptoms, active surveillance for CRV disease, and isolation of symptomatic patients with recognition that viral shedding is prolonged in this patient population.129, 130

reventing contact from symptomatic health care workers and family members, daily screening of health care workers and visitors to inpatient units for symptoms, active surveillance for CRV disease, and isolation of symptomatic patients with recognition that viral shedding is prolonged in this patient population.129, 130 Summary The profound and prolonged immunosuppression experienced by patients undergoing HCT and intensive chemotherapy for HM results in rates of viral pneumonia that far surpass the incidence in the general population. Patients with viral pneumonia generally present with fever; hypoxia; and often bilateral, patchy nodular infiltrates with or without surrounding ground glass opacities on high-resolution CT imaging. Because the imaging findings do not help distinguish among different viral etiologies, and these patients commonly have other viral, bacterial, and fungal coinfections, a microbiologic diagnosis typically requires fiberoptic bronchoscopy with bronchoalveolar lavage.

ground glass opacities on high-resolution CT imaging. Because the imaging findings do not help distinguish among different viral etiologies, and these patients commonly have other viral, bacterial, and fungal coinfections, a microbiologic diagnosis typically requires fiberoptic bronchoscopy with bronchoalveolar lavage. Although CMV remains the most common cause of viral pneumonia in this population, efforts to treat CMV replication early in its course and changes in transplant practices have resulted in improvements in the incidence and associated mortality of CMV pneumonia. Taken together, CRV infections also occur commonly in this patient population. However, the rates of progression to pneumonia vary depending on the virus with influenza, RSV, and PIV causing lower tract disease more commonly than adenovirus, human metapneumovirus, and rhinovirus, and patient risk factors relating to the degree of immunosuppression (ie, lymphopenia, early posttransplant, steroid use). Once established, however, pneumonia caused by these infections is associated with high mortality rates in part because of the lack of direct-acting antiviral agents for most of these viruses. Infection prevention practices that limit the acquisition of CRVs by patients with HCT/HM and decrease the risk of spread within the clinics and inpatient settings are of particular importance. Disclosure: Dr M.L. Green has received research funding from Merck and Astellas Global Pharma. No other relationships to disclose.

DEFINITIONS AND OVERVIEW OF EPIDEMIOLOGY Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction caused by chronic bronchitis or emphysema. This airflow obstruction is generally progressive, may be accompanied by airway hyperreactivity, and often is partially reversible.7 Declining lung function is almost universally caused by decades of tobacco smoke exposure and develops insidiously so that patients often do not complain of exertional dyspnea until their 1-second forced expiratory volume (FEV1) is between 40% and 59% of its predicted value.20 When the FEV1 falls below 1 L, patients are disabled in the activities of daily living and have a 5-year survival of approximately 50%.3 Forced expiratory volume in 1 second declines by about 30 mL/year in healthy nonsmokers, whereas the average decline is approximately 45 mL/year in smokers.20 Approximately 15% of smokers are susceptible to the airway effects of smoking and will develop COPD. These patients show accelerated rates of decline in FEV1 of between 50 and 90 mL/year.24

1 second declines by about 30 mL/year in healthy nonsmokers, whereas the average decline is approximately 45 mL/year in smokers.20 Approximately 15% of smokers are susceptible to the airway effects of smoking and will develop COPD. These patients show accelerated rates of decline in FEV1 of between 50 and 90 mL/year.24 In 1994, approximately 16 million Americans suffered from COPD, an estimated increase of 60% since 1982.6 It ranks fourth among leading causes of death in North America and is the only leading cause of death that is rising in prevalence.6, 28 According to 1993 estimates made by the National Heart Lung and Blood Institute, the annual total cost arising from COPD was nearly $24 billion dollars. This amount includes almost $15 billion in direct health care expenditures, nearly $5 billion in indirect morbidity costs, and $4.5 billion in indirect mortality costs.6 Periods of relative clinical stability during the course of COPD are interrupted by recurrent exacerbations. The definition of an acute exacerbation of COPD (AECOPD) is imprecise but is generally considered clinically as an episode of increased dyspnea, sputum production, and sputum purulence in a patient with COPD.11 When these symptoms are severe and accompanied by significant hypoxemia or hypercapnia, patients may require hospitalization. This article focuses primarily on the management of hospitalized patients with AECOPD outside of the intensive care unit and reviews the evidence supporting the available therapies for COPD exacerbations.

symptoms are severe and accompanied by significant hypoxemia or hypercapnia, patients may require hospitalization. This article focuses primarily on the management of hospitalized patients with AECOPD outside of the intensive care unit and reviews the evidence supporting the available therapies for COPD exacerbations. PATHOPHYSIOLOGY OF EXACERBATIONS Smoking-related Lung Damage and Pathobiology of Bacterial Colonization Cigarette smoking is the most important cause of COPD.109 Smoking compromises local airway defense mechanisms by damaging ciliated airway epithelium, increasing mucus viscosity, and slowing mucociliary clearance. These conditions promote bacterial colonization of the lower respiratory tract. The three major bacterial pathogens isolated from patients with COPD during periods of both clinical stability and exacerbation are nontypeable Haemophilus influenzae, Streptococcus pneumoniae, and Moraxella catarrhalis.83 When FEV1 is severely reduced, Enterobacteriaceae and Pseudomonas aeruginosa are also commonly detected.42 These organisms possess a wide array of virulence factors that allow them to evade clearance from the lower airways. Although a detailed discussion of the bacterial mechanisms of colonization and infection is beyond the scope of this article, several concepts are noteworthy.

nas aeruginosa are also commonly detected.42 These organisms possess a wide array of virulence factors that allow them to evade clearance from the lower airways. Although a detailed discussion of the bacterial mechanisms of colonization and infection is beyond the scope of this article, several concepts are noteworthy. Smokers prone to acute episodes of bronchitis have a greater degree of bacterial adherence to oropharyngeal airway epithelial cells compared with nonsmokers.101, 123 After adhering to mucus or epithelial cells, pathogenic bacteria elaborate exoproducts that stimulate excess mucous production,1 disorganize and slow ciliary beating,95 damage epithelial cells,94 and impair immune effector-cell function.23 Furthermore, bacterial proteases destroy local immunoglobulins.45 When these bacteria loiter in the airways, a host inflammatory response is stimulated. With the movement of large numbers of neutrophils and their subsequent release of proteases and toxic oxygen radicals, epithelial surface damage may be enhanced. After the inciting impact of smoking, bacterial colonization therefore begets airway damage which, in turn, begets further inflammation and bacterial colonization. This event is the vicious circle hypothesis that has been proposed to explain how the bacteria–host interaction establishes the insidious loss of lung function.83, 135

nciting impact of smoking, bacterial colonization therefore begets airway damage which, in turn, begets further inflammation and bacterial colonization. This event is the vicious circle hypothesis that has been proposed to explain how the bacteria–host interaction establishes the insidious loss of lung function.83, 135 Mechanisms of Disordered Gas Exchange Disordered pulmonary gas exchange is characteristic of acute exacerbations of COPD. Patients typically are found to have severe hypoxemia with or without hypercarbia. A variety of infectious and noninfectious insults result in inflammation, bronchospasm, and mucous hypersecretion. These lead to acute airway narrowing that aggravates ventilation-perfusion (/) mismatching and can worsen existing hyperinflation. Although / inequality is the most important determinant of hypoxemia, low mixed venous oxygen tension (Pvo 2) is a contributing factor.16 During exacerbations, the work of breathing increases to overcome increased airway resistance and dynamic hyperinflation. Oxygen utilization by the respiratory muscles therefore is markedly increased, resulting in lower Pvo 2. Fortunately, among patients with adequate cardiac reserve, increases in cardiac output partly compensate for diminished Pvo 2 to defend arterial oxygenation.

e increased airway resistance and dynamic hyperinflation. Oxygen utilization by the respiratory muscles therefore is markedly increased, resulting in lower Pvo 2. Fortunately, among patients with adequate cardiac reserve, increases in cardiac output partly compensate for diminished Pvo 2 to defend arterial oxygenation. Among the mechanisms leading to hypercarbia, / mismatch is probably more important than hypoventilation, at least among patients who recover from their exacerbations without needing mechanical ventilation. This concept is supported by the observation that, during exacerbations, patients are often hypercarbic despite increased minute ventilation.16, 131 Hypoventilation may be an additional mechanism of hypercarbia if respiratory muscle fatigue and acute respiratory failure ensue.

ns without needing mechanical ventilation. This concept is supported by the observation that, during exacerbations, patients are often hypercarbic despite increased minute ventilation.16, 131 Hypoventilation may be an additional mechanism of hypercarbia if respiratory muscle fatigue and acute respiratory failure ensue. SPECIFIC CAUSES OF EXACERBATIONS Bacteria The relationship between bacterial infection and COPD exacerbations is not precisely understood. Several lines of evidence, however, have established an important role for bacterial infection in many exacerbations. High titers of antibody against nontypeable H. influenzae 84, 97, 114 and M. catarrhalis 31 are found following AECOPD. Although bacterial colonization of the distal airways is common in stable COPD, patients with exacerbations often have higher numbers of organisms.44, 80, 115 Table 1 summarizes studies using protected specimen brushes to define the microflora of the distal airways in COPD exacerbations. Monsó et al55 found positive bacterial cultures in 52% of outpatients with AECOPD. Compared with stable patients, exacerbated patients were twice as likely to have positive cultures (i.e., ≥ 1000 CFU/mL) and five times as likely to have bacterial counts greater than 10,000 CFU/mL.80 Likewise, S. pneumoniae is more likely to be found in sputum during exacerbations than remission.Table 1 PROTECTED SPECIMEN BRUSH STUDIES OF BACTERIAL INFECTION DURING CHRONIC OBSTRUCTIVE PULMONARY DISEASE EXACERBATIONS

i.e., ≥ 1000 CFU/mL) and five times as likely to have bacterial counts greater than 10,000 CFU/mL.80 Likewise, S. pneumoniae is more likely to be found in sputum during exacerbations than remission.Table 1 PROTECTED SPECIMEN BRUSH STUDIES OF BACTERIAL INFECTION DURING CHRONIC OBSTRUCTIVE PULMONARY DISEASE EXACERBATIONS Monsó et al80Outpatients (n = 29) PSB Cultures Fagon et al44Ventilated Patients (n = 54) PSB Cultures Soler et al115Ventilated Patients (n = 50) PSB, TBA, BAL Cultures Isolates (n) Haemophilus influenzae 10 6 11 Streptococcus pneumoniae 3 7 4 Moraxella catarrhalis 2 3 4 Pseudomonas aeruginosa – 3 9 Strenotrophomonas maltophilia – – 2 Enterobacteriaceae – 5 4 Staphylococcus aureus – 4 – Other (nonpathogenic)* – 19 (11–H. parainfluenzae) 30 (13–S. viridans) Patients with positive cultures† 15/29 27/54 36/50‡ AECOPD with positive cultures 52% 50% 72% PSB = Protected specimen brush; TBA = tracheobronchial aspirate; BAL = bronchoalveolar lavage fluid; AECOPD = acute exacerbation of COPD. * Streptococcus spp, Corynebacterium spp, H. parainfluenzae, S. epidermidis, Neisseria spp, Candida spp † PSB ≥ 102, BAL ≥ 103, TBA ≥ 105CFU/mL. ‡ Includes patients with positive serology for C. pneumoniae and respiratory viruses.

Monsó et al80Outpatients (n = 29) PSB Cultures Fagon et al44Ventilated Patients (n = 54) PSB Cultures Soler et al115Ventilated Patients (n = 50) PSB, TBA, BAL Cultures Isolates (n) Haemophilus influenzae 10 6 11 Streptococcus pneumoniae 3 7 4 Moraxella catarrhalis 2 3 4 Pseudomonas aeruginosa – 3 9 Strenotrophomonas maltophilia – – 2 Enterobacteriaceae – 5 4 Staphylococcus aureus – 4 – Other (nonpathogenic)* – 19 (11–H. parainfluenzae) 30 (13–S. viridans) Patients with positive cultures† 15/29 27/54 36/50‡ AECOPD with positive cultures 52% 50% 72% PSB = Protected specimen brush; TBA = tracheobronchial aspirate; BAL = bronchoalveolar lavage fluid; AECOPD = acute exacerbation of COPD. * Streptococcus spp, Corynebacterium spp, H. parainfluenzae, S. epidermidis, Neisseria spp, Candida spp † PSB ≥ 102, BAL ≥ 103, TBA ≥ 105CFU/mL. ‡ Includes patients with positive serology for C. pneumoniae and respiratory viruses. Fagon44 et al found evidence of bacterial infection in 50% of patients who required mechanical ventilation. Streptococcus pneumoniae, H. influenzae, M. catarrhalis, and enteric gram-negative organisms collectively accounted for 55% of the isolates. The investigators did not attempt to identify M. pneumoniae, C. pneumoniae, or respiratory viruses. Interestingly, gram-negative bacteria accounted for 64% of isolates, and nearly half of these were H. parainfluenzae. Yet, similar studies employing protected brush specimens have not detected H. parainfluenzae among patients with COPD exacerbations.80, 115 Moreover, Smith et al114 were unable to demonstrate rises in antibody against H. parainfluenzae following COPD-related acute respiratory illnesses. It seems justified therefore to consider H. parainfluenzae as generally nonpathogenic.

ns have not detected H. parainfluenzae among patients with COPD exacerbations.80, 115 Moreover, Smith et al114 were unable to demonstrate rises in antibody against H. parainfluenzae following COPD-related acute respiratory illnesses. It seems justified therefore to consider H. parainfluenzae as generally nonpathogenic. In contrast to the findings of Fagon et al,115 a more comprehensive microbiologic survey of patients with severe exacerbations found a higher incidence of potential pathogens. Overall, 72% of patients had at least one positive bacterial culture or positive serology for C. pneumoniae or respiratory viruses. All cultures were obtained within 48 hours of admission, thereby reducing the likelihood of nosocomial infection. Streptococcus pneumoniae, H. influenzae, and M. catarrhalis accounted for 56% of potential pathogens. Strikingly, gram-negative enteric bacteria and Pseudomonas or Stenotrophomonas represented 39% of potential pathogens. Positive serology for acute infection with C. pneumoniae or respiratory viruses (almost exclusively influenza) was found in 26% of the patients.115 This study suggests that a broader profile of potential pathogens may be present among patients with COPD with severe exacerbations. Although these findings should be confirmed in a larger study, the choice of empiric antibiotics for patients with AECOPD should be based in part on the degree of exacerbation severity, and broader-spectrum initial coverage may be warranted for patients with AECOPD who present with respiratory failure and require mechanical ventilation.

dings should be confirmed in a larger study, the choice of empiric antibiotics for patients with AECOPD should be based in part on the degree of exacerbation severity, and broader-spectrum initial coverage may be warranted for patients with AECOPD who present with respiratory failure and require mechanical ventilation. The source of bacterial infection during a COPD exacerbation may be endogenous or exogenous. In a small group of patients followed for 3 years, exacerbations coincided with reinfection by strains of H. influenzae having either the same (i.e., endogenous) or different (i.e., exogenous) DNA fingerprint. Strains of H. influenzae were shown to persist for several months and antibiotic treatment was not effective in eradicating the bacteria.49 Viruses Estimates of the proportion of COPD exacerbations associated with viral infection range from 7%117 to 63%.67 This large discrepancy is because of significant differences in study design.25 Several studies lacked adequate control by failing to record the frequency of viral infection during exacerbation-free periods.40, 75 Others, such as those by Sommerville and Stenhouse,116, 118 attempted to detect only selected pathogens. Variability in the definition of an exacerbation is another factor that may affect the percentage of exacerbations caused by viral illness. Finally, different serologic and isolation techniques account for some of the variety in study results.55

rville and Stenhouse,116, 118 attempted to detect only selected pathogens. Variability in the definition of an exacerbation is another factor that may affect the percentage of exacerbations caused by viral illness. Finally, different serologic and isolation techniques account for some of the variety in study results.55 The three most rigorous studies are summarized in Table 2 .25, 55, 113 The proportion of exacerbations attributed to viral (or mycoplasma) illness ranges from 18% to 34%. Influenza, parainfluenza, and coronavirus were the most frequent pathogens to be significantly associated with exacerbations.Table 2 HIGHEST-QUALITY STUDIES OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE EXACERBATIONS ASSOCIATED WITH VIRAL AND MYCOPLASMA PNEUMONIAE INFECTION Study Patients(n) Exacerbations (n) Viral Exacerbationsn(%) Identified Agent (n) Influenza A & B Para-influenza 1,2,3 Corona-virus Rhino-virus Adeno-virus HSV RSV Mycoplasma pneumoniae Gump et al55 25 116 39 (34) 15 9 6 4 3 11 5 1 Buscho et al25 46 166 50 (25) 17 10 8 NR 2 NR 0 4 Smith et al113 150 1030 186 (18) 50 29 17 44 7 21 8 5 HSV = Herpes simplex virus; RSV = respiratory syncytial virus; NR = not reported.

Para-influenza 1,2,3 Corona-virus Rhino-virus Adeno-virus HSV RSV Mycoplasma pneumoniae Gump et al55 25 116 39 (34) 15 9 6 4 3 11 5 1 Buscho et al25 46 166 50 (25) 17 10 8 NR 2 NR 0 4 Smith et al113 150 1030 186 (18) 50 29 17 44 7 21 8 5 HSV = Herpes simplex virus; RSV = respiratory syncytial virus; NR = not reported. More recently, Goh et al48 performed a prospective etiologic study of 90 inpatients with AECOPD. They collected paired sera for influenza A, B, and parainfluenza viruses as well as Legionella, Mycoplasma, and Chlamydia. Positive serology was found in 31 patients (34%), of whom 26 patients (28%) had viral infections. The most common organism was influenza A, with 18 patients demonstrating positive serology (20%). Five patients had positive serology for Legionella, whereas no evidence was found for infections caused by Mycoplasma or Chlamydia.48

serology was found in 31 patients (34%), of whom 26 patients (28%) had viral infections. The most common organism was influenza A, with 18 patients demonstrating positive serology (20%). Five patients had positive serology for Legionella, whereas no evidence was found for infections caused by Mycoplasma or Chlamydia.48 Gump et al55 observed 25 patients every 2 weeks for 4 years and documented 116 exacerbations. They derived a striking correlation of infection with exacerbations by interpreting their data in a time-weighted analysis. They found that the incidence of infection was 32% per patient week of exacerbation but only 0.9% per patient week in remission. In their 5-year analysis, Buscho et al25 found that 25% of exacerbations were associated with viral infection. This rate was twice that of viral infections during remission detected as an asymptomatic fourfold rise in antiviral antibody titers. In the largest study, Smith et al113 followed 150 patients over 8 years and analyzed more than 1000 acute respiratory illnesses. They associated nonbacterial infections with approximately 20% of acute respiratory illnesses but only 6% of illness-free periods. In contrast with the work by Buscho and Gump, Smith et al113 noted a high rate of rhinoviral infection. Uncontrolled studies by McNamara and Eadie40, 75 also reported rhinoviral infections to be associated with COPD exacerbations in 43% and 20% of cases, respectively. Common colds may have a deleterious effect upon lung function29 and patients with COPD are more likely to develop increased cough and lower respiratory tract symptoms during rhinoviral infections than healthy subjects.57, 113

ral infections to be associated with COPD exacerbations in 43% and 20% of cases, respectively. Common colds may have a deleterious effect upon lung function29 and patients with COPD are more likely to develop increased cough and lower respiratory tract symptoms during rhinoviral infections than healthy subjects.57, 113 Smith et al112 performed a 7-year observational study of 120 patients to assess potential interactions between viral, mycoplasmal, and bacterial infections in patients with COPD. They calculated the ratio of number of observed exacerbations to number of expected viral or bacterial associations. Haemophilus influenzae and S. pneumoniae were isolated more than twice as often as expected following influenza virus infection. Marked rises in titers of antibodies against H. influenzae were associated with preceding viral or Mycoplasma infections, suggesting that viral infection promotes increased invasiveness of H. influenzae and subsequent infection. There are no other rigorously performed clinical studies of the interaction between viral and bacterial infections in AECOPD. Although the concept that viruses promote secondary bacterial infection seems biologically plausible and is supported by animal research, it remains unknown how often bacterial infection in AECOPD follows an inciting viral infection.

clinical studies of the interaction between viral and bacterial infections in AECOPD. Although the concept that viruses promote secondary bacterial infection seems biologically plausible and is supported by animal research, it remains unknown how often bacterial infection in AECOPD follows an inciting viral infection. Air Pollution Suspended particulate matter less than 10 μm in diameter (PM10) is produced by vehicle exhaust and many industrial processes. Several epidemiologic studies8, 105, 106 have associated elevated PM10 levels with a wide range of respiratory outcomes, including reduced pulmonary function and increased chronic respiratory symptoms, rates of hospitalization, and mortality. Similar associations exist for other pollutants, notably sulfur dioxide (SO2) and nitrogen dioxide. A 5-year study in Barcelona121 reported that small increases of SO2 and airborne particles produced adjusted increases of 6% in emergency room admissions for COPD in winter and 9% in summer. Similar rates of excess hospitalizations for COPD have been reported from Sydney, Australia (4%); Detroit, Michigan (6%); and Birmingham, Alabama (7%).81, 105, 106 Although small, these effects represent a significant public health concern, particularly because they are demonstrable at pollution levels below current air-quality standards.

ess hospitalizations for COPD have been reported from Sydney, Australia (4%); Detroit, Michigan (6%); and Birmingham, Alabama (7%).81, 105, 106 Although small, these effects represent a significant public health concern, particularly because they are demonstrable at pollution levels below current air-quality standards. In summary, the foregoing studies imply that bacterial pathogens can be identified in approximately half of COPD exacerbations. Viral pathogens are identifiable in about 25% of such episodes. Poor air quality may account for slightly more than 5% of episodes of AECOPD. In many COPD exacerbations, no obvious pathogen or precipitating cause is found. It is not known how frequently other potential factors, such as medication noncompliance or coincidental events such as pulmonary embolism and myocardial infarction, play an inciting role in AECOPD. Indeed, it is often difficult to distinguish clinically between exacerbations with and without an infectious cause. This distinction is discussed subsequently in the section on antibiotic therapy.

noncompliance or coincidental events such as pulmonary embolism and myocardial infarction, play an inciting role in AECOPD. Indeed, it is often difficult to distinguish clinically between exacerbations with and without an infectious cause. This distinction is discussed subsequently in the section on antibiotic therapy. PREVENTION Smoking Prevention, Influenza and Pneumococcal Vaccination, Immunostimulating Medication Smoking cessation is the most important intervention in the management of patients with COPD. The landmark Lung Health Study confirmed that smoking cessation greatly reduces the rate of FEV1 decline.10 The benefit of smoking cessation is seen even in patients over the age of 60 years.56 Chronic sputum production often clears within 4 weeks of stopping smoking.138 Although nicotine replacement therapy is an effective approach to smoking cessation, counseling by a physician has been shown to be the most potent intervention.124

fit of smoking cessation is seen even in patients over the age of 60 years.56 Chronic sputum production often clears within 4 weeks of stopping smoking.138 Although nicotine replacement therapy is an effective approach to smoking cessation, counseling by a physician has been shown to be the most potent intervention.124 Each year, influenza and its complications are responsible for hundreds of thousands of excess hospitalizations, tens of thousands of excess deaths, and billions of dollars in health care costs.85, 86 Those with chronic lung disease are at especially high risk for the consequences of influenza. Despite recommendations for annual influenza vaccination, recent studies have documented inadequate vaccination rates in this risk group.91, 92 Among elderly persons, including those with chronic lung disease, Nichol et al86 overwhelmingly demonstrated the efficacy and cost effectiveness of influenza vaccination. In a serial cohort study of more than 25,000 patients,86 vaccination was associated with a 30% to 40% reduction in the rate of hospitalization for all acute and chronic respiratory conditions. Another study by the same authors85 found that influenza vaccination was associated with a 70% reduction in the risk for death from any cause (odds ratio [OR] = 0.3; 95% confidence interval [CI] = 0.21–0.43). A recent meta-analysis of 20 cohort studies concluded that the estimates of vaccine efficacy for preventing respiratory illness, hospitalization, and death were 56%, 50% and 68%, respectively.107 These data clearly affirm that influenza vaccination is an indispensable part of the care of all elderly persons, especially those with COPD.

lysis of 20 cohort studies concluded that the estimates of vaccine efficacy for preventing respiratory illness, hospitalization, and death were 56%, 50% and 68%, respectively.107 These data clearly affirm that influenza vaccination is an indispensable part of the care of all elderly persons, especially those with COPD. The value of pneumococcal vaccination for elderly patients with COPD has been controversial. Two randomized controlled trials evaluating the vaccine's efficacy among patients with COPD were unable to show statistically significant protective benefit.36, 69 A recent meta-analysis concluded that the vaccine provides partial protection against bacteremic pneumococcal pneumonia but not against other important outcomes, including bronchitis or mortality caused by pneumococcal infection. This protective benefit was seen only in low-risk groups and not among those with COPD or other high-risk patients.46 Nevertheless, pneumococcal vaccination continues to be strongly recommended for patients with COPD because it is safe and has been found to provide significant benefit for patients in case-control and indirect cohort studies26, 93, 108 as well as in a more recent randomized population-based trial.64

gh-risk patients.46 Nevertheless, pneumococcal vaccination continues to be strongly recommended for patients with COPD because it is safe and has been found to provide significant benefit for patients in case-control and indirect cohort studies26, 93, 108 as well as in a more recent randomized population-based trial.64 Several additional novel strategies for reducing acute exacerbations of COPD are under investigation. For example, OM-85 BV is an oral immunostimulating agent containing lyophylized fractions of the eight most common respiratory pathogens. Its use was associated with a 40% reduction in the incidence of acute exacerbations of chronic bronchitis and a 28% decrease in antibiotic use among elderly institutionalized patients in one trial, and the same frequency of exacerbations but less than half as many days in hospital as those given placebo in another.32, 90 There was a trend toward fewer hospitalizations for respiratory reasons among those receiving the immunostimulating drug. Although not available in North America, OM-85 BV has been used for many years in Europe. Further trials are required to properly define its role.

n hospital as those given placebo in another.32, 90 There was a trend toward fewer hospitalizations for respiratory reasons among those receiving the immunostimulating drug. Although not available in North America, OM-85 BV has been used for many years in Europe. Further trials are required to properly define its role. Another approach has been the subcutaneous administration of hyaluronic acid (HA), a glycosaminoglycan with neutrophil-regulating functions, to patients with chronic bronchitis. Fewer acute exacerbations of bronchitis and reduced antibiotic use were noted among HA-treated patients in a placebo-controlled crossover study.130 Other investigators found that continous administration of carbocysteine lysine salt monohydrate during winter months was effective in preventing AECOPD and reducing antibiotic consumption in patients with chronic bronchitis.4 Despite these recent intriguing data suggesting that immunomodulatory agents may attenuate the development of AECOPD, randomized, controlled trials are needed to clarify their potential roles in the routine management of patients with COPD. TREATMENTS The following sections provide an overview of the merits (or lack thereof) of common therapeutic interventions for AECOPD. Controlled oxygen (O2), bronchodilators, antibiotics, corticosteroids, noninvasive ventilation, nutritional support, and chest physiotherapy are discussed. When available, evidence from randomized controlled trials is presented.

an overview of the merits (or lack thereof) of common therapeutic interventions for AECOPD. Controlled oxygen (O2), bronchodilators, antibiotics, corticosteroids, noninvasive ventilation, nutritional support, and chest physiotherapy are discussed. When available, evidence from randomized controlled trials is presented. Controlled Oxygen Hypoxemia is the most immediate threat to life for patients with AECOPD. Hypercapnia is a well-recognized consequence of O2 therapy. In the past, the risk of hypoventilation or even apnea resulting from O2 administration has been vastly overestimated, and the notion that O2 commonly induces clinically important hypercarbia and acidosis has been discredited.12, 13, 39, 104 The traditional concept is that correction of hypoxemia with supplemental O2 removes the hypoxic drive to breathe and leads to a fall in minute ventilation and subsequent carbon dioxide (CO2) retention.47 Aubier et al12 found that although administration of supplemental O2 does reduce minute ventilation and increase arterial partial pressure of carbon dioxide (Paco 2), mouth occlusion pressure (an indicator of central respiratory drive) was significantly higher during acute exacerbations than during stable conditions. The drive to breathe therefore remained very high in spite of oxygen treatment. In a later study by the same authors,13 patients with COPD and acute respiratory failure received 100% O2 for 15 minutes to abolish hypoxic drive. Minute ventilation fell by only 7% and could not account for the entire increase in Paco 2. The authors concluded that the rise in CO2 was caused by increased Vd/Vt) (i.e., increased deadspace ventilation) and that the primary mechanism of O2-induced hypercarbia is / mismatching, perhaps through the loss of hypoxic vasoconstriction. More recent work by Dunn et al39 and Stradling119 has challenged Aubier's conclusions and defended the traditional concept that hypoventilation, rather than / mismatching, is to blame for O2-induced hypercarbia. One further mechanism that appears to contribute, albeit to a minor extent, is the Haldane effect, whereby CO2 is displaced from hemoglobin by O2, causing a rise in Paco 2.119 Nevertheless, the risks for acute hypoxemia far outweigh the risks for severe hypercarbia. As such, supplemental O2 administration is recommended for hypoxemic patients with AECOPD.

contribute, albeit to a minor extent, is the Haldane effect, whereby CO2 is displaced from hemoglobin by O2, causing a rise in Paco 2.119 Nevertheless, the risks for acute hypoxemia far outweigh the risks for severe hypercarbia. As such, supplemental O2 administration is recommended for hypoxemic patients with AECOPD. Oxygen initially should be given to any hypoxemic patient with AECOPD by nasal cannulae or Venturi mask. If nasal cannulae are used, flow rates of 1 to 2 L/minute generally suffice. The inspired concentration of oxygen (Fio 2) usually increases by 3% to 4% for each increase of 1 L/minute in flow but varies according to the patient's own inspiratory flow rate. Venturi masks are more precise and should initially be set to deliver a Fio 2 of 24% to 28%. On average, the Pao 2 increases by 10 mm Hg when the Fio 2 is increased from room air to 24%, and by 20 mm Hg when the Fio 2 is increased to 28%.17 The target Pao 2 is between 60 and 70 mm Hg, corresponding to the Pao 2 at which there is near-complete saturation of hemoglobin with O2. Rarely, overzealous O2 administration produces progressive hypercapnia and respiratory acidosis.20, 34, 74 For this reason, response to O2 must be assessed according to arterial blood gas and pH measurements. These should be obtained at baseline and within 60 minutes of starting or changing the O2 concentration. If Pao 2 remains intractably low or the pH drops as a result of increasing Paco 2, alternate strategies to improve hypoxemia and respiratory acidosis must be devised. These include maximizing bronchodilation and the use of assisted ventilation.

aseline and within 60 minutes of starting or changing the O2 concentration. If Pao 2 remains intractably low or the pH drops as a result of increasing Paco 2, alternate strategies to improve hypoxemia and respiratory acidosis must be devised. These include maximizing bronchodilation and the use of assisted ventilation. Bronchodilators The role of bronchodilators in the management of stable COPD is discussed in the article by Ferguson on pharmacologic therapy for COPD. Bronchodilator agents, specifically β2-agonists and ipratropium bromide (IB), also play a central role in the management of patients with AECOPD, with and without respiratory failure. These agents are generally given by inhalation to reduce side-effects and, in the acute setting, nebulizers have been traditionally preferred over metered-dose inhalers (MDIs) for ease of drug administration.68, 74, 127 Although there are few data to support the choice of either β2-agonists or IB as first-line therapy for acute exacerbations, β2-agonists are usually given as the first step, perhaps because of the longer time to peak effect for IB.7, 73 When given in recommended doses (two puffs), IB generally produces greater bronchodilation than β2-agonists.19, 122

upport the choice of either β2-agonists or IB as first-line therapy for acute exacerbations, β2-agonists are usually given as the first step, perhaps because of the longer time to peak effect for IB.7, 73 When given in recommended doses (two puffs), IB generally produces greater bronchodilation than β2-agonists.19, 122 Ipratropium bromide and β2-agonists are often used together in the acute setting, despite a lack of evidence from randomized-controlled trials that they are more efficacious in tandem than either agent alone in that setting.79, 89, 96 In a doubleblind, randomized study involving 51 patients with AECOPD, the effects of 0.5 mg of IB, 1.25 mg of fenoterol, or a combination of the two agents were compared. All three regimens resulted in improved spirometric function at 45 and 90 minutes post-treatment, but combination therapy was no better than either agent alone. Similarly, O'Driscoll et al89 compared nebulized salbutamol (10 mg) with and without IB (0.5 mg) in 47 patients with AECOPD. One hour following treatment, peak expiratory flow rates were not significantly different between regimens.89 Others have examined clinical, rather than spirometric, outcomes following combined therapy in AECOPD.79, 110 Patients randomly assigned to a combination of isoetharine and IB were discharged from the emergency department an average of 90 minutes sooner than those who received only isoetharine. The authors attributed this time saving to approximately five puffs of IB. Interestingly, mean discharge FEV1 was not different between the two groups.110 Because these studies followed patients for only 60 to 90 minutes, Moayyedi et al79 recently attempted to capture longer-term benefits of combination therapy. Comparing nebulized treatments of salbutamol with and without IB among inpatients with AECOPD, they found no difference in duration of stay, subjective breathlessness, or spirometric values over a 14-day assessment period.

utes, Moayyedi et al79 recently attempted to capture longer-term benefits of combination therapy. Comparing nebulized treatments of salbutamol with and without IB among inpatients with AECOPD, they found no difference in duration of stay, subjective breathlessness, or spirometric values over a 14-day assessment period. Considerable effort has been made to establish the most effective method for delivering bronchodilator drugs to patients with acute airflow obstruction. Delivery methods include MDIs with or without a spacer device, nebulizers (hand-held or attached to a face mask), and dry powder inhalers. A recent systematic review of 12 randomized studies comparing MDIs and nebulizers for administration of β2-agonists in acute exacerbations of asthma and COPD found the two methods to be equivalent.128 A reasonable approach advocated by several workers is to begin with nebulized treatments among patients who are too dyspneic to use an MDI and spacer device correctly.74, 111 As early as is feasible patients can then be switched to MDIs, which can result in considerable cost savings.60, 120

methods to be equivalent.128 A reasonable approach advocated by several workers is to begin with nebulized treatments among patients who are too dyspneic to use an MDI and spacer device correctly.74, 111 As early as is feasible patients can then be switched to MDIs, which can result in considerable cost savings.60, 120 Theophylline Theophylline has been used for decades to ameliorate symptoms in patients with airflow obstruction.129 The use of theophylline in the management of stable COPD is discussed in the article by Ferguson in this issue. Theophylline has now been relegated to having a minor role in the acute setting because of the development of safer and more potent bronchodilators.41, 74 The lack of convincing, well-designed trials showing its efficacy has further contributed to the decline in its use.70, 99, 137 There are only two published studies that relate specifically to the role of theophylline in the treatment of COPD exacerbations. The first, by Rice et al,99 was a small, yet rigorously controlled trial in which patients were randomized to aminophylline infusion or placebo during hospitalization for AECOPD. The drug conferred no incremental benefit over standard care in either subjective (dyspnea scores) or objective (spirometry) outcome measures. Gastrointestinal side effects were more common in the aminophylline group.

which patients were randomized to aminophylline infusion or placebo during hospitalization for AECOPD. The drug conferred no incremental benefit over standard care in either subjective (dyspnea scores) or objective (spirometry) outcome measures. Gastrointestinal side effects were more common in the aminophylline group. An emergency department-based study randomized patients with acute bronchospasm to receive either aminophylline or placebo.137 The trial included patients with asthma and COPD but failed to report the number of each. Unexpectedly, there was a threefold decrease in the hospital admission rate for patients treated with aminophylline (P = 0.016). The authors argued that aminophylline should be considered in selected patients with acute exacerbations of COPD and asthma because reduced hospitalizations would decrease costs. Although their study illustrates a potentially important clinical benefit of theophylline, a cautious approach is necessary. The effect of theophylline on admission rates was not the primary outcome variable and the reduction in admission rates did not quite reach statistical significance after adjusting for multiple comparisons. Even though the magnitude of the clinical benefit was large, theophylline produced no objective improvement in pulmonary function as measured by spirometry. Despite these reservations, similar reports of clinical benefit in the absence of statistically significant spirometric improvement have been found in studies of corticosteroids in acute asthma71 and bronchodilator therapy in acute COPD.110

duced no objective improvement in pulmonary function as measured by spirometry. Despite these reservations, similar reports of clinical benefit in the absence of statistically significant spirometric improvement have been found in studies of corticosteroids in acute asthma71 and bronchodilator therapy in acute COPD.110 Antibiotics Bacterial infections' contribution to exacerbations of COPD has been inferred from studies demonstrating clinical benefit as a result of antibiotic therapy. Antibiotics have been employed for prophylaxis and acute treatment of AECOPD. In the 1950s and 1960s, attention was given to preventing exacerbations with antibiotics. Murphy and Sethi83 reviewed nine prospective, placebocontrolled trials of antibiotic prophylaxis. Of these, five failed to show any reduction in the frequency of exacerbations, although two demonstrated significantly less time lost from work among patients receiving antibiotics. In contrast, compared with placebo, antibiotic prophylaxis significantly reduced the frequency of exacerbations in four studies. In these investigations, prophylaxis seemed to benefit patients suffering the largest annual number of exacerbations. Some authorities therefore have recently suggested that antibiotic prophylaxis may be appropriate for individuals prone to frequent exacerbations,14 although this practice is not recommended as part of the regular care of all patients with COPD.

to benefit patients suffering the largest annual number of exacerbations. Some authorities therefore have recently suggested that antibiotic prophylaxis may be appropriate for individuals prone to frequent exacerbations,14 although this practice is not recommended as part of the regular care of all patients with COPD. The prescription of antibiotics to facilitate early recovery in AECOPD has become routine despite unresolved questions about their true benefit.14, 51, 52, 83 Nicotra et al87 randomized 40 inpatients with AECOPD to either tetracycline or placebo for 1 week. At 7 days, there was no difference between groups in terms of oxygenation or lung function. In a similar, but larger, study of outpatients with uncomplicated AECOPD, Jorgensen61 also could not demonstrate a clinically important advantage of amoxicillin over placebo.

AECOPD to either tetracycline or placebo for 1 week. At 7 days, there was no difference between groups in terms of oxygenation or lung function. In a similar, but larger, study of outpatients with uncomplicated AECOPD, Jorgensen61 also could not demonstrate a clinically important advantage of amoxicillin over placebo. These null trials notwithstanding, the highest-quality clinical study to date, by Anthonisen et al,11 concluded that antibiotics improved outcomes. They randomized 173 outpatients to receive either placebo or broad-spectrum antibiotic (amoxicillin, trimethoprim-sulfamethoxazole, or doxycycline) during 362 COPD exacerbations. Exacerbations were classified according to severity. Type 1 (the most severe) was defined as an increase in dyspnea, sputum volume, and sputum purulence. Type 2 involved the presence of only two of the three symptoms, and type 3 was defined as the presence of one of the three symptoms in addition to one other finding (sore throat, rhinorrhea, fever, increased wheeze, or increased cough). Compared with placebo, antibiotics shortened the duration of exacerbations by about 2 days and accelerated recovery of peak expiratory flow rate (P < 0.02 for both). Treatment success, defined as resolution of symptoms within 21 days, occurred in 55% of the placebo group and 68.1% of patients receiving active treatment (P < 0.05). Significantly, among patients presenting with type 1 exacerbations, clinical deterioration was more than twice as common with placebo as with antibiotic. A clinician therefore would need to treat roughly eight exacerbations with antibiotics to achieve one treatment success beyond chance, or two in order to avoid a single deterioration. The authors concluded that avoidance of deleterious outcomes is the strongest reason to offer antibiotics to patients with AECOPD. Furthermore, they stated that antibiotics are clearly indicated in type 1 exacerbations, of no benefit in type 3 exacerbations, and probably justifiable for patients with type 2 presentations.11 Similar findings were reported in another large-scale, randomized trial comparing amoxicillin/clavulanate to placebo.5

ECOPD. Furthermore, they stated that antibiotics are clearly indicated in type 1 exacerbations, of no benefit in type 3 exacerbations, and probably justifiable for patients with type 2 presentations.11 Similar findings were reported in another large-scale, randomized trial comparing amoxicillin/clavulanate to placebo.5 Saint and associates103 systematically reviewed the clinical efficacy of antibiotics for AECOPD. They identified nine randomized, placebo-controlled trials of antibiotics in COPD exacerbations in which the patients were followed for at least 5 days. Because no outcome measure was common to all nine studies, the authors derived an overall effect size to quantify the efficacy of antibiotics and concluded that a small but statistically significant improvement could be expected among patients receiving antibiotics. From the six trials that reported peak expiratory flow rates, an overall improvement of 10.75 liters per minute favoring the antibiotic group was noted. Although small, such an effect may be clinically important for patients with severely compromised baseline lung function by preventing respiratory failure and hospital or intensive care unit (ICU) admission.51

iratory flow rates, an overall improvement of 10.75 liters per minute favoring the antibiotic group was noted. Although small, such an effect may be clinically important for patients with severely compromised baseline lung function by preventing respiratory failure and hospital or intensive care unit (ICU) admission.51 To reduce the risk for treatment failure, antibiotics should be selected according to pertinent clinical data and the potential for antimicrobial resistance. Several schemes have been proposed to stratify the patient's risk and select the most appropriate therapy.14, 53, 72, 134 The simplest, and most recent, classification system is presented in Table 3 . Grossman53 has classified acute exacerbations into four groups. Group 1 patients have acute simple bronchitis, likely of viral origin, for which antibiotics are not recommended at the outset. If symptoms persist for longer than 1 week, a macrolide or tetracycline is suggested to treat suspected pathogenic organisms (i.e. M. pneumoniae, C. pneumoniae). Group 2 patients have simple chronic bronchitis with minimally impaired lung function and no additional risk factors for treatment failure. In these patients, amoxicillin, tetracycline, or trimethoprim/sulfamethoxazole are the proposed first-line agents because the consequences of treatment failure in this group are generally few.11 Probable pathogens are H. influenzae, M. catarrhalis, and S. pneumoniae. Group 3 patients have moderate to severe COPD and other risk factors for treatment failure, such as frequent exacerbations and comorbid conditions, including congestive heart failure, diabetes mellitus, chronic renal insufficiency, or chronic liver disease. The probable bacterial pathogens are similar to those in group 2, although gram-negative organisms are more likely in patients with severely impaired lung function. Because the costs of treatment failure are high in this group, and because β-lactamase-producing strains of H. influenzae and M. catarrhalis are increasingly prevalent, fluoroquinolones are the suggested first-line treatment. Group 4 patients have chronic suppurative lung disease, particularly bronchiectasis. Antibiotic therapy is directed at P. aeruginosa and other commonly drug-resistant gram-negative bacteria (see Table 3).

influenzae and M. catarrhalis are increasingly prevalent, fluoroquinolones are the suggested first-line treatment. Group 4 patients have chronic suppurative lung disease, particularly bronchiectasis. Antibiotic therapy is directed at P. aeruginosa and other commonly drug-resistant gram-negative bacteria (see Table 3). As noted, approaches to antibiotic therapy based upon a rational appraisal of patient risk factors and likely pathogens reduce the risk for treatment failure and avoid unnecessary medical and economic expense.Table 3 RECOMMENDATIONS FOR CLASSIFICATION AND ANTIBIOTIC TREATMENT

influenzae and M. catarrhalis are increasingly prevalent, fluoroquinolones are the suggested first-line treatment. Group 4 patients have chronic suppurative lung disease, particularly bronchiectasis. Antibiotic therapy is directed at P. aeruginosa and other commonly drug-resistant gram-negative bacteria (see Table 3). As noted, approaches to antibiotic therapy based upon a rational appraisal of patient risk factors and likely pathogens reduce the risk for treatment failure and avoid unnecessary medical and economic expense.Table 3 RECOMMENDATIONS FOR CLASSIFICATION AND ANTIBIOTIC TREATMENT Group Clinical State Risk Factors Probable Pathogens First Choice Alternatives I Acute tracheobronchitis None Viral, rarelyM. pneumoniae orC. pneumoniae No antibiotics Macrolide or tetracycline (for persistent symptoms) II Acute exacerbation of chronic bronchitis None H. influenzae, Haemophilus spp,M. catarrhalis,S. pneumoniae Amoxicillin, tetracycline, TMP/SMX Second generation cephalosporin, second generation macrolide, amoxicillin/clavulanate,fluoroquinolone III Acute exacerbation of chronic bronchitis with risk factors Multiple* Same as above. Also consider gram-negatives especially in patients with severely impaired lung function. Fluoroquinolone Amoxicillin/clavulanate, oral second or third generation cephalosporin, or second generation macrolide IV Chronic suppurative airway disease Most have bronchiectasis Same as group IIIplus multiresistantgram-negatives, particularlyP. aeruginosa Antipseudomonal fluoroquinolone (ciprofloxacin) Consider parenteral therapy with antipseudomonal agents * FEV1 < 50% predicted, frequent exacerbations, significant comorbid conditions, malnutrition, chronic steroid use, mucous hypersecretion, duration of COPD >10 years, previous pneumonia. TMP/SMX = Trimethoprim/sulphamethoxazole.

nal fluoroquinolone (ciprofloxacin) Consider parenteral therapy with antipseudomonal agents * FEV1 < 50% predicted, frequent exacerbations, significant comorbid conditions, malnutrition, chronic steroid use, mucous hypersecretion, duration of COPD >10 years, previous pneumonia. TMP/SMX = Trimethoprim/sulphamethoxazole. There are outcome data to suggest this approach leads to improved clinical outcomes, with reduced overall costs.37 A retrospective study by Destache and colleagues37 demonstrated that, compared with the usual first-line antibiotics in the treatment of acute exacerbations of chronic bronchitis, the use of newer antibiotics reduced both the hospitalization rate and failure rate. Although the acquisition cost of newer antibiotics (cephalosporins, macrolides and fluoroquinolones) was higher, the overall costs of the treated patients given these drugs were lower. In particular, the group receiving amoxicillin/clavulanate, azithromycin, or ciprofloxacin had the lowest hospitalization rate, clinical failure rate, and costs compared with cephalosporins or first-line therapy.

s and fluoroquinolones) was higher, the overall costs of the treated patients given these drugs were lower. In particular, the group receiving amoxicillin/clavulanate, azithromycin, or ciprofloxacin had the lowest hospitalization rate, clinical failure rate, and costs compared with cephalosporins or first-line therapy. The hypothesis that aggressive antibiotic therapy should be offered to high-risk patients was tested in a recent, prospective, health economic study.54 Patients with at least three treated exacerbations of chronic bronchitis in the past year were randomized to receive either ciprofloxacin or any nonquinolone-based therapy for their next acute exacerbation. Clinical endpoints (days of illness, hospitalizations, time to next exacerbation) were blended with quality-of-life measurements (Nottingham Health Profile, St. George's Hospital Respiratory Questionnaire, Health Utility Index), and total respiratory costs from a societal perspective. Although the overall results indicated no advantage for either treatment arm, in patients with risk factors (severe underlying lung disease, more than four exacerbations per year, duration of bronchitis greater than 10 years, elderly, significant comorbid illness) the use of ciprofloxacin led to improved clinical outcome, higher quality of life, and fewer costs. The results of this study would suggest that aggressive antimicrobial therapy directed especially toward resistant organisms in high-risk patients is a more effective strategy than no therapy or therapy with older antimicrobials that would not be effective against the usual target organisms, particularly β-lactamase-producing H. influenzae.

udy would suggest that aggressive antimicrobial therapy directed especially toward resistant organisms in high-risk patients is a more effective strategy than no therapy or therapy with older antimicrobials that would not be effective against the usual target organisms, particularly β-lactamase-producing H. influenzae. Further studies are needed to clarify the optimal antibiotic treatment regimens for subgroups of patients with AECOPD. Corticosteroids Randomized, controlled trials of the efficacy of corticosteroids for acute exacerbations of COPD are summarized in Table 4 . Their role in outpatient exacerbations has been evaluated in only one small study.125 Compared with placebo, oral prednisone (60 mg tapered to 0 mg over 9 days) significantly improved airflow and oxygenation and resulted in fewer treatment failures. FEV1 improved on average by only 50 mL per day among patients receiving prednisone.99 These findings support earlier retrospective data that suggest that, among patients with AECOPD presenting to an emergency department (ED), the incidence of revisit to the ED within 48 hours is significantly reduced if corticosteroids are prescribed.82 Table 4 SUMMARY OF RANDOMIZED CONTROLLED TRIALS OF CORTICOSTEROIDS FOR ACUTE EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE

hat, among patients with AECOPD presenting to an emergency department (ED), the incidence of revisit to the ED within 48 hours is significantly reduced if corticosteroids are prescribed.82 Table 4 SUMMARY OF RANDOMIZED CONTROLLED TRIALS OF CORTICOSTEROIDS FOR ACUTE EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE Study Population Intervention n Outcome P Niewoehner et al88 1999 Inpatients Steroid × 8 wks 80 Faster recovery of FEV1 <0.05 FEV1 < 0.8 L Steroid × 2 wks 80 Fewer treatment failures <0.05 Placebo 111 No difference between 2 and 8 wks ns Davies et al (A)35 1997 Inpatients Prednisolone × 2 wks 28 Faster recovery of FEV1 <0.05 FEV1 = 0.61 L Placebo 22 Thompson et al125 1996 Outpatients Prednisone × 9 d 13 Improved FEV1 and oxygenation <0.05 FEV1 = 1.3 L Placebo 14 Fewer treatment failures <0.05 Bullard et al22 1996 ER patients Steroid × 5 d 60 Faster recovery of FEV1 by 6 hr <0.05 FEV1 < 0.55 L Placebo 52 Rostom et al (A)102 1994 Inpatients Steroid × 30 d Total No difference in FEV1 at 30 d ns Placebo 30 20% dropout rate Emerman et al43 1989 ER patients MPS 100 mg IV × 1 52 No difference in FEV1 ns FEV1 < 30% Placebo 44 No difference in admission rates Albert et al3 1980 Inpatients MPS × 3 d 22 Faster recovery of FEV1 <0.01 FEV1 < 0.64 Placebo 22 No difference in ABGs ns FEV1 = Forced expiratory volume in one second; A = published in abstract form only; ER = emergency room; MPS = methylprednisolone; ABG = arterial blood gas; IV = intravenously; ns = not significant.

Albert et al3 1980 Inpatients MPS × 3 d 22 Faster recovery of FEV1 <0.01 FEV1 < 0.64 Placebo 22 No difference in ABGs ns FEV1 = Forced expiratory volume in one second; A = published in abstract form only; ER = emergency room; MPS = methylprednisolone; ABG = arterial blood gas; IV = intravenously; ns = not significant. Corticosteroids are often given initially by ED physicians and several trials have examined this practice.22, 43 Emerman et al43 studied the effect of a single dose of methylprednisolone (100 mg intravenously [IV]) upon pulmonary function and hospitalization rates for 98 patients in the ED with COPD exacerbations. They failed to show any improvement in spirometry or decrease in the rate of hospitalization. Patients were treated for approximately 4.5 hours and the single steroid dose and short period of observation, however, have been postulated to account for the apparent lack of efficacy of steroids in this study.74 More recently, Bullard et al22 demonstrated a beneficial effect of steroids upon flow rates as early as 6 hours after initiation of treatment.

ly 4.5 hours and the single steroid dose and short period of observation, however, have been postulated to account for the apparent lack of efficacy of steroids in this study.74 More recently, Bullard et al22 demonstrated a beneficial effect of steroids upon flow rates as early as 6 hours after initiation of treatment. In 1980, Albert3 provided the initial justification for the routine use of systemic steroids in the care of hospitalized patients with COPD exacerbations. Forty-four patients with COPD and acute respiratory insufficiency were randomized to placebo or methylprednisolone 0.5 mg/kg intravenously every 6 hours for 3 days. Those treated with steroids were significantly more likely to achieve a 40% or greater increase in FEV1 over their baseline. This effect was observed by 12 hours following the start of treatment and persisted for 72 hours. Corticosteroids improved postbronchodilator lung function more than placebo but had minimal effect upon total symptoms in another small trial in which hospitalized patients were randomized to receive placebo or 30 mg of oral prednisolone for 14 days.35

hours following the start of treatment and persisted for 72 hours. Corticosteroids improved postbronchodilator lung function more than placebo but had minimal effect upon total symptoms in another small trial in which hospitalized patients were randomized to receive placebo or 30 mg of oral prednisolone for 14 days.35 Although the studies described have established that corticosteroids significantly increase FEV1 over the short term, no study was explicitly designed to capture longer-term endpoints or the adverse consequences associated with steroid therapy.88, 102 Rostom102 studied hospitalized patients with AECOPD given placebo or methylprednisolone (40 mg tapered to 0 mg over 1 month) and followed for 1 month after discharge. Mean FEV1 and FVC values were no different between the treatment groups at the end of the study. More recently, Niewoehner et al88 published the important Systemic Corticosteroids in Chronic Obstructive Pulmonary Disease Exacerbations trial. They performed a three-way randomization whereby 80 patients received an 8-week course of glucocorticoids, 80 received a 2-week course, and 111 received placebo. Steroids resulted in faster recovery of FEV1 and shortened hospital stay by 1 day (P < 0.05). At both 30 and 90 days, steroid therapy reduced treatment failures (defined as death from any cause, need for intubation, readmission, or intensification of drug therapy) by approximately 10%. There was no difference between 2 and 8 weeks of treatment with respect to spirometry or treatment failure rates, however. The dose of methylprednisolone was high (125 mg every 6 hours for 3 days) and resulted in significantly more hyperglycemia and, possibly, increased secondary infection rates.106

approximately 10%. There was no difference between 2 and 8 weeks of treatment with respect to spirometry or treatment failure rates, however. The dose of methylprednisolone was high (125 mg every 6 hours for 3 days) and resulted in significantly more hyperglycemia and, possibly, increased secondary infection rates.106 In summary, the evidence from randomized, controlled trials supports the conclusion that among patients with acute exacerbations, oral or intravenous corticosteroids significantly increase the FEV1 for up to 72 hours and likely reduce the risk for treatment failure. There is no proved benefit for treatment longer than 2 weeks. Hyperglycemia is the most common short-term complication of steroid treatment. As further studies become available, it will be possible to better understand the risk–benefit ratio for corticosteroids and, through meta-analysis, to better define the optimum dose and duration of therapy.136 It is also important to investigate the long-term risk for adverse effects of intermittent corticosteroids in patients who require them for recurrent exacerbations over many years time.

d the risk–benefit ratio for corticosteroids and, through meta-analysis, to better define the optimum dose and duration of therapy.136 It is also important to investigate the long-term risk for adverse effects of intermittent corticosteroids in patients who require them for recurrent exacerbations over many years time. Noninvasive Positive-Pressure Ventilation Noninvasive positive-pressure ventilation (NPPV) is arguably the most significant recent advance in the care of patients with COPD with acute respiratory failure. It avoids the complications of endotracheal intubation and preserves airway defense mechanisms while allowing patients to eat, speak, and expectorate secretions. Acute respiratory failure in COPD is often characterized by a vicious circle wherein the respiratory muscles must meet ever-increasing ventilatory demands under conditions of worsening hypoxemia, hypercapnia, and acidosis. When the increased metabolic requirements of the respiratory muscles cannot be matched by a commensurate rise in the cardiac output, further acidosis and muscle fatigue ensue. By allowing the muscles to rest, NPPV interrupts this process, thereby preventing respiratory arrest and death.77 Table 5 summarizes the randomized, controlled trials of NPPV in AECOPD.Table 5 SUMMARY OF RANDOMIZED CONTROLLED TRIALS OF NONINVASIVE POSITIVE-PRESSURE VENTILATION FOR ACUTE EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE Rights were not granted to include this data in electronic media. Please refer to the printed journal.

Noninvasive Positive-Pressure Ventilation Noninvasive positive-pressure ventilation (NPPV) is arguably the most significant recent advance in the care of patients with COPD with acute respiratory failure. It avoids the complications of endotracheal intubation and preserves airway defense mechanisms while allowing patients to eat, speak, and expectorate secretions. Acute respiratory failure in COPD is often characterized by a vicious circle wherein the respiratory muscles must meet ever-increasing ventilatory demands under conditions of worsening hypoxemia, hypercapnia, and acidosis. When the increased metabolic requirements of the respiratory muscles cannot be matched by a commensurate rise in the cardiac output, further acidosis and muscle fatigue ensue. By allowing the muscles to rest, NPPV interrupts this process, thereby preventing respiratory arrest and death.77 Table 5 summarizes the randomized, controlled trials of NPPV in AECOPD.Table 5 SUMMARY OF RANDOMIZED CONTROLLED TRIALS OF NONINVASIVE POSITIVE-PRESSURE VENTILATION FOR ACUTE EXACERBATIONS OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE Rights were not granted to include this data in electronic media. Please refer to the printed journal. Bott et al18 randomized 60 patients with COPD and hypoxemic–hypercarbic respiratory failure to either conventional treatment or volume-cycled nasal NPPV. Patients in both groups had similar pretreatment blood gas and spirometric abnormalities. At 1 hour, there was a significant rise in pH and drop in Paco 2 in the NPPV group compared with conventional treatment. NPPV also resulted in significantly less breathlessness by day 3. Most importantly, however, intention-to-treat analysis revealed a trend toward lower 30-day mortality in the NPPV group (3/30 versus 9/30, relative risk = 0.33, 95% CI 0.1–1.11, P = ns). This effect became significant after excluding the four patients randomized to NPPV who did not receive it (two were confused, one was unable to breathe through his nose, one had all active treatment withdrawn). None of the patients randomized to NPPV required intubation and no serious complications of NPPV were reported. The study has been criticized for the lack of standardized treatment in both groups and for the unusually high mortality in the control group.76

athe through his nose, one had all active treatment withdrawn). None of the patients randomized to NPPV required intubation and no serious complications of NPPV were reported. The study has been criticized for the lack of standardized treatment in both groups and for the unusually high mortality in the control group.76 Kramer et al65 investigated the impact of NPPV on need for intubation among 31 patients with severe hypercarbic respiratory failure, most with COPD exacerbations. Sixteen patients (11 with COPD) were randomized to pressure-limited nasal NPPV in addition to standard care and 15 patients (12 with COPD) received standard care alone. Clear a priori indications for intubation were given. Significantly, only five patients (31%) in the NPPV group required intubation, compared with 11 (73%) in the standard therapy arm. Furthermore, maximal inspiratory pressures increased significantly in the NPPV arm over 6 hours, indicating a rapid reversal of diaphragmatic fatigue. In contrast to work by Bott,18 however, there were no significant differences in Paco 2 between the treatment groups at any time over the first 24 hours. The study was underpowered to detect differences in mortality.

eased significantly in the NPPV arm over 6 hours, indicating a rapid reversal of diaphragmatic fatigue. In contrast to work by Bott,18 however, there were no significant differences in Paco 2 between the treatment groups at any time over the first 24 hours. The study was underpowered to detect differences in mortality. A more recent European multicenter trial21 randomized 85 patients to standard therapy or pressure-limited NPPV by face mask. All patients required admission to an intensive care unit and were followed until death or hospital discharge. Noninvasive ventilation markedly reduced the need for intubation (controls 74% versus NPPV 26%). Compared with standard care, NPPV also significantly reduced mortality (9% versus 29%), complication rates (16% versus 48%), and mean duration of hospital stay (23 days versus 35 days) (all P < 0.05).21

hospital discharge. Noninvasive ventilation markedly reduced the need for intubation (controls 74% versus NPPV 26%). Compared with standard care, NPPV also significantly reduced mortality (9% versus 29%), complication rates (16% versus 48%), and mean duration of hospital stay (23 days versus 35 days) (all P < 0.05).21 More recently, Çelikel et al30 compared pressure-limited NPPV by face mask with usual care among patients with moderately severe hypercarbic acute respiratory failure and COPD. Noninvasive positive-pressure ventilation resulted in significantly fewer treatment failures, defined as need for intubation in the NPPV group and need for NPPV or intubation in the control group. Hospital stays were significantly shorter in the NPPV group. In contrast, Barbé et al,15 however, were unable to demonstrate any statistically significant benefit of nasal NPPV over conventional treatment in terms of duration of hospitalization, dyspnea scores, arterial blood gas measurements, or maximal inspiratory pressures. Patients in this trial, however, were clearly not as ill as those in other studies. Indeed, no patient in either group required intubation.

ficant benefit of nasal NPPV over conventional treatment in terms of duration of hospitalization, dyspnea scores, arterial blood gas measurements, or maximal inspiratory pressures. Patients in this trial, however, were clearly not as ill as those in other studies. Indeed, no patient in either group required intubation. Randomized, controlled trials of NPPV for the treatment of AECOPD with hypercarbic respiratory failure recently were reviewed systematically.63 The pooled odds ratio for intubation following NPPV is 0.12 (95% CI, 0.05–0.29). More importantly, however, the trials that included mortality as an outcome collectively demonstrate a strong survival benefit for NPPV.2, 18, 21 The pooled odds ratio for death is 0.22 (95% CI, 0.09–0.54). Therefore, at worst, NPPV increases the patient's chance of surviving by nearly 50%; at best, the chance of survival is 90% better than that of a similar patient not receiving NPPV. Improvements in pH and Paco 2 within 1 hour of initiating NPPV and good level of consciousness at the beginning of NPPV are associated with successful responses to NPPV in patients with AECOPD and respiratory acidosis.9

0%; at best, the chance of survival is 90% better than that of a similar patient not receiving NPPV. Improvements in pH and Paco 2 within 1 hour of initiating NPPV and good level of consciousness at the beginning of NPPV are associated with successful responses to NPPV in patients with AECOPD and respiratory acidosis.9 Nutritional Support and Physiotherapy Malnutrition is common among patients with COPD and increases the morbidity and mortality associated with the disease. The Veterans Administration Cooperative Study of Pulmonary Function98 showed that patients with FEV1 less than or equal to 0.5 L weighed less than 82% of their ideal body weight (IBW), compared with near normal body weight in less severely impaired patients. In another study,133 43% of patients with emphysema were found to weigh less than 90% of their IBW. Furthermore, Hunter58 reported that more than 70% of hospitalized patients with COPD had evidence of weight loss. Pingleton91 found that, among ventilated patients, mortality was significantly higher in those who were poorly nourished than in those with better nutritional status. Poorly nourished patients also had a significantly higher frequency of hypercapnia.

n 70% of hospitalized patients with COPD had evidence of weight loss. Pingleton91 found that, among ventilated patients, mortality was significantly higher in those who were poorly nourished than in those with better nutritional status. Poorly nourished patients also had a significantly higher frequency of hypercapnia. The principal effects of malnutrition upon the respiratory system are thought to be worsened respiratory muscle function, impairment of ventilatory drive, and immune dysfunction. Malnutrition impairs muscle function by reducing the availability of energy substrates such as glycogen and phosphate and by altering the structure of muscle fibers. When combined with intercurrent airway infection and the mechanical disadvantage of the diaphragm in COPD, malnutrition may have a profound effect on respiratory muscle mechanics. Experimental evidence for blunted hypoxic drive in response to semistarvation suggests another mechanism by which patients with COPD may be predisposed to respiratory failure.91, 133

ection and the mechanical disadvantage of the diaphragm in COPD, malnutrition may have a profound effect on respiratory muscle mechanics. Experimental evidence for blunted hypoxic drive in response to semistarvation suggests another mechanism by which patients with COPD may be predisposed to respiratory failure.91, 133 No randomized, controlled trial has demonstrated reduced morbidity and mortality as a result of nutritional support during acute respiratory failure. Nevertheless, clinicians should be able to identify malnourished patients and understand the goals of nutritional therapy. Particular attention should be paid to patients with hypercatabolic states that increase the risk for protein-calorie malnutrition. The Subjective Global Assessment and the Harris-Benedict equation are two valid clinical instruments for assessing malnutrition and planning nutritional therapy. They are reviewed elsewhere.38, 91 In general, the goals of nutritional supplementation among patients with acute respiratory failure consist of maintaining body weight and preventing protein breakdown. The effects of malnutrition and nutritional supplementation in patients with COPD are discussed in more detail in the article by Schols and Wouters in this issue.

, the goals of nutritional supplementation among patients with acute respiratory failure consist of maintaining body weight and preventing protein breakdown. The effects of malnutrition and nutritional supplementation in patients with COPD are discussed in more detail in the article by Schols and Wouters in this issue. The value of chest physiotherapy (postural drainage with or without chest percussion) in AECOPD has not been demonstrated. Studies of patients with AECOPD have failed to demonstrate a beneficial effect of chest physiotherapy upon sputum volume, gas exchange, or spirometry.66 One trial27 documented a transient but significant decrease in FEV1 as a result of bronchoconstriction following chest percussion and vibration. Some evidence suggests that patients with larger volumes of airway secretions (>25 mL/d), particularly those with bronchiectasis, may benefit. In some guidelines, therefore, chest physiotherapy has been advocated in this situation.7 In general, however, it is not recommended in the routine management of AECOPD.20

tion. Some evidence suggests that patients with larger volumes of airway secretions (>25 mL/d), particularly those with bronchiectasis, may benefit. In some guidelines, therefore, chest physiotherapy has been advocated in this situation.7 In general, however, it is not recommended in the routine management of AECOPD.20 OUTCOMES Although outcome data are important, caution is required in their interpretation. Because data are generated by observing large numbers of patients, typically in tertiary referral centers, the pertinence of these data to individual patients may be limited, especially outside of major centers where most COPD exacerbations are treated.33 Studies of prognosis in patients with COPD and acute respiratory failure performed during previous decades are less relevant by today's standards. Current outcomes appear to be better than those of the past, in part because of the widespread use of controlled O2 therapy, corticosteroids and IB, availability of better β2-agonists, reduced use of methylxanthines, and increased use of noninvasive ventilatory support.33 Indeed, there is evidence to support a trend toward improved survival for hypercapnic respiratory failure over the past 20 years. The mortality rates in studies of survival of acute respiratory failure in COPD conducted from 1968 to 1973 ranged from 22% to 34%, with an overall mortality of 26%. For similar studies between 1978 and 1992, the range is 6% to 12%, with an overall mortality of 10%.132

capnic respiratory failure over the past 20 years. The mortality rates in studies of survival of acute respiratory failure in COPD conducted from 1968 to 1973 ranged from 22% to 34%, with an overall mortality of 26%. For similar studies between 1978 and 1992, the range is 6% to 12%, with an overall mortality of 10%.132 The most recent and comprehensive evaluation of outcomes following AECOPD was published by Connors et al as a component of the landmark Study to Understand Prognoses for Outcomes and Risks of Treatment (SUPPORT) trial.33 They prospectively studied more than 1000 patients admitted to five US tertiary care hospitals with severe hypercarbic COPD exacerbations (initial Paco 2≥50 mm Hg). Baseline FEV1 was not available for most patients. Half the patients required intensive care unit admission and 35% required mechanical ventilation. Hospital mortality was 11%. More striking, however, was the finding that following discharge, one third of the patients died in within 6 months and one half within 2 years. Not surprisingly, patients who survived hospitalization had a substantial risk of discharge to a facility other than their home (20%) and of readmission to acute care over the ensuing 6 months (50%). Higher acute physiology score (Acute Physiology and Chronic Health Evaluation; APACHE III), older age, and poor functional status prior to admission independently increased risk of death. Improved survival was predicted by greater BMI and albumin level, higher Pao 2/Fio 2, and, surprisingly, the presence of cor pulmonale and congestive heart failure as the cause of the exacerbation. The latter findings may be explained by the good response of these two disorders to acute therapy.

ncreased risk of death. Improved survival was predicted by greater BMI and albumin level, higher Pao 2/Fio 2, and, surprisingly, the presence of cor pulmonale and congestive heart failure as the cause of the exacerbation. The latter findings may be explained by the good response of these two disorders to acute therapy. A study by Dewan and colleagues38a also supports the finding that host factors are principal determinants of outcomes of AECOPD. In their retrospective analysis of 232 exacerbations in 107 patients with COPD, severity of airflow obstruction, use of home O2, frequency of exacerbation, history of previous pneumonia or sinusitis, and use of maintenance corticosteroids each were independently associated with treatment failure. Surprisingly, age, choice of antibiotics, and presence of comorbid conditions did not affect the treatment outcome in that study.

tion, use of home O2, frequency of exacerbation, history of previous pneumonia or sinusitis, and use of maintenance corticosteroids each were independently associated with treatment failure. Surprisingly, age, choice of antibiotics, and presence of comorbid conditions did not affect the treatment outcome in that study. Several investigators62, 78, 100, 107 have evaluated the prognosis of patients with COPD who require ICU admission for acute exacerbations. The results have been somewhat contradictory. Kaelin et al,62 for example, using several easily obtainable indices, were unable to discriminate between patients surviving more or less than 6 months following intubation. In their analysis of 39 consecutive acute COPD exacerbations, neither age nor spirometric, blood gas, or nutritional indices predicted survival. In contrast, Menzies et al78 reported that, among their 95 patients, higher baseline FEV1 and serum albumin were significantly associated with improved 1-year survival following mechanical ventilation. These contradictory findings are especially curious given that both studies had similar inclusion and exclusion criteria and periods of observation. Moreover, their populations both had a mean percent-predicted FEV1 of 35% and nearly identical baseline values for serum albumin.

urvival following mechanical ventilation. These contradictory findings are especially curious given that both studies had similar inclusion and exclusion criteria and periods of observation. Moreover, their populations both had a mean percent-predicted FEV1 of 35% and nearly identical baseline values for serum albumin. More recently, Rieves et al100 tried to identify clinically useful variables that predict successful weaning from mechanical ventilation and short-term survival in patients with COPD with acute respiratory failure. They observed episodes of acute respiratory failure in 19 and 33 patients with baseline FEV1 of greater and less than 1 L respectively. Only 56% of the cohort with severe COPD survived weaning and spontaneous breathing for 72 hours. Furthermore, in the same group, 1-year survival was only 27%. Absence of infiltrates on chest radiograph was the most influential predictor of survival in patients with severe COPD. Pneumonia accounted for most of the infiltrates that were seen in this group. Baseline FEV1 obtained during a period of clinical stability prior to the episode of acute respiratory failure was available for all patients. The extent of baseline obstruction alone was not statistically correlated with short-term survival in either group, but the combination of severe baseline obstruction and pulmonary infiltrates markedly increased the risk for death. Outcomes following AECOPD associated with ICU admission and respiratory failure are discussed further in the article by Sethi and Siegel in this issue.

correlated with short-term survival in either group, but the combination of severe baseline obstruction and pulmonary infiltrates markedly increased the risk for death. Outcomes following AECOPD associated with ICU admission and respiratory failure are discussed further in the article by Sethi and Siegel in this issue. Seneff et al107 recently refined the discussion over the relative prognostic value of different clinical variables following COPD exacerbation. They analyzed 362 admissions for acute COPD exacerbation from the APACHE III database. Hospital mortality was 24%. For patients aged 65 and older, hospital and 1-year mortality rates were 30% and 59% respectively. Their report emphasizes that individual clinical variables have different value for predicting short- and long-term survival. Patient age, for example, was a statistically significant determinant of 6-month survival but not influential for hospital mortality after accounting for nonrespiratory organ dysfunction. Similarly, the presence of hypercarbia was of no value in predicting hospital mortality but became important over the long term; 1-year mortality rates for patients with admission Paco 2 of less than 50 mm Hg versus greater than 50 mm Hg were 54% and 70% respectively. The most significant predictors of short and long-term mortality are development and severity of multiple organ dysfunction syndrome. Respiratory dysfunction is more important over the longer term. As the authors state: “In most cases, the acute, life-threatening components of the exacerbations can be reversed and short-term death avoided by mechanical ventilation and other appropriate treatments. However, because abnormalities in respiratory physiology reflect underlying severity of lung disease, patients with greater abnormality who survive hospitalization are at greater risk of subsequent death.”107

ations can be reversed and short-term death avoided by mechanical ventilation and other appropriate treatments. However, because abnormalities in respiratory physiology reflect underlying severity of lung disease, patients with greater abnormality who survive hospitalization are at greater risk of subsequent death.”107 SUMMARY Chronic obstructive pulmonary disease is the only leading cause of death with a rising prevalence. The medical and economic costs arising from acute exacerbations of COPD are therefore expected to increase over the coming years. Although exacerbations may be initiated by multiple factors, the most common identifiable associations are with bacterial and viral infections. These are associated with approximately 50% to 70% and 20% to 30% of COPD exacerbations, respectively. In addition to smoking cessation, annual influenza vaccination is the most important method for preventing exacerbations. Controlled O2 is the most important intervention for patients with acute hypoxic respiratory failue. Evidence from randomized, controlled trials justifies the use of corticosteroids, bronchodilators (but not theophylline), noninvasive positive-pressure ventilation (in selected patients), and antibiotics, particularly for severe exacerbations. Antibiotics should be chosen according to the patient's risk for treatment failure and the potential for antibiotic resistance. In the acute setting, combined treatment with β-agonist and anticholinergic bronchodilators is reasonable but not supported by randomized controlled studies. Physicians should identify and, when possible, correct malnutrition. Chest physiotherapy has no proven role in the management of acute exacerbations.

resistance. In the acute setting, combined treatment with β-agonist and anticholinergic bronchodilators is reasonable but not supported by randomized controlled studies. Physicians should identify and, when possible, correct malnutrition. Chest physiotherapy has no proven role in the management of acute exacerbations. Address reprint requests to Ronald F. Grossman, MD, Division of Respiratory Medicine, Department of Medicine, Mount Sinai Hospital, Suite 640, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5, e-mail: ronaldf.grossman@utoronto.ca

VIRAL INFECTIONS AND THE INCEPTION OF ASTHMA Infections with respiratory syncytial virus (RSV) or parainfluenza virus (PIV) have received much attention because of their predilection to produce a pattern of symptoms termed bronchiolitis that parallels many of the features of childhood and adult asthma.67 Respiratory syncytial virus causes about 70% of these episodes and it is estimated that, by age 1 year, 50% to 65% of children will have been infected with this virus77 and 40% of these infections involve the lower respiratory tract.92 By age 2, nearly all children will have been infected with RSV at least once. Children aged 3 to 6 months are most prone to develop lower respiratory tract symptoms, suggesting that a developmental component (e.g., lung or immunologic maturation) may be involved as well. 77, 83 Figure 1 Mechanisms by which viruses may influence either the inception of asthma or exacerbations of the underlying disease process once it has been established. RSV = respiratory syncytial virus; PIV = parainfluenza virus.

developmental component (e.g., lung or immunologic maturation) may be involved as well. 77, 83 Figure 1 Mechanisms by which viruses may influence either the inception of asthma or exacerbations of the underlying disease process once it has been established. RSV = respiratory syncytial virus; PIV = parainfluenza virus. The relationship between RSV infections during the first few years of life and the subsequent development of the asthmatic phenotype has been the subject of much interest as well as controversy. Variations in reporting longitudinal outcomes (e.g., recurrent wheezing, measurements of airway hyperresponsiveness, diagnosis of asthma) appear to be influenced mostly by the criteria used to define “bronchiolitis.” These criteria include the type of virus producing the symptoms (in addition to RSV, viruses that may contribute to the development of bronchiolitis in this age group could be PIV, coronavirus, influenzavirus, and rhinovirus33); the age at the time of infection; the nature and severity of symptoms required for inclusion; and, finally, the characteristics of both the study population (community versus hospital-based) and the study design (retrospective versus prospective). A number of long-term prospective studies of children admitted to a hospital with documented RSV-induced bronchiolitis have shown that about 75% experience wheezing in the first 2 years after the initial illness, more than 50% still wheeze 3 years later, and approximately 40% continue to wheeze after 5 years. 42, 49, 73, 91, 117, 121

g-term prospective studies of children admitted to a hospital with documented RSV-induced bronchiolitis have shown that about 75% experience wheezing in the first 2 years after the initial illness, more than 50% still wheeze 3 years later, and approximately 40% continue to wheeze after 5 years. 42, 49, 73, 91, 117, 121 Additional insight into these areas recently was provided by the results of an 11-year prospective study involving 880 children who were enrolled at birth, followed for the development of lower respiratory tract illnesses (LRIs) in the first 3 years of life, and then evaluated for the presence or absence of physician-diagnosed asthma or a history of current wheezing at ages 6 and 11 years.14 Most importantly, lung function was evaluated in the first few months of life in a subset of these children prior to the development of a documented LRI. During the first 3 years of life, 7.4% had pneumonia documented radiographically and 44.7% had a significant LRI without pneumonia. Respiratory syncytial virus and PIV were identified in 36.4% and 7.3%, respectively, in the subjects with pneumonia, and in 35.6% and 15.2%, respectively, of the subjects with a LRI. At age 6, physician-diagnosed asthma was present in 13.6% (OR = 3.3), 10.2% (OR = 2.4), and 4.6% of the subjects with pneumonia, LRI, and no LRI, respectively. By age 11, these values increased to 25.9% (OR = 2.8), 16.1% (OR = 1.6), and 11%, respectively. Mean maximum volume at functional residual capacity values before any LRI were lower in children with pneumonia and with LRIs than in children with no LRIs. These values continued to be lower at age 6 and by age 11, when forced expiratory volume in 1 second (FEV1) and FEF25–75 were recorded, similar group relationships persisted. Interestingly, despite the persistence of lowered baseline lung function in both the pneumonia and LRI groups, many of these deficits were markedly (but not completely) reduced following administration of albuterol.

expiratory volume in 1 second (FEV1) and FEF25–75 were recorded, similar group relationships persisted. Interestingly, despite the persistence of lowered baseline lung function in both the pneumonia and LRI groups, many of these deficits were markedly (but not completely) reduced following administration of albuterol. In a second report, further follow-up of this large cohort of children demonstrated that the risk for both frequent (more than three episodes of wheezing per year) and infrequent (three episodes of wheezing per year) wheezing in relation to RSV lower respiratory illnesses decreased markedly with age and became nonsignificant by age 13.104 These data suggest that, although RSV infections contribute substantially to the expression of the asthmatic phenotype, other factors (e.g., genetic, environmental, developmental) appear to contribute as well, either in terms of its initial expression or the modification of the phenotype over time. CONTRIBUTION OF ATOPY In addition to premorbid lung function, the influence of atopy on the development of the asthmatic phenotype in relationship to viral infections has also been evaluated. Interactions between these two factors appear to be bidirectional and dynamic, in that the atopic state can influence the lower airway response to viral infections, 8, 71 viral infections can influence the development of allergen sensitization, 28, 29, 99 and interactions can occur when individuals are exposed simultaneously to both allergens and viruses. 12, 68, 100

be bidirectional and dynamic, in that the atopic state can influence the lower airway response to viral infections, 8, 71 viral infections can influence the development of allergen sensitization, 28, 29, 99 and interactions can occur when individuals are exposed simultaneously to both allergens and viruses. 12, 68, 100 Atopy can be defined as the genetic predisposition to the preferential development of an immunoglobulin (Ig)E antibody response to a variety of environmental allergens. As stated previously, atopy has been considered to be a risk factor for the development of childhood asthma and its influence on the pattern of responses following viral infections has been of interest to many investigative groups. It has also been suggested that atopy could be a significant predisposing factor for the development of acute bronchiolitis during RSV epidemics.64 Although some have found that children most likely to have persistent wheezing were those born to atopic parents, 64, 91, 121 others have not. 14, 73, 84 Some have found that personal atopy is not more prevalent in symptomatic children after bronchiolitis14, 73; others have found that documented RSV bronchiolitis significantly increases a child's chances (32% versus 9% in controls) of subsequently developing IgE antibody99 or lymphocyte proliferative responses75 to both food and aeroallergens.

sonal atopy is not more prevalent in symptomatic children after bronchiolitis14, 73; others have found that documented RSV bronchiolitis significantly increases a child's chances (32% versus 9% in controls) of subsequently developing IgE antibody99 or lymphocyte proliferative responses75 to both food and aeroallergens. RESPIRATORY SYNCYTIAL VIRUS AND THE IMMUNE RESPONSE Respiratory syncytial virus infections may interact with immunoinflammatory mechanisms involved in immediate hypersensitivity responses in a number of ways.18 First, it has been suggested that viruses capable of infecting lower airway epithelium may lead to enhanced absorption of aeroallergens across the airway wall, predisposing to subsequent sensitization. 27, 94 Second, RSV-specific IgE antibody formation may lead to mast-cell–mediator release within the airway, resulting in the development of bronchospasm and the ingress of eosinophils. 32, 60, 85, 115, 119, 120 Third, airway resident and inflammatory cell generation of various cytokines (tumor necrosis factor [TNF], interleukin [IL]-1, IL-6, IL-8), 4, 81, 106, 109 chemokines (MIP-1-, RANTES, MCP-1), 47, 76 leukotrienes, 113 and adhesion molecules (intercellular adhesion molecule)81 may further upregulate the ongoing inflammatory response. Finally, similar to various allergenic proteins, 17 the processing of RSV antigens and their subsequent presentation to lymphocyte subpopulations may provide a unique mechanism of interaction to promote a T-helper 2 (Th2)-like response in a predisposed host.

)81 may further upregulate the ongoing inflammatory response. Finally, similar to various allergenic proteins, 17 the processing of RSV antigens and their subsequent presentation to lymphocyte subpopulations may provide a unique mechanism of interaction to promote a T-helper 2 (Th2)-like response in a predisposed host. Respiratory syncytial virus belongs to the family Paramyxoviridae, the genera Pneumovirus, and can be differentiated into two serologic subgroups, A and B. 44, 77 It has 10 genes, with 12 potential gene products. The G (attachment) and F (fusion) proteins are the major surface glycoproteins against which neutralizing antibody is directed. Interestingly, in both murine2 and human51 in vitro experiments, it has been noted that the G protein elicits a predominant Th2 response, whereas the F protein produces a predominant Th1 response. In mice, to test the activities of T cells recognizing individual RSV proteins in vivo, virus-specific T-cell lines have been produced using recombinant vaccinia viruses that express either the G or F proteins. Following passive transfer of these cell lines to naive recipients and subsequent intranasal inoculation with RSV, mice receiving G-specific cells have more severe illnesses, characterized by lung hemorrhage, pulmonary neutrophil recruitment, and intense pulmonary eosinophilia.1 These experiments are of interest based on the adverse clinical response noted in many infants who received a formalin-inactivated RSV vaccine and subsequently became infected with RSV.77

have more severe illnesses, characterized by lung hemorrhage, pulmonary neutrophil recruitment, and intense pulmonary eosinophilia.1 These experiments are of interest based on the adverse clinical response noted in many infants who received a formalin-inactivated RSV vaccine and subsequently became infected with RSV.77 These intriguing observations regarding RSV and its influence on Th1/Th2 responses have recently been expanded. Roman et al evaluated 15 hospitalized infants (1–15 months) with an acute lower respiratory tract infection caused by RSV. Compared with control infants, peripheral blood cells from infected children had suppressed IFN-γ production ex vivo and, although IL-4 production was also decreased, the IL-4/IFN-γ ratio was significantly increased. Renzi et al88 prospectively followed 26 infants hospitalized with bronchiolitis by obtaining blood samples at the time of illness and 5 months later, and found that immune responses during the acute infection correlated with long-term pulmonary outcomes. Blood lymphocytes, obtained during the time of bronchiolitis, produced less IFN-γ ex vivo in response to IL-2 and more IL-4 in response to D. farinae antigen in children who went on to develop a pattern of recurrent wheezing.88 Finally, lower IFN-γ production at the time of bronchiolitis has been demonstrated to be an indicator of reduced pulmonary function and increased responsiveness to histamine 5 months after bronchiolitis, and was related to the development of asthma after bronchiolitis in infants.87 In contrast, other groups have noted increased levels of IFN-γ respiratory tract secretions during RSV illnesses in infants and children with bronchiolitis and recurrent wheezing compared with those with upper respiratory tract symptoms only.113 Unfortunately, in all of the studies reported thus far, the pattern of cytokine response these infants had prior to infection was not evaluated, begging the question as to which of the observed results may be cause and which effect.

d recurrent wheezing compared with those with upper respiratory tract symptoms only.113 Unfortunately, in all of the studies reported thus far, the pattern of cytokine response these infants had prior to infection was not evaluated, begging the question as to which of the observed results may be cause and which effect. ANIMAL MODELS To more comprehensively evaluate the relationships among virus infection, atopy (cytokine dysregulation of Th1/Th2 imbalance), and immune system or lung developmental components, a rat model of virus-induced airway dysfunction has been studied extensively.111 In this model, infection with PIV type 1 during a critical developmental time period (when the animals are weaning [3–4 weeks of age] as opposed to when they are neonates [4–5 days] or adults) produces chronic (8–12 weeks following infection), episodic, reversible airway inflammation and remodeling with associated alterations in airway physiology (increased resistance and methacholine responsiveness) that resemble human asthma in high (brown Norway strain) but not low (F344 strain) IgE-antibody producing rats.62 The temporal progression of this asthma-like syndrome is associated with a Th1/Th2 imbalance within the lung, and its development can be significantly attenuated by the exogenous administration of IFN-8 just prior to and during the viral infection in the brown Norway responder strain.102 This model further supports the concept of both genetic (atopy; cytokine dysregulation or imbalance) and environmental factors (virus infection) being important in the inception of the asthmatic phenotype, as well as a developmental component contributing.

ng the viral infection in the brown Norway responder strain.102 This model further supports the concept of both genetic (atopy; cytokine dysregulation or imbalance) and environmental factors (virus infection) being important in the inception of the asthmatic phenotype, as well as a developmental component contributing. EFFECT OF VIRAL INFECTIONS IN PATIENTS WITH ASTHMA Respiratory viruses are common causes of asthma exacerbations in asthmatic subjects of different age groups. 57, 74, 86 Serology or culture detection methods of viruses initially indicated an association during asthma exacerbations82 despite the fact that these detection methods are relatively insensitive for viruses such as rhinovirus (RV). The use of reverse transcription polymerase chain reaction (RT-PCR) assays that are more sensitive for detection of RV have confirmed and expanded these initial observations.58 Indeed, Johnston et al57 found that 80% to 85% of school-aged children with acute wheezing episodes tested positive for a virus using RT-PCR and other standard virologic techniques. The virus most often detected was RV. Seasonal patterns of upper respiratory virus infections correlate closely with hospital admissions for asthma, particularly in pediatric age groups.55 These studies indicate that RV infections are the most common cause of asthma exacerbations in children, especially during spring and fall. Similar studies, performed in adults, 74 found that about half of asthma exacerbations were associated with RV infection.

ssions for asthma, particularly in pediatric age groups.55 These studies indicate that RV infections are the most common cause of asthma exacerbations in children, especially during spring and fall. Similar studies, performed in adults, 74 found that about half of asthma exacerbations were associated with RV infection. As discussed previously, in infancy, atopy may define a susceptibility of the host to wheezing with respiratory infections. Duff et al, 22 for example, studied children who presented to an emergency department with wheezing. Children over 2 years of age were more likely to have respiratory allergies or a confirmed respiratory viral infection compared with children with no wheezing. Children with the highest risk for wheezing were those who had respiratory allergies and respiratory viral infection, implying that respiratory viral infections and respiratory allergies may have synergistic effects on lower airway physiology and enhance the likelihood of wheezing with respiratory infection. In children less than 2 years of age, wheezing was also noted, but risk factors for wheezing were quite different. These infants were not allergic, had RSV as the major viral isolate, and had passive tobacco smoke exposure as a major risk factor for wheezing.

hance the likelihood of wheezing with respiratory infection. In children less than 2 years of age, wheezing was also noted, but risk factors for wheezing were quite different. These infants were not allergic, had RSV as the major viral isolate, and had passive tobacco smoke exposure as a major risk factor for wheezing. MECHANISMS OF VIRAL-INDUCED AIRWAY OBSTRUCTION AND ASTHMA Development of Variable Airway Obstruction Available epidemiologic data in children and adults have shown that episodic drops in peak flow measurements are associated with RV infections. This was found to correlate with an increase in asthma symptoms and nonspecific airway hyperresponsiveness following experimentally infecting asthmatic subjects with RV. 15, 41 Further studies by Grünberg et al40 demonstrated that experimental RV16 infection leads to a transient drop in daily home recordings of FEV1 in subjects with asthma. This variable airway obstruction correlated significantly with cold symptoms, asthma symptoms, and the increase in airway hyperresponsiveness to histamine. Such daily variability in FEV1 reflects the inflammatory changes within the airway wall, which can be induced by the natural RV infection.

in subjects with asthma. This variable airway obstruction correlated significantly with cold symptoms, asthma symptoms, and the increase in airway hyperresponsiveness to histamine. Such daily variability in FEV1 reflects the inflammatory changes within the airway wall, which can be induced by the natural RV infection. Increased Bronchial Hyperresponsiveness Increased bronchial responsiveness has been found in normal and asthmatic subjects following infections with RV68 and influenza A. 65, 66, 72 In a study by Cheung et al15 14 subjects with mild asthma were inoculated with RV16 or placebo. The maximal contractile response to inhaled methacholine was significantly greater during the RV16 infection and remained elevated for up to 15 days after the acute infection. This study indicates that an upper respiratory viral infection can enhance the reactivity of the lower airway and the magnitude of bronchonstriction changes, which can persist for weeks after the acute infection. Respiratory viral infections' effect on lower airway responses are also influenced by host factors. In particular, allergic subjects experience greater changes in airway responsiveness after viral infection than nonallergic control subjects. 9, 34 Furthermore, subjects with lower FEV1 values tend to have greater changes in airway responsiveness during viral infection.34 These studies suggest that effects of pre-existing conditions such as allergy and intrinsic lower airway function on caliber are likely to contribute to airway hyperresponsiveness during respiratory viral infection.

s with lower FEV1 values tend to have greater changes in airway responsiveness during viral infection.34 These studies suggest that effects of pre-existing conditions such as allergy and intrinsic lower airway function on caliber are likely to contribute to airway hyperresponsiveness during respiratory viral infection. Neural Control of the Airways Potential mechanisms through which viral infections could potentially cause bronchoconstriction and increased airway responsiveness include enhancing parasympathetic bronchoconstrictive responses, stimulation of airway sensory nerves, and interference with the bronchodilatory functions of the nonadrenergic, noncholinergic neurons (Table 1) . Because of difficulties in assessing dysfunction of pulmonary neural regulation in humans, most data that support these proposed mechanisms were derived in animal models of acute respiratory viral infection. Further definition of these pathways in humans will depend upon the development of new experimental techniques or inhibition of specific neural pathways.Table 1 NEURAL MECHANISMS IMPLICATED IN VIRUS-INDUCED AIRWAY DYSFUNCTION

these proposed mechanisms were derived in animal models of acute respiratory viral infection. Further definition of these pathways in humans will depend upon the development of new experimental techniques or inhibition of specific neural pathways.Table 1 NEURAL MECHANISMS IMPLICATED IN VIRUS-INDUCED AIRWAY DYSFUNCTION Effect of Virus Potential Mechanisms References Heightened • Increased efferent activity of efferent cholinergic nerves Buckner et al11 parasympathetic • Viral neuraminidase Fryer et al30, 31 responses • Eosinophil cationic protein-induced M2 dysfunction Jacoby et al52 • M2-independent mechanisms Sorkness et al101 Bronchoconstriction Enhanced contractile responses to neurokinins Jacoby et al53 secondary to sensory Ladenius et al63 C-fibers Roberts et al89 Saban et al93 Inhibition of nonadrenergic–noncholinergic neurons Reduced production of nitric oxide Colasurdo et al16

coby et al52 • M2-independent mechanisms Sorkness et al101 Bronchoconstriction Enhanced contractile responses to neurokinins Jacoby et al53 secondary to sensory Ladenius et al63 C-fibers Roberts et al89 Saban et al93 Inhibition of nonadrenergic–noncholinergic neurons Reduced production of nitric oxide Colasurdo et al16 Structural Effects on the Small Airways Changes in small airways structure and function may also contribute significantly to the severity of hyperinflation and gas exchange abnormalities noted in acute asthma exacerbations. The maximal airway contractile response to methacholine in mild asthmatic subjects is increased during a cold, which is probably secondary to excessive airway narrowing attributable to airway wall thickening, airway parenchymal uncoupling, or abnormalities in smooth muscle contraction.15 Significant changes in airway morphology are noticed in animals with acute viral respiratory illness that leads to marked bronchiolar narrowing and plugging. These changes include bronchiolar airway edema and cell infiltration, epithelial hyperplasia, and folding and sloughing of airway epithelial surfaces. In addition, rats with mild increases in pulmonary resistance and methacholine sensitivity during acute viral respiratory illness have evidence of air trapping and ventilation–perfusion mismatches.101 These latter findings indicate that viruses can induce significant changes in the peripheral airways that have significant functional outcomes in the absence of marked changes in measurements of airway obstruction and hyperresponsiveness.

ry illness have evidence of air trapping and ventilation–perfusion mismatches.101 These latter findings indicate that viruses can induce significant changes in the peripheral airways that have significant functional outcomes in the absence of marked changes in measurements of airway obstruction and hyperresponsiveness. Effects of Respiratory Viruses on Airway Inflammation Respiratory viruses can cause inflammation and injury to healthy airways and can worsen injury in airways that are already inflamed, as demonstrable in asthma. Respiratory viruses can induce an inflammatory process by direct cytopathic effects on the airway epithelium (e.g., RSV bronchiolitis) and can induce an immune response to stop viral replication and eradicate the virus. The immune response to viral infection may be a double-edged sword, however, as virus-induced inflammation can also contribute to airway obstruction and respiratory symptoms. Indeed, although many common cold viruses (e.g., RV) do not produce significant cytopathic effects, possibly because few cells are infected, the immunoinflammatory response to the virus is probably the major cause of respiratory symptoms. In this section, the association between virus-induced immune responses and respiratory symptoms is explored.

cold viruses (e.g., RV) do not produce significant cytopathic effects, possibly because few cells are infected, the immunoinflammatory response to the virus is probably the major cause of respiratory symptoms. In this section, the association between virus-induced immune responses and respiratory symptoms is explored. Role of Epithelial Cells Respiratory viruses replicate primarily in airway epithelial cells. In addition to serving as host cells, it is now well documented that epithelial cells also initiate the immune response to infections through the secretion of cytokines and chemokines. In vitro studies of epithelial cells or cell lines have demonstrated that respiratory viruses such as RV, RSV, and parainfluenzavirus can induce the secretion of many different proinflammatory cytokines (IL-1, TNF-α, GM-CSF, IL-6, IL-11) and chemokines (RANTES, IL-8, MIP-1α). 10, 20, 23, 96, 98, 105 Epithelial-derived chemokines are likely to be an important signal in initiating antiviral responses through the recruitment of leukocytes to the airway. In support of this concept, IL-8, a potent neutrophil chemoattractant, is found in high levels in nasal secretions of children with virus-induced asthma, and levels of IL-8 correlate with the number of airway neutrophils and neutrophil myeloperoxidase levels (suggesting neutrophil activation).108 There is also evidence, however, that enhanced airway inflammation caused by chemokine secretion may also disturb normal airway physiology. Chemokine levels in nasal secretions correlate closely with cold symptoms, 110 for example, and IL-8 levels correlate with virus-induced changes in airway responsiveness.41 Levels of epithelial-derived cytokines such as IL-6 and IL-11 also correlate with respiratory symptoms, 23 and animal studies indicate that overexpression of IL-11 can cause bronchial hyperresponsiveness. 23, 107

symptoms, 110 for example, and IL-8 levels correlate with virus-induced changes in airway responsiveness.41 Levels of epithelial-derived cytokines such as IL-6 and IL-11 also correlate with respiratory symptoms, 23 and animal studies indicate that overexpression of IL-11 can cause bronchial hyperresponsiveness. 23, 107 In addition to stimulating cytokine production, RV can upregulate epithelial cell surface expression of intercellular adhesion molecule-1, 79 which, in addition to facilitating cell–cell adhesion, is the receptor for the major group of RV. 38, 103 This enhanced expression of adhesion proteins may contribute to the persistence and severity of inflammation in asthmatic subjects and, possibly, the greater susceptibility of asthmatic children to colds compared with nonasthmatic children. Mechanisms for the activation of cytokine genes in epithelial cells and adhesion molecules are under investigation. It is known that nuclear factor-κ B activation is important in virus-induced transcriptional regulation of IL-650 and, possibly, for the synthesis of a variety of inflammatory cytokines.7 In addition, nitric oxide may regulate virus-induced chemokine production through a posttranscriptional mechanism and by inhibiting viral replication, 95 although a clinical study did not find a relationship between IL-8 and nitrate levels in nasal secretions.59

or the synthesis of a variety of inflammatory cytokines.7 In addition, nitric oxide may regulate virus-induced chemokine production through a posttranscriptional mechanism and by inhibiting viral replication, 95 although a clinical study did not find a relationship between IL-8 and nitrate levels in nasal secretions.59 Effect on Granulocytes Granulocyte recruitment and activation seem to have an important role in the pathogenesis of virus-induced asthma exacerbations. Grünberg et al, 41 for example, experimentally inoculated 35 atopic asthma subjects with either RV16 or placebo and found that neutrophil counts in the peripheral blood correlated with the cold and asthma symptom scores and cold-induced changes in airway hyperresponsiveness. In addition, eosinophil granular proteins and leukotriene C4 have been detected in the nasal secretions of infants and children with virus-induced wheezing illnesses. 32, 86, 97, 116 Increased concentrations of sputum eosinophil cationic protein found during the acute phase of RV infection correlated with increases in airway responsiveness in a group of adults with asthma after experimental inoculation with RV16.39 In vitro experiments indicate that RV does not activate eosinophils directly45; it is more likely that inflammatory mediators and cytokines, secreted by virus-activated cells in the lung, contribute to eosinophil activation. Finally, guinea pigs infected with PIV develop airway eosinophils and airway hyperresponsiveness25 and this outcome is blocked if the guinea pigs are pretreated with IL-5–neutralizing antibody.112

t inflammatory mediators and cytokines, secreted by virus-activated cells in the lung, contribute to eosinophil activation. Finally, guinea pigs infected with PIV develop airway eosinophils and airway hyperresponsiveness25 and this outcome is blocked if the guinea pigs are pretreated with IL-5–neutralizing antibody.112 Role of Mononuclear Cells Most respiratory viruses replicate quickly and, within a few days of inoculation, the quantity of viruses and viral proteins is sufficient to activate mononuclear cells in the airway. In vitro infection of human monocytes with respiratory viruses, for example, leads to a potent proinflammatory cytokine response by release of IL-8, IL-1, and TNF-α. 35, 54, 90 Interleukin-1 and TNF-α can increase cell recruitment into the airway by enhancing adhesion molecule expression on endothelial cells. In addition, TNF-α has been associated with wheezing illnesses in infancy6 and the development of late-phase allergic reaction and asthma. 3, 37 Monocytes and macrophages also produce interferon (INF), and its appearance in nasal secretions coincides with the onset of the recovery process. In addition to cytokine production, macrophages incubated with RSV or PIV produce lipid mediators such as prostaglandin E2, platelet-activating factor, and thromboxane B2 48, 78, 114 that can augment airway inflammation.

ron (INF), and its appearance in nasal secretions coincides with the onset of the recovery process. In addition to cytokine production, macrophages incubated with RSV or PIV produce lipid mediators such as prostaglandin E2, platelet-activating factor, and thromboxane B2 48, 78, 114 that can augment airway inflammation. Lymphocytes, including natural killer cells, CD8+ cytotoxic T cells, and CD4+ T cells, are involved in limiting viral replication and viral clearance. To test the possibility that variations in lymphocyte responses might account for variability in the ability to clear viral infections, Parry et al80 measured in vitro lymphocyte responses in a group of allergic subjects who were then inoculated with RV 16. Vigorous virus-induced responses (lymphocyte proliferation or IFN-γ secretion) before inoculation correlated with reduced viral shedding after inoculation. These results suggest that factors related to the host cellular response help determine the degree of viral replication during respiratory viral infections. Further characterization of these host factors may lead to new therapeutic strategies for respiratory infections, a goal that is particularly important for people with asthma.

s suggest that factors related to the host cellular response help determine the degree of viral replication during respiratory viral infections. Further characterization of these host factors may lead to new therapeutic strategies for respiratory infections, a goal that is particularly important for people with asthma. Several studies have shown that viral infections activate a wide range of T cells. Evidence for this comes from experiments in mice, in which most of the T cells found in the lung after an acute viral infection are not virus-specific, 21 and in vitro studies, in which 25% to 50% of human peripheral blood T cells express the early activation marker CD69 after 24 hours in culture with RV.36 RANTES, induced by respiratory viruses, at high concentrations can also induce antigen-independent T-cell activation.5 These studies suggest that respiratory viruses can induce early, nonspecific T-cell activation and recruitment that could significantly increase the intensity of airway inflammation, resulting in airway dysfunction and respiratory symptoms. This hypothesis is supported by studies of volunteers infected with rhinovirus. Respiratory virus infections usually cause peripheral lymphopenia and increased numbers of lymphocytes in the upper and lower airways, for example. The degree of peripheral blood lymphopenia and lymphocytic infiltration of the airway epithelium has been correlated with the increases in airway responsiveness. 15, 26

us. Respiratory virus infections usually cause peripheral lymphopenia and increased numbers of lymphocytes in the upper and lower airways, for example. The degree of peripheral blood lymphopenia and lymphocytic infiltration of the airway epithelium has been correlated with the increases in airway responsiveness. 15, 26 Interactions Between Viral Infections and Responses to Allergen Although viral infections cause similar upper respiratory symptoms in allergic and nonallergic individuals, 46, 100 there is evidence of interactions between virus- and allergen-induced responses in the lower airway. Lemanske and colleagues, 69 for example, identified 10 patients with allergic rhinitis and experimentally infected them with RV16. The viral infection increased airway reactivity to both inhaled allergen and histamine, and also increased the frequency of a late allergic reaction to inhaled antigens. Moreover, Calhoun and colleagues13 used bronchoscopy to study the inflammatory response to allergen in individual lung segments before, during, and 1 month after RV16 infection. RV infection enhanced the immediate antigen-induced release of histamine, and also increased eosinophil recruitment of eosinophils to the lung.

oreover, Calhoun and colleagues13 used bronchoscopy to study the inflammatory response to allergen in individual lung segments before, during, and 1 month after RV16 infection. RV infection enhanced the immediate antigen-induced release of histamine, and also increased eosinophil recruitment of eosinophils to the lung. SUMMARY Respiratory infections can have dual effects related to asthma. First, there is increasing evidence that severe infections with RSV and PIV in infancy can alter lung development and physiology to increase the risks of subsequent wheezing and asthma. Second, infections with common cold viruses and influenza commonly precipitate wheezing symptoms in children and adults who already have established asthma, and RV appears to be the most important virus in producing exacerbations of the disease. The principal mechanisms by which this occurs appears to be viral replication in epithelial cells, triggering a cascade of inflammation involving granulocytes, macrophages, T cells, and secreted cytokines and mediators. The inflammatory process, although essential to clear the infection, augments pre-existing airway inflammation in asthma, leading to increased airway obstruction and lower respiratory tract symptoms. Greater understanding of virus-induced changes in inflammation and corresponding changes in airway physiology may lead to new therapeutic approaches to the treatment and prevention of virus-induced airway dysfunction.

inflammation in asthma, leading to increased airway obstruction and lower respiratory tract symptoms. Greater understanding of virus-induced changes in inflammation and corresponding changes in airway physiology may lead to new therapeutic approaches to the treatment and prevention of virus-induced airway dysfunction. Address reprint requests to Robert F. Lemanske, Jr, MD, Department of Pediatrics, University of Wisconsin, Children's Hospital, 600 Highland Avenue H4/432, Madison, WI 53792 Supported by NIH Grants Al34891, HL56396, and 1RO1HL61879.

Transfusion of blood products and acute lung injury and acute respiratory distress syndrome Clinical spectrum of lung injury secondary to hemotherapy The development of lung injury from the transfusion of blood products occurs on a clinical spectrum, and therefore the proper recognition of lung injury must consider these possible scenarios. Transfusion-related acute lung injury (TRALI), in its classic and most recognized form, is the fulminant development of new lung injury in a patient who has just received or is receiving a blood-product transfusion. Often, severe respiratory failure develops, and frothy pulmonary edema fluid is suctioned from the endotracheal tube. This catastrophic and fulminant form of TRALI is easily diagnosed by most health care providers. Like most pathologic processes, however, TRALI extends on spectrum from mild to severe, and the milder forms of TRALI can go unrecognized even by highly trained and experienced health care providers. For example, TRALI in the stable patient on the general medical ward who is receiving packed red blood cells (PRBCs) for anemia and develops a mild decrease in oxygen saturation may go unrecognized, or the cause of the oxygen desaturation may be blindly assigned to volume overload. A recent retrospective study that tracked the recipients of blood products from a donor implicated in a case of TRALI exemplifies this point [1]. This highly motivated donor was linked to 15 cases of TRALI over a 2-year period, and many of the cases were associated with mild symptoms or mild oxygen desaturation. Most of these cases were not initially recognized as TRALI, nor were they reported to the blood bank as a potential adverse event. It is essential for the health care provider to maintain a high degree of vigilance for adverse reactions to blood products and to investigate thoroughly any oxygen desaturation temporally related to blood-product transfusion.

y recognized as TRALI, nor were they reported to the blood bank as a potential adverse event. It is essential for the health care provider to maintain a high degree of vigilance for adverse reactions to blood products and to investigate thoroughly any oxygen desaturation temporally related to blood-product transfusion. Another important category of potential lung injury related to blood-product transfusion is the worsening of pre-existing acute lung injury (ALI) by subsequent hemotherapy. It often is difficult to make a diagnosis of TRALI in critically ill patients who have existing ALI/acute respiratory distress syndrome (ARDS); however, if there is a sudden worsening of oxygenation and pulmonary compliance in a patient who is receiving or in the past 6 hours has received a blood-product transfusion (in the absence of signs of volume overload), a probable diagnosis of TRALI can be made. There are a few studies in the medical literature that support a contribution of blood-product transfusion to the worsening of pre-existing ALI/ARDS. Using the largest prospective study on the incidence of ALI in the pediatric population, Church and colleagues [2] retrospectively assessed the role of blood-product transfusions on the clinical outcomes of this patient population. These investigators discovered that the transfusion of fresh frozen plasma (FFP) was an independent risk factor for mortality in these pediatric ALI patients at 30 days. In an adult prospective investigation of the risk factors, incidence, and outcomes of ALI/ARDS at a single center, Gong and colleagues [3] reported that PRBC transfusion was independently associated with the initial development of ALI/ARDS and was an independent risk factor for mortality in ALI/ARDS, with a dose-dependent response.

rospective investigation of the risk factors, incidence, and outcomes of ALI/ARDS at a single center, Gong and colleagues [3] reported that PRBC transfusion was independently associated with the initial development of ALI/ARDS and was an independent risk factor for mortality in ALI/ARDS, with a dose-dependent response. Potential lessons also can be learned from the seminal Transfusions Requirements in Critical Care (TRICC) trial by the Canadian Care Trials Group [4]. In this randomized, controlled equivalency trial comparing restrictive and liberal transfusion thresholds, the patients transfused with a hemoglobin trigger of less than 7 g/dL had at least as good outcomes as the patients in the liberal transfusion arm. In an analysis of the ICU complications that developed in the two groups of patients, 32 patients (7.7%) in the restrictive transfusion group versus 48 patients (11.4%) in the liberal transfusion group developed ARDS during their ICU stay (P = .06). Unfortunately, it was not determined what proportion of these patients who had ARDS developed overt TRALI; however, it is provocative to consider that a proportion of ARDS may be preventable by adhering to lower transfusion thresholds. This potential benefit could result from the reduction of ALI/ARDS directly caused by the transfusion of blood products and ALI/ARDS resulting from the combination of blood transfusions and other causes (the two-hit model). In the TRICC trial, decreased infectious complications (eg, pneumonia, sepsis) did not seem to be a benefit of the restrictive transfusion group, and thus the decreased incidence of ALI/ARDS probably cannot be attributed to decreased transfusion-related immunomodulation. One other notable point to consider regarding the TRICC trial was the statistically significant decrease in pulmonary edema in the restrictive transfusion group. Pulmonary edema was listed as a cardiac complication, although it is unclear how rigorously noncardiogenic pulmonary edema was excluded. This decrease in pulmonary edema could potentially comprise both transfusion-associated circulatory overload and TRALI.

crease in pulmonary edema in the restrictive transfusion group. Pulmonary edema was listed as a cardiac complication, although it is unclear how rigorously noncardiogenic pulmonary edema was excluded. This decrease in pulmonary edema could potentially comprise both transfusion-associated circulatory overload and TRALI. Definition of transfusion-related acute lung injury The term “transfusion-related acute lung injury” was first coined in 1983 by Popovsky and Moore [5] and has been variably defined in the ensuing years. Recently, the field has been aided significantly by a consensus definition developed by a panel investigators convened by the National heart, Lung, and Blood Institute (NHLBI) [6]. Simply put, TRALI remains a clinical diagnosis, similar to the clinical definition used for ALI/ARDS [7]. In fact, if a patient meets clinical criteria for ALI/ARDS and has received a blood-product transfusion during the previous 6 hours (in the absence of another credible risk factor for ARDS), a clinical diagnosis of TRALI can be made. The blood bank and laboratory work-up of TRALI is an important component for the possible prevention of future TRALI from implicated blood donors, but it is not required to make a diagnosis of TRALI.

e previous 6 hours (in the absence of another credible risk factor for ARDS), a clinical diagnosis of TRALI can be made. The blood bank and laboratory work-up of TRALI is an important component for the possible prevention of future TRALI from implicated blood donors, but it is not required to make a diagnosis of TRALI. The use of the 6-hour window to implicate a blood-product transfusion in TRALI is not an arbitrary designation. The multitude of case reports in the medical literature confirms a close temporal association with the transfusion of blood products. In fact, it is common for TRALI to develop during the first 30 to 60 minutes of a transfusion [8]. Experimental evidence from animal models supports this temporal association. In at least four different animal models of TRALI using anti-neutrophil antibodies, anti–major histocompatibility class (MHC) I antibodies, and the plasma and lipid fractions from day 42 PRBCs and day 5 platelets, lung injury develops within 6 hours of challenge with these experimental agents [9]. In fact, in the MHC I antibody model, lung injury develops within 2 hours and often within 15 to 30 minutes [10]. The speed with which lung injury develops in a given patient is probably a complex interplay of host susceptibility and dose or titer of the injurious blood product.

enge with these experimental agents [9]. In fact, in the MHC I antibody model, lung injury develops within 2 hours and often within 15 to 30 minutes [10]. The speed with which lung injury develops in a given patient is probably a complex interplay of host susceptibility and dose or titer of the injurious blood product. The clinical diagnosis of TRALI is potentially complicated by the existence of other major risk factors for ALI/ARDS, clinical risk factors that often are present in critically ill patients receiving hemotherapy. The consensus definition of TRALI addresses this point by assigning a definite diagnosis of TRALI when no other ALI/ARDS risk factors are present and a probable diagnosis of TRALI when these other major risk factors are present [6]. The clinical course of these probable TRALI patients can be followed over time, and the credibility of the relationship of the risk factors to ALI/ARDS can be assessed. If hemotherapy remains the most credible risk factor for ALI/ARDS, a diagnosis of TRALI can be made.

en these other major risk factors are present [6]. The clinical course of these probable TRALI patients can be followed over time, and the credibility of the relationship of the risk factors to ALI/ARDS can be assessed. If hemotherapy remains the most credible risk factor for ALI/ARDS, a diagnosis of TRALI can be made. The issue of massive transfusions as a risk factor for ALI/ARDS is a potential source of confusion in assigning the diagnosis of TRALI. Massive transfusion is defined as the replacement by transfusion of more than 50% of a patient's blood volume over 12 to 24 hours [11]. Massive transfusion has been implicated as a major risk factor for ALI/ARDS in multiple studies and is probably the fourth most common cause of ALI/ARDS, accounting for approximately 20% of cases. Even with the experimental models of massive blood transfusion, it is not entirely clear how lung injury is produced by this insult. Because many of these patients, if not the majority, are involved in trauma, considerations include ischemia-reperfusion lung injury from shock and potentially a component of direct thoracic trauma. Older investigations using animal models of “shock lung” disproved the microaggregate or particulate theory of lung injury from transfusions but focused attention on the plasma fraction of blood products [9]. Is it possible that the development of ALI/ARDS from massive blood transfusion is a function of the cumulative risk of TRALI from individual blood products? This supposition may explain many of the cases; receiving multiple blood products places the patient at higher risk of receiving an incompatible product containing either a matched antibody or priming or activating bioactive lipids. If one considers the two-hit model of TRALI, then the underlying medical condition of the critically ill patient who is requiring multiple transfusions also places the patient at risk for TRALI. In summary, the certainty by which a diagnosis of TRALI can be made in a patient who has other risk factors for ALI or pre-existing ALI is a complicated issue that may be aided by following the clinical course of the patient and also by focused laboratory investigation of implicated blood products.

t at risk for TRALI. In summary, the certainty by which a diagnosis of TRALI can be made in a patient who has other risk factors for ALI or pre-existing ALI is a complicated issue that may be aided by following the clinical course of the patient and also by focused laboratory investigation of implicated blood products. Incidence and clinical outcomes The most consistent incidence of TRALI reported in the medical literature is 1 in 5000 blood products transfused. [5]. This figure is probably a significant underestimation given the evidence for the under-recognition and under-reporting of TRALI [1]. The new consensus definition and the recent proliferation of case reports and clinical reviews of TRALI should aid in the greater recognition and reporting of this condition in the future. In addition, the NHLBI has recently funded a Specialized Centers of Clinically Oriented Research (SCCOR) grant to the University of California, San Francisco and the Mayo Clinic, Rochester to conduct a prospective observational cohort study on the incidence and outcomes of TRALI at these two medical centers. This study promises to yield the largest cohort of TRALI patients in the medical literature and should provide a reliable estimate of the incidence of TRALI.

Francisco and the Mayo Clinic, Rochester to conduct a prospective observational cohort study on the incidence and outcomes of TRALI at these two medical centers. This study promises to yield the largest cohort of TRALI patients in the medical literature and should provide a reliable estimate of the incidence of TRALI. Medical providers are required to report fatalities related to TRALI to the Food and Drug Administration (FDA). During the past few years the FDA has received between 8 and 21 reports per year of fatal TRALI, and TRALI has now emerged as the primary cause of transfusion-associated mortality, surpassing infectious complications and ABO mismatch [12], [13]. The United Kingdom's Serious Hazards of Transfusion Annual Report for 2004 evaluated 23 cases that had been reported as TRALI events and concluded that 13 cases were either highly likely or probable TRALI, and 4 of these cases were possible TRALI [14].

y, surpassing infectious complications and ABO mismatch [12], [13]. The United Kingdom's Serious Hazards of Transfusion Annual Report for 2004 evaluated 23 cases that had been reported as TRALI events and concluded that 13 cases were either highly likely or probable TRALI, and 4 of these cases were possible TRALI [14]. The clinical outcomes of patients who have TRALI differ considerably from the outcomes of patients who have all causes of ALI/ARDS. One of the largest case series in the TRALI literature reported the clinical outcomes of 36 patients at the Mayo Clinic in the mid-1980s, predating the use of low tidal volume ventilation in the management of patients who have ALI/ARDS [15]. In this investigation, three quarters of the patients required mechanical ventilation, and the vast majority of the patients had rapid clearance (<96 hours) of the pulmonary edema. Two of the 36 patients died, yielding a mortality of 6%. The rapid clearance of the pulmonary edema and mortality differ considerably from all-cause ALI/ARDS, which has an estimated mortality of 30% to 40% [16].

l ventilation, and the vast majority of the patients had rapid clearance (<96 hours) of the pulmonary edema. Two of the 36 patients died, yielding a mortality of 6%. The rapid clearance of the pulmonary edema and mortality differ considerably from all-cause ALI/ARDS, which has an estimated mortality of 30% to 40% [16]. Implicated blood products in transfusion-related acute lung injury All plasma-containing blood products have been implicated in TRALI. This list includes whole blood, PRBCs, FFP, whole blood platelets, and apheresis platelets, with less frequent associations with intravenous immunoglobulin and cryoprecipitate. Controversy exists concerning which blood product is most commonly implicated in TRALI, but the two blood products with the largest plasma fraction, FFP and platelets, are probably the top offenders. In the reports of TRALI fatalities reported to the FDA, FFP was the most commonly implicated product, followed by red blood cells, platelets, and cryoprecipitate [13]. The association of platelets with TRALI is supported by a single-center investigation reporting that whole blood platelets caused TRALI in 72 of 90 cases [17].

reports of TRALI fatalities reported to the FDA, FFP was the most commonly implicated product, followed by red blood cells, platelets, and cryoprecipitate [13]. The association of platelets with TRALI is supported by a single-center investigation reporting that whole blood platelets caused TRALI in 72 of 90 cases [17]. Pathogenesis of transfusion-related acute lung injury Classically, the pathogenesis of TRALI has been explained by the passive transfusion of a plasma-containing blood product with a HLA class I/II or neutrophil antibody that recognizes specific HLA or neutrophil antigens in the recipient. Numerous case reports and case series have documented the presence of these antibodies and the matching antigens in the recipient, and in animal models MHC I and anti-neutrophil antibodies have produced acute lung injury [8], [10], [18], [19]. In rarer circumstances, roles can be reversed, and recipient antibodies can react with antigens on donor leukocytes. In the FDA's report on fatalities secondary to TRALI, anti-HLA I antibodies were the most frequently implicated antibody, followed by anti-granulocyte and anti-HLA II antibodies [13]. In an anti–MHC I mouse model that reproduces many features of clinical TRALI, lung injury was produced secondary to the recognition of endothelial-bound MHC I antibody by circulating neutrophils and their Fc gamma receptors [10]. In another experimental model of TRALI, evidence was presented that a mouse monoclonal anti-neutrophil antibody directly activated human neutrophils, leading to lung injury [19]. Common to all antibody models, however, is the central role of the neutrophil in producing disease, either from direct antibody activation or indirectly through Fc gamma receptor activation. Other investigators have shown that monocytes may be important in TRALI pathogenesis [20].

trophils, leading to lung injury [19]. Common to all antibody models, however, is the central role of the neutrophil in producing disease, either from direct antibody activation or indirectly through Fc gamma receptor activation. Other investigators have shown that monocytes may be important in TRALI pathogenesis [20]. Although the antibody theory of TRALI is supported by both clinical and experimental evidence, solid clinical data and animal models also implicate biologically active lipids that accumulate in older cellular blood products [21], [22], [23]. These biologically active lipids, thought to be breakdown products of cellular membranes from stored blood products, can prime and activate neutrophils in vitro. Cellular blood products (PRBCs, platelets) have been shown to accumulate these lipids over time, and the plasma and lipid fractions from these older blood products have been used to produce pulmonary edema in isolated, perfused rat lung models. Older blood products have been implicated in TRALI cases [17] but recently, red blood cell storage time was not associated with the development of new ALI/ARDS [24].

time, and the plasma and lipid fractions from these older blood products have been used to produce pulmonary edema in isolated, perfused rat lung models. Older blood products have been implicated in TRALI cases [17] but recently, red blood cell storage time was not associated with the development of new ALI/ARDS [24]. A phenomenon that has not been adequately explained is the inconsistent development of lung injury even when a recipient is transfused with a matched HLA or neutrophil antibody. For example, a retrospective study of TRALI involving an antibody to a neutrophil antigen expected to be present in more than 90% of recipients produced TRALI in only a minority of patients transfused with this antibody [1]. This lack of a consistent relationship between antibody-antigen matches and TRALI has been explained by host susceptibility and specifically by the two-hit model of TRALI. This hypothesis states that the recipient must have an underlying medical condition that contributes to immune priming and that with the transfusion of HLA or neutrophil antibody or biologically active lipids (or both), TRALI is produced. Evidence supporting this hypothesis includes the frequent occurrence of TRALI in the ICU or operating room and epidemiologic associations with cardiopulmonary bypass and hematologic malignancies [17]. In addition, the biologically active lipid model involves a necessary systemic endotoxin-priming step to produce ALI [22], [23]. Host susceptibility factors such as genetic polymorphisms also may be contributing factors.

room and epidemiologic associations with cardiopulmonary bypass and hematologic malignancies [17]. In addition, the biologically active lipid model involves a necessary systemic endotoxin-priming step to produce ALI [22], [23]. Host susceptibility factors such as genetic polymorphisms also may be contributing factors. Clinical manifestations, diagnosis, and management of transfusion-related acute lung injury Patients who develop TRALI often develop dyspnea, tachypnea, and hypoxia. Both hypotension and hypertension have been reported in patients who have TRALI, as has hyper- and hypothermia. In mechanically ventilated patients or at the time of endotracheal intubation, frothy pulmonary edema fluid is sometimes elaborated in fulminant TRALI cases. The same criteria used by clinicians and researchers to diagnose other causes of ALI/ARDS are used to make a diagnosis of TRALI [7]. Chest imaging reveals bilateral pulmonary opacities in a pattern consistent with noncardiogenic pulmonary edema ( Fig. 1). Adjunctive tests that aid in a TRALI diagnosis include echocardiography, serial white blood cell counts, and the protein analysis of undiluted pulmonary edema fluid [8]. Echocardiography can aid in the work-up by helping exclude volume overload and cardiogenic dysfunction. Often, patients who have fulminant TRALI have evidence of low cardiac filling pressures. Measurement of brain natriuretic peptide may also be an adjunctive test that can be helpful. There are several case reports of leukopenia and neutropenia temporally associated with the onset of pulmonary edema in TRALI [25], [26], [27], [28]. This laboratory finding often is dynamic, with leukocyte counts recovering a few hours after the initiation of TRALI. There is even one case report of leukopenia heralding the onset of TRALI [29]. The mouse MHC I antibody model of TRALI also has documented this phenomenon, which speaks to the association of leukopenia with disease pathogenesis [10]. Other causes of ALI/ARDS (eg, pneumonia, sepsis) also might produce leukopenia, so this finding is not specific to TRALI. Thrombocytopenia and decreased complement have been reported also, but much less frequently. Finally, if present, the measurement of the protein concentration of undiluted pulmonary edema fluid and a matched plasma sample can be helpful in documenting a permeability pulmonary edema and thus helping exclude transfusion-associated circulatory overload or cardiogenic pulmonary edema [30].Fig.

uch less frequently. Finally, if present, the measurement of the protein concentration of undiluted pulmonary edema fluid and a matched plasma sample can be helpful in documenting a permeability pulmonary edema and thus helping exclude transfusion-associated circulatory overload or cardiogenic pulmonary edema [30].Fig. 1 (A) Anterior-posterior chest radiograph in a 43-year-old woman recovering from knee surgery who developed respiratory distress and hypoxia during a PRBC transfusion. Bilateral pulmonary opacities are present. (B) Chest CT in the same patient revealing bilateral ground-glass opacities and interstitial septal thickening. CT was done 24 hours after the chest radiograph in Fig. 1A. There is no unique or specific treatment for TRALI, but the proper recognition of this syndrome leads to provision of the same supportive care given to any patient who has ALI/ARDS. Additionally, correctly identifying TRALI allows one to avoid potentially injurious interventions, such as diuretics, in these patients, who often are volume depleted. Paradoxically, these patients, who sometimes have severe pulmonary edema, may require intravenous fluids to support blood pressure. Colloid and vasopressors may be required in some instances. Corticosteroids have no role in the treatment of TRALI patients. It is important to consider that most patients who have TRALI improve fairly rapidly, and overall mortality is low; thus, with patience and the avoidance of any unnecessary blood-product transfusions, most patients will do well.

equired in some instances. Corticosteroids have no role in the treatment of TRALI patients. It is important to consider that most patients who have TRALI improve fairly rapidly, and overall mortality is low; thus, with patience and the avoidance of any unnecessary blood-product transfusions, most patients will do well. Prevention of transfusion-related acute lung injury TRALI is best prevented by avoiding unnecessary blood-product transfusions using evidence-based transfusion triggers. Use of erythropoietin [31] and recombinant factor VII may help reduce transfusion requirements in selected populations. Most experts agree that if a blood donor is implicated in a TRALI case, that donor and the donor's existing blood products should be removed permanently from the donor pool. Given the incidence of HLA sensitization in multiparous females [32] and the association of TRALI with FFP transfusion, the United Kingdom has excluded all women from donating FFP, directing their donations instead to plasma-poor blood products [14]. In addition, platelet pools are suspended in male plasma as much as possible. There has not been ample time to determine the benefit of this regulatory action on the incidence of TRALI; however, the number of highly likely or probable TRALI cases decreased from 22 in 2003 to 13 cases in 2004. (The FFP policy was implemented in October 2003.) Given time delays in the work-up of TRALI cases and the long shelf-life of stored FFP, the true impact of the male-only FFP policy change will not be known until the next reporting years. Leukoreduction of PRBCs is now an almost universal blood bank procedure, but it is unlikely that it will significantly reduce the incidence of TRALI, because recipient antibody recognition of antigens on donor leukocytes is a rare mechanism in TRALI.

y FFP policy change will not be known until the next reporting years. Leukoreduction of PRBCs is now an almost universal blood bank procedure, but it is unlikely that it will significantly reduce the incidence of TRALI, because recipient antibody recognition of antigens on donor leukocytes is a rare mechanism in TRALI. Severe acute respiratory syndrome Clinical importance and epidemiology of severe acute respiratory syndrome The severe acute respiratory syndrome (SARS) burst on the international scene in 2002 and 2003 causing much concern because of its rapid, global dissemination and fears of a pandemic. From late 2002 to mid-2003, more than 8000 people developed probable SARS in many different countries across five continents, with the predominance of cases in China, Hong Kong, Viet Nam, Taiwan, Singapore, and Canada. Approximately one in four patients who had SARS become critically ill, with ALI occurring in 80% of these patients (16% of all patients). In fact, ALI is the most common organ-system dysfunction in SARS patients. Of the patients who become critically ill with SARS, approximately 50% die, with mortality rates increased in the elderly [33].

four patients who had SARS become critically ill, with ALI occurring in 80% of these patients (16% of all patients). In fact, ALI is the most common organ-system dysfunction in SARS patients. Of the patients who become critically ill with SARS, approximately 50% die, with mortality rates increased in the elderly [33]. Much has been learned about the epidemiology of SARS since its emergence in the Guangdong Province of China. Remarkably, through collaborations with multiple laboratories in different countries, a novel coronavirus was identified as the causative agent of SARS just 4 to 5 months after the initial reports of the SARS outbreak [34], [35]. A similar coronavirus was also isolated from wild animals (eg, Himalayan palm civets, raccoon dogs) sold in the “wet” markets in Guangdong Province, supporting a zoonotic origin of this novel virus [36]. The incubation period for SARS seems to range between 2 and 10 days, although longer incubation times have been documented. Unlike influenza, in which transmissibility peaks soon after the onset of clinical symptoms, SARS is transmitted most efficiently after 10 days of illness. The primary route of transmission is contact (direct or indirect) with respiratory droplets or fomites. Fecal–oral spread also may play a role, given the presence of SARS coronavirus in stool specimens and the presence of watery diarrhea in many affected patients. The spread of SARS seems to involve so-called “super-spreaders” who disproportionately infect many persons, and it also prominently involves transmission in the health-care setting [37].

play a role, given the presence of SARS coronavirus in stool specimens and the presence of watery diarrhea in many affected patients. The spread of SARS seems to involve so-called “super-spreaders” who disproportionately infect many persons, and it also prominently involves transmission in the health-care setting [37]. Pathogenesis of severe acute respiratory syndrome A Clinical SARS Working Group, comprised of experts from Canada and the United States, determined that patients admitted to ICUs with respiratory failure secondary to SARS met the established clinical definition of ALI/ARDS [33]. Lung pathology obtained from SARS nonsurvivors reveals a pattern of disease that is indistinguishable from other causes of ALI/ARDS, namely diffuse alveolar damage [38]. Much has been learned about the potential cellular and molecular mechanisms of lung injury secondary to SARS coronavirus infection. For example, the tropism of the SARS coronavirus for the lung and the gastrointestinal tract can be explained by the epithelial distribution of its cellular receptor, ACE-2, which is found on the surface of alveolar type I and II epithelial cells and also on small bowel enterocytes [39], [40]. Therefore, the severe pulmonary damage observed in SARS can be explained by viral–alveolar epithelial cell interaction.

an be explained by the epithelial distribution of its cellular receptor, ACE-2, which is found on the surface of alveolar type I and II epithelial cells and also on small bowel enterocytes [39], [40]. Therefore, the severe pulmonary damage observed in SARS can be explained by viral–alveolar epithelial cell interaction. Diagnosis, management, and prevention of severe acute respiratory syndrome SARS presents clinically with a nonspecific viral syndrome of fever, myalgia, chills, fatigue, and cough; upper respiratory symptoms (eg, rhinorrhea, sore throat) are often absent. Watery diarrhea can be present in some patients. Severely affected patients develop shortness of breath, tachypnea, and tachycardia. Routine laboratory investigation often reveals lymphopenia, thrombocytopenia, and elevated transaminases. Chest imaging is nonspecific with high-resolution CT being abnormal in nearly all patients showing ground-glass attenuation and focal consolidation with some cases of pneumomediastinum [37].

ypnea, and tachycardia. Routine laboratory investigation often reveals lymphopenia, thrombocytopenia, and elevated transaminases. Chest imaging is nonspecific with high-resolution CT being abnormal in nearly all patients showing ground-glass attenuation and focal consolidation with some cases of pneumomediastinum [37]. The diagnosis of SARS relies on consideration of key epidemiologic data in a patient who has a viral syndrome or idiopathic respiratory failure. Because the clinical syndrome, routine laboratory testing, and chest imaging are nonspecific, diagnosis relies on detection of the SARS coronavirus in clinical specimens. Real-time polymerase chain reaction (PCR) is used to test samples from multiple potential sites, including the upper and lower respiratory tracts, stool, urine, and plasma. The virus concentrates in the lower respiratory tract and is present in low quantities early in the disease course; thus negative results early in the clinical course, especially from upper respiratory specimens, should be interpreted with caution. The standard diagnostic test for SARS is seroconversion, but this testing is rarely helpful prospectively.

respiratory tract and is present in low quantities early in the disease course; thus negative results early in the clinical course, especially from upper respiratory specimens, should be interpreted with caution. The standard diagnostic test for SARS is seroconversion, but this testing is rarely helpful prospectively. The evidence-based management of SARS has been reviewed by the NHLBI/Centers for Disease Control and Prevention/National Institute of Allergy and Infections Diseases Clinical SARS Working Group [33]. Generically, because SARS produces a clinical syndrome consistent with other causes of ALI/ARDS, respiratory failure should be managed in a similar fashion, with a pressure-limited, low tidal volume strategy. The roles of antiviral agents (eg, ribavirin and interferon-α) and corticosteroids are controversial and suffer from the lack of quality data. Anecdotal and small case series have supported the role of corticosteroids for severe cases [41], and thus, in the absence of placebo-controlled trials, steroid therapy should be considered, especially in patients who have clinical deterioration.

rticosteroids are controversial and suffer from the lack of quality data. Anecdotal and small case series have supported the role of corticosteroids for severe cases [41], and thus, in the absence of placebo-controlled trials, steroid therapy should be considered, especially in patients who have clinical deterioration. Several factors contributed to the halt of SARS transmission and its current disappearance from the clinical landscape. The involvement by the World Health Organization in issuing a global health alert, the quarantine of infected and exposed individuals, and, importantly, the ban on the sale of wildlife in the wet markets of Guangdong, China all helped to stop the spread of this potential pandemic. Phase I trials are now in progress on vaccine strategies that could prove to be important should SARS re-emerge on the global scene [42].

f infected and exposed individuals, and, importantly, the ban on the sale of wildlife in the wet markets of Guangdong, China all helped to stop the spread of this potential pandemic. Phase I trials are now in progress on vaccine strategies that could prove to be important should SARS re-emerge on the global scene [42]. Avian influenza (H5N1) Clinical importance and epidemiology of H5N1 infection Influenza pandemics have been marked by the emergence of epizootic strains of influenza for which there is no immunologic memory in humans. The Spanish influenza (H1N1) pandemic of 1918 killed an estimated 40 to 50 million people worldwide. Subsequent pandemics in 1957 (H2N2) and 1968 (H3N2) also killed many thousands. Attention and trepidation are now focused on the H5N1 epizootic strain and its potential to cause worldwide disease and mortality. The H5N1 virus first emerged in 1959 in chickens in Scotland and now is causing a pandemic among chickens in Southeast Asia [43]. According to World Health Organization statistics accessed on June 5, 2006, 224 humans have been infected with H5N1 virus in 10 different countries, and 127 have died, yielding a mortality of 57%, many of which are children and young adults [44]. Almost all the persons infected with H5N1 have been linked epidemiologically with exposure to sick birds or, in a very small number of cases, to exposure to another human who had H5N1 infection. It is unknown just how widespread and lethal a pandemic of H5N1 infection would be, but the Congressional Budget Office estimates that 200 million people could be infected in the United States alone.

ly with exposure to sick birds or, in a very small number of cases, to exposure to another human who had H5N1 infection. It is unknown just how widespread and lethal a pandemic of H5N1 infection would be, but the Congressional Budget Office estimates that 200 million people could be infected in the United States alone. H5N1 disease pathogenesis The H5N1 influenza A subtype contains a new H5 hemagglutinin to which humans have little immunity. The hemagglutinin molecules attach to epithelial cells and macrophages in the lungs by interaction with cell-surface sialic acid residues. A true influenza pandemic can develop only if the avian H5N1 virus can spread efficiently from human to human, a scenario that has occurred only rarely thus far. Recent reports might help explain why the avian H5N1 virus currently is inefficient in human-to-human infection. It seems that established human influenza viruses (eg, H1N1) and avian flu viruses target different regions of the human respiratory tract. Human flu viruses preferentially recognize sialic acid residues in the proximal respiratory tract (trachea and bronchi), whereas H5N1 infects alveolar type II cells, macrophages, and the nonciliated cuboidal epithelium of the terminal bronchi [45], [46]. This differential tropism may help explain the clinical presentation of ALI/ARDS in humans infected with H5N1, and perhaps more importantly, it may help explain why H5N1 is not transmitted efficiently among humans: human influenza is transmitted easily from its proximal location in the respiratory tract, whereas H5N1 is a more deeply seated infection. There is even some concern from animal models of H5N1 infection that viremia can lead to fecal shedding of the virus and the potential for fecal–oral transmission.

tly among humans: human influenza is transmitted easily from its proximal location in the respiratory tract, whereas H5N1 is a more deeply seated infection. There is even some concern from animal models of H5N1 infection that viremia can lead to fecal shedding of the virus and the potential for fecal–oral transmission. Diagnosis, management, and prevention of H5N1 infection Children and young adults seem to be disproportionately affected by H5N1 infections, with median ages of affected individuals ranging from 9.5 to 22 years [47]. H5N1 infection has typical influenza-like symptoms but prominently involves the lower respiratory tract and often is accompanied by a watery diarrhea. Like SARS, lymphopenia, thrombocytopenia, and increased transaminase levels are often present. The most common radiographic findings are multifocal consolidations, and progression to respiratory failure occurs in the majority of hospitalized individuals within 48 hours of admission. Diagnosis relies on consideration of the appropriate epidemiologic context in conjunction with reverse transcriptase PCR of respiratory samples, with pharyngeal samples having a higher yield than nasal specimens.

ion to respiratory failure occurs in the majority of hospitalized individuals within 48 hours of admission. Diagnosis relies on consideration of the appropriate epidemiologic context in conjunction with reverse transcriptase PCR of respiratory samples, with pharyngeal samples having a higher yield than nasal specimens. From what is currently known, the treatment of H5N1 infection does not deviate substantially from the treatment of severe human influenza infections. Supportive care for patients who have H5N1-associated ALI/ARDS should include a pressure-limited, low tidal volume ventilatory strategy. For antiviral treatment, some data indicate that many of the H5N1 strains isolated from humans are resistant to the adamantanes [48], to which the current circulating human influenza strains also have recently become highly resistant. The H5N1 strains isolated from humans have proven susceptible to the neuraminidase inhibitors oseltamivir (Tamiflu) and zanamivir (Relenza), although there have been case reports of isolates resistant to oseltamivir [49]. Prophylaxis of health care workers and postexposure prophylaxis would be essential elements of the response to a pandemic, provided that adequate supplies of neuraminidase inhibitors are available. The potential role of corticosteroids in the treatment of influenza-associated ALI/ARDS is not known.

to oseltamivir [49]. Prophylaxis of health care workers and postexposure prophylaxis would be essential elements of the response to a pandemic, provided that adequate supplies of neuraminidase inhibitors are available. The potential role of corticosteroids in the treatment of influenza-associated ALI/ARDS is not known. Like the primary focus in the annual influenza season, prevention of H5N1 infection is justly receiving a great deal of attention. Researchers from throughout the world are racing to develop a vaccine that is highly effective, has low side effects, and can be mass produced in a short period of time. Multiple strategies are being used to produce an immunogenic vaccine. A recent report used two doses of an inactivated subvirion H5 vaccine administered intramuscularly to humans and showed that, at the highest dose of the vaccine administered, approximately half of the patients developed neutralizing antibody titers [50]. The 50% effectiveness of this vaccine contrasts with the 70% to 90% effectiveness of human influenza vaccines in current use. It has been proposed that the use of an adjuvant may increase H5 vaccine effectiveness, because new hemagglutinin proteins in humans may be poorly immunogenic. Other groups are testing live attenuated influenza vaccines and also adenovirus-based immunization strategies, which can elicit a strong T-cell immunity response.

has been proposed that the use of an adjuvant may increase H5 vaccine effectiveness, because new hemagglutinin proteins in humans may be poorly immunogenic. Other groups are testing live attenuated influenza vaccines and also adenovirus-based immunization strategies, which can elicit a strong T-cell immunity response. Summary TRALI, SARS, and H5N1 influenza are recently described causes of ALI/ARDS from which much has been learned, but many questions remain unanswered. The biggest impact on decreasing the incidence of TRALI will be from adherence to evidence-based transfusion guidelines and potentially from regulatory action that limits exposure to blood products or donors that have been consistently implicated in TRALI. The outcomes of recent initiatives by the United Kingdom limiting exposure to female plasma will be followed closely to determine if similar action should be taken in the United States. With the threat of SARS temporarily under control, attention is intensely focused on the pandemic threat of H5N1 influenza, which has the potential to overwhelm existing critical care resources for the treatment of respiratory failure. Undoubtedly, new infectious and noninfectious causes of ALI/ARDS will emerge in the future, mandating vigilance by health care providers, providing a challenge to public health officials and clinical and basic science investigators, and requiring transparent communication among the members of today's global society. This work was supported by Grant No. HL81027 from the National Institutes of Health.

Key points • The burden of pneumonia, including that due to respiratory viruses, is markedly higher in the very young (<5 years) and older adults (≥50 years). • Respiratory viruses substantially contribute to pneumonia in both adults and children, and when systematically tested for, are more commonly detected than bacteria in both adults and children. • The most commonly detected respiratory viruses in adults and children are adenoviruses, coronaviruses, human metapneumovirus, human rhinoviruses, influenza viruses, parainfluenza viruses, and respiratory syncytial virus. • It is difficult to distinguish between viruses by clinical presentation, and the exact clinical implication of viral detections among patients with pneumonia depends on the pathogen detected; however, there is increasing evidence of their importance in pneumonia. • The circulation of respiratory viruses varies from region to region around the world, demonstrating seasonal variation in different parts of the world, which affects the prevalence and incidence of viral pneumonia globally.

• It is difficult to distinguish between viruses by clinical presentation, and the exact clinical implication of viral detections among patients with pneumonia depends on the pathogen detected; however, there is increasing evidence of their importance in pneumonia. • The circulation of respiratory viruses varies from region to region around the world, demonstrating seasonal variation in different parts of the world, which affects the prevalence and incidence of viral pneumonia globally. Introduction Worldwide, 900,000 children aged less than 5 years die from pneumonia every year.1 Pneumonia is a leading infectious cause of hospitalization and death among US adults, resulting in more than $10 billion annual expenses.2 Despite advances in clinical diagnostic methods, especially molecular-based methods, a cause is not always ascertained in a patient with pneumonia. Recent prospective pneumonia etiology studies have failed to detect a pathogen in greater than 50% of adults and approximately 20% of children hospitalized with pneumonia.3, 4, 5, 6, 7 In these same studies, viruses were more commonly detected than bacteria in both adults and children, accounting for greater than 25% of detections in adults and greater than 70% in children.3, 7 The exact implications of viral detections among patients with pneumonia depend on the pathogen detected, but there is increasing evidence of their importance in pneumonia.8

detected than bacteria in both adults and children, accounting for greater than 25% of detections in adults and greater than 70% in children.3, 7 The exact implications of viral detections among patients with pneumonia depend on the pathogen detected, but there is increasing evidence of their importance in pneumonia.8 US prevalence/incidence The Etiology of Pneumonia in the Community (EPIC) study was a large prospective multicenter US population-based active surveillance study in which viruses were more commonly detected than bacteria in both adults and children hospitalized with community-acquired pneumonia when systematic testing was used.3, 7 Detailed study details have been previously described,3, 7 but in brief, community-acquired pneumonia was defined as evidence of acute infection, acute respiratory illness, and radiographic evidence of pneumonia; patients with severe immunosuppression and recent hospitalization were excluded. Multiple modalities for pathogen detection of bacteria and viruses were used, including culture, polymerase chain reaction (PCR), serology, and antigen-based diagnostic assays.3, 7

spiratory illness, and radiographic evidence of pneumonia; patients with severe immunosuppression and recent hospitalization were excluded. Multiple modalities for pathogen detection of bacteria and viruses were used, including culture, polymerase chain reaction (PCR), serology, and antigen-based diagnostic assays.3, 7 The results of the EPIC study demonstrated that prevalence and incidence of different pathogens varied by age. Among children less than 18 years old enrolled in the EPIC study, 70% of pneumonia hospitalizations occurred among children less than 5 years old.7 Overall annual incidence of community-acquired pneumonia hospitalization in children was 15.7/10,000 children, and incidence was highest in children less than 2 years old (62.2/10,000 children), decreased in children 2 to 4 years old (23.8/10,000), and further decreased with increasing age. These rates were slightly lower than the 2009 national Kids’ Inpatient Database, which reported 22.4 hospitalized pneumonia cases per 10,000 children less than 18 years old.9 There are methodologic differences that likely explain these differences, including nonoverlapping years of analysis, distinctions between the populations studied, and varying case definitions, including exclusion of the severely immunocompromised in the EPIC study. Nonetheless, there were similar trends indicating that pneumonia burden is highest among the youngest children.

se differences, including nonoverlapping years of analysis, distinctions between the populations studied, and varying case definitions, including exclusion of the severely immunocompromised in the EPIC study. Nonetheless, there were similar trends indicating that pneumonia burden is highest among the youngest children. In the EPIC study, among 2222 children with clinical and radiographic pneumonia who had specimens available for bacterial and viral diagnostic testing, a pathogen was detected in 1802 (81%) children with one or more viruses in 1472 (66%), bacteria in 175 (8%), and both bacteria and viruses in 155 (7%). Among these 2222 children, the most commonly detected viruses were respiratory syncytial virus (RSV, 28%), human rhinoviruses (HRV, 27%), human metapneumovirus (HMPV, 13%), adenoviruses (AdV, 11%), parainfluenza 1 to 3 viruses (PIV, 7%), influenza A and B viruses (7%), and coronaviruses (CoV, 5%) (Fig. 1 B, codetections are indicated by the lighter shading).7 Compared with older children, RSV, AdV, and HMPV were all more commonly detected among children less than 5 years old (Fig. 1C). The incidence of RSV, HRV, HMPV, AdV, influenza viruses, PIV, and CoV was all higher among children less than 5 years old than among older children but was highest among children less than 2 years old.7 Fig. 1 (A) Numbers (above the bars) and percentages of all adults in whom a specific pathogen was detected in the adult component of the EPIC study. The proportions of viral, viral-viral, bacterial-viral, bacterial, fungal or mycobacterial pathogens detected, and no pathogen detected are shown in the pie chart. (B) Numbers (above the bars) and percentages of all children in whom a specific pathogen was detected in the pediatric component of the EPIC study. (C) Proportions of pathogens detected, according to age group in the pediatric component of the EPIC study.

s detected, and no pathogen detected are shown in the pie chart. (B) Numbers (above the bars) and percentages of all children in whom a specific pathogen was detected in the pediatric component of the EPIC study. (C) Proportions of pathogens detected, according to age group in the pediatric component of the EPIC study. (From [A] Jain S, Self WH, Wunderink RG, et al. Community-acquired pneumonia requiring hospitalization among U.S. adults. N Engl J Med 2015;373:420, with permission from Massachusetts Medical Society; and [B, C] Jain S, Williams DJ, Arnold SR, et al. Community-acquired pneumonia requiring hospitalization among U.S. children. N Engl J Med 2015;372:840; with permission from Massachusetts Medical Society.) In adults enrolled in the EPIC study, overall annual incidence of community-acquired pneumonia hospitalization was 24.8/10,000 adults.3 The overall and pathogen-specific incidences increased with age with rates highest among adults 50 years of age and older. The EPIC study rates and trends are similar to previous pneumonia etiology studies conducted in the 1990s despite methodologic differences,10 but the EPIC study hospitalization rates were lower than more recent estimates based on hospitalization claims data likely due to certain excluded groups in the EPIC study, including those with severe immunosuppression.11

to previous pneumonia etiology studies conducted in the 1990s despite methodologic differences,10 but the EPIC study hospitalization rates were lower than more recent estimates based on hospitalization claims data likely due to certain excluded groups in the EPIC study, including those with severe immunosuppression.11 Among the 2259 adults enrolled in the EPIC study with clinical and radiographic pneumonia who had specimens available for bacterial and viral diagnostic testing, a pathogen was detected in 853 (38%) with one or more viruses in 530 (23%), bacteria in 247 (11%), bacteria and viruses in 59 (3%), and a fungal or mycobacterial pathogen in 17 (1%). Among the 2259 adults, the most commonly detected viruses were HRV (9%), influenza A and B viruses (6%), HMPV (4%), RSV (3%), PIV (2%), CoV (2%), and AdV (1%) (Fig. 1A).3 Importantly, the incidence of pneumonia hospitalization with influenza was almost 5 times higher among adults 65 years and older than among younger adults, and the incidence of HRV was almost 10 times as high. Interestingly, the overall incidence of pneumonia hospitalization with influenza (1.5/10,000) was similar to that of pneumococcus (1.2/10,000), a well-known bacterial cause of community-acquired pneumonia.

r among adults 65 years and older than among younger adults, and the incidence of HRV was almost 10 times as high. Interestingly, the overall incidence of pneumonia hospitalization with influenza (1.5/10,000) was similar to that of pneumococcus (1.2/10,000), a well-known bacterial cause of community-acquired pneumonia. Worldwide/regional prevalence, incidence, and mortalities It is well known that respiratory viruses contribute to acute respiratory infections, including those involving the lower respiratory tract and leading to bronchiolitis, pneumonia, and other complications. Although some global estimates of respiratory virus burden have been derived, including some from low- and middle-income countries, these data remain sparse because little surveillance for respiratory viruses is systematically carried out in many countries. In addition, most surveillance and thus estimates are not specific to pneumonia, and definitions of pneumonia vary widely between studies, making comparisons difficult. In a 2005 study, RSV was associated with 22% of acute lower respiratory infections in children less than 5 years old worldwide with 3.4 (2.8–4.3) million hospitalizations and 66,000 to 199,000 deaths; 99% of deaths occurred in developing countries.12 Data from this same analysis demonstrated that most RSV deaths in high-income countries were in children less than 1 year old, whereas in low- and middle-income countries, these deaths extended into the second year of life.

hospitalizations and 66,000 to 199,000 deaths; 99% of deaths occurred in developing countries.12 Data from this same analysis demonstrated that most RSV deaths in high-income countries were in children less than 1 year old, whereas in low- and middle-income countries, these deaths extended into the second year of life. Similar analyses have been done for the burden of influenza virus infection, again not necessarily limited to pneumonia. According to the World Health Organization (WHO), influenza occurs globally with an annual attack rate estimated at 5% to 10% in adults and 20% to 30% in children.13 Worldwide, these annual epidemics are estimated to result in about 3 to 5 million cases of severe illness, and about 250,000 to 500,000 deaths. Although hospitalizations and deaths occur in healthy people, certain groups are at higher risk for complications, and thus, influenza vaccines are targeted for these high-risk groups, including children 6 months to 5 years old, elderly 65 years and older, people with chronic medical conditions, pregnant women, and health care workers.

izations and deaths occur in healthy people, certain groups are at higher risk for complications, and thus, influenza vaccines are targeted for these high-risk groups, including children 6 months to 5 years old, elderly 65 years and older, people with chronic medical conditions, pregnant women, and health care workers. As part of the Pneumonia Research for Child Health (PERCH) project, 7 low- and middle-income countries have conducted research on pneumonia etiology among children less than 5 years old. In the PERCH project, the pneumonia case-definition per the WHO definitions was of severe (lower chest-wall indrawing in a child with history of cough or difficulty breathing) or very severe (cyanosis, oxygen saturation <90%, inability to feed, head nodding, or impaired consciousness in a child with history of cough or difficulty in breathing) pneumonia. Two types of outpatient controls without pneumonia included asymptomatic children and children with upper respiratory tract infection. In a preliminary analysis from one study site in rural Kenya, respiratory viruses were detected in most (60%) children less than 5 years old with pneumonia but also in controls (47%).14 Of the viruses detected, RSV was the most commonly detected virus in case-patients but not controls with a statistically significant association between virus detection and pneumonia hospitalization.14

ratory viruses were detected in most (60%) children less than 5 years old with pneumonia but also in controls (47%).14 Of the viruses detected, RSV was the most commonly detected virus in case-patients but not controls with a statistically significant association between virus detection and pneumonia hospitalization.14 In other pneumonia studies conducted in high-, low-, and middle-income countries, although the prevalence of specific viruses varies greatly, viruses are more commonly detected than bacteria, particularly in children. Prevalence of viruses can vary by geography and other factors, such as immunization coverage, as well as study design, including case definitions, specimen collection methods, and diagnostic tools applied; however, in most of these studies, certain viruses predominate, including RSV but also HMPV, AdV, and PIV. In a study of severe and very severe pneumonia conducted in Kenya and using multiplex PCR, a virus was detected in 56% of children less than 12 years old; RSV was most common and detected in 34% of children.15 In a different study conducted in Mozambique, viruses were detected in 49% of children with severe pneumonia, and in this case, HRV (41%), AdV (21%), and RSV (11%) were the most common.16 In similar studies, HMPV, AdV, PIV, and CoV combined account for 25% to 40% of pathogens detected in children when using PCR methods.17, 18 In many studies, codetections (viral and bacterial) in children with pneumonia have been demonstrated in more than one-quarter of cases.7, 14, 19

RSV (11%) were the most common.16 In similar studies, HMPV, AdV, PIV, and CoV combined account for 25% to 40% of pathogens detected in children when using PCR methods.17, 18 In many studies, codetections (viral and bacterial) in children with pneumonia have been demonstrated in more than one-quarter of cases.7, 14, 19 The role of viruses in adults has had increasing attention because viruses like RSV and HMPV have been commonly detected in systematic studies of hospitalized adults.20, 21 Although the same viruses that circulate in children also affect adults, the prevalence of the viruses differs between children and adults with pneumonia and also compared with data from controls. For example, HRV has been commonly detected in adult pneumonia patients, including from sterile lower respiratory tract specimens; in contrast with children, HRV is not commonly detected among adult asymptomatic controls and is often detected as the sole pathogen in adults, whereas, in children, it is often codetected.3, 4, 8, 22, 23, 24, 25 Influenza viruses are a known contributor to viral pneumonia,25 as well as a precursor to bacterial pneumonia, and are a common cause of pneumonia among persons 65 years and older. The range of other virus detections, including HMPV, RSV, PIV, CoV, and AdV, in adults with pneumonia is broad, ranging from 11% to 28% depending on the study location, design, and diagnostic tools.4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 Bacterial or viral codetections are less frequently detected in adults than in children.3

irus detections, including HMPV, RSV, PIV, CoV, and AdV, in adults with pneumonia is broad, ranging from 11% to 28% depending on the study location, design, and diagnostic tools.4, 5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 Bacterial or viral codetections are less frequently detected in adults than in children.3 Worldwide/regional seasonality and circulation patterns The circulation of respiratory viruses varies from region to region around the world and demonstrates seasonal variation in different parts of the world, which affects the prevalence and incidence of viral pneumonia globally. In the United States, similar to other Northern Hemisphere countries, there are distinct peaks and troughs for different viruses. Generally, except for the 2009 H1N1 pandemic period from 2008 to 2009, there have been very clear peaks of influenza virus circulation annually in the United States that occur in the late fall or early winter, although it is never predictable exactly when the influenza season will start, and there is also regional variation. US influenza virus circulation data are reported weekly at the national and regional level on the Centers for Disease Control and Prevention (CDC) FluView Web site: http://www.cdc.gov/flu/weekly/fluactivitysurv.htm. Similarly, influenza circulation varies worldwide and surveillance methods vary from nation to nation, but data on global influenza circulation can be accessed on the WHO FluNet Web site: http://www.who.int/influenza/gisrs_laboratory/flunet/en/. Weekly global update reports are also available here: http://www.who.int/influenza/surveillance_monitoring/updates/latest_update_GIP_surveillance/en/.

ary from nation to nation, but data on global influenza circulation can be accessed on the WHO FluNet Web site: http://www.who.int/influenza/gisrs_laboratory/flunet/en/. Weekly global update reports are also available here: http://www.who.int/influenza/surveillance_monitoring/updates/latest_update_GIP_surveillance/en/. In the United States, RSV circulation usually starts in late October and lasts until late January but can shift, starting in late January and lasting until early April depending on the year and circulation of other respiratory pathogens. Like influenza viruses, there is also regional variation with an earlier season starting in Florida and lasting longer than other US regions. National and regional RSV surveillance trends can be monitored at the CDC National Respiratory and Enteric Virus Surveillance System (NREVSS) Web site: http://www.cdc.gov/surveillance/nrevss/rsv/natl-trend.html. Through NREVSS, national and regional data are available for HMPV (http://www.cdc.gov/surveillance/nrevss/hmpv/natl-trend.html) and national data are also available for AdV (http://www.cdc.gov/surveillance/nrevss/adeno/natl-trend.html) and PIV (http://www.cdc.gov/surveillance/nrevss/human-paraflu/natl-trend.html), although specimens are not routinely tested for these viruses.

or HMPV (http://www.cdc.gov/surveillance/nrevss/hmpv/natl-trend.html) and national data are also available for AdV (http://www.cdc.gov/surveillance/nrevss/adeno/natl-trend.html) and PIV (http://www.cdc.gov/surveillance/nrevss/human-paraflu/natl-trend.html), although specimens are not routinely tested for these viruses. Globally, RSV surveillance efforts and methods vary from country to country. Data from 7 countries (Bangladesh, China, Egypt, Guatemala, Kenya, South Africa, and Thailand) over 8 years (2004–2012) demonstrated that RSV infection had 1 to 2 epidemic periods each year in each country; in general, seasonality patterns were similar within country but differed between countries from year to year. Likely factors affecting circulation were weather, geography, precipitation, and temperature.29 Similar reports from tropical and subtropical areas of southern and southeastern Asia have also shown that influenza virus circulation is highly dependent on weather patterns, especially rainfall and monsoons, and affects the seasonality of influenza virus circulation regionally, which has direct implications for influenza vaccination timing in these regions.30, 31

reas of southern and southeastern Asia have also shown that influenza virus circulation is highly dependent on weather patterns, especially rainfall and monsoons, and affects the seasonality of influenza virus circulation regionally, which has direct implications for influenza vaccination timing in these regions.30, 31 Clinical correlation and risk factors Many pneumonia etiology studies rely on naso/oropharyngeal specimens for pathogen detection in addition to blood, serum, sputum, and urine (only in adults) because lower respiratory specimens are not practical or possible unless clinically necessary. Because most of these specimens do not come not directly from the lung, it is difficult to discern the association of a pathogen detection with pneumonia. Thus, many studies have enrolled asymptomatic controls along with pneumonia cases to help determine the possible contribution of respiratory viruses (attributable risk) to pneumonia at a population level. However, studies show variable prevalence of viruses among controls, possibly due to differences in definitions and methods for ascertainment of controls.32 Different strategies for control enrollment may be applicable to different study objectives and settings.32

(attributable risk) to pneumonia at a population level. However, studies show variable prevalence of viruses among controls, possibly due to differences in definitions and methods for ascertainment of controls.32 Different strategies for control enrollment may be applicable to different study objectives and settings.32 In the EPIC study, all pathogens except for HRV were detected in 3% or less of asymptomatic controls; HRV was detected in 17% of pediatric controls compared with 22% of children with pneumonia (P = .04).7, 8 In asymptomatic adult controls, only 2% had any pathogen detection, compared with 27% among the patients hospitalized with pneumonia; interestingly, HRV was rarely detected in adult controls (1%).3, 8 In the EPIC study, the attributable fraction (AF) was calculated by comparing prevalence of viruses in pneumonia cases with asymptomatic controls, and adjusting for age, enrollment month, and enrollment city. In this analysis, the AF indicated that the detection of influenza, RSV, and HMPV among all patients, both children and adults, indicated an etiologic role. However, the detections of PIV, CoV, and AdV, particularly in children, did not demonstrate high AF for pneumonia. HRV was associated with pneumonia in adults but not children.8 The exact role of HRV in pneumonia remains unclear and controversial, even when detected as a single pathogen, particularly because HRV can shed for greater than 2 weeks after a primary infection, making its detection at time of pneumonia challenging to interpret with respect to the current clinical illness.22, 23, 24

The exact role of HRV in pneumonia remains unclear and controversial, even when detected as a single pathogen, particularly because HRV can shed for greater than 2 weeks after a primary infection, making its detection at time of pneumonia challenging to interpret with respect to the current clinical illness.22, 23, 24 Data from the EPIC study are similar to other US data from Singleton and colleagues,33 which compared asymptomatic Alaskan children less than 3 years old from the community with children hospitalized with respiratory infections. In this study, RSV, PIV, HMPV, and influenza viruses were all significantly more common in hospitalized children than controls, but HRV, AdV, and CoV were not; interestingly, children with RSV only or HMPV only had more severe illness compared with children with the other viruses.

pitalized with respiratory infections. In this study, RSV, PIV, HMPV, and influenza viruses were all significantly more common in hospitalized children than controls, but HRV, AdV, and CoV were not; interestingly, children with RSV only or HMPV only had more severe illness compared with children with the other viruses. Data from the recent US studies, including the EPIC study, are somewhat in contrast to data from the PERCH project site in Kenya, which tested for multiple pathogens (bacteria and viruses) in children less than 5 years old with pneumonia and in comparison to community controls (who could have had an upper respiratory infection). In the Kenya site for the PERCH project, compared with controls, RSV was the only virus determined to have a statistically significant association with hospitalization.14 The differences between the study results are likely due to variations in case and control definitions, prevalence of pathogens in the different studies, geography, and sociocultural characteristics that determine access to health care, threshold for hospitalization, and vaccination coverage for available immunizations.

he differences between the study results are likely due to variations in case and control definitions, prevalence of pathogens in the different studies, geography, and sociocultural characteristics that determine access to health care, threshold for hospitalization, and vaccination coverage for available immunizations. In addition to nuances around etiology, the clinical spectrum of illness due to respiratory viruses is broad and encompasses asymptomatic infection, upper respiratory infection, and lower respiratory tract infection, resulting in pneumonia as well as other complications (eg, acute respiratory distress or secondary bacterial infection). Clinically, symptoms due to any specific virus infection, including in relation to pneumonia, largely overlap; thus, there are few clinical clues to distinguish between illnesses due to different pathogens. Some epidemiologic clues such as age and seasonality may be helpful. In addition, greater than 25% of children and less than 5% of adults are found to have multiple pathogens, potentially including both viruses and bacteria, and on this basis alone could be expected to have a mixed clinical presentation.3, 7

pathogens. Some epidemiologic clues such as age and seasonality may be helpful. In addition, greater than 25% of children and less than 5% of adults are found to have multiple pathogens, potentially including both viruses and bacteria, and on this basis alone could be expected to have a mixed clinical presentation.3, 7 There are some data from comparisons of patients with illness of varying severity that suggest that bacterial pneumonia contributes to more severe illness. For example, in the EPIC study, Streptococcus pneumoniae, Staphylococcus aureus, and Enterobacteriaceae combined accounted for 16% of detected pathogens among adults admitted to the intensive care unit (ICU) as compared with 6% among adults not admitted to the ICU.3 Thus, when bacteria were detected, it was more likely in a severely ill patient. However, it is important to note that viruses were detected in 22% of adults admitted to the ICU compared with 24% not admitted to the ICU; so although there were no statistically significant differences between the severe and nonsevere groups, viral pneumonia contributed to ICU admissions, mechanical ventilation, and also death.

is important to note that viruses were detected in 22% of adults admitted to the ICU compared with 24% not admitted to the ICU; so although there were no statistically significant differences between the severe and nonsevere groups, viral pneumonia contributed to ICU admissions, mechanical ventilation, and also death. Influenza viruses are some of the more commonly recognized viruses that cause pneumonia and can lead to a primary viral pneumonia, secondary bacterial pneumonia, or mixed viral-bacterial pneumonia.25, 34, 35 Much of the influenza-associated pneumonia literature has focused on pandemics,36, 37 including the most recent 2009 H1N1 pandemic.38, 39, 40 During interpandemic years, influenza virus circulation varies, and thus, rates of influenza-associated pneumonia vary from year to year. Data from these reports indicate that in comparison to patients with influenza virus infection without pneumonia, patients with influenza virus infection and radiographic evidence of pneumonia have a more severe course of illness in terms of longer length of stay, ICU admission, mechanical ventilation, acute respiratory distress syndrome, sepsis, and death.40

comparison to patients with influenza virus infection without pneumonia, patients with influenza virus infection and radiographic evidence of pneumonia have a more severe course of illness in terms of longer length of stay, ICU admission, mechanical ventilation, acute respiratory distress syndrome, sepsis, and death.40 Although RSV is commonly associated with bronchiolitis, it is also a well-known cause of the clinically and radiographically defined pneumonia, with RSV burden highest among children less than 2 years old. Most children are infected with RSV by 2 years of age, and the first infection, which may not confer immunity, is usually most severe. Prematurity and young age have been shown to be independent risk factors for hospitalization.41 In countries with a high HIV prevalence, RSV-associated acute lower respiratory tract infection has led to an increased risk of hospitalization and death, and longer hospital stay in children with HIV infection compared with children without HIV infection.42 RSV has also been shown to lead to acute respiratory infection in adults, including hospitalization, with the highest burden among adults greater than 50 years old.20 In addition to older age, other risk factors for RSV infection in adults include immunocompromised states and chronic lung conditions.43

HIV infection.42 RSV has also been shown to lead to acute respiratory infection in adults, including hospitalization, with the highest burden among adults greater than 50 years old.20 In addition to older age, other risk factors for RSV infection in adults include immunocompromised states and chronic lung conditions.43 Pneumonia was reported in almost half of the children who were hospitalized with HMPV infection in one multisite study of acute respiratory illness conducted in the United States.44 Similar to RSV, prematurity and asthma have been shown to be more frequent among hospitalized children with HMPV infection than children without HMPV infection.44 Risk factors for HMPV infection and subsequent complications, including pneumonia and hospitalization in adults, include older age, and underlying conditions, including asthma, cancer, and chronic obstructive pulmonary disease.21

nt among hospitalized children with HMPV infection than children without HMPV infection.44 Risk factors for HMPV infection and subsequent complications, including pneumonia and hospitalization in adults, include older age, and underlying conditions, including asthma, cancer, and chronic obstructive pulmonary disease.21 In addition to annual epidemics of respiratory viruses, it is important to be vigilant for new and emerging viruses that can lead to severe illness including pneumonia, hospitalization, and death. Recent examples include Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)45 and Middle-East Respiratory Syndrome Coronavirus (MERS-CoV).46 SARS-CoV was first recognized in China in 2002 and caused a worldwide outbreak with 8098 probable cases and 774 deaths from 2002 to 2003; however, no cases have been reported since 2004. MERS-CoV was first reported in 2012 in Saudi Arabia and has since caused outbreaks in multiple countries in the region. Most MERS cases have been in older men and those with chronic medical conditions, but all ages have been affected, including those in contact with a person who had MERS, especially health care workers.47

S-CoV was first reported in 2012 in Saudi Arabia and has since caused outbreaks in multiple countries in the region. Most MERS cases have been in older men and those with chronic medical conditions, but all ages have been affected, including those in contact with a person who had MERS, especially health care workers.47 Avian influenza viruses are circulating in birds, both wild and domesticated, throughout the world. Although many viruses are considered low pathogenic avian influenza A viruses, there are highly pathogenic virus strains as well that have been detected in humans. Illness can range from mild to severe, including pneumonia, and cases are often sporadic and after contact with infected birds or their fluids; closing of live bird markets has been shown to reduce transmission. From 2003 to 2016, there have been 854 infections of avian influenza A (H5N1) virus reported to WHO, among which there were 450 (53%) deaths.48 Human infections with avian influenza A (H7N9) virus were first reported in China in 2013.49 Since then, there have been 798 laboratory-confirmed infections in China; most infections are sporadic with a few clusters of infection. There is no evidence of sustained human-to-human transmission.50 Since 2014, there have also been outbreaks in North America and elsewhere among wild and domesticated birds with influenza A (H5Nx and H7N8), which are considered highly pathogenic viruses in birds, but thus far, there have not been any human cases (http://www.oie.int/en/animal-health-in-the-world/update-on-avian-influenza/2016/).

there have also been outbreaks in North America and elsewhere among wild and domesticated birds with influenza A (H5Nx and H7N8), which are considered highly pathogenic viruses in birds, but thus far, there have not been any human cases (http://www.oie.int/en/animal-health-in-the-world/update-on-avian-influenza/2016/). Gaps in knowledge about the epidemiology of respiratory virus–associated pneumonia Although active and systematic surveillance for all respiratory viruses has increased globally and is strongest for influenza viruses, it is still lacking throughout much of the world.51 Many questions remain about the seasonality of influenza and other respiratory viruses in tropical countries where there is increasing evidence of year-round circulation of influenza in warmer, more humid climates.29, 30, 31, 52 Country-specific studies to better understand the contribution of viruses to pneumonia are still required, especially from low- and middle-income countries. The PERCH project will further the understanding of pneumonia in children less than 5 years old,14 but data on older adults and pneumonia remain scant.

30, 31, 52 Country-specific studies to better understand the contribution of viruses to pneumonia are still required, especially from low- and middle-income countries. The PERCH project will further the understanding of pneumonia in children less than 5 years old,14 but data on older adults and pneumonia remain scant. Country-specific data on pneumonia, from high, low, and middle income are continually needed because the prevalence of respiratory viruses varies due to seasonal and geographic differences. Incidence also varies, including by age but also due to socioeconomic and sociocultural factors. In addition, access to prevention and control methods that may be in place in some but not all countries (ie, vaccination coverage for S pneumoniae or Haemophilus influenzae, antibiotics, or antivirals) can affect the epidemiology of viral pneumonia. Risk factors for pneumonia, such as malnutrition, air pollution, tuberculosis, or coexisting malarial infection, may be more relevant in low- and middle-income countries than in more developed countries, where underlying conditions or tobacco exposure may be more prevalent among patients with pneumonia.34, 35

neumonia. Risk factors for pneumonia, such as malnutrition, air pollution, tuberculosis, or coexisting malarial infection, may be more relevant in low- and middle-income countries than in more developed countries, where underlying conditions or tobacco exposure may be more prevalent among patients with pneumonia.34, 35 In addition, most pneumonia studies have been performed in hospital settings. However, in many parts of the world, including developed and developing nations, viral pneumonia does not always result in hospitalization; deaths are missed because they occur at home.53 Thus, more studies conducted in the community and also outpatient settings are needed to more fully understand the burden and epidemiology of respiratory viral pneumonia. Summary The burden of pneumonia, including that due to respiratory viruses, is markedly higher in the very young (<5 years) and older adults (≥50 years). Viruses are commonly detected, and clinically, it is difficult to distinguish between viral and bacterial pneumonia for most patients at presentation. Further development of new rapid diagnostic tests that can accurately distinguish among potential pathogens is urgently needed to better inform clinical care and public health practice.54 Treatment and vaccination are only currently available for influenza despite the high burden of RSV, HMPV, and other viruses. Development of effective vaccines and treatments for these viruses of importance could reduce the burden of pneumonia and their complications in both children and adults around the world.

ice.54 Treatment and vaccination are only currently available for influenza despite the high burden of RSV, HMPV, and other viruses. Development of effective vaccines and treatments for these viruses of importance could reduce the burden of pneumonia and their complications in both children and adults around the world. Disclaimer: The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Key points • Most community-acquired respiratory viruses are RNA viruses except for adenovirus and human bocavirus, which are DNA viruses. • Using molecular techniques, respiratory viruses are identified in approximately 25% of patients with community-acquired pneumonia. • In addition to the community-acquired respiratory viruses, immunocompromised patients are particularly susceptible to viruses of the Herpesviridae family. • It is difficult to diagnose influenza or other viral infection on clinical grounds. • Patients with influenza pneumonia should be treated with a neuraminidase inhibitor. For other viruses, treatment options are limited.

• In addition to the community-acquired respiratory viruses, immunocompromised patients are particularly susceptible to viruses of the Herpesviridae family. • It is difficult to diagnose influenza or other viral infection on clinical grounds. • Patients with influenza pneumonia should be treated with a neuraminidase inhibitor. For other viruses, treatment options are limited. Introduction Respiratory viral infections cause substantial burden. They are prevalent and tend to affect those who are more vulnerable, such as children, elderly, and people living in developing areas, such as sub-Saharan Africa and Southeast Asia.1 The advent of molecular techniques has facilitated the identification of respiratory viruses in patients with pneumonia and has shed a light on how commonly these viruses occur in patients with pneumonia. With the currently available diagnostic tools, viral pathogens are more often identified than bacterial pathogens in community-acquired pneumonia.2 A large amount of effort is currently being dedicated to elucidate the pathogenicity of respiratory viruses and the interaction between viruses and bacteria in the setting of pneumonia. Since the last century, a number of devastating pandemics and outbreaks related to respiratory viruses have occurred.3, 4 Recently, there has been a growing interest in the development of new antiviral medications for respiratory infection. In this article, we provide an overview of pneumonia caused by influenza and other respiratory viruses from the practicing clinician perspective and with a focus on the adult population.

occurred.3, 4 Recently, there has been a growing interest in the development of new antiviral medications for respiratory infection. In this article, we provide an overview of pneumonia caused by influenza and other respiratory viruses from the practicing clinician perspective and with a focus on the adult population. Microbiology overview Human influenza is an RNA virus that belongs to the Orthomyxoviridae family and is categorized into types A, B, and C based on its nucleoprotein and matrix protein. Influenza A virus is subcategorized into subtypes such as H1N1, H1N2, and H3N2 based on hemagglutinin and neuraminidase. Influenza B is subcategorized into the B/Yamagata and the B/Victoria lineages.3, 5, 6 Most influenza infections are caused by types A and B.7 The gene mutation that influenza undergoes every year is called antigenic drift and is responsible for seasonal outbreaks. Conversely, influenza pandemics are caused by antigenic shift, which occurs when new hemagglutinin or neuraminidase subtypes are acquired.7

6 Most influenza infections are caused by types A and B.7 The gene mutation that influenza undergoes every year is called antigenic drift and is responsible for seasonal outbreaks. Conversely, influenza pandemics are caused by antigenic shift, which occurs when new hemagglutinin or neuraminidase subtypes are acquired.7 Most community-acquired respiratory viruses are RNA viruses except for adenovirus and human bocavirus, which are DNA viruses.8, 9, 10, 11, 12, 13, 14, 15 The Paramyxoviridae family includes respiratory syncytial virus, parainfluenza, and human metapneumovirus. A distinctive feature of the Paramyxoviridae family viruses is the presence of a fusion protein.9, 12, 14 The fusion protein, which enables the integration of the virus with the cell membrane, allowing the introduction of the viral genome into the cell cytoplasm, is a potential target for vaccines and antivirals.16 The Picornaviridae family of virus, which includes enterovirus and human rhinovirus, are characterized by a capsid that contains the viral genome. The capsid has a large cleft (or canyon) that binds to adhesion molecules on the cell surface, leading to the eventual entry of the viral genome into the cell. The capsid and the adhesion molecules are potential targets of antivirals17, 18 (Table 1 ).Table 1 Characteristics and taxonomy of commonly identified respiratory viruses in patients with community-acquired pneumonia

dhesion molecules on the cell surface, leading to the eventual entry of the viral genome into the cell. The capsid and the adhesion molecules are potential targets of antivirals17, 18 (Table 1 ).Table 1 Characteristics and taxonomy of commonly identified respiratory viruses in patients with community-acquired pneumonia Virus Genome Family Important Antigenic Structures Influenza RNA Orthomyxoviridae Surface glycoproteins hemagglutinin (HA) and the neuraminidase (NA).8 Respiratory syncytial virus RNA Paramyxoviridae Attachment glycoprotein (G) and fusion (F) glycoprotein.9 Human rhinovirus RNA Picornaviridae Viral capsid proteins VP1, VP2, VP3, and VP4.10 Adenovirus DNA Adenoviridae Capsid major structures: hexon (the building block of the capsid), penton base, and polypetides.11 Parainfluenza RNA Paramyxoviridae Surface glycoproteins hemagglutinin-neuraminidase and fusion protein. Membrane protein.12 Coronavirus RNA Coronaviridae Membrane glycoprotein and spike protein.13 Human metapneumovirus RNA Paramyxoviridae Virus fusion (F) glycoprotein.14 Human bocavirus DNA Parvoviridae Capsid viral proteins (VPs), VP1, and VP2.15

idae Surface glycoproteins hemagglutinin-neuraminidase and fusion protein. Membrane protein.12 Coronavirus RNA Coronaviridae Membrane glycoprotein and spike protein.13 Human metapneumovirus RNA Paramyxoviridae Virus fusion (F) glycoprotein.14 Human bocavirus DNA Parvoviridae Capsid viral proteins (VPs), VP1, and VP2.15 Incidence and epidemiology Epidemiology of Viral Respiratory Infection in Community-Acquired Pneumonia A systematic review included 31 observational studies that enrolled patients with community-acquired pneumonia who underwent viral polymerase chain reaction testing. The pooled proportion of patients with viral infection was 24.5% (95% confidence interval [CI] 21.5%–27.5%; I2 = 92.9%).19 Most of these studies were performed in the inpatient setting and viral polymerase chain reaction was obtained mostly from nasal or oropharyngeal swab. In the only study that was performed in the outpatient setting, the proportion of viral infection was 12.1% (95% CI 7.7%–16.5%; I2 = 0.0%).20 The pooled proportion of viral infection was 44.2% (95% CI 35.1%–53.3%; I2 = 0%) from studies in which a lower respiratory sample was obtained in more than half of the patients.21, 22 The proportion of dual bacterial and viral infection was 10% (95% CI 8%–11%; I2 = 93.1%). Although the presence of a viral infection did not significantly increase the risk of short-term death, patients with dual bacterial-viral infection had twice the risk of death as compared with patients without dual infection.19 It is important to note that the identification of a viral pathogen in a patient with pneumonia does not necessarily mean that the virus has a pathogenic effect, particularly if the identification is via nasopharyngeal swab (Fig. 1 , Table 2 ).Fig. 1 Number of studies according to most commonly identified viral pathogen. RSV, respiratory syncytial virus.

ntification of a viral pathogen in a patient with pneumonia does not necessarily mean that the virus has a pathogenic effect, particularly if the identification is via nasopharyngeal swab (Fig. 1 , Table 2 ).Fig. 1 Number of studies according to most commonly identified viral pathogen. RSV, respiratory syncytial virus. (Data from Burk M, El-Kersh K, Saad M, et al. Viral infection in community-acquired pneumonia: a systematic review and meta-analysis. Eur Respir Rev 2016;25(140):178–88.)Table 2 Different scenarios for the effect of an identified viral pathogen in the setting of pneumonia Virus is a “bystander” and does not have a pathogenic effect. Although uncommon in adults, asymptomatic carriage of respiratory viruses occurs.126 Virus has a pathogenic effect and is causing pneumonia in isolation. Potential mechanisms include dysregulation of cytokines and chemokines, infection of epithelial cells in the lungs, and apoptosis.127 Virus has a pathogenic effect and is causing pneumonia along with a bacterial pathogen. A study showed that the mortality for patients with community-acquired pneumonia and bacterial and viral coinfection is higher.19 Virus caused a recent infection that prompted a secondary bacterial infection. This occurs particularly with Streptococcus pneumoniae or Staphylococcus aureus infection following influenza infection.128 Lag time of 2–4 wk between the viral and bacterial infection.129 Polymerase chain reaction test may remain positive for up to 5 wk after a viral infection.130

pted a secondary bacterial infection. This occurs particularly with Streptococcus pneumoniae or Staphylococcus aureus infection following influenza infection.128 Lag time of 2–4 wk between the viral and bacterial infection.129 Polymerase chain reaction test may remain positive for up to 5 wk after a viral infection.130 Epidemiology of Viral Respiratory Infection in Immunocompromised Patients In immunocompromised patients with pneumonia, infection by respiratory viruses is exceedingly common. Surveillance studies show that a respiratory viral pathogen is identified in close to a third of hospitalized patients with leukemia or hematopoietic stem cell transplantation and respiratory symptoms. Pneumonia occurs in most immunosuppressed patients infected with a respiratory viral pathogen.23 Immunocompromised patients are commonly infected by the same respiratory viruses that cause infection in immunocompetent patients. However, viruses of the Herpesviridae family also tend to cause infection in immunocompromised patients. As an example, in an early series of patients who underwent allogeneic bone marrow transplantation, cytomegalovirus was the most common viral pathogen.24 Varicella zoster virus reactivation can occur in patients after hematopoietic stem cell transplantation with early series reporting incidences ranging from 22% to 41%.25, 26 It is not unusual for the infection to present in a disseminated form in these patients, and pneumonia is one of the complications.25, 26, 27

n.24 Varicella zoster virus reactivation can occur in patients after hematopoietic stem cell transplantation with early series reporting incidences ranging from 22% to 41%.25, 26 It is not unusual for the infection to present in a disseminated form in these patients, and pneumonia is one of the complications.25, 26, 27 Epidemiology of Hospital-Acquired Viral Respiratory Infection Traditionally, hospital-acquired respiratory viral infection has been thought to be limited to immunocompromised patients. However, it is now known that this can also commonly occur in immunocompetent patients. This was highlighted by a prospective cohort study that included 262 patients with hospital-acquired pneumonia. The proportion of viral infection was 36.1% in immunocompromised patients and 11.2% in non-immunocompromised patients. The identified viruses were respiratory syncytial virus (6.1%), parainfluenza virus (6.1%), influenza virus (3.8%), cytomegalovirus (1.9%), human coronavirus (1.5%), bocavirus (0.8%), human metapneumovirus (0.8%), and adenovirus (0.4%).28 These data underscore the importance of infection control measures in patients with pneumonia.

ratory syncytial virus infection was diagnosed in 8% to 13% of these patients depending on the year. Of the 132 hospitalized patients with respiratory syncytial virus infection, 41 (31%) had an infiltrate on chest radiograph, 20 (15%) required ICU admission, 17 (13%) required mechanical ventilation, and 10 (8%) died.45 Epidemiology of Other Respiratory Viruses Rhinovirus • Most common cause of common cold, a self-limited acute illness that occurs 2 to 4 times per year in adults. • This infection is characterized by sneezing, nasal discharge, sore throat, and low-grade fever.47 • Rhinovirus tends to occur more often in the early fall or spring.48 • Rhinovirus is commonly identified in the upper respiratory tract of patients with community-acquired pneumonia via molecular techniques. In fact, rhinovirus was the most commonly identified pathogen in a large cohort of adult patients hospitalized with community-acquired pneumonia conducted in the United States.2 Coronavirus • Occurs more commonly in the winter and follows a seasonal pattern that resembles that of influenza.49 • Coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 have ubiquitous circulation and are a usual etiology of common cold.35 • Coronaviruses have also been commonly associated with lower respiratory tract symptoms.49 • Adult hospitalized patients with coronavirus infection are often immunocompromised, and pneumonia is a common occurrence.50

d viruses were respiratory syncytial virus (6.1%), parainfluenza virus (6.1%), influenza virus (3.8%), cytomegalovirus (1.9%), human coronavirus (1.5%), bocavirus (0.8%), human metapneumovirus (0.8%), and adenovirus (0.4%).28 These data underscore the importance of infection control measures in patients with pneumonia. Pandemics and Outbreaks Since the past century, there have been 5 influenza pandemics: 1918 to 1919 Spanish influenza, 1957 H2N2 Asian influenza, 1968 H3N2 Hong Kong influenza, 1977 H1N1 Russian influenza, and the 2009 H1N1 pandemic.3, 4 It is estimated that the 2009 H1N1 pandemic caused 201,200 respiratory deaths and 83,000 cardiovascular deaths. Most of these deaths occurred in patients younger than 65 years old.29 In 2003, a major outbreak of atypical pneumonia was reported. The cases initially clustered in China but were subsequently reported worldwide. The pneumonia often resulted in acute respiratory failure and was named severe acute respiratory syndrome.30 Subsequently, the etiologic agent of this disease was identified as a novel coronavirus,31, 32 which was named the Urbani strain of severe acute respiratory syndrome–associated coronavirus.31 In 2012, another novel coronavirus was isolated from a patient with pneumonia in Saudi Arabia.33 The virus was subsequently named Middle East respiratory syndrome coronavirus.34 Infection by this virus causes an illness that is clinically similar to that caused by severe acute respiratory syndrome–associated coronavirus but with higher mortality.35 Cases of Middle East respiratory syndrome coronavirus were initially reported in Saudi Arabia but were subsequently reported in other countries, including the United States, typically in persons who had traveled from the Arabian Peninsula.36, 37, 38

spiratory syndrome–associated coronavirus but with higher mortality.35 Cases of Middle East respiratory syndrome coronavirus were initially reported in Saudi Arabia but were subsequently reported in other countries, including the United States, typically in persons who had traveled from the Arabian Peninsula.36, 37, 38 Influenza The incidence of influenza can vary substantially in different seasons. As an example, using online surveillance data, it was estimated that the influenza attack rate for adults aged 20 to 64 years old was 30.5% (95% CI 4.4–49.3) in the 2012 to 2013 season and 7.1 (95% CI −5.1 to 32.5) in the 2013 to 2014 season.39 The rates of influenza-associated hospitalization per 100,000 persons varied from 4.8 to 18.7 in 3 different seasons in the United States.40

hat the influenza attack rate for adults aged 20 to 64 years old was 30.5% (95% CI 4.4–49.3) in the 2012 to 2013 season and 7.1 (95% CI −5.1 to 32.5) in the 2013 to 2014 season.39 The rates of influenza-associated hospitalization per 100,000 persons varied from 4.8 to 18.7 in 3 different seasons in the United States.40 Different studies showed that approximately one-third of hospitalized patients with laboratory-confirmed influenza have pneumonia.41, 42, 43 In a study that included 4765 patients hospitalized with influenza, those with pneumonia were older than those without pneumonia (median age of 74 years vs 69 years; P < .01). In a multivariate analyses, the following factors were significant predictors of pneumonia in hospitalized patients with influenza: age older than 75 years (odds ratio [OR] 1.27; 95% CI 1.10–1.46), white race (OR 1.24; 95% CI 1.03–1.49), nursing home residence (OR 1.37; 95% CI 1.14–1.66), chronic lung disease (OR 1.37; 95% CI 1.18–1.59), and immunosuppression (OR 1.45; 95% CI 1.19–1.78). Asthma was associated with lower odds of pneumonia (OR 0.76; 95% CI 0.62–0.92).42 In another study of 579 adult patients hospitalized with laboratory-confirmed influenza, a multivariate analyses showed that the following factors were significantly associated with pneumonia: older age (OR 1.026; 95% CI 1.013–1.04), higher C-reactive protein, mg/dL (OR 1.128; 95% CI 1.088–1.17), smoking (OR 1.818; 95% CI 1.115–2.965), low albumin level (OR 2.518; 95% CI 1.283–4.9), acute respiratory failure (OR 4.525; 95% CI 2.964–6.907), and productive cough (OR 8.173; 95% CI 3.674–18.182).43

th pneumonia: older age (OR 1.026; 95% CI 1.013–1.04), higher C-reactive protein, mg/dL (OR 1.128; 95% CI 1.088–1.17), smoking (OR 1.818; 95% CI 1.115–2.965), low albumin level (OR 2.518; 95% CI 1.283–4.9), acute respiratory failure (OR 4.525; 95% CI 2.964–6.907), and productive cough (OR 8.173; 95% CI 3.674–18.182).43 During an influenza season, the attributed mortality to pneumonia and influenza in the United States ranges from 5.6% to 11.1%.44 In a cohort study that included laboratory-confirmed cases of influenza admitted to the hospital, those with pneumonia, as compared with those without pneumonia, were more likely to require intensive care unit (ICU) admission (27% vs 10%) and mechanical ventilation (18% vs 5%), and to die (9% vs 2%)42 (Fig. 2 ).Fig. 2 Proportion of pneumonia and associated outcomes in patients admitted to the hospital with influenza infection. (Data from Garg S, Jain S, Dawood FS, et al. Pneumonia among adults hospitalized with laboratory-confirmed seasonal influenza virus infection-United States, 2005-2008. BMC Infect Dis 2015;15:369.)

During an influenza season, the attributed mortality to pneumonia and influenza in the United States ranges from 5.6% to 11.1%.44 In a cohort study that included laboratory-confirmed cases of influenza admitted to the hospital, those with pneumonia, as compared with those without pneumonia, were more likely to require intensive care unit (ICU) admission (27% vs 10%) and mechanical ventilation (18% vs 5%), and to die (9% vs 2%)42 (Fig. 2 ).Fig. 2 Proportion of pneumonia and associated outcomes in patients admitted to the hospital with influenza infection. (Data from Garg S, Jain S, Dawood FS, et al. Pneumonia among adults hospitalized with laboratory-confirmed seasonal influenza virus infection-United States, 2005-2008. BMC Infect Dis 2015;15:369.) Respiratory Syncytial Virus In older subjects, the burden of respiratory syncytial virus infection is similar to that of influenza. A study prospectively followed 2 outpatient cohorts during 4 seasons: 608 heathy elderly patients and 540 high-risk adults. High-risk status was defined as the presence of congestive heart failure or chronic pulmonary disease. Respiratory syncytial virus infection was diagnosed in 3% to 7% of healthy elderly subjects and 4% to 10% of high-risk subjects. This accounted for 1.5 respiratory syncytial virus infections per 100 person-months in high-risk adults and 0.9 in healthy elderly subjects.45 In an analysis of hospitalization and viral surveillance data that encompassed several years, it was estimated that the respiratory syncytial virus–associated hospitalization rate per 100,000 person-years in the United States was 12.8 (95% CI 2.4–73.9) for patients age 50 to 64 years old and 86.1 (95% CI 37.3–326.2) for patients aged ≥65 years old. In contrast to influenza-associated hospitalizations, the rates of respiratory syncytial virus–associated hospitalizations were relatively similar across the years.46 In a cohort of 1388 hospitalized adults older than 65 years or with underlying cardiopulmonary diseases, respiratory syncytial virus infection was diagnosed in 8% to 13% of these patients depending on the year. Of the 132 hospitalized patients with respiratory syncytial virus infection, 41 (31%) had an infiltrate on chest radiograph, 20 (15%) required ICU admission, 17 (13%) required mechanical ventilation, and 10 (8%) died.45

• Coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 have ubiquitous circulation and are a usual etiology of common cold.35 • Coronaviruses have also been commonly associated with lower respiratory tract symptoms.49 • Adult hospitalized patients with coronavirus infection are often immunocompromised, and pneumonia is a common occurrence.50 • Severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus caused outbreaks and pandemics of an acute respiratory illness, often leading to respiratory failure.35 Adenovirus • Adenovirus is a common cause of upper respiratory tract symptoms and conjunctivitis.51 • Adult patients with adenovirus pneumonia are relatively young. • Different studies have reported that patients with community-acquired pneumonia and adenovirus infection have mean age that ranges from 30 to 38 years old.52, 53 • Adenovirus also causes serious infection in immunocompromised patients. The adenovirus species found in immunocompromised patients are not typically found in the community, which indicates endogenous viral reactivation in these patients.54 • No clear seasonality, although cases may spike in some months.55 • A number of outbreaks caused by adenovirus have been reported. Some examples include reports of outbreaks in military personnel,56 psychiatric care facility,57 and ICU.58 Parainfluenza • Most infections are caused by parainfluenza 1 and 3.59 Parainfluenza 2 is less commonly identified, and parainfluenza 4 is a rare cause of respiratory infection.

• A number of outbreaks caused by adenovirus have been reported. Some examples include reports of outbreaks in military personnel,56 psychiatric care facility,57 and ICU.58 Parainfluenza • Most infections are caused by parainfluenza 1 and 3.59 Parainfluenza 2 is less commonly identified, and parainfluenza 4 is a rare cause of respiratory infection. • In adults, influenzalike symptoms are a common manifestation of parainfluenza infection.60 In children, common presentations are croup and bronchiolitis.59 • In a population-based study of adults hospitalized for lower respiratory tract infection in 2 counties in Ohio, parainflueza-1 and parainfluenza-3 were detected in 2.5% to 3.1% of tested patients. Parainfluenza-1 epidemic season spanned the summer-autumn. Parainfluenza-3 epidemic season spanned the spring-summer. Median age was 61.5 years for parainfluenza-1–infected patients and 77.5 years for parainfluenza-3–infected patients. Of those infected by parainfluenza-3, 59% had an infiltrate on chest radiograph, 23% required ICU stay, and none died.61 Metapneumovirus • It has been identified in 4.5% of acute respiratory illnesses of adults prospectively followed as outpatients.62 • It has been identified in 4% of patients with community-acquired pneumonia.63 • Among outpatient adults, those of younger age tend to be more commonly infected by metapneumovirus, which has been presumably attributed to their closer contact with children; however, hospitalized patients with metapneumovirus infection are older.62 • Mean age in a series of community-acquired pneumonia and metapneumovirus infection: 62 years.63

• Among outpatient adults, those of younger age tend to be more commonly infected by metapneumovirus, which has been presumably attributed to their closer contact with children; however, hospitalized patients with metapneumovirus infection are older.62 • Mean age in a series of community-acquired pneumonia and metapneumovirus infection: 62 years.63 • In the outpatient setting, cough and nasal congestion are the most common symptoms.62 • In patients with metapneumovirus infection and pneumonia, common symptoms are cough with sputum production, dyspnea, and fatigue.63 Human bocavirus • Commonly identified in symptomatic and asymptomatic children but it seems to be a less common cause of respiratory symptoms in adults.64 • Human bocavirus infection is more common in the winter.65 • Common clinical presentations include upper respiratory tract symptoms, bronchiolitis. and pneumonia.66 Cases of encephalitis have been reported.67, 68 • It has been detected in acute respiratory illness of adults with immunosuppression and chronic lung disease.69, 70 • A study showed that it can be often identified in the sinus tissue specimens of adult patients with chronic sinusitis.71

• Common clinical presentations include upper respiratory tract symptoms, bronchiolitis. and pneumonia.66 Cases of encephalitis have been reported.67, 68 • It has been detected in acute respiratory illness of adults with immunosuppression and chronic lung disease.69, 70 • A study showed that it can be often identified in the sinus tissue specimens of adult patients with chronic sinusitis.71 Clinical presentation Clinical Manifestations Patients with influenza infection in general (not just pneumonia) commonly present with cough, fever, fatigue, myalgia, runny nose, and sweating. Wheezing as a symptom can occur in close to half of the patients.72 Patients with influenza pneumonia tend to have the same symptoms as patients with nonpneumonic influenza infection but an important distinction is that patients with pneumonia more often have dyspnea.73 Perhaps the greatest clinical clue for influenza in a patient with acute respiratory symptoms (or pneumonia) is whether the patient is presenting during an influenza epidemic. As an example, the absence of coughing and temperature higher than 37.8°C make influenza very unlikely in patients presenting with influenzalike illness outside an influenza epidemic but has a lesser impact on the likelihood of influenza if the same patient presenting during an epidemic. On the other hand, the presence of these symptoms during an epidemic substantially increases the probability of influenza but has a lesser impact outside of an epidemic.74

like illness outside an influenza epidemic but has a lesser impact on the likelihood of influenza if the same patient presenting during an epidemic. On the other hand, the presence of these symptoms during an epidemic substantially increases the probability of influenza but has a lesser impact outside of an epidemic.74 Studies have assessed the accuracy of clinical manifestations for the diagnosis of influenza in patients with acute respiratory symptoms. Some of the earlier studies were limited by retrospective design, leading to potential classification bias, or by the reliance on clinical manifestations for the final diagnosis of influenza, leading to incorporation bias.75 More recent studies used a prospective design and viral polymerase chain reaction test as the reference standard. A prospective study enrolled 100 patients with influenzalike illness who presented to 3 different clinics. Viral polymerase chain reaction test was used for the diagnosis of influenza. The accuracy of a number of symptoms was tested. On multivariate analysis, only cough and temperature remained significant predictors of influenza.76 In a prospective study of 258 patients who presented to the emergency department with acute respiratory symptoms, a symptom inventory and influenza polymerase chain reaction test was applied to the patients. Using polymerase chain reaction test as the reference standard, the accuracy of clinical judgment, decision rule, and rapid influenza test was provided. The presence of cough and fever had a positive likelihood ratio of 5.1 and a negative likelihood ratio of 0.7.72 In a prospective study of 270 high-risk patients who presented to an emergency department with acute respiratory illness, clinicians were asked whether they thought the patient had influenza. Viral polymerase chain reaction was the reference standard. A clinician diagnosis of influenza had a positive likelihood ratio of 1.63 and negative likelihood ratio of 0.82.77 Likelihood ratios are an interesting way of providing the accuracy of symptoms or clinical diagnosis because they allow for the estimate of the probability of a disease after taking into account the pre-test probability78 (Fig. 3 ). See Table 3 for a summary of these studies.Fig. 3 Probability of influenza according to presence of combined cough and fever in patients presenting during influenza season (A) and outside the influenza season (B).

e estimate of the probability of a disease after taking into account the pre-test probability78 (Fig. 3 ). See Table 3 for a summary of these studies.Fig. 3 Probability of influenza according to presence of combined cough and fever in patients presenting during influenza season (A) and outside the influenza season (B). (Data for likelihood ratios from Stein J, Louie J, Flanders S, et al. Performance characteristics of clinical diagnosis, a clinical decision rule, and a rapid influenza test in the detection of influenza infection in a community sample of adults. Ann Emerg Med 2005;46(5):412–9.)Table 3 Characteristics of studies that prospectively assessed the accuracy of symptoms for the diagnosis of influenza infection Author, Year Design Setting Sample Inclusion Criteria Reference Results Boivin et al,76 2000 Prospective cohort Patients presenting to 3 outpatient clinics 100 Flulike illness of <72 h duration PCR and culture from nasopharyngeal swab Cough and fever (>38°C):Sens of 77.6% Spec of 55.0% PPV of 86.8% NPV of 39.3% Stein et al,72 2005 Prospective cohort Adult patients presenting to the emergency department 258 New illness within the past 3 wk associated with cough, fever, or upper respiratory tract symptoms Clinician judgment:Sens of 29% (95% CI 18%–43%) Spec of 92% (95% CI 87%–95%) PLR of 3.8 (95% CI 1.9–7.5) NLR of 0.8 (95% CI 0.6–0.9) Decision rule (cough and fever):Sens of 40% (95% CI 27%–54%) Spec of 92% (95% CI 87%–95%) PLR of 5.1 (95% CI 2.7–9.6) NLR of 0.7 (95% CI 0.5–0.8)

Stein et al,72 2005 Prospective cohort Adult patients presenting to the emergency department 258 New illness within the past 3 wk associated with cough, fever, or upper respiratory tract symptoms Clinician judgment:Sens of 29% (95% CI 18%–43%) Spec of 92% (95% CI 87%–95%) PLR of 3.8 (95% CI 1.9–7.5) NLR of 0.8 (95% CI 0.6–0.9) Decision rule (cough and fever):Sens of 40% (95% CI 27%–54%) Spec of 92% (95% CI 87%–95%) PLR of 5.1 (95% CI 2.7–9.6) NLR of 0.7 (95% CI 0.5–0.8) Dugas et al,77 2015 Prospective cohort Adult patients presenting to the emergency department 270 Fever or any respiratory-related symptom PCR from nasopharyngeal swab Clinical judgment:Sens of 36% (95% CI 22%–52%) Spec of 78% (95% CI 72%–83%) PLR of 1.63 (95% CI 1.01–2.62) NLR of 0.82 (95% CI 0.65–1.04) Influenzalike illness (fever ≥37.8°C with either cough or sore throat):Sens of 31% (95% CI 18%–47%) Spec of 88% (95% CI 83%–92%) PLR of 2.61 (95% CI 1.47–4.64) NLR of 0.78 (95% CI 0.64–0.96) Abbreviations: CI, confidence interval; NLR, negative likelihood ratio; NPV, negative predictive value; PCR, polymerase chain reaction; PLR, positive likelihood ratio; PPV, positive predictive value; Sens, sensitivity; Spec, specificity.

Spec of 88% (95% CI 83%–92%) PLR of 2.61 (95% CI 1.47–4.64) NLR of 0.78 (95% CI 0.64–0.96) Abbreviations: CI, confidence interval; NLR, negative likelihood ratio; NPV, negative predictive value; PCR, polymerase chain reaction; PLR, positive likelihood ratio; PPV, positive predictive value; Sens, sensitivity; Spec, specificity. Overall, the previously described studies indicate that the predictive value of symptoms, combination of symptoms, or clinical impression for the diagnosis of influenza is only modest for patients presenting with acute illness. Symptoms or clinical impression are not enough to rule in or rule out influenza. In fact, clinicians failed to clinically diagnose influenza in approximately two-thirds of influenza-confirmed patients in a prospective series.77 Ultimately, clinicians need to pay close attention to surveillance data, and if there is evidence of influenza activity in the area where they practice, any acute febrile respiratory illness should place influenza as a high possibility in the differential diagnosis. In the United States, the Centers for Disease Control and Prevention provide weekly data on influenza activity according to regions in the country. This is available at https://www.cdc.gov/flu/weekly/index.htm. Other important aspects of clinical history include close contact with persons with acute febrile illness, and recent travel. Additionally, it is important to realize that in some tropical countries, influenza circulates throughout the year.79

the country. This is available at https://www.cdc.gov/flu/weekly/index.htm. Other important aspects of clinical history include close contact with persons with acute febrile illness, and recent travel. Additionally, it is important to realize that in some tropical countries, influenza circulates throughout the year.79 A hallmark of respiratory syncytial virus infection is the presence of wheezing, which occurs in a higher frequency as compared with patients with influenza. Hospitalized patients with respiratory syncytial virus infection may present with clinical-radiological dissociation, in which patients may appear toxemic despite mild radiological abnormalities. In a cohort of 118 hospitalized patients with respiratory syncytial virus infection, the most common symptoms were cough (97%), dyspnea (95%), wheezing (73%), and nasal congestion (68%). On physical examination, wheezing was present in 82% of the patients. A temperature higher than 39°C was present in only 13% of the patients. It should be noted, however, that these percentages are for all hospitalized patients with respiratory syncytial virus infection. When assessing only those hospitalized patients with respiratory syncytial virus infection and pneumonia, wheezing and nasal congestion were less common.80 In another study of 57 patients with respiratory syncytial virus infection and clinical diagnosis of pneumonia, the most common symptoms were cough (88%), dyspnea (82%), wheezing (79%), fever (61%), and runny nose (58%). On physical examination, the most common findings were wheezing (53%), rhonchi (46%), and crackles (40%).81

er study of 57 patients with respiratory syncytial virus infection and clinical diagnosis of pneumonia, the most common symptoms were cough (88%), dyspnea (82%), wheezing (79%), fever (61%), and runny nose (58%). On physical examination, the most common findings were wheezing (53%), rhonchi (46%), and crackles (40%).81 Just as in pneumonia caused by influenza or respiratory syncytial virus, there are no specific clinical manifestations of pneumonia caused by other respiratory viruses. In fact, symptoms and signs are not specific enough to differentiate viral from bacterial pneumonia.82 The usual clinical manifestations of pneumonia, including fever higher than 37.8°C, heart rate faster than 100 beats per minute, crackles, and decreased breath sounds,83 are to be expected in pneumonia caused by any of the respiratory viruses. In the end, the diagnosis of viral infection in patients with pneumonia relies on the recognition that respiratory viruses are a common etiology of pneumonia, and on the systematic performance of viral microbiology studies on these patients.

s,83 are to be expected in pneumonia caused by any of the respiratory viruses. In the end, the diagnosis of viral infection in patients with pneumonia relies on the recognition that respiratory viruses are a common etiology of pneumonia, and on the systematic performance of viral microbiology studies on these patients. Radiological Manifestations The chest radiograph of patients with viral pneumonia can show different patterns, including ground-glass opacities, consolidation, and nodular opacities. In general, patients present with faint opacities, commonly described as a ground-glass pattern. The second most commonly reported pattern is consolidation. Nodular opacities are less common but can occur. The opacities are often patchy in distribution.80, 84, 85, 86, 87 Bilateral involvement is fairly common, and some series in influenza pneumonia show that bilateral involvement is slightly more common than unilateral involvement.84 On the other hand, other series in respiratory syncytial virus or coronavirus pneumonia show that unilateral involvement is more common.80, 85 Pleural effusions are not usual but have been reported.87 On computed tomography of the chest, the most common pattern, ground-glass opacity, becomes even more noticeable, often in a patchy and bilateral distribution. Other patterns, such as consolidation, nodular opacities, and interlobular thickening, also can be present86 (Figs. 4 and 5 ).Fig. 4 Chest radiograph and computed tomography of the chest of a 42-year-old male patient admitted with pneumonia and 2009 H1N1 influenza infection leading to acute respiratory failure. Chest radiograph (A) reveals diffuse consolidation, and the computed tomography of the chest (B) reveals bilateral patchy ground-glass opacities and dense consolidation in the dorsal areas.

hest of a 42-year-old male patient admitted with pneumonia and 2009 H1N1 influenza infection leading to acute respiratory failure. Chest radiograph (A) reveals diffuse consolidation, and the computed tomography of the chest (B) reveals bilateral patchy ground-glass opacities and dense consolidation in the dorsal areas. Fig. 5 Computed tomography of the chest revealing diffuse ground-glass opacities and small bilateral pleural effusion in a 62-year-old female patient with respiratory syncytial virus infection who developed pneumonia and acute respiratory distress syndrome. Similar to the clinical manifestations, the radiological findings are not specific and do not allow for the differentiation of viral from bacterial infection in patients with pneumonia, let alone the identification of a specific virus. The radiological findings, however, can help corroborate the diagnosis of viral pneumonia. For instance, in a patient in whom a viral pathogen has been identified by oropharyngeal swab, the demonstration of patchy ground-glass opacities in the lung are suggestive of a viral pneumonic infiltrate.

on of a specific virus. The radiological findings, however, can help corroborate the diagnosis of viral pneumonia. For instance, in a patient in whom a viral pathogen has been identified by oropharyngeal swab, the demonstration of patchy ground-glass opacities in the lung are suggestive of a viral pneumonic infiltrate. Pathogen-directed therapy Influenza The 2 main classes of antiviral drugs for treatment of influenza include neuraminidase inhibitors and adamantanes.7 Influenza viruses infect cells through the binding of its surface glycoprotein hemagglutinin to the sialic acid receptor. The attached virus is then released into the cells by another surface glycoprotein, neuraminidase, which is the target of neuraminidase inhibitors.88 The adamantanes, which include amantadine and rimantadine, block the M2 protein, a membrane protein with ion channel activity.89 They exhibit activity against influenza A but not against influenza B. The antiviral drugs currently approved by the US Food and Drug Administration are the neuraminidase inhibitors oral oseltamivir, inhaled zanamivir, and intravenous peramivir.90 The adamantanes are not recommended for the treatment of influenza because of high resistance of influenza A against these drugs.90

nfluenza B. The antiviral drugs currently approved by the US Food and Drug Administration are the neuraminidase inhibitors oral oseltamivir, inhaled zanamivir, and intravenous peramivir.90 The adamantanes are not recommended for the treatment of influenza because of high resistance of influenza A against these drugs.90 There are a number of clinical trials that assessed the effect of oseltamivir for influenza. A comprehensive systematic review summarized the effect of oseltamivir for prophylaxis and treatment in adults and children. For the assessment of time to alleviation of symptoms in adults with influenza, 8 studies were pooled, totaling 2208 patients in the oseltamivir group and 1746 in the placebo group. Oseltamivir led to earlier relief of symptoms (16.8 hours; 95% CI 8.4–25.1 hours; P < .001). For the assessment of pneumonia prevention in adults with influenza, 8 studies were pooled, which included 2694 patients in the oseltamivir group and 1758 in the placebo group. Oseltamivir led to a reduction in pneumonia (risk difference of 1% [0.22%–1.49%]). For the assessment of hospitalization prevention in adults with influenza, 7 studies were pooled that included 2663 patients in the oseltamivir group and 1731 in the placebo group. There was no difference in need for hospitalization (risk ratio 0.92; 95% CI 0.57–1.5; P = .73). The pooling of 8 studies in adults, which included 2694 patients in the oseltamivir group and 1758 in the control group, showed that oseltamivir led to more nausea (risk ratio 1.57; 95% CI 1.14–2.15; P = .005) and more vomiting (risk ratio 2.43; 95% CI 1.75–3.38; P<.001]).91 In aggregate, these meta-analyses indicate that influenza-infected patients treated with oseltamivir have a modest benefit in relief of symptoms and prevention of pneumonia. This comes at the expense of more nausea and vomiting. It should be noted, however, that the patients included in these trials did not appear ill. For instance, studies that enrolled patients with immunosuppressive conditions such as human immunodeficiency virus infection or malignancy were not included in the meta-analyses. The inclusion criteria for the pooled studies were the presence of influenzalike illness rather than pneumonia. Additionally, only 1 death was reported among all trials that included the adult population.

ive conditions such as human immunodeficiency virus infection or malignancy were not included in the meta-analyses. The inclusion criteria for the pooled studies were the presence of influenzalike illness rather than pneumonia. Additionally, only 1 death was reported among all trials that included the adult population. An earlier systematic review included observational studies that evaluated antiviral therapy versus no therapy or other antiviral therapy in patients with laboratory-confirmed or a clinical diagnosis of influenza. This review of observational studies had important distinctions from the review of randomized clinical trials. First, here the investigators pooled studies that included hospitalized patients, a high-risk population. The pooling of 3 studies (total of 681 patients) that adjusted for confounders showed that oseltamivir, as compared with no antiviral therapy, was associated with a reduction in mortality (OR 0.23; CI 0.13–0.43).92 The quality of the evidence generated by this review was generally low because it relied on observational studies, which are at risk of confounding despite adjustment in the analyses. However, these observational studies and their meta-analyses fill in important knowledge gaps that were not and likely will not be addressed by clinical trials.

nce generated by this review was generally low because it relied on observational studies, which are at risk of confounding despite adjustment in the analyses. However, these observational studies and their meta-analyses fill in important knowledge gaps that were not and likely will not be addressed by clinical trials. The Centers for Disease Control and Prevention recommends that treatment be initiated as soon as possible for those hospitalized; patients with severe, complicated, or progressive disease; and those at higher risk for influenza complications.90 We agree with the Centers for Disease Control and Prevention recommendations and as such we submit that all influenza-infected patients with pneumonia, a complication from influenza, should receive antiviral therapy, which currently should be a neuraminidase inhibitor. In the absence of a sensitive point-of-care polymerase chain reaction, clinicians have to decide whether to initiate empiric treatment for influenza pneumonia. Strong consideration should be given to surveillance data and risk factors for influenza. It is important to note that not only an influenza diagnosis is often missed but also clinicians often fail to prescribe antiviral influenza treatment when a clinical diagnosis of influenza is made and there is indication for treatment.93, 94 The benefit from treatment is greatest when it is started early but a survival benefit has been demonstrated with treatment up to 5 days after symptom initiation95 (Fig. 6 ).Fig. 6 Treatment approach in patients presenting with community-acquired pneumonia.

influenza is made and there is indication for treatment.93, 94 The benefit from treatment is greatest when it is started early but a survival benefit has been demonstrated with treatment up to 5 days after symptom initiation95 (Fig. 6 ).Fig. 6 Treatment approach in patients presenting with community-acquired pneumonia. Other Respiratory Viruses For the treatment of pneumonia caused by respiratory viruses other than influenza, defining whether the patient is immunocompetent or immunosuppressed is important. In immunocompetent patients, current antiviral treatment options are limited, generally reserved for severely ill patients, and based on anecdotal data. For instance, case reports and series have reported the use of cidofovir for the treatment of severe pneumonia caused by adenovirus in non-immunocompromised patients.96, 97 Even though patients had clinical improvement in these series, those studies were uncontrolled and thus do not allow a firm conclusion as to the efficacy of cidofovir. Antiviral treatment for pneumonia caused by viruses of the Herpesviridae family in immunocompetent hosts has been reported in severe cases.98, 99 In pregnant women with varicella zoster virus pneumonia, the mortality is high, and treatment with intravenous acyclovir is indicated.100

ion as to the efficacy of cidofovir. Antiviral treatment for pneumonia caused by viruses of the Herpesviridae family in immunocompetent hosts has been reported in severe cases.98, 99 In pregnant women with varicella zoster virus pneumonia, the mortality is high, and treatment with intravenous acyclovir is indicated.100 In immunosuppressed patients, aerosolized ribavirin, oral ribavirin, intravenous immunoglobulin, hyperimmunoglobulin, and palivizumab are treatment options that have been used in respiratory syncytial virus infection, particularly in patients with hematological malignancy or transplant recipients.101 For cytomegalovirus pneumonia, treatment includes intravenous ganciclovir.102 The addition of cytomegalovirus immunoglobulin to ganciclovir appears to lead to improved survival according to a case series.103 An alternative treatment for cytomegalovirus pneumonia is intravenous foscarnet.104 For the treatment of varicella pneumonia, the indicated treatment is intravenous acyclovir.105 Similarly, herpes simplex virus pneumonia is treated with intravenous acyclovir.106 The evidence for the use of these therapies is weak and comes in the form of observational studies (Fig. 7 ).Fig. 7 Viral pathogen-directed therapy. CMV, cytomegalovirus; HSV, herpes simplex virus; IV, intravenous; RSV, respiratory syncytial virus; VZV, varicella zoster virus.

is treated with intravenous acyclovir.106 The evidence for the use of these therapies is weak and comes in the form of observational studies (Fig. 7 ).Fig. 7 Viral pathogen-directed therapy. CMV, cytomegalovirus; HSV, herpes simplex virus; IV, intravenous; RSV, respiratory syncytial virus; VZV, varicella zoster virus. Discontinuation of antibiotic therapy The identification of a viral pathogen in pneumonia should not in itself prompt a clinician to discontinue the initial empirical antibiotics because dual bacterial-viral infection is common. In fact, the recognition that dual bacterial-viral is common seems to be reflected in clinical practice. In an observational study, most patients with respiratory tract infection admitted to the hospital who turned out to have an identified viral pathogen did not have their antibiotics discontinued.107 On the other hand, the use of a clinical pathway integrating the results of viral microbiology testing with clinical findings and procalcitonin testing could have a role in the safe discontinuation of antibiotics. It is now well established that use of procalcitonin to guide initiation and discontinuation of antibiotic in patients with acute respiratory tract infection leads to less use of antibiotics without worsening the outcomes.108

ndings and procalcitonin testing could have a role in the safe discontinuation of antibiotics. It is now well established that use of procalcitonin to guide initiation and discontinuation of antibiotic in patients with acute respiratory tract infection leads to less use of antibiotics without worsening the outcomes.108 In a randomized clinical trial of 300 hospitalized patients with lower respiratory tract infection, the use of combined procalcitonin and viral polymerase chain reaction tests was compared with standard care. Both groups had similar antibiotic exposure. However, a lower proportion of patients with a positive viral polymerase chain reaction test and low procalcitonin received antibiotic on discharge as compared with standard care.109 This study suggests that the result of a viral polymerase chain reaction test has the impact to further influence decision making even after procalcitonin and clinical evolution are factored in. It should be noted, however, that this was a feasibility study and patients with pneumonia were excluded. Additionally, viral polymerase chain reaction test result may not influence antibiotic decision in the absence of a protocol. This was shown in an observational, retrospective study in which only 10.5% of patients had antibiotic discontinued within 48 hours of a positive viral respiratory panel and a low procalcitonin result.110

ly, viral polymerase chain reaction test result may not influence antibiotic decision in the absence of a protocol. This was shown in an observational, retrospective study in which only 10.5% of patients had antibiotic discontinued within 48 hours of a positive viral respiratory panel and a low procalcitonin result.110 Another randomized clinical trial assessed the effect of point-of-care respiratory viral panel in patients with acute respiratory illness or fever. The study enrolled 720 patients. There was no difference in the primary endpoint, which was the proportion of patients treated with antibiotics. However, the relevance of the primary outcome was impaired because many patients received antibiotics before the results of the point-of-care test. A significantly greater proportion of patients in the point-of-care group received only a single dose of antibiotics (10% vs 3%) or antibiotics for less than 48 hours (17% vs 9%).111 In summary, there is weak but mounting evidence that the use of nucleic acid amplification tests have the potential to aid in the decision to discontinue antibiotics in patients with respiratory infection (including pneumonia) but it is more likely to do so if integrated with clinical findings and procalcitonin. Additionally, continuing clinician education will be important to ensure implementation of strategies to minimize antibiotic exposure.

e decision to discontinue antibiotics in patients with respiratory infection (including pneumonia) but it is more likely to do so if integrated with clinical findings and procalcitonin. Additionally, continuing clinician education will be important to ensure implementation of strategies to minimize antibiotic exposure. Corticosteroid therapy An exuberant inflammatory response can play a major role in the morbidity and mortality of patients with pneumonia. Corticosteroid has been used as a way of mitigating the exacerbated inflammatory response in these patients. A systematic review has synthesized the results of clinical trials assessing systemic corticosteroids. The clinical trials are mostly small with sample sizes ranging from 30 to 784 patients. Although no statistically significant improvement in mortality was observed in general, corticosteroids led to a reduction in mortality in patients with severe community-acquired pneumonia (risk ratio 0.39; CI 0.20–0.77).112 Corticosteroids may be particularly beneficial in patients with community-acquired pneumonia and heightened inflammatory state, as demonstrated in a trial that enrolled patients with severe community-acquired pneumonia and a C-reactive protein greater than 150 mg/L.113 In summary, despite the small sample size of most trials, the weight of evidence currently favors the use of systemic corticosteroids in patients with community-acquired pneumonia admitted to the hospital, particularly in patients with a high inflammatory state and severe pneumonia. Our approach currently is to reserve the use of corticosteroids for patients with community-acquired pneumonia with C-reactive protein greater than 150 mg/L and a lactic acid greater than 4 nmol/L or acidosis with pH <7.30.114

o the hospital, particularly in patients with a high inflammatory state and severe pneumonia. Our approach currently is to reserve the use of corticosteroids for patients with community-acquired pneumonia with C-reactive protein greater than 150 mg/L and a lactic acid greater than 4 nmol/L or acidosis with pH <7.30.114 The 2009 H1N1 pandemic brought to light the use of systemic corticosteroid in influenza pneumonia. Some studies revealed that 40% to 50% of patients with severe influenza pneumonia received corticosteroid during the pandemic.115, 116 Unfortunately, although corticosteroid appears to be beneficial in patients with severe community-acquired pneumonia, the same does not hold true for patients with influenza pneumonia, a condition in which corticosteroids may actually be detrimental, as demonstrated in the systematic review. In this study, the investigators pooled 10 observational studies (total of 1497 patients) and found that corticosteroid therapy was associated with higher odds of death (OR 2.12; 95% CI 1.36–3.29). Of note, the studies included in the meta-analysis were predominantly conducted during the 2009 H1N1 influenza pandemic and in the ICU setting.117

tigators pooled 10 observational studies (total of 1497 patients) and found that corticosteroid therapy was associated with higher odds of death (OR 2.12; 95% CI 1.36–3.29). Of note, the studies included in the meta-analysis were predominantly conducted during the 2009 H1N1 influenza pandemic and in the ICU setting.117 A clinical trial designed to evaluate the effect of systemic corticosteroid in ICU patients with the 2009 H1N1 influenza pneumonia was unable to enroll the planned number of patients, highlighting the difficulties in conducting a clinical trial during a pandemic.116 A limitation of the observational studies assessing corticosteroid therapy in influenza pneumonia is the possibility of confounding by indication; that is, the possibility that sicker patients are more often prescribed systemic corticosteroid. This has the potential to cause the false impression that corticosteroid therapy leads to worse outcomes in influenza pneumonia. Some studies adjusted for confounding factors, but residual confounding can still occur. In the absence of randomized clinical trials, and in view of the results of observational studies, it is our opinion that currently corticosteroid therapy should not be administered in influenza pneumonia. The effect of corticosteroid in patients with noninfluenza viral pneumonia is unclear.

onfounding can still occur. In the absence of randomized clinical trials, and in view of the results of observational studies, it is our opinion that currently corticosteroid therapy should not be administered in influenza pneumonia. The effect of corticosteroid in patients with noninfluenza viral pneumonia is unclear. Future research The advent of nucleic acid amplification tests improved our understanding of the epidemiology of viral infections in pneumonia, and enables an etiologic diagnosis of viral infection in a large proportion of patients with pneumonia. However, one of the downsides of nucleic acid amplifications tests was a relatively long turnaround, limiting its clinical utility. This has been overcome by the development of “point-of-care” polymerase chain reaction tests that have a turnaround time of approximately 1 hour.118 The assessment of these point-of-care tests in clinical pathways is a promising venue for clinical investigation. As these tests are being rapidly integrated into clinical practice, it is important to study their cost-effectiveness and whether they influence outcomes or decision making.

time of approximately 1 hour.118 The assessment of these point-of-care tests in clinical pathways is a promising venue for clinical investigation. As these tests are being rapidly integrated into clinical practice, it is important to study their cost-effectiveness and whether they influence outcomes or decision making. Ongoing research on antiviral treatment is promising. Just as for bacterial infection, combination therapy has been studied in influenza infection with different goals, such as preventing pathogen resistance,119, 120 mitigating the inflammatory response,121 or achieving synergy.122, 123 There has been development of new compounds for the treatment of respiratory syncytial virus. These include a fusion inhibitor, which prevents the fusion of respiratory syncytial virus viral envelope with the host cell membrane, and a nucleoside analog, which prevents respiratory syncytial virus replication.124, 125

23 There has been development of new compounds for the treatment of respiratory syncytial virus. These include a fusion inhibitor, which prevents the fusion of respiratory syncytial virus viral envelope with the host cell membrane, and a nucleoside analog, which prevents respiratory syncytial virus replication.124, 125 Summary Viral respiratory infection is common in pneumonia and is present in approximately 25% of patients with community-acquired pneumonia. It is also common in immunosuppressed patients, but the latter are susceptible not only to the usual community-acquired respiratory viruses but also to viruses of the Herpesviridae family. Recent data show that respiratory viruses are also identified in hospital-acquired infections. The clinical diagnosis of viral infection is challenging. Clinical prediction rules have been developed for the diagnosis of influenza infection but they showed only modest accuracy. Similarly, radiological studies are nonspecific. In the end, the diagnosis of viral infection relies on the recognition that respiratory viruses are commonly present in pneumonia, and on the systematic performance of viral microbiology studies, particularly nucleic acid amplifications tests. The treatment of influenza pneumonia is currently with a neuraminidase inhibitor. The treatment options for pneumonia caused by other viruses in immunocompetent patients with pneumonia are limited, and the data are largely anecdotal. In immunosuppressed patients with infection by respiratory syncytial virus or a virus of the Herpesviridae family, there are antiviral treatments available. There is ongoing research involved with the development and testing of new treatment strategies both for influenza and noninfluenza viruses.

are largely anecdotal. In immunosuppressed patients with infection by respiratory syncytial virus or a virus of the Herpesviridae family, there are antiviral treatments available. There is ongoing research involved with the development and testing of new treatment strategies both for influenza and noninfluenza viruses. Conflict of Interest: R. Cavallazzi was a site investigator for a clinical trial investigating a new antiviral for adults with respiratory syncytial virus infection. The study was led by Gilead. R. Cavallazzi was a site investigator for a clinical trial investigating a new drug for influenza. The study was led by GlaxoSmithKline. Funding: None.