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Introduction and definition The incidence of incurable disease and disability has been increasing in the Western world in recent years. Medical and technological advances have certainly reduced neonatal and pediatric mortality rates, but they have also led to a longer survival of patients with severe and potentially lethal diseases, without always succeeding in curing them, thereby increasing in absolute terms the number of pediatric patients with incurable diseases who continue to suffer from life-threatening problems. Children with life-limiting and life-threatening illnesses that lead to death or a life of severe disability deserve a profound cultural and organizational reappraisal of how we care for them when the aim of care is not to make them recover, but to offer the best possible "health" and "quality of life", despite their disease. Infants, children and adolescents with life-limiting and life threatening illnesses need support and long-term care in a palliative setting.

Children with life-limiting and life-threatening illnesses that lead to death or a life of severe disability deserve a profound cultural and organizational reappraisal of how we care for them when the aim of care is not to make them recover, but to offer the best possible "health" and "quality of life", despite their disease. Infants, children and adolescents with life-limiting and life threatening illnesses need support and long-term care in a palliative setting. The World Health Organization defines palliative care for children as "the active total care of the child's body, mind and spirit, ... also involves giving support to the family. It begins when illness is diagnosed, and continues regardless of whether or not a child receives treatment directed at the disease" [1]. Pediatric palliative care is concerned with the medical, psycho-social, spiritual and economic needs of patients and their families, providing complex patient care solutions involving all aspects of the health care system, from hospital to hospice, to community, to home, and it involves an interdisciplinary team of caregivers. It is important to draw a distinction between palliative care and terminal care: the latter refers to looking after children and their parents during a time closely related to their death (within weeks, days or hours). Terminal care is not palliative care, but palliative care includes terminal care [1].

The World Health Organization defines palliative care for children as "the active total care of the child's body, mind and spirit, ... also involves giving support to the family. It begins when illness is diagnosed, and continues regardless of whether or not a child receives treatment directed at the disease" [1]. Pediatric palliative care is concerned with the medical, psycho-social, spiritual and economic needs of patients and their families, providing complex patient care solutions involving all aspects of the health care system, from hospital to hospice, to community, to home, and it involves an interdisciplinary team of caregivers. It is important to draw a distinction between palliative care and terminal care: the latter refers to looking after children and their parents during a time closely related to their death (within weeks, days or hours). Terminal care is not palliative care, but palliative care includes terminal care [1]. The gross misconception relating to this difference heavily influences the errors made in establishing the related eligibility criteria, the needs and the best way to provide adequate solutions, especially in the pediatric setting. For a long time, pediatric patients were not offered palliative care and, even nowadays, only a minimal part of the children with incurable disease can benefit from palliative care in Europe [2].

The gross misconception relating to this difference heavily influences the errors made in establishing the related eligibility criteria, the needs and the best way to provide adequate solutions, especially in the pediatric setting. For a long time, pediatric patients were not offered palliative care and, even nowadays, only a minimal part of the children with incurable disease can benefit from palliative care in Europe [2]. Numerous studies have confirmed that symptom management at the end of a pediatric patient's life is often still largely inadequate and any treatment provided is scarcely effective [3-5]. Like the clinical problems, the psychological, social and spiritual problems also receive little attention. The global situation is one of undertreatment and a scarce availability of proper patient care. Several studies have provided important information on the multiple needs of children with life-limiting and life-threatening illnesses and their families, and on the problem of finding adequate solutions [2]. These studies have all revealed a surprisingly similar picture, despite geographical, cultural, organizational and social differences [1], i.e. - families want their child to be treated at home and to be able to stay there until they die; - the child wants to stay at home; - insufficient resources currently dedicated to pediatric palliative care; - the availability of "support" services is essential and currently inadequate; - access to pediatric palliative care services often depends on where a child lives and the type of disease involved (they are more readily available for cancer patients);

- the child wants to stay at home; - insufficient resources currently dedicated to pediatric palliative care; - the availability of "support" services is essential and currently inadequate; - access to pediatric palliative care services often depends on where a child lives and the type of disease involved (they are more readily available for cancer patients); - communication between the various professionals taking care of children with incurable disease is limited and needs to be improved; - there is an urgent need to provide training for professionals and volunteers involved in caring for the children and their families. Various cultural, affective, educational and organizational reasons have probably given rise to and influenced the persistence of this shortcoming in patient care. Moreover, it is by no means easy to deal with this complex problem, which demands an interdisciplinary expertise to come up with effective, realistic, applicable solutions. As is often the case, the very complexity of the problem delays the search for adequate solutions and generates doubts and controversies as to the real needs. Problems and peculiarities Children make very special patients: this is already true when it comes to deciding a course of treatment, but even more so when we move into the palliative care setting. There are numerous issues to consider:

Various cultural, affective, educational and organizational reasons have probably given rise to and influenced the persistence of this shortcoming in patient care. Moreover, it is by no means easy to deal with this complex problem, which demands an interdisciplinary expertise to come up with effective, realistic, applicable solutions. As is often the case, the very complexity of the problem delays the search for adequate solutions and generates doubts and controversies as to the real needs. Problems and peculiarities Children make very special patients: this is already true when it comes to deciding a course of treatment, but even more so when we move into the palliative care setting. There are numerous issues to consider: - the limited numbers: the number of pediatric cases of chronic and/or terminal disease requiring palliative care is fortunately limited, but the patients involved are scattered over a wide geographical area and this is bound to pose problems of an organizational nature, influencing the levels of available expertise, training and costs [1]; - the variable types of disease and their duration: the spectrum of conditions requiring pediatric palliative care is broad and heterogeneous (including neurological, oncological, metabolic, chromosomal, cardiological, respiratory and infectious diseases, the effects of prematurity and trauma, etc.), and the duration and complexity of the patient care required are equally varied [1]. Many pediatric diseases are also rare, and a fair proportion of cases remain without a diagnosis;

l, oncological, metabolic, chromosomal, cardiological, respiratory and infectious diseases, the effects of prematurity and trauma, etc.), and the duration and complexity of the patient care required are equally varied [1]. Many pediatric diseases are also rare, and a fair proportion of cases remain without a diagnosis; - the specificity and complexity of the measures required: despite their limited numerosity, these patients need very different types of approach, for various periods of time and entail an emotional involvement that amplify the energy demands and call for a multidisciplinary action of a highly complex nature. Children are in continuous physical, emotional and cognitive evolution, and this affects every aspects of their care, from the dosage of medication to the choice of methods for communication, containment and support; - the novelty of the problem: in some cases, the need to extend palliative care to the pediatric setting is a consequence of technological advances enabling the survival (even in the longer term) of patients with complex diseases that led relatively rapidly to the patient's death up until not many years ago. The novelty of the problem gives rise to cultural shortcomings and training inadequacies and helps to explain why it is so difficult to provide the specific expertise needed by the healthcare and other operators;

ients with complex diseases that led relatively rapidly to the patient's death up until not many years ago. The novelty of the problem gives rise to cultural shortcomings and training inadequacies and helps to explain why it is so difficult to provide the specific expertise needed by the healthcare and other operators; - the role of family: all children are members of a close-knit unit consisting of their family, which has a very special role when young patients develop incurable, chronic disease. Parents legally represent their offspring in all clinical, therapeutic, ethical and social decisions. The family has a pivotal role in communications with healthcare providers and institutions, and if the child remains at home, then patient care and treatment is delegated to the family. Other members of the expanded family also play an important part in creating a sharing and affective support network with a cascade effect on the sick children's and their parents' quality of life [1]; - the emotional involvement: the emotional and affective involvement accompanying the course of disease when it is a child who is dying is inescapable, making it more difficult for family members and healthcare operators to accept the failure of treatment, the irreversibility of the disease and death, and it often becomes easier to slip into situations of overtreatment or the refusal of treatment;

g the course of disease when it is a child who is dying is inescapable, making it more difficult for family members and healthcare operators to accept the failure of treatment, the irreversibility of the disease and death, and it often becomes easier to slip into situations of overtreatment or the refusal of treatment; - the ethical and legal issues: children have a very particular ethical and social position in our society. When the patient is a child, it is not always easy to speak of freedom of choice, respect for the patients' wishes, and their right to honest communications. The legal reference is the child's parent or guardian. In the pediatric setting, more than in any other, there may be a marked dichotomy between the dictates of professional ethics and deontology, and those of legislation. All these issues combine to influence the type and entity of very particular needs, that can only be met by implementing highly specific organizational decisions and patient care models. Eligibility criteria According to the literature, life-limiting and life-threatening diseases are eligible for pediatric palliative care [1]. The term life-limiting defines a condition where premature death is usual (e.g. Duchenne muscular dystrophy, trisomy 18, trisomy 22), while a life-threatening condition carries a high likelihood of premature death due to severe illness, but there is also a chance of long-term survival into adulthood (e.g. cancer).

Eligibility criteria According to the literature, life-limiting and life-threatening diseases are eligible for pediatric palliative care [1]. The term life-limiting defines a condition where premature death is usual (e.g. Duchenne muscular dystrophy, trisomy 18, trisomy 22), while a life-threatening condition carries a high likelihood of premature death due to severe illness, but there is also a chance of long-term survival into adulthood (e.g. cancer). Four different categories of childhood diseases have been identified by the Association for Children with Life-threatening or Terminal Conditions and the Royal College of Paediatrics and Child Health [6]: 1. life-threatening conditions for which curative treatment may be feasible but can fail, where palliative care is provided together with attempts at curative treatment. Examples: cancer, irreversible organ failure; 2. diseases which are life-threatening at an early age, where appropriate treatment may prolong life and provide an adequate quality of life. Examples: cystic fibrosis; 3. progressive conditions without curative treatment options, where treatment is exclusively palliative. Examples: some chromosomal diseases, muscular dystrophy, rare metabolic diseases; 4. non-progressive, irreversible conditions, with complex healthcare needs, that give rise to many complications and premature death. Examples: severe cerebral palsy, brain or spinal cord injuries due to trauma or infection.

3. progressive conditions without curative treatment options, where treatment is exclusively palliative. Examples: some chromosomal diseases, muscular dystrophy, rare metabolic diseases; 4. non-progressive, irreversible conditions, with complex healthcare needs, that give rise to many complications and premature death. Examples: severe cerebral palsy, brain or spinal cord injuries due to trauma or infection. Clearly, therefore, how long palliative care may be needed in children with incurable disease is an extremely variable parameter and hard to predict: in some cases, it is limited to the early years of life (congenital diseases); in others, it may be needed for far longer (neurological, cardiological or autoimmune diseases), and in other cases again, it may be concentrated in a brief period before death. In all these cases, however, no clear distinction is made between curative attempts to improve the patient's quality of life and extend its duration, and purely "palliative" measures; both approaches coexist and one prevails over the other depending on the stage of disease and the situation. For the time being, the absence of a curative therapy and the presence of complex clinical, psycho-relational, social and spiritual needs demanding a multi-specialistic approach are the elements that define eligibility and drive the implementation of specialist palliative care.

Clearly, therefore, how long palliative care may be needed in children with incurable disease is an extremely variable parameter and hard to predict: in some cases, it is limited to the early years of life (congenital diseases); in others, it may be needed for far longer (neurological, cardiological or autoimmune diseases), and in other cases again, it may be concentrated in a brief period before death. In all these cases, however, no clear distinction is made between curative attempts to improve the patient's quality of life and extend its duration, and purely "palliative" measures; both approaches coexist and one prevails over the other depending on the stage of disease and the situation. For the time being, the absence of a curative therapy and the presence of complex clinical, psycho-relational, social and spiritual needs demanding a multi-specialistic approach are the elements that define eligibility and drive the implementation of specialist palliative care. Epidemiological data There are currently no published data on the incidence and prevalence of life-limiting and life-threatening pediatric disease in the majority of European countries, but it is essential to obtain information on the numbers, diagnostic categories, age ranges and location of children with life-limiting or life-threatening conditions in order to design and organize adequate patient care solutions. Some degree of variability is evident in analyses conducted to date on the mortality rates for life-limiting and terminal pediatric illness.

Epidemiological data There are currently no published data on the incidence and prevalence of life-limiting and life-threatening pediatric disease in the majority of European countries, but it is essential to obtain information on the numbers, diagnostic categories, age ranges and location of children with life-limiting or life-threatening conditions in order to design and organize adequate patient care solutions. Some degree of variability is evident in analyses conducted to date on the mortality rates for life-limiting and terminal pediatric illness. A study by the Association for Children with Life-Threatening or Terminal Conditions and their Families, and by the Royal College of Paediatrics and Child Health in England [6] identified an annual mortality due to incurable disease of 1/10,000 children aged from one to 17 years. An Italian study [7] identified a mortality rate of 1100–1200 pediatric cases a year, corresponding to 1/10,000 0–17 year-olds. Only 40% of these patients die at home, the percentage being marginally higher for cancer victims (41%) than for other types of disease (38%). In Italy, the proportion of patients dying at home varies considerably by geographical region, the figure being 60–70% in the south and 10–15% in the north of the country. A study on mortality in children with life-threatening or terminal disease in the Republic of Ireland identified an annual death rate of 3.6/10,000 patients aged from 0 to 19 years [8].

dying at home varies considerably by geographical region, the figure being 60–70% in the south and 10–15% in the north of the country. A study on mortality in children with life-threatening or terminal disease in the Republic of Ireland identified an annual death rate of 3.6/10,000 patients aged from 0 to 19 years [8]. Different mortality rates are reported in infancy, childhood and adolescence. In all studies, many childhood deaths occur in the first year of life, the majority of them caused by congenital anomalies, deformities and chromosomal anomalies. Deaths occurring after the first year of life are more likely to be caused by diseases of the nervous or circulatory systems, or neoplasms [6-8]. Available reports on the prevalence of life-limiting and terminal illness in pediatric age also paint a rather heterogeneous picture. The analysis conducted by the Association for Children with Life-Threatening or Terminal Conditions and their Families and by the Royal College of Paediatrics and Child Health in England [6] identified a prevalence of incurable diseases among 0–17 year-olds amounting to 10/10,000/year. Forty per cent of these are cases of cancer, the other 60% comprising a miscellany of conditions, particularly neurodegenerative, metabolic and genetic diseases. An Italian study currently in press analyzed the hospital discharge reports and mortality data on pediatric patients for the years 2002–2004, recording 12,000 children with life-limiting and terminal illness, corresponding to an annual prevalence of 10/10,000 children (0–17 year-olds).

The analysis conducted by the Association for Children with Life-Threatening or Terminal Conditions and their Families and by the Royal College of Paediatrics and Child Health in England [6] identified a prevalence of incurable diseases among 0–17 year-olds amounting to 10/10,000/year. Forty per cent of these are cases of cancer, the other 60% comprising a miscellany of conditions, particularly neurodegenerative, metabolic and genetic diseases. An Italian study currently in press analyzed the hospital discharge reports and mortality data on pediatric patients for the years 2002–2004, recording 12,000 children with life-limiting and terminal illness, corresponding to an annual prevalence of 10/10,000 children (0–17 year-olds). The estimated prevalence in the Irish Republic of children and young people likely to require palliative care is 16/10,000 population aged 0–19 (15/10,000 if neonatal deaths are excluded) [8]. The differences emerging in the data collected are due to differences in the age ranges considered and in the eligibility criteria adopted.

The estimated prevalence in the Irish Republic of children and young people likely to require palliative care is 16/10,000 population aged 0–19 (15/10,000 if neonatal deaths are excluded) [8]. The differences emerging in the data collected are due to differences in the age ranges considered and in the eligibility criteria adopted. Needs Children with life-limiting and life-threatening disease and their families have diverse and mutable priorities that include clinical needs (symptom assessment and management, patient care planning and sharing clinical, organizational and social decisions) [4,9], psychosocial needs (of the children and their families) [1], social needs (education, play, economic support and services), and spiritual needs (of the children and their families) [1]. To provide adequate patient care, however, it is also important to contextualize and assess the needs of the team taking care of these patients and their families, and of the institutions that have to meet these new needs and find new ways to provide patient care.

iritual needs (of the children and their families) [1]. To provide adequate patient care, however, it is also important to contextualize and assess the needs of the team taking care of these patients and their families, and of the institutions that have to meet these new needs and find new ways to provide patient care. The child's needs The priority is symptom control. The literature [3,5] confirms major shortcomings in the provision and efficacy of treatment for patients with terminal disease. Even now, most children with life-threatening and terminal illness have many, often severe symptoms that have a dramatic, negative impact on their quality of life: about 90% of them experience generalized suffering; more than 70% suffer pain, which is controlled in less than 30% of cases. Little is known about how dyspnea and respiratory distress are controlled in these patients, but the proportion of cases given effective treatment is limited to around 20%. There are numerous options available, but their application in daily clinical practice is wholly inadequate. Needs relating to the psychological, communication, social exchange and spiritual spheres are also often not satisfied: these problems are delegated to the family, and only rarely considered part of a global patient care project [9]. In every single case, the patient's needs are continuously changing, in both intensity and prevalence, in relation to the child's natural psychological, physical and emotional development, and to the trend of the disease and its effects on the child's growth and developmental progress.

Needs relating to the psychological, communication, social exchange and spiritual spheres are also often not satisfied: these problems are delegated to the family, and only rarely considered part of a global patient care project [9]. In every single case, the patient's needs are continuously changing, in both intensity and prevalence, in relation to the child's natural psychological, physical and emotional development, and to the trend of the disease and its effects on the child's growth and developmental progress. However short the patients' future life expectancy might be, and however severely these lives are affected or underdeveloped, the acquisition of new functions and abilities, and the child's development and maturity are core issues that must guide any action undertaken, from the more traditional healthcare measures (pharmacological treatments, supplementation or substitution of vital functions or organs, etc.) to those involving other sectors of society, to assure an adequate personal growth, provide an education, deliver cultural and creative input, and support the patients' spirituality and their social role in the community.

s (pharmacological treatments, supplementation or substitution of vital functions or organs, etc.) to those involving other sectors of society, to assure an adequate personal growth, provide an education, deliver cultural and creative input, and support the patients' spirituality and their social role in the community. The family's needs The family is a fundamental part of any pediatric palliative care program: it is actively involved in providing care and has a great deal of responsibility for caring for the child, having to make often difficult decisions in the child's best interest, and paying the social and economic price of incurable disease; without adequate support, its burden is often too heavy to bear [1,10]. The needs of the family are numerous and include: - educational needs, i.e. training on various aspects of patient care and support; - psychological needs: they require assessment, support and treatment for sentiments such as guilt, rage, depression, anticipatory grief, and escape; - spiritual needs: a competent source of answers, open to an exchange of ideas and respectful of people's cultural background and religious beliefs; - financial and social needs: practical proposals for coping with a situation of isolation, loss of identity and financial insecurity for families whose members often lose their jobs, as well as supporting the cost of patient treatment and care.

- spiritual needs: a competent source of answers, open to an exchange of ideas and respectful of people's cultural background and religious beliefs; - financial and social needs: practical proposals for coping with a situation of isolation, loss of identity and financial insecurity for families whose members often lose their jobs, as well as supporting the cost of patient treatment and care. The grief associated with the incurable disease and death of a child has devastating, long-term implications for the whole family. The child's siblings have special needs during the child's illness and after their death. These brothers and sisters are at high risk of subsequent problems at school and in their relationship with their parents, as well as other psychological and social problems after their sibling's death. These problems are less evident if the disease and death are managed at home rather than in hospital [11]. Other members of the expanded family (grandparents, uncles and aunts, and friends) can play an important part in creating a supportive, sharing and affective network during the child's disease and after their death: these people also need support and supervision.

The grief associated with the incurable disease and death of a child has devastating, long-term implications for the whole family. The child's siblings have special needs during the child's illness and after their death. These brothers and sisters are at high risk of subsequent problems at school and in their relationship with their parents, as well as other psychological and social problems after their sibling's death. These problems are less evident if the disease and death are managed at home rather than in hospital [11]. Other members of the expanded family (grandparents, uncles and aunts, and friends) can play an important part in creating a supportive, sharing and affective network during the child's disease and after their death: these people also need support and supervision. The needs of the team - Education and training for healthcare professionals: several studies conducted in Europe and the United States have emphasized an important shortage of know-how among public health operators as regards the fundamentals of palliative care [12,13]. Various types of expertise are required: in addition to the technical abilities needed for diagnosis and treatment, it is indispensable to have skills relating to communication (with the children and their families), teamwork and service organization.

th operators as regards the fundamentals of palliative care [12,13]. Various types of expertise are required: in addition to the technical abilities needed for diagnosis and treatment, it is indispensable to have skills relating to communication (with the children and their families), teamwork and service organization. - Supervision: the emotional impact and stress are undeniable and are often considered responsible for situations of severe burnout, leading to a rapid turnover of the professionals involved at the expense of the acquisition of the necessary experience and professional skills. These people needs support, sharing and supervision to help them cope with the problems of death and incurable disease, so that they can be useful to others, without losing their own identity and personality. - Resources: little interest has been paid so far to pediatric palliative care as a need that healthcare organizations must meet, and the professional expertise of people working in this field has received scarce acknowledgement. It is now clear to all, from the literature and in daily clinical practice, that pediatric palliative care is a fundamental issue and warrants the allocation of specific resources, and that it is currently falling very short of meeting the real needs. - Public education: people need to learn that palliative care should be seen as serving a right to quality of life: well-informed users know what to ask for, and this facilitates the work of healthcare operators and enables better forms of cooperation and a sharing of the problems and potential solutions.

- Resources: little interest has been paid so far to pediatric palliative care as a need that healthcare organizations must meet, and the professional expertise of people working in this field has received scarce acknowledgement. It is now clear to all, from the literature and in daily clinical practice, that pediatric palliative care is a fundamental issue and warrants the allocation of specific resources, and that it is currently falling very short of meeting the real needs. - Public education: people need to learn that palliative care should be seen as serving a right to quality of life: well-informed users know what to ask for, and this facilitates the work of healthcare operators and enables better forms of cooperation and a sharing of the problems and potential solutions. The needs of institutions Institutions are faced with an entirely new and complex demand for care, as regards both the type of patient and how their needs can be met, in a context where little information is available, research has been limited and much remains to be done in terms of social and healthcare programs, the availability of monitoring tools, and cost analyses [1]. It is consequently important to produce: - epidemiological data on the numbers, diagnostic categories, age ranges and locations of children with life-limiting or life-threatening conditions, in the light of current patient care methods and the related costs; - tools and indicators/standards for monitoring the quality of care and the quality of life of such young patients and their families;

- epidemiological data on the numbers, diagnostic categories, age ranges and locations of children with life-limiting or life-threatening conditions, in the light of current patient care methods and the related costs; - tools and indicators/standards for monitoring the quality of care and the quality of life of such young patients and their families; - assessments of the best practices in pediatric palliative care in all settings and situations in which is it needed. Healthcare models Responding to these needs is by no means easy and, throughout the course of the disease, from diagnosis to death and beyond, it requires a multi-specialistic, shared action on the part of various services and institutions, that can come together to act as a single reference. Home management is the goal of pediatric palliative care, much appreciated by patients and their families, influencing the patient's quality of life, reducing the feelings of fear, isolation and helplessness, giving the child a chance to stay involved in the family's routines, and affording more opportunities for communication [1,14,15]. Home management is not always feasible, however, and particularly complex clinical problems, exhaustion, emotional stress, logistic and organizational factors sometimes make temporary periods in institutional settings unavoidable. From an organizational standpoint, there are theoretically four possible solutions for children with life-limiting or life-threatening disease, two domestic and two institutional:

Home management is the goal of pediatric palliative care, much appreciated by patients and their families, influencing the patient's quality of life, reducing the feelings of fear, isolation and helplessness, giving the child a chance to stay involved in the family's routines, and affording more opportunities for communication [1,14,15]. Home management is not always feasible, however, and particularly complex clinical problems, exhaustion, emotional stress, logistic and organizational factors sometimes make temporary periods in institutional settings unavoidable. From an organizational standpoint, there are theoretically four possible solutions for children with life-limiting or life-threatening disease, two domestic and two institutional: - institutionalized pediatric palliative care, provided in structures specifically for incurable children (hospices or nursing homes dedicated to patients with specific diseases) or in hospital departments dealing with acute conditions; - pediatric palliative care at home, where patients are cared for in their own homes by a team from the hospital, or from the territorial health care services, or a combination of the two (integrated home care schemes). None of these solutions is ideal; in practice, they all have their pros and cons.

- institutionalized pediatric palliative care, provided in structures specifically for incurable children (hospices or nursing homes dedicated to patients with specific diseases) or in hospital departments dealing with acute conditions; - pediatric palliative care at home, where patients are cared for in their own homes by a team from the hospital, or from the territorial health care services, or a combination of the two (integrated home care schemes). None of these solutions is ideal; in practice, they all have their pros and cons. The institutional solution, in a hospice or dedicated nursing home, has the advantage of focusing expertise in the management of rare and complex cases, assuring large enough numbers to enable the necessary professional competence and economically sustainable dedicated resources to be provided, but it has the disadvantage of separating the children from their environments. This approach to providing care is in contrast with the patients' and their families desire to return home and it cannot be the only solution, especially in the case of a very lengthy course of disease. The same problems apply to managing palliative care in hospitals dealing with acute conditions; in addition, numerous reports in the literature have confirmed that hospitals for dealing with acute conditions is far removed, in terms of its mission and aptitude, its organization and the opportunities it offers, from the ideal setting for providing palliative care for children.

pitals dealing with acute conditions; in addition, numerous reports in the literature have confirmed that hospitals for dealing with acute conditions is far removed, in terms of its mission and aptitude, its organization and the opportunities it offers, from the ideal setting for providing palliative care for children. While hospitalization at home can certainly assure the necessary competence and continuity in patient management, it has its drawbacks relating to the fact that the operators' expertise is disease-specific and it can only afford a limited territorial coverage. Since the number of users involved is always limited, it is difficult – in organizational and economic terms – to implement these solutions outside large towns where the density of the population is greatest. Keeping patients at home under an integrated home care system restores children to their families and social settings, enabling ample territorial areas to be covered and multidisciplinary services to be offered, but this approach often suffers from shortcomings in the continuity of the treatment and healthcare program involving the hospitals and limited resources and experience, and the burden of patient management and support frequently lies mainly with the family.

s to be covered and multidisciplinary services to be offered, but this approach often suffers from shortcomings in the continuity of the treatment and healthcare program involving the hospitals and limited resources and experience, and the burden of patient management and support frequently lies mainly with the family. Because none of the organizational options considered is without its drawbacks, almost all pediatric palliative care models currently adopted use a combination of the four types described above, considering them almost as organizational modules within a single support network, in which various public health and other institutions are involved at different times in the course of the patient's disease, giving priority to one or other type of solution depending on a patient's conditions and specific situations. The various experiences gained in different countries and in some parts of Italy show that the organization of a dedicated pediatric palliative care network combining home care with institutional solutions (hospices) stands as a reference in terms of its efficacy and efficiency, and its feasibility.

Because none of the organizational options considered is without its drawbacks, almost all pediatric palliative care models currently adopted use a combination of the four types described above, considering them almost as organizational modules within a single support network, in which various public health and other institutions are involved at different times in the course of the patient's disease, giving priority to one or other type of solution depending on a patient's conditions and specific situations. The various experiences gained in different countries and in some parts of Italy show that the organization of a dedicated pediatric palliative care network combining home care with institutional solutions (hospices) stands as a reference in terms of its efficacy and efficiency, and its feasibility. Since the pediatric patients requiring palliative care are fortunately few and far between, and pose complex management issues, the trials currently underway would suggest the need to create specific care networks on fairly large scale (regional or even supra-regional) with the support of a dedicated team of pediatric palliative care specialists who manage the numerous and varied needs of children with incurable disease and their families in close cooperation with other territorial and hospital care providers.

ific care networks on fairly large scale (regional or even supra-regional) with the support of a dedicated team of pediatric palliative care specialists who manage the numerous and varied needs of children with incurable disease and their families in close cooperation with other territorial and hospital care providers. An indispensable goal of this network is to develop a capable, multidisciplinary pediatric palliative care team (including physicians, nurses, psychologists, physiotherapists, occupational therapists, and social support workers) who will serve as a unifying reference throughout the course of the disease and after the child's death, assuring availability and help around the clock, and the opportunity to access appropriate respite and immediate hospice care if necessary [1-9]. In the UK, evidence in the literature shows that using the community services to care for children is far more cost-effective than having children spend inappropriate amounts of time in hospitals or attending hospital outpatient clinics [16]. Home is the best place to manage terminal disease and the pediatric patient's social role makes this easier to do for them than for an adult. Families want their children to stay at home, partly to preserve an impression of normality, partly because that is what their child want, but they also need to feel safe and secure, they want to be in control but they also need support [17].

ric patient's social role makes this easier to do for them than for an adult. Families want their children to stay at home, partly to preserve an impression of normality, partly because that is what their child want, but they also need to feel safe and secure, they want to be in control but they also need support [17]. The pediatric hospice is an important link in the pediatric palliative care chain: it is a highly complex structure designed with the child in mind, open-space, with areas suited to specific age groups, giving the children a chance to socialize, and providing the expertise and social interactions that can help the patient lead a "normal life". It can be the starting point that leads families to manage their "special" child at home, or it can temporarily give the family a chance to take a rest from the burden of care, when the family needs help for some reason, or when the patient's clinical management becomes too complex. It is not a place where patients go just before they die; it is a reference continually interacting in the care network on clinical, training and research levels. Although a child's care may focus more and more on palliation as the disease progresses, active treatment is still part of palliative care in the hospice and both aspects play an important part throughout a child's illness. With this type of model, the number of beds needed in pediatric hospices is distinctly limited (4–5 beds per 3 million population) [1,9,18,19].

The pediatric hospice is an important link in the pediatric palliative care chain: it is a highly complex structure designed with the child in mind, open-space, with areas suited to specific age groups, giving the children a chance to socialize, and providing the expertise and social interactions that can help the patient lead a "normal life". It can be the starting point that leads families to manage their "special" child at home, or it can temporarily give the family a chance to take a rest from the burden of care, when the family needs help for some reason, or when the patient's clinical management becomes too complex. It is not a place where patients go just before they die; it is a reference continually interacting in the care network on clinical, training and research levels. Although a child's care may focus more and more on palliation as the disease progresses, active treatment is still part of palliative care in the hospice and both aspects play an important part throughout a child's illness. With this type of model, the number of beds needed in pediatric hospices is distinctly limited (4–5 beds per 3 million population) [1,9,18,19]. Practical experience Various solutions are currently being tested. On the international scene, it is worth mentioning the experience of the network of integrated hospices linked to a satellite home care program currently underway in the United States, in a vast territorial setting that includes a number of states, and covers a large population. This network of centers is characterized by functional links, shared protocols and behavior patterns, and the pooling of information.

d hospices linked to a satellite home care program currently underway in the United States, in a vast territorial setting that includes a number of states, and covers a large population. This network of centers is characterized by functional links, shared protocols and behavior patterns, and the pooling of information. An Australian experience likewise focuses on the fundamental role of home care. Despite the country's vast dimensions, there are reference centers specializing in pediatric palliative care, generally based at a few pediatric hospices and working in close contact with family pediatricians, hospitals and territorial services, working as essential partners to enable very complex patients demanding high levels of care to be managed at home. Direct telecommunications between the home and the center facilitate this process and enable the child to stay at home. The computer-based infrastructure also helps them to continue with their schooling and contributes to reducing the isolation and solitude of these children and their families. In Italy, a few regional experiences are demonstrating the feasibility and validity of patient care models based on a network of services that include specialist care and intermediate care levels.

An Australian experience likewise focuses on the fundamental role of home care. Despite the country's vast dimensions, there are reference centers specializing in pediatric palliative care, generally based at a few pediatric hospices and working in close contact with family pediatricians, hospitals and territorial services, working as essential partners to enable very complex patients demanding high levels of care to be managed at home. Direct telecommunications between the home and the center facilitate this process and enable the child to stay at home. The computer-based infrastructure also helps them to continue with their schooling and contributes to reducing the isolation and solitude of these children and their families. In Italy, a few regional experiences are demonstrating the feasibility and validity of patient care models based on a network of services that include specialist care and intermediate care levels. In the Veneto region, there is a pediatric palliative care network with a pediatric hospice in Padua serving as its specialist reference center, where a multiprofessional team of palliative care specialists provide supervision, training and care integrated with the territorial and hospital services for children with cancer and other diseases in need of palliative care throughout the region (with a population of 7.5 million). In the Veneto, a diagnosis of incurable disease and complex needs in a pediatric patient (whatever the type of disease or the patient's age and place of residence) coincides with the activation of this regional centre for pediatric palliative care. This can be done by the family pediatrician or the hospital physician dealing with the case. The specialist palliative care team at the regional centre (physician, nurse, psychologist) meets the child and his family, and all the public health and other figures involved in the case. At a first meeting, the family pediatrician, the territorial services of the local public health office, the physician specializing in the patient's disease, the reference person at the pediatrics department of the hospital serving the area where the child lives and representatives from the local authority and from the specialist palliative care team assess the case, the resources available and the feasibility of meeting the clinical and other needs of the child and his family. An integrated patient care program is prepared and shared with the child (depending on his age and disease) and his parents. The home care program involves the territorial health care personnel undergoing a period of training and sharing of procedures and methods for dealing with acute problems, predictable or otherwise, which is provided at the reference centre or at the specialist department that diagnosed the child's disease.

his parents. The home care program involves the territorial health care personnel undergoing a period of training and sharing of procedures and methods for dealing with acute problems, predictable or otherwise, which is provided at the reference centre or at the specialist department that diagnosed the child's disease. Once the child is at home, the person responsible for the case is the family pediatrician who takes care of the patient with the cooperation of the territorial services and the regional centre for specialist palliative care, which coordinates the services required, and assures technical supervision and support for all members of the expanded team to guarantee a round-the-clock medical/nursing service. Meetings are also held between the specialist palliative care team, the family pediatrician, the territorial services and all the other institutions directly and/or indirectly involved in managing these young patients and their families, to assess the efficacy of the service being provided, any changes needed, organizational problems to solve, and whether the needs of the team have been met. If a child goes into hospital or the hospice, the reference team continues its work within the residential institution too.

hese young patients and their families, to assess the efficacy of the service being provided, any changes needed, organizational problems to solve, and whether the needs of the team have been met. If a child goes into hospital or the hospice, the reference team continues its work within the residential institution too. Albeit using different organizational methods, there are numerous other pediatric palliative care experiences ongoing in Italy and around the world: the diversity of care solutions provided (due mainly to local issues and the different availability of resources in the areas involved) is a factor that should stimulate comparisons, research and experimentation with different practical approaches to ensure that these very "special" patients are guaranteed the competent, unequivocal response and global care they need. Currently legislation in Italy In legal terms, the last two years have seen considerable advances and many issues have been brought up for debate.

Albeit using different organizational methods, there are numerous other pediatric palliative care experiences ongoing in Italy and around the world: the diversity of care solutions provided (due mainly to local issues and the different availability of resources in the areas involved) is a factor that should stimulate comparisons, research and experimentation with different practical approaches to ensure that these very "special" patients are guaranteed the competent, unequivocal response and global care they need. Currently legislation in Italy In legal terms, the last two years have seen considerable advances and many issues have been brought up for debate. - In the decree of the President of the Republic of 7 April 2006 on the adoption of the Italian national public health plan for 2006–2008 (published in the Official Gazette n.139 of 17 June 2006), the strategic objective 3.10 states that, " particular attention must be paid to the need for palliative care in neonatal, pediatric and adolescent age, bearing in mind the considerable diversity of the problems involved by comparison with those of adult age and the elderly, the great variety and fragmentation of the diseases in question, which are often rare and demand highly-specialized measures, and the often very lengthy and unpredictable time interval during which such care is needed. Given the above considerations, it is indispensable to organize dedicated palliative care networks for this age group that can guarantee the quality and specialization of the action needed together with a global and multi-dimensional care for the children and their families."

interval during which such care is needed. Given the above considerations, it is indispensable to organize dedicated palliative care networks for this age group that can guarantee the quality and specialization of the action needed together with a global and multi-dimensional care for the children and their families." - The Technical document on palliative care for the newborn, children and adolescents, issued by the Italian Ministry of Public Health in December 2006, defines the context and the particular features, the care models currently adopted in Italy and internationally, and the necessary resources. - The document on Institutional and semi-institutional care provision, approved by the Essential Assistance Levels Commissionon 13 May 2007, includes a specific section on the pediatric setting. - The Agreement between the State and the Regions, approved on 27 June 2007 at the conference on palliative care in neonatal, pediatric and adolescent age, lays the foundations for the nationwide implementation of action and programs designed to assure young people with incurable disease and their families the same availability of pediatric palliative care that, whatever the patient's age and type of disease, offers practical solutions, multi-specialistic expertise, continuity of treatments and goals, support and sharing of the burden. - Protocol of Intent between the Italian Ministry of Public Health and the Maruzza Lefebvre d'Ovidio Foundation, approved on 26 September 2007, for the implementation of the "Progetto Bambino" for a National Network of pediatric palliative care services.

- The Agreement between the State and the Regions, approved on 27 June 2007 at the conference on palliative care in neonatal, pediatric and adolescent age, lays the foundations for the nationwide implementation of action and programs designed to assure young people with incurable disease and their families the same availability of pediatric palliative care that, whatever the patient's age and type of disease, offers practical solutions, multi-specialistic expertise, continuity of treatments and goals, support and sharing of the burden. - Protocol of Intent between the Italian Ministry of Public Health and the Maruzza Lefebvre d'Ovidio Foundation, approved on 26 September 2007, for the implementation of the "Progetto Bambino" for a National Network of pediatric palliative care services. - The Technical document on the State-Regions agreement, approved on 20/3/2008, defines the public health and social support settings for providing practical support in the process for implementing pediatric palliative care in all Italian regions. This is an area of public health programming and implementation that is undeniably difficult, where there is still much to be done. For the children involved, palliative care also represents an indispensable goal so that pediatric medicine does not stop at establishing that a treatment has failed or that a condition is incurable, but must go on to recommend the best way for the child to live with disease.

Sir, Mastroianni et al. [1] report high prevalence (100%) of respiratory symptoms in their cohort of 19 children who received surgery for esophageal atresia. In contrast with the results of other studies [2], they found gastro-esophageal reflux incidence was low (26,3%). They therefore emphasize anti-reflux therapy could not be effective for treatment of respiratory symptoms in this group of patients. We agree with the speculation of the authors, as this was also our experience. Indeed, children who received surgical intervention for esophageal atresia have high incidence of respiratory function abnormalities [3] and bronchial hyperreactivity [4]. These conditions may be multifactorial in origin and are related to recurrent inhalation, epithelial damage, bronchial obstruction and tracheomalacia. Treatment of underlying respiratory disease instead of anti-reflux therapy may be warranted in children with a history of surgical repair of esophageal atresia and tracheoesophageal fistula, especially when lung function studies confirm respiratory involvement.

Introduction Significant advances in the diagnosis and treatment of inborn errors of metabolism have occurred in recent years. Expanded newborn screening using tandem mass spectrometry has led to the ability to identify and treat neonates who have metabolic conditions before symptoms appear [1]. Developments in nutritional support and pharmacological treatments with vitamin cofactors, end-product replacement, and drugs inducing specific enzymes or alternative pathways have also led to better outcomes. Cell therapy, primarily orthotopic liver transplantation (OLT), has significantly changed the prognosis of some of these diseases. Patients undergoing solid organ transplantation have benefited from innovative surgical techniques and novel, less toxic nonsteroidal immunosuppressive regimens. However, pharmacological treatments are often insufficient in the face of the activation of catabolic states, many patients succumb while waiting for a donor organ (approximately 15%), and short-term peri-transplant morbidity and long-term morbidity associated with lifelong immunosuppression continue to be significant issues [2-5]. Therefore, a risk/benefit assessment could make gene therapy an acceptable option for several inborn errors of metabolism.

e waiting for a donor organ (approximately 15%), and short-term peri-transplant morbidity and long-term morbidity associated with lifelong immunosuppression continue to be significant issues [2-5]. Therefore, a risk/benefit assessment could make gene therapy an acceptable option for several inborn errors of metabolism. Progress in the direction of clinical application of gene replacement therapy has been scarce so far despite extensive investigations for over 20 years. A general skepticism toward gene therapy was raised by the death of one patient in the ornithine transcarbamylase deficiency (OTCD) clinical trial [6] and by the recent report of leukemia occurred in few patients with severe combined immunodeficiencies (SCID) treated with retroviral ex vivo gene therapy [7]. However, with regard to the SCID trial, it is important to emphasize that despite the adverse events, it clearly demonstrated the benefits of gene therapy as treated patients can now cope with environmental microorganisms and live a normal life in the absence of any specific therapy [8].

viral ex vivo gene therapy [7]. However, with regard to the SCID trial, it is important to emphasize that despite the adverse events, it clearly demonstrated the benefits of gene therapy as treated patients can now cope with environmental microorganisms and live a normal life in the absence of any specific therapy [8]. Several different types of vectors, both viral and nonviral, have been developed for liver-directed gene therapy and have resulted in phenotypic correction in numerous animal disease models. The optimal vector for in vivo liver-directed gene therapy should be able to transfer genes to a high percentage of hepatocytes with limited toxicity. However, the available vectors have all shown some limitations (Table 1). In aiming at the treatment of liver metabolic diseases an important issue is how much of the liver needs to be corrected (i.e. percentage of hepatocyte) to achieve clinically relevant improvements. The percentage of hepatocyte transduction required for phenotypic correction is generally low in non-cell autonomous disorders such as hemophilia A and B or mucopolysaccharidoses and higher in cell autonomous defects such as urea cycle disorders. As a general principle, maximizing therapeutic gene expression per cell and minimizing the vector dose for a clinical effect are desirable. However, the potential adverse effects of over-expression of the therapeutic protein should also be taken into account. Table 1 Overview of gene therapy vectors.

Several different types of vectors, both viral and nonviral, have been developed for liver-directed gene therapy and have resulted in phenotypic correction in numerous animal disease models. The optimal vector for in vivo liver-directed gene therapy should be able to transfer genes to a high percentage of hepatocytes with limited toxicity. However, the available vectors have all shown some limitations (Table 1). In aiming at the treatment of liver metabolic diseases an important issue is how much of the liver needs to be corrected (i.e. percentage of hepatocyte) to achieve clinically relevant improvements. The percentage of hepatocyte transduction required for phenotypic correction is generally low in non-cell autonomous disorders such as hemophilia A and B or mucopolysaccharidoses and higher in cell autonomous defects such as urea cycle disorders. As a general principle, maximizing therapeutic gene expression per cell and minimizing the vector dose for a clinical effect are desirable. However, the potential adverse effects of over-expression of the therapeutic protein should also be taken into account. Table 1 Overview of gene therapy vectors. Genetic material Packaging capacity Vector genome forms Advantages Disadvantages Retrovirus RNA 8 kb Integrated - High efficiency integration - Transduction only in dividing cells - No viral immune response - Insertional carcinogenesis - Long-term expression Lentivirus RNA 8 kb Integrated - Non-dividing cells - Integration into active genes - Long-term expression - Risk of replication competent HIV Adenovirus dsDNA Up to 35 kb (HDAd) Episomal - Non-dividing cells - Acute toxicity - Large cloning capacity - High transduction levels - Long-term expression (HDAd) Adeno-associated vectors ssDNA 5–9 kb Episomal (> 90%) - Non-dividing cells - Limited cloning capacity Integrated (< 10%) - Long-term expression - CTL-mediated immune reaction Naked plasmid DNA dsDNA Unlimited Episomal - Non dividing cells - Low efficiency of transduction - No inflammatory response - Efficient and clinically relevant delivery method still to be developed - Large cloning capacity - Long-term expression - Ease preparation dsDNA = double stranded DNA; ssDNA = single stranded DNA; HDAd = helper-dependent adenoviral vector; CTL = cytotoxic T lymphocyte.

iency of transduction - No inflammatory response - Efficient and clinically relevant delivery method still to be developed - Large cloning capacity - Long-term expression - Ease preparation dsDNA = double stranded DNA; ssDNA = single stranded DNA; HDAd = helper-dependent adenoviral vector; CTL = cytotoxic T lymphocyte. Vectors for liver directed gene therapy The number of different vectors that are under development for liver-directed gene therapy is continuously increasing. However, five main classes of vectors have been more extensively investigated and each of these classes is characterized by different strengths and weaknesses (Table 1). Retrovirus Retroviral vectors (RV) were the first vectors used for gene therapy. They can efficiently integrate into the chromatin of target cells. However, they require the target cells to be mitotically active for an efficient transduction. Therefore, induction of liver division or liver regeneration through manipulations such as partial hepatectomy or hapatocyte growth factor treatment have been required for efficient hepatocyte transduction [9-11]. More recently, it has been shown that RV can transduce hepatocytes from newborn mice [12] and dogs [13] without an exogenous stimulation of cell division. However, as shown by the SCID trial experience, the risk of insertional mutagenesis is still a major consideration for RV.

efficient hepatocyte transduction [9-11]. More recently, it has been shown that RV can transduce hepatocytes from newborn mice [12] and dogs [13] without an exogenous stimulation of cell division. However, as shown by the SCID trial experience, the risk of insertional mutagenesis is still a major consideration for RV. Lentivirus Lentivirus vectors (LV) offer similar advantages to the RV, in that they mediate long-term integration of the therapeutic transgene, but unlike RV, they do not require cellular mitosis to gain access to the host genome for integration. They also are thought to share the potential for insertional mutagenesis with subsequent carcinogenesis, although this has not been observed yet in animal models. Following systemic LV delivery, the majority of transduced liver cells are of nonparenchymal origin and therefore, the efficiency of hepatocyte transduction is relatively low.

ght to share the potential for insertional mutagenesis with subsequent carcinogenesis, although this has not been observed yet in animal models. Following systemic LV delivery, the majority of transduced liver cells are of nonparenchymal origin and therefore, the efficiency of hepatocyte transduction is relatively low. Adenovirus Adenovirus (Ad) vectors are well suited for liver-directed gene therapy because they can transduce hepatocytes with high efficiency. When tested in vivo, first generation of Ad (FGAd) vectors, which are replication-defective but can still express viral genes at low levels, cause acute and chronic toxicity. Helper-dependent adenoviral (HDAd) vectors, which are devoid of all viral genes, offer a better safety profile and can provide long-term transgene expression with negligible chronic toxicity [14]. Several preclinical studies have shown that HDAd results in long-term phenotypic correction in several genetic diseases [14]. However, similar to FGAd they can still cause an acute toxic reaction due to activation of the host innate immune system when they are administered at high dose systemically [15]. A clinical trial for OTCD using an early generation Ad vector bearing the human OTC gene was interrupted when the second subject at the highest dose suffered fatal complications. The trial involved 18 subjects divided into 6 cohorts of 1/2 log dose escalations between cohorts until two subjects were enrolled at the highest dose [16]. As predicted from preclinical models, clinical findings were mild and transient in the initial 17 patients [16]. However, unlike the previous subjects, the last patient enrolled developed within 24 hours after vector infusion a lethal reaction characterized by acute respiratory distress syndrome, hepatitis, disseminated intravascular coagulopathy, hyperammonemia, and high levels of serum IL-6 [6]. Several mechanisms have been proposed to be responsible for the activation of this acute response [17]. However, regardless of the multiple mechanisms involved, systemic administration of Ad results in an acute toxic reaction which is triggered by the Ad capsid proteins in a dose-dependent fashion [15]. This acute toxicity is currently the main obstacle preventing clinical application of HDAd and strategies to overcome this problem are currently under investigation [18-20].

d, systemic administration of Ad results in an acute toxic reaction which is triggered by the Ad capsid proteins in a dose-dependent fashion [15]. This acute toxicity is currently the main obstacle preventing clinical application of HDAd and strategies to overcome this problem are currently under investigation [18-20]. Adeno-associated virus (AAV) AAV vectors are derived from a non-pathogenic human parvovirus that can infect non-dividing cells and remains latent for prolonged periods, predominantly in an episomal state. AAV vectors appear to persist in infected cells and do not trigger a robust innate response following in vivo administration. A wide repertoire of different AAV serotypes with different tissue tropisms is now available for several disease applications [21]. AAV vectors have a limited packaging capacity which precludes applications in diseases requiring large therapeutic genes. However, novel AAV serotypes with larger cloning capacity are emerging and they may at least in part overcome this problem [22]. In the clinical study for liver-directed gene therapy of hemophilia B, a recombinant AAV vector expressing human Factor IX (FIX) was infused through the hepatic artery in subjects with severe hemophilia B in an open label, dose-escalation study. Two subjects in the higher dose cohorts achieved measurable FIX levels at 2 weeks after vector infusion but, in contrast to the results generated in animal models, they exhibited a gradual decline in factor levels to < 1% by 10 weeks after vector infusion. This was accompanied in both subjects by an asymptomatic transaminase elevation beginning 4 weeks after vector infusion, with a gradual decline to baseline normal levels coinciding with the loss of FIX expression [23]. This reaction is due to the rejection of transduced hepatocytes by AAV capsid-specific memory CD8(+) T cells reactivated by AAV [24] and intense investigations are currently ongoing to overcome this problem. Another problem of the AAV vectors is as yet theoretical risk of insertional mutagenesis in humans. Studies in mice suggest that AAV vectors are predominantly nonintegrating [25], and a wealth of experience in the field had failed to uncover any evidence of tumor formation as a result of AAV transduction, except for a mouse disease model of mucopolysaccharidosis type VII [26]. A recent study has reported that, in the tumor tissue, the vector appeared to have integrated in a region rich in microRNA sequences on mouse chromosome 12 [27].

ad failed to uncover any evidence of tumor formation as a result of AAV transduction, except for a mouse disease model of mucopolysaccharidosis type VII [26]. A recent study has reported that, in the tumor tissue, the vector appeared to have integrated in a region rich in microRNA sequences on mouse chromosome 12 [27]. On the other hand, follow-up periods ranging up to 9 years in several hemophilic dogs have failed to reveal any evidence of tumor formation [28]. Nonviral vectors Nonviral vectors offer a number of advantages over viral-based strategies, including minimal toxicity from the vector, long-term transgene expression, lack of a humoral response against the vector, and the consequent ability to repeat dose [29]. A major advance in the field has been the development of the hydrodynamic injection technique which involves, in mice, the rapid injection of a large volume of naked plasmid DNA (pDNA) and typically results in 10–15% of hepatocyte transfection [30,31]. Systemic hydrodynamic procedure as practiced in the rodents is neither safe nor practical in larger animals or humans. However, clinically relevant methods using balloon catheters for regional hydrodynamic delivery of pDNA have been developed [32,33]. These studies demonstrate the feasibility of intravascular delivery to the liver using minimally invasive approaches, and are a step in the direction of human clinical trials.

or humans. However, clinically relevant methods using balloon catheters for regional hydrodynamic delivery of pDNA have been developed [32,33]. These studies demonstrate the feasibility of intravascular delivery to the liver using minimally invasive approaches, and are a step in the direction of human clinical trials. Pre-clinical and clinical studies Experimental gene therapy has been used to correct several metabolic diseases. We will discuss two diseases (Crigler-Najjar syndrome type I and OTCD) as representative examples to illustrate the potential and the limitations of currently available strategies for liver-directed gene therapy.

linical and clinical studies Experimental gene therapy has been used to correct several metabolic diseases. We will discuss two diseases (Crigler-Najjar syndrome type I and OTCD) as representative examples to illustrate the potential and the limitations of currently available strategies for liver-directed gene therapy. Crigler-Najjar syndrome Crigler-Najjar syndrome is an autosomal recessive condition characterized by non-hemolytic unconjugated hyperbilirubinaemia due to mutations bilirubin-uridinediphosphoglucuronate glucuronosyltransferase (UGT1A1). Patients with Crigler-Najjar syndrome type I (MIM 218800) are refractory to phenobarbital treatment, have life-threatening elevations of bilirubin, and are generally managed with phototherapy throughout childhood and adolescence. Although effective, phototherapy is cumbersome, inconvenient, and its efficacy may diminish with age because of increased skin thickness and decreased surface/mass ratio. Moreover, despite this treatment, patients remain at risk of brain damage when intercurrent infections may increase production of bilirubin above that which can be controlled by the phototherapy [34]. Therefore, patients with Crigler-Najjar type I are often advised to consider liver transplantation, most frequently in the range of 18 – 25 years of age. Crigler-Najjar syndrome has long been considered a paradigm for developing gene therapies for metabolic liver diseases for several reasons: (a) the underlying defect is well characterized at the biochemical and molecular level; (b) the fraction of corrected hepatocytes required for clinical benefit is small, as deduced from hepatocyte transplantation studies [35]; (c) the UGT1A1 does not require strict gene regulation for normal activity; (d) an animal model, the Gunn rat, recapitulating the human disease is available; (e) the outcome of the experimental therapies can be easily determined by measuring bilirubin fractions in serum and bile; (f) the UGT1A1 can be produced from skeletal muscle other than liver, its natural production site, and still retain the ability to transform bilirubin into water-soluble derivatives [36]. For these several reasons, Crigler-Najjar syndrome type I is very attractive as a gene therapy disease candidate and its correction has been the goal of several studies using different vector systems including RV, LV, Ad, AAV, and nonviral vectors.

the ability to transform bilirubin into water-soluble derivatives [36]. For these several reasons, Crigler-Najjar syndrome type I is very attractive as a gene therapy disease candidate and its correction has been the goal of several studies using different vector systems including RV, LV, Ad, AAV, and nonviral vectors. RV expressing UGT1A1 injected in newborns [37] or in conjunction with partial hepatectomy [38] have achieved long-term correction of the hyperbilirubinemia in the Gunn rats. As previously discussed, LV can also transduce nonproliferating cells and, in the Gunn rats, they resulted in stable reduction of bilirubin levels to near normal levels for over 1 year after treatment [39]. Impressive lifelong correction of hyperbilirubinemia has been also reported in the Gunn rats following a single intravenous injection of HDAd vector encoding UGT1A1 with negligible chronic toxicity [40]. Among different serotypes, AAV serotype 1 was found to be the most efficient in correcting the hyperbilirubinemia of the Gunn rats although large hepatic macroscopic lipid lesions of unclear etiology were found in AAV-treated animals [41]. A reduction of hyperbilirubinemia has also been reported following hydrodynamic injection of pDNA [42]. However, as discussed in previous sections, each of the vectors used in this disease model has some limitations which are currently preventing clinical applications.

r etiology were found in AAV-treated animals [41]. A reduction of hyperbilirubinemia has also been reported following hydrodynamic injection of pDNA [42]. However, as discussed in previous sections, each of the vectors used in this disease model has some limitations which are currently preventing clinical applications. Urea cycle disorders Urea cycle disorders typically present in the first few days after birth with poor feeding, vomiting, lethargy, and coma due to hyperammonemia. Despite aggressive pharmacotherapy, patients are at high risk for repeated episodes of hyperammonemia and cumulative neurological morbidity and mortality [43,44]. Given these significant problems, gene-replacement therapy could represent a viable alternative to OLT for long-term correction. Several studies over the past decade have found the therapeutic effect of several different FGAd vectors to be transient in the OTCD mouse models and lasting no longer than 2 months [45]. HDAd instead can mediate long-term correction of the OTCD animal model without chronic toxicity [46,47]. The novel AAV serotypes (AAV7, 8, 9), with higher efficiency of hepatocyte transduction, have also resulted in long-term phenotypic correction [48]. The application of LV and nonviral vectors for OTCD has not been reported to date and these vectors are likely to be inefficient in these diseases due to the high percentage of hepatocyte correction required.

with higher efficiency of hepatocyte transduction, have also resulted in long-term phenotypic correction [48]. The application of LV and nonviral vectors for OTCD has not been reported to date and these vectors are likely to be inefficient in these diseases due to the high percentage of hepatocyte correction required. Conclusion Gene therapy for liver metabolic diseases as an alternative or adjunctive treatment to cell therapy is a logical target given the problems with the available treatment modalities. Disorders such as Crigler-Najjar syndrome and urea cycle disorders are excellent candidates because of their poor prognosis. However, each of the available vector transfer technologies offers strengths and weaknesses. Integrating vectors such as LV and RV may be associated with long-term risk of genotoxicity and potential life long correction; AAV have a lower risk regarding integration, but are limited by cloning capacity and potential adaptive immune response to viral antigens. HDAd are associated with a dose-related innate immune response but offers efficient transduction without risk of genome integration. None of these obstacles are conceptually immovable and novel strategies need to be investigated to improve the safety profile of these vectors. Based on the significant progress to date, in spite of the expected setbacks of all drug development efforts, gene therapy for liver metabolic disorders may soon become a clinical reality. Competing interests The author declares that she has no competing interests.

Conclusion Gene therapy for liver metabolic diseases as an alternative or adjunctive treatment to cell therapy is a logical target given the problems with the available treatment modalities. Disorders such as Crigler-Najjar syndrome and urea cycle disorders are excellent candidates because of their poor prognosis. However, each of the available vector transfer technologies offers strengths and weaknesses. Integrating vectors such as LV and RV may be associated with long-term risk of genotoxicity and potential life long correction; AAV have a lower risk regarding integration, but are limited by cloning capacity and potential adaptive immune response to viral antigens. HDAd are associated with a dose-related innate immune response but offers efficient transduction without risk of genome integration. None of these obstacles are conceptually immovable and novel strategies need to be investigated to improve the safety profile of these vectors. Based on the significant progress to date, in spite of the expected setbacks of all drug development efforts, gene therapy for liver metabolic disorders may soon become a clinical reality. Competing interests The author declares that she has no competing interests. Acknowledgements NB-P is supported by the National Institutes of Health (K99 DK077447), the Texas Affiliate of the American Heart Association (0765032Y), and the Texas Medical Center Digestive Disease Center. The financial support of Telethon – Italy (Fellowship GFP04008) to NB-P is gratefully acknowledged.

Introduction Scientific studies show that even very premature newborns may experience sensation of distress which could unfavourably influence many clinical and behavioural parameters of their present and future [1]. Pain control in newborns is so primary importance, also stressed by the American Academy of Paediatrics [2]. The purpose of our study was comparing the analgesic effects of sucking own mother milk, versus alternative chances like caressing and/or pacifier, during routine invasive procedures in full-term newborns. The most painful routine invasive procedures in full-term newborns include venous blood sample and capillary heel stick blood sampling [3]. The analgesic effect of oral glucose 24% solution [4], pacifiers [5] and skin-to-skin contact [6] have already been demonstrated. The use of sucrose and/or pacifier for analgesia may interfere with a correct beginning of breastfeeding [7], so it may be an interesting alternative to test the analgesic effect of breastfeeding during painful procedures. In a recent review by Shah PS et al [8] breastfeeding is associated with changes in heart rate, duration of cry, percentage of crying time and a decrease of measured pain. Breastfeeding instead, does not seem to be favourable, if compared with higher glucose concentrations, with regards for crying duration, PIPP score and DAN score [9-11]. This suggests that neonates undergoing painful procedures may be breastfed or given expressed breast milk to obtain analgesic effect. This special power of breast-sucking may be linked to relational factors (skin-to-skin contact, nearness to mother, entertainment) [12,13] and to specific components of human milk like sugar [14] and triptophane [15] a melatonine precursor that enhances in neonates the production of beta endorphins [16], or the endogenous opioids like galattorphins [17]. This practice may be useful for driving on breastfeeding by frequent sucking and using mother's breast for comfort.

ic components of human milk like sugar [14] and triptophane [15] a melatonine precursor that enhances in neonates the production of beta endorphins [16], or the endogenous opioids like galattorphins [17]. This practice may be useful for driving on breastfeeding by frequent sucking and using mother's breast for comfort. Methods We enlisted 200 full term healthy neonates (100 cases and 100 controls). We suggested to all mothers of completely or partial breastfed neonates the execution of metabolic screening from heel puncture during breast sucking, explaining to mothers the advantages of this practice. The magnitude of pain in neonates was measured by DAN scale (Douleur Aigue Nouveau-nè scale – Figure 1), reckoning suffering in full-term healthy neonates by observing facial changes, limb movements and vocal expression [18]. This evaluation regarded both newborns undergoing capillary heel stick for screening while breastfed and control ones, i.e. children refusing breast during puncture, or formula fed ones, or babies who's mother had refused the procedure. In control cases, analgesia was provided by caressing and/or pacifier. Figure 1 DAN scale.

The magnitude of pain in neonates was measured by DAN scale (Douleur Aigue Nouveau-nè scale – Figure 1), reckoning suffering in full-term healthy neonates by observing facial changes, limb movements and vocal expression [18]. This evaluation regarded both newborns undergoing capillary heel stick for screening while breastfed and control ones, i.e. children refusing breast during puncture, or formula fed ones, or babies who's mother had refused the procedure. In control cases, analgesia was provided by caressing and/or pacifier. Figure 1 DAN scale. Every child was taken to his own mother and the puncture was performed after minimum two minutes of effective sucking. Capillary puncture was made on the postero-lateral area of the heel, using sterile click prick lancet (the kind of lancets we used were AMES MINILET LANCETS by Bayer). Every heel puncture was performed by dedicated nurses of Neonatology Division. Every nurse was trained about enlistment of neonates, about information of mothers, and especially about evaluation of pain according the standards of Dan Scale and attended a course of pain evaluation of newborn. For every enroled newborn, was filled a form including his/her name, date of birth, gestational age, Apgar score and way of punture (while breastfeeding or pacifier) (Figure 1).

of mothers, and especially about evaluation of pain according the standards of Dan Scale and attended a course of pain evaluation of newborn. For every enroled newborn, was filled a form including his/her name, date of birth, gestational age, Apgar score and way of punture (while breastfeeding or pacifier) (Figure 1). A score from 0 (no pain) to 10 (highest pain) was attributed to each child, according to the intensity of pain [3,19]. After discharge, mothers were called to verify the outcome in breastfeeding at one month of life in the two groups. Scores obtained in the two groups were statistically analysed by χ2 test. Dates confirming homogeneity of groups were statistically compared with t-Student test. We used test for relating two proportions in feed evaluation of one month aged children. We did not collect differences between caressing and pacifier in the control group. Statistics data give signs of homogeneity of champions, in fact there are not elements influencing mothers in the choice of way of puncture, except for formula-fed neonates.

A score from 0 (no pain) to 10 (highest pain) was attributed to each child, according to the intensity of pain [3,19]. After discharge, mothers were called to verify the outcome in breastfeeding at one month of life in the two groups. Scores obtained in the two groups were statistically analysed by χ2 test. Dates confirming homogeneity of groups were statistically compared with t-Student test. We used test for relating two proportions in feed evaluation of one month aged children. We did not collect differences between caressing and pacifier in the control group. Statistics data give signs of homogeneity of champions, in fact there are not elements influencing mothers in the choice of way of puncture, except for formula-fed neonates. Results Description of the studied population The case group included 52 female neonates and 48 males tested with heel puncture while breastfed. The control group included 41 female and 59 male neonates. In the case group, 67 were born by vaginal delivery, 28 by caesarean section and 5 with use of vacuum extractor. In the control group, 59 were born by vaginal delivery and 41 by caesarean section. The mean gestational age in the case group was 39.42 weeks (DS1.27 and CI95 = 38.16–40.69), in controls one was 39.35 weeks (DS 1.31 and CI95 = 38.04–40.67). The mean first and fifth minute Apgar score in case group was 8.78 (DS 0.69 and CI 95 = 8.09–9.47) and 9.75 (DS 0.52 and CI 95 = 9.23–10); in the control group they were 8.76 (DS 0.85 and CI 95 = 7.90–9.61) and 9.69 (DS 0.71 and CI 95 = 8.98–10).

40.69), in controls one was 39.35 weeks (DS 1.31 and CI95 = 38.04–40.67). The mean first and fifth minute Apgar score in case group was 8.78 (DS 0.69 and CI 95 = 8.09–9.47) and 9.75 (DS 0.52 and CI 95 = 9.23–10); in the control group they were 8.76 (DS 0.85 and CI 95 = 7.90–9.61) and 9.69 (DS 0.71 and CI 95 = 8.98–10). The mean birth weight in the first group was 3335.2 g (DS 434.82 and CI 95 = 2900.38 – 3770.02), in the second one 3333.45 g (DS 443.89 and CI 95 = 2889.55–3777.34). In the first group, at discharge 93 neonates were being breastfed and 7 were receiving breast milk and formula; at 72 hours after-discharge check-up, only one child receiving mixed feeding began to receive only formula. In the control group at discharge 90 neonates were fed by breast and 4 neonates were fed with human milk and formula. At the age of one month, in the examined group 65 neonates were exclusively breast fed, 14 were mainly breastfed, 13 partially breastfed, 8 were exclusively fed with formula. In controls group instead, at one month 63 neonates were exclusively breastfed, 13 were mainly breastfed, 7 were partially breastfed, 17 were fully formula fed. There is no evident statistical significant difference between the two groups regarding this last specific parameter, even if in control group, a larger number of neonates left own mother's milk. Rooming-in during hospital stay, was practised by 47 pairs of mother-child in the first group, and by 37 pairs in the second one. The mother's age was concerned, the mean age in the cases group was 32.18 years (DS 5.37 and CI 95 = 26.81–37.55). For half (50%) of the women it was their first pregnancy. Eleven percent of women had previous abortions and 5% voluntary interruption of pregnancy. Between pluriparous mothers, 78% had previously breastfed. In the control group, the mean age of mothers was 31.36 years (DS 4.93 and CI95 = 26.43–36.29); 43% of women were primiparous, 19% had previous abortions and 4% had voluntary pregnancy interruption. In pluriparous women, 78.95% had previously breastfed.

gnancy. Between pluriparous mothers, 78% had previously breastfed. In the control group, the mean age of mothers was 31.36 years (DS 4.93 and CI95 = 26.43–36.29); 43% of women were primiparous, 19% had previous abortions and 4% had voluntary pregnancy interruption. In pluriparous women, 78.95% had previously breastfed. Score for pain perception in neonates (DAN scale) The mean DAN score in the case group (neonates with puncture during breastfeeding) was 2.65 (DS 2.31 and CI 95 = 0.34 – 4.96), for control group 5.15 (DS 2.07 and CI95 = 3.08–7.22). In the case group, 20 neonates obtained score 0, while no neonates in the control group got this score (Table 1 and Figure 2). From statistical analysis a significant difference resulted between the score obtained in the two groups (p = 0.000), even when the single parameters of the DAN scale were considered independently: face expression (p = 0.000) (Figure 3), limb movements (p = 0.000) (Figure 4), vocal expression (p = 0.000) (Figure 5). Table 1 DAN scale total scores Total 0 1 2 3 4 5 6 7 8 9 10 cases 100 20 13 20 21 9 8 1 2 2 3 1 controls 100 0 4 1 16 23 17 13 14 3 6 3 Figure 2 Graph comparing DAN scale total scores for cases and controls. Figure 3 Facial expressions. Figure 4 Limb movements. Figure 5 Vocal expression.

Score for pain perception in neonates (DAN scale) The mean DAN score in the case group (neonates with puncture during breastfeeding) was 2.65 (DS 2.31 and CI 95 = 0.34 – 4.96), for control group 5.15 (DS 2.07 and CI95 = 3.08–7.22). In the case group, 20 neonates obtained score 0, while no neonates in the control group got this score (Table 1 and Figure 2). From statistical analysis a significant difference resulted between the score obtained in the two groups (p = 0.000), even when the single parameters of the DAN scale were considered independently: face expression (p = 0.000) (Figure 3), limb movements (p = 0.000) (Figure 4), vocal expression (p = 0.000) (Figure 5). Table 1 DAN scale total scores Total 0 1 2 3 4 5 6 7 8 9 10 cases 100 20 13 20 21 9 8 1 2 2 3 1 controls 100 0 4 1 16 23 17 13 14 3 6 3 Figure 2 Graph comparing DAN scale total scores for cases and controls. Figure 3 Facial expressions. Figure 4 Limb movements. Figure 5 Vocal expression. Discussion Our results strengthen the already well-known analgesic effect of breastfeeding. This analgesic effect may be successfully exploited for minor distressing procedures in full term healthy neonates. The choice of this easy and effective method, is useful in reducing all external interferences with a beginning of breastfeeding, while other analgesic systems such as pacifiers or sucrose might disrupt a good start at breastfeeding. Moreover, stressing the curative effects of human milk, may be an important confidence boost for mothers, hopefully rendering breastfeeding easier to pursue (regarding this point, we need a longer follow-up). In our study, doing a heel lance without sucking, does not seem to influence the kind of feeding at one month of age. In our next collection of data, we would include a further group of newborns – the ones pacified with sucrose.

endering breastfeeding easier to pursue (regarding this point, we need a longer follow-up). In our study, doing a heel lance without sucking, does not seem to influence the kind of feeding at one month of age. In our next collection of data, we would include a further group of newborns – the ones pacified with sucrose. In our opinion, this analgesic system may easily become a routine way of performing heel lance in maternity wards. This procedure may be suggested to available mothers of newborns who are sucking effectively, and possibly also extended to other painful minor procedures like intramuscular injection or venous puncture. Further studies might consider this method in older children, for instance during vaccination. Competing interests The authors declare that they have no competing interests. Authors' contributions EU and SP conceived of the study and participated in its design and coordination. AP, VA, GA, MT, AMZ, MBR, DZ, CT and MA participated in the collection of data. MV and LG carried out the analyses. MC participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.

Introduction Purpura fulminans (PF) is an ominous cutaneous condition usually associated with meningococcemia [1-9]. PF in the newborn is rarely reported [6-12] We report the case of a female preterm infant with extensive PF due to group B streptococcus (GBS) septicemia and discussion issues of management of this rare but often fatal condition.

nans (PF) is an ominous cutaneous condition usually associated with meningococcemia [1-9]. PF in the newborn is rarely reported [6-12] We report the case of a female preterm infant with extensive PF due to group B streptococcus (GBS) septicemia and discussion issues of management of this rare but often fatal condition. Case Purupura fulminans (PF) was immediately evident in a moribund 2.7 kg newborn girl delivered by emergency caesarean section for fetal tachycardia (200/minute by cardiotocography) at 35 week gestation 1. There was no family history of bleeding disorder. The membranes were ruptured 3 hours prior to delivery. The mother developed intrapartum fever (38.9°C) with chills and rigors and was given intravenous ampicillin and gentamicin 23 minutes before delivery by emergency caesarean section. At birth, the baby was apneic with heart rate of 80/minute. She cried and the heart rate responded upon bag and mask ventilation for 1 minute. Apgars were 8 and 10 at 1 and 5 minutes, respectively. On arrival at the NICU, the baby developed further apneas with cyanosis followed by tachypnea, insucking chest and grunting. Her mean arterial blood pressure was 30 mmHg and heart rate 190/minute. Arterial blood gas analysis showed a pH of 7.19, pCO2 8.03 kPa, pO2 2.25 kPa, and base excess of -6.9 mmol/L. Respiratory support (nasal continuous positive airway pressure of 5 cm H2O with 8 L/min of oxygen), normal saline bolus, and intravenous penicillin plus gentamicin were administered within the first hour of resuscitation. In the next 2 hours, she remained hypotensive despite further saline boluses, dopamine infusion and mechanical ventilation. Group B streptococcus, sensitive to penicillin, was isolated from the blood cultures of the mother and the infant. She was aggressively treated with broad antibiotic coverage, cardiopulmonary support with mechanical ventilation and multiple inotropes, and peritoneal dialysis (Table 1). The purpuric rash became more extensive and she developed progressive multi-organ system failure despite full intensive care support and succumbed 9 days later

ressively treated with broad antibiotic coverage, cardiopulmonary support with mechanical ventilation and multiple inotropes, and peritoneal dialysis (Table 1). The purpuric rash became more extensive and she developed progressive multi-organ system failure despite full intensive care support and succumbed 9 days later Figure 1 Purupura fulminans (PF) was immediately evident in a moribund 2.7 kg newborn girl delivered by emergency caesarean section for fetal tachycardia (200/minute by cardiotocography) at 35 week gestation. Table 1 Multi-organ system failure in the neonate with group B streptococcal septicemia

ressively treated with broad antibiotic coverage, cardiopulmonary support with mechanical ventilation and multiple inotropes, and peritoneal dialysis (Table 1). The purpuric rash became more extensive and she developed progressive multi-organ system failure despite full intensive care support and succumbed 9 days later Figure 1 Purupura fulminans (PF) was immediately evident in a moribund 2.7 kg newborn girl delivered by emergency caesarean section for fetal tachycardia (200/minute by cardiotocography) at 35 week gestation. Table 1 Multi-organ system failure in the neonate with group B streptococcal septicemia Organ system Abnormal findings Management Cardiovascular Cardiogenic and distributive shock; poor perfusion; ejection fraction 54% and fractional shortening 26% . Highest creatine phosphokinase 1033 U/l and cardiac troponin 0.45 ug/l Intravenous saline boluses, dopamine, dobutamine, epinephrine, hydrocortisone, milrinone, vasopressin Respiratory Respiratory failure with hypercarbnia and diffuse haziness on chest radiograph Mechanical ventilation, FiO2 1.0, surfactant, vecuronium Renal Passed urine at 10 hours of life; persistent oliguria; anuria 4 days later. Highest creatinine 153 umol/l Intravenous frusemide; peritoneal dialysis; gentamicin stopped Septicemic Group B streptococcus, sensitive to penicillin, isolated on surface swabs, and in baby and mother's blood cultures; highest C-reactive protein 12.9 mg/l Intravenous penicillin and gentamicin initially; ampicillin; cefotaxime; meropenim and vancomycin empirically; intravenous immunoglobulin Subsequently on high-dose penicillin and cefotaxime when group B streptococcus and sensitivity were available. Hematologic Disseminated intravascular coagulopathy with lowest hemoglobin 8.6 g/dl, thrombocytopenia 13 × 109/l, D-dimer 9735 ng/ml, prothrombin time 60 seconds, and activated plasma thromboplastin time 120 seconds Packed red cell, fresh frozen plasma, cryoprecipitate, platelet Metabolic Metabolic acidosis (worst pH 6.87), hypoglycemia (glucose 1.0 mmol/l), hypocalcemia (0.63 mmol/l) Dextrose and NaHCO3 infusion; calcium supplementation Neurologic Convulsion Anticonvulsant Hepatic Deranged liver function with worst total bilirubin of 125 umol/l and alanine aminotransferase 574 IU/l Supportive and treating underlying infection Discussion This report illustrates that PF is an ominous cutaneous sign of fulminant neonatal GBS septicemia. Childhood PF is often associated with meningococcaemia [1-3]. In the neonatal period group B streptococcus is the major cause of PF but gram negative organisms such as Escherichia coli and Enterobacter have been described [7-11].

ort illustrates that PF is an ominous cutaneous sign of fulminant neonatal GBS septicemia. Childhood PF is often associated with meningococcaemia [1-3]. In the neonatal period group B streptococcus is the major cause of PF but gram negative organisms such as Escherichia coli and Enterobacter have been described [7-11]. Nolan et al reported two cases of PF associated with meningococcal and chickenpox, respectively, and reviewed various treatment modalities [1]. Protein C and antithrombin III have been given if these factors are deficient [1,5]. In PF associated with meningococcemia and septic shock, Rivard et al described severe acquired protein C deficiency successfully treated with conventional therapy and high-volume plasma exchange as a source of protein C [2]. Many other therapies have been described in case reports that claimed to arrest the progression of neonatal PF, such as the use of heparin [8], Protein C [5,12], Antithrombin III, recombinant tissue plasminogen activator (rtPA), epoprostenol (prostacyclin) [6], topical nitroglycerin, intravenous dextran, and plasmapheresis [1-13]. Nevertheless, there is no strong evidence in favor of one particular therapy due to the small number of cases.

use of heparin [8], Protein C [5,12], Antithrombin III, recombinant tissue plasminogen activator (rtPA), epoprostenol (prostacyclin) [6], topical nitroglycerin, intravenous dextran, and plasmapheresis [1-13]. Nevertheless, there is no strong evidence in favor of one particular therapy due to the small number of cases. One limitation of our case was that the coagulopathy was immediately treated empirically before the endogenous activities of the anticoagulant factors protein C, protein S, and Antithrombin III (AT III) were assessed. In case any of these factors are found to be affected, human protein C or recombinant human activated protein C may be considered, and protein C, protein S, and AT III genes should have been analyzed in the patient and her parents. There was no family history of coagulopathy and group B streptococcus was identified to be the pathogen, making syndromes of congenital anticoagulant factor deficiency unlikely in this patient.

tein C may be considered, and protein C, protein S, and AT III genes should have been analyzed in the patient and her parents. There was no family history of coagulopathy and group B streptococcus was identified to be the pathogen, making syndromes of congenital anticoagulant factor deficiency unlikely in this patient. Regardless of the pathogen, neonatal PF must be promptly recognized and aggressively treated in children [1-6]. Early goal-directed therapy provides significant benefits with respect to outcome in adult patients with severe sepsis and septic shock [14]. The therapy involves adjustments of cardiac preload, afterload, and contractility to balance oxygen delivery with oxygen demand. Guidelines were proposed through the Surviving Sepsis Campaign to improve outcome in septic patients [15]. They are difficult to apply routinely and validate in neonates. As Claessens et al has commented, attempts to apply all of the procedures recommended by the experts, despite the apparent pragmatism of those procedures, have varied widely; diagnosis may be problematic because of atypical or unspecific presentations, biomarkers are of little help at the start of treatment and are unspecific, supportive treatment often depends on local supply of resources, and specific devices are often absent for initial therapy and monitoring [15]. Resuscitation policy for septic shock in neonates generally includes prompt treatment of the underlying infection with broad spectrum antibiotics, replacement of fluids or blood for preload, appropriate usage of inotropes for cardiac contractility and afterload, support of oxygenation and ventilation with mechanical ventilation, and support of individual multi-organ system failure [16].

prompt treatment of the underlying infection with broad spectrum antibiotics, replacement of fluids or blood for preload, appropriate usage of inotropes for cardiac contractility and afterload, support of oxygenation and ventilation with mechanical ventilation, and support of individual multi-organ system failure [16]. Despite full intensive care support, PF is often associated with multiple organ failure and high mortality in children [4]. PF has a reported mortality of 50 per cent secondary to multiple organ failure which commonly accompanies the syndrome and is associated with major long-term morbidity in those who survive [4]. In 3 cases of early neonatal PF, all the babies survived but had markedly compromised neurologic outcomes [7]. Consent Written informed consent was obtained from the patient's next of kin for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal. Competing interests The authors declare that they have no competing interests. Authors' contributions All authors participated in the care of the patient. KH is the principal author who drafts the manuscript. KS, WW and KC ensure that the clinical data is accurate. WW provides dialysis care. All authors read and approved the final manuscript.

Aim The non-organic Childhood constipation is a widespread problem and a recent systematic review has estimated the prevalence to be 0.7% to 29.6% [1]. A multifactorial pathophysiology is more accepted among researchers. Low fiber intake, psychiatric factors and positive family history [2-4] as well as experiencing stressful events in family and instability in the child-parent relationships are the reported explanations [5]. The stool-withholding behavior is known to be the major cause for the development and/or persistence of constipation in childhood [6]. Defining the associated psychiatric factors will improve the challenging treatment especially in chronic and recurrent situations.

arent relationships are the reported explanations [5]. The stool-withholding behavior is known to be the major cause for the development and/or persistence of constipation in childhood [6]. Defining the associated psychiatric factors will improve the challenging treatment especially in chronic and recurrent situations. The parental behavior strongly influences the mental and physical situation of their children such as they may have an effect on the manifestations of a disorder. Functional abdominal pain in children has been described to be increased by more "attention" from their parents [7]. Again, they are the ones who decide help seeking and influence the treatment decisions. Training the parents is an important part of the standard pediatric care for children with functional constipation [8]. The mother may be the parent to play the most important role in early childhood. Studies report that secure attachment and maternal secure base support are related to higher levels of positive mood, more constructive coping and better regulation of emotion [9]. However; the mother-child relationship has not been studies in details concerning its effect on defecation behavior of the children. The parenting style of a mother is defined by her personal characteristics. We hypothesized that the control on defecation in a child is influenced by the common manners of mother; e.g. her personality. The aim of this study is to examine the relations between the personality dimensions in mothers and existence of the functional constipation in their children.

The parental behavior strongly influences the mental and physical situation of their children such as they may have an effect on the manifestations of a disorder. Functional abdominal pain in children has been described to be increased by more "attention" from their parents [7]. Again, they are the ones who decide help seeking and influence the treatment decisions. Training the parents is an important part of the standard pediatric care for children with functional constipation [8]. The mother may be the parent to play the most important role in early childhood. Studies report that secure attachment and maternal secure base support are related to higher levels of positive mood, more constructive coping and better regulation of emotion [9]. However; the mother-child relationship has not been studies in details concerning its effect on defecation behavior of the children. The parenting style of a mother is defined by her personal characteristics. We hypothesized that the control on defecation in a child is influenced by the common manners of mother; e.g. her personality. The aim of this study is to examine the relations between the personality dimensions in mothers and existence of the functional constipation in their children. Methods Study population was recruited continuously from the university pediatric clinic (Tabriz University of medical sciences, Iran) during September 2007–September 2008. All of the patients with the complaint of constipation were fully evaluated by a same pediatric gastroenterologist and children with functional constipation (based on ROME III criteria for pediatric functional constipation) were enrolled in the study. Another group including mothers of children attending to the same clinic and without functional constipation was invited to participate as the control group. Mothers then were invited for a psychiatric assessment. A history and/or current symptoms related to psychiatric disorders on Axis I (based on DSM-IV) [10] or seizure disorders in mothers led to exclusion from the study. The research procedure was compatible with Helsinki Declaration and all of the participants gave written consent.

ere invited for a psychiatric assessment. A history and/or current symptoms related to psychiatric disorders on Axis I (based on DSM-IV) [10] or seizure disorders in mothers led to exclusion from the study. The research procedure was compatible with Helsinki Declaration and all of the participants gave written consent. Personality dimensions in both groups were evaluated by the NEO Five-Factor Inventory (NEO-FFI) which provides a dimensional account of the structure of normal personality traits, dividing the personality into five broad dimensions which are: extraversion, agreeableness, conscientiousness, neuroticism and openness to experience [11]. This 60-item standard questionnaire usually requires 15 minutes to complete and is rated on a five-point scale to yield scores in five major domains of personality. Means (standard deviations) were used to describe continuous variables and proportions for categorical data. Conditions were met for using Two-tailed Student's t test and Chi-square test, which was applied when appropriate and the overall significance was set at 0.05. Results One hundred and fifty children were evaluated in each group. No significant difference was noted between the two groups regarding their age (p = 0.240) and gender (p = 0.354). Mean age (SD) was 28.8(18.6) months in constipated children and 20.0(19.3) months in controls, 54.6% of constipated children and 56.7% of controls were male.

nd fifty children were evaluated in each group. No significant difference was noted between the two groups regarding their age (p = 0.240) and gender (p = 0.354). Mean age (SD) was 28.8(18.6) months in constipated children and 20.0(19.3) months in controls, 54.6% of constipated children and 56.7% of controls were male. Mean age (SD) was 30.9(7.1) years in mothers of children with functional constipation and 30.1(7.6) years in controls (p = 0.348). In both groups; near to 50% had a diploma degree, about 30% were educated under diploma and about 20% were postgraduates (p = 0.777). The scores of five personality dimensions assessed by NEO inventory are described in figure 1 by error bars. These two groups had significant differences. Mothers of children with functional constipation scored lower in neuroticism [(24.9(8.1) vs. 26.7(7.2), p = 0.046] and scored higher in extraversion [29.1(6.2) vs. 27.1(6.2), p = 0.005], conscientiousness [36.2(5.3 vs. 34.9(5.7), p = 0.049] and agreeableness [31.9(5.7) vs. 29.8(5.1), p = 0.002]. No significant difference was observed in the score of openness to experiences [23.9(4.9) vs. 24.2(3.7), p = 0.512]. Figure 1 Error bars comparing scores of five personality dimensions in mothers of children with functional constipation and controls. Personality of mothers of children with functional constipation was assembled of significantly lower neuroticism and higher extraversion, conscientiousness and agreeableness compared to mothers of children without functional constipation.

lity dimensions in mothers of children with functional constipation and controls. Personality of mothers of children with functional constipation was assembled of significantly lower neuroticism and higher extraversion, conscientiousness and agreeableness compared to mothers of children without functional constipation. We also compared two groups according to their dominant personality dimension. The dominant personality dimension was not statistically different between the two groups. The most common was conscientiousness (58.5%) followed by neuroticism (18.1%). Discussion Studies have documented the associations between motility disorders of the gastrointestinal tract (like chronic constipation) and psychological stresses like anxiety and depression [12,13]. We have previously reported specific overstated personality dimensions and the personality profile to be similar in constipation dominant variant of irritable bowel syndrome [14]. Likewise; the basis of pediatric functional constipation may be explained by psychoanalysis of the family events and relationships. Parents of children with idiopathic constipation may have no psychological problems [15] however cultural and social pressures are described to result in constipation in children as an "over-control problem" [16]. According to these results; we suggested and checked up the personality variations of mothers; which are not a "disorder" but may influence the child-mother relationship and child's behavior.

15] however cultural and social pressures are described to result in constipation in children as an "over-control problem" [16]. According to these results; we suggested and checked up the personality variations of mothers; which are not a "disorder" but may influence the child-mother relationship and child's behavior. The frequency of "hiding to stool" and "asking for pull-ups" in constipated children indicates full bowel control and their social awareness [17] so maternal expectations may design her child's reactions. While the extreme variation of a personality dimension (personality disorder) [18] in mothers was excluded by an interview, we believe that this study could trace the differences between them (extent of each dimension) in a way which is describing their manner and relationships. This study benefited using the five-factor model of personality which is considered to be the most comprehensive experimental enquiry into personality.

n interview, we believe that this study could trace the differences between them (extent of each dimension) in a way which is describing their manner and relationships. This study benefited using the five-factor model of personality which is considered to be the most comprehensive experimental enquiry into personality. Our results generally supported a personality difference between mothers with or without constipation in their children. Lower score in neuroticism, besides higher score in extraversion, conscientiousness and agreeableness is compatible with a character described by forcefulness, dutifulness, self conscientious, orderliness and discipline who is optimist, proficient, inflexible with less humiliation, anxious and restlessness. This restriction, force and orderliness may result in resistance and withholding as a response of child to the maternal behavior. This response may demonstrate a messy, careless or destructive character against the inflexible approach of the mother up to an orderly, rigid and obsessive personality. In conclusion, a considerable personality was obvious in mothers of constipated children in the current study which is compatible with a character that may rise up the stool-withholding behavior in their children. Future researches must include personality of mothers as a noteworthy factor in evaluating the treatment options.

lusion, a considerable personality was obvious in mothers of constipated children in the current study which is compatible with a character that may rise up the stool-withholding behavior in their children. Future researches must include personality of mothers as a noteworthy factor in evaluating the treatment options. Authors' contributions AF had primary responsiblity for protocol development, patient screening, enrollment, outcome assessment. MR and SK participated in the development of the protocol and were responsible for patient screening. SF supervised the design and execution of the study, performed the data analyses and writing the manuscript. All authors have read and approved the final manuscript.

Background Primary ciliary dyskinesia (PCD; MIM #244400) is a rare (1:15–30 000 live births) and usually autosomal recessive disease associated with situs viscerum inversus (Kartagener syndrome) in nearly half the cases [1]. Impaired mucociliary clearance due to defective motility of cilia is the hallmark of the condition [2]. Early clinical events are represented by continuous rhinorrhoea from the first days of life, respiratory distress or neonatal pneumonia with no obvious predisposing cause, and chronic or recurrent lower and upper airway infections, e.g. otitis media and purulent rhinosinusitis [3]. Three lower airway diseases are associated with PCD: pneumonia, bronchiectasis, and asthma [3].

rhoea from the first days of life, respiratory distress or neonatal pneumonia with no obvious predisposing cause, and chronic or recurrent lower and upper airway infections, e.g. otitis media and purulent rhinosinusitis [3]. Three lower airway diseases are associated with PCD: pneumonia, bronchiectasis, and asthma [3]. Computed tomography (CT) of the chest, particularly high-resolution CT (HRCT), has become the method of choice to evaluate chronic lung disease at any age [4,5]. Nevertheless, CT entails exposure to ionizing radiation, thereby increasing the risk of cancer in exposed individuals [6,7]. Magnetic resonance imaging (MRI) of the chest has been proposed as a potential radiation-free technique in several chest disorders [8-13]. However, its application in lung disease has long been limited by technical problems, namely a low signal-to-noise ratio because of the low proton density of the lung, and artifacts due to cardiac and breathing motion or to air/soft tissue transition [14]. Nevertheless, the results of chest MRI in assessment of lung disease were found to be comparable with those of conventional chest X-ray and CT, mainly in patients with cystic fibrosis (CF) [15-17]. Lung disease in PCD is similar to lung disease in CF, although changes can be milder in PCD [18]. Recent studies showed that CT scoring systems adequately describe the extent and severity of PCD lung changes [18-20]. Patients with PCD may develop chronic lower airways symptoms and/or signs at any time [21]. Therefore, a sensitive radiation-free imaging tool is highly desirable for the longitudinal assessment of structural lung damage.

d that CT scoring systems adequately describe the extent and severity of PCD lung changes [18-20]. Patients with PCD may develop chronic lower airways symptoms and/or signs at any time [21]. Therefore, a sensitive radiation-free imaging tool is highly desirable for the longitudinal assessment of structural lung damage. To our knowledge, no study has compared chest CT to MRI in PCD. The primary aim of this pilot study was to assess whether MRI is as effective as CT in identifying pulmonary abnormalities in patients with PCD. Our secondary aim was to investigate the relationships between the severity and extension of lung disease, identified with HRCT and MRI, and pulmonary function tests (PFTs).

. The primary aim of this pilot study was to assess whether MRI is as effective as CT in identifying pulmonary abnormalities in patients with PCD. Our secondary aim was to investigate the relationships between the severity and extension of lung disease, identified with HRCT and MRI, and pulmonary function tests (PFTs). Methods Patients Thirteen subjects (8 children/5 adults; median age, 15.2 yrs; range: 10.4–29.3 yrs) with PCD followed at the Department of Paediatrics, University of Naples Federico II, Naples, Italy, were prospectively enrolled in the study. PCD was suspected on the basis of clinical features and/or situs viscerum inversus [3]. The clinical characteristics of the study population are summarized in Table 1. Diagnosis was confirmed by light microscopy (LM) and by electron microscopy (EM) analysis of cilia ultrastructure on nasal brushing at a median age of 7.3 yrs (range, 0.1–17.1 yrs). In all cases LM revealed dysmotility or immotility [see Additional file 1]. All patients were undergoing the following treatment: daily airway clearance therapy constituted by nebulized saline prior to chest physiotherapy; physical exercise; and aggressive treatment of upper and lower airway infections by antibiotics. Table 1 Clinical characteristics of the study population

Methods Patients Thirteen subjects (8 children/5 adults; median age, 15.2 yrs; range: 10.4–29.3 yrs) with PCD followed at the Department of Paediatrics, University of Naples Federico II, Naples, Italy, were prospectively enrolled in the study. PCD was suspected on the basis of clinical features and/or situs viscerum inversus [3]. The clinical characteristics of the study population are summarized in Table 1. Diagnosis was confirmed by light microscopy (LM) and by electron microscopy (EM) analysis of cilia ultrastructure on nasal brushing at a median age of 7.3 yrs (range, 0.1–17.1 yrs). In all cases LM revealed dysmotility or immotility [see Additional file 1]. All patients were undergoing the following treatment: daily airway clearance therapy constituted by nebulized saline prior to chest physiotherapy; physical exercise; and aggressive treatment of upper and lower airway infections by antibiotics. Table 1 Clinical characteristics of the study population All (n = 13) Children (n = 8) Adults (n = 5) Clinical data Male/Female 9/4 6/2 3/2 Situs viscerum inversus (%) 61 75 40 Age at study entry (yrs) 15.2 (10.4–29.3) 13.1 (10.4–17.7) 23.8 (20.9–29.3) Age at onset of respiratory symptoms (yrs) 0.1 (0.1–4) 0.1 (0.1–4) 0.1 (0.1–1) Presenting respiratory symptoms Chronic cough (%) 46 50 40 Pneumonia (%) 31 12.5 60 Persistent wheezing (%) 8 12.5 0 Neonatal respiratory distress (%) 15 25 0 Age at PCD diagnosis (yrs) 7.3 (0.1–17.1) 7 (0.3–15.5) 10.1 (0.1–17.1) Exhaled nitric oxide (ppb) 4.3 (1.5–8.2) 5 (2.6–8.2) 3.9 (1.5–6.9) Nasal nitric oxide (ppb) 15.8 (2.6–34.3) 12 (4.4–34.3) 16.9 (2.6–30.1) Atopy (%) 23 25 20 Lobectomy or segmentectomy (%) 31 12 60 Values in parentheses are ranges.

stress (%) 15 25 0 Age at PCD diagnosis (yrs) 7.3 (0.1–17.1) 7 (0.3–15.5) 10.1 (0.1–17.1) Exhaled nitric oxide (ppb) 4.3 (1.5–8.2) 5 (2.6–8.2) 3.9 (1.5–6.9) Nasal nitric oxide (ppb) 15.8 (2.6–34.3) 12 (4.4–34.3) 16.9 (2.6–30.1) Atopy (%) 23 25 20 Lobectomy or segmentectomy (%) 31 12 60 Values in parentheses are ranges. Patients underwent chest HRCT for clinical reasons, namely persistence of chronic cough and/or focal abnormality at chest X-ray unresponsive to medical treatment, and/or discrepancy between lung function or clinical status and chest X-ray. In all patients chest MRI and HRCT were performed on the same day. The ethics review board of the Medical School, University of Naples Federico II, Naples, Italy, approved the study, and informed, written consent was obtained from the parent/legal guardian of each child and from adult patients.

Patients underwent chest HRCT for clinical reasons, namely persistence of chronic cough and/or focal abnormality at chest X-ray unresponsive to medical treatment, and/or discrepancy between lung function or clinical status and chest X-ray. In all patients chest MRI and HRCT were performed on the same day. The ethics review board of the Medical School, University of Naples Federico II, Naples, Italy, approved the study, and informed, written consent was obtained from the parent/legal guardian of each child and from adult patients. HRCT scanning The HRCT scan was performed with a 4-slice CT scanner (Aquilion, Toshiba, Japan) and a bodyweight adapted protocol (adults: 120 kV, 140 mAs; children over 45 kg: 120 kV, 65 mAs; children over 35 kg: 120 kV, 45 mAs; children below 35 kg: 120 kV, 30 mAs), with 1 × 4 mm collimation, 10 mm gap, 0.5 sec rotation time, automatic exposure control, multiple inspiratory breath holds of 3 sec each, with the patient in a supine position. The field of view of each sequence was patient-adapted. Images were reconstructed using a high-resolution algorithm. The total time for acquisition of the images was about 5 minutes, including positioning of the patient. Contrast medium was not administered. A lung window setting (+1500/-500 Hounsfield unit) was used for image analysis.

of each sequence was patient-adapted. Images were reconstructed using a high-resolution algorithm. The total time for acquisition of the images was about 5 minutes, including positioning of the patient. Contrast medium was not administered. A lung window setting (+1500/-500 Hounsfield unit) was used for image analysis. MR scanning MRI was performed with a 3T MR scanner (Magnetom Trio, Siemens Erlangen, Germany). We used a dedicated 12-element integrated matrix coil system, covering the whole thorax, for signal reception. It consisted of one anterior and one posterior flexible phased-array coil, each containing a set of six receiver elements. We applied the following sequences: 1) a half-Fourier single-shot turbo spin-echo (HASTE) sequence; 2) true-Fast Imaging with Steady Precession (true-FISP or TRUFI); and 3) volume-interpolated breath-hold examination (VIBE) sequence before and after (25, 60, and 180 sec) injection of contrast medium (0.1 mmol/kg gadopentetate dimeglumine). The field of view of each sequence was patient-adapted. HASTE and true-FISP/TRUFI sequences were performed using an electrocardiograph-gating to reduce cardiac motion artefacts, and respiratory-gating by a navigator signal that monitored the diaphragm position. Sequence parameters were: 1) HASTE: repetition time/echo time/flip angle, infinite/92 ms/150°; parallel acquisition factor, 2; slice thickness, 5 mm; distance factor, 20%; transversal (matrix, 380 × 256) and coronal (matrix, 400 × 320) orientation; acquisition time, approximately 90 sec; 2) True-FISP/TRUFI: repetition time/echo time/flip angle, 364.8–477.8 ms/1.2–4.3 ms/41–52°; parallel acquisition factor, 2; slice thickness, 5 mm; distance factor, 20%; transversal, coronal, and sagittal orientation (matrix, 380–480 × 320); acquisition time, 14–22 sec; 3) VIBE: repetition time/echo time/flip angle, 3.3 ms/1.2 ms/11.5°; parallel acquisition factor, 2; slice thickness, 3 mm; distance factor, 20%; transversal orientation (matrix, 350 × 256); acquisition time, 19 sec approximately.

l, coronal, and sagittal orientation (matrix, 380–480 × 320); acquisition time, 14–22 sec; 3) VIBE: repetition time/echo time/flip angle, 3.3 ms/1.2 ms/11.5°; parallel acquisition factor, 2; slice thickness, 3 mm; distance factor, 20%; transversal orientation (matrix, 350 × 256); acquisition time, 19 sec approximately. No patient required sedation. Door-to-door time was approximately 15 min. All HRCT and MRI studies were of diagnostic quality and were well tolerated.

l, coronal, and sagittal orientation (matrix, 380–480 × 320); acquisition time, 14–22 sec; 3) VIBE: repetition time/echo time/flip angle, 3.3 ms/1.2 ms/11.5°; parallel acquisition factor, 2; slice thickness, 3 mm; distance factor, 20%; transversal orientation (matrix, 350 × 256); acquisition time, 19 sec approximately. No patient required sedation. Door-to-door time was approximately 15 min. All HRCT and MRI studies were of diagnostic quality and were well tolerated. Image evaluation All identifying information was removed from the scans. To compare chest MRI and HRCT results, we scored HRCT and MR scans using a modified version of the scoring system developed by Helbich et al. for CF [22]. The original Helbich score includes severity and extent of bronchiectasis, severity of peribronchial wall thickening, extent of mucous plugging, generation of bronchial divisions involved by bronchiectasis or plugging, extent of sacculations or abscesses, severity of bullae, severity of emphysema, severity of collapse or consolidation, and severity of mosaic perfusion [22]. We excluded the severity of mosaic perfusion from imaging evaluation because it cannot be assessed by morphological MRI [16]. Therefore, the maximum total score was 25 points and not 27 (Table 2). We evaluated HRCT and MRI using the modified Helbich CT score as follows: 1) for the categories "severity of bronchiectasis" and "severity of peribronchial wall thickening", we recorded the most prevalent degree of severity; 2) it was not possible to assess peribronchial wall thickening in the presence of mucous plugging; 3) hyperintensity on HASTE images had to be present for an MRI diagnosis of mucous plugging; 4) if mucous plugging was seen within the periphery of a lung segment, bronchiectasis was scored also in that segment; 5) sacculations/abscesses were defined as circular structures with a minimum diameter of 1.5 cm that were air-filled or showed an air-fluid level; 6) a size of 2 cm was required for a diagnosis of collapse/consolidation; 7) emphysema was defined as an area of decreased signal (compared with the surrounding lung parenchyma) due to a reduction of vessel and parenchymal density; and 8) in case of lobectomy or segmentectomy, the maximum scores for "severity of bronchiectasis" and "severity of collapse/consolidation" were arbitrarily assigned to the missing lobe/segments. The assessment of "extent of bronchiectasis" took into account the number of missing segments.

essel and parenchymal density; and 8) in case of lobectomy or segmentectomy, the maximum scores for "severity of bronchiectasis" and "severity of collapse/consolidation" were arbitrarily assigned to the missing lobe/segments. The assessment of "extent of bronchiectasis" took into account the number of missing segments. Table 2 Modified Helbich scoring system for HRCT and MRI Score Category 0 1 2 3 Severity of bronchiectasis Absent Mild (lumen slightly greater than diameter of adjacent blood vessel) Moderate (lumen 2 to 3 times the diameter of the vessel) Severe (lumen > 3 times the diameter of the vessel) Severity of peribronchial wall thickening Absent Mild (wall thickness equal to diameter of adjacent vessel) Moderate (wall thickness greater than and up to twice the diameter of adjacent vessel) Severe (wall thickness more than twice the diameter of adjacent vessel) Extent of bronchiectasis Absent 1–5# 6–9# > 9# Extent of mucous plugging Absent 1–5# 6–9# > 9# Extent of sacculations or abscesses Absent 1–5# 6–9# > 9# Generation of bronchial divisions involved (bronchiectasis or plugging) Absent Up to the 4th generation Up to the 5th generation Up to the 6th generation and distal Severity of bullae Absent Unilateral (not > 4) Bilateral (not > 4) > 4 Severity of emphysema Absent 1–5# > 5# Not applicable Severity of collapse or consolidation Absent Subsegmental Segmental or lobar Not applicable # Numbers of bronchopulmonary segments.

on Up to the 5th generation Up to the 6th generation and distal Severity of bullae Absent Unilateral (not > 4) Bilateral (not > 4) > 4 Severity of emphysema Absent 1–5# > 5# Not applicable Severity of collapse or consolidation Absent Subsegmental Segmental or lobar Not applicable # Numbers of bronchopulmonary segments. Six lobes were examined; the lingula was scored separately. In patients with situs viscerum inversus, the right lung was the lung in which the middle lobar bronchus and the corresponding middle lobe were identified at scans. The images were evaluated in consensus by two experienced observers (one radiologist with an 8-years experience and one paediatric pulmonologist with a 15-years experience in HRCT and MRI evaluation). Raters were blinded to the patients' history and to any clinical data that could have biased their interpretation of the images. Disagreement between the two observers occurred in only one case, and then the debated abnormality was scored by the most trained rater. MRI and HRCT scans were presented to the raters in a random, independent order. HRCT scans were scored 8 weeks after the MR images, so that the HRCT findings would not influence the raters' judgments of the MRI findings. MRI scores resulted from the combined evaluation of the three image sets (HASTE, True-FISP/TRUFI, and VIBE), because the information provided by each set is complementary to that deriving from the others.

weeks after the MR images, so that the HRCT findings would not influence the raters' judgments of the MRI findings. MRI scores resulted from the combined evaluation of the three image sets (HASTE, True-FISP/TRUFI, and VIBE), because the information provided by each set is complementary to that deriving from the others. Lung function and microbiological evaluation Forced vital capacity (FVC) and forced expiratory volume at 1 second (FEV1) were measured, on the same day as chest imaging, with spirometry according to ATS criteria [23]. A FEV1 > 85% predicted was considered normal. Deep throat or sputum cultures were also obtained in all patients at the time of HRCT and MRI evaluation. Statistical analysis Results are expressed as median and range values. Spearman's rank correlation coefficient (rho) assessed correlations among the variables and agreement between HRCT and MRI scores. A coefficient of > 0.8 represents good agreement. A two-sided p ≤ 0.05 was significant. Data were analyzed with SPSS-PC, release 13.0, SPSS Inc. (Chicago, IL). Results Bronchiectasis, peribronchial wall thickening, mucous plugging and collapse/consolidation were the most frequent lung changes at HRCT and at MRI in the entire study population, and in children and adults (Table 3). MRI failed to detect bullae in two patients, but identified mucous plugging more frequently than HRCT. There were no other differences in the prevalences of lung abnormalities identified by the two techniques. Table 3 Prevalence of abnormalities at HRCT and MRI in the study population HRCT MRI

Results Bronchiectasis, peribronchial wall thickening, mucous plugging and collapse/consolidation were the most frequent lung changes at HRCT and at MRI in the entire study population, and in children and adults (Table 3). MRI failed to detect bullae in two patients, but identified mucous plugging more frequently than HRCT. There were no other differences in the prevalences of lung abnormalities identified by the two techniques. Table 3 Prevalence of abnormalities at HRCT and MRI in the study population HRCT MRI All (n = 13) Children (n = 8) Adults (n = 5) All (n = 13) Children (n = 8) Adults (n = 5) Bronchiectasis (%) 92 87 100 92 87 100 Peribronchial wall thickening (%) 100 100 100 100 100 100 Mucous plugging (%) 92 87 100 100 100 100 Sacculations or abscesses (%) 8 0 20 8 0 20 Bullae (%) 15 12 20 0 0 0 Emphysema (%) 15 12 20 15 12 20 Collapse or consolidation (%) 92 87 100 92 87 100 Median HRCT and MRI scores are summarized in Table 4. Since only one or two patients had sacculations/abscesses, bullae or emphysema, agreement between HRCT and MRI scores was not calculated for these categories. Moreover, agreement for generation of bronchial divisions involved by bronchiectasis or plugging was not computable because of the constant value assigned to this category at HRCT and at MRI in all the subjects. However, the same score for generation of bronchial divisions involved by the above mentioned changes was attributed in each patient to both HRCT and MRI scans. For all the other categories, agreement between the two techniques was good or excellent. Figure 1 shows the excellent correlation (r = 0.95) between HRCT and MRI total scores.

score for generation of bronchial divisions involved by the above mentioned changes was attributed in each patient to both HRCT and MRI scans. For all the other categories, agreement between the two techniques was good or excellent. Figure 1 shows the excellent correlation (r = 0.95) between HRCT and MRI total scores. Table 4 Median HRCT and MRI scores of the whole study population and their agreement HRCT MRI r Severity of bronchiectasis 2 (0–3) 2 (0–3) 0.87 Severity of peribronchial wall thickening 2 (1–2) 2 (0–2) 1 Extent of bronchiectasis 2 (0–3) 2 (0–3) 0.97 Extent of mucous plugging 2 (0–3) 2 (1–3) 0.94 Extent of sacculations or abscesses 0 (0–1) 0 (0–1) NA Generation of bronchial divisions involved (bronchiectasis or plugging) 3* 3* NA Severity of bullae 0 (0–3) 0* NA Severity of emphysema 0 (0–1) 0 (0–1) NA Severity of collapse or consolidation 2 (0–2) 2 (0–2) 1 Total score 12 (6–20) 12 (5–17) 0.95 NA: not applicable (see text). Values in parentheses are ranges. * No range values are given because all patients had the same score. For generation of bronchial divisions involved by bronchiectasis or plugging, the same score was attributed in each patient to both HRCT and MRI scans. Figure 1 Agreement between MRI and HRCT total scores in the whole population. Twelve points are shown because of the overlap of one value.

* No range values are given because all patients had the same score. For generation of bronchial divisions involved by bronchiectasis or plugging, the same score was attributed in each patient to both HRCT and MRI scans. Figure 1 Agreement between MRI and HRCT total scores in the whole population. Twelve points are shown because of the overlap of one value. Median FVC and FEV1 were 96% predicted (range, 57–121) and 85% predicted (range, 47–108), respectively. Children had higher values than adults for both FVC (96% predicted [range, 82–120] versus 80% predicted [range, 57–121], respectively) and FEV1 (89% predicted [range, 72–108] versus 60% predicted [range, 47–103], respectively). Total HRCT and MRI scores were significantly related to FVC (r = -0.5, p = 0.05; and r = -0.7, p = 0.009, respectively) and FEV1 (r = -0.6, p = 0.03; and r = -0.7, p = 0.009, respectively). Fifty-four percent of patients had a normal FEV1. The most common pathogen at deep throat or sputum culture was Haemophilus influenzae in both adults and children (60% and 50%, respectively). Pseudomonas aeruginosa was isolated only in one children and in one adult, whereas Staphylococcus aureus was found in one boy. In three cases no pathogens were cultured. Figure 2 shows an area of consolidation at HRCT (panel A) and MRI (panel B) scans of a boy with PCD and situs viscerum inversus (Kartagener syndrome). Figure 2 Transversal CT image (A) and transversal MRI HASTE (B) sequence of a 10.4-year-old boy with Kartagener syndrome. The scans demonstrate an area of consolidation in the middle lobe.

Figure 2 shows an area of consolidation at HRCT (panel A) and MRI (panel B) scans of a boy with PCD and situs viscerum inversus (Kartagener syndrome). Figure 2 Transversal CT image (A) and transversal MRI HASTE (B) sequence of a 10.4-year-old boy with Kartagener syndrome. The scans demonstrate an area of consolidation in the middle lobe. Discussion In this study, we compared the efficacy of chest HRCT and high-field MRI in the assessment of the severity and extent of lung abnormalities in children and young adults with PCD. The agreement between the two techniques was good or excellent for most of the abnormalities considered. Bronchiectasis, peribronchial wall thickening, mucous plugging, and collapse/consolidation were the most frequent abnormalities. HRCT and MRI total scores were significantly related to PFTs.

d young adults with PCD. The agreement between the two techniques was good or excellent for most of the abnormalities considered. Bronchiectasis, peribronchial wall thickening, mucous plugging, and collapse/consolidation were the most frequent abnormalities. HRCT and MRI total scores were significantly related to PFTs. At present, the management of patients with PCD is based on clinical assessment and regular sputum cultures combined with pulmonary function evaluation [3]. Pulmonary function measurement is generally used to assess lung disease in PCD. It has been previously reported that lung function assessment, and particularly static lung volumes evaluation, may provide suggestive information about peripheral airway disease and its evolution over time [3,21,24-26]. However, PFTs are relatively insensitive markers of early disease and fail to detect regional structural changes because these tools reflect the function of the lung as a whole and gives no information about localized abnormalities. Chest CT provides a more accurate picture of the type, distribution and nature of lung changes than either PFTs or chest radiography [4,27]. In fact, we recently found that 40% of PCD patients had a normal FEV1 despite CT evidence of significant structural lung damage [18]. Our MRI results reported herein confirm this finding in that pulmonary abnormalities were easily recognized also in patients with normal lung function.

PFTs or chest radiography [4,27]. In fact, we recently found that 40% of PCD patients had a normal FEV1 despite CT evidence of significant structural lung damage [18]. Our MRI results reported herein confirm this finding in that pulmonary abnormalities were easily recognized also in patients with normal lung function. Chest CT has the substantial advantage of revealing early and/or localized structural changes [14,28]. Nevertheless, despite its potential benefits, CT is not regularly used in PCD, and there is no consensus as to when it is best obtained [3]. However, although the outcome of PCD is not universally unfavourable, complications of bronchiectasis and severe pulmonary impairment become more severe with age [21]. Early assessment of structural lung changes is then crucial in PCD, especially in candidates for lobectomy and/or lung transplantation [29]. CT has been criticized for its ionizing radiation burden and the possible consequences of cumulative doses, particularly when used for frequent follow-up examinations in patients with chronic lung diseases, in pregnancy, or in children [6,7]. Several surveys on the effects of repeated CT examinations have demonstrated that lifetime cancer risks are cumulative and not negligible, even though the consequences of low levels of exposure have not been clearly elucidated [30,31]. It has been hypothesized that, at low doses, there is a linear dose-response relationship between the exposure to ionizing radiation and the development of solid cancers in humans [32]. It is unlikely that there is a threshold below which cancers are not induced, but at low doses the number of radiation-induced cancers might be small. In addition to this, other health end-points have been linked to radiation exposure. In particular, statistically significant associations have been found with heart diseases, stroke, and diseases of the digestive, respiratory, and hematopoietic systems [32]. In this scenario, a sensitive technique that could assess PCD lung disease without ionizing radiation is highly desirable.

to radiation exposure. In particular, statistically significant associations have been found with heart diseases, stroke, and diseases of the digestive, respiratory, and hematopoietic systems [32]. In this scenario, a sensitive technique that could assess PCD lung disease without ionizing radiation is highly desirable. To determine the clinical value of chest MRI scanning and its potential as a surrogate outcome measure in PCD, we assessed the comparability of MR and HRCT images using a quantitative scoring system, i.e. a modified Helbich score [16]. Although the Helbich score was devised for the CF setting [22], we used it because there is no scoring system specific for PCD and because we found it to be easily adaptable to MRI. Here we show that in PCD patients the results of chest MRI coincide with chest CT findings. Indeed, we found an excellent agreement between HRCT and MRI for most of the categories of the scoring system, thereby supporting the concept that MRI might be an alternative, radiation-free method for pulmonary assessment in PCD as well as in CF [15-17]. This issue is particularly relevant in PCD because of the presumed long life expectancy of these patients.

tween HRCT and MRI for most of the categories of the scoring system, thereby supporting the concept that MRI might be an alternative, radiation-free method for pulmonary assessment in PCD as well as in CF [15-17]. This issue is particularly relevant in PCD because of the presumed long life expectancy of these patients. Chest MRI has several advantages over CT. First, being radiation-free, it can be repeated (e.g., in case of artifacts), and is thus suitable for long-term disease monitoring. Second, MRI identifies various characteristics of lung tissue, allows a precise characterization of the lesion and assesses function, e.g. lung perfusion and/or ventilation, and respiratory mechanics [14]. Thus, structural information and functional data are obtained in a single examination, thereby reducing imaging costs and increasing the patient's compliance [33,34]. Finally, unlike CT, chest MRI easily distinguishes between mucous plugging and bronchial wall thickening even in the peripheral airways [15]. This finding is relevant because peripheral mucous plugging is a sensitive marker of early small airway disease in PCD [18]. None of these features is found (or is only partly found) in spirometry and other surrogate outcome measures, including CT.

ging and bronchial wall thickening even in the peripheral airways [15]. This finding is relevant because peripheral mucous plugging is a sensitive marker of early small airway disease in PCD [18]. None of these features is found (or is only partly found) in spirometry and other surrogate outcome measures, including CT. On the other hand, the spatial resolution of MRI is lower than that of CT and slight morphological changes such as peripheral bronchiectasis without bronchial wall thickening are not consistently visualized by MRI [15]. However, this did not seem to affect our findings as shown by the comparable HRCT and MRI scores. Other potential drawbacks of MRI are limited access to the technology, long acquisition times and high costs. Long acquisition times represented a concern in our study, because our population included children who were unable to hold their breath for a long time, despite training. Therefore, we used respiratory-gating to overcome the lack of cooperation in children, and applied it also to adults for the sake of uniformity. We believe that the benefits of MRI outweigh the greater discomfort for the patient and the higher costs. Furthermore, technical progress will hopefully lead to lower costs, shorter examination times and higher image resolution.

lack of cooperation in children, and applied it also to adults for the sake of uniformity. We believe that the benefits of MRI outweigh the greater discomfort for the patient and the higher costs. Furthermore, technical progress will hopefully lead to lower costs, shorter examination times and higher image resolution. To our knowledge, this is the first time that 3T MRI has been used for the quantitative analysis of pulmonary abnormalities in children and adults with PCD. Moreover, this study is the first to compare typical PCD findings at chest CT with MRI. It is also the first evaluation of chest high-field 3T morphological MRI in patients with lung disorders due to abnormal mucociliary clearance. Although a field strength of 1.5T is generally used in clinical practice, MR whole-body units operating at field strengths of 3T and beyond are increasingly being installed in research institutions and in clinical facilities [35-37]. The scanner we used has several advantages over 1.5T: 1) it has a high-speed and a high-strength gradient system; 2) it is equipped with multiple phased-array coils and receiver channels; and 3) it has acquisition acceleration techniques, such as parallel imaging. Parallel acquisition techniques improve image quality by shortening the echo times of single-shot sequences [38,39]. Despite the high-field used, shorter echo times result in decreased blurring artifacts and less signal decay caused by T2* effects. Another advantage of the system we used is that cardiac- and respiratory-gating reduce artifacts due to heart/great vessels/chest wall motion thereby overcoming the need for sedation even in poorly cooperating subjects. Thus, patient's discomfort was less with this system. However, our study is limited by the fact that CT scans were performed for clinical purposes, images were read in consensus, functional MRI data were not provided, infants were not included, and longitudinal data were not obtained. In addition to this, MRI failed to detect bullae in two of our patients. In both of them these lesions were small in size, and it has been previously reported that small pulmonary bullae might pose a problem to chest MRI [40]. However, the prevalence of bullae in our study population was very low, and other authors failed to detect them at HRCT in larger PCD populations [19,20].

r patients. In both of them these lesions were small in size, and it has been previously reported that small pulmonary bullae might pose a problem to chest MRI [40]. However, the prevalence of bullae in our study population was very low, and other authors failed to detect them at HRCT in larger PCD populations [19,20]. Given the very low prevalence of bullae in PCD and the small size of the lesions found in our patients, the failure in the detection of bullae unlikely entails a significant clinical impact. Nevertheless, further studies on chest MRI in larger PCD populations are needed. Conclusion Chest high-field 3T MRI appears to be as effective as HRCT in the assessment of extent and severity of PCD lung disease. Therefore, it might be considered a reliable radiation-free option to HRCT in PCD and be proposed for follow-up examinations. This non-ionizing radiation technique could prove to be a useful tool in monitoring lung disease, guiding clinical management and assessing the consequences of therapy in PCD as well as in other pulmonary disorders in children, adults and the elderly. Abbreviations PCD: Primary ciliary dyskinesia; CT: Computed tomography; HRCT: High-resolution computed tomography; MRI: Magnetic resonance imaging; CF: Cystic fibrosis; PFT: Pulmonary function test; LM: Light microscopy; EM: Electron microscopy; HASTE: Half-Fourier single-shot turbo spin-echo; True-FISP/TRUFI: True-Fast Imaging with Steady Precession; VIBE: Volume-interpolated breath-hold examination; FVC: Forced vital capacity; FEV1: Forced expiratory volume at 1 second.

ic fibrosis; PFT: Pulmonary function test; LM: Light microscopy; EM: Electron microscopy; HASTE: Half-Fourier single-shot turbo spin-echo; True-FISP/TRUFI: True-Fast Imaging with Steady Precession; VIBE: Volume-interpolated breath-hold examination; FVC: Forced vital capacity; FEV1: Forced expiratory volume at 1 second. Competing interests All authors declare that they have not been funded and they disclose any involvement with organisation(s) with financial interest in the subject matter or materials discussed in the submitted manuscript. All authors declare that they have no potential conflict of interest, real or perceived. Authors' contributions SM participated in the design of the study, performed the statistical analysis, and drafted the manuscript. FS conceived of the study, participated in its design and coordination, and scored HRCT and MR images. MS conceived of the study and participated in its design. MM and PI participated in the collection of data and helped to draft the manuscript. MMDS participated in the design of the study and performed the electron microscopy analysis of cilia ultrastructure. CM carried out the imaging studies, scored the HRCT and MR images, and helped to draft the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Cilia ultrastructure at EM and motion pattern at LM of the study population. The data provided in the table derive from individual cilia ultrastructural analysis at EM and evaluation of ciliary motion pattern at LM. Click here for file

Authors' contributions SM participated in the design of the study, performed the statistical analysis, and drafted the manuscript. FS conceived of the study, participated in its design and coordination, and scored HRCT and MR images. MS conceived of the study and participated in its design. MM and PI participated in the collection of data and helped to draft the manuscript. MMDS participated in the design of the study and performed the electron microscopy analysis of cilia ultrastructure. CM carried out the imaging studies, scored the HRCT and MR images, and helped to draft the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Cilia ultrastructure at EM and motion pattern at LM of the study population. The data provided in the table derive from individual cilia ultrastructural analysis at EM and evaluation of ciliary motion pattern at LM. Click here for file Acknowledgements The authors are grateful to Jean Ann Gilder for text editing.

Introduction Acute lympphoblastic leukaemia accounts for around 77% of childhood leukemia. Hemophilia is a congenital bleeding disorder characterized by deficiency of coagulation factor VIII or IX out of which 85% is due to factor VIII deficiency and 15% due to factor IX deficiency. The coincidence of these two diseases together is rare which has led to challenges in developing treatment strategies. Case Report 12 yr old boy known case of hemophilia (factor VIII deficiency) diagnosed at 6 month of age presented with intermittent fever (on and off) of 2 month without chills and rigors, significant weight loss of 3 kilograms in 1 one month, generalized weakness of 1 month and joint swelling right knee of 7 days duration. The swelling was spontaneous associated with restriction of movements. There was no family history of malignancy or bleeding disorders.

and off) of 2 month without chills and rigors, significant weight loss of 3 kilograms in 1 one month, generalized weakness of 1 month and joint swelling right knee of 7 days duration. The swelling was spontaneous associated with restriction of movements. There was no family history of malignancy or bleeding disorders. On examination, the anthropometric measurements were appropriate for age. There was pallor, petechiae and ecchymoses on his legs, along with gingival bleeding, and haematoma on right knee. Systemic examination revealed hepatosplenomegaly. Peripheral blood counts revealed hemoglobin levels of 7.6 g%, white cell counts of 41 × 109/L (blasts 85%), and platelet counts of 100 × 109/L. After receiving 40 U/kg FVIII concentrates, bone marrow aspiration was performed, which showed lymphoblast of 85%. His immunophenotype consisted of CD3 87%, CD7 99%, CD8 10%, CD5 3%, CD10 98%, CD19 18%, CD22 20% and HLA DR negative The cells were negative with MPO staining. RT-PCR for BCR abl negative. A hepatitis viruses screening test found the patient to be hepatitis B and C negative. HIV serological test was negative and cytogenetic test revealed 46, XY. Coagulation screening showed a prolonged activated partial thromboplastin time (APTT) (test 46 seconds, control 28 seconds), with normal prothrombin time. The ultrasound scan of abdomen revealed hepatosplenomegaly, Echocardiography and Xray chest was normal.

gical test was negative and cytogenetic test revealed 46, XY. Coagulation screening showed a prolonged activated partial thromboplastin time (APTT) (test 46 seconds, control 28 seconds), with normal prothrombin time. The ultrasound scan of abdomen revealed hepatosplenomegaly, Echocardiography and Xray chest was normal. After admission to our center, the patient received 30 U/kg of factor VIII. The swelling and pain in his right knee subsided. The patient was placed on induction therapy on the second day, which included oral prednisolone, intrathecal methotrexate, vincristine, daunorubicin. There was no CNS involvement. He was given treatment as per CCG guidelines(Children Cancer Group) which includes 4 weeks of induction, 8 weeks of consolidation, 8 weeks of interim maintenance cycle I (8 weeks), delayed intensification I (8 weeks), delayed intensification II (8 weeks) and maintenance period of 12 weeks. The patient was given intrathecal methotrexate under the cover of factor VIII. During the induction phase there was period of febrile neutropenia, thrombocytopenia and swelling of right knee joint which was managed with antibiotics, platelet transfusion and factor VIII. His peripheral blood counts gradually returned to normal during the treatment, and the patient did not have bleeding complications subsequently. After twenty-eight days of treatment, the patient achieved complete remission. The bone marrow done revealed blasts cells of less than 5% which was consistent with remission. He was discharged from the hospital on the 31st day of induction therapy. He has remained in good condition since then.

mplications subsequently. After twenty-eight days of treatment, the patient achieved complete remission. The bone marrow done revealed blasts cells of less than 5% which was consistent with remission. He was discharged from the hospital on the 31st day of induction therapy. He has remained in good condition since then. Discussion In literature, 11 cases of hemophilia with acute leukemia have been documented [1-5] with only 5 pediatric patients. Other authors have reported an increased risk of lymphoma in hemophilia patients, mainly among HIV positive patients [6,7]. Further it has also been observed that hemophiliac patients have long-term abnormalities of immune function from factor concentrate usage [8-10]. Thus, probable factor contributing to a high risk of lymphocytic malignancy could be immunological dysfunction caused by factor concentrate use and/or HIV. However, as of now, only 5 hemophilia patients with acute non-lymphocytic leukemia have been described. It is likely that the association of these two disorders is merely accidental, although further confirmation is needed to bring about more facts under the scanner.

unction caused by factor concentrate use and/or HIV. However, as of now, only 5 hemophilia patients with acute non-lymphocytic leukemia have been described. It is likely that the association of these two disorders is merely accidental, although further confirmation is needed to bring about more facts under the scanner. The coincidence of leukemia and hemophilia led to a challenge in developing a treatment strategy. Thrombocytopenia, a complication associated with chemotherapy, can exacerbate bleeding diathesis in severe hemophilia patients and may result in life-threatening bleeding, therefore the threshold for platelet transfusion is kept high. The use of blood products including plasma-derived Factor VIII and platelets may be associated with a risk of virus transmission, in addition to increasing the cost of treatment, leading to a financial burden on the patient and his family. Conclusion The coincidence of hemophilia and leukemia is rare, more so in pediatric age group. This is the sixth case in the literature, which is required to be mentioned for its rarity. The mechanism leading to the combination of these diseases needs further investigation. While both hemophilia and leukemia are severe blood diseases, proper treatment can bring about favorable results. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. Competing interests The authors declare that they have no competing interests.

Conclusion The coincidence of hemophilia and leukemia is rare, more so in pediatric age group. This is the sixth case in the literature, which is required to be mentioned for its rarity. The mechanism leading to the combination of these diseases needs further investigation. While both hemophilia and leukemia are severe blood diseases, proper treatment can bring about favorable results. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. Competing interests The authors declare that they have no competing interests. Authors' contributions RS: Manuscript preparation. KS: Evaluation and editing of the manuscript. BJ: Evaluation and editing of the manuscript. DS: Evaluation and editing of the manuscript. All authors read and approved the final manuscript.

Introduction A recent scientific statement from the American Heart Association and other related Committees focussed the topic of the Metabolic Syndrome (MetS) in pediatric age [1]. The paper provided a set of fundamental questions about what the pediatric MetS means in clinical or research setting. The Authors concluded defining limits of our current knowledge and providing suggestions for needed future research [1]. This position paper outlined most of the concerns that pediatricians feel about the usefulness of MetS diagnosis in day by day clinical practice. Concerns also are referred to which MetS definition has to be used for children. Controversies are mainly related to two diverging approaches: one adapting the definition of MetS from adults [2-4], the other considering the peculiarities of children and adolescents [5,6]. Aim of the present paper is to discuss difficulties found by pediatricians facing to MetS definition and usefulness.

Introduction A recent scientific statement from the American Heart Association and other related Committees focussed the topic of the Metabolic Syndrome (MetS) in pediatric age [1]. The paper provided a set of fundamental questions about what the pediatric MetS means in clinical or research setting. The Authors concluded defining limits of our current knowledge and providing suggestions for needed future research [1]. This position paper outlined most of the concerns that pediatricians feel about the usefulness of MetS diagnosis in day by day clinical practice. Concerns also are referred to which MetS definition has to be used for children. Controversies are mainly related to two diverging approaches: one adapting the definition of MetS from adults [2-4], the other considering the peculiarities of children and adolescents [5,6]. Aim of the present paper is to discuss difficulties found by pediatricians facing to MetS definition and usefulness. Discussion Metabolic syndrome definition Metabolic syndrome is the clustering of specific metabolic abnormalities found in overweight and obese subjects, but present also in some normal weight subjects. In adults, the presence of three among clinical (obesity or abdominal obesity, hypertension) and metabolic parameters (hyperglycemia, high triglycerides, low HDL-cholesterol) is used to define MetS [2-4]. In the last decade, many studies in paediatrics derived MetS definition from those used for adults, mostly adapting cut-off points for each parameter to children or adolescents. For this purpose, the percentile methodology was generally used. Recently, the International Diabetes Federation (IDF) proposed a new pediatric definition according to age groups [7]. In particular, IDF suggested that the MetS should not be diagnosed in children younger than 10 yrs, while for subjects 10 to 16 years they proposed the use of adult IDF MetS definition [3] with the only difference represented by waist 90th percentile instead of absolute values. Therefore, the IDF defined a 10-16 yrs old subject as having the MetS if waist circumference was ≥ the 90th percentile and if two other of the following items were above or under a single cut-point: triglycerides ≥ 150 mg/dl, HDL < 40 mg/dl, glucose ≥ 100 mg/dl, systolic or diastolic blood pressure (BP) ≥ 130 or ≥ 85 mmHg, respectively [7].

ined a 10-16 yrs old subject as having the MetS if waist circumference was ≥ the 90th percentile and if two other of the following items were above or under a single cut-point: triglycerides ≥ 150 mg/dl, HDL < 40 mg/dl, glucose ≥ 100 mg/dl, systolic or diastolic blood pressure (BP) ≥ 130 or ≥ 85 mmHg, respectively [7]. Despite the pediatric IDF definition is becoming to be used in epidemiological studies [8,9], pediatricians face these proposed new limits. Glucose cut-point corresponds to that proposed by the American Diabetes Association few years ago with a general agreement [10], as well as the 90th waist circumference percentile is a worldwide considered and accepted limit [11]. On the contrary, the use of single cut-points for high triglycerides and low HDL, independently from the known age- and gender-related variations in this age group, is particularly striking due to the pubertal impact on lipid parameters [12].

t circumference percentile is a worldwide considered and accepted limit [11]. On the contrary, the use of single cut-points for high triglycerides and low HDL, independently from the known age- and gender-related variations in this age group, is particularly striking due to the pubertal impact on lipid parameters [12]. Hypertension diagnosis in pediatric age The most discussed limit among those proposed by IDF is the single blood pressure (BP) cut-point (i.e., 130 for systolic or 85 for diastolic) instead of those related to age, gender and height. Up to now, the risk for hypertension was generally set at the 95th percentile level according to National Health Blood Pressure Education Program (NHBPEP) chart [13]. It appears that the difference between the two limits can be conspicuous, especially for younger subjects with lowest height percentiles, and therefore that a certain degree of underestimation of hypertension could derive from the application of IDF proposal. The main question is whether the pediatrician should prefer the use of single BP limit (130/85), derived from adult clinical surveys and linked to health consequences [14], instead of the use of multiple (up to 84 couple of values in this age interval) age, gender, and height-related limits [13]. However, the latter cumbersome approach seems to be justified by the known variation of BP values in pediatric subjects due to the mentioned variables. At the same time it is striking that an unique BP limit for hypertension risk could work in adult population with subjects of different ages and height ranging even from 150 to 200 cm. In other words, is there an excessive complication of hypertension diagnosis by pediatricians, or a simplification of it for adult doctors, or maybe both? The question remains open. Recently, the discussion on hypertension diagnosis in pediatrics was stimulated by the publication of a simplified table of BP values to be used for screening hypertension risk. This proposal suggested to use, for each age group and gender, the 90th percentile usually applied for shorter children (5th height percentile) [15] in order to avoid undiagnosed hypertension, an approach which seems to take the opposite direction than that of the IDF definition [7]. At the same time, a recent paper together with the accompanying editorial [16,17] rose other doubts on pediatric hypertension diagnosis, up to now based on normative values instead of on health-related ones.

gnosed hypertension, an approach which seems to take the opposite direction than that of the IDF definition [7]. At the same time, a recent paper together with the accompanying editorial [16,17] rose other doubts on pediatric hypertension diagnosis, up to now based on normative values instead of on health-related ones. In any case, until this point will be clarified by further longitudinal surveys, it seem preferable for pediatric hypertension diagnosis the use of the more prudent NHBPEP strategy instead of the IDF proposal [7].

gnosed hypertension, an approach which seems to take the opposite direction than that of the IDF definition [7]. At the same time, a recent paper together with the accompanying editorial [16,17] rose other doubts on pediatric hypertension diagnosis, up to now based on normative values instead of on health-related ones. In any case, until this point will be clarified by further longitudinal surveys, it seem preferable for pediatric hypertension diagnosis the use of the more prudent NHBPEP strategy instead of the IDF proposal [7]. Metabolic Syndrome usefulness in clinical practice In Table 1 we show comparison between 2 obese boys 12 years old with similar excessive BMI and increased waist circumference. The diagnosis of MetS can be done only in the case #1 according to the pediatric definition [7], while in the case #2 we can found the presence of only 2 abnormal parameters. Nevertheless, if we look to other variables not included in the set for MetS, we note that the case #2 shows a clustering of both familial and individual factors which can be considered closely associated with metabolic risk. Recently it was demonstrated the relevant importance of positive family history for type 2 diabetes and other CVD-related diseases in the prediction of future health and risk for MetS [18,19]. Therefore, we feel that we cannot estimate an higher risk for case #1 respect to case #2 on the basis of the presence of MetS diagnosis alone. Even in the absence of an overt metabolic pattern, as represented by the MetS, the paediatrician should extend the analysis by looking for the presence of other factors (familial or individual) which could confer to that child a potential future risk. We should also highlight the limitations of a dichotomous definition of the MetS, by preferring a continuous indicator [6,20], or the use of a multiple-items screening form [5].

d the analysis by looking for the presence of other factors (familial or individual) which could confer to that child a potential future risk. We should also highlight the limitations of a dichotomous definition of the MetS, by preferring a continuous indicator [6,20], or the use of a multiple-items screening form [5]. Table 1 Comparison between 2 cases of 12 years old boys for parameters related to metabolic syndrome and other family or individual risk factors. Case #1 Case #2 BMI* 26.5 26.5 Waist circumference** (cm) 86 85 Glucose (mg/dl) 86 85 Triglycerides (mg/dl) 142 115 HDL cholesterol (mg/dl) 38 42 Blood pressure (mmHg) 136/78 132/86 MetS diagnosis, according to [7] yes No Family history for CVD related diseases (in 1st or 2nd degree) negative DM2, hypertension, dyslipidemia Birth weight status normal small for gestational age Early adiposity rebound no yes (at 4 years of age) *BMI as obese according to Cole et al [30] ** Waist circumference values > 90th percentile according to Fernandez et al [11] Values over or under the cut-points for MetS according to Zimmet et al [7] are reported in bold character.

Birth weight status normal small for gestational age Early adiposity rebound no yes (at 4 years of age) *BMI as obese according to Cole et al [30] ** Waist circumference values > 90th percentile according to Fernandez et al [11] Values over or under the cut-points for MetS according to Zimmet et al [7] are reported in bold character. Taking into consideration MetS diagnosis, we could suggest that its presence in a child could be useful for rule-in but not for rule-out a risk for future health. In fact, there is the possibility of having a false negative estimation in those children not (yet) achieving MetS diagnosis but having other potential factors. In other words, the presence of one cardiovascular risk factor should raise suspicion that additional risk factors may also be present and encourage further investigation [21]. Moreover, it has been proposed that hepatic steatosis should be considered as an additional diagnostic criterion to be added in future approach to this problem [22].

esence of one cardiovascular risk factor should raise suspicion that additional risk factors may also be present and encourage further investigation [21]. Moreover, it has been proposed that hepatic steatosis should be considered as an additional diagnostic criterion to be added in future approach to this problem [22]. On the other hand we need to consider the clinical value of MetS diagnosis for subject's management. MetS can be considered the clustering of specific conditions and metabolic disorders, but none of them is required to be clinically severe. In fact, the diagnosis can be done by the presence of an increased abdominal adiposity together with pre-hypertension or mild lipid abnormalities, and does not necessarily imply the presence of hypertension, dyslipidemia or reduced glucose tolerance, diseases for which a specific diagnosis is already available. As a consequence of this statement, no specific treatment is strictly necessary in order to manage these findings, except for lifestyle and nutritional changes needed for a correction of excess weight. Therefore, the MetS diagnosis seems not useful for individual treatment, but it can be considered helpful for understanding the possible underlying pathogenesis and for addressing further investigations. Emphasis should be given to the presence of physical inactivity when aiming to estimate the individual metabolic risk, as an impressive mass of data are now confirming the effect of the degree of physical fitness for its prevention in childhood [23-27].

underlying pathogenesis and for addressing further investigations. Emphasis should be given to the presence of physical inactivity when aiming to estimate the individual metabolic risk, as an impressive mass of data are now confirming the effect of the degree of physical fitness for its prevention in childhood [23-27]. Finally, a certain degree of instability has been described in adolescents with MetS during a 3-year observational study in which almost half of previously affected subjects lost their condition [28]. Another recent study confirmed the MetS variability in pediatric age [29]. Conclusions We must support the need for consensus and further studies on MetS, as stated by the American Heart Association [1]. The main questions to be solved are: 1) is it really safe that a child younger than 10 years should not be diagnosed? 2) which is the clinical cost of considering a 10-16 years adolescent like an adult? 3) is it mandatory to identify cut-points related with future health risk instead of normative ones in pediatric age? Until these points will be ascertained, we strongly suggest to face the topic of MetS taking into consideration that, at present time, MetS in pediatric age suffers from important limitations (i.e., adult derived definition, possibility to rule-in but not to rule-out the individual metabolic risk, instability of MetS during adolescence, poor usefulness of the diagnosis for specific treatment).

c of MetS taking into consideration that, at present time, MetS in pediatric age suffers from important limitations (i.e., adult derived definition, possibility to rule-in but not to rule-out the individual metabolic risk, instability of MetS during adolescence, poor usefulness of the diagnosis for specific treatment). We think that this should be the best and honest position that a paediatrician can now apply to MetS in childhood, waiting for long term, longitudinal follow-up studies that could clarify the entire question. List of Abbreviations used MetS: metabolic syndrome; HDL: high density lipoprotein cholesterol; IDF: International Diabetes Federation; BP: blood pressure; NHBPEP: National Health Blood Pressure Education Program; CVD: cardiovascular disease; BMI: body mass index; DM2: type 2 diabetes; Competing interests The authors declare that they have no competing interests.

Introduction In Iran BCG sub strain Pasteur vaccine, is administered to all the newborns at birth to prevent tuberculosis. Adverse reactions induced by BCG vaccination are rare (ranging from zero to 23.8%) [1,2]. The most frequent complications are purulent regional lymphadenitis (0.9 per 1000 vaccinated children) [2]. Bone BCG infection is the second most frequent (0.39 to 46 per million vaccinated children) [3-5]. Disseminated infections are even rarer and their estimated incidence is 0.1 to 4.3 per one million vaccinated children but is lethal in 50 to 71% of the cases [6-8]. The death rate is especially higher in cases of immunodepression (83%) and it is important that a temporary or permanent immune deficiency observed in 86% of the cases [6-9]. Disseminated BCG infections have occurred in children with immunodeficiency disorders such as severe combined immunodeficiency (SCID), chronic granulomatous disease (CGD), complete Di George syndrome, AIDS and idiopathic immunodeficiency of genetic origin or mendelian susceptibility to mycobacterial disease(MSMD) with underlying genetic defects, but only rarely in apparently normal individuals[6,8-10]. The aim of this study was to determine the role of immunodeficiency disorders in exciting disseminated BCG infection.

Introduction In Iran BCG sub strain Pasteur vaccine, is administered to all the newborns at birth to prevent tuberculosis. Adverse reactions induced by BCG vaccination are rare (ranging from zero to 23.8%) [1,2]. The most frequent complications are purulent regional lymphadenitis (0.9 per 1000 vaccinated children) [2]. Bone BCG infection is the second most frequent (0.39 to 46 per million vaccinated children) [3-5]. Disseminated infections are even rarer and their estimated incidence is 0.1 to 4.3 per one million vaccinated children but is lethal in 50 to 71% of the cases [6-8]. The death rate is especially higher in cases of immunodepression (83%) and it is important that a temporary or permanent immune deficiency observed in 86% of the cases [6-9]. Disseminated BCG infections have occurred in children with immunodeficiency disorders such as severe combined immunodeficiency (SCID), chronic granulomatous disease (CGD), complete Di George syndrome, AIDS and idiopathic immunodeficiency of genetic origin or mendelian susceptibility to mycobacterial disease(MSMD) with underlying genetic defects, but only rarely in apparently normal individuals[6,8-10]. The aim of this study was to determine the role of immunodeficiency disorders in exciting disseminated BCG infection. Materials and methods This prospective study was performed through 24- month period by TB and lung disease research center of Tabriz because of in Iran all children at birth immunize with BCG vaccine. So, Children with BCG lymphadenitis and criteria as follows were referred to children's hospital from health centers by convenience sampling.

This prospective study was performed through 24- month period by TB and lung disease research center of Tabriz because of in Iran all children at birth immunize with BCG vaccine. So, Children with BCG lymphadenitis and criteria as follows were referred to children's hospital from health centers by convenience sampling. Inclusion criteria were: Lymphadenitis, Abscesses or fistula in the site of BCG vaccination or another site or BCG ulceration with 2 or more than of following such as: 1. Fever >38°c more than 2 weeks 2. Anemia (Hb<10) 3. Recurrent or persistent oral Candida 4. Organomegaly (Hepatosplenomegaly) 5. Bone diseases (Pain or arthritis) 6. Weight loss 7. Recurrent or persistent diarrhea 8. Related parents 9. Family history of immunodeficiency Then physical examination and laboratory studies were performed based on: 1. A systemic syndrome compatible with mycobacterial disease. Typical manifestations include fever, weight loss, anemia and death. 2. Evidence of infection by positive acid-fast bacilli on smears and growth of M. bovis, BCG strain at two or more anatomic sites beyond the region of vaccination. The competency of the immune system were evaluated by different tests including measurement of Igs, Isohemaglotination test, phagocyte activity by NBT (slide test), T and B cell counts by flowcytometry, delayed type hypersensitivity test (DHT). HIV performed by Elisa test. These tests were performed by standard procedures and by trained laboratory personnel in the specialized laboratories.

cluding measurement of Igs, Isohemaglotination test, phagocyte activity by NBT (slide test), T and B cell counts by flowcytometry, delayed type hypersensitivity test (DHT). HIV performed by Elisa test. These tests were performed by standard procedures and by trained laboratory personnel in the specialized laboratories. Clinical manifestations and hematological and immunologic changes were compared between two different groups of cases: Patients (who are children with disseminated BCG infection) and control group (who are children with complications of BCG vaccination but without disseminated BCG infection). Then frequency of patients with disseminated BCG infection who had immunodeficiency was reported. The children and their parents were informed about the necessary clinical and laboratory examination procedures and their consent was taken before the start of the study. Static Sings, symptoms and laboratory findings were compared between patients and control by Chi-square test and fisher exact test. Also, we used student t-test for comparation between T, B, NK cells and SPSS 14 for data analysis. Results In this survey from 122093 BCG vaccinated children through 24 month period,48 infants with BCG lymphadenitis, within range of 2-62 months (mean ± SD: 9.9 ± 9.85) were selected. 28 infants were male (58.3%) and 20 infants were female (41.7%).

Static Sings, symptoms and laboratory findings were compared between patients and control by Chi-square test and fisher exact test. Also, we used student t-test for comparation between T, B, NK cells and SPSS 14 for data analysis. Results In this survey from 122093 BCG vaccinated children through 24 month period,48 infants with BCG lymphadenitis, within range of 2-62 months (mean ± SD: 9.9 ± 9.85) were selected. 28 infants were male (58.3%) and 20 infants were female (41.7%). Onset age was between 1-21 months (mean ± SD: 6.36 ± 4.62). Twenty four cases had multiple lymphadenitis near to BCG incubation such as cervical, axiliary, and supraclaviculs, 11 cases had suppurative lymphadenitis with fistula and abscesses and 5 cases had ulcer of BCG incubation. Osteomyelitis due to BCG vaccination was detected in 2 cases and disseminated BCG infection in 11 cases (22.9%) with multi organ involvement and systemic symptoms was detected. Family history of BCG infection or immunodeficiency was positive in 22.9% of control group and the rate of consanguineous marriages in the parents of them was 37.5% (18 cases). A chi-square test comparing the clinical manifestations in children with disseminated BCG infection with control group showed statistically significant differences for the two groups of children (Table 1). Table 1 Comparison of clinical and laboratory findings in patients and control groups

Onset age was between 1-21 months (mean ± SD: 6.36 ± 4.62). Twenty four cases had multiple lymphadenitis near to BCG incubation such as cervical, axiliary, and supraclaviculs, 11 cases had suppurative lymphadenitis with fistula and abscesses and 5 cases had ulcer of BCG incubation. Osteomyelitis due to BCG vaccination was detected in 2 cases and disseminated BCG infection in 11 cases (22.9%) with multi organ involvement and systemic symptoms was detected. Family history of BCG infection or immunodeficiency was positive in 22.9% of control group and the rate of consanguineous marriages in the parents of them was 37.5% (18 cases). A chi-square test comparing the clinical manifestations in children with disseminated BCG infection with control group showed statistically significant differences for the two groups of children (Table 1). Table 1 Comparison of clinical and laboratory findings in patients and control groups Clinical and laboratory findings Patient Number (%) Control Number (%) P. value Lymphadenopathy 5(45) 10(27) 0.28 Organomegaly 5(45) 0 <0.001 Bone involvement 3(27.3) 1(2.7) 0.033 Lung involvement 9(81.8) 3(8.1) <0.001 Thymus atrophy 9(81.8) 0 <0.001 Recurrent diarrhea 8(72.7) 9(24.3) 0.009 Recurrent infection 8(72.7) 3(8.1) <0.001 Oral Candida 9(81.8) 5(13.5) <0.001 Fever 11(100) 15(41.7) 0.005 Lymphopenia 8(72.7) 3(8.1) <0.001 Anemia 11(100) 19(51.4) 0.003 Definitive immunodeficiency was detected in approximately all of children with disseminated BCG infection including: SCID in 7 cases with homozygous mutation and homozygous polymorphism in Rag2 in one patient with heterozygous for the Rag2 mutation and polymorphism in the both parents of him and exclusion of Rag1, Rag2 and Artemis defect in another patients.

in approximately all of children with disseminated BCG infection including: SCID in 7 cases with homozygous mutation and homozygous polymorphism in Rag2 in one patient with heterozygous for the Rag2 mutation and polymorphism in the both parents of him and exclusion of Rag1, Rag2 and Artemis defect in another patients. Other cases were CGD, IL12RB1 deficiency and MSMD. In one cases of MSMD we could exclude an IL12RB1 deficiency. So he had a normal expression of IL12RB1on cell surface with two different antibodies. HIV was not identified in any of the cases (Table 2). Table 2 summary of data on children with disseminated BCG infection Number Age/sex Site(s) of dissemination Immune defect Outcome Consanguinity Family history immunodeficiency 1 9 mo/M Chest, Spleen MSMD Survived - 2 3year/M As cite, CNS Bone CGD died - 3 4 mo/M DLN, Liver SCID Rag2 deficiency T-B-NK+ died + + 4 4 mo/M Bone, Pericardia Liver Omen syn T+B-NK+ died + 5 6 mo/F Gastric aspirate, DLN T-B-NK+ SCID died - 6 5 mo/F DLN, Liver T-B-NK+ SCID died + + 7 4/5 mo/M Eye, DLN, Lung (Miliary) *T-B-NK+ SCID died + 8 5 mo/F As cite, Liver, DLN SCID T-B+NK+ died + 9 3.5 years/M Bone, Skin, DLN MSMD Survived - + 10 4 mo/F DLN, Gastric aspiration IL12 R deficiency Survived + 11 4 mo/F Urine, Liver SCID T-B+NK+ died + * Exclusion of Rag1, Rag2 and Artemis defect CMID = cell mediated immune defect, CGD = chronic granulomatous disease, SCID = severe combined immunodeficiency, DLN = distal lymph node.

Number Age/sex Site(s) of dissemination Immune defect Outcome Consanguinity Family history immunodeficiency 1 9 mo/M Chest, Spleen MSMD Survived - 2 3year/M As cite, CNS Bone CGD died - 3 4 mo/M DLN, Liver SCID Rag2 deficiency T-B-NK+ died + + 4 4 mo/M Bone, Pericardia Liver Omen syn T+B-NK+ died + 5 6 mo/F Gastric aspirate, DLN T-B-NK+ SCID died - 6 5 mo/F DLN, Liver T-B-NK+ SCID died + + 7 4/5 mo/M Eye, DLN, Lung (Miliary) *T-B-NK+ SCID died + 8 5 mo/F As cite, Liver, DLN SCID T-B+NK+ died + 9 3.5 years/M Bone, Skin, DLN MSMD Survived - + 10 4 mo/F DLN, Gastric aspiration IL12 R deficiency Survived + 11 4 mo/F Urine, Liver SCID T-B+NK+ died + * Exclusion of Rag1, Rag2 and Artemis defect CMID = cell mediated immune defect, CGD = chronic granulomatous disease, SCID = severe combined immunodeficiency, DLN = distal lymph node. Comparison of immunologic markers showed that significant differences were seen between two groups of children. The mean value of CD3 in patients was (P = 0.005) in comparison with control group and the mean value of CD4 in patients was (P < 0.001) in comparison with control group (Table 3). Table 3 The mean of immunologic markers in patients and control groups Immunologic markers Patients (%) Mean ± SD Control (%) Mean ± SD P. value CD3 30.82 ± 28.98 62.11 ± 8.77 0.005 CD4 13.36 ± 14.21 42.12 ± 11.34 0.001 CD8 19.00 ± 13.61 22.30 ± 6.06 0.452 CD19 23.91 ± 15.40 21.19 ± 8.26 0.586 CD56 35.18 ± 30.46 10.57 ± 4.90 0.029 Although antimycobacterial regimens used for the treatment of patients with 4 drugs, but 8 of the patients died despite aggressive management.

± 28.98 62.11 ± 8.77 0.005 CD4 13.36 ± 14.21 42.12 ± 11.34 0.001 CD8 19.00 ± 13.61 22.30 ± 6.06 0.452 CD19 23.91 ± 15.40 21.19 ± 8.26 0.586 CD56 35.18 ± 30.46 10.57 ± 4.90 0.029 Although antimycobacterial regimens used for the treatment of patients with 4 drugs, but 8 of the patients died despite aggressive management. Discussion BCG is a live attenuated bacterial vaccine that protects children from miliary tuberculosis and tuberculosis meningitis [10]. It is considered a safe vaccine with a low incidence of complications, such as purulent lymphadenitis and Bone BCG infection [2,3,9,11,12]. A disseminated infection is even rarer [6-8] but in recent years has increased up 0-1/100000 vaccinated children [6-8,13]. Lotte et al identified 60 cases of dissemination for which the mortality rate was %50 and cellular immunodeficiency was identified as the chief risk factors for fatal outcome [14,15]. Additionally the estimated incidence of disseminated disease in the 5.5 million vaccinated infants in six European countries was 2 cases per 1 million vaccinated children, and the mortality rate was 80% [14]. Disseminated BCG infections have occurred following vaccination of children with immunodeficiency disorders but only rarely in apparently normal individuals [6,8-10,16-20].

in the 5.5 million vaccinated infants in six European countries was 2 cases per 1 million vaccinated children, and the mortality rate was 80% [14]. Disseminated BCG infections have occurred following vaccination of children with immunodeficiency disorders but only rarely in apparently normal individuals [6,8-10,16-20]. Also in 28 cases of definite disseminated BCG disease in 13 countries; immune defects were identified in 24(86%) of cases. Of 20 patients who died of disseminated BCG disease, all had an immune defect whereas the mortality rate among patients without an identified immunodeficiency was zero [6]. In Canada, 21 BCG vaccinated child with adverse reactions have detected with 6 disseminated BCG infection which 5 of them were died [21]. In our study 2/3 of our patients with disseminated BCG infection, had immunodeficiency that all of them died. Our results are compatible with our pilot study that was done in children's hospital of Tabriz. In this study we could identify 8 cases of immunodeficiency that all of them had severe combined immunodeficiency and died due to disseminated BCG infection with mortality rate100% (in press). Also in retrospective study of 17 cases with disseminated BCG in Tehran impaired immunity was detected in 10 cases that all of them died [22]. Complications of BCG vaccine usually develops 5 months to 5 years after vaccination [6,9,22]. Also review over 5000 reports showed that 71%(20) of 28 cases occurred in patients younger than 2 years of age [6]. In our study, except 2 cases that were within 3 years old, all of them were younger than one year old.

Our results are compatible with our pilot study that was done in children's hospital of Tabriz. In this study we could identify 8 cases of immunodeficiency that all of them had severe combined immunodeficiency and died due to disseminated BCG infection with mortality rate100% (in press). Also in retrospective study of 17 cases with disseminated BCG in Tehran impaired immunity was detected in 10 cases that all of them died [22]. Complications of BCG vaccine usually develops 5 months to 5 years after vaccination [6,9,22]. Also review over 5000 reports showed that 71%(20) of 28 cases occurred in patients younger than 2 years of age [6]. In our study, except 2 cases that were within 3 years old, all of them were younger than one year old. The most commonly reported symptoms in the definite cases of disseminated BCG disease were fever, diarrhea, cough, Lymphadenopathy and weight loss, FTT and Hepatosplenomegaly, were also common [6,15,19,22,23]. In our study fever was in 100% and oral Candida was in 81.8%. Also thymus atrophy in 81.8% of patients was found. Anemia was the most common sign in our patients which was found in 100% of patients. On the other hands, more than half (7 of 11 cases) of our patients had related parents which is compatible with study of Casanova et al. that parental consanguinity was found in 30% of the families [8] and also in study by Afshar et al. that related parents was found in 82/35% of patients [22].

The most commonly reported symptoms in the definite cases of disseminated BCG disease were fever, diarrhea, cough, Lymphadenopathy and weight loss, FTT and Hepatosplenomegaly, were also common [6,15,19,22,23]. In our study fever was in 100% and oral Candida was in 81.8%. Also thymus atrophy in 81.8% of patients was found. Anemia was the most common sign in our patients which was found in 100% of patients. On the other hands, more than half (7 of 11 cases) of our patients had related parents which is compatible with study of Casanova et al. that parental consanguinity was found in 30% of the families [8] and also in study by Afshar et al. that related parents was found in 82/35% of patients [22]. Conclusion AT present BCG inoculation is necessary in countries with high incidence of tuberculosis, but has a high mortality in patients with primary immunodeficiency, so it seems to make an effort in development of a more safer, inoffensive vaccine with minimum side effects are a necessity because infants with immunodeficiency are vaccinated at birth prior to diagnosis. On the other hands, inoculation of BCG vaccine should be prohibited for a few months in the families with history of inherited immunodeficiency. Also, screening of newborns at birth for SCID especially in region with high incidence of SCID such as in the North West Iran [24] can be useful for identify high risk children. Finally BCG lymphadenitis with concomitant signs and symptoms that we mentioned in our study, are requisite for evaluation of underlying disorders such as immunodeficiency.

On the other hands, inoculation of BCG vaccine should be prohibited for a few months in the families with history of inherited immunodeficiency. Also, screening of newborns at birth for SCID especially in region with high incidence of SCID such as in the North West Iran [24] can be useful for identify high risk children. Finally BCG lymphadenitis with concomitant signs and symptoms that we mentioned in our study, are requisite for evaluation of underlying disorders such as immunodeficiency. Competing interests The authors declare that they have no competing interests. Authors' contributions Ms sh: examination of patients, diagnosis and treatment of them, and corresponding author, KA: Head of TB and lung disease research center and supporting by a grant from the TB and lung disease research center, help in drafting the proposal, SM: laboratory analysis (flowcytometry) of samples, MR: participate in draft the manuscript, ZP: analysis of data, RB: examination of patients, AK: the manager of public health center, referral of cases, JC: involving in genetic studies, participate in the design of the study and revising it, JF: involving in genetic studies of Samples, JV: involving in genetic studies of samples. All authors read and approved the final manuscript. Acknowledgements This study was supported by a grant from the TB and lung disease research center of Tabriz; we thank health assistance of Tabriz University (medical sciences) and dr. khayatzadeh for referral of patients, dr Jabarpoor bonyadi for DNA extraction of patient's samples and Mr. Amini a colleague of TB research laboratory.

Phenotyping of the wheezing Wheezing and cough are frequently observed in preschool children, in many cases related to a respiratory tract infection. These situations represent a major challenge in the practice of paediatricians since, at present, it is very hard to distinguish between the different phenotypes which underlie an apparently similar clinical presentation. In fact, among the preschool children who wheeze, at least two different groups can be identified, i.e. children who wheeze on the basis of a viral trigger and those who respond to multiple triggers, the latter being somehow referable to the asthmatic wheezing [1]. The temporal pattern for viral wheezing is commonly reported as episodic, with periods free of symptoms between episodes, and is reported to be concomitant with a viral infection of the airways. In contrast, children with multiple trigger wheezing, though being highly responsive to viral infections which represent the most common trigger for their symptoms, also wheeze when exposed to a variety of other triggers, such as exercise or allergens [1]. Cohort studies have suggested that some clinical characteristics can help in distinguishing among the different phenotypes of preschool wheezers [2], but the application of such parameters cannot be considered conclusive for the clinical purposes. More recently, studies based upon a latent class analysis have demonstrated that considering clusters of symptoms may contribute to the identification of different phenotypes among children with respiratory disorders [3].

Cohort studies have suggested that some clinical characteristics can help in distinguishing among the different phenotypes of preschool wheezers [2], but the application of such parameters cannot be considered conclusive for the clinical purposes. More recently, studies based upon a latent class analysis have demonstrated that considering clusters of symptoms may contribute to the identification of different phenotypes among children with respiratory disorders [3]. The clinical approach to preschool wheezers From a practical point of view, the difficulty in allocating an individual patient within a specific phenotype represent a major task for paediatricians not only in terms of diagnostic procedures but consequently also, and even more importantly, in terms of treatment strategy. In particular, in the last few months, a body of discussion has been raised around the issue of the use of corticosteroids, either systemic or inhaled in the treatment of acute wheezing in preschool children. The use of inhaled or oral corticosteroids in preschool wheezers is widely diffuse in the paediatric practice, mainly based upon the recommendation of guidelines for elder asthmatic children. Some recent publications has looked for a deeper insight into the challenging issue of the use of either systemic or inhaled corticosteroids in younger children with wheezing. In particular, Panickard et al have studied the role of oral prednisolone in wheezing induced by viral infection [4].

The use of inhaled or oral corticosteroids in preschool wheezers is widely diffuse in the paediatric practice, mainly based upon the recommendation of guidelines for elder asthmatic children. Some recent publications has looked for a deeper insight into the challenging issue of the use of either systemic or inhaled corticosteroids in younger children with wheezing. In particular, Panickard et al have studied the role of oral prednisolone in wheezing induced by viral infection [4]. They have randomly treated 687 preschool children with an attack of wheezing associated with viral infection with either prednisolone or placebo for 5-days. The results of this study definitely showed that oral corticosteroids cannot be proposed as an effective treatment in children with viral induced wheezing and, therefore, it no longer allows the indiscriminate use of such a common treatment in this situation, at least in mild-to-moderate conditions. As clearly pointed in the accompanying editorial by Bush [5], the results of this study are to be considered in the frame of the characteristics of the study population, i.e. preschool children who didn't present with the classic atopic asthma phenotype, which, on the other hand, is positively recognized to be responsive to corticosteroid treatment. Therefore, even if it has to be taken into consideration that also severe episodic viral wheezing may need a treatment with oral steroid, the evidence from the study by Panickar and co-workers no longer justify the use of oral steroids in non-atopic children for whom a severe prognosis is not anticipated [5]

As clearly pointed in the accompanying editorial by Bush [5], the results of this study are to be considered in the frame of the characteristics of the study population, i.e. preschool children who didn't present with the classic atopic asthma phenotype, which, on the other hand, is positively recognized to be responsive to corticosteroid treatment. Therefore, even if it has to be taken into consideration that also severe episodic viral wheezing may need a treatment with oral steroid, the evidence from the study by Panickar and co-workers no longer justify the use of oral steroids in non-atopic children for whom a severe prognosis is not anticipated [5] Inhaled corticosteroid courses are proposed even more frequently than oral ones in children presenting with wheeze. Ducharme et al. have demonstrated that high dose inhaled fluticasone (750 mcg/bid) can be somewhat effective in preventing the use of oral corticosteroids in preschool children with moderate-to-severe virus -induced wheezing [6]. The apparent paradox of a better response to the administration of an inhaled corticosteroid in a population with clinical characteristics very similar to those of the group receiving oral prednisone in the Panickar's study could be speculatively explained by a vasoconstrictive effect which is potentially resulting in an anti-edema effect at the site of the bronchial mucosa [5], rather than due to an anti-inflammatory effect.

with clinical characteristics very similar to those of the group receiving oral prednisone in the Panickar's study could be speculatively explained by a vasoconstrictive effect which is potentially resulting in an anti-edema effect at the site of the bronchial mucosa [5], rather than due to an anti-inflammatory effect. Nevertheless, the Authors observed a potential risk of side effects on growth in children treated with a such high dose of fluticasone and clearly warned about the use of this therapeutic strategy in children. On the basis of the hypothesis of an anti-oedema effect of high doses of inhaled corticosteroid, other drugs with a vasoconstrictive effect could be even more effective in controlling viral wheezing, with potentially lower risk of side effects, thus warranting further studies with the aim to address this specific question. If the issue of using corticosteroid in acute episodes of wheezing in preschool children deserved the publication of the two above reported studies, a meta-analysis aiming to evaluate the efficacy of prolonged treatment with inhaled corticosteroids in preschool children with recurrent episodes of wheezing or asthma was recently published in the Pediatrics [7]. The Authors considered 29 randomized trials, including 3.592 children who were receiving for at least four weeks of inhaled corticosteroids for their frequent wheezing episodes. They concluded that ICS are useful in such paediatric population in terms of reduction of exacerbations, symptoms and lung function improvement.

The Authors considered 29 randomized trials, including 3.592 children who were receiving for at least four weeks of inhaled corticosteroids for their frequent wheezing episodes. They concluded that ICS are useful in such paediatric population in terms of reduction of exacerbations, symptoms and lung function improvement. Nevertheless, though the conclusions of this analysis are presented for the pooled group of wheezing and asthmatic children, they are mostly driven by studies including patients with pronounced asthmatic features as previously described by Castro-Rodriguez et al. [2], for whom the effectiveness of ICS treatment is widely accepted. In particular, the study by Guilbert et al [8], which weighted 22,8% for the RR of wheeze/asthma exacerbation (WAE), was designed to select and treat children with a positive asthma predictive index. Also in the study by Baker et al. [9], which weighted for 15.1% in the meta-analysis by Castro Rodriguez and Rodrigo [7], were considered only children with a specific diagnosis of persistent asthma. In the study by Roorda et al [10], accounting for 13.7% in the meta-analysis, it is clearly indicated that a significant response to ICS treatment was observed in the children with frequent symptoms, a family history of asthma, or both, but not in those without a family history. Similar consideration can be extended to a number of further studies with lighter weight in the analysis.

ta-analysis, it is clearly indicated that a significant response to ICS treatment was observed in the children with frequent symptoms, a family history of asthma, or both, but not in those without a family history. Similar consideration can be extended to a number of further studies with lighter weight in the analysis. Therefore, the conclusion that ICSs are useful in infants and preschool children with wheeze/asthma can represent a misleading message to the reader regarding the potential usefulness of a maintenance treatment with ICSs in the group of transient wheezers. To the best of our knowledge, for this group, at present, there is no indication that a maintenance treatment with ICCs can be effective in reducing the number or the severity of wheezing [11]. Conclusions The main clinical dilemma for paediatricians dealing with preschool children with wheezing is represented by the difficulty to predict the future development of the disease in the individual patients. At the present we know that the response to systemic and inhaled steroids in this age group is poor, due to the prevalence of viral wheeze in these children[12]. Therefore, at present, the therapeutic options potentially available for preschool children with episodic non-atopic wheeze are β2-agonists to control the re-exacerbations, along with intermittent or prophylactic leukotriene receptors antagonists, whereas high dose inhaled corticosteroids are not recommended and oral corticosteroids should be considered only in severe children in hospital setting [5].

hildren with episodic non-atopic wheeze are β2-agonists to control the re-exacerbations, along with intermittent or prophylactic leukotriene receptors antagonists, whereas high dose inhaled corticosteroids are not recommended and oral corticosteroids should be considered only in severe children in hospital setting [5]. We agree with Saglani and Bush that asthma in preschool children represents the next challenge for pediatricians and that there is a desperate need for both a more efficient phenotypic process and new therapeutic options for these children [13].

Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is largely employed for treating paediatric patients affected by many haematological conditions of both malignant and non-malignant origin, as well as by several hereditary disorders of the immune system and metabolism [1,2]. More than 40 years have elapsed since the first successful allo-HSCT, then performed using bone marrow (BM) cells [3,4], and through this procedure thousands of children have been cured of their original disease [1,2].

lignant origin, as well as by several hereditary disorders of the immune system and metabolism [1,2]. More than 40 years have elapsed since the first successful allo-HSCT, then performed using bone marrow (BM) cells [3,4], and through this procedure thousands of children have been cured of their original disease [1,2]. All this long history has taught that the success of allo-HSCT is strictly dependent on the balance between the detrimental, sometimes fatal, attack of donor immune cells against recipient tissues (i.e. graft-versus-host disease, GVHD) and the favourable reaction of donor lymphocytes towards malignant cells (i.e. graft-versus-tumor, GVT, effect) and/or pathogens capable of causing life-threatening infections during the period of profound neutropenia and lymphopenia which follows the conditioning regimen. While in the context of classical allo-HSCT from an HLA-identical sibling large part of the GVT effect is related to the attack of donor T lymphocytes against non-shared minor histocompatibility antigens expressed on tumor cells, there is clear evidence that also a selective effect of donor innate immunity (i.e. not dependent from previous encounter with the antigen) can contribute to the eradication of the malignant clone [5]. The role played by the donor innate immunity has attracted the interest of the scientific community only in recent years, when significant advances in the use of allo-HSCT have occurred. In particular, while for many years an HLA-matched sibling was the only type of donor routinely employed, in the last decade, matched unrelated volunteers and unrelated umbilical cord blood (UCB) units are being largely utilized to transplant patients lacking an HLA-identical relative [6]. The most recent, and advanced, frontier of allo-HSCT is represented by transplantation from an HLA-partially matched relative and, in this very context, the contribution of donor innate immunity to the success of the procedure has become evident. Indeed, in order to perform transplantation from an HLA-disparate donor, it is mandatory to remove from the allograft donor T cells, which, otherwise, could attack recipient-specific, either major (in large part) or minor histocompatibility, antigens, this resulting in extremely severe, often fatal, GVHD [6].

evident. Indeed, in order to perform transplantation from an HLA-disparate donor, it is mandatory to remove from the allograft donor T cells, which, otherwise, could attack recipient-specific, either major (in large part) or minor histocompatibility, antigens, this resulting in extremely severe, often fatal, GVHD [6]. The removal of T lymphocytes, coupled with the infusion of high-doses of haematopoietic progenitors, collected from donor peripheral blood after mobilization with cytokines, has rendered possible what for many years was considered a sort of holy grail, namely the engraftment of donor haematopoiesis across the HLA barrier, without leading to the development of GVHD [7,8]. However, in view of the role played by donor T cells in mediating the GVT effect, it could be expected that a relevant proportion of patients given this type of allograft experience leukaemia recurrence. This expectation was only partly confirmed by the clinical results, as it became evident, mainly in adult patients affected by acute myeloid leukaemia (AML), that there was a subgroup of patients given T-cell depleted allo-HSCT from an HLA-disparate relative who had a particularly low risk of leukaemia relapse [9]. These patients belonged to the group transplanted from a donor having natural killer cells (NK) that are alloreactive towards recipient targets. This concept of NK-cell alloreactivity has represented a sort of revolution in the field of allo-HSCT, underlining for the first time that not only adaptive immunity, but also innate immunity is a crucial element for guaranteeing a successful patient's outcome.

ells (NK) that are alloreactive towards recipient targets. This concept of NK-cell alloreactivity has represented a sort of revolution in the field of allo-HSCT, underlining for the first time that not only adaptive immunity, but also innate immunity is a crucial element for guaranteeing a successful patient's outcome. NK cells are key members of the natural immune system, which each single individual possesses, and are of fundamental importance to limit or eradicate pathogens during the early phases of a primary infection, i.e. before T and B cells can mount efficient responses [5,10]. Indeed, NK cells and phagocytes do not require clonal expansion and can enter and defend a tissue almost as soon as it becomes infected. NK cells, derived from the haematopoietic progenitors through a common lymphoid progenitor [10], play a pivotal role in the defense against viral infections and transformed cells. The molecular mechanisms that allow NK cells to spare normal cells and kill tumor or virus-infected cells are represented by the interplay between an array of surface receptors with either activating or inhibitory function [5,10]. Indeed, NK-cell function results from the net balance between activatory and inhibitory signals (see also Figure 1). The role played by inhibitory signals represents a peculiarity of NK cells in comparison to the function of both T and B lymphocytes which is regulated only by activatory signals, and the signals delivered by inhibitory receptors present on the surface of NK lymphocytes are even more important than the activatory signals [5,10,11]. NK receptors are known as Killer Immunoglobulin-like receptors (KIR), and the inhibitory ones specifically interact with determinants that are shared by different HLA-class I molecules (referred to as KIR ligands). In a self environment, to ensure self tolerance, each individual NK cell expresses at least one inhibitory receptor specific for autologous HLA-class I and an NK-mediated attack towards autologous cells occurs only when these do not express at all or express low amounts of HLA class-I molecules.

red to as KIR ligands). In a self environment, to ensure self tolerance, each individual NK cell expresses at least one inhibitory receptor specific for autologous HLA-class I and an NK-mediated attack towards autologous cells occurs only when these do not express at all or express low amounts of HLA class-I molecules. Figure 1 Schematic representation of the main interactions occurring between normal natural killer (NK) cells (expressing both HLA class I-specific inhibitory receptors and activating receptors) and potential target cells. Normal tissues deliver inhibitory signals, which block NK cells (a). Normal stressed tissues deliver simultaneously both activatory and inhibitory signals, this also resulting in block of the lytic capacity of NK cells (b). The lack of inhibitory signals, by contrast, permits NK cells to kill their targets, activatory signals playing a facilitating role (c). Modified from Moretta et al. Immunology Today 2004.

deliver simultaneously both activatory and inhibitory signals, this also resulting in block of the lytic capacity of NK cells (b). The lack of inhibitory signals, by contrast, permits NK cells to kill their targets, activatory signals playing a facilitating role (c). Modified from Moretta et al. Immunology Today 2004. In an allogeneic setting, NK cells can kill non-self cells and the molecular basis for NK alloreactivity also involves NK-cell KIR inhibitory receptors that are specific for determinants that are shared by different KIR ligands (i.e. HLA-class I alleles). In detail, KIR2DL1 recognizes HLA-C alleles characterized by Lys at position 80 (HLA-CLys80, also named C2 group), KIR2DL2/3 recognize HLA-C alleles characterized by Asn at position 80 (HLA-CAsn80, C1 group), KIR3DL1 recognizes HLA-B alleles sharing the Bw4 supertypic specificity (HLA-BBw4, Bw4 group) [12]. This kind of inhibitory KIR receptor repertoire can lead to alloreactivity in haplo-HSCT through the mechanism of "missing self" recognition [13], provided that the donor: i) expresses a KIR-ligand which is missing in the recipient HLA genotype; and ii) expresses the specific KIR, leading to a KIR/KIR-ligand mismatch in graft-versus-host (GvH) direction. In other words, NK alloreactivity can be predicted to occur when analysis of the HLA-class I genotypes reveals that a KIR ligand is expressed in the donor but is missing in the recipient (KIR ligand-mismatch).

and ii) expresses the specific KIR, leading to a KIR/KIR-ligand mismatch in graft-versus-host (GvH) direction. In other words, NK alloreactivity can be predicted to occur when analysis of the HLA-class I genotypes reveals that a KIR ligand is expressed in the donor but is missing in the recipient (KIR ligand-mismatch). The existence of human "alloreactive" NK cells was originally suggested two decades ago by the hybrid resistance phenomenon in which NK cells proved to be responsible for the rejection of parental bone marrow grafts in F1 hybrid mice [14]. However, the clinical relevance of the corresponding phenomenon in human beings was not recognized till the emergence of results in adults given haplo-HSCT, demonstrating the reduction in the risk of leukaemia recurrence observed when the donor showed an NK alloreactivity with respect to the recipient HLA typing [15]. This striking finding significantly changed the criteria for choosing the HSCT donor in the context of T-cell depleted allograft from an HLA-disparate relative.

onstrating the reduction in the risk of leukaemia recurrence observed when the donor showed an NK alloreactivity with respect to the recipient HLA typing [15]. This striking finding significantly changed the criteria for choosing the HSCT donor in the context of T-cell depleted allograft from an HLA-disparate relative. One of the most intriguing clinical questions related to donor NK alloreactivity involves the observation that this peculiar GVT effect is completely separated by the occurrence of GVHD, this indicating that NK cells are able to kill leukaemia targets, while sparing normal tissues. Clearly, the selectivity of the GVT effect displayed by NK cells cannot be attributed to an attack directed against antigens with a restricted expression on tumor cells, as the ligands of NK-cell KIR inhibitory receptors are, as mentioned above, determinants of HLA-class I molecules which are expressed on all nucleated cells of the body. Thus, the preferential lysis of tumor cells by NK lymphocytes can be interpreted only in view of the role played by activatory signals. Indeed, there is large experimental evidence that killing of allogeneic cells also depends on the surface density of activating receptors on NK cells and on the expression of their ligands on target cells [16,17]. It is currently accepted that leukaemia targets express on their cell surface ligands able to efficiently engage activatory KIR receptors [18]. Support to this interpretation is provided by the observation that, in the animal model, alloreactive NK cells not only lyse leukaemia blasts, but also kill recipient dendritic cells (DC) and T lymphocytes [10], all these targets belonging to cells deriving from the haematopoietic tissue, which, evidently, has a particular capacity to render NK cells efficiently active. This biological observation translates into other two relevant clinical advantages, namely prevention of GVHD, whose occurrence is certainly facilitated by the role played by recipient DC [19], and prevention of graft rejection, which is a complication mainly due to the effect of recipient cytotoxic T lymphocytes surviving the preparative regimen (see also Figure 2 for details).

t clinical advantages, namely prevention of GVHD, whose occurrence is certainly facilitated by the role played by recipient DC [19], and prevention of graft rejection, which is a complication mainly due to the effect of recipient cytotoxic T lymphocytes surviving the preparative regimen (see also Figure 2 for details). Figure 2 Donor-derived alloreactive NK cells are able to kill leukaemia targets (this preventing disease recurrence), dendritic cells (DC) of the recipient (this preventing graft-versus-host disease recurrence) and cytotoxic T-lymphocytes of the recipient (this preventing graft rejection). Modified from Ruggeri et al. Science 2002.

r-derived alloreactive NK cells are able to kill leukaemia targets (this preventing disease recurrence), dendritic cells (DC) of the recipient (this preventing graft-versus-host disease recurrence) and cytotoxic T-lymphocytes of the recipient (this preventing graft rejection). Modified from Ruggeri et al. Science 2002. If the relevance of NK alloreactivity has been demonstrated in the setting of adult myeloid leukaemia, one could argue which is the interest of this phenomenon for paediatricians. The answer comes from recent results that our groups have obtained [12]. In fact, we have demonstrated that the favourable effect mediated by donor NK alloreactivity is of crucial relevance in paediatric patients with acute lymphoblastic leukaemia (ALL), which, by far, represents the most common malignancy of childhood. In a study involving 21 children transplanted from an NK-alloreactive parent, we have documented that NK cells of donor origin able to kill leukaemia blasts emerge since the 4-5th month after the allograft, persist for years and significantly contribute to the eradication of the malignant clone [12]. These results emphasize the concept that the role of innate immunity in allo-HSCT must be completely revised and re-considered also by pediatricians and, once again, demonstrated that what has been obtained in adults (namely an NK effect in AML, but not in ALL) cannot automatically be translated to children, this finding being in keeping with the old aphorism that a child is not an adult in miniature.

st be completely revised and re-considered also by pediatricians and, once again, demonstrated that what has been obtained in adults (namely an NK effect in AML, but not in ALL) cannot automatically be translated to children, this finding being in keeping with the old aphorism that a child is not an adult in miniature. Is the mechanisms of NK alloreactivity in childhood leukaemia completely understood and the role of NK cells in transplantation fully defined? The answer is obviously no. Indeed, it remains to be addressed the unsolved problem of the need of an efficient NK alloreactive effect also in the first months after the allograft. Isolation of mature donor NK cells and their proper activation with specifically active cytokines, such as interleukin-15, could be the right solution for filling this gap and further ameliorating the clinical results of haplo-HSCT in childhood ALL. Many other biological and clinical issues also deserve critical attention for the future. Among them, the observation that, in the presence of the same kind of NK alloreactivity, mothers are better donors than fathers [20]. So far, we do not precisely know the biological explanation of this finding. What we can certainly conclude is that, also in the field of transplantation of haematopoietic progenitors, what happens during pregnancy matters.....

Background Regurgitation is defined as the passage of refluxed gastric content into the oral pharynx whilst vomiting is defined as expulsion of the refluxed gastric content from the mouth. The frequency of regurgitation may vary largely in relation to age and younger infants up to first month of age are more frequently affected by regurgitation. Gastroesophageal reflux (GER) is the backward flow of stomach contents up into the esophagus or the mouth. It happens to everyone. In babies, a small amount of GER is normal and almost always goes away by the time a child is 18 months old. The consensus statements that comprise the definition of gastroesophageal reflux disease (GERD) in the pediatric population were developed through a rigorous process [1]. Consensus items of particular note were: (i) GERD is present when reflux of gastric contents causes troublesome symptoms and/or complications, but this definition is complicated by unreliable reporting of symptoms in children under the age of approximately 8 years; (ii) histology has limited use in establishing or excluding a diagnosis of GERD; its primary role is to exclude other conditions; (iii) Barrett's esophagus should be defined as esophageal metaplasia that is intestinal metaplasia positive or negative; and (iv) extraesophageal conditions may be associated with GERD, but for most of these conditions causality remains to be established. The prevalence and natural history of gastroesophageal reflux in infants have been poorly documented. In a recent pediatric prospective survey, the 12% of Italian infants satisfied the Rome II criteria for infant regurgitation. Eighty-eight percent of the infants who had completed two-years follow-up period had improved at the age of 12 months. Only one apart 210 infants turned out to have GERD [2].

poorly documented. In a recent pediatric prospective survey, the 12% of Italian infants satisfied the Rome II criteria for infant regurgitation. Eighty-eight percent of the infants who had completed two-years follow-up period had improved at the age of 12 months. Only one apart 210 infants turned out to have GERD [2]. Diagnostic investigation of infants who regurgitate, but gain weight satisfactorily and do not exhibit other signs or symptoms is not indicated in clinical practice. The North American Society for Pediatric Gastroenterology, Hepatology and Nutrition (NASPGHAN) [3] recommends that, once other causes of vomiting have been ruled out, infants presenting regurgitation and irritability should undergo a two-week therapeutic test involving a hypoallergenic diet and acid suppression, either sequentially or simultaneously. If no improvement is seen, examinations (pH measurement or endoscopy with biopsy) would be indicated after this period [4]. The non-erosive or exclusively histological reflux esophagitis responds well to treatment based on conservative measures and histamine-2 receptor antagonists (H2RAs), of which the most often used in pediatrics is ranitidine [5].

nations (pH measurement or endoscopy with biopsy) would be indicated after this period [4]. The non-erosive or exclusively histological reflux esophagitis responds well to treatment based on conservative measures and histamine-2 receptor antagonists (H2RAs), of which the most often used in pediatrics is ranitidine [5]. Clinical Approach In children is important distinguishing between normal, physiologic reflux and pathological one. Most infants with physiologic regurgitation are happy and healthy even if they frequently spit up or vomit, and babies usually outgrow GER by their first birthday. These patients have no underlying predisposing factors or conditions, growth and development are normal, and pharmacologic treatment is typically not necessary. Patients with pathologic gastroesophageal reflux or GERD frequently experience complications noted above, requiring careful evaluation and treatment.

birthday. These patients have no underlying predisposing factors or conditions, growth and development are normal, and pharmacologic treatment is typically not necessary. Patients with pathologic gastroesophageal reflux or GERD frequently experience complications noted above, requiring careful evaluation and treatment. Symptoms and signs associated with GER are non-specific. Regurgitation, irritability, and vomiting are common both in infants with physiologic GER or GERD [6] and in infant with other diseases such as food allergy [7], persistent crying [8] and so on. Cough and anorexia/feeding refusal were more common in children 1 to 5 years of age than in older children [9]. Several attempts have been made to introduce specific questionnaire in order to evaluate the role of single gastrointestinal symptoms or cluster of symptoms, calculating the discriminative power of the symptom score in patients and controls. In a recent study the items of a validated questionnaire were tested against the pH esophageal 24-h study in children with suspected GERD. Regurgitation/vomiting yielded the best symptom discrimination, and was reported by 46% with abnormal versus 24% with normal pH-study results. A weighted score including the five best discriminating symptoms was positive in 75% versus 44% [10]. Comparing children with abnormal pH studies and healthy controls, a correct diagnosis based on five symptoms could be obtained in 75% and 94%, respectively. Overall, questionnaires are poorly predictive for the severity of gastroesophageal reflux disease, as they do not correlate with esophageal acid exposure as measured by pH-metry and with esophagitis as evaluated by histology of esophageal biopsies [10,11]. The role of the history and physical examination of a child suspected to have GER(D) is to exclude other disorders that present with the same gastrointestinal symptoms and to identify complications of GERD [12].

osure as measured by pH-metry and with esophagitis as evaluated by histology of esophageal biopsies [10,11]. The role of the history and physical examination of a child suspected to have GER(D) is to exclude other disorders that present with the same gastrointestinal symptoms and to identify complications of GERD [12]. Diagnostic Instrumental Approach As reported above, a diagnostic approach for the "happy spitter" infants, gaining weight satisfactorily and without other signs or symptoms, is not needed. However, diagnostic instrumental tests can be performed in infants frequently present complications, in which it is not easy to identify individuals who truly have GERD. The list of current available diagnostic tools in the management of GERD are reported in table 1. Table 1 List of current available diagnostic tools in the management of GERD Exam Advantages Disadvantage 24 h Esophageal pH monitoring Gold standard for acid reflux Reference data available Reproducibility Portability The probe is often disconfortable Non acid or gas reflux are not detected Esophageal manometry Identification of the GER mechanisms Evaluation of the esophageal and sphincter motility pattern Measurement of esophageal length Portability Limited availability Trained personnel Endoscopy Description of esophageal mucosal damage Biopsy allows histological description Anesthesia is needed Trained personnel Rx series Fine definition of anatomy Poor information on the GER mechanism Possible aspiration Rx exposure Not portable

Esophageal manometry Identification of the GER mechanisms Evaluation of the esophageal and sphincter motility pattern Measurement of esophageal length Portability Limited availability Trained personnel Endoscopy Description of esophageal mucosal damage Biopsy allows histological description Anesthesia is needed Trained personnel Rx series Fine definition of anatomy Poor information on the GER mechanism Possible aspiration Rx exposure Not portable Scintigraphy Study of gastric emptying Radiation exposure Not portable Esophageal pH monitoring Ambulatory 24-h esophageal pH monitoring is currently the best available test for quantifying esophageal acid exposure, particularly in patients presenting with atypical symptoms. Esophageal pH recording provides quantitative data on both esophageal acid exposure and on the correlation between patient symptoms and reflux events. Esophageal acid exposure is defined by the percentage of the 24-h recording time that the pH is < 4.0. Values > 3.5% are considered abnormal. However, pH monitoring is of limited use in preterm infants whose gastric pH is >4 for the 90% of the time making it almost impossible to detect GER by this technique [13,14]. Wireless pH monitoring has superior sensitivity to catheter studies for detecting pathological esophageal acid exposure because of the extended period of recording (48 hours) and has also shown superior recording accuracy compared with catheter equipment. The American Gastroenterological Association (AGA) reported that ambulatory impedance-pH, catheter pH, or wireless pH monitoring (proton pump inhibitor (PPI) therapy withheld for 7 days) is useful to evaluate patients with a suspected esophageal GERD syndrome who have not responded to an empirical trial of PPI therapy, have normal findings on endoscopy, and have no major abnormality on manometry [15].

e-pH, catheter pH, or wireless pH monitoring (proton pump inhibitor (PPI) therapy withheld for 7 days) is useful to evaluate patients with a suspected esophageal GERD syndrome who have not responded to an empirical trial of PPI therapy, have normal findings on endoscopy, and have no major abnormality on manometry [15]. Although important contributions have been made to assess the diagnostic value of the long-term pH monitoring in any age pediatric groups, only few reports in children have attempted to correlate the pH pattern of reflux with the clinical severity of gastro-oesophageal reflux disease and to determine the ability of the test to differentiate normal subjects from patients with various degrees of reflux disease [16,17]. The reproducibility of the intraluminal oesophageal pH test to discriminate patients with various degrees of reflux disease have produced contradictory results [18,19]. The 24 hour intraesophageal pH monitoring may present false negative results that limit overall sensitivity of the test. Several scoring systems for pH monitoring studies have been developed [20,21] but any system is clearly better than reflux index (RI) [22].

reflux disease have produced contradictory results [18,19]. The 24 hour intraesophageal pH monitoring may present false negative results that limit overall sensitivity of the test. Several scoring systems for pH monitoring studies have been developed [20,21] but any system is clearly better than reflux index (RI) [22]. Development of esophagitis was associated with increased acid exposure of the esophagus. The number of reflux episodes lasting more than five minutes was the most significant variable that differentiated patients with esophagitis from those with simple gastro-esophageal reflux disease. The five minute value is currently regarded as the most accurate variable in predicting the occurrence of esophagitis because it reflects the mechanisms of esophageal acid clearing. However, symptoms may not correlate with acid exposure or the presence of esophagitis. This may be because symptoms may result from nonacidic as well as acidic refluxate [23]. A surprising finding relates to the fact that reflux during sleep was not implicated in the occurrence of esophagitis. It is commonly assumed that reflux occurring during sleep can be more dangerous to the esophagus than the awake acid exposure as acid clearing is usually impaired during sleep [24]. Reports on adults have produced strong evidence that nighttime heartburn and GER represent a distinct clinical entity which deserves specific attention in the diagnosis and optimal treatment of GERD [25]. The discriminating power of the pH test is optimal for long lasting recording, even the postprandial esophageal integrated acidity provides a robust estimation of esophageal acid exposure and may predict symptoms in gastro-esophageal reflux disease patients [26]. However, in infants milk or formula feeding can neutralize gastric acidity, so reflux of non-acid gastric content might not be detected by pH test [23].

dial esophageal integrated acidity provides a robust estimation of esophageal acid exposure and may predict symptoms in gastro-esophageal reflux disease patients [26]. However, in infants milk or formula feeding can neutralize gastric acidity, so reflux of non-acid gastric content might not be detected by pH test [23]. Multiple intraluminal esophageal impedance Gastroesophageal reflux can be acid, nonacid, pure liquid, or a mixture of gas and liquid. Esophageal pH and impedance were used to identify acid reflux (pH drop below 4.0), minor acid reflux (pH drop above 4.0), nonacid reflux (pH drop less than 1 unit + liquid reflux in impedance), and gas reflux [27]. Non-acid reflux is a particular problem in pediatrics because children are fed more frequently than adults and the majority of non-acid reflux occurs in the post-prandial period when stomach content is neutralized.

above 4.0), nonacid reflux (pH drop less than 1 unit + liquid reflux in impedance), and gas reflux [27]. Non-acid reflux is a particular problem in pediatrics because children are fed more frequently than adults and the majority of non-acid reflux occurs in the post-prandial period when stomach content is neutralized. Additionally, there are many children that are continuously fed through gastrostomy tubes such that the pH of the stomach is neutral for the majority of the day. Other factors can explain a negative pH monitoring in subjects with gastro-esophageal reflux disease. First, episodes of alkaline gastroesophageal reflux might be overlooked using the standard routine pH measurement. Increased flow/volume of saliva can reduce the exposure acid time of the esophagus neutralising the acidity of the refluxed content [28]. Esophageal bile reflux seems to play an additional role in the pathophysiology of gastroesophageal reflux disease [29]. A third possible explanation for a negative pH result in patients with gastro-esophageal reflux disease lies in the variability of the prolonged intraesophageal pH monitoring. In fact, milk fed infants had been reported to have a low reflux index reflecting prolonged buffering of gastric acidity rather than the absence of reflux [30,31].

planation for a negative pH result in patients with gastro-esophageal reflux disease lies in the variability of the prolonged intraesophageal pH monitoring. In fact, milk fed infants had been reported to have a low reflux index reflecting prolonged buffering of gastric acidity rather than the absence of reflux [30,31]. Previous pediatric studies have shown that between 30-88% of reflux in children is non-acid [32]. The literature has focused on the role that acid reflux plays and currently, it is thought that non-acid reflux may be involved in the pathogenesis of respiratory diseases [33]. Children under the age of 18 months have the highest rates of acute respiratory diseases of any age group. Many of these acute illnesses progress to chronic respiratory diseases such as asthma, which result in significant morbidity and mortality [34]. Despite excellent medical therapy, the prevalence rates of chronic respiratory disease remain high.

nths have the highest rates of acute respiratory diseases of any age group. Many of these acute illnesses progress to chronic respiratory diseases such as asthma, which result in significant morbidity and mortality [34]. Despite excellent medical therapy, the prevalence rates of chronic respiratory disease remain high. A recent work have attempted to characterize the proportion of acid and nonacid esophageal reflux events in young infants with suspected GER using combined pH-multichannel intraluminal impedance (pH-MII) monitoring. To determine the symptom index correlation with nonacid reflux and acid reflux events in children, aged 2 weeks to 1 year, 1890 reflux events were detected by pH-MII, and 588 reflux events were detected by pH probe alone. The percent of reflux that was acid was 47% versus 53% of nonacid reflux events. The proportion of nonacid reflux decreased with age and with increasing time elapsed from last meal. The most frequently reported symptom was fussiness/pain, which correlated with nonacid reflux events 24.6% and acid reflux 25.2%. The proportion of nonacid reflux to acid reflux events in infants was more similar to adults than previously reported. Combined pH-MII esophageal monitoring identifies more reflux events and improves clinical correlation with symptoms [35].

/pain, which correlated with nonacid reflux events 24.6% and acid reflux 25.2%. The proportion of nonacid reflux to acid reflux events in infants was more similar to adults than previously reported. Combined pH-MII esophageal monitoring identifies more reflux events and improves clinical correlation with symptoms [35]. The pH-MII catheter is a small tube that is inserted through the nose into the esophagus and is identical in size to the standard pH probe. The catheter remains in place for 24 hours during which it continuously measures the amount of both acid and non-acid reflux that is entering the esophagus from the stomach. Another significant advantage to pH-MII is the ability of the catheter to measure the height of the refluxed stomach contents; impedance sensors are positioned throughout the esophagus so reflux extends along the entire length of the esophagus, and even up into the mouth and potentially the airway, can be determined. Pediatric studies have suggested that the pH-MII catheter is as sensitive as the pH probe in the detection of reflux.

contents; impedance sensors are positioned throughout the esophagus so reflux extends along the entire length of the esophagus, and even up into the mouth and potentially the airway, can be determined. Pediatric studies have suggested that the pH-MII catheter is as sensitive as the pH probe in the detection of reflux. This tool has been very useful in the evaluation of patients with atypical reflux symptoms (such as asthma, chronic cough, laryngitis, chest pain) and in patients who continue to have symptoms while taking acid blocking medicines. Studies in adults and children have shown that the addition of pH-MII monitoring significantly improves the physicians' ability to diagnose reflux-related disease. In studies of infants, the use of pH-MII has been particularly important in clarifying the relationship between respiratory diseases. While the association between apnea and reflux in infants has been debated, there is some evidence that non-acid reflux may be associated with breathing problems in these young patients. In a study of infants with primarily respiratory symptoms who underwent pH-MII testing, the standard pH probe failed to detect 88% of reflux episodes that were associated with breathing problems [36]. There is also literature that suggests that non-acid reflux in children, as well in adults, may be associated with other respiratory symptoms. In particular, in children with severe respiratory disease who were taking acid blocking medicine, non-acid reflux seems more likely to be associated with respiratory symptoms than acid reflux. In pediatrics, pH-MII has been used to evaluate other reflux therapies such as body positioning [37], apnea of premature infants [38], and thickening of feeds [39]. All of the therapeutic studies have involved a small number of patients and additional data on the treatment of non-acid reflux are needed.

an acid reflux. In pediatrics, pH-MII has been used to evaluate other reflux therapies such as body positioning [37], apnea of premature infants [38], and thickening of feeds [39]. All of the therapeutic studies have involved a small number of patients and additional data on the treatment of non-acid reflux are needed. Because the understanding of the role of non-acid reflux is in its infancy, very few studies have addressed the treatment options for patients with pathologic non-acid reflux. Adult and pediatric studies suggest that proton pump inhibitors such as omeprazole and lansoprazole do not decrease the total amount of reflux in patients. Instead, they convert the reflux from acid to non-acid reflux which may explain why some patients continue to have symptoms despite therapy with proton pump inhibitors [40]. Adult studies have suggested that therapy with the drug baclofen may effectively treat non-acid reflux [41]. Baclofen is a gamma-aminobutyric acid (GABA) agonist which decreases the amount of esophageal sphincter (LES) relaxations, the main cause of reflux. However, because of its evident side effects on the central nervous system (CNS) (drowsiness, confusion or mental depression, mood or mental changes, seizures) baclofen is undesirable for use as a treatment for GERD. Further development work has yielded a number of novel GABA type B receptor agonists with reduced CNS side effect profiles, and clinical trials are currently being performed with several agents. Compounds that target esophageal sphincter relaxations may therefore present a new add-on treatment for patients with persistent GERD symptoms despite PPI therapy.

the esophageal body and sphincters; (2) details of post-prandial state, including the response to wet and dry swallows; (3) response to esophageal provocation; and (4) identification of esophageal-protective reflexes. Such information may be useful in understanding the pathophysiology of esophageal motor function [55]. Manometric study is useful in identifying transient relaxations of the LES as a pathophysiological mechanism of GERD [56] and for the diagnosis of achalasia or other motor disorders of the esophagus which may present itself as reflux. Esophageal motor abnormalities are commonly found in children with esophagitis [57] and in children with developmental delay and neurologic impairment, with GERD recurred after Nissen funduplication [58]. As regard the discriminating role of manometric studies, a recent study point out that manometry assess only resting LES pressure and its length in children with acid GER but do not clearly differentiate GER into primary and secondary refluxes to cow's milk allergy [59]. Gastric emptying studies have shown prolonged half-emptying times in children with gastroesophageal reflux. The significance of this phenomenon is not clear. Tests of gastric emptying are not routinely performed in patients with suspected GERD, but may become worthy gastric retention is suspected (see scintigraphy and ultrasonography).

number of novel GABA type B receptor agonists with reduced CNS side effect profiles, and clinical trials are currently being performed with several agents. Compounds that target esophageal sphincter relaxations may therefore present a new add-on treatment for patients with persistent GERD symptoms despite PPI therapy. Upper endoscopy and histology Endoscopy associated with histology is a reliable and accurate method to demonstrate esophageal damage induced by GERD, such as inflammation and strictures. However, up today optimization and standardization of pediatric endoscopy procedure have not yet realized [42]. The findings of erythema, edema, loss of shine and friability in the distal esophagus are aspecific, and the introducing of controversial parameters for esophagitis diagnosis, with interpretations varying greatly from one endoscopist to another, have increased the disagreement between macroscopic and histological findings. In contrast, the presence of esophageal erosion is less subject to observer interpretation [43]. Some authors observed that enanthema of the esophageal mucosa may not have any histological correspondence with reflux esophagitis. Studies have shown the predominance of disagreement between endoscopic and histological results in milder cases, while agreement between the two diagnostic tests predominates in more severe forms [44]. Hiatal hernia is the only endoscopic observation that predicts erosive esophagitis [45]. The use of the Tytgat classification, which does not take into account the presence or absence of Barrett's esophagus, but describes non-erosive abnormalities observed in the discrete esophagitis (commonly observed among infants), may report the endoscopic diagnosis of level I esophagitis associated with normal histology [46].

of the Tytgat classification, which does not take into account the presence or absence of Barrett's esophagus, but describes non-erosive abnormalities observed in the discrete esophagitis (commonly observed among infants), may report the endoscopic diagnosis of level I esophagitis associated with normal histology [46]. All patients with erosive esophagitis presented reflux esophagitis on histology. The esophageal biopsy plays an important role, as much in cases of normal examinations or mild abnormalities as in cases of erosive esophagitis. If the edema, erythema and friability commonly observed in children are non-specific, findings from histological examination and morphometric studies of the esophageal mucosa allow an etiologic diagnosis of eosinophilic esophagitis if characteristic alterations such as eosinophil infiltrates, increased total epithelial and basal cell thickness, and elongation of stromal papillae are seen [47]. Furthermore, histopathology allows the investigation of other diagnostic possibilities such as infectious esophagitis (Herpes virus, Cytomegalovirus, Candida), Barrett's esophagus, dysplasia, adenocarcinoma, Crohn disease, and others. Microscopic evaluation of biopsy samples from the distal esophagus, but avoiding the most distal area to minimize the false positive findings at LES, demonstrated abnormalities in many patients who have symptoms but no endoscopically evident erosions. Infiltration of the epithelium with inflammatory cells, the changes recognizable in esophageal epithelium regardless of orientation of the specimen, received early attention. Neuthrophils and eosinophils are not normally present in the ephitelium of the children and can be used as marker of GERD even though they may be fairly insensitive [48]. Intraepitelial limphocites are more sensitive than other inflammatory cells but they are very common and so their specificity for GERD remains unclear.

uthrophils and eosinophils are not normally present in the ephitelium of the children and can be used as marker of GERD even though they may be fairly insensitive [48]. Intraepitelial limphocites are more sensitive than other inflammatory cells but they are very common and so their specificity for GERD remains unclear. Eosinophilic oesophagitis results in inflammation the esophagus, and in most cases are seen in people with allergies such as hay fever and asthma. There is some evidence that this may be an unusual form of food allergy. It is important to rule out it since eosinophilic esophagitis can progress to esophageal stenosis, and not responding well to anti-GER treatment, corticoid therapy being indicated instead. In such cases, the high eosinophil density (> 20 per high power field) and the presence of eosinophils in the proximal esophagus favor the hypothesis of eosinophilic esophagitis [49]. To determine the clinical, endoscopic, and histologic criteria that distinguish children with eosinophilic esophagitis (EE) from those with non-EE diagnoses, a retrospective case-control study was performed for children with any degree of esophageal eosinophilic inflammation who underwent esophageal biopsy [50]. Although EE and non-EE patients complained of vomiting and abdominal pain at equivalent rates, EE patients were 3 times more likely to complain of dysphagia and twice as likely to have stricture formation. On endoscopy, patients with EE were 19-times more likely than non-EE patients to have endoscopic abnormalities. Histologically, EE patients were more likely to have basal zone hyperplasia and degranulated eosinophils [50]. Although the above mentioned findings, the histologic distinction between EE and GERD cannot be reliably made on histopathologic evidence alone in children with upper aerodigestive symptoms. Despite the recent gastroenterology consensus statement regarding the clinic-pathologic diagnosis of EE, children with primary airway symptoms in whom EE is suspected represent a diagnostic dilemma [51].

EE and GERD cannot be reliably made on histopathologic evidence alone in children with upper aerodigestive symptoms. Despite the recent gastroenterology consensus statement regarding the clinic-pathologic diagnosis of EE, children with primary airway symptoms in whom EE is suspected represent a diagnostic dilemma [51]. Motility studies Motility disorders are postulated to potentially cause reflux since an association between diminished LES tone, transient LES relaxations, delayed gastric emptying and GER have been recognized. Esophageal manometry measures movement and pressure in the esophagus. In particular, it measures esophageal motility pattern and coordinated peristalsis, and the upper and lower esophageal sphincter pressures. There are two main types of manometric recording systems: perfused and solid state. Both have strengths and weaknesses, and the choice of any particular system depends on how these strengths and weaknesses are viewed. Esophageal motility develops during infancy and early childhood, and may be influenced by various factors, including maturation, dietary and postural habits, arousal state, ongoing illnesses, congenital anomalies, and effects of medical or surgical interventions. Esophageal motility is particularly important because it regulates the movement of a bolus during swallowing or during GER. Infantile reflux is different from adult reflux in that regurgitation or vomiting is quite common, even in normal infants [52]. Despite its common occurrence, the mechanisms of esophageal and airway protection during episodes of GER in infants are relatively poorly understood. None of the current approaches [3] for the evaluation of GER in infants evaluates the protective mechanisms. To date, there is not much evidence of esophageal defense mechanisms against GER in children, although data exist from adult studies [53,54]. In summary, carefully performed esophageal manometric studies in infants and children should include (1) basal measurements of the esophageal body and sphincters; (2) details of post-prandial state, including the response to wet and dry swallows; (3) response to esophageal provocation; and (4) identification of esophageal-protective reflexes. Such information may be useful in understanding the pathophysiology of esophageal motor function [55].

studies have shown prolonged half-emptying times in children with gastroesophageal reflux. The significance of this phenomenon is not clear. Tests of gastric emptying are not routinely performed in patients with suspected GERD, but may become worthy gastric retention is suspected (see scintigraphy and ultrasonography). Imaging Radiography Plain radiographic findings are not useful in evaluating patients for GERD, but they are helpful in evaluating pulmonary status and basic anatomy. Esophageal inflammatory and neoplastic diseases are better detected with double-contrast techniques [60]. Conversely, single-contrast techniques are more sensitive for structural defects such as hiatal hernias and strictures or esophageal rings [61]. Various techniques are used, and each has relative strengths and weaknesses in the ability to detect specific abnormalities or disease processes. A typical barium esophagram is performed in multiple steps or phases. A high-density barium suspension is administered, and double-contrast views are used for images taken with the patient in the upright position. Prone-positioned images are typically obtained with single contrast and a lower-density barium suspension. Mucosal relief images can be made to complement these techniques.

es. A high-density barium suspension is administered, and double-contrast views are used for images taken with the patient in the upright position. Prone-positioned images are typically obtained with single contrast and a lower-density barium suspension. Mucosal relief images can be made to complement these techniques. Early esophagitis is not well demonstrated and decreases the overall sensitivity of barium swallows [62]. This is why many clinicians reserve barium swallow for the evaluation of patients with GERD and symptoms that include dysphagia. Barium swallow is not sensitive in the detection of actual reflux, except in the occasional patient who has a wide-open LES and free reflux. Radiographic series are neither sensitive nor specific for diagnosing GERD especially compared to tests such as 24-hour pH monitoring. The presence of Barrett esophagus occasionally is detected as a reticular mucosal pattern. As expected, the more advanced the esophageal disease, the more sensitive is barium swallow at detecting it [63]. Barium swallow is a very important study in the investigation and detection of postoperative complications following fundoplication. Recurrent hiatal hernia, disruption or slippage of the fundoplication, and other structural abnormalities can be identified [64]. Late postoperative dysphagia can be investigated by a combination of manometry and esophageal fluoroscopic examination. Increases in esophago-gastric transit time of liquid barium and solid boluses correlate positively with the presence of postoperative dysphagia [65].

r structural abnormalities can be identified [64]. Late postoperative dysphagia can be investigated by a combination of manometry and esophageal fluoroscopic examination. Increases in esophago-gastric transit time of liquid barium and solid boluses correlate positively with the presence of postoperative dysphagia [65]. Ultrasonography Conventional ultrasonography have reported to be a reliable non invasive method to detect reflux events and as well to describe anatomical conditions such as hiatal hernia, length and position of the LES and the magnitude of the gastro-esophageal angle of His. Although conventional sonography is not a diagnostic tool for achalasia, it provides interesting sonographic information. It cannot reveal each layer of the wall of the lumen as endoscopic ultrasound does, but it may tentatively differentiate achalasia from malignancies and assists clinicians when endoscopic ultrasound is not available [66]. Few improvements have been introduced for studying esophageal function, i.e high-frequency intraluminal ultrasound, whereas conventional techniques, such as manometry, have undergone substantial upgrades because of advances in transducer technology, computerization, and graphic data presentation. Although this techniques provide both novel and more detailed information regarding the measure of the esophageal contractility and the thickness of esophageal muscle, it is still unclear whether they have improved the ability to diagnose and treat patients more effectively [67,68]. Ultrasonography is not recommended as a test for GERD for its low sensitivity and specificity.

led information regarding the measure of the esophageal contractility and the thickness of esophageal muscle, it is still unclear whether they have improved the ability to diagnose and treat patients more effectively [67,68]. Ultrasonography is not recommended as a test for GERD for its low sensitivity and specificity. Last, dinamic ultrasound may be useful for the study of the gastric emptying time [69,70]. Antral measurements are made before and immediately after the end of the test meal (time 0), and at regular 30-min intervals up to 180 min after the meal. In each patient, the gastric emptying rate was expressed as percent reduction in antral cross sectional area from time 0 to 120 min after meal ingestion [71]. Gastric emptying assessed by a non-invasive technique as ultrasonography is particularly suitable for young patients even if it is time consuming and investigator dependent [72].

the gastric emptying rate was expressed as percent reduction in antral cross sectional area from time 0 to 120 min after meal ingestion [71]. Gastric emptying assessed by a non-invasive technique as ultrasonography is particularly suitable for young patients even if it is time consuming and investigator dependent [72]. In young children suspected of GERD, the gastroesophageal junction was examined with ultrasonography directly after a feeding while these children were on overnight extended esophageal pH monitoring (EEpHM). The two tests showed 81% to 84% agreement in the detection of the presence or absence of GER, depending on whether the whole period of EEpHM or only the part of it covering the ultrasound observation period [73]. The two studies probably measure different aspects of clinically significant reflux and must be correlated with the clinical symptoms. Morphological findings associated with significant reflux were: (1) a short intra-abdominal part of the esophagus, (2) a rounded gastroesophageal angle, and (3) a "beak" at the gastroesophageal junction. Barium meal findings confirmed these sonographic signs, indicating a sliding hiatal hernia of the distal esophagus, either fixed or intermittent. Ultrasonography can be recommended as a useful and physiological screening test to demonstrate clinically significant GER and a predisposing hiatal hernia of the esophagus in symptomatic children but it is not routinarily used in the diagnosis of GERD.

iatal hernia of the distal esophagus, either fixed or intermittent. Ultrasonography can be recommended as a useful and physiological screening test to demonstrate clinically significant GER and a predisposing hiatal hernia of the esophagus in symptomatic children but it is not routinarily used in the diagnosis of GERD. Scintigraphy Gastroesophageal reflux and clearance of the refluxed material can be measured by plotting a time-activity curve from an esophageal area of interest after 1 mCi of99mTc sulfur colloid is placed in the stomach. Control subjects do not have peaks exceeding a value twice that of the baseline count levels. Reflux patients exceed this value, either spontaneously or after Valsalva maneuvers. This technique has a sensitivity which is greater than that of barium and equal to the sensitivity of a pH probe in patients with both moderate and severe reflux. Scintigraphic reflux was shown in 62% of moderate refluxes and 85% of those with severe reflux as defined clinically. This test can be performed rapidly with minimal radiation exposure and is noninvasive [74]. The sensitivity of the milk scan compared to pH probe for diagnosis of esophageal reflux is 15-59% that is low whilst specificity is much higher since it is 83-100% [75]. Scintigraphy in children with GERD can provide information on postprandial reflux and delayed gastric emptying [76]. Besides, the 1-hr scintigraphic study formatted in 60-sec frames provides a quantitative representation of postprandial gastroesophageal reflux for children, particularly if they do not have rapid gastric emptying [77]. Even its ability to identify reflux and gastric emptying time, the routine diagnosis and management of GERD in infants and children does not comprise scintigraphy.

s provides a quantitative representation of postprandial gastroesophageal reflux for children, particularly if they do not have rapid gastric emptying [77]. Even its ability to identify reflux and gastric emptying time, the routine diagnosis and management of GERD in infants and children does not comprise scintigraphy. Treatment The treatment of GER/GERD should be individually tailored according to the clinical manifestation and possible complications. Treatment options for regurgitation and GERD include conservative measures, dietary management, pharmacologic therapy and surgery. Table 2 contains the strategy step and the grade of recommendation for each of them [78]. Table 2 Therapeutic options in gastro-oesophageal reflux in neonates, infants and children according with the strategy steps and the grade of recommendation Therapeutic option Strategy Step Grade of recommendation Positioning 1 GRADE B (the left lateral position) Feed frequency 1 GRADE D Thickened formula or feed 2 GRADE B (for reducing vomiting) Domperidone 3 GRADE C/D Ranitidine/cimetidine + PPI 3 GRADE B/C (in relieving esophagitis)

Table 2 Therapeutic options in gastro-oesophageal reflux in neonates, infants and children according with the strategy steps and the grade of recommendation Therapeutic option Strategy Step Grade of recommendation Positioning 1 GRADE B (the left lateral position) Feed frequency 1 GRADE D Thickened formula or feed 2 GRADE B (for reducing vomiting) Domperidone 3 GRADE C/D Ranitidine/cimetidine + PPI 3 GRADE B/C (in relieving esophagitis) Surgery 4 Surgical intervention is rarely necessary in case of severe complications Conservative measures Because most cases are functional GER, reassurance is the only treatment needed [79]. Conservative measures may include upright positioning after feeding, elevating the head of the bed, prone positioning (infants >6 mo), and providing small, frequent feeds thickened with cereal [80]. Older children benefit from a diet that avoids tomato and citrus products, fruit juices, peppermint, chocolate, and caffeine-containing beverages. Smaller, more frequent feeds are recommended, as well a relatively lower fat diet because lipid retards gastric emptying [81]. Prone positioning may be recommended, at least for the first postprandial hour [82]. Clearly, the use of the prone position during infancy must be based on a careful risk-to-benefit analysis. When it is advised, only very firm bedding material (no pillows) must be used. Bed elevations offer no added advantage to the prone position, and seated positions are not recommended.

first postprandial hour [82]. Clearly, the use of the prone position during infancy must be based on a careful risk-to-benefit analysis. When it is advised, only very firm bedding material (no pillows) must be used. Bed elevations offer no added advantage to the prone position, and seated positions are not recommended. Dietary management Although some authors consider conservative therapy to be an efficient first choice for improving regurgitation even compared with thickened formula [79], the latter has been considered a reliable dietary management for decreasing recurrent regurgitation and/or vomiting in young infants [83]. Several thickening agents, i.e. rice cereal, gelatin, carob bean gum or galactomannan, have been successfully administered for the treatment of regurgitation in infants [84,85] and they provide a therapeutic advantage, particularly when excessive vomiting is associated with suboptimal weight gain [86]. Even for infants with normal weight gain, thickened and reduced volume feedings may reduce the frequency and amount of vomiting episodes, ameliorating the concerns of an anxious caregiver. Formula thickened with carob flour, locust bean gum, rice cereal or rice starch have been found to decrease episodes of regurgitation and vomiting as well as esophageal acid exposure [83,87]. Undesiderable side effects may occur, however, with various thickening agents. Orestein et al. reported an increase in coughing after infants were fed a formula enriched with rice cereal [88] and Takahashi et al. reported that soybean fibre decreased food consumption and weight gain in an animal model [89]. Clarke and Robinson [90] reported some cases of fatal necrotising enterocolitis in infants fed carob thickened milk.

n increase in coughing after infants were fed a formula enriched with rice cereal [88] and Takahashi et al. reported that soybean fibre decreased food consumption and weight gain in an animal model [89]. Clarke and Robinson [90] reported some cases of fatal necrotising enterocolitis in infants fed carob thickened milk. As a result, the last European Society for Pediatric Gastroenterology Hepatology and Nutrition (ESPGHAN) guidelines suggested avoidance of formula thickened with locust bean in infants up to six months because the possible risk of enterocolitis [91]. Thus, there is a need for alternative interventions to thickening agents in infants with recurrent regurgitation. Probiotic formulas have been shown to promote a regression of symptoms [71] without adverse growth or behavioral effects whilst the earlier demonstration of the safety and tolerance of probiotics in full term infants makes it suitable for use in this population [92]. Further understanding and elucidation of the mechanisms underlying the beneficial effects of probiotics on gastrointestinal symptoms and motility should provide new regimens for prevention and treatment of illness in infants. Another possible diet option could be feeding the infant with a formula supplemented with prebiotics. Prebiotics stimulate the gastric emptying so improving tolerance to enteral feeding, and this would be of clinical relevance. However, this hypothesis needs further evaluation [93].

and treatment of illness in infants. Another possible diet option could be feeding the infant with a formula supplemented with prebiotics. Prebiotics stimulate the gastric emptying so improving tolerance to enteral feeding, and this would be of clinical relevance. However, this hypothesis needs further evaluation [93]. The leading symptoms of GERD are present in the case of cow milk allergy. This disorder should be considered in preterm infants with recurrent vomiting and irritability [94]. Confirmation of this diagnosis and treatment consists of a trial of cow milk protein free formula. In some cases infants are also allergic to hydrolysate and so the only treatment is amino-acid based formula [13].

ergy. This disorder should be considered in preterm infants with recurrent vomiting and irritability [94]. Confirmation of this diagnosis and treatment consists of a trial of cow milk protein free formula. In some cases infants are also allergic to hydrolysate and so the only treatment is amino-acid based formula [13]. Pharmacologic therapy In the case of pharmacologic intervention, "step-up" therapy involves progression from diet and lifestyle changes to H2RAs and to PPI [3]. Both classes of acid antisecretory have proven safe and effective for both infants and children in reducing gastric acid output [95]. A specific target may be children with moderate-to-severe neurodevelopmental disabilities who typically have manifest dysphagia and gastroesophageal reflux, and present a high risk for aspiration [96,97]. In these patients, conservative therapy alone may not be sufficient in preventing reflux-associated complications. Overall, the therapeutic approach of GERD disease in infants and children needs to be well-balanced, considering therapeutic efficacy and side effects of the different therapeutic options [98]. Last, careful monitoring under optimal nonsurgical therapy should be conducted before considering operative intervention [96,99].

rall, the therapeutic approach of GERD disease in infants and children needs to be well-balanced, considering therapeutic efficacy and side effects of the different therapeutic options [98]. Last, careful monitoring under optimal nonsurgical therapy should be conducted before considering operative intervention [96,99]. H2RAs decrease acid secretion by inhibiting H2 receptors on gastric parietal cells [100]. The fairly rapid tachyphylaxis that develops with H2RAs is a drawback to chronic use. In some infants, H2RA therapy causes irritability, head banging, headache, somnolence and other side effects which, if interpreted as persistent symptoms of GER, could result in an inappropriate increase in dosage [101]. H2RAs, particularly ranitidine, are associated with an increased risk of liver disease, and cimetidine with gynecomastia [102].

y causes irritability, head banging, headache, somnolence and other side effects which, if interpreted as persistent symptoms of GER, could result in an inappropriate increase in dosage [101]. H2RAs, particularly ranitidine, are associated with an increased risk of liver disease, and cimetidine with gynecomastia [102]. PPIs inhibit acid secretion by blocking Na+, K+ ATP-ase, the final common pathway of parietal cell acid secretion, often called the proton pump. PPIs currently approved for use in children in North America are omeprazole, lansoprazole and esomeprazole. In Europe, only omeprazole is approved. No PPI has been approved for use in infants < 1 year of age. Most studies of PPIs in children have demonstrated the efficacy of PPIs in the controlling of symptoms and haeling of erosive esofagitis [103,104]. Children 1-10 yrs of age appear to have a greater metabolic capacity for some PPIs than adolescents and adults; that is, they require higher per kilogram doses to attain the same acid blocking effect, or area-under-the-curve [105]. There are few pharmacokinetic data for PPIs in infants, i.e. lansoprazole displays pharmacokinetic and pharmacodynamic parameters in children between 13 and 24 months of age similar to those observed in older children and adults [106]. Infants < 6 months may have a lower per kilogram dose requirement than older children and adolescents. In preterm infants and term neonates esomeprazole produces no change in bolus reflux characteristics despite significant acid suppression [107]. Last, a recent study detected no difference in efficacy between lansoprazole and placebo for symptoms attributed to GERD in infants age 1 to 12 months. Severe adverse events, particularly lower respiratory tract infections, occurred more frequently with lansoprazole than with placebo [108].

Finally, on the basis of our Italian experience we are confident that, as proposed by other Authors [2], the possible availability of a central European wide database on CH would represent a potent tool of surveillance, epidemiological research and knowledge on CH, and that the INRICH could strongly contribute to achieve this goal. Competing interests The authors declare that they have no competing interests. Authors' contributions AO prepared and wrote the manuscript. All the member of the SGCH contributed by giving their data on infants with congenital hypothyroidism to the Italian National Register of Infants with Congenital Hypothyroidism. All the authors read and approved the final manuscript. Acknowledgements The skillful technical assistance of Mrs F. Latini and Mrs M. Bocci are gratefully acknowledged.

cant acid suppression [107]. Last, a recent study detected no difference in efficacy between lansoprazole and placebo for symptoms attributed to GERD in infants age 1 to 12 months. Severe adverse events, particularly lower respiratory tract infections, occurred more frequently with lansoprazole than with placebo [108]. Surgery When medical therapy has failed, or when complications of gastroesophageal reflux are present [109], the antireflux operations may include partial or complete fundoplication and, if possible, the reduction of the hiatal hernia [110]. As pharmacotherapy has improved, the need for surgical therapy has markedly decreased. Nevertheless, antireflux surgery remains one of the most common surgical procedures performed during infancy and early childhood for refractory erosive oesophagitis or reflux aspiration [111]. Current guidelines from NASPGHAN [3] have reported the conditions in which surgery may be suggested. GERD with an atypical presentation, especially respiratory, whose symptoms are clearly associated with gastroesophageal reflux (i.e. obstructive apnea temporally associated with reflux during pH monitoring) should be considered for surgical treatment. However, a period of medical therapy (including acid blockade) under close monitoring conditions should be attempted in many cases prior to recommending a surgical approach. Besides, patients with complications of gastroesophageal reflux, such as aspiration, stricture of the esophagus, or Barrett esophagus should be considered for surgical treatment. In particular, children with pathologic reflux and neurologic impairment, that requires feeding gastrostomy and continuous medication should also be considered for surgery. For those infants who fail medical therapy, continuous intragastric administration of feeds via nasogastric tube is an option [112]. It is often used in preterm infants because of the significantly greater surgical risk in such patients. In these cases, adequate nutritional management, in conjunction with appropriate medical therapy, may permit the infant to "outgrow" reflux while optimizing weight gain.

eeds via nasogastric tube is an option [112]. It is often used in preterm infants because of the significantly greater surgical risk in such patients. In these cases, adequate nutritional management, in conjunction with appropriate medical therapy, may permit the infant to "outgrow" reflux while optimizing weight gain. There are no controlled studies of fundoplication versus medical therapy and studies evaluating different surgical treatments. In fact, there is no randomization of children undergoing partial versus complete wraps, even if some studies suggest that the results of partial one was better than those of Nissen fundoplication [112]; there are no clinical trials comparing laparoscopic antireflux surgery versus open antireflux ones. Only retrospective reviews and case series have been performed demonstrating laparoscopic antireflux procedures safe and effective once the learning curve has been achieved [113]. Complications of fundoplication include dysphagia for solid food, gas bloat syndrome, wrap herniation and dumping syndrome.

antireflux ones. Only retrospective reviews and case series have been performed demonstrating laparoscopic antireflux procedures safe and effective once the learning curve has been achieved [113]. Complications of fundoplication include dysphagia for solid food, gas bloat syndrome, wrap herniation and dumping syndrome. Conclusion Standard approaches to infants who regurgitate gastric contents (often the overflow from an overly generous feeding) differ from that recommended for children who reflux and have resultant disease manifestations (GERD). For infants with functional GER, a rational and conservative approach is to reassure the parents of the benign nature of the "spitting". Pathologic gastroesophageal reflux or gastroesophageal reflux disease refers to infants with regurgitation and vomiting associated with poor weight gain, respiratory symptoms, esophagitis. In such case clinical and instrumental diagnosis are needed. Among the latter upper radiology, pH-testing and MII testing are useful for diagnosis. Endoscopy and biopsy are performed in the case of esophagitis. The therapy with H2 receptor antagonist is currently suggested.

ight gain, respiratory symptoms, esophagitis. In such case clinical and instrumental diagnosis are needed. Among the latter upper radiology, pH-testing and MII testing are useful for diagnosis. Endoscopy and biopsy are performed in the case of esophagitis. The therapy with H2 receptor antagonist is currently suggested. List of Abbreviations AGA: American Gastroenterology Association; EE: Eosinophilic esophagitis; ESPGHAN: European Society for Pediatric Gastroenterology Hepatology and Nutrition; EEpHM: Extended esophageal pH monitoring; H2RA: Histamine2 receptor antagonist; LES: Lower esophageal sphincter; GABA: Gamma-aminobutyric acid; GER: Gastroesophageal reflux; GERD: Gastroesophageal reflux disease; NASPGHAN: North American Society for Pediatric Gastroenterology Hepatology and Nutrition; pH-MII: pH-Multichannel intraluminal impedance; PPI: Proton pump inhibitor; RI: Reflux index. Competing interests The authors declare that they have no competing interests. Authors' contributions FI: Conceived the study and helped draft the manuscript. GR: Drafted the manuscript. FR: Participated in the drafting and polishing the manuscript in the diagnostic approach section. LC: Participated in its design and coordination RF: Participated in its design and coordination. All authors read and approved the final manuscript.

Introduction The main objective of neonatal screening, the eradication of mental retardation after congenital hypothyroidism (CH), has been achieved in all the countries where nationwide screening programmes have been established [1-3]. In Italy the nationwide newborn screening programme for CH began in 1977 as a pilot program in 7 laboratories and then progressively developed all over the country. The 100% coverage of neonatal population has been achieved since the 90's thanks to an efficient network of 26 regional and inter-regional Screening and Follow-up Centres active in our country. At present in all the Italian Screening Centers a biochemical assessment of TSH, and in 11 of the 26 centres also of T4, is performed on dried spot blood within a few days from birth. In all the Centers positive results of screening tests are confirmed by definitive tests of thyroid function on serum. These include TSH, free T4 (FT4) and/or T4. Thyroid ultrasound and/or scintigraphy are generally performed to complete the CH diagnosis. Infants with confirmed primary CH are then referred to the Follow-up Center of their own region for starting replacement therapy. According to international guidelines [1,4,5], when the definitive diagnosis is not established in the neonatal period and a suspicion of transient primary hypothyroidism is present, a reevaluation of diagnosis is performed at the age of 3 years after a withdrawal of the replacement therapy to ascertain the persistence of CH. At that time, serum T4, FT4, and TSH levels are measured, and ultrasound imaging, scintigraphy, and clinical evaluation are performed to establish the definitive diagnosis.

t, a reevaluation of diagnosis is performed at the age of 3 years after a withdrawal of the replacement therapy to ascertain the persistence of CH. At that time, serum T4, FT4, and TSH levels are measured, and ultrasound imaging, scintigraphy, and clinical evaluation are performed to establish the definitive diagnosis. All the Italian Centres in charge of screening, diagnosis and follow-up of infants with CH participate in the Italian National Register of Infants with Congenital Hypothyroidism (INRICH), which performs the nationwide surveillance of the disease. The INRICH was established in 1987 as a program of the Health Ministry [6] and is coordinated by the Istituto Superiore di Sanità. The aim of the INRICH is to monitor efficiency and effectiveness of neonatal screening, to provide disease surveillance and to allow identification of possible aetiological risk factors for CH. Information on new cases with CH are collected in the INRICH by means 3 questionnaires filled in at diagnosis. These include anonymous data concerning CH infants such as screening and confirmatory laboratory tests, information on demographic data, details on clinical state in neonatal period, diagnostic investigations (biochemical determinations, radiography of the knee, thyroid scintigraphy, and ultrasound), information regarding pregnancy, birth, and family background, starting and dose of the replacement therapy. It is important to note that babies with transient hyperthyrotropinemia on the basis of spontaneous normalization of TSH between screening and diagnosis are not recorded in the Register. Since 1991, the INRICH started collecting specific data on the occurrence of congenital anomalies (detected during neonatal period) other than those of the thyroid gland by using a specific reporting form. The Screening Centres are responsible for collecting information in the questionnaires and for the accuracy of their compilation. Data are coded and stored in an informed database at the Istituto Superiore di Sanità and results of the Register are reported in a web site , presented yearly in a national conference, and published in international scientific journals.

cting information in the questionnaires and for the accuracy of their compilation. Data are coded and stored in an informed database at the Istituto Superiore di Sanità and results of the Register are reported in a web site , presented yearly in a national conference, and published in international scientific journals. Discussion During the past twenty years the active and continuous collaboration between the INRICH and the Italian Screening and Follow up Centres allowed to perform a standardization of screening procedures and considerable improvements in the time at starting treatment and in the dose of therapy. In fact, while the median value of infant's age at starting therapy was 23 days between 1987 and 1999, the last analysis of the INRICH data (performed on data referred to babies born between 2000 and 2004) confirmed a reduction of this value (19 days) with significant differences among the 3 diagnoses: agenesia 16 days; ectopia: 15 days; in situ thyroid: 23 days. Similar improvements have been also obtained in dose of L-T4 at starting therapy. The median value of L-T4 dose was 8.0 μg/Kg/day between 1987 and 1999, and 9.6 μg/Kg/day between 2000 and 2004. An analysis on data referring to 2005–2008 is going on and the results are expected further improved.

n situ thyroid: 23 days. Similar improvements have been also obtained in dose of L-T4 at starting therapy. The median value of L-T4 dose was 8.0 μg/Kg/day between 1987 and 1999, and 9.6 μg/Kg/day between 2000 and 2004. An analysis on data referring to 2005–2008 is going on and the results are expected further improved. The INRICH has also allowed to well characterize the Italian population of babies with CH. As expected, in Italy the frequency of the disease is higher in female than in male babies with a F/M sex ratio = 1.7. However, when thyroid disgenesis is considered separately from in situ thyroid, the sex ratio results: F/M = 2.0 among babies with thyroid disgenesis and F/M = 1.0 among those with in situ thyroid. Moreover, the INRICH data have shown that scintigraphy and/or ultrasonography is performed in the 64% of CH babies before starting therapy and that the different diagnoses are distributed as follows among babies with permanent CH: 40% ectopy, 26% agenesis, 34% in situ thyroid .

M = 1.0 among those with in situ thyroid. Moreover, the INRICH data have shown that scintigraphy and/or ultrasonography is performed in the 64% of CH babies before starting therapy and that the different diagnoses are distributed as follows among babies with permanent CH: 40% ectopy, 26% agenesis, 34% in situ thyroid . The high number of CH infants with confirmed diagnosis recorded in the INRICH (about 3800 at the end of 2007) and the fact that the INRICH is a population-based register have allowed to perform a robust estimation of the CH incidence in our country. This results to be 1:2400 live borns (1995–2003). It is important to note that this estimation is based only on cases with permanent forms of CH. In fact, all the cases with transient hypothyroidism, ascertained by means a re-evaluation of the diagnosis after a withdrawal of the replacement therapy at 3 years of age, are not considered in the incidence estimation. Moreover, to avoid the possibility of including some cases with transient hypothyroidism not re-evaluated yet, only data regarding children older than 3 years at the time of analysis are used. This methodology avoids the danger of drawing conclusions from an overestimation of the CH incidence and allows to ascertain the real impact of this condition on the Italian newborn population. On this regard, the INRICH data have also shown a high frequency of twins in the CH population with a proportion 3-fold higher in the CH population (3.5%) than in the Italian general population (1.1%) [7,8]. For the first time the INRICH data have allowed to estimate the CH incidence in multiple and single deliveries separately. It was found 3-fold higher in multiple (10.1 per 10,000 live births) than in single deliveries (3.2 per 10,000 live births) with a relative risk of CH occurrence in twin deliveries of 3.1 (95% CI, 2.5–3.9) [7]. Moreover, the analysis of re-evaluated infants with high suspicion of transient hypothyroidism recorded in the INRICH has shown a twin prevalence of 1.9% among infants who were affected by permanent CH and 13.2% in those who resulted affected by transient CH. Taken together these findings have demonstrated an increased risk for both permanent and transient CH in multiple than in single deliveries.

hypothyroidism recorded in the INRICH has shown a twin prevalence of 1.9% among infants who were affected by permanent CH and 13.2% in those who resulted affected by transient CH. Taken together these findings have demonstrated an increased risk for both permanent and transient CH in multiple than in single deliveries. This increased CH risk in multiple pregnancies has important implications in terms of public health given the high number of induced pregnancies, in Italy as well as in other Western countries, because of the increasing use of techniques of assisted reproduction and drugs inducing ovulation [9,10].

hypothyroidism recorded in the INRICH has shown a twin prevalence of 1.9% among infants who were affected by permanent CH and 13.2% in those who resulted affected by transient CH. Taken together these findings have demonstrated an increased risk for both permanent and transient CH in multiple than in single deliveries. This increased CH risk in multiple pregnancies has important implications in terms of public health given the high number of induced pregnancies, in Italy as well as in other Western countries, because of the increasing use of techniques of assisted reproduction and drugs inducing ovulation [9,10]. It is important to underline that, beside an efficient nationwide surveillance of the disease, the large amount and the high quality of information collected in the INRICH during these twenty years provided a unique opportunity for research into this condition. This because data collected in the INRICH are highly representative as referred to the entire Italian population of infants with CH. One of the most important results concerns the well known association between congenital malformations and CH [11]. Given the large CH population recorded in the INRICH, it has been possible to study a high number of CH infants with extrathyroidal malformations and compare these data with those of the International Clearinghouse for Birth Defects, the worldwide database collecting information on infants born with congenital malformations [12]. By using this approach, it has been possible to demonstrate that not all congenital extrathyroidal malformations but only anomalies of heart, nervous system, eyes (representing precocious structures in the developing embryo) and multiple congenital malformations are significantly associated to CH [13]. These findings have strongly suggested a very early impairment in the first stages of embryo development with a consequent involvement of different organs and structures. For what specifically concerns cardiac congenital malformations, the comparison of the INRICH data with other national and/or regional registries of congenital malformations included in the database of the International Clearinghouse for Birth Defects, have demonstrated that the most frequent cardiac malformations in the CH population were represented by the atrial septal defects with a rate of 13.8 of 1000 [10]. This finding is different from what found in the general population in which the most frequent cardiac anomalies (3 of 1000) are represented by ventricular septal defects [14]. These peculiar results oriented molecular biologists to focus their investigations on genes involved in both heart and thyroid development.

0]. This finding is different from what found in the general population in which the most frequent cardiac anomalies (3 of 1000) are represented by ventricular septal defects [14]. These peculiar results oriented molecular biologists to focus their investigations on genes involved in both heart and thyroid development. Therefore, as many point mutations have had identified in NKX2.5 trascription factor in families with atrial septal defects [15], the possible involvement of NKX2.5 mutations in thyroid disgenesis was investigated both in vivo model and humans. It was found that mutations in this gene can contribute to thyroid disgenesis phenotype [16].

point mutations have had identified in NKX2.5 trascription factor in families with atrial septal defects [15], the possible involvement of NKX2.5 mutations in thyroid disgenesis was investigated both in vivo model and humans. It was found that mutations in this gene can contribute to thyroid disgenesis phenotype [16]. It has been demonstrated that CH is a multigenic disease [17-20]. However, the occurrence of mutations in genes known to be involved in the development of the disease have been observed only in a small proportion of the CH patients. Moreover, the aetiological role of specific environmental risk factors has not completely elucidated yet. These considerations imply that the etiology of CH is still largely unknown and that further efforts to identify new genetic markers and modifiable (environmental) risk factors are needed to allow an efficient primary prevention of the disease. To this end a population-based case control study was carried out to identify the most important risk factors for permanent and transient forms of CH on the basis of information collected in the INRICH questionnaires [21]. This study showed that many risk factors contribute to the aetiology of CH suggesting a multifactorial origin of the disease in which genetic and environmental (especially iodine deficiency and maternal diabetes) risk factors play a role in the development of the disease. The multifactorial origin of CH was also supported by results obtained in the above mentioned study on CH twins recorded in the INRICH between 1989 and 2000 [7]. This study showed that, despite a low concordance rate (4.3%) for permanent CH observed among twins at birth, a high recurrence risk for the disease was present among siblings of CH cases (Sibling Recurrence Risk = 35.4; 95%CI: 4.7 – 269.3). These findings strongly suggested the occurrence of non-inheritable postzygotic events in the aetiology of CH and that environmental risk factors may act as a trigger on a susceptible genetic background in the aetiology of the disease.

ent among siblings of CH cases (Sibling Recurrence Risk = 35.4; 95%CI: 4.7 – 269.3). These findings strongly suggested the occurrence of non-inheritable postzygotic events in the aetiology of CH and that environmental risk factors may act as a trigger on a susceptible genetic background in the aetiology of the disease. Conclusion The surveillance of CH carried out by the INRICH together with the early diagnosis made by the nationwide screening programme, the prompt treatment and the appropriate clinical management of the patients performed by the Italian Follow-up Centres for CH, are elements of an integrated approach to CH which has been successfully established in our country. However, despite the important results obtained in terms of standardization of screening procedures and improvements in time and dose at starting treatment, we are conscious that further efforts have to be made to improve the diagnosis of CH and to make more precocious the therapy establishment in all affected infants. To this end national recommendations on diagnosis and follow-up of CH, resulting from the integration of available guidelines [1,5,22], the Italian Screening and Follow-up Centres experience, and information derived from the INRICH surveillance activity, are needed. These would help to carry out a process of harmonization and optimization of the Italian Screening programme with a consequent guarantee of an optimal quality of life to all CH infants.

2], the Italian Screening and Follow-up Centres experience, and information derived from the INRICH surveillance activity, are needed. These would help to carry out a process of harmonization and optimization of the Italian Screening programme with a consequent guarantee of an optimal quality of life to all CH infants. For what concerns the INRICH research activity, the results derived from the epidemiological studies performed in these years have contributed to deepen knowledge of CH, to start identifying the most important risk factors for the disease, and to orient molecular biologists towards the identification of new genes involved in the aetiology of the disease. At present, further collaborative research studies based on the INRICH database are going on and our efforts in the field of CH risk factors identification are continuing with the aim of making possible, in a near future, an efficient primary prevention of a disease which still represents the most frequent endocrinopathy in infancy. Finally, on the basis of our Italian experience we are confident that, as proposed by other Authors [2], the possible availability of a central European wide database on CH would represent a potent tool of surveillance, epidemiological research and knowledge on CH, and that the INRICH could strongly contribute to achieve this goal. Competing interests The authors declare that they have no competing interests.

Authors' contributions AO prepared and wrote the manuscript. All the member of the SGCH contributed by giving their data on infants with congenital hypothyroidism to the Italian National Register of Infants with Congenital Hypothyroidism. All the authors read and approved the final manuscript. Acknowledgements The skillful technical assistance of Mrs F. Latini and Mrs M. Bocci are gratefully acknowledged. The Study Group for Congenital Hypothyroidism includes: R. Altamura (Brindisi), U. Angeloni (Roma), I. Antonozzi (Roma), M. Baserga (Catanzaro), R. Berardi (Siena), S. Bernasconi (Parma), G. Bona (Novara), I. Bucci (Chieti), M. Burroni (Fano), F. Calaciura (Catania), R. Caldarera (Messina), M. Cappa (Roma), U. Caruso (Genova), M. R. Casini (Cagliari), A. Cassio (Bologna), L. Cavallo (Bari), R. Cerone (Genova), G. Cesaretti (Pisa), V. Cherubini (Ancona), F. Chiarelli (Chieti), G. Chiumello (Milano), M. Cicchetti (Campobasso), M. P. Ciccio' (Messina), A. Cicognani (Bologna), A. Coppola (Napoli), C. Corbetta (Milano), R. Cordova (Potenza), A. Correra (Napoli), P. Costa (Roma), F. Dammacco (Bari), F. De Luca (Messina), C. De Santis (Torino), S. Di Maio (Napoli), G. Gallicchio (Potenza), R. Gastaldi (Genova), G. Grasso (Caltanissetta), R. Gurrado (Taranto), L. Lasciarrea (Bari), A. Lelli (Roma), D. Leonardi (Catania), A. Liotta (Palermo), S. Loche (Cagliari), R. Lorini (Genova), G. Manente (Taranto), G. Minelli (Foggia), F. Monaco (Chieti), L. Moschini (Roma), M. A. Musaro' (Siena), T. Narducci (Foggia), S. Pagliardini (Torino), L. Palillo (Palermo), G. Parlato (Catanzaro), E. Pasquini (Firenze), L. Peruzzi (Siena), A. Pinchera (Pisa), M. Pizzolante (Lecce), G. Radetti (Bolzano), F. Righetti (Bologna), A. Rizzo (Lecce), G. Saggese (Pisa), D. Sala (Napoli), M.C. Salerno (Napoli), R. Salti (Firenze), L. Sava (Catania), D. Scognamiglio (Napoli), V. Stoppioni (Fano), L. Tato' (Verona), M. Tonacchera (Pisa), R. Vigneri (Catania), G. Vignola (Potenza), M.C. Vigone (Milano), C. Volta (Parma), and G. Weber (Milano).

ologna), A. Rizzo (Lecce), G. Saggese (Pisa), D. Sala (Napoli), M.C. Salerno (Napoli), R. Salti (Firenze), L. Sava (Catania), D. Scognamiglio (Napoli), V. Stoppioni (Fano), L. Tato' (Verona), M. Tonacchera (Pisa), R. Vigneri (Catania), G. Vignola (Potenza), M.C. Vigone (Milano), C. Volta (Parma), and G. Weber (Milano). E. Medda, C. Fazzini, S. De Angelis, M.A. Stazi, M. Sorcini at the Istituto Superiore di Sanità, Roma.

Background Italy has traditionally been a country of emigration, with immigration being a relatively recent phenomenon beginning in the mid-eighties. Nowadays, immigrants in Italy are 3.432.651, representing 5.8% of total population [1]. Mainly, immigrants come from developing countries that are economically disadvantaged compared with Italy. One of the most worrisome political issues regarding immigration is the ability of healthcare institutions to serve the increasing number of immigrants and to provide equity of access to healthcare services among this population. The Italian National Health System provides universal access to healthcare; nevertheless, often the utilisation of primary and specialized care might present several barriers for foreign people, including lack of knowledge about how to access these services and difficulties in making an appointment (language, time schedules, waiting lists, etc.). Therefore, it is possible that foreign people tend to access to emergency departments more easily than to primary and specialised care services. It has to be taken into account that economic indicators of poverty have been related to poorer health and higher emergency units utilisation [2]. Previous studies describing healthcare services use by immigrants tended to highlight their overall under-utilisation of services compared with non-immigrants, the preponderant access to public units over the private sector, and the greater use of emergency rooms to counteract access barriers to other health services [3-5]. Few studies analysing worldwide paediatric healthcare services utilisation showed a similar disadvantaged condition for foreign children, too, resulting in an excessive and often improper use of emergency units [6,7]. Up to now, paediatric emergency services utilisation in Italy has not yet been studied in depth. We aimed to quantify the amount of accesses to emergency units by immigrant and Italian population analysing one-year activity of ten Italian public paediatric hospitals.

ive and often improper use of emergency units [6,7]. Up to now, paediatric emergency services utilisation in Italy has not yet been studied in depth. We aimed to quantify the amount of accesses to emergency units by immigrant and Italian population analysing one-year activity of ten Italian public paediatric hospitals. Therefore, we evaluated admissions to paediatric emergency units of foreign and Italian children, in order to test the hypothesis that the emergency department utilisation would be higher, and consequently often inappropriate, among immigrants than among the host population. Secondary aim was to identify the causes of consultation for foreign and Italian patients and to verify whether immigrants suffer from specific import diseases. Methods Between January and December 2007 a multicentric cross-sectional study was undertaken in the paediatric emergency department of ten Italian public hospitals. Clinical notes for all foreign patients from developing countries outside the European Union admitted to the paediatric emergency departments were recorded by a medical interviewer. A patient was considered as foreign if one or both parents were born outside Italy and the European Union. Each foreign patient was matched with the following Italian patient admitted to the same paediatric emergency department, whose clinical notes were collected, too.

partments were recorded by a medical interviewer. A patient was considered as foreign if one or both parents were born outside Italy and the European Union. Each foreign patient was matched with the following Italian patient admitted to the same paediatric emergency department, whose clinical notes were collected, too. Data about age, gender, country of birth, maternal and paternal nationality, time of admission, cause of consultation and severity code were recorded for each child enrolled. Patients were divided into five groups according to age: newborns if <1 month, infants if 1 month – 1 year, preschool-aged children if 1 – 5 year, school-aged children if 6 – 10 years, adolescents if >11 years. Children and their parents' nationality was classified into nine groups to simplify the analysis: 1. Italy, 2. non-EU Eastern Europe, 3. Latin America, 4. Nile Valley, 5. Northern Africa, 6. sub-Saharan Africa, 7. India, Pakistan and Sri-Lanka, 8. China and the rest of Asia, 9. nomads and Gypsies. Time of admission was recorded as day-time if admission to the emergency department took place between 8 a.m. and 8 p.m. and night-time if between 8 p.m. and 8 a.m. Cause of consultation was synthesized with the main symptom complained at admission. Evaluating the severity of the symptoms complained, patients were sorted into four categories identified by four different colours according to Canadian Pediatric Triage and Acuity Scale Guidelines [8]: white if non-urgent (cases and situations that could also be managed by the family Paediatrician – these patients will be examined only after the most serious cases have been managed), green if semi-urgent (cases with postponable health troubles – there is no risk of death and the patient will be cared for after more urgent cases), yellow if urgent (patients with the risk of fast prejudice of vital functions), red if emergency (cases with immediate risk of death). The diagnosis at discharge was analyzed according to a broad spectrum of variables: respiratory diseases, injuries, gastroenteric diseases, skin affections, fever, localized infections, childhood infectious diseases, genitourinary infections, foreign bodies ingestion or inhalation, poisoning, surgical problems and burns. Discharge-related circumstances (discharge to home, further outpatient investigations, temporary observation, hospital admission, voluntary discharge against the physician's recommendation or death) were also evaluated.

ourinary infections, foreign bodies ingestion or inhalation, poisoning, surgical problems and burns. Discharge-related circumstances (discharge to home, further outpatient investigations, temporary observation, hospital admission, voluntary discharge against the physician's recommendation or death) were also evaluated. The study has been approved by the local Ethical Committee and informed consent was signed by the parents. Statistical analysis was performed with SPSS 16.0 software (SPSS Inc, Chicago, IL). Results Study population In the period analyzed in the survey, 2437 foreign citizens referred to the paediatric emergency departments of the hospitals taking part to this multicentric study and were enrolled for the survey. As many Italian patients admitted to the same paediatric emergency department were enrolled. Among immigrant patients 1409 (57.8%) were males and 1028 (42.2%) females; among Italian ones, 1368 (56.1%) were males and 1069 (43.9%) females. Out of the 2437 foreign subjects, 35 (1.4%) were newborns, 519 (21.3%) infants, 1110 (45.6%) preschool-aged children, 447 (18.3%) school-aged children and 326 (13.4%) adolescents. Out of the 2437 Italian subjects, 34 (1.4%) were newborns, 336 (13.8%) infants, 1193 (49%) preschool-aged children, 424 (17.4%) school-aged children and 450 (18.4%) adolescents.

(1.4%) were newborns, 519 (21.3%) infants, 1110 (45.6%) preschool-aged children, 447 (18.3%) school-aged children and 326 (13.4%) adolescents. Out of the 2437 Italian subjects, 34 (1.4%) were newborns, 336 (13.8%) infants, 1193 (49%) preschool-aged children, 424 (17.4%) school-aged children and 450 (18.4%) adolescents. Analyzing the country of birth of foreign patients, 51.2% of subjects were born in Italy, 17.4% in non-UE Eastern European countries, 15.3% in Northern Africa, 7% in Latin America, 3% in sub-Saharan Africa, 1.7% in the Nile Valley/Arabian countries, 1.9% in China or other Asian countries, 0.9% in India, Pakistan or Sri-Lanka and 1.6% were nomads or Gypsies. Fathers came in most cases from Northern Africa (30.3%), followed by non-UE Eastern Europe (28%), Latin America (14.4%), sub-Saharan Africa (6.6%), China or other Asian countries (4.2%), the Nile Valley/Arabian countries (3.6%), Indian area (1.9%), Italy (0.7%). 9.4% of fathers had no fixed abode/were nomads; father nationality was unknown in 0.9%. Similarly, among the mothers the most frequent country of origin was Northern Africa (28.1%), followed by non-UE Eastern Europe (27.3%), Latin America (14.5%), sub-Saharan Africa (6.4%), China or other Asian countries (4.1%), the Nile Valley/Arabian countries (3.4%), Indian area (1.8%), Italy (0.7%). 9.1% of mothers had no fixed abode/were nomads; mother nationality was unknown in 4.6%.

Northern Africa (28.1%), followed by non-UE Eastern Europe (27.3%), Latin America (14.5%), sub-Saharan Africa (6.4%), China or other Asian countries (4.1%), the Nile Valley/Arabian countries (3.4%), Indian area (1.8%), Italy (0.7%). 9.1% of mothers had no fixed abode/were nomads; mother nationality was unknown in 4.6%. Access characteristics Both among immigrant and Italian patients most of paediatric emergency department visits took place during day-time, between 8 a.m. and 8 p.m. (76.4% and 78.8%, respectively). According to the severity of symptoms complained most of foreign patients (72.5%) were assigned a green code (semi-urgent), 25.2% of immigrant patients' admissions were sorted as white codes (non-urgent), 2.1% as yellow codes (urgent) and only 0.2% as red codes (emergency). Among the Italian patients, green codes accounted for 87.8% of total consultations, followed by white codes (9.8%), yellow codes (2.3%) and red codes (0.1%).

mi-urgent), 25.2% of immigrant patients' admissions were sorted as white codes (non-urgent), 2.1% as yellow codes (urgent) and only 0.2% as red codes (emergency). Among the Italian patients, green codes accounted for 87.8% of total consultations, followed by white codes (9.8%), yellow codes (2.3%) and red codes (0.1%). Analyzing the diagnosis at discharge, most of foreign patients had respiratory diseases (36.3%); 17.8% showed gastroenteric diseases, 12.8% were injured, 5% had skin affections, 5.9% fever, 4.3% localized infections, 3.2% childhood infectious diseases and 1.5% displayed genitourinary infections. Only a small proportion of foreign patients referred to the emergency department for foreign bodies ingestion or inhalation, poisoning, surgical problems or burns. Similarly, among Italian patients the most frequent diagnosis at discharge was a respiratory disease (32.7%), followed by accidental trauma or injuries (17.4%), gastroenteric diseases (15.1%), skin affections (6.6%), fever (6.2%), localized infections (4%), childhood infectious diseases (2.5%), genitourinary infections (1.9%), and by fewer cases of foreign bodies ingestion or inhalation, poisoning, surgical problems or burns. Given the high rate of respiratory diseases, these were analysed separately considering the subgroup pathologies (Table 1). At the end of consultation process 87% of immigrants were discharged to home, 11.9% were hospitalized. Only 7 (0.3%) foreign subjects required further outpatient investigations and 9 (0.4%) a temporary observation, 11 (0.4%) were voluntary discharged against the physician's recommendation. Among Italian patients, discharge to home occurred in 86.7%, hospital admission in 12.6%, temporary observation in 0.5%, voluntary discharge in 0.1% and further outpatients visits in 0.1%. Both between foreign and Italian patients no death was recorded.

were voluntary discharged against the physician's recommendation. Among Italian patients, discharge to home occurred in 86.7%, hospital admission in 12.6%, temporary observation in 0.5%, voluntary discharge in 0.1% and further outpatients visits in 0.1%. Both between foreign and Italian patients no death was recorded. Table 1 Respiratory diseases. RESPIRATORY DISEASES FOREIGN PATIENTS ITALIAN PATIENTS n % n % UPPER-AIRWAYS DISEASES 250 28.2 178 22.3 SINUSITIS 4 0.5 5 0.6 ACUTE OTITIS MEDIA 129 14.6 141 17.7 PHARYNGOTONSILLITIS 252 28.5 200 25.1 LARYNGITIS 36 4.1 44 5.5 INFLUENZAL SYNDROME 39 4.4 16 2.0 BRONCHITIS 64 7.2 94 11.8 BRONCHOSPASM 47 5.3 54 6.8 BRONCHIOLITIS 17 1.9 22 2.8 POLMONITIS 47 5.3 43 5.4 Total 885 100.0 798 100.0 Specific respiratory diagnoses in foreign and Italian patients.

TIS MEDIA 129 14.6 141 17.7 PHARYNGOTONSILLITIS 252 28.5 200 25.1 LARYNGITIS 36 4.1 44 5.5 INFLUENZAL SYNDROME 39 4.4 16 2.0 BRONCHITIS 64 7.2 94 11.8 BRONCHOSPASM 47 5.3 54 6.8 BRONCHIOLITIS 17 1.9 22 2.8 POLMONITIS 47 5.3 43 5.4 Total 885 100.0 798 100.0 Specific respiratory diagnoses in foreign and Italian patients. Discussion The most relevant result of our survey is the high rate of accesses to paediatric emergency departments for non-urgent or semi-urgent medical problems, both between foreign and Italian patients. Indeed, more than 95% of foreign and Italian children were assigned green or white codes. This finding suggests that a large proportion of the demand for emergency departments utilisation often may be attributed to visits for medical problems that do not require emergency treatment. Accident and emergency departments have been created in hospitals with the primary function of providing immediate care for patients with life-threatening medical conditions, trauma, or injuries, but not to treat minor illnesses or provide primary care. In recent years, the emergency department has increasingly become a major provider of health care and this overcrowding has become problematic. People tend to use emergency department as a substitute for general practitioner to treat minor illness. In the context of limited inpatient hospital resources, it is acknowledged that the phenomenon of non-urgent emergency departments visits, which can be managed alternatively and appropriately in general practice, has raised serious concerns among healthcare planners, both because of its magnitude and because the appropriate utilisation of hospital care can significantly improve health outcomes. In particular, the over-utilisation of emergency departments by paediatric patients has become an important problem that must be solved. Medical, social, economic and psychological factors mainly influence the parents' decision to visit the emergency department rather than manage their children at home, or prior to making an unscheduled visit to an emergency department their demands could have been satisfactorily met by an appropriate visit at a different health care level. Reports in literature indicate similar presenting problems in other studies performed in children attending paediatric accident and emergency departments in USA and UK [9-11], in Malaysia [12] and also in Italy [13-15].

demands could have been satisfactorily met by an appropriate visit at a different health care level. Reports in literature indicate similar presenting problems in other studies performed in children attending paediatric accident and emergency departments in USA and UK [9-11], in Malaysia [12] and also in Italy [13-15]. It is noteworthy that in our survey immigrants didn't access to emergency departments more than Italian children, in contrast with evidences reported in previous study [4,16,17]. Our finding might reflect a well-integrated foreign population. This may be due, on the one hand, to the fact that in the last twenty years of immigration foreign citizens learned how to utilize all the services offered by the Italian National Health System and when to access to primary and specialized care rather than to the emergency departments. On the other hand, well-integrated immigrants could be the result of a proper management by medical staff, trained to deal with migrants' specific health problems and able to overcome linguistic and logistic barriers. Immigrants' health concerns are strongly correlated with their country of origin, so that a broad spectrum of variables should be carefully taken into account, when considering both the migration phenomenon and the individual foreign citizen (i.e. nationality, reason of migration such as poverty, political and ethnic persecution or civil war, health standards in the country of origin, social and hygienic conditions in Italy).

variables should be carefully taken into account, when considering both the migration phenomenon and the individual foreign citizen (i.e. nationality, reason of migration such as poverty, political and ethnic persecution or civil war, health standards in the country of origin, social and hygienic conditions in Italy). Remarkably infectious diseases, initially thought to be a potential prominent problem in these immigrant populations, actually seem to be quite infrequent, when compared with overall morbidity. Immigrant populations are often considered as a source of many known and unknown infectious diseases, such as Ebola, SARS, TBC, malariae, etc. occasionally resulting in unjustified prejudice [18]. As a whole, in our survey infectious diseases accounted for 3.2% of foreign children visits and for 2.5% Italian children visits. Therefore immigrants should not be considered as infectious diseases carriers. Both among immigrant and Italian patients the most frequent presenting problems for visits to emergency departments were respiratory diseases, injury, and digestive symptoms as observed in other cross-sectional study [13], demonstrating that foreign patients and Italian ones suffer by the same pathologies.

es carriers. Both among immigrant and Italian patients the most frequent presenting problems for visits to emergency departments were respiratory diseases, injury, and digestive symptoms as observed in other cross-sectional study [13], demonstrating that foreign patients and Italian ones suffer by the same pathologies. Conclusion In conclusion, it is not possible to identify a specific "migrant type", remaining the same from a health care, social, economic, and anthropologic point of view. Several different features are typical of each immigrant, and they are generally consistent with the area of origin. Other differences emerge after the immigration process, and they are strictly related to life-style in Italy (i.e. hygienic conditions, nutritional status, etc). Therefore, specific knowledge is needed to face immigrants' different health problems. Under the perspective to better understand foreign patients health needs many instruments, such as translators and multilingual fliers concerning health problems, have to be used in emergency departments. Competing interests The authors declare that they have no competing interests.

Conclusion In conclusion, it is not possible to identify a specific "migrant type", remaining the same from a health care, social, economic, and anthropologic point of view. Several different features are typical of each immigrant, and they are generally consistent with the area of origin. Other differences emerge after the immigration process, and they are strictly related to life-style in Italy (i.e. hygienic conditions, nutritional status, etc). Therefore, specific knowledge is needed to face immigrants' different health problems. Under the perspective to better understand foreign patients health needs many instruments, such as translators and multilingual fliers concerning health problems, have to be used in emergency departments. Competing interests The authors declare that they have no competing interests. Authors' contributions ECG participated in the design of the study, performed the statistical analysis and draft the manuscript. CG participated in the design of the study, carried out the data and performed the statistical analysis. AM performed the statistical analysis and draft the manuscript. PDP participated in the design of the study and collection of data. GB conceived of the study and participated in its coordination and helped to draft the manuscript. All authors read and approved the final version of the manuscript.

Authors' contributions ECG participated in the design of the study, performed the statistical analysis and draft the manuscript. CG participated in the design of the study, carried out the data and performed the statistical analysis. AM performed the statistical analysis and draft the manuscript. PDP participated in the design of the study and collection of data. GB conceived of the study and participated in its coordination and helped to draft the manuscript. All authors read and approved the final version of the manuscript. Acknowledgements The authors would like to thank the coworkers of the multicentric study SIMEUP-GLNBI (Italian Society of Paediatrics), (Zampogna S. and Masciari P from Department of Pediatrics, Azienda Ospedaliera Pugliese, Ciaccio, Catanzaro, Italy; Piccotti E and Calcagno A from Emergency Room and Emergency Medicine Division, G. Gaslini Institute, Genova, Italy; Bartolini M and Acutis M. S from Department of Pediatrics, Ospedale Galliera, Genova, Italy; Zaffaroni M., Stasi I., Capelli A. and Acucella G. from Department of Pediatrics, AOU Maggiore della Carità, University of Piemonte Orientale, Novara, Italy; Calligari G. C. from Department of Pediatrics, Ospedale Fatebenefratelli, Erba (CO), Italy; Valentini P. from Department of Pediatrics, Policlinico Agostini Gemelli, Roma, Italy; Zavarise G. from Department of Pediatrics, Ospedale Sacro Cuore, Negrar (VR), Italy; Visci G from Department of Pediatrics, Ospedale Regionale Spirito Santo, Pescara, Italy: Guala A. from Department of Pediatrics, Ospedale SS. Pietro e Paolo, Borgosesia (VC), Italy; Lo Coco G. from Department of Pediatrics, Ospedale A. Ajello, Mazara del Vallo (TP), Italy) who contributed towards the study by the acquisition of data and the critical revision of the manuscript.

Background Respiratory Syncytial Virus (RSV) is the most common cause of viral respiratory tract infections in infancy. [1] The common presentation of RSV on infants are lower respiratory tract infections such as pneumonias and bronchiolitis occurring usually during the first two years of life. [2-6] In Italy about 4–5,000 RSV-infected, high-risk, preterm infants (gestational age < 36 weeks, with or without bronchopulmonary dysplasia [BPD]) are hospitalized every year. A proportion of these infants require admission to intensive care units due to severity of the condition and the level of care needed. [7] Mortality rates in hospitalized infants are high, reaching almost 4% during the first year of life. [8] Several prospective clinical studies have moreover demonstrated a strong correlation between RSV and recurrent wheezing/asthma episodes. These studies have more specifically noted that the airway hyperreactivity rate is higher by 50%–100% in RSV-infected infants compared to non-infected children.[9] Presence of recurrent wheezing episodes was observed through the age of 11 years, possibly extending throughout early adolescence.[4,6] In these past few years, the launch of the intramuscular humanized monoclonal antibody, palivizumab, has added to the therapeutic options available for RSV prophylaxis. This molecule demonstrated clinical efficacy and satisfactory tolerability. [10-13]

Several prospective clinical studies have moreover demonstrated a strong correlation between RSV and recurrent wheezing/asthma episodes. These studies have more specifically noted that the airway hyperreactivity rate is higher by 50%–100% in RSV-infected infants compared to non-infected children.[9] Presence of recurrent wheezing episodes was observed through the age of 11 years, possibly extending throughout early adolescence.[4,6] In these past few years, the launch of the intramuscular humanized monoclonal antibody, palivizumab, has added to the therapeutic options available for RSV prophylaxis. This molecule demonstrated clinical efficacy and satisfactory tolerability. [10-13] In consideration of the above, Simoes et al. [14] conducted a double blind cohort study with prospective follow-up to verify whether administration of palivizumab in high-risk preterm infants was capable not only of preventing RSV infections but also of reducing the number of possible subsequent recurrent wheezing episodes. Incidence rates of physician-diagnosed recurrent wheezing episodes over the 2-year duration of the study in preterm patients not requiring RSV hospitalization were noted to be significantly lower in the group of preterm infants receiving palivizumab prophylaxis rather than in children with no prophylaxis, showing rates of 8% versus 16%, respectively (p = 0,011).

rent wheezing episodes over the 2-year duration of the study in preterm patients not requiring RSV hospitalization were noted to be significantly lower in the group of preterm infants receiving palivizumab prophylaxis rather than in children with no prophylaxis, showing rates of 8% versus 16%, respectively (p = 0,011). In consideration of the findings obtained by Simoes et al. [14], it was considered appropriate to update the results of a prior cost-effectiveness analysis [15] comparing palivizumab versus absence of prophylaxis in the prevention of RSV infections in preterm infants of different gestational ages (less than 33 weeks, and 33 to 35 weeks) with or without complications (BPD). The present study intended to assess quality-adjusted and non-adjusted incremental cost-effectiveness ratios per year of life for palivizumab versus non-prophylaxis in the prevention of RSV infections in high-risk preterm infants. Methods Background The incremental cost-effectiveness analysis (Incremental Cost Effectiveness Ratio – ICER) was conducted from the perspective of the National Health System (NHS) taking into account the direct health care expenditure assessed in 2007 euros. Life-years (LY) and QALYs (Quality-Adjusted Life-Years) are the two outcomes assessed by the decision-making model. As a lifetime horizon was used in the study, a 3% discount rate was applied both to expenditure and outcomes. In particular, costs associated with prophylaxis and absence of prophylaxis were assessed for the first two years after enrollment.

justed Life-Years) are the two outcomes assessed by the decision-making model. As a lifetime horizon was used in the study, a 3% discount rate was applied both to expenditure and outcomes. In particular, costs associated with prophylaxis and absence of prophylaxis were assessed for the first two years after enrollment. Model In order to estimate cost-effectiveness ratios for palivizumab versus non-prophylaxis, a pre-existing decision-making tree model [15] (Figure 1) was updated based on the results published by Simoes et al. [14] Figure 1 General structure of the decision-making tree model. Figure 2 describes the development of only one of the possible branches foreseen by the decision-making model ("Prophylaxis – < 33 weeks"), since branches are all equivalent in terms of structure and are differentiated only in terms of the different odds assigned to the probabilistic nodes and the different health care expenditure. Figure 2 Detail of the "Prophylaxis, < 33 weeks" branch.

Figure 2 describes the development of only one of the possible branches foreseen by the decision-making model ("Prophylaxis – < 33 weeks"), since branches are all equivalent in terms of structure and are differentiated only in terms of the different odds assigned to the probabilistic nodes and the different health care expenditure. Figure 2 Detail of the "Prophylaxis, < 33 weeks" branch. The course of the "Prophylaxis, < 33 weeks" branch foresees that infants may or may not develop an RSV infection with ensuing hospitalization within 12 months post administration of prophylaxis. Based on the severity of the pediatric patient's conditions, he/she may be hospitalized in the ordinary ward (OW) or in the Intensive Care Unit (ICU). In either case, OW or ICU, preterm infants might die due to RSV. Subsequently, after the first hospitalization and once again within the 12 months following administration of prophylaxis, survivors might experience infection-related sequelae requiring rehospitalization or leading to death. Finally, patients who survive are followed for further 12 months to assess chances of developing recurrent wheezing episode 24 months after prophylaxis with palivizumab. On the other hand, in the event pediatric patients are not immediately hospitalized due to RSV infection, the model foresees the possibility of hospitalizations (and the relating respiratory infection-related mortality rate) within the 12-month period following enrollment (administration of prophylaxis) and of recurrent wheezing episodes in the subsequent 24 months.

ts are not immediately hospitalized due to RSV infection, the model foresees the possibility of hospitalizations (and the relating respiratory infection-related mortality rate) within the 12-month period following enrollment (administration of prophylaxis) and of recurrent wheezing episodes in the subsequent 24 months. Probability of events The probabilities of occurrence of events considered in the present study (Tables 1 and 2) are equal to those used in the original model [15], except for the probability of developing recurrent wheezing in the 24 months of follow-up after the enrolment. Table 1 Risk class and probability of hospitalization Prophylaxis Non prophylaxis Probability of occurrence 33–35 wks < 33 wks BPD 33–35 wks < 33 wks BPD Risk class - patient rate 11.0% 70.0% 19.0% 11.0% 70.0% 19.0% Probability of hospitalization - ordinary ward 1.5% 2.0% 5.6% 9.8% 10.3% 18.4% - Intensive therapy unit 1.3% 1.3% 1.3% 3.0% 3.0% 3.0% From: Ravasio R et al. [15] Table 2 Probability of events following the first hospitalization and number of events RSV-hospitalized subjects Subjects without RSV-hospitalization Probability of occurrence 33–35 wks < 33 wks BPD 33–35 wks < 33 wks BPD Probability of events following the first hospitalization - hospitalization during the first 12 months after enrolment 100.0% 100.0% 100.0% 60.0% 60.0% 60.0% - mortality throughout the first 12 months 4.0% 4.0% 4.0% 0.8% 0.8% 0.8% - probability of developing recurrent wheezing throughout the 24 months following enrolment with palivizumab [14] 17.0%* 17.0%* 17.0%* 8.0% 8.0% 8.0%

Probability of occurrence 33–35 wks < 33 wks BPD 33–35 wks < 33 wks BPD Probability of events following the first hospitalization - hospitalization during the first 12 months after enrolment 100.0% 100.0% 100.0% 60.0% 60.0% 60.0% - mortality throughout the first 12 months 4.0% 4.0% 4.0% 0.8% 0.8% 0.8% - probability of developing recurrent wheezing throughout the 24 months following enrolment with palivizumab [14] 17.0%* 17.0%* 17.0%* 8.0% 8.0% 8.0% - probability of developing recurrent wheezing throughout the 24 months following enrolment without palivizumab [14] 17.0% 17.0% 17.0% 16.0% 16.0% 16.0% Events - no. of hospitalizations due to respiratory causes during the first 12 months after enrollment 2.89 5.40 5.40 1.28 1.00 1.00 From: Ravasio R et al. [15]; *Conservative assumption based on study data published

- probability of developing recurrent wheezing throughout the 24 months following enrolment without palivizumab [14] 17.0% 17.0% 17.0% 16.0% 16.0% 16.0% Events - no. of hospitalizations due to respiratory causes during the first 12 months after enrollment 2.89 5.40 5.40 1.28 1.00 1.00 From: Ravasio R et al. [15]; *Conservative assumption based on study data published The latter clinical finding was calculated based on the results published by Simoes et al. [14]. The probability of developing recurrent wheezing in patients who were not hospitalized for RSV was noted to be 8% for palivizumab-treated subjects and 16% in subjects who did not receive prophylaxis. The data published by Simoes et al. [14] made it possible to calculate a 17% probability of developing recurrent wheezing in subjects who where hospitalized for RSV and who did not receive prophylaxis (13/76 patients). In order to complete the population of the present simulation model, the decision was taken to set at a conservative 17% the odds of developing recurrent wheezing in the 24 months of follow-up after the enrolment also for RSV-hospitalized patients treated with palivizumab; the odds being equal to the probability for patients without prophylaxis (Table 2). Use of Resources As with the original model, the use of resources refers to administration of prophylaxis, any hospitalizations required to treat RSV infections and treatment for recurrent wheezing.

0) was higher (+28.3%) compared to the estimated cost per patient without prophylaxis (€ 4,867.70). Therefore, due to the greater efficacy and greater costs associated with palivizumab versus the comparator (absence of prophylaxis), it was necessary to determine the incremental cost-effectiveness ratio per LY and QALY. The calculation of the incremental cost per life year gained was based on an overall mean cost per patient of € 6,244.20 for palivizumab and of € 4,867.70 for absence of prophylaxis, with a mean survival of 29.842 years and 29.754 years, respectively. The incremental cost for prophylaxis (€ 1,376.50) was thus compared to an incremental efficacy of 0.088 years, leading to a cost per life year gained of € 15,568.65. In the case of the incremental cost per QALY the same mean total costs were recorded per patient (€ 6,244.20 and € 4,867.70), with however different outcomes. Palivizumab is characterized by 29.202 QALYs and absence of prophylaxis is associated with 29.043 QALYs. Incremental efficacy in this case is equal to 0.159 QALYs; therefore the incremental cost per QALY is € 8,676.74. Taking into account patient subgroups, the incremental cost effectiveness ratio per LY ranges between € 4,332.29 and € 28,417.08, whereas those per QALY range between € 2,731.81 and € 14,937.32.

The latter clinical finding was calculated based on the results published by Simoes et al. [14]. The probability of developing recurrent wheezing in patients who were not hospitalized for RSV was noted to be 8% for palivizumab-treated subjects and 16% in subjects who did not receive prophylaxis. The data published by Simoes et al. [14] made it possible to calculate a 17% probability of developing recurrent wheezing in subjects who where hospitalized for RSV and who did not receive prophylaxis (13/76 patients). In order to complete the population of the present simulation model, the decision was taken to set at a conservative 17% the odds of developing recurrent wheezing in the 24 months of follow-up after the enrolment also for RSV-hospitalized patients treated with palivizumab; the odds being equal to the probability for patients without prophylaxis (Table 2). Use of Resources As with the original model, the use of resources refers to administration of prophylaxis, any hospitalizations required to treat RSV infections and treatment for recurrent wheezing. Outcome The present decision-making model, in accordance with the original study [15], has provided an estimate of life-years based on the various mortality odds associated with the different probabilistic nodes for patients with or without prophylaxis. Even quality-adjusted life-years (QALY) were estimated based on the method used in the original study. [15] They were calculated by adding, for the expected average life span, quality-weighted duration of the disease associated with the presence or absence of RSV [16,17](Table 3).

Outcome The present decision-making model, in accordance with the original study [15], has provided an estimate of life-years based on the various mortality odds associated with the different probabilistic nodes for patients with or without prophylaxis. Even quality-adjusted life-years (QALY) were estimated based on the method used in the original study. [15] They were calculated by adding, for the expected average life span, quality-weighted duration of the disease associated with the presence or absence of RSV [16,17](Table 3). Table 3 Quality of life Variable Value Health-related QoL score* (range 0–1) No RSV hospitalization 0.950 RSV hospitalization 0.880 From: Greenough A et al. [16], calculations based on Health Utility Index 2: Torrance GW et al. [17]. *QoL = Quality of Life Costs The € 3,099.84 cost for prophylaxis with palivizumab was calculated based on NHS reimbursements rates [18], and on the administration scheme foreseeing a recommended dosage of 15 mg per kilogram. The model is based on a palivizumab administration scheme of one 50 mg vial/month for the first three months and one 100 mg vial/month for the subsequent two months, for an overall cost of € 3,099.84. The overall dosage was calculated based on a conservative usage hypothesis, in other words expenditure includes also any unused active principle.

livizumab administration scheme of one 50 mg vial/month for the first three months and one 100 mg vial/month for the subsequent two months, for an overall cost of € 3,099.84. The overall dosage was calculated based on a conservative usage hypothesis, in other words expenditure includes also any unused active principle. Appreciation of hospitalization-related costs was based on the Interregional tariff agreement (Tariffa Unica Convenzionale). [19] In particular, with respect to RSV infection hospitalization in ordinary wards, DRG 98 was taken into account – Bronchitis and asthma, age < 18 years – equal to € 1,328.22, whereas for RSV admission to Intensive Care, reference was made to DRG 475- Diagnoses relating to the respiratory system with mechanical ventilation – equal to € 8,158.90. Finally for hospitalizations occurring for lower respiratory tract infections subsequent to the first hospitalization, DRG 81 – Respiratory infections and inflammations, age < 18 year – equal to € 3,729.54. [15] Since no data are available in published studies, the annual mean cost for the treatment of a pediatric patient with recurrent wheezing was appreciated based on the results of the SIRIO study [20], which had calculated the annual mean cost of an adult patient suffering from recurrent wheezing with respect to the Italian clinical setting (Table 4). Table 4 Annual mean cost for a patient with asthma Expenditure Breakdown Mean cost per Patient (€) Pharmacological therapy 457.82 Hospitalizations (ordinary admission and Day Hospital) 461.87 Admissions to Emergency Room 5.11 Visits 112.26 Examinations 127.76

Since no data are available in published studies, the annual mean cost for the treatment of a pediatric patient with recurrent wheezing was appreciated based on the results of the SIRIO study [20], which had calculated the annual mean cost of an adult patient suffering from recurrent wheezing with respect to the Italian clinical setting (Table 4). Table 4 Annual mean cost for a patient with asthma Expenditure Breakdown Mean cost per Patient (€) Pharmacological therapy 457.82 Hospitalizations (ordinary admission and Day Hospital) 461.87 Admissions to Emergency Room 5.11 Visits 112.26 Examinations 127.76 Specific immune therapy 31.86 Other 30.21

Since no data are available in published studies, the annual mean cost for the treatment of a pediatric patient with recurrent wheezing was appreciated based on the results of the SIRIO study [20], which had calculated the annual mean cost of an adult patient suffering from recurrent wheezing with respect to the Italian clinical setting (Table 4). Table 4 Annual mean cost for a patient with asthma Expenditure Breakdown Mean cost per Patient (€) Pharmacological therapy 457.82 Hospitalizations (ordinary admission and Day Hospital) 461.87 Admissions to Emergency Room 5.11 Visits 112.26 Examinations 127.76 Specific immune therapy 31.86 Other 30.21 Annual mean cost 1,226.88 Sensitivity analyses In order to test the robustness of the estimates resulting from the decision-making model [21] a series of univariate analyses were performed on the variables producing the greatest impact on the results of the simulation model: palivizumab dosage scheme, annual mean cost for a patient with recurrent wheezing and probability of developing recurrent wheezing within 24 months post-enrollment. In the first case (palivizumab dosage) the univariate analysis was conducted based on the assumption of a prophylaxis scheme with five monthly 100 mg injections, contrary to the base case scenario (three 50 mg administrations and two 100 mg administrations). The annual mean cost for a patient with recurrent wheezing changed based on the limits of the respective Confidence Interval (CI 95%, € 1,054.34 – € 1,399.42), whereas the probability of developing recurrent wheezing within 24 months post-enrolment was adjusted by ± 10% compared to its base value [21] assuming the worst case scenario for palivizumab (+10% [least advantageous] compared to the base value for the "Prophylaxis" branch and -10% [more advantageous] for the "Non prophylaxis" branch).

ility of developing recurrent wheezing within 24 months post-enrolment was adjusted by ± 10% compared to its base value [21] assuming the worst case scenario for palivizumab (+10% [least advantageous] compared to the base value for the "Prophylaxis" branch and -10% [more advantageous] for the "Non prophylaxis" branch). Finally, a threshold analysis was conducted on the probability of an RSV hospitalization occurring in patients receiving palivizumab prophylaxis. This analysis intended to identify which value of this parameter would show an incremental cost per life year (quality weighted and non-quality weighted) equal to € 50.000 in the simulation model, considering the latter value to represent the threshold below which a therapy would be considered acceptable. [22] Results Life years (LY) and QALY (3% discount) Patients receiving palivizumab prophylaxis show better efficacy results measured in terms both of LYs (+ 0.088) and of QALYs (+ 0.159) [Table 5]. Such an advantage, versus the absence of prophylaxis comparator, is maintained even when analyzing the results for the comparisons on patient subgroups (BPD, < 33 weeks, 33–35 weeks) [Table 5]. Table 5 Results: LY and QALY (3% discount) Parameter Prophylaxis Non prophylaxis Difference LY Total 29.842 29.754 0.088 - BPD 29.813 29.694 0.119 - < 33 weeks 29.849 29.771 0.078 - 33–35 weeks 29.854 29.776 0.078 QALY Total 29.202 29.043 0.159 - BPD 29.173 28.985 0.188 - < 33 weeks. 29.209 29.060 0.150

Results Life years (LY) and QALY (3% discount) Patients receiving palivizumab prophylaxis show better efficacy results measured in terms both of LYs (+ 0.088) and of QALYs (+ 0.159) [Table 5]. Such an advantage, versus the absence of prophylaxis comparator, is maintained even when analyzing the results for the comparisons on patient subgroups (BPD, < 33 weeks, 33–35 weeks) [Table 5]. Table 5 Results: LY and QALY (3% discount) Parameter Prophylaxis Non prophylaxis Difference LY Total 29.842 29.754 0.088 - BPD 29.813 29.694 0.119 - < 33 weeks 29.849 29.771 0.078 - 33–35 weeks 29.854 29.776 0.078 QALY Total 29.202 29.043 0.159 - BPD 29.173 28.985 0.188 - < 33 weeks. 29.209 29.060 0.150 - 33–35 weeks 29.214 29.065 0.149 Total treatment costs (3% discount) The annual mean cost per patient receiving prophylaxis (€ 6,244.20) exceeds by € 1,376.50 the cost estimated for a non-prophylaxis patient (€ 4,867.70) [Table 6]. The same result is apparent also in comparisons across patient subgroups, with differences between the two therapeutic options ranging from € 513.47 to € 2,220.09. Table 6 Results: mean cost per patient (3% discount) Mean cost per treated patient Parameter Prophylaxis Non prophylaxis Difference - BPD 6,517.26 6,003.79 513.47 - < 33 weeks 5,819.65 4,429.13 1,390.52 - 33–35 weeks 6,199.79 3,979.70 2,220.09 Total 6,244.20 4,867.70 1,376.50 Incremental cost-effectiveness ratio In terms of overall data ("total mean"), the incremental cost effectiveness ratio per LY estimated by the model was equal to € 15,568.65, and that per QALY was € 8,676.74 (Table 7). Table 7 Results: ICER (3% discounted costs and outcomes)

- 33–35 weeks 6,199.79 3,979.70 2,220.09 Total 6,244.20 4,867.70 1,376.50 Incremental cost-effectiveness ratio In terms of overall data ("total mean"), the incremental cost effectiveness ratio per LY estimated by the model was equal to € 15,568.65, and that per QALY was € 8,676.74 (Table 7). Table 7 Results: ICER (3% discounted costs and outcomes) Parameter ICER LY Total (mean) 15,568.65 - BPD 4,332.29 - < 33 weeks 17,885.86 - 33–35 weeks 28,417.08 QALY Total (mean) 8,676.74 - BPD 2,731.81 - < 33 weeks 9,380.00 - 33–35 weeks 14,937.32 With respect to the secondary analysis conducted for the three subgroups, ICERs were observed to range between € 4,332.29 – € 28,417.08 per LY, and from € 2,731.81 to € 14,937.32 for QALYs. Sensitivity analysis Univariate analysis Table 8 illustrates the results of the sensitivity analysis conducted by adjusting the base values as per the palivizumab dosage scheme, the annual mean cost of a patient with recurrent wheezing patient and the probabilities of developing recurrent wheezing within 24 months post-enrolment. The incremental cost effectiveness ratios per LY or per QALY estimated by all the univariate analyses show values lower than the internationally accepted € 50,000 threshold [22]. Table 8 Univariate sensitivity analysis

Sensitivity analysis Univariate analysis Table 8 illustrates the results of the sensitivity analysis conducted by adjusting the base values as per the palivizumab dosage scheme, the annual mean cost of a patient with recurrent wheezing patient and the probabilities of developing recurrent wheezing within 24 months post-enrolment. The incremental cost effectiveness ratios per LY or per QALY estimated by all the univariate analyses show values lower than the internationally accepted € 50,000 threshold [22]. Table 8 Univariate sensitivity analysis Parameters ICER per LY (€) ICER per QALY (€) Palivizumab dosage scheme Total 25,352.07 14,129.26 BPD 12,532.56 7,902.66 < 33 weeks 30,387.26 15,936.20 33–35 weeks 40,857.49 21,476.58 Annual mean cost per patient with recurrent wheezing Total 15,437.90 – 15,699.62 8,603.88 – 8,749.74 BPD 4,225.56 – 4,438.94 2,664.51 – 2,799.06 < 33 weeks 17,717.48 – 18,054.36 9,291.70 – 9,468.37 33–35 weeks 28,248.76 – 28,585.65 14,848.85 – 15,025.93 Probability of developing recurrent wheezing within 24 months from enrollment Total 15,857.51 8,837.74 BPD 4,577.05 2,886.15 < 33 weeks 18,253.99 9,573.06 33–35 weeks 28,782.64 15,129.48 Amongst those taken into account, the parameter that most affects the variability of incremental cost-effectiveness ratios is the palivizumab dosage scheme. When mean data ("Total") are referred to, the univariate analyses conducted on this parameter show an increase of ICER versus base values by 62.8% both per LY and QALY (Table 8).

aken into account, the parameter that most affects the variability of incremental cost-effectiveness ratios is the palivizumab dosage scheme. When mean data ("Total") are referred to, the univariate analyses conducted on this parameter show an increase of ICER versus base values by 62.8% both per LY and QALY (Table 8). Threshold analyses Figures 3, 4 and 5 illustrate the results of the threshold analyses. The three analyses were based on a simulation reducing efficacy of palivizumab, therefore increasing the probabilities of an RSV hospitalization in prophylaxis-treated patients, differentiating between preterm infants with BPD, gestational age < 33 weeks and gestational age between 33 and 35 weeks. Figure 3 Threshold analysis: group of preterm BPD infants. Figure 4 Threshold analysis: group of infants with gestational < 33 weeks. Figure 5 Threshold analysis: group of infants with gestational age between 33 and 35 weeks. In the first case (preterm BPD infants) (figure 3) the threshold level (for hospitalizations) at which palivizumab shows an incremental cost per LY of € 50,000 is 13.3% (increased by 137.5% compared to the 5.6% base value), whereas for QALYs such value rises to 18.6% (+ 232.1% compared to the base value). In the second case (infants with gestational age < 33 weeks) (figure 4) the threshold level at which palivizumab shows incremental values per LY of € 50,000 is 5.6% (increased by 180% compared to the 2% base value), rising to 10.8% for QALYs (+440% compared to base values).

In the first case (preterm BPD infants) (figure 3) the threshold level (for hospitalizations) at which palivizumab shows an incremental cost per LY of € 50,000 is 13.3% (increased by 137.5% compared to the 5.6% base value), whereas for QALYs such value rises to 18.6% (+ 232.1% compared to the base value). In the second case (infants with gestational age < 33 weeks) (figure 4) the threshold level at which palivizumab shows incremental values per LY of € 50,000 is 5.6% (increased by 180% compared to the 2% base value), rising to 10.8% for QALYs (+440% compared to base values). Finally, the third case (children whose gestational age ranges between 33 and 35 weeks) (figure 5) points out a 4.3% threshold level at which Palivizumab shows an incremental cost per LY of € 50,000 (increased by 186.7% compared to the 1.5% base value), whereas said rate rises to 10.4% for QALYs (+ 593.3% compared to base value).

e third case (children whose gestational age ranges between 33 and 35 weeks) (figure 5) points out a 4.3% threshold level at which Palivizumab shows an incremental cost per LY of € 50,000 (increased by 186.7% compared to the 1.5% base value), whereas said rate rises to 10.4% for QALYs (+ 593.3% compared to base value). Discussion The present analysis assessed efficacy and costs of prophylaxis with palivizumab versus absence of prophylaxis in the prevention of RSV infections in high-risk preterm infants. The (incremental) cost-effectiveness analysis was conducted from the perspective of the National Health System, updating and adapting a pre-existing lifetime decision-making model [15]. We wish to point out that as in the previously published model, RSV infection mortality during the first hospitalization was estimated based on the results of the IMPACT study: mortality rates were noted to be 0.2% and 0.0% in subjects who received palivizumab prophylaxis and subjects who did not receive prophylaxis, respectively [15]. Other probability rates of occurrence of the events investigated in the present study are also the same ones used in the original model [15], with the exception of the odds of developing recurrent wheezing within 24 months post-enrolment, data deriving from Simoes study [14].

not receive prophylaxis, respectively [15]. Other probability rates of occurrence of the events investigated in the present study are also the same ones used in the original model [15], with the exception of the odds of developing recurrent wheezing within 24 months post-enrolment, data deriving from Simoes study [14]. We wish to briefly describe the changes made to the original model. Costs associated with high-risk preterm patients refer to 24 months after enrollment instead of 14 years so as to uniform the financial evaluation to the new data available in literature [14]. Palivizumab administration scheme was changed, with three 50 mg vials and two 100 mg vials instead of the five 100 mg vials administered in the first model. Finally, all of the health care resources used by the patient were revaluated based on year 2007 costs and fees. As regards general results, patients receiving palivizumab prophylaxis show better efficacy in terms both of LY (+ 0.088) and QALY (+ 0.159) compared to those who did not receive any prophylaxis. The best efficacy result achieved by palivizumab is confirmed also when differentiating patients per gestational age and presence of BPD.

neral results, patients receiving palivizumab prophylaxis show better efficacy in terms both of LY (+ 0.088) and QALY (+ 0.159) compared to those who did not receive any prophylaxis. The best efficacy result achieved by palivizumab is confirmed also when differentiating patients per gestational age and presence of BPD. On the other hand, in terms of the health care resources used, the mean cost per patient receiving prophylaxis (€ 6,244.20) was higher (+28.3%) compared to the estimated cost per patient without prophylaxis (€ 4,867.70). Therefore, due to the greater efficacy and greater costs associated with palivizumab versus the comparator (absence of prophylaxis), it was necessary to determine the incremental cost-effectiveness ratio per LY and QALY.

.043 QALYs. Incremental efficacy in this case is equal to 0.159 QALYs; therefore the incremental cost per QALY is € 8,676.74. Taking into account patient subgroups, the incremental cost effectiveness ratio per LY ranges between € 4,332.29 and € 28,417.08, whereas those per QALY range between € 2,731.81 and € 14,937.32. All incremental costs calculated in this paper were finally compared against internationally acknowledged threshold values, which reflect the decision-making bodies' willingness to pay in order to achieve additional health units. Several international studies addressed the issue and established threshold values [22-25] and € 50,000 were set as the threshold below which a therapy is acceptable. Incremental costs per life year gained and per QALY calculated for palivizumab versus the absence of prophylaxis were always noted to be below the aforesaid threshold. The incremental cost per LY or per QALY of palivizumab was quite different observing the results of the three infant groups. The 33–35 weeks infant group showed the higher ICER per LY (€ 28,417.08) and per QALY (€ 14,937.32). If we considered a more restricted threshold value (< € 50,000), this third infant group (33–35 weeks) could be included in the prophylaxis programs, provided additional risk factors are present.

he three infant groups. The 33–35 weeks infant group showed the higher ICER per LY (€ 28,417.08) and per QALY (€ 14,937.32). If we considered a more restricted threshold value (< € 50,000), this third infant group (33–35 weeks) could be included in the prophylaxis programs, provided additional risk factors are present. The univariate analyses conducted on several clinical and financial parameters taken into consideration in the model confirmed the robustness of the results, producing ICERs that were always below € 50,000. Also the threshold analysis conducted on the probability of an RSV hospitalization occurring for a patient receiving palivizumab prophylaxis estimated that to reach an incremental cost per LY or per QALY exceeding € 50,000 the efficacy of palivizumab would have to drop to non-realistic levels, i.e., down to efficacy levels equal to or even lower than those for patients without prophylaxis.

talization occurring for a patient receiving palivizumab prophylaxis estimated that to reach an incremental cost per LY or per QALY exceeding € 50,000 the efficacy of palivizumab would have to drop to non-realistic levels, i.e., down to efficacy levels equal to or even lower than those for patients without prophylaxis. These results must be compared with those generated by other studies. Nuijten et al. [26] conducted in the United Kingdom an incremental cost effectiveness analysis for palivizumab in the prophylaxis of RSV infections in preterm infants with gestational age < 36 weeks and the possible presence of complications such as BPD or CHD (Congenital Heart Disease). The assessment was conducted from the perspective of the UK NHS (National Health Service), comparing administration of palivizumab to absence of prophylaxis. Applying a discount (3.5%) both to costs and outcome, an incremental cost per QALY equal to £ 16,720 for children with gestational age < 36 weeks and for children with BPD and an ICER per QALY of £ 6,664 for children with CHD. The results of the study conducted by Nuijten et al. [26] suggest that from the NHS viewpoint, palivizumab is cost effective compared to the therapeutic alternative represented by absence of prophylaxis.

ren with gestational age < 36 weeks and for children with BPD and an ICER per QALY of £ 6,664 for children with CHD. The results of the study conducted by Nuijten et al. [26] suggest that from the NHS viewpoint, palivizumab is cost effective compared to the therapeutic alternative represented by absence of prophylaxis. Lazaro et al. [27] assessed efficacy and costs of palivizumab in the prevention of RSV infections in preterm children with gestational age in the 32–35 week range with two or more risk factors. The study was conducted from the perspective of the Spanish health care system, with absence of prophylaxis as the comparator. The Authors, thanks to a decision-making model, have calculated (with a 3% discount applied to efficacy and costs) an incremental cost effectiveness ratio per QALY of € 13,849. Once again, palivizumab was noted to be cost-effective versus absence of prophylaxis.

re system, with absence of prophylaxis as the comparator. The Authors, thanks to a decision-making model, have calculated (with a 3% discount applied to efficacy and costs) an incremental cost effectiveness ratio per QALY of € 13,849. Once again, palivizumab was noted to be cost-effective versus absence of prophylaxis. The study conducted in the USA by Elhassan et al. [28] intended to assess both the cost-effectiveness ratio of palivizumab as RSV prophylaxis in preterm children without BPD and the impact of decreasing recurrent wheezing risks in patients receiving prophylaxis on the cost effectiveness ratio. The Authors constructed two decision-making models, one of which took into account also risks of recurrent wheezing after an RSV infection and another one that did not consider this risk. The patients included in the model were preterm children with gestational age ranging from 26 to 32 weeks. Prophylaxis with palivizumab was compared against absence of prophylaxis. The results of the study conducted by Elhassan et al. [28] estimated that administration of palivizumab was cost effective only when taking into account also the benefits yielded by a decreased risk of recurrent wheezing in RSV infection patients.

rophylaxis with palivizumab was compared against absence of prophylaxis. The results of the study conducted by Elhassan et al. [28] estimated that administration of palivizumab was cost effective only when taking into account also the benefits yielded by a decreased risk of recurrent wheezing in RSV infection patients. The results of the present review present limitations and therefore several considerations must be taken into account when interpreting the data. As the study conducted by Simoes et al.[14] has estimated the probabilities of developing recurrent wheezing episodes during 24 months of follow-up after the enrollment in preterm children with gestational age equal or less than 35 weeks, with or without RSV hospitalization, the present model is based on the assumption that such probability was identical for all children, without differentiating based on gestational age and the presence of BPD. Moreover, since the study performed by Simoes et al.[14] did not report the odds of developing recurrent wheezing in preterm children receiving prophylaxis with RSV hospitalization, for this group of patients the same percentage (17%) calculated for preterm children with RSV hospitalization not receiving prophylaxis was conservatively adopted. To assess the impact of this double assumption, a sensitivity analysis was performed to test the probability of developing recurrent wheezing during the 24 months of follow-up post-enrollment, with a ± 10% change of the base value, assuming the worst case scenario for palivizumab; also in this case, the incremental cost effectiveness ratios per LY and per QALY show values lower than € 50.000.

performed to test the probability of developing recurrent wheezing during the 24 months of follow-up post-enrollment, with a ± 10% change of the base value, assuming the worst case scenario for palivizumab; also in this case, the incremental cost effectiveness ratios per LY and per QALY show values lower than € 50.000. Another limitation of the study might be that a pre-existing simulation model was adopted for the comparison between the two alternatives, and was adapted with clinical data deriving from international literature. This choice might be justified by the fact that to date no studies are available providing national data suitable for constructing the model population. The dosage scheme (three 50 mg vials + two 100 mg vials) adopted herein for prophylaxis administration is different from that used in the original model.[15] In support thereof, a dosage of 15 mg of active principle per kg of weight was considered, assuming that average weight of the preterm infant in the first three months of life was less than 3.4 kg, thus such as to justify the administration of a 50 mg vial; moreover the model conservatively included also the non-administered drug. Moreover, by means of the sensitivity analysis, also the five 100 mg vials administration assumption was taken into consideration, and also in this case ICERs per LY and per QALY remained lower than the € 50,000 threshold.

ation of a 50 mg vial; moreover the model conservatively included also the non-administered drug. Moreover, by means of the sensitivity analysis, also the five 100 mg vials administration assumption was taken into consideration, and also in this case ICERs per LY and per QALY remained lower than the € 50,000 threshold. Finally, as already pointed out by the original study, a further limitation might be the use of average costs referring to an adult sample for the appreciation of the annual mean cost for recurrent wheezing of pediatric patients. However, comparing the cost included in the present model (€ 1,226.88) against that of children suffering from asthma (US$ 1,129) within a study conducted in the United Stated in 1999 [29], we have reason to believe that the data we used are conservative. Conclusion Based on the results of the present cost-effectiveness assessment it therefore appears possible to state that compared to absence of prophylaxis, the administration of palivizumab in preterm infants of varying gestational ages, with or without complications, does improve survival (quality-weighted or not) at reasonable costs in terms of resources covered by the NHS, when compared to internationally accepted threshold values. However, there is not general consensus concerning palivizumab prophylaxis for preterm infants born between 32 and 35 weeks of gestational age without chronic lung disease and haemodynamically significant congenital heart disease. [22].

ces covered by the NHS, when compared to internationally accepted threshold values. However, there is not general consensus concerning palivizumab prophylaxis for preterm infants born between 32 and 35 weeks of gestational age without chronic lung disease and haemodynamically significant congenital heart disease. [22]. Abbreviations BPD: Bronchopulmonary dysplasia; ICER: Incremental Cost Effectiveness Ratio; ICU: Intensive Care Unit; LY: Life-years; NHS: National Health System; OW: Ordinary ward; QALY: Quality-Adjusted Life-Years; RSV: Respiratory Syncytial Virus. Competing interests The authors declare that they have no competing interests. Authors' contributions All the authors (GC, RR and US) contributed in the conceiving, design and realization of the manuscript. All authors read and approved the final manuscript. Acknowledgements This paper was supported thanks to a non-binding contribution provided by Abbott.

Sir, Greco et al. described a case of a 12-year-old girl who presented symptoms of stroke after diving into the sea [1]. An ischemic lesion in the lenticular nucleus, in the posterior limb of internal capsula and in the caudate nucleus of the right hemisphere was found at a Diffusion Weighted Imaging. No abnormality of coagulation parameters was revealed. Surprisingly, there is no mention of investigation of congenital heart disease, which is recognised as a major cause of stroke in children [2]. Persistence of patent foramen ovale (PFO) is present in up to 25% of general population. Under normal circumstances, this is of little relevance as the left atrial pressure is higher than that of the right and the PFO is functionally closed most of the time. However, during coughing, sneezing, diving, and other conditions of increased endothoracic pressure, the PFO can open allowing potential for right-to-left shunt. This opens the cerebral circulation to the systemic venous circulation and has been implicated in the pathogenesis of stroke [3]. In adults, the presence of PFO is associated with cryptogenic stroke [4]. It is not known whether children with such condition have a similar increase in PFO, but a causal link has been suggested [5].

the cerebral circulation to the systemic venous circulation and has been implicated in the pathogenesis of stroke [3]. In adults, the presence of PFO is associated with cryptogenic stroke [4]. It is not known whether children with such condition have a similar increase in PFO, but a causal link has been suggested [5]. SCUBA diving is popular between adolescents. There is an association between PFO and neurological decompression illness, the onset of which is soon after surfacing. Divers with this type of decompression illness have a much higher prevalence of significant right-to-left shunt than do controls [6]. An increased prevalence of brain lesions has also been reported in divers, with a right-to-left shunt present in those with multiple brain lesions [7]. Although rare, cryptogenic stroke in childhood is devastating, and investigation of PFO-associated right-to-left shunt is largely warranted in such condition [8].

Background The continuous advances in intensive care of preterm newborns have led to a progressive decline of mortality in Institutions where facilities and expertise for respiratory resuscitation and respiratory distress syndrome are available. Infant mortality dropped among all races between 1980 and 2000. The survival rate depends on the gestational age of the newborn; actually the survival rates for very low birth weight (VLBW) are the following: for those weighing 501 – 750 g is 56% and for the ones above 750 is 88% [1]. However, the success in the survival achieved through an aggressive intensive care is not always paralleled by a subsequent fully healthy development of the newborn. Among the common conditions of morbidity due to the prematurity (cerebral impairment, bronchopulmonary dysplasia, growth failure, retinopathy...) a growing interest is focusing now on the metabolic bone disease of the prematurity (MBD), also called osteopenia of prematurity. This condition is characterised by a reduction in bone mineral content (osteopenia), with or without rachitic changes, and is caused by several nutritional and biomechanical factors. An inadequate supply of nutrients (vitamin D, calcium and phosphorus), a prolonged period of total parenteral nutrition, immobilisation and the intake of some drugs are the main factors involved in the pathogenesis of osteopenia [2]. The MBD usually occurs between tenth and sixteenth week of life, but it may remain silent until severe demineralisation (a reduction of BMD of 20 – 40%) occurs.

An inadequate supply of nutrients (vitamin D, calcium and phosphorus), a prolonged period of total parenteral nutrition, immobilisation and the intake of some drugs are the main factors involved in the pathogenesis of osteopenia [2]. The MBD usually occurs between tenth and sixteenth week of life, but it may remain silent until severe demineralisation (a reduction of BMD of 20 – 40%) occurs. The clinical picture is various, ranging from a totally silent condition to a clinical picture of overt rickets, with multiple fractures and other alterations, when the demineralisation is severe. The purpose of this review is to focus on the recent advances in the understanding of the bone tissue metabolism and on the nutritional approach to prevent and to treat the MBD. Magnitude of the problem The prevalence of MBD varies depending on gestational age, birthweight and kind of alimentation. It occurs in up to 55% of babies born with weight under 1000 g [3] and 23% of infants weighing < 1500 g at birth [4] and it is especially frequent in babies under 28 weeks of gestation. The prevalence is 40% in premature infants who are breastfed, in contrast to 16% of those fed with a formula designed for preterm infants and supplemented with calcium and phosphorus [5,6]. Preterm infants with a complicated medical course and delayed nutrition are also at high risk for MBD. Actually in western countries there is a trend of decrease of gestational age and birthweight, so the frequency of the MBD is expected to further increase.

It occurs in up to 55% of babies born with weight under 1000 g [3] and 23% of infants weighing < 1500 g at birth [4] and it is especially frequent in babies under 28 weeks of gestation. The prevalence is 40% in premature infants who are breastfed, in contrast to 16% of those fed with a formula designed for preterm infants and supplemented with calcium and phosphorus [5,6]. Preterm infants with a complicated medical course and delayed nutrition are also at high risk for MBD. Actually in western countries there is a trend of decrease of gestational age and birthweight, so the frequency of the MBD is expected to further increase. Homeostasis of calcium -phosphorus The homeostasis of calcium, phosphorus and magnesium is fundamental for structural matrix of the bone. Calcium and phosphate represent the major inorganic constituents of bone. The highest amount of calcium (99%) and of phosphorus (80%) of the whole body is in the bone as microcrystalline apatite. Only 1% of the total body calcium is within the extracellular fluids and soft tissues. About the 50% of total serum calcium is in the ionised form and represents the biologically active part. A further 8–10% is bounded to organic and inorganic acid and the remaining percentage of calcium is protein-bound (80% to albumin, 20% to globulin). The formation of the apatite takes place if calcium and phosphorus are simultaneously available in optimal proportions. Also magnesium is part of the bone matrix and the 60% of total body magnesium is in the bone.

Only 1% of the total body calcium is within the extracellular fluids and soft tissues. About the 50% of total serum calcium is in the ionised form and represents the biologically active part. A further 8–10% is bounded to organic and inorganic acid and the remaining percentage of calcium is protein-bound (80% to albumin, 20% to globulin). The formation of the apatite takes place if calcium and phosphorus are simultaneously available in optimal proportions. Also magnesium is part of the bone matrix and the 60% of total body magnesium is in the bone. Calcium and phosphorus homeostasis is a function of hormones, vitamin D and dietary intake, and depends on the intestinal absorption, skeletal accretion and reabsorption, and urinary excretion. [7] Parathyroid hormone (PTH) is synthesised and secreted from the parathyroid glands in response to a reduction of serum level of ionised calcium. PTH regulates mineral metabolism and skeletal homeostasis through its action on target cells in bone and kidneys. It stimulates the reabsorption of calcium and excretion of phosphorus in the kidney and bone reabsorption of calcium. PTH also is able to activate the synthesis of calcitriol via stimulation of renal 25 (OH) D3-1-alpha-hydroxylase activities. In its active form, 1, 25(OH) 2 vitamin D, stimulates the renal reabsorption of calcium and phosphorus. The synthesis of calcitriol is inhibited by elevated serum levels of calcium and phosphorus. The combined actions of PTH and calcitriol maintain the adequate concentration of calcium in the extracellular fluids.

Parathyroid hormone (PTH) is synthesised and secreted from the parathyroid glands in response to a reduction of serum level of ionised calcium. PTH regulates mineral metabolism and skeletal homeostasis through its action on target cells in bone and kidneys. It stimulates the reabsorption of calcium and excretion of phosphorus in the kidney and bone reabsorption of calcium. PTH also is able to activate the synthesis of calcitriol via stimulation of renal 25 (OH) D3-1-alpha-hydroxylase activities. In its active form, 1, 25(OH) 2 vitamin D, stimulates the renal reabsorption of calcium and phosphorus. The synthesis of calcitriol is inhibited by elevated serum levels of calcium and phosphorus. The combined actions of PTH and calcitriol maintain the adequate concentration of calcium in the extracellular fluids. Kidneys contribute to maintain homeostasis of calcium; urinary calcium is one third derived from diet and the remaining from body stores, mostly bone. Diuretics, as furosemide, increase renal calcium excretion. Prenatal bone physiology The amounts of minerals required for a correct accretion of the skeleton are widely different depending on the age of the babies. The period of greater skeletal development is during the intrauterine life and specifically during the last trimester. The bone volume increases significantly with gestational age and the high net bone formation activity is mainly due to modelling, with a rapidly increasing trabecular thickness (the trabecular thickening rate being approximately 240 times faster in the foetus than in the children).

ecifically during the last trimester. The bone volume increases significantly with gestational age and the high net bone formation activity is mainly due to modelling, with a rapidly increasing trabecular thickness (the trabecular thickening rate being approximately 240 times faster in the foetus than in the children). The mineralization process is determined by synthesis of the organic bone matrix by osteoblasts (osteoid) onto which calcium and phosphate salts are deposited. This process increases exponentially between 24 and 37 weeks of gestation, reaching the 80% of mineral accretion in the third trimester [8]. During gestation the developing fetus receives supplies of energy, protein and mineral for adequate growth (1.2 cm/week) and bone development. At term the newborn skeleton has a high physical density (expressed as bone mass divided by bone volume). The foetal accretion of calcium and phosphate during the last three months of gestation is about 20 g and 10 g respectively, which represents accretion rates of 100–120 mg/kg/day for calcium and 50–65 mg/kg/day for phosphate [9]. A very important role in skeletal accretion of the foetus is played by the placenta. In fact the transfer of calcium from the mother to the foetus through the placenta occurs via an active transport done by the calcium pump in the basal membrane [10]. There is a 1:4 maternal to foetal calcium gradient [11]. Moreover, the placenta is able to convert vitamin D to 1,25-dihydrocholecalciferol which is fundamental for transferring phosphate to the foetus [12].

A very important role in skeletal accretion of the foetus is played by the placenta. In fact the transfer of calcium from the mother to the foetus through the placenta occurs via an active transport done by the calcium pump in the basal membrane [10]. There is a 1:4 maternal to foetal calcium gradient [11]. Moreover, the placenta is able to convert vitamin D to 1,25-dihydrocholecalciferol which is fundamental for transferring phosphate to the foetus [12]. The foetus is maintained hypercalcemic in a high calcitonin and estrogen environment which promotes the modelling/remodelling ratio in favour of modelling and thus increasing the endocortical bone [13]. As a result, infants born prematurely will be deprived of the intrauterine supply of calcium and phosphorus affecting bone mineralization. It is well known that a chronic damage to the placenta may alter the phosphate transport; this explains why babies with intrauterine growth restriction may be osteopenic. Demineralization is also observed in infants born from mother with chorioamniositis and placental infection [14]. Maternal dietary intake of calcium is a factor implied in foetal bone accretion. A supplement of calcium (2 g from before 22 weeks of gestation) to women with a low dietary calcium intake resulted in higher bone mineral content (BMC) of the total body in infants born at term [15].

Demineralization is also observed in infants born from mother with chorioamniositis and placental infection [14]. Maternal dietary intake of calcium is a factor implied in foetal bone accretion. A supplement of calcium (2 g from before 22 weeks of gestation) to women with a low dietary calcium intake resulted in higher bone mineral content (BMC) of the total body in infants born at term [15]. Post-natal bone physiology After birth the physical density of term newborns bones decreases by 30% in the first 6 months of life [13]. This is mostly due to an enlargement of the marrow cavity size, which occurs faster than the increase in the cross-sectional area of the bone cortex [16]. In term infants these postnatal changes are not accompanied by an increase in bone fragility and occur because bone is exposed to different conditions before and after birth. First, there are important changes of hormonal environment: the reduction of maternal estrogens [17] and a postnatal increase of PTH level mainly due to a reduction of the calcium supply by the placenta [18]. As the serum calcium levels falls in the first day of life, PTH secretion is stimulated. During this transition the response of the parathyroid gland to falling levels of ionised calcium is blunted, as emphasized in a recent review article [19]. This finally results in a physiological nadir in neonatal serum calcium levels within the first 48 hours of life. Of note, PTH level is still within the normal range for term babies or adult, but represents a decrease from foetal levels.

vels of ionised calcium is blunted, as emphasized in a recent review article [19]. This finally results in a physiological nadir in neonatal serum calcium levels within the first 48 hours of life. Of note, PTH level is still within the normal range for term babies or adult, but represents a decrease from foetal levels. Many factors affect calcium absorption including the maternal vitamin D status, solubility and bioavailability of calcium salts, quality and quantity of calcium, amount and type of lipids and, obviously, gut function. Calcium absorption from the intestine occurs both passively and through a vitamin-D dependent active transport mechanism. In a newly born preterm the low mineral content of human milk associated with a poorly efficient absorption of the developing gut determine a net reduction of calcium and phosphorus supply. Absorption of phosphorus takes place in the jejunum and depends on the dietary intake. The phosphorus supply regulates calcium absorption and retention: the higher is the phosphorus content of the diet, the higher is the calcium retention. However, an excessive amount of one decreases the absorption of the other.

Calcium absorption from the intestine occurs both passively and through a vitamin-D dependent active transport mechanism. In a newly born preterm the low mineral content of human milk associated with a poorly efficient absorption of the developing gut determine a net reduction of calcium and phosphorus supply. Absorption of phosphorus takes place in the jejunum and depends on the dietary intake. The phosphorus supply regulates calcium absorption and retention: the higher is the phosphorus content of the diet, the higher is the calcium retention. However, an excessive amount of one decreases the absorption of the other. Moreover, while in utero fetus experiments mechanical stimulation by kicking against the uterine wall, this kind of training is missing during the extrauterine life since preterm babies usually stay in the incubator [20,21]. Inactivity due to immobilisation stimulates bone reabsorption by osteoclasts and urinary calcium excretion; furthermore the reduced muscle activity prevents the addition of new bone tissue [22]. Conditio sine qua non for the physical activity to be beneficial is that an adequate mineral intake is guaranteed [23]. The figure 1 shows that during the third trimester of gestation, bone mineral apparent density (BMAD) increases at a faster rate in utero (term infants) than ex utero (preterm infants) according to gestational age.

Moreover, while in utero fetus experiments mechanical stimulation by kicking against the uterine wall, this kind of training is missing during the extrauterine life since preterm babies usually stay in the incubator [20,21]. Inactivity due to immobilisation stimulates bone reabsorption by osteoclasts and urinary calcium excretion; furthermore the reduced muscle activity prevents the addition of new bone tissue [22]. Conditio sine qua non for the physical activity to be beneficial is that an adequate mineral intake is guaranteed [23]. The figure 1 shows that during the third trimester of gestation, bone mineral apparent density (BMAD) increases at a faster rate in utero (term infants) than ex utero (preterm infants) according to gestational age. Figure 1 Physiological evolution of DEXA apparent bone mineral density during the last trimester of gestation (filled squares) and during the first year of life in healthy term infants (upper triangles) compared to that observed in preterm infants (open squares and lower triangles). (With permission from Ref [12]).

The figure 1 shows that during the third trimester of gestation, bone mineral apparent density (BMAD) increases at a faster rate in utero (term infants) than ex utero (preterm infants) according to gestational age. Figure 1 Physiological evolution of DEXA apparent bone mineral density during the last trimester of gestation (filled squares) and during the first year of life in healthy term infants (upper triangles) compared to that observed in preterm infants (open squares and lower triangles). (With permission from Ref [12]). BMAD is an estimation of volumetric BMD (g/cm3) calculated as bone mineral content/bone area (BMC/BA). The figure 1 also shows that there is a sharp reduction in BMAD in neonatal age followed by a stabilization that lasts all the first year of life ("black triangles"). A similar event occurs in preterm babies: from birth to the term, mineral retention sharply diminishes comparing with the foetal life, while the skeletal growth remains high. This leads to a reduction of bone density ("white squares"). A catch up mineralization occurs after discharge of VLBW so BMC spontaneously improves ("white rhombs"). Among the other pathogenic factors, also problems related to inadequate supply of calcium to babies, which require parenteral nutrition and interference of several drugs, may contribute to determine preterm osteopenia with an increasing risk of bones fractures.

BMAD is an estimation of volumetric BMD (g/cm3) calculated as bone mineral content/bone area (BMC/BA). The figure 1 also shows that there is a sharp reduction in BMAD in neonatal age followed by a stabilization that lasts all the first year of life ("black triangles"). A similar event occurs in preterm babies: from birth to the term, mineral retention sharply diminishes comparing with the foetal life, while the skeletal growth remains high. This leads to a reduction of bone density ("white squares"). A catch up mineralization occurs after discharge of VLBW so BMC spontaneously improves ("white rhombs"). Among the other pathogenic factors, also problems related to inadequate supply of calcium to babies, which require parenteral nutrition and interference of several drugs, may contribute to determine preterm osteopenia with an increasing risk of bones fractures. The drugs mostly implied in pathogenesis of MBD include steroids, methylxanthines and diuretics. They stimulate osteoclasts activation, decrease calcium absorption, reduce osteoblasts proliferation and increase calcium renal excretion and hence increase the risk of poor bone mineralization [24-26]. Neonatal mineral requirements The requirements of calcium and phosphorus are based on demands for matching intrauterine bone mineral accretion rates.

The drugs mostly implied in pathogenesis of MBD include steroids, methylxanthines and diuretics. They stimulate osteoclasts activation, decrease calcium absorption, reduce osteoblasts proliferation and increase calcium renal excretion and hence increase the risk of poor bone mineralization [24-26]. Neonatal mineral requirements The requirements of calcium and phosphorus are based on demands for matching intrauterine bone mineral accretion rates. Supplying calcium and phosphorus in parenteral nutrition is a challenge because of limited solubility of these two minerals. Calcium and phosphorus's solubility in nutrition admixtures depends on temperature, type and concentration of aminoacid, glucose concentration, pH, type and concentration of calcium salts, and presence of lipid and so on... In parenteral nutrition calcium is administered as inorganic salt and phosphorus may be administered as inorganic sodium and potassium phosphate or sodium-glucose phosphate or glycerolphosphate, which are quite soluble in water. The addition of cystein to lower pH of the parenteral admixtures improves the solubility of calcium and phosphorus. For all such reasons it is not possible to supply these minerals according to the physiologic requirements of the preterm to reach an adequate bone mineralization.

In parenteral nutrition calcium is administered as inorganic salt and phosphorus may be administered as inorganic sodium and potassium phosphate or sodium-glucose phosphate or glycerolphosphate, which are quite soluble in water. The addition of cystein to lower pH of the parenteral admixtures improves the solubility of calcium and phosphorus. For all such reasons it is not possible to supply these minerals according to the physiologic requirements of the preterm to reach an adequate bone mineralization. In the transition period, most of VLBW neonates receive full or partial parenteral nutrition with the goal to maintain normal levels of calcium and phosphorus. Hypocalcaemia, in fact, is a common event during the first days of life because of the sharp decrease of the calcium supply by the placenta and the delayed release of PTH due to the immature response of the parathyroid glands. Parenteral administration of 50–75 mg of calcium/kg/day can prevent early neonatal hypocalcaemia in preterm infants. Through the parenteral administration of calcium and phosphorus (40–70 mg/kg/day and of 25–45 mg/kg/day respectively) it is possible to achieve 60 – 70% of intrauterine mineralization [27]. The best calcium to phosphorus ratio for bone mineralization is 1.7:1 [28-30]. In preterm babies receiving parenteral nutrition only limited amounts of vitamin D are required since calcium is given by vein and there is no need of calcitriol to facilitate the intestinal uptake.

Through the parenteral administration of calcium and phosphorus (40–70 mg/kg/day and of 25–45 mg/kg/day respectively) it is possible to achieve 60 – 70% of intrauterine mineralization [27]. The best calcium to phosphorus ratio for bone mineralization is 1.7:1 [28-30]. In preterm babies receiving parenteral nutrition only limited amounts of vitamin D are required since calcium is given by vein and there is no need of calcitriol to facilitate the intestinal uptake. Moreover only the parent compound needs to be administered since the preterm infant is able to hydroxylate the inactive form to the active one since the 24th week of gestation. It is now generally accepted the daily recommended dose of vitamin D is 400 U.I./day [18]. For the transitional period, when infants are weaned from parenteral nutrition to the enteral one, the aim usually is to maintain an adequate serum level of calcium and phosphorus. However the serum level of calcium is not a good marker of adequacy of calcium intake since the level is maintained stable at the expense of the bone. Therefore the clinicians should be aware that a normal serum level of both calcium and phosphorus are not guarantee for an adequate whole body accretion as in intrauterine life. The enteral administration of calcium is fraught with many problems as regards the calcium bioavailability. Vomiting, large gastric aspirates, constipation and abdominal distension are quite common in preterm babies and the gut absorption capacity is impaired due to the immaturity of the gastrointestinal mucosa.

For the transitional period, when infants are weaned from parenteral nutrition to the enteral one, the aim usually is to maintain an adequate serum level of calcium and phosphorus. However the serum level of calcium is not a good marker of adequacy of calcium intake since the level is maintained stable at the expense of the bone. Therefore the clinicians should be aware that a normal serum level of both calcium and phosphorus are not guarantee for an adequate whole body accretion as in intrauterine life. The enteral administration of calcium is fraught with many problems as regards the calcium bioavailability. Vomiting, large gastric aspirates, constipation and abdominal distension are quite common in preterm babies and the gut absorption capacity is impaired due to the immaturity of the gastrointestinal mucosa. Calcium absorption depends on vitamin D status, solubility of calcium salts, quality and quantity of lipid intake. Moreover, in preterm babies, vitamin D demands are influenced by body contents at birth which depends on the duration of gestation and maternal vitamin status. Current estimates of requirements for calcium, phosphorus and vitamin D in growing premature infants vary among international sources of recommendations [31-34] (Table 1). Table 1 Minerals and vitamin D recommended intakes in growing preterm infants. Requirements ESPGAN [32] LSRO [31] Atkinson [33] Rigo [34] 1987 2002 2005 2007 Calcium (mg/kg/day) 70–140 150–220 120–200 100–160 Phosphorus (mg/kg/day) 50–90 100–130 60–140 60–90

Current estimates of requirements for calcium, phosphorus and vitamin D in growing premature infants vary among international sources of recommendations [31-34] (Table 1). Table 1 Minerals and vitamin D recommended intakes in growing preterm infants. Requirements ESPGAN [32] LSRO [31] Atkinson [33] Rigo [34] 1987 2002 2005 2007 Calcium (mg/kg/day) 70–140 150–220 120–200 100–160 Phosphorus (mg/kg/day) 50–90 100–130 60–140 60–90 Vitamin D (I.U./day) 800–1600 200–1000 800–1000 (I.U./kg/day) 90–225 150–400 The human milk content is inadequate for preterm requirements since the content of calcium and phosphorus in preterm human milk is 31 mg/100 kcal and 20 mg/100 kcal [18] while the Life Science Research Office [31] suggests, for premature formulas, a dose approximately 4–6 times higher (123 to 185 mg Ca/100 kcal and 80 to 110 mg P/100 kcal). Even when VLBW are fed at high feeding volumes (180–200 mL/Kg), assuming calcium and phosphorus absorption of 70% and 80% respectively, this would provide only one-third of the in utero level of absorbed calcium and phosphorus [6]. Formula milk is richer in calcium and phosphorus than human one, but bioavailability is quite different. In formula fed infants, calcium absorption is usually less than with human milk, ranging from 35 to 60% of the intake. Hence the human milk intake has to be promoted, but a fortification with mineral and protein fortifier is necessary to achieve adequate nutrient intake.

man one, but bioavailability is quite different. In formula fed infants, calcium absorption is usually less than with human milk, ranging from 35 to 60% of the intake. Hence the human milk intake has to be promoted, but a fortification with mineral and protein fortifier is necessary to achieve adequate nutrient intake. With the current human milk fortifiers, containing highly soluble calcium glycerolphosphate, calcium retention reaches a level of 90 mg/kg/day (88% of the overall intake). However the new human milk fortifiers available in the market still do not allow intakes of calcium comparable with the values achieved during the last trimester of gestation (100–120 mg/kg/day) which are considered the target mineral accretion for preterm infants, nevertheless the use of multinutrient fortification of human milk for premature infants is currently recommended. A Cochrane systematic review and metaanalysis of human milk fortifiers, which however included studies on children who were not extremely preterm (the class at major risk) stated that the effects on bone mineralization were not conclusive [35]. Finally, it must be noted that high calcium supplementation of milk is not well tolerated; it is associated with high faecal calcium, prolonged gastrointestinal transit time and impaired fat absorption. All these effects are potential risk factors for developing necrotizing enterocolitis. (See table 1)

A Cochrane systematic review and metaanalysis of human milk fortifiers, which however included studies on children who were not extremely preterm (the class at major risk) stated that the effects on bone mineralization were not conclusive [35]. Finally, it must be noted that high calcium supplementation of milk is not well tolerated; it is associated with high faecal calcium, prolonged gastrointestinal transit time and impaired fat absorption. All these effects are potential risk factors for developing necrotizing enterocolitis. (See table 1) Clinical features and diagnosis MBD remains silent until a severe demineralisation occurs. The most evident clinical findings of osteopenia are deformity of the skull (diastasis of the suture, enlargement of the sagittal fontanelle and frontal bosses, craniotabe), thickening of the chondrocostal junctions and of the wrists, rib and long bones fractures. Softening and/or fractures of the ribs can cause pulmonary changes and respiratory distress, typically between 5 and 11 weeks of age [36]. Diagnosis of osteopenia is mainly done by serum analysis. Biochemically osteopenia is characterised by low serum levels of phosphorus and by an increase in serum levels of alkaline phosphatase that can reach values 5 times higher than the upper reference range used for adults [37]. It is useful dosing the isoenzimes of alkaline phosphatase since this enzyme is synthetised also by the liver and by the gut.

aracterised by low serum levels of phosphorus and by an increase in serum levels of alkaline phosphatase that can reach values 5 times higher than the upper reference range used for adults [37]. It is useful dosing the isoenzimes of alkaline phosphatase since this enzyme is synthetised also by the liver and by the gut. Backstrom and colleagues suggested that serum alkaline phosphatase levels higher than 900 U.I/l associated with a serum phosphate level lower than 1.8 mmol/l have a diagnostic sensitivity of 100% and specificity of 70% [38]. However the opinions in literature about the reliability of alkaline phosphatase to predict the status of bone mineralization are still conflicting [39,40]

hatase levels higher than 900 U.I/l associated with a serum phosphate level lower than 1.8 mmol/l have a diagnostic sensitivity of 100% and specificity of 70% [38]. However the opinions in literature about the reliability of alkaline phosphatase to predict the status of bone mineralization are still conflicting [39,40] Serum level of calcium is usually within the normal range due to effects of PTH on the bone. Low concentrations of calcium and phosphorus in the urine suggest an inadequate intake. This is manly due by an increase of the tubular reabsorption of phosphate because of the low dietary intake and by an increase of PTH level that stimulates the reabsorption of calcium. Markers of nutritional status should be assessed baseline, and then weekly during the initial phase; once the newborn is stable, assessment must be done at the starting of total enteral nutrition and successively every 2–3 weeks. If MBD is diagnosed and nutritional supplementation is started, a periodic assessment of laboratory data is necessary to evaluate the response to treatment also when babies are discharged from hospital. The key clinical goal is to maintain normocalcemia and normophosphatemia and to avoid an excessive calciuria. Once levels of ALP, calcium and phosphorus normalize, serum analysis can be performed monthly up to 6 months of age and then every 3 months. X-rays examination may show fractures, thin bones and other alterations as reduction of thickness of the cortical, enlargement of the epiphysis, irregular border between growth cartilage and bony metaphysis [41].

Once levels of ALP, calcium and phosphorus normalize, serum analysis can be performed monthly up to 6 months of age and then every 3 months. X-rays examination may show fractures, thin bones and other alterations as reduction of thickness of the cortical, enlargement of the epiphysis, irregular border between growth cartilage and bony metaphysis [41]. Dual energy X-ray absorbitometry (DEXA) is able to determine the bone mass content of neonates and can predict the risk of fractures [39,42] since it is sensitive in detecting small changes in BMC and BMD. Its use is now validated in neonates both term and preterm ones. DEXA reflects most accurately the state of bone mineralization in preterm infants [43] but the examination involves radiations for the baby and the device is not portable. Quantitative ultrasound is simpler than DEXA and is non-invasive; it can be used bedside without moving the baby. Reference values are now available for infants. Quantitative ultrasound gives information about structure of the bone and about bone density [44]. Osteopenia has a good prognosis since the disease is self-resolving, provided that calcium, phosphates and vitamin D are appropriately administered to the babies. It is still controversial the need for high calcium and phosphorus intakes in preterm infants after hospital discharge. Few data are available about the optimal length, quantity and methods of providing supplemental minerals for preterm infants who are in stable growth.

Osteopenia has a good prognosis since the disease is self-resolving, provided that calcium, phosphates and vitamin D are appropriately administered to the babies. It is still controversial the need for high calcium and phosphorus intakes in preterm infants after hospital discharge. Few data are available about the optimal length, quantity and methods of providing supplemental minerals for preterm infants who are in stable growth. There are studies that show increased bone mineral mass in infants who receive formulas containing more minerals that the traditional ones up to 9 months [45,46]. It has been shown, with studies assessing bone mineralization with quantitative ultrasound and DEXA, that preterm infants show a catch-up mineralization for the first year of life. There is no difference in late childhood of bone mineralization between term and ex-preterm infants [47] even though the biochemical evidence of metabolic bone disease during the neonatal period may have a long-term stunting effect which continues up to 12 years later. A recent study published on Journal of Perinatology [48] stated that children who were born prematurely with birth weights less than 1.5 kg tend to be significantly smaller for age and have lower lumbar spinal bone mineral content and density compared with children born at term gestation. The long duration of this complication provides further rationale for implementing any practice that can prevent this condition [49].

It has been shown, with studies assessing bone mineralization with quantitative ultrasound and DEXA, that preterm infants show a catch-up mineralization for the first year of life. There is no difference in late childhood of bone mineralization between term and ex-preterm infants [47] even though the biochemical evidence of metabolic bone disease during the neonatal period may have a long-term stunting effect which continues up to 12 years later. A recent study published on Journal of Perinatology [48] stated that children who were born prematurely with birth weights less than 1.5 kg tend to be significantly smaller for age and have lower lumbar spinal bone mineral content and density compared with children born at term gestation. The long duration of this complication provides further rationale for implementing any practice that can prevent this condition [49]. In the case of BMD of prematurity nutrition is both therapy and prevention. An adequate intake of minerals and of vitamin D, with breast milk fortifier or formula with a content of minerals suitable for preterm infant's requirements, are necessary for a correct bone mineralization. A regular physical stimulation, when the preterm infant is clinically stable and is receiving adequate doses of calcium, phosphate and vitamin D, should also be included in the standard preventive approach.

In the case of BMD of prematurity nutrition is both therapy and prevention. An adequate intake of minerals and of vitamin D, with breast milk fortifier or formula with a content of minerals suitable for preterm infant's requirements, are necessary for a correct bone mineralization. A regular physical stimulation, when the preterm infant is clinically stable and is receiving adequate doses of calcium, phosphate and vitamin D, should also be included in the standard preventive approach. Conclusion An adequate nutritional intake of calcium, phosphorus and vitamin D and passive physical exercise may prevent abnormal bone-remodelling activity during first weeks of life and may optimize growth potential of preterm infants. It is important to recognize the biochemical signs of osteopenia in an early stage in order to be able to precociously implement the dietary intake and reduce the risk of bones fractures. The determination of alkaline phosphatase and of phosphoraemia seems to be useful in assessing the risk of metabolic bone disease and serum analysis need to be performed periodically in order to assess response to nutritional treatment. Through DEXA and quantitative ultrasound it is also possible to determine the state of bone mineralization and therefore to plan a nutritional intervention. Competing interests The authors declare that they have no competing interests. Authors' contributions VB and PT equally contributed at the article, analyzing the literature and writing the paper. All authors read and approved the final manuscript.

Conclusion An adequate nutritional intake of calcium, phosphorus and vitamin D and passive physical exercise may prevent abnormal bone-remodelling activity during first weeks of life and may optimize growth potential of preterm infants. It is important to recognize the biochemical signs of osteopenia in an early stage in order to be able to precociously implement the dietary intake and reduce the risk of bones fractures. The determination of alkaline phosphatase and of phosphoraemia seems to be useful in assessing the risk of metabolic bone disease and serum analysis need to be performed periodically in order to assess response to nutritional treatment. Through DEXA and quantitative ultrasound it is also possible to determine the state of bone mineralization and therefore to plan a nutritional intervention. Competing interests The authors declare that they have no competing interests. Authors' contributions VB and PT equally contributed at the article, analyzing the literature and writing the paper. All authors read and approved the final manuscript. Acknowledgements We are grateful to Prof. G. Weber, University Vita-Salute, San Raffaele Scientific Institute of Milan, Italy, for her helpful advice.

Introduction Nitric oxide (NO) is formed in biological systems from L-arginine and oxygen by the enzyme nitric oxide synthases (NOS), of which 3 isoforms have been described: type I and III, which are constitutive (cNOS) and type II which is inducible (iNOS) [1-3] (figure 1). NO is a mediator with a multitude of important regulatory functions and is present in exhaled air in humans [4]. The measurement of nitric oxide in exhaled air (FeNO) is a useful tool to monitor eosinophilic bronchial inflammation in atopic asthma [5]. FeNO measurement has been standardized in cooperative children and adults [6], and normative values have been published for children aged 4 to 17 years [7]. Guidelines for the measurement of FeNO in young children are also available and a number of methodological issues related to the FeNO measurement in infants have been addressed [8]. However, to date there is no standardized technique to measure FeNO in the first 2 years of life. Figure 1 Synthesis of nitric oxide (NO) from L-arginine. iNOS: inducible nitric oxide synthases; cNOS: constitutive nitric oxide synthases.

Introduction Nitric oxide (NO) is formed in biological systems from L-arginine and oxygen by the enzyme nitric oxide synthases (NOS), of which 3 isoforms have been described: type I and III, which are constitutive (cNOS) and type II which is inducible (iNOS) [1-3] (figure 1). NO is a mediator with a multitude of important regulatory functions and is present in exhaled air in humans [4]. The measurement of nitric oxide in exhaled air (FeNO) is a useful tool to monitor eosinophilic bronchial inflammation in atopic asthma [5]. FeNO measurement has been standardized in cooperative children and adults [6], and normative values have been published for children aged 4 to 17 years [7]. Guidelines for the measurement of FeNO in young children are also available and a number of methodological issues related to the FeNO measurement in infants have been addressed [8]. However, to date there is no standardized technique to measure FeNO in the first 2 years of life. Figure 1 Synthesis of nitric oxide (NO) from L-arginine. iNOS: inducible nitric oxide synthases; cNOS: constitutive nitric oxide synthases. FeNO measurement in infants A method appropriate for the measurement of FeNO in infants should be safe, easy to perform and non-invasive. The tidal breathing and the single breath methods have been proposed for the measurement of FeNO in infants and both techniques have showed a good reproducibility and a high success rate [9-12]. The single breath method is often combined with other lung function tests, but requires the sedation of the infant, specialized equipment and well-trained personnel [9,13]. Therefore, it is less suitable for routine testing or large epidemiological studies. The tidal breathing method has been successfully used both on-line (FeNO testing with a real-time display of NO breath profiles) [10] and off-line (collection of exhaled air into receptacles for delayed analysis) [11] (figure 2). This is a simple technique that has the advantages of being non-invasive and can be applied without the use of sedatives [14,15]. However, as FeNO is flow-dependent, with tidal breathing there is a scatter of data depending on the variation in flow rates [6]. The influence of variable breathing pattern on FeNO could be limited by correcting the FeNO values for tidal flow parameters measured during the FeNO sampling [16]. Another option would be the computation of NO output, which takes into account also expiratory flow (NO output = FeNO × tidal flow). However, it has been shown that NO output differentiates children with and without smoke exposure as well as FeNO [10], hinting that the correction for expiratory flow might not be necessary.

her option would be the computation of NO output, which takes into account also expiratory flow (NO output = FeNO × tidal flow). However, it has been shown that NO output differentiates children with and without smoke exposure as well as FeNO [10], hinting that the correction for expiratory flow might not be necessary. Figure 2 Off-line FeNO measurement during tidal breathing. Mixed (oral and nasal) expired air is sampled via a face mask, connected to an NO-inert balloon via a nonrebreathing valve. To reduce contamination by ambient NO, the inspiratory port can be connected to a NO-free air reservoir (not shown). The measurement should be considered successful if the infants maintains a quite tidal breathing during the whole procedure, if the facemask is tightly fitted to nose and mouth and if at least five tidal breaths are collected. The contamination of exhaled air samples with ambient NO has been shown to occur even for low ambient NO concentration, particularly with the tidal-breathing method [15,17]. Hence, it has been suggested that infants always inhale NO free air prior to the FeNO measurement [6,15].

Figure 2 Off-line FeNO measurement during tidal breathing. Mixed (oral and nasal) expired air is sampled via a face mask, connected to an NO-inert balloon via a nonrebreathing valve. To reduce contamination by ambient NO, the inspiratory port can be connected to a NO-free air reservoir (not shown). The measurement should be considered successful if the infants maintains a quite tidal breathing during the whole procedure, if the facemask is tightly fitted to nose and mouth and if at least five tidal breaths are collected. The contamination of exhaled air samples with ambient NO has been shown to occur even for low ambient NO concentration, particularly with the tidal-breathing method [15,17]. Hence, it has been suggested that infants always inhale NO free air prior to the FeNO measurement [6,15]. In children and adults the nasal mucosa and the paranasal sinuses are important sources of NO production [6,8]. However, the extent of nasal NO contamination on the orally exhaled NO in infants is unknown. Although oral FeNO values are lower than mixed (oral + nasal) FeNO [11], either nasal or oral NO levels reflect mixed exhaled NO in infants [17]. The use of a two-compartment facemask could reduce nasal contamination, without disturbing the breathing pattern of infants. Nevertheless, the advantages of excluding the nasal contamination during the FeNO measurement in infants have still to be shown.

nasal or oral NO levels reflect mixed exhaled NO in infants [17]. The use of a two-compartment facemask could reduce nasal contamination, without disturbing the breathing pattern of infants. Nevertheless, the advantages of excluding the nasal contamination during the FeNO measurement in infants have still to be shown. Spirometric maneuvers have been shown to transiently reduce FeNO levels in asthmatic children [18] and adults [19] and current guidelines recommend to measure FeNO prior to lung function testing [6]. The limited data available in infants have demonstrate no effect of forced expiratory manoeuvres immediately preceding FeNO measurements on FeNO values in sedated infants with airway diseases [11]. Hence, it seems unnecessary to standardize the sequence of these tests. Several foods and beverages, such as nitrate-reach meals [20] and water consumption [21] have been shown to transiently influence FeNO. In healthy infants it has been show that there is no evidence of an effect on FeNO values of breastfeeding immediately preceding the FeNO measurements [11]. However, such finding should be confirmed also in infants with airways diseases.

als [20] and water consumption [21] have been shown to transiently influence FeNO. In healthy infants it has been show that there is no evidence of an effect on FeNO values of breastfeeding immediately preceding the FeNO measurements [11]. However, such finding should be confirmed also in infants with airways diseases. Factors not related to the measurement conditions, such as gender, gestational age and anthropometrics have been associated with FeNO also in infants [16,22]. Therefore, these factors should always be assessed and a possible association with FeNO in the specific population under study should be investigated and, eventually, controlled for in multivariable analyses. Increased FeNO values have been found in infants of atopic parents [13]. Also, it has been shown that infants exposed to maternal smoking during pregnancy have lower FeNO than unexposed [10], but this association was subsequently confirmed only in infants of mothers without atopic disease [16]. These findings suggest that maternal atopy modifies the association between FeNO and prenatal smoke exposure. A cross-sectional study by Franklin et al. [23] showed that FeNO was higher in infants exposed to environmental tobacco smoke after birth than in unexposed, suggesting a direct irritant effect of smoke on the infants' airways. However, a prospective birth cohort study recently evaluated the separate effects of pre- and postnatal tobacco smoke exposure in infants. This study showed that infants continuously exposed to smoke both in utero and after birth had lower FeNO than never exposed infants and infants exposed only postnatally, suggesting that the influence of smoking on FeNO depends on the timing and intensity of the exposure [16].

stnatal tobacco smoke exposure in infants. This study showed that infants continuously exposed to smoke both in utero and after birth had lower FeNO than never exposed infants and infants exposed only postnatally, suggesting that the influence of smoking on FeNO depends on the timing and intensity of the exposure [16]. Several methodological issues related to the FeNO measurements in infants have been addressed and further studies in large cohorts of healthy and diseased infants are needed for standardization. Results of recent studies support the hypothesis that the eosinophilic inflammation seen in asthmatic adults may be present already in wheezy preschool children [24] and in atopic infants [25]. Therefore, the development of a non-invasive bed-side test that could help predicting which infants will develop persistent symptoms would be of great value.

rt the hypothesis that the eosinophilic inflammation seen in asthmatic adults may be present already in wheezy preschool children [24] and in atopic infants [25]. Therefore, the development of a non-invasive bed-side test that could help predicting which infants will develop persistent symptoms would be of great value. FeNO and wheezing Only few studies evaluated FeNO in relation to specific pulmonary diseases early in life and the majority of them were performed with a cross-sectional design in selected populations of infants with a high risk of developing asthma. Baraldi et al. [14] showed that infants with recurrent wheeze had increased FeNO levels during an asthma exacerbation, which rapidly decreased after a course of corticosteroid treatment. Other studies have shown high FeNO levels in infants at increased risk of asthma, such as infants of atopic parents and infants with recurrent episodes of wheezing [13,16,17]. Also, in infants with multiple-trigger wheeze, a reduction of FeNO values has been shown after a course of montelukast or inhaled corticosteroid treatment [26-28]. FeNO has been shown to differentiate infants with various airways diseases already in the first 2 years of life, with the highest levels of FeNO in infants with recurrent wheezing and with atopic predisposition [29]. Such finding was subsequently replicated by Moeller et al. [30] who showed that preschoolers with frequent recurrent wheeze and a stringent index for the prediction of asthma at school age had increased FeNO compared with children with recurrent cough but no history of wheeze. Overall, these findings suggest that the eosinophilic bronchial inflammation seen in asthmatic children and adults could be a characteristic of multiple-trigger wheezy infants, as recently suggested also by a bronchial biopsy study [24]. Hence, FeNO might provide a useful tool to monitor eosinophilic bronchial inflammation early in life. It is noteworthy that previous studies evaluating the association between FeNO and wheezing reached similar conclusions, although the method used to measure FeNO differed between the studies.

onchial biopsy study [24]. Hence, FeNO might provide a useful tool to monitor eosinophilic bronchial inflammation early in life. It is noteworthy that previous studies evaluating the association between FeNO and wheezing reached similar conclusions, although the method used to measure FeNO differed between the studies. Only few prospective studies evaluated the role of FeNO in the development of wheezing and asthma-like symptoms. Latzin et al. [31] showed that a high FeNO after birth was associated with severe respiratory symptoms in the first year of life if the mother had an atopic disease or had been smoking during pregnancy, with the strongest association when both factors were present. These findings would support the hypothesis that only infants who are at higher risk of developing asthma, either due to genetic factors, exposures or both, have higher FeNO. In fact, in the same study a trend towards a negative association between FeNO and severe respiratory symptoms in infants of nonatopic mothers was found [31]. This is in agreement with a recent prospective birth cohort study, which showed that 2 month-old infants with respiratory symptoms had lower FeNO than asymptomatic infants, and that these associations were independent and not modified by maternal atopy [15]. A possible explanation for the negative association between FeNO and respiratory symptoms is that infants participating in these longitudinal studies were not selected because of their health status and therefore did not represent a population of infants at increased risk of developing asthma, who would be more likely to have increased FeNO. Another explanation is that the symptoms assessed in these studies were grouped in one variable, which included not only wheezing, but also other respiratory symptoms that may be due to other mechanisms, including infection. Indeed low FeNO have been found in infants with first time viral-induced acute wheezy bronchitis [32], suggesting that acute viral infection may downregulate NO production [32]. An alternative hypothesis could be that impaired NO diffusion into the airway leading to reduced FeNO might be due to epithelial damage and increased airway secretions in infants with upper respiratory infections [33]. As episodic wheezing associated to viral infections is mostly related to neutrophilic airway inflammation [34], this might explain the reduced FeNO found in infants with viral-induced wheezing.

uced FeNO might be due to epithelial damage and increased airway secretions in infants with upper respiratory infections [33]. As episodic wheezing associated to viral infections is mostly related to neutrophilic airway inflammation [34], this might explain the reduced FeNO found in infants with viral-induced wheezing. The limited follow-up of recent prospective studies does not allow at the moment to evaluate whether FeNO measured in the first year of life predicts the development of asthma at school age. Also, the differences in FeNO values reported in previous studies comparing infants with and without symptoms are rather small. Furthermore, it is not possible to evaluate whether the FeNO values obtained in infants correlate with the FeNO measured with a standardized method (on-line or off-line with constant flow) at school age. As children in these cohort studies will grow up, a more clear wheezing pattern will become evident and the diagnosis of asthma will be supported also by the measurement of lung function. Then, it will be possible to evaluate with more precision the predictive role of FeNO measured in infants on the development of asthma at school age. FeNO and other pulmonary diseases FeNO has been explored as a marker of pulmonary diseases that begin early in life, such as primary ciliary dyskinesia, bronchopulmonary dysplasia and cystic fibrosis.

The limited follow-up of recent prospective studies does not allow at the moment to evaluate whether FeNO measured in the first year of life predicts the development of asthma at school age. Also, the differences in FeNO values reported in previous studies comparing infants with and without symptoms are rather small. Furthermore, it is not possible to evaluate whether the FeNO values obtained in infants correlate with the FeNO measured with a standardized method (on-line or off-line with constant flow) at school age. As children in these cohort studies will grow up, a more clear wheezing pattern will become evident and the diagnosis of asthma will be supported also by the measurement of lung function. Then, it will be possible to evaluate with more precision the predictive role of FeNO measured in infants on the development of asthma at school age. FeNO and other pulmonary diseases FeNO has been explored as a marker of pulmonary diseases that begin early in life, such as primary ciliary dyskinesia, bronchopulmonary dysplasia and cystic fibrosis. Primary ciliary dyskinesia Primary ciliary dyskinesia (PCD) is an autosomal recessive disease characterized by the lack of effective ciliary motility, causing abnormal mucociliary clearance. This leads to recurrent or persistent respiratory infections in the upper and lower airways [35]. Lower levels of nasal NO have been found in children with PCD as compared to children with bronchiectasis, CF, asthma and healthy controls [36,37]. A recent study has proposed the measurement of nasal NO as a screening test for the diagnosis of PCD, showing that values above a cut-off level of 105 parts per billion could exclude PCD [38]. Children with PCD have low FeNO, but overlap in FeNO values between normal children and those with PCD has been documented [39]. Therefore, FeNO cannot be used to discriminate patients with PCD from healthy subject as clearly as measurements of nasal NO [40]. Larger studies are needed in order to evaluate whether and to what extent FeNO and nasal NO measurements can be helpful for the screening or the diagnosis of PCD in the first years of life.

Therefore, FeNO cannot be used to discriminate patients with PCD from healthy subject as clearly as measurements of nasal NO [40]. Larger studies are needed in order to evaluate whether and to what extent FeNO and nasal NO measurements can be helpful for the screening or the diagnosis of PCD in the first years of life. Bronchopulmonary dysplasia and chronic lung disease Bronchopulmonary dysplasia (BPD) is defined as clinical signs of respiratory distress, chest radiograph abnormalities, and oxygen dependence at 28 days [41]. BPD accounts for the majority of chronic lung disease (CLD) and the airways inflammation of patients with BPD seems to be mostly mediated by neutrophilic granulocytes [42]. It has been shown that school-age BPD survivors have lower FeNO than asthmatics with comparable airflow obstruction, than preterm non-BPD and than healthy children [43]. It has been hypothesized that the pulmonary damage occurring in the early stage of BPD or the reduction of the vascular bed [44] could be responsible of such findings. However, little data is available on the association between FeNO and BPD in the first years of life. Leipala et al. [45] measured FeNO on-line at constant flow in 1-year old infants and showed higher FeNO values in infants with CLD compared to preterm infants without CLD and to infants born at term. Roiha et al. [46] measured on-line tidal FeNO in infants at 1 year and showed that NO output could differentiate between CLD and non-CLD infants better than FeNO. Also, the current authors measured FeNO off-line during tidal breathing in large groups of infants with different airways diseases below the age of 2 years. BPD infants had higher FeNO than CF infants, but lower than atopic infants with recurrent wheezing, taking lung function parameters, tidal volume, and breathing frequency into account [29]. Further studies with longer follow-up are warranted in order to evaluate whether FeNO can be useful to monitor the inflammatory pattern of the airways of BPD infants.

infants, but lower than atopic infants with recurrent wheezing, taking lung function parameters, tidal volume, and breathing frequency into account [29]. Further studies with longer follow-up are warranted in order to evaluate whether FeNO can be useful to monitor the inflammatory pattern of the airways of BPD infants. Cystic fibrosis Cystic fibrosis (CF) is a hereditary disease that affects the lungs, digestive system, sweat glands and male reproductive organs. CF lung disease is characterized by chronic neutrophilic airway inflammation, mucus plugging and bronchial infections with specific bacterial pathogens, leading to progressive bronchiectasis and lung damage from which most CF patients die at a median age of 35–40 years. Low FeNO values have been found in both adults and children with CF [47,48] and FeNO has also been shown to correlate with CF severity in children [49]. Although one study previously showed not reduced FeNO levels in CF patients as compared with healthy infants [50], there is a large body of evidence suggesting that infants with CF have lower FeNO values compared with infants with other respiratory diseases and healthy controls [29,51]. It has been hypothesized that the mechanisms underlying the reduced FeNO values in CF patients are related to excess secretions in CF airways that might impair the NO diffusion through the airways [52] or to a primary defect in NO production [53,54]. A recent study by Zetterquist et al. [55] supports the latter hypothesis, by showing that the discrepancy of elevated exhaled NO metabolites and low levels of FeNO are explained by an impaired function or expression of the enzymes involved in the NO metabolism. The differences in FeNO values between CF, healthy infants and infants with other respiratory diseases are relatively small. However, the findings of lower FeNO in CF patients are consistent between studies and the data available seem to suggest that FeNO measurements might be useful in alerting for the diagnosis of CF.

he differences in FeNO values between CF, healthy infants and infants with other respiratory diseases are relatively small. However, the findings of lower FeNO in CF patients are consistent between studies and the data available seem to suggest that FeNO measurements might be useful in alerting for the diagnosis of CF. Conclusion Practical recommendations for the measurement of FeNO in the first 2 years of life have been published, but to date no standardized technique is available for this age group. Despite different methods to measure FeNO in infants, most findings are comparable among study groups and show consistently high FeNO values in infants at increased risk of developing asthma. Also, specific patterns of FeNO have been shown for groups of infants with different airways diseases. Hence, FeNO might provide a helpful tool for the differential diagnosis of airways diseases in the first years of life. Further follow-up studies in larger groups of infants are needed in order to assess whether and to what extent the measurement of FeNO can predict the subsequent development of airways diseases. Competing interests CG and FMdB declare they have no competing interest. JCdJ received a research grant from Aerocrine AG, Solna, Sweden (manufacturer of NO analyzers) in 2005 and 2006. Authors' contributions CG drafted the manuscript. FMdB and JCdJ critically revised the manuscript. All authors read and approved the final manuscript.

Competing interests CG and FMdB declare they have no competing interest. JCdJ received a research grant from Aerocrine AG, Solna, Sweden (manufacturer of NO analyzers) in 2005 and 2006. Authors' contributions CG drafted the manuscript. FMdB and JCdJ critically revised the manuscript. All authors read and approved the final manuscript. Authors' information CG worked at the Department of Pediatric Respiratory Medicine, Sophia Children's Hospital – Erasmus University Medical Centre (head Prof. JC de Jongste) in Rotterdam, The Netherlands, between 2003 and 2008. During this period he developed several research projects focusing on the measurements of exhaled nitric oxide in infants. This study period will culminate with the dissertation of the PhD thesis to be held at the Erasmus University in 2009. CG currently works as attending physician at the Department of Pediatrics, Salesi Children's Hospital, Azienda Ospedaliero-Universitaria, Ancona, Italy (head Prof. FM de Benedictis). Acknowledgements Written informed consent was obtained from the relatives of the patient for publication of the accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

Dear Editor, Recent studies suggested a link between type 1 diabetes mellitus and pervasive developmental disorder (PDD) and hypothesized a common autoimmune pathogenesis or shared genetic factors [1]. Confirmation of an association between PDD and type 1 diabetes mellitus is still lacking. On the contrary permanent neonatal diabetes mellitus (PNDM) due to pancreas agenesis is an extremely rare condition, associated with intra-uterine growth retardation (IUGR), cardiac defects, gall bladder or cerebellum agenesis [2]. No association with psychiatric symptoms has been described up to now. We describe a patient with association of PNDM due to pancreatic agenesis, mild mental retardation and PDD. The patient, a boy who is currently aged 6, is the first child of healthy unrelated parents (father from Italy and mother from Spain). He was born at 35 weeks of gestation, with severe IUGR (weight 1620 g, <3rd percentile and length 45 cm, <3rd percentile) [3]. In the first day of life he developed hyperglycemia: neonatal diabetes was diagnosed and treated with insulin, initially intravenously and then subcutaneously. Atrial septal defect was discovered, in absence of other dysmorphic features. Pancreas agenesis was detected by ultrasound, and then confirmed by magnetic resonance imaging (MRI). Pancreatic exocrine insufficiency was treated with enzymes. Glycemic control was characterized during the first two years of life by fluctuations despite strict dietary regimen and frequent variation of insulin dosages. When 3 month-old the child presented a severe episode of hypoglicemia (plasma glucose 30 mg%); intramuscular glucagon was administrated and no neurological damage was evident at subsequent neuroimaging studies. Karyotype was normal and search for Fragile-X Syndrome was negative, as well as genetic testing for the most common forms of neonatal diabetes [4]. Brain MRI and neurophysiologic evaluations (auditory brainstem evoked potential, nerve conduction velocities and electroencephalogram) were normal.

nt neuroimaging studies. Karyotype was normal and search for Fragile-X Syndrome was negative, as well as genetic testing for the most common forms of neonatal diabetes [4]. Brain MRI and neurophysiologic evaluations (auditory brainstem evoked potential, nerve conduction velocities and electroencephalogram) were normal. Because of developmental delay with behavioural abnormalities (impaired language functions with echolalia, isolation, stereotypies, no emotional sharing, hyperactivity) he underwent neuropsychiatric evaluation when 4 year-old. No focal signs were observed at neurological examination. Global motricity and coordination resulted normal. Griffiths' mental developmental scales revealed mild mental retardation, with global developmental quotient of 70. Neuropsychological testing with Childhood Autism Rating Scale (CARS), Autism Diagnostic Interview (ADI) and Autism Diagnostic Observation Schedule (ADOS) lead to diagnosis of Pervasive Developmental Disorder Non Otherwise Specified (PDD-NOS), according to Diagnostic and Statistical Manual of Mental Disorders IV-R (DSM IV-R) criteria [5]. This is the first case of PDD diagnosed in a child with pancreas agenesis. Four patients from two consanguineous Pakistan families with pancreatic agenesis associated with cerebellar agenesis have been reported, so that association of both pancreatic system and central nervous system developmental failure has been previously described [6]. Cerebellum is considered to have an important role in cognitive network as well as in the development of PDD [7]; our patient might have had abnormalities of cerebellar development not detected by standard neuroimaging but sufficient to impair cognition and behaviour. Nevertheless, we cannot exclude that in our case other perinatal risk factors for PDD should have influenced the behavioural disorder, as reported for IUGR and congenital malformations [8]. We want to emphasize the potential association between PNDM due to pancreatic agenesis and PDD, for prompt identification and appropriate care. A possible syndromic association needs further cases to be established. The existence of a link between PNDM and PDD may provide important insights into the pathogenesis of both conditions.

phasize the potential association between PNDM due to pancreatic agenesis and PDD, for prompt identification and appropriate care. A possible syndromic association needs further cases to be established. The existence of a link between PNDM and PDD may provide important insights into the pathogenesis of both conditions. Competing interests The authors declare that they have no competing interests. Authors' contributions All Authors participated in the data collection and read and approved the final manuscript. AG and GdA conceived of the study, and participated in its design and coordination, MP and MMM partecipated in clinical evaluation, FMB stated neuropsychological analysis, EV and RL partecipated in writing this manuscript.

Introduction Cow's milk protein allergy (CMPA) is a common disease occurring in childhood and its prevalence approximates 3% during the first 3 years of life [1]. Clinical manifestations of CMPA are atopic dermatitis (AD), urticaria/angioedema, gastrointestinal symtoms, and less frequently, respiratory disorders, such as wheezing and asthma. The therapy for CMPA consists in eliminating cow's milk proteins (CMP) from the child's diet. Extensively hydrolyzed formulas (eHF) are currently the most used substitutes of CMP. They have a good nutritional value, but are not tolerated by all patients with CMPA, are quite expensive and present poor palatability. Also amino-acid formulas have an unpleasant taste, and should be considered in severe clinical manifestations of food allergy or when eHF are not effective [2]. Soy protein-based formulas have a moderate palatability and provide appropriate nutrition. However there are not reccommended for the tratment of young children with CMPA because of the potential to evoke allergic reactions [3]. For these reasons, other mammalian milks have been considered to replace cow's milk (CM), including ass's milk (AM). The nutritional composition of AM is similar to that of human milk [4,5]. Furthermore, AM has an acceptable taste. There are in literature few clinical Italian studies concerning the allergenicity and tolerability of AM, and their results are not conclusive [6-8]. Recently, Monti et al. have documented that the tolerability of AM was 82.6% in their selected cohort of children with CMPA, without other alternative to the use of common CMP substitutes [6]. Other Authors have emphasized the risk of potential cross-reactivity between CMP and AM proteins, suggesting that more in vivo and in vitro studies are required to optimize dietary needs for infants with CMPA [9]. Furthermore, some authors have found an impaired growth in infants affected by CMPA, who received different type of CMP substitutes [10]. We aimed to investigate the AM tolerability and nutitional adequacy in children with CMPA from Apulia, a Southern Italy region where AM is readily available and frequently used also in healthy children.

thors have found an impaired growth in infants affected by CMPA, who received different type of CMP substitutes [10]. We aimed to investigate the AM tolerability and nutitional adequacy in children with CMPA from Apulia, a Southern Italy region where AM is readily available and frequently used also in healthy children. Methods Between January and July 2008 we recruited 30 consecutive children who attended our University Department of Pediatric Allergy and Immunology. Inclusion criteria were: i) suspected clinical history of CMPA; ii) absence of concomitant chronic diseases; iii) antihistamine therapy and/or systemic corticosterois not administred in the 4 week preceeding the study. The local Ethics Committee approved the study protocol. Informed consent to partecipate to the study was obtained from the participants or legal guardians. Patients underwent a full history and physical examination at entry. The diagnosis of CMPA was made on the basis of a CMP elimination diet (for 4 weeks), followed by bouble-blind, placebo controlled food challenge (DBPCFC). The DBPCFCs were performed at the clinic, with full facilities for resuscitation available. Fresh CM was administred at increasing doses of 0.1, 0.3, 1.0, 3.0, 10.0, 30.0, and 100 ml, using Neocate® (Nutricia, Liverpool, UK) as placebo. Pear juice was mixed to both CM and placebo in equal parts to mask the taste. The time interval between each dose was 20 min [11]. After completing the DBPCFC, children were kept under observation for at least 6 h and then discharged. Before food challenge, skin prick tests (SPT) were also performed using fresh CM, AM, pear juice and other common food- and aero-allergens, according to a protocol described elsewhere (positive if wheal diameter > 3 mm) [12]. Serum levels of specific IgE (alpha-lactalbumin, beta-lactoglobulin, casein, AM) were determined by the automated Pharmacia CAP System FEIA (Pharmacia Diagnostics AB, Uppsala, Sweden), with a cut-off point for positivity set at 0.35 KU/l.

rding to a protocol described elsewhere (positive if wheal diameter > 3 mm) [12]. Serum levels of specific IgE (alpha-lactalbumin, beta-lactoglobulin, casein, AM) were determined by the automated Pharmacia CAP System FEIA (Pharmacia Diagnostics AB, Uppsala, Sweden), with a cut-off point for positivity set at 0.35 KU/l. After confirming the diagnosis of CMPA by DBPCFC, patients received fresh AM (ass-farming of Alberobello, Bari, Italy) in a open challenge, as described for CM. Fresh AM was analyzed for sterility criteria by the Experimental Zooprophylactic Institute of Putignano, Bari, Italy. In case of clinical tolerance, AM was included into the child's diet, which was appropriately balanced depending on age requirements. The study design included follow-up in the form of clinical check-up and auxological evaluation at entry (T0), and after 4–6 months (T1) of AM consumption. Standing height (H) was evaluated with a wall-mounted Harpenden Stadiometer. With patients in underwear, weight (W) was measured with an electronic scale with digital readings accurate to 0.1 kg. Body mass index (BMI) was calculated dividing W in kilograms by the square of H in meters, and it was expressed as centiles according to the Italian growth charts [13]. Blood levels of biochemical and metabolic parameters (iron, calcium, proteins, cholesterol, triglycerides, glycaemia, folic acid, etc.) from all subjects, were assessed at each observational time period. Also hidden faecal blood was sought at the beginning and at the end of the study.

Standing height (H) was evaluated with a wall-mounted Harpenden Stadiometer. With patients in underwear, weight (W) was measured with an electronic scale with digital readings accurate to 0.1 kg. Body mass index (BMI) was calculated dividing W in kilograms by the square of H in meters, and it was expressed as centiles according to the Italian growth charts [13]. Blood levels of biochemical and metabolic parameters (iron, calcium, proteins, cholesterol, triglycerides, glycaemia, folic acid, etc.) from all subjects, were assessed at each observational time period. Also hidden faecal blood was sought at the beginning and at the end of the study. Statistical analysis Statistical analyses were performed using SPSS® for Windows software (SPSS Inc., version 15.0, Chicago, IL). Data were expressed as the mean ± SD. Mann-Whitney tests were used to compare groups, and correlations between variables were performed using the Sperman rank correlation test. Paired-samples t test was used to compare data before and after AM intake for the group of CMPA patients. Significance was defined as p < 0.05.

Data were expressed as the mean ± SD. Mann-Whitney tests were used to compare groups, and correlations between variables were performed using the Sperman rank correlation test. Paired-samples t test was used to compare data before and after AM intake for the group of CMPA patients. Significance was defined as p < 0.05. Results All enrolled children fulfilled the inclusion criteria. Symptoms at first observation were cutaneous (atopic dermatitis, 24/20; urticaria/angioedema, 14/30), gastrointestinal (12/30), and/or respiratory (8/30). Patient's clinical data are summarized in Table 1. Twenty-seven children (90%) were affected by an IgE-mediated form of CMPA, while only two subjects (7%), who presented gastrointestinal manifestations, had a non IgE-mediated CMPA, with negative SPT and CMP-specific IgE. All patients were SPT-negative for pear juice. Table 1 Characteristics of the patients enrolled in the study Patient's Characteristics Male/Female ratio 1:1 Age (y), median (range) 4.5 (6.0 mo-10 y) Total serum IgE at baseline (IU/mL) 335,93 Positive Skin Prick Tests to: (n) Cow's milk 27 Ass's milk 2 Wheat 2 Hen's egg 7 Soy 4 Pollens 5 Dust mite 7 Molds 2 Pets with fur 4

Table 1 Characteristics of the patients enrolled in the study Patient's Characteristics Male/Female ratio 1:1 Age (y), median (range) 4.5 (6.0 mo-10 y) Total serum IgE at baseline (IU/mL) 335,93 Positive Skin Prick Tests to: (n) Cow's milk 27 Ass's milk 2 Wheat 2 Hen's egg 7 Soy 4 Pollens 5 Dust mite 7 Molds 2 Pets with fur 4 Positive specific IgE to: (n) alpha-lactalbumin 21 beta-lactoglobulin 17 casein 9 Ass's milk 2 The CMPA was confirmed with DBPCFC in 28 children. The parents of one out of two children with SPT positive for AM and the concomitant presence of serum specific IgE for AM refused to give their consent to the ass's milk challenge. In two other cases parents of childs with severe reactions to CM challege did not consent to continue the study. The AM was tolerated by 24 out of 25 children who were tested with the food challenge (22 with IgE-mediated CMPA and 2 with non-IgE-mediated disease). The patient with CMPA and positive AM specific IgE and SPT for AM had a systemic reaction at the food challenge with cutaneous lesions, cought and vomiting. The auxological evaluations at enrolement (T0) and at the end of the study (T1) are shown in Table 2. All children had an improvement of their W and H at T1. Moreover, at entry all children presented a BMI corresponding to the normalweight (50° centile), which was kept unchanged by the end of the study. Table 2 Auxological parameters of the study population by sex and age at enrolement (T0) and at the end of the study (T1)

The auxological evaluations at enrolement (T0) and at the end of the study (T1) are shown in Table 2. All children had an improvement of their W and H at T1. Moreover, at entry all children presented a BMI corresponding to the normalweight (50° centile), which was kept unchanged by the end of the study. Table 2 Auxological parameters of the study population by sex and age at enrolement (T0) and at the end of the study (T1) W (Kg) mean ± SD H (cm) mean ± SD BMI mean ± SD (centiles) All children T0 17,3 ± 9,6 T0 100,3 ± 14,9 T0 16,1 ± 1,5 (50°) T1 19,4 ± 11,6 T1 105,4 ± 23,2 T1 16,0 ± 2,1 (50°) p = 0.02 p < 0.001 p = ns Boys T0 16,9 ± 6,7 T0 102,2 ± 23,7 T0 15,8 ± 1,1 (50°) T1 18,3 ± 7,2 T1 106,3 ± 20,8 T1 15,6 ± 0,8 (50°) p < 0.01 p = 0.03 p = ns Girls T0 17,9 ± 13,3 T0 98,1 ± 29,0 T0 16,5 ± 2,0 (50°) T1 20,6 ± 16,4 T1 104,2 ± 28,2 T1 16,6 ± 3,0 (50°) p = ns p < 0.01 p = ns *p significance for Paired T-test ns = not significant Any relevant variation of blood biochemical and metabolic parameters was observed (data not shown), and also the faecal tests resulted negative.

W (Kg) mean ± SD H (cm) mean ± SD BMI mean ± SD (centiles) All children T0 17,3 ± 9,6 T0 100,3 ± 14,9 T0 16,1 ± 1,5 (50°) T1 19,4 ± 11,6 T1 105,4 ± 23,2 T1 16,0 ± 2,1 (50°) p = 0.02 p < 0.001 p = ns Boys T0 16,9 ± 6,7 T0 102,2 ± 23,7 T0 15,8 ± 1,1 (50°) T1 18,3 ± 7,2 T1 106,3 ± 20,8 T1 15,6 ± 0,8 (50°) p < 0.01 p = 0.03 p = ns Girls T0 17,9 ± 13,3 T0 98,1 ± 29,0 T0 16,5 ± 2,0 (50°) T1 20,6 ± 16,4 T1 104,2 ± 28,2 T1 16,6 ± 3,0 (50°) p = ns p < 0.01 p = ns *p significance for Paired T-test ns = not significant Any relevant variation of blood biochemical and metabolic parameters was observed (data not shown), and also the faecal tests resulted negative. Discussion In this study we report an experience on clinical tolerability of AM, which was high at the food challenge (24/25, 96%), made in an Italian Region, Apulia, where AM is readily available and frequently used because of the presence of several ass's farming. AM was tolerated by our patients either with the IgE- and the non-IgE-mediated CMPA. All enrolled subjects found it acceptable due to its palatability, and did not interrupted the study. AM has been considered an alternative to CM considering that its protein composition is similar to human milk [14]. However, it is a low-calory food and thus in our study we enrolled children older than 6 months who did not have an exclusive milk diet. It is noteworthy to highlight the fact that patients with severe reaction to CM refused to continue this study, thus our results are referred to subjects with a mild to moderate form of CMPA. Only one recruited patient had a systemic food reaction during the AM challenge. He was the only tested child who had SPT positive for AM and high serum levels of AM specific IgE, suggesting the predictive positive value of these tests also for AM allergy.

are referred to subjects with a mild to moderate form of CMPA. Only one recruited patient had a systemic food reaction during the AM challenge. He was the only tested child who had SPT positive for AM and high serum levels of AM specific IgE, suggesting the predictive positive value of these tests also for AM allergy. Other Italian authors have documented the efficacy of this food in treating children with CMPA. Vita et al. showed that the rate of tolerability of AM in patients with AD and CMPA was 88%, and that AM improved child's eczema [7]. Similarly, Monti et al. assessed the tolerance of AM (82.6%) in a selected population of children with CMPA, for whom it was not possible to use any other cow's milk substitute [6]. However, it must be taken into account the potential cross-reactivity of AM proteins with CMP, also suggested by the above mentioned studies that reported some severe reactions to AM in their study cohorts [6-9].

population of children with CMPA, for whom it was not possible to use any other cow's milk substitute [6]. However, it must be taken into account the potential cross-reactivity of AM proteins with CMP, also suggested by the above mentioned studies that reported some severe reactions to AM in their study cohorts [6-9]. Furthermore, our data show an adequate increase in weight and length/stature in all recruited children. Their BMI centiles measured after 4–6 months of AM administration were stable compared to those at the beginning of the study. Similar observations were reported by Monti in the early months of AM intake [6]. These Authors concluded that the effect on growth of AM is related to its ability to fill some nutritional gaps present in the diet of the subjects treated. Other authors have found an impaired growth in infants affected by CMPA, who received different type of CMP substitutes [10]. We suggest that a longer follow-up study is needed in order to reach reliable results. We also verified that patients' biochemical and metabolic parameters in the blood did not vary during the study period. Taken toghether these results suggest that AM might be considered nutritionally adequate in children with a varied diet. Competing interests The authors declare that they have no competing interests.

Furthermore, our data show an adequate increase in weight and length/stature in all recruited children. Their BMI centiles measured after 4–6 months of AM administration were stable compared to those at the beginning of the study. Similar observations were reported by Monti in the early months of AM intake [6]. These Authors concluded that the effect on growth of AM is related to its ability to fill some nutritional gaps present in the diet of the subjects treated. Other authors have found an impaired growth in infants affected by CMPA, who received different type of CMP substitutes [10]. We suggest that a longer follow-up study is needed in order to reach reliable results. We also verified that patients' biochemical and metabolic parameters in the blood did not vary during the study period. Taken toghether these results suggest that AM might be considered nutritionally adequate in children with a varied diet. Competing interests The authors declare that they have no competing interests. Authors' contributions TR partecipated in the study design and interpretations of results, and performed statistical analysis and the preparation of the manuscript. PC partecipated in the data collection and helped to draft the manuscript. BS has provided contribution to the data collection. AL conceived the study, partecipated in its design and coordination and helped in the interpretation of results. All authors read and approved the final manuscript. Acknowledgements We thanks Dr Domenica Rizzi and Carolina Desiderato for technical support.

Background Inherited hemoglobin disorders are an increasing global health problem[1]. Italy is a natural reservoir of Sickle Cell Disease (SCD) due to the high frequency of the S gene in the Sicilian population (2–13%) [2] and to the high frequency of beta thalassemia mutations in the Italian population (6–11%)[3]. HbS/HbS and HbS/βthalassemia forms of SCD are therefore already present in Italy. Globalization, with its population movements, brought to the Northern Regions of Italy immigrants from areas where other SCD variants are even more frequent (HbS/HbS with different aplotypes or HbS/HbC) [1]. This led to an increase of SCD patients, mainly African immigrants [4] and raises the need to organize a network of care tailored to the specific needs of our population of SCD patients. In fact, SCD shows extreme phenotypic variability among individuals and among populations [1,2,5]. Both environmental and genetic factors are likely to contribute to most manifestations of HbS/HbS SCD disease that develops with different patterns in various ethnic groups and individuals [5,6]. Stroke is a frequent complication of SCD. Approximately 10% of African-american patients have a clinical stroke before 20 years of age [7] and another 22% have a silent infarction on magnetic resonance imaging [8]. Stroke has also been described in Mediterranean [6,9] and African SCD patients [10-13] even though with lesser frequencies (4.1% and 6.7% respectively).

ximately 10% of African-american patients have a clinical stroke before 20 years of age [7] and another 22% have a silent infarction on magnetic resonance imaging [8]. Stroke has also been described in Mediterranean [6,9] and African SCD patients [10-13] even though with lesser frequencies (4.1% and 6.7% respectively). Screening programs for stroke prevention in SCD children have been implemented in the United States and in some European countries where comprehensive services for SCD patients are part of routine care [14-17] using Transcranial Doppler (TCD). TCD can measure flow velocities in the large intracranial arteries. The narrowing of these arteries, which leads to cerebral infarction is characterized by an increased velocity of flow. Long term prospective studies have demonstrated the validity of TCD in identifying children at risk of stroke due to increased cerebral velocities[14]. These studies have also demonstrated that prevention of the first stroke (primary prevention) can be done with success with chronic transfusion in children at risk for stroke according to abnormal blood flow TCD velocities [15]. Although TCD cannot predict all strokes, TCD and TCCS offer an opportunity to apply an effective therapy (chronic transfusion) for patients in the risk group [7,14,15].

mary prevention) can be done with success with chronic transfusion in children at risk for stroke according to abnormal blood flow TCD velocities [15]. Although TCD cannot predict all strokes, TCD and TCCS offer an opportunity to apply an effective therapy (chronic transfusion) for patients in the risk group [7,14,15]. TCD/TCCS Reference peak-flow velocity values defining normal, conditional and at-risk patients (TAMM <170, 170–200, >200 cm/sec respectively) have been defined through long-term prospective studies performed in African-american patients with HbS/HbS or HbS/HbC disease in the United States [7,14]. The same reference values have also been used in Caucasian patients with HbS/βthalassemia in Greece demonstrating an equal validity in predicting stroke[16]. But TCD or TCCS studies are not yet performed in African countries and normal reference values or baseline data for African SCD patients or for healthy Africans are therefore not yet available. The stroke predictive power of TCD in African populations has therefore not been demonstrated. In Italy a National Comprehensive Program for the care and treatment for SCD is still missing and a routine screening with TCD able to reach the majority of SCD pediatric patients has still to be implemented. Italian national reported data on TCD values are mainly from Caucasian adult patients during acute stroke or from the healthy adult female population [18,19]. A survey on the healthy pediatric Italian population or the healthy first generation of African immigrant children is missing.

ients has still to be implemented. Italian national reported data on TCD values are mainly from Caucasian adult patients during acute stroke or from the healthy adult female population [18,19]. A survey on the healthy pediatric Italian population or the healthy first generation of African immigrant children is missing. One of the major concerns in TCD/TCCS screening programs in US and UK is the limited access that patients and family have to routine TCD/TCCS exams. Several studies have in fact outlined that even though TCD screening is included in the American National Institute of Health (NIH) guidelines [20] and British National Health System (NHS) [21] guidelines of standard health maintenance for SCD children, wide application of this procedure is uncommon [21-24]. Overburdening children and families with too many appointments and not including TCD/TCCS in routine hematological visits are one of the causative factors of non adherence to TCD screening. Others include poor understanding of TCD/TCCS usefulness to prevent serious complications by the parents. These issues are of particular concern when dealing with a population of first-generation immigrants – as the one that mainly constitutes the group of SCD pediatric patients in Italy- and must be taken into account in the design of a primary stroke prevention program in our setting.

erious complications by the parents. These issues are of particular concern when dealing with a population of first-generation immigrants – as the one that mainly constitutes the group of SCD pediatric patients in Italy- and must be taken into account in the design of a primary stroke prevention program in our setting. In order to set up a Primary Stroke Prevention Program in our region, we performed a pilot study to determine the feasibility of TCD and TCCS in a group of African children with SCD routinely followed at the Clinic of Pediatric Hematology-Oncology of the University of Padova. With the objective of increasing acceptance of TCD/TCCS screening and compliance to follow-up we stressed the educational part, performed in different languages With the aim of verifying if internationally accepted reference values could also apply to our population and could be used as reference screening values, we compared cerebral velocities, pulsatility indexes and depth of the patient's group to those of two groups of healthy age-matched controls of Caucasian and African origin respectively.

ying if internationally accepted reference values could also apply to our population and could be used as reference screening values, we compared cerebral velocities, pulsatility indexes and depth of the patient's group to those of two groups of healthy age-matched controls of Caucasian and African origin respectively. Materials and methods Patients enrolment TCD and TCCS screening were proposed to the parents of African children with SCD routinely followed at the Clinic of Pediatric Hematology-Oncology of the University of Padova. After explaining the neurological complications of SCD and the possibility to identify children at risk of stroke with TCD/TCCS, the opportunity to perform the screening during one of the scheduled routine hematology visits was offered. To be sure of proper understanding, explanations to parents were performed not only in Italian, but also in English or French, according to the need. Age-matched controls were recruited among healthy Caucasian and African children known by the physicians. TCD-TCCS TCD and TCCS were performed on the same day of a scheduled hematology visit and in the same hospital complex were the Pediatric Hematology-Oncology Clinic is located. TCD and TCCS were performed using a 2 MHz pulsed Doppler ultrasonograph (EME TCD 2000/S) and a ATL HDI 3000/S Echo Doppler system respectively. Patients were not sedated during the examination.

TCD-TCCS TCD and TCCS were performed on the same day of a scheduled hematology visit and in the same hospital complex were the Pediatric Hematology-Oncology Clinic is located. TCD and TCCS were performed using a 2 MHz pulsed Doppler ultrasonograph (EME TCD 2000/S) and a ATL HDI 3000/S Echo Doppler system respectively. Patients were not sedated during the examination. We have measured peak systolic blood flow velocities (PSV), end diastolic blood flow velocities (EDV), time-averaged mean velocity of maximum blood flow (TAMM) and mean blood flow velocities (MV), at the level of the Basilar Artery (BA) and of the Middle Cerebral Artery (MCA) on both sides of the brain. We used the Stroke Prevention Trial in Sickle Cell Anemia Study (STOP) criteria [7,14,15], which are based on the intracranial velocities (TAMM) measured in the MCAs, by the TCD method, to assign stroke risk as low (TAMM < 170 cm/sec), conditional (TAMM ≥ 170 – 199 cm/sec), or high (TAMM ≥ 200 cm/sec). In our study we decided to assess also the blood flow velocities of the BA as we wanted to explore the intracranial hemodynamics of the Vertebro Basilar circulation in SCD (an arterial territory not evaluated in the STOP Trial). The intracranial blood flow velocities can have a wide variations in different normal subjects depending on several factors (age, angle correction, diameter of the vessel). Statistical Analyses The Kolmogorov-Smirnov test was used to preliminary check the normality of the variables. The One-Way Analysis of Variance was then performed. The significance level was set at p < 0.05.

In our study we decided to assess also the blood flow velocities of the BA as we wanted to explore the intracranial hemodynamics of the Vertebro Basilar circulation in SCD (an arterial territory not evaluated in the STOP Trial). The intracranial blood flow velocities can have a wide variations in different normal subjects depending on several factors (age, angle correction, diameter of the vessel). Statistical Analyses The Kolmogorov-Smirnov test was used to preliminary check the normality of the variables. The One-Way Analysis of Variance was then performed. The significance level was set at p < 0.05. Results Thirty-six children were enrolled: group A with 12 SCD HbS/HbS African patients, Group B with 12 healthy African age-matched controls, Group C with 12 healthy Caucasian age-matched controls. Each group had 8 Females and 4 Males. Mean age was 4.51 ± 2.87 years (range: 1.06–9.86), 5.75 ± 3.38 (range:1.21–12.55), 6.84 ± 3.6 (range: 1.41–12.07) for GROUP A, B and C respectively with no significantly statistical difference (p = 0.55). TCD and TCCS were performed in all patients and an adequate temporal window could be obtained in all of them. Pulsatility index and depth values in both the MCA and the BA (Fig 1, 2) were similar at TCD and TCCS evaluation in the three groups. TAMM, PSV and EDV in the MCA and BA (Fig. 3, 4) were significantly higher in group A patients on both TCD and TCCS evaluation (p = 0.0001 and p = 0286, respectively). No difference was observed between the right and left side. TCCS values were slightly higher that TCD values, with no statistical difference.

ree groups. TAMM, PSV and EDV in the MCA and BA (Fig. 3, 4) were significantly higher in group A patients on both TCD and TCCS evaluation (p = 0.0001 and p = 0286, respectively). No difference was observed between the right and left side. TCCS values were slightly higher that TCD values, with no statistical difference. Figure 1 Middle Cerebral Artery: Mean of Depth (circle) and Pulsatility index (square) in TCD (narrow line) and TCCS (thick line) in the three groups of patients A, B, C. Figure 2 Basilar Artery: Mean of Depth (circle) and Pulsatility index (square) in TCD (narrow line) and TCCS (thick line) in the three groups of patients A, B, C. Figure 3 Middle Cerebral Artery: Mean of PSV (circle), TAMM (square) EDV (triangle) in TCD (narrow line) e TCCS (thick line) in the three groups of patients A, B, C. Figure 4 Basilar Artery: Mean of PSV (circle), TAMM (square) EDV (triangle) in TCD (narrow line) e TCCS (thick line) in the three groups of patients A, B, C. Three GROUP A patients had conditional velocities, the remaining being normal. In our population none had abnormal velocity, even though the overall mean velocities were higher than control ones.

Figure 4 Basilar Artery: Mean of PSV (circle), TAMM (square) EDV (triangle) in TCD (narrow line) e TCCS (thick line) in the three groups of patients A, B, C. Three GROUP A patients had conditional velocities, the remaining being normal. In our population none had abnormal velocity, even though the overall mean velocities were higher than control ones. Discussion Evaluating immigrant children with SCD using TCD and TCCS for primary stroke prevention in our setting is feasible, even if neurosonology examinations are not yet routinely performed for children or adolescents. All parents accepted to perform TCD and TCCS and both exams were feasible in all children. Performing TCD and TCCS during a routine visit at the Hematology Clinic without over burning families with other appointments, might have helped in assuring a positive answer and in assuring compliance for successive controls as happened in other countries [21-24]. The education of parents on neurological complications of SCD and the benefits of screening, as well as the reassurance on the lack of risk of the TCD procedure, emerges as a fundamental component of a successful stroke prevention program in our setting. Performing education in a language that parents can understand, feeling free to expose doubts and ask questions without the linguistic barriers that immigrant parents and patients generally experience, is surely a simple way to increase adherence and participation.

a successful stroke prevention program in our setting. Performing education in a language that parents can understand, feeling free to expose doubts and ask questions without the linguistic barriers that immigrant parents and patients generally experience, is surely a simple way to increase adherence and participation. Greater variability of TCCS compared to TCD in our population correlates with previous reports [25]. TCD was easily performed in pediatric patients than TCCS due to the less time needed to perform the exam. Extreme phenotypic variability is a landmark of SCD [1,6]. Various reports describe different clinical patterns in different populations, according to haplotype of the βglobin genes and related genes [26]. Various reports indicate also cerebrovascular complications of SCD as happening with different prevalence in different populations. Even though excess stroke risk exists in blacks [27,28], it has been postulated that stroke occurs with less frequency in African SCD children compared to Caucasians or African-americans [10,11,29], questioning the need to perform regular screening in these populations. A 11% prevalence of stroke has been observed in the American Cooperative Study of Sickle Cell Disease [7,8] while a 6.7% prevalence of clinically evident stroke has been reported in first generation SCD African immigrants living in France [30]. In the French cohort of homozygous HbS/HbS african children, abnormal TCD has been reported in 9.6% of cases [30,31], while abnormal TCD was present in 9.5% to 12% of the American cohorts [14,15].

prevalence of clinically evident stroke has been reported in first generation SCD African immigrants living in France [30]. In the French cohort of homozygous HbS/HbS african children, abnormal TCD has been reported in 9.6% of cases [30,31], while abnormal TCD was present in 9.5% to 12% of the American cohorts [14,15]. In our study, Caucasian healthy and African healthy children had similar low values of all parameters measured on TCD and TCCS. SCD African patients had higher mean values of velocities, but pulsatility and depth were similar to healthy controls, both white and black. These data suggest that ethnic background does not influence baseline normal values of pulsatility, depth and velocities parameters and that SCD is the only factor influencing and determining velocities increase. Pulsatility index, indicating vascular resistance, does not seem to be influenced by SCD being the same in the three groups of children.

c background does not influence baseline normal values of pulsatility, depth and velocities parameters and that SCD is the only factor influencing and determining velocities increase. Pulsatility index, indicating vascular resistance, does not seem to be influenced by SCD being the same in the three groups of children. In our group of patients, at the time of the analysis, none presented abnormal velocities even thought mean velocities were higher that in controls. Three children (25%), however, had conditional values. The lower Mean age in our group compared to the one of the French cohort (mean 10.1 ± 5.8 years and median 9.3 years) [30,31] could partially be taken as an explanation of this data. Longer follow up and a larger population is needed to precisely define the risk of stoke and the percentage of abnormal TCD in African children with SCD. The three children that had conditional velocities (170–200 cm/sec) at initial evaluation, developed abnormal high velocities (>200 cm/sec) after six months and had to be placed on a chronic transfusion program; one child developed ischemic stroke while waiting for the first transfusion. These preliminary data suggest that a primary stroke prevention program for our population is necessary and set the base to further evaluate the implementation of such a program in our setting of HbS/HbS African immigrants and HbS/beta thalassemia Italians.

loped ischemic stroke while waiting for the first transfusion. These preliminary data suggest that a primary stroke prevention program for our population is necessary and set the base to further evaluate the implementation of such a program in our setting of HbS/HbS African immigrants and HbS/beta thalassemia Italians. With appropriate training and coordination between Pediatric Sickle Cell Groups and Neurosonology Services, the costs of the screening could be very limited. The Adult Neurosonology Services that are already developed across Italy for the evaluation of Adult Stroke could use the equipment and the personnel already available in the Adult Stroke Team to perform TCD screening in pediatric sickle cell patients. For the present being, the small number of pediatric sickle cell patients is not likely to over burn the neurosonology services and to raise the need of adjunctive pediatric neurosonology services Investigating multiple cerebral vascular districts, besides the MCA, could aid in defining the ones that can be easily monitored and that could better predict future stroke. Conclusion Evaluation of blood flow in the MCAs and in the BA through two different intracranial ultrasonographic techniques is feasible in pediatric SCD African immigrants. A routine primary stroke prevention program should be implemented as part of comprehensive standard health evaluation for SCD children in our setting and parent education-possibly in the native language-should be a necessary part of the program.

trasonographic techniques is feasible in pediatric SCD African immigrants. A routine primary stroke prevention program should be implemented as part of comprehensive standard health evaluation for SCD children in our setting and parent education-possibly in the native language-should be a necessary part of the program. The definitive validity in our setting of the internationally accepted reference cut-off values already in use in other Caucasian and African-american SCD pediatric patients should be confirmed in a wide prospective study. Competing interests The authors declare that they have no competing interests. Authors' contributions All the authors contributed equally to the research and to the manuscript. RC designed the study, supervised data acquisition and interpretation; wrote the manuscript and gave the final approval. GM designed the study, performed the analysis supervising data interpretation and revised the manuscript. ME performed data analysis, participated in data interpretation and revised the manuscript. MP designed the study, collected the data and revised the manuscript. LS designed the study, supervised data acquisition and interpretation; revised the manuscript and gave the final approval Acknowledgements The research was performed with the grant 07/04 of the Fondazione Città della Speranza

Introduction The T wave is variable in shape under physiological or pathological conditions, but rarely shows a bifid configuration in adults; this phenomenon can be observed in patients with some forms of long QT syndrome [1], in subjects taking Class 3 antiarrhythmic drugs, particularly Amiodarone [2], in patients with alcoholic cardiomyopathy [3,4] or in the presence of central nervous system lesions [5]. At times, a U wave partially superimposed upon the T wave is responsible for an apparently bifid T wave: in such a situation, often associated with electrolyte imbalance [6], the second wave hump is a U wave rather than part of the T wave. Bifid T waves, in contrast, have been described in the past in normal children [7-9], but the phenomenon has not been emphasized in recent years, to the extent that it is not mentioned in current textbooks of paediatric electrocardiography. The present study was aimed at exploring the incidence and clinical counterpart of bifid T waves in children, namely whether they are merely a normal variant or can be associated with any disease. Methods At the cardiology outpatient clinic of our Paediatric Department, 604 consecutive children were selected from March to May 2008, on the basis of the following: 1) absence of clinically detectable heart disease; 2) good quality electrocardiogram (ECG), with at least 4 consecutive QRS complexes in each lead; 3) absence of ECG abnormalities such as arrhythmias (apart from respiratory sinus arrhythmia), bundle branch block, atrial enlargement, ventricular hypertrophy, preexcitation.

absence of clinically detectable heart disease; 2) good quality electrocardiogram (ECG), with at least 4 consecutive QRS complexes in each lead; 3) absence of ECG abnormalities such as arrhythmias (apart from respiratory sinus arrhythmia), bundle branch block, atrial enlargement, ventricular hypertrophy, preexcitation. Whenever a bifid T wave was detected, a complete clinical and echocardiographic examination was performed using an Acuson Sequoia machine. The ECGs of 110 consecutive adult healthy subjects observed at the cardiology outpatient clinic of the Department of Medicine and Pharmacology, have also been examined. In any ECG, a search was done for the presence of bifid T waves; in addition, the heart rate and the QTc interval were measured. A T wave was defined as bifid whenever it was notched, being the 2 peaks separated from each other by a notch with duration ≥ 0.02 sec and voltage ≥ 0.05 mV. Moreover, in 7 consecutive children with bifid T wave in lead V2 further precordial recordings were obtained; a small electrode was gradually moved from V1 to V3, and 4 additional leads were recorded: 2 between V1 an V2, and 2 between V2 and V3.

In any ECG, a search was done for the presence of bifid T waves; in addition, the heart rate and the QTc interval were measured. A T wave was defined as bifid whenever it was notched, being the 2 peaks separated from each other by a notch with duration ≥ 0.02 sec and voltage ≥ 0.05 mV. Moreover, in 7 consecutive children with bifid T wave in lead V2 further precordial recordings were obtained; a small electrode was gradually moved from V1 to V3, and 4 additional leads were recorded: 2 between V1 an V2, and 2 between V2 and V3. Results The mean age of the 604 children was 7.3 ± 3.2 years (range 3 months-16 years). The subjects were divided in 17 groups, each corresponding to 1 year of age. A bifid T wave was observed in 110 children (18,3%), with a relatively age-related incidence; Figure 1 reflects the prevalence of bifid T wave in the age classes: the phenomenon is relatively rare in infants and in teen-agers, whereas the highest rate of bifid T wave (53%) occurs in the 5-year-old children group. Examples of bifid T waves are shown in Figure 2; in some cases the T wave has a "dome and dart" configuration, with a second component peaked and clearly separated from the first one (panels D, E). Figure 1 Incidence of bifid T wave in the age classes. Figure 2 Elettrocardiograms (leads V1–V3) recorded in 5 healthy children. The bottom row shows an magnification of the T wave in lead V2.

Results The mean age of the 604 children was 7.3 ± 3.2 years (range 3 months-16 years). The subjects were divided in 17 groups, each corresponding to 1 year of age. A bifid T wave was observed in 110 children (18,3%), with a relatively age-related incidence; Figure 1 reflects the prevalence of bifid T wave in the age classes: the phenomenon is relatively rare in infants and in teen-agers, whereas the highest rate of bifid T wave (53%) occurs in the 5-year-old children group. Examples of bifid T waves are shown in Figure 2; in some cases the T wave has a "dome and dart" configuration, with a second component peaked and clearly separated from the first one (panels D, E). Figure 1 Incidence of bifid T wave in the age classes. Figure 2 Elettrocardiograms (leads V1–V3) recorded in 5 healthy children. The bottom row shows an magnification of the T wave in lead V2. The bifid T wave was detected only in lead V2 in 51 cases (46,4%), only in lead V3 in 5 cases (4,6%), in both leads V2 and V3 in 50 cases (45,4%), and in leads other than V2 and V3 in 4 cases (3,6%). In the adult group, none of the examined ECGs showed bifid T waves in any lead.

Figure 2 Elettrocardiograms (leads V1–V3) recorded in 5 healthy children. The bottom row shows an magnification of the T wave in lead V2. The bifid T wave was detected only in lead V2 in 51 cases (46,4%), only in lead V3 in 5 cases (4,6%), in both leads V2 and V3 in 50 cases (45,4%), and in leads other than V2 and V3 in 4 cases (3,6%). In the adult group, none of the examined ECGs showed bifid T waves in any lead. The additional leads recorded in 7 subjects (2 recordings between V1 an V2, as well as between V2 and V3) showed always the pattern represented in Figure 3. The T wave was totally negative in lead V1, but became less negative, with a terminal minimally positive deflection, when the electrode was slightly displaced towards the left (panel A). A further displacement of the electrode in the same direction (panel B) resulted in reduction of the negative T wave component and increase of the positive part, so that a bifid T wave appeared. Lead V2, in turn, showed again a typical bifid T wave, and the same morphology was once more recorded with the electrode minimally shifted leftward (panel C); a further displacement (panel D) resulted in increase of the first positive component of the T wave, and finally V3 showed either a bifid or an entirely positive T wave (panel E). Figure 3 Precordial recordings obtained displacing gradually the electrode from V1 to V3. A e B are recordings intermediate between V1 and V2; C e D are intermediate between V2 and V3.

The additional leads recorded in 7 subjects (2 recordings between V1 an V2, as well as between V2 and V3) showed always the pattern represented in Figure 3. The T wave was totally negative in lead V1, but became less negative, with a terminal minimally positive deflection, when the electrode was slightly displaced towards the left (panel A). A further displacement of the electrode in the same direction (panel B) resulted in reduction of the negative T wave component and increase of the positive part, so that a bifid T wave appeared. Lead V2, in turn, showed again a typical bifid T wave, and the same morphology was once more recorded with the electrode minimally shifted leftward (panel C); a further displacement (panel D) resulted in increase of the first positive component of the T wave, and finally V3 showed either a bifid or an entirely positive T wave (panel E). Figure 3 Precordial recordings obtained displacing gradually the electrode from V1 to V3. A e B are recordings intermediate between V1 and V2; C e D are intermediate between V2 and V3. In the bifid T wave population, the echocardiogram did not reveal any abnormality, apart from 3 subjects which had an asymptomatic mitral valve prolapse; a trivial mitral and/or tricuspid regurgitation detected by color Doppler, as well as a patent foramen ovale in infants, were not considered as abnormal findings.

Figure 3 Precordial recordings obtained displacing gradually the electrode from V1 to V3. A e B are recordings intermediate between V1 and V2; C e D are intermediate between V2 and V3. In the bifid T wave population, the echocardiogram did not reveal any abnormality, apart from 3 subjects which had an asymptomatic mitral valve prolapse; a trivial mitral and/or tricuspid regurgitation detected by color Doppler, as well as a patent foramen ovale in infants, were not considered as abnormal findings. A U wave clearly separated from the bifid T wave (Figure 2, panels A and B) has been detected in leads V2 and/or V3 in 31 children (28%). The QTc interval was normal in all of the subjects; the average QTc interval was not different in the bifid T wave population (402 ± 46 msec) with respect to the control group (407 ± 39 msec). Discussion The reported data show that a bifid T wave in leads V2 and V3 is a normal pattern in children, particularly in those aged 5 to 8 years; in healthy adults, in contrast, no bifid T wave is detectable in any lead. The statement that a bifid T wave in leads V2 and V3 is a normal finding in children is reinforced by the absence of detectable heart disease in all of our subjects; in addition, the normal QTc interval denies that such a phenomenon expresses a repolarization abnormality.

ast, no bifid T wave is detectable in any lead. The statement that a bifid T wave in leads V2 and V3 is a normal finding in children is reinforced by the absence of detectable heart disease in all of our subjects; in addition, the normal QTc interval denies that such a phenomenon expresses a repolarization abnormality. Origin of the bifid T wave The bifid T wave was present only in leads V2 and V3, and never in the limb leads or in leads V1, V5 or V6. This finding suggests that the phenomenon depends on the peculiar orientation of the electrical forces generated by ventricular repolarization on the horizontal plane. In order to point out the mechanism underlying the bifid T wave in V2 and V3, we obtained in 7 subjects additional recordings by moving the electrode from V1 to V3. A progressive T wave change from a negative to a positive shape was always observed, and the T wave showed a bifid configuration during transition from negativity to positivity. This observation has led us to the following explanation, represented in Figure 4. Figure 4 Genesis of the bifid T wave. Vectorcardiographic T loop on the horizontal plane; T waves recorded in V1, V2, and V4.

In order to point out the mechanism underlying the bifid T wave in V2 and V3, we obtained in 7 subjects additional recordings by moving the electrode from V1 to V3. A progressive T wave change from a negative to a positive shape was always observed, and the T wave showed a bifid configuration during transition from negativity to positivity. This observation has led us to the following explanation, represented in Figure 4. Figure 4 Genesis of the bifid T wave. Vectorcardiographic T loop on the horizontal plane; T waves recorded in V1, V2, and V4. In subjects with bifid T wave, the T wave vectorcardiographic loop in the horizontal plane shows a figure-of-eight shape with rotation initially counterclockwise and subsequently clockwise: the first vectors are directed leftward and slightly anteriorly, and then the electrical forces move posteriorly. The second part of the loop rotates in a clockwise direction, being displaced anteriorly, and only the final section of the loop is again counterclockwise with the terminal forces directed posteriorly. In accordance with this sequence of electrical forces, the T wave is negative in lead V1 because all the vectors project on the negative part of the lead line. Lead V2, whose positive pole is at +90°, in turn, "sees" as positive the first T wave vectors, projecting on the positive part of the lead line, while the ensuing vectors, directed posteriorly, are "seen" as negative. When the late section of the loop is again displaced anteriorly, a second T wave positivity occurs.

e. Lead V2, whose positive pole is at +90°, in turn, "sees" as positive the first T wave vectors, projecting on the positive part of the lead line, while the ensuing vectors, directed posteriorly, are "seen" as negative. When the late section of the loop is again displaced anteriorly, a second T wave positivity occurs. The two T wave positive components in lead V2, thus, are due to the 2 groups of anterior forces, whereas the intermediate notch expresses the vectors directed posteriorly. When the recording electrode is displaced towards the left, the amount of vectors projecting on the positive part of the lead line increases, and the notch between the two cusps becomes less and less pronounced until the T waves becomes entirely positive, as it happens in lead V4, whose positive pole is at +60°. Clinical relevance of the bifid T wave The incidence of bifid T waves in leads V2 and V3 in normal children is high, and awareness of this phenomenon avoids possible misinterpretations leading to a diagnosis of ECG abnormalities.

The two T wave positive components in lead V2, thus, are due to the 2 groups of anterior forces, whereas the intermediate notch expresses the vectors directed posteriorly. When the recording electrode is displaced towards the left, the amount of vectors projecting on the positive part of the lead line increases, and the notch between the two cusps becomes less and less pronounced until the T waves becomes entirely positive, as it happens in lead V4, whose positive pole is at +60°. Clinical relevance of the bifid T wave The incidence of bifid T waves in leads V2 and V3 in normal children is high, and awareness of this phenomenon avoids possible misinterpretations leading to a diagnosis of ECG abnormalities. The first diagnostic problem, in the presence of a bifid T wave, is distinction from a U wave more or less coinciding with the T wave. Some drugs, particularly antiarrhythmic drugs, as well electrolyte imbalance such as hypokalemia, prolong the QT interval and give rise to a prominent U wave that is partially superimposed upon the T wave, simulating a bifid morphology of this. Drug or hypokalemia-induced QT prolongation and related T wave change, however, are not limited to leads V2 and V3 but occur in several leads, including the limb leads. In our normal children, in contrast, bifid T waves have been observed only in V2 or V2 and V3; in addition, the simultaneous presence of bifid T wave and U wave, observed in several cases, rules out the possibility that the second "hump" of the bifid T wave was indeed a U wave.

several leads, including the limb leads. In our normal children, in contrast, bifid T waves have been observed only in V2 or V2 and V3; in addition, the simultaneous presence of bifid T wave and U wave, observed in several cases, rules out the possibility that the second "hump" of the bifid T wave was indeed a U wave. Another possible abnormal condition simulated by the bifid T wave is a non-conducted P wave superimposed upon the T wave and responsible for the bifid configuration: in our series, however, no atrial extrasystoles were evident in any lead, and furthermore no bifid T wave was followed by a pause, as it generally occurs in non-conducted atrial extrasystoles. Accordingly, this hypothesis was ruled out. A bifid T wave in leads V2 and/or V3 should be considered a normal phenomenon in children; the absence of this pattern in adults demonstrates that the ventricular repolarization process changes with aging, in such a way that the T loop, that has a figure-of-eight shape in very young subjects, becomes entirely counterclockwise in adults, as it normally occurs [10]. Study limitations A vectorcardiogram would have provided a definite demonstration of the relationship between a figure-of-eight T loop in the horizontal plane and the bifid T wave in leads V2 and V3. In our Institution, however, no vectorcardiographic machine was available, and no vectorcardiographic examination was performed. Competing interests The authors declare that they have no competing interests.

Study limitations A vectorcardiogram would have provided a definite demonstration of the relationship between a figure-of-eight T loop in the horizontal plane and the bifid T wave in leads V2 and V3. In our Institution, however, no vectorcardiographic machine was available, and no vectorcardiographic examination was performed. Competing interests The authors declare that they have no competing interests. Authors' contributions MPC carried out the clinical examination of subjects, and coordinated the study. IB participated in data analysis and drafted the manuscript. ALM recorded the ECG tracings in the paediatric population, particularly those with additional precordial leads. MCT carried out the ECG analysis. FLDL participated in the design of the study. LO contributed in analyzing the Electrocardiograms. MSR performed the echocardiograms. MC recorded and analyzed the electrocardiograms in the adult population. LB performed the Echocardiograms. GO conceived of the study, participating in its design and coordination, and revised the manuscript. All authors read and approved the final manuscript.

Background Perinatal Hypoxic-Ischemic Encephalopathy (H.I.E.), secondary to asphyxia, is one of the most important causes of acute perinatal mortality and neurological sequelae[1] which include severe disability (e.g. cerebral palsy, mental retardation, epilepsy) and clinical situations of moderate severity where the cerebral impairment establishes a neurodevelopmental delay, including for example a deficit of postural-motor acquirements or a reduced school performance.[2] Our work aims to find: - predictive factors concerning the risk of both Cerebral Palsy (C.P.) and Epilepsy, secondary to HIE, at one year of age. These factors should be searched among clinical-instrumental parameters registered at birth. - Correlations between the clinical-instrumental parameters registered at birth and the variable severity of the above mentioned sequelae. We suppose that having early prognostic factors for these important neurological sequelae of HIE should enable: - the application of strategies to limit cerebral impairment by means of neuroprotection protocols[3]; - the application of early diagnosis protocols and the formulation of a rehabilitative and therapeutic strategy to improve the infants' quality of life [4,5].

We suppose that having early prognostic factors for these important neurological sequelae of HIE should enable: - the application of strategies to limit cerebral impairment by means of neuroprotection protocols[3]; - the application of early diagnosis protocols and the formulation of a rehabilitative and therapeutic strategy to improve the infants' quality of life [4,5]. Methods From June 2005 to June 2007 we have recruited 32 subjects between 1 and 8 years of age (among the patients attending our neurological Day Hospital1) with a clinical-instrumental diagnosis of HIE. The diagnosis was made on the basis of their general and neurological clinical picture and confirmed by cranial Ultrasonography and cerebral MRI images which showed hypoxic-ischemic cerebral impairments. The exclusion criteria were: - age below 1 year - infective encephalopathy - metabolic encephalopathy - encephalopathy secondary to genetic or chromosomal diseases - cerebral malformations The analysed parameters at birth were: - gender: male (m) or female (f); - gestational age (GA): term infants (t.i.) and preterm infants (p.i.), that is infants <37 weeks GA; - type of delivery: spontaneous vaginal delivery (s.v.d.), cesarean section delivery (c.s.d), emergency cesarean section delivery (e.c.s.d.) due to pregnancy or labour complications which could endanger the mother's or foetus' health. - birth weight: normal birth weight(NBW ≥ 2,5 kg), low birth weight (LBW ≥ 1,5–2,5 kg<), very low birth weight (VLBW ≥ 1–1,5 kg<), extremely low birth weight (ELBW < 1 kg);

- type of delivery: spontaneous vaginal delivery (s.v.d.), cesarean section delivery (c.s.d), emergency cesarean section delivery (e.c.s.d.) due to pregnancy or labour complications which could endanger the mother's or foetus' health. - birth weight: normal birth weight(NBW ≥ 2,5 kg), low birth weight (LBW ≥ 1,5–2,5 kg<), very low birth weight (VLBW ≥ 1–1,5 kg<), extremely low birth weight (ELBW < 1 kg); - 5' Apgar Score (5'AS): infants were subdivided into <5 5'AS group and ≥ 5 5'AS group, this parameter plays not only an important role in the early clinical assessment of neonates at birth but it is also a significant prognosis factor[4,6]; - neonatal clinical pattern: infants with prevalent neurological symptoms attributable to Sarnat 2nd/3rd stadium of HIE[6], infants with prevalent severe systemic phenomena of asphyxia (e.g. multiorgan involvement), infants with bland phenomena of asphyxia with neurologic symptoms attributable to Sarnat 1st stadium of HIE[6]; - EEG characteristics at early hours/days (early EEG): this item is very important for the prognosis of neonates with HIE[4,7,8]; we have defined early EEG reports as pathological (presence of abnormalities in EEG background rhythm and/or presence of pathologic patterns) (Figure 1) and not pathological [9]; Figure 1 electroencephalografical seizures and significantly altered EEG background rhythm in a two-day-old subject with diffuse post ischemic cerebral atrophy (Jasper montage).

- EEG characteristics at early hours/days (early EEG): this item is very important for the prognosis of neonates with HIE[4,7,8]; we have defined early EEG reports as pathological (presence of abnormalities in EEG background rhythm and/or presence of pathologic patterns) (Figure 1) and not pathological [9]; Figure 1 electroencephalografical seizures and significantly altered EEG background rhythm in a two-day-old subject with diffuse post ischemic cerebral atrophy (Jasper montage). - type of encephalic hypoxic-ischemic injuries on the basis of cranial ultrasonography(US) reports at early hours/days of life: evidence of persistent periventricular hyperechogenicity (PVH), evidence of periventricular/intraventricular hemorrhage (PV-IVH), evidence of focal-multifocal parenchymal hyperechogenicity with/without cystic formation; - type of encephalic hypoxic-ischemic injuries on the basis of MRI reports[1] at early hours/days of life: selective necrosis including lesions of basal ganglia (S.N.), parasagittal injury (P.S.I.), periventricular Leucomalacia(P.V.L.) and focal multifocal ischemic necrosis (F./M.I.N.). At one year of age we have classified the patients on the basis of their neurologic outcome: we have identified patients with both secondary epilepsy and CP (which is one of the main objectives of our research), patients with only CP or secondary epilepsy and patients with only a transitory deficit of postural-motor acquirements (motor delay) with full recovery within the first few years of life. Afterwards we have evaluated secondary epilepsy and CP severity.

At one year of age we have classified the patients on the basis of their neurologic outcome: we have identified patients with both secondary epilepsy and CP (which is one of the main objectives of our research), patients with only CP or secondary epilepsy and patients with only a transitory deficit of postural-motor acquirements (motor delay) with full recovery within the first few years of life. Afterwards we have evaluated secondary epilepsy and CP severity. Secondary epilepsy was classified as: - highly severe: presence of partial/secondary generalized seizures, pluriweekly frequency of crisis, drug refractoriness of seizures, active or very active EEG pathological patterns; - moderately severe: presence of partial seizures, 1 or <1 monthly frequency of crisis, good pharmacological control of seizures with crisis frequency reduction or disappearance, less active or absent EEG pathological patterns. Furthermore we decided to register the epileptic onset age in patients with 2 severest sequelae (both CP and secondary epilepsy), supposing that it could be an useful parameter to better evaluate the outcome of the epileptic clinical picture. CP was analyzed on the basis of neurologic examination, clinical observation and medical history in order to ascertain trunk control and sitting abilities within the first year of life [10].

Furthermore we decided to register the epileptic onset age in patients with 2 severest sequelae (both CP and secondary epilepsy), supposing that it could be an useful parameter to better evaluate the outcome of the epileptic clinical picture. CP was analyzed on the basis of neurologic examination, clinical observation and medical history in order to ascertain trunk control and sitting abilities within the first year of life [10]. This stage of psychomotor development is of crucial importance to determine the future prognosis of CP patients, especially in relation to their functional limitations (e.g. walking) and their performance in the various social contexts (home, school, community setting) [11,12]. CP was classified as: - highly severe CP: at 1 year of life patients do not have trunk control and cannot maintain floor sitting. - moderately severe CP: at 1 year of life patients have trunk control and can maintain floor sitting. After collecting all data, we carried out a descriptive statistical analysis and a correlation study among the examined variables. The correlation study wanted to establish a statistical significance (indicated by a < 0,05 P-value) and it was carried out using the analysis of variance (ANOVA) for continuous variables and the M-L Chi Square Test for categorical variables. Results The patient study group had the following characteristics: - 20 m and 12 f; - 19 t.i.(59%) and 13 p.i. (41%), average GA 35 weeks (range 24–41 weeks, median GA 39 weeks.); - 18 NBW (56%), 6 LBW (19%), 2 VLBW(6%) and 6 ELBW(19%), average birth weight 2425 g (r. 680-4370 g, median BW 2615 g);

After collecting all data, we carried out a descriptive statistical analysis and a correlation study among the examined variables. The correlation study wanted to establish a statistical significance (indicated by a < 0,05 P-value) and it was carried out using the analysis of variance (ANOVA) for continuous variables and the M-L Chi Square Test for categorical variables. Results The patient study group had the following characteristics: - 20 m and 12 f; - 19 t.i.(59%) and 13 p.i. (41%), average GA 35 weeks (range 24–41 weeks, median GA 39 weeks.); - 18 NBW (56%), 6 LBW (19%), 2 VLBW(6%) and 6 ELBW(19%), average birth weight 2425 g (r. 680-4370 g, median BW 2615 g); - 11 infants (34%) with 5'AS <5 (these subjects had signs of neonatal suffering due to severe or moderate asphyxia) and 21 infants (66%) with 5'AS ≥ 5. - 12 s.v.d.(38%), 10 c.s.d.(31%) and 10 e.c.s.d. (31%) - 10 patients (31%) with prevalent neurological symptoms (in particular neonatal seizures), 11 patients (34%) with prevalent severe systemic phenomena of asphyxia (essentially premature infants where the asphyxia was mainly expressed by symptoms such as sepsis, necrotizing enterocolite, respiratory distress etc...); 11 patients (34%) with bland phenomena of asphyxia (including neurological symptoms ascribable to Sarnat 1ststadium of HIE); - 14 infants(44%) with normal and 18 patients (56%) with pathological early EEG; - Pathological Cranial U.S in all 32 infants: PVH (38%), PV-IVH (16%), evidence of focal-multifocal parenchymal hyperechogenicity with/without cystic formation (47%);

- 10 patients (31%) with prevalent neurological symptoms (in particular neonatal seizures), 11 patients (34%) with prevalent severe systemic phenomena of asphyxia (essentially premature infants where the asphyxia was mainly expressed by symptoms such as sepsis, necrotizing enterocolite, respiratory distress etc...); 11 patients (34%) with bland phenomena of asphyxia (including neurological symptoms ascribable to Sarnat 1ststadium of HIE); - 14 infants(44%) with normal and 18 patients (56%) with pathological early EEG; - Pathological Cranial U.S in all 32 infants: PVH (38%), PV-IVH (16%), evidence of focal-multifocal parenchymal hyperechogenicity with/without cystic formation (47%); - Pathological cerebral MRI in all 32 infants: S.N. (19%), P.S.I.(19%), P.V.L. (41%), F./M.I.N (22%); The neurologic outcome within the first year of age showed the following results:

- Pathological Cranial U.S in all 32 infants: PVH (38%), PV-IVH (16%), evidence of focal-multifocal parenchymal hyperechogenicity with/without cystic formation (47%); - Pathological cerebral MRI in all 32 infants: S.N. (19%), P.S.I.(19%), P.V.L. (41%), F./M.I.N (22%); The neurologic outcome within the first year of age showed the following results: - 9 patients developed both secondary epilepsy and CP (28%) [see Additional file 1]: 7 were term infants (only 2 pre-term infants); 6 were NW (only 3 LBW); 7 with early pathologic EEG and neuroimages pointing out cortex injuries (typical of term infants[1]), these lesions are consistent with the generation of both an epileptogenical focus and a CP syndrome; the average age of epileptic onset was 5 months (median age 6 months) with 2 cases of neonatal exordium (neonatal seizures directly developed in a structured epileptic syndrome); 6 of these patients developed a highly severe epilepsy and a neuromotor clinical picture essentially characterized by highly severe CP, while 3 subjects developed a moderately severe epilepsy and a moderately severe CP. - 10 patients developed only a motor delay (31%). - 1 patient developed only secondary epilepsy, this infant was t.i., NBW with a ≥5 5'AS, with neonatal seizures and neuroimages indicating a P.S.I. and the patient manifested a moderately severe epilepsy at 1 year of age; - 12 patients showed only CP (38%): 4 subjects developed highly severe CP and 8 developed moderately severe CP; 6 of these infants were T.I., 6 were P.T. and 7 were NBW with ≥5 5'AS.

- 1 patient developed only secondary epilepsy, this infant was t.i., NBW with a ≥5 5'AS, with neonatal seizures and neuroimages indicating a P.S.I. and the patient manifested a moderately severe epilepsy at 1 year of age; - 12 patients showed only CP (38%): 4 subjects developed highly severe CP and 8 developed moderately severe CP; 6 of these infants were T.I., 6 were P.T. and 7 were NBW with ≥5 5'AS. It is worth to underline that all patients showing a moderately severe CP at one year of age, after their second birthday are able to walk, even if with some functional limitations, and do not need permanent assistive mobility devices. Whereas all patients showing a highly severe CP at one year of age, after their second birthday cannot walk autonomously, show important functional limitations and need assistive mobility devices. These figures could find a correspondence with the GMFCS (Gross Motor Function Classification System), which was the assessment scale showing the best validity and clinical utility [11,12]. The statistic study showed the following correlations: - birth weight and neurologic outcome (χ2 = 14,03; p = 0,04) - neonatal clinical pattern and CP severity (χ2 = 14,03; p = 0,0009) - early EEG and CP severity (χ2 = 4,32; p = 0,04) - epilepsy onset age and CP and Epilepsy severity (F = 16,01; p = 0,005)

These figures could find a correspondence with the GMFCS (Gross Motor Function Classification System), which was the assessment scale showing the best validity and clinical utility [11,12]. The statistic study showed the following correlations: - birth weight and neurologic outcome (χ2 = 14,03; p = 0,04) - neonatal clinical pattern and CP severity (χ2 = 14,03; p = 0,0009) - early EEG and CP severity (χ2 = 4,32; p = 0,04) - epilepsy onset age and CP and Epilepsy severity (F = 16,01; p = 0,005) Discussion Our study pointed out that birth weight is a predictive factor for neurologic outcome. We have observed that all ELBW and VLBW developed either CP or motor delay, therefore <1,5 kg birth weight neonates are not at risk of both epilepsy and CP at one year of age (Figure 2): in these subjects, typically preterm infants, the post-asphyxial injury involves essentially the white matter but also basal ganglia and thalamus[1], therefore it is possible to detect a neuromotor involvement compatible with a postural motor syndrome (such as cerebral palsy) but not compatible with an epileptic syndrome where the damage involves essentially the cerebral cortex[13]. Figure 2 birth weight and neurologic outcome. < 1500 birth weight do not involve the risk of developing both CP and epilepsy within the first year of life.

Discussion Our study pointed out that birth weight is a predictive factor for neurologic outcome. We have observed that all ELBW and VLBW developed either CP or motor delay, therefore <1,5 kg birth weight neonates are not at risk of both epilepsy and CP at one year of age (Figure 2): in these subjects, typically preterm infants, the post-asphyxial injury involves essentially the white matter but also basal ganglia and thalamus[1], therefore it is possible to detect a neuromotor involvement compatible with a postural motor syndrome (such as cerebral palsy) but not compatible with an epileptic syndrome where the damage involves essentially the cerebral cortex[13]. Figure 2 birth weight and neurologic outcome. < 1500 birth weight do not involve the risk of developing both CP and epilepsy within the first year of life. An epileptic onset within the first 6 months of age predicts a worse paretic and epileptic outcome (Figure 3): the patients were essentially term infants; neuroimaging revealed cortex injuries in most of them; at one year cut off they showed a highly severe epilepsy and CP clinical picture characterized by highly severe CP. Figure 3 Epilepsy onset within the first 6 months of life is an adverse prognosis factor for the neurologic outcome in patients with both epilepsy and CP within the first year of life.

An epileptic onset within the first 6 months of age predicts a worse paretic and epileptic outcome (Figure 3): the patients were essentially term infants; neuroimaging revealed cortex injuries in most of them; at one year cut off they showed a highly severe epilepsy and CP clinical picture characterized by highly severe CP. Figure 3 Epilepsy onset within the first 6 months of life is an adverse prognosis factor for the neurologic outcome in patients with both epilepsy and CP within the first year of life. Neonatal clinical pattern or early EEG, as predictive factors of outcome, have been cited in many studies[4,5,7], which showed the key role of a major involvement of the central nervous system as a decisive factor for an abnormal outcome. The results of our study confirm these evidences (Figure 4, 5), indeed the presence of neurological symptoms ascribable to Sarnat 2nd/3rd stadium of HIE[7], in particular neonatal seizures, and an early pathological neonatal EEG represent adverse prognostic factors in terms of CP severity: most of the subjects with highly severe CP, essentially term infants, have a typical neuropathological picture of very important lesions of cortex and/or basal ganglia, while most of the patients with moderately severe CP were preterm infants with bland phenomena of asphyxia at birth (with a neurological status ascribable to Sarnat 1st stadium of perinatal H.I.E.), white matter injury(sometimes also basal ganglia injury) and a not pathological early EEG.

f cortex and/or basal ganglia, while most of the patients with moderately severe CP were preterm infants with bland phenomena of asphyxia at birth (with a neurological status ascribable to Sarnat 1st stadium of perinatal H.I.E.), white matter injury(sometimes also basal ganglia injury) and a not pathological early EEG. Figure 4 neonatal clinical pattern and CP severity. a severe neurologic pattern at birth is an adverse prognosis factor in terms of CP severity. Figure 5 early EEG and CP severity. a pathologic early EEG is an adverse prognosis factor in terms of CP severity. Conclusion From a clinical point of view, it is of crucial importance to have some parameters which enable to discriminate patients at early risk of more severe sequelae from those at early risk of moderately severe outcome, in this way it is possible obtain a more manageable epilepsy and a CP susceptible of rehabilitation in order to improve the quality of life. This result could be achieved through the use of protocols for early diagnosis (e.g. including EEG and neurological examination follow up) or prevention (e.g. by the continuation or rational discontinuation of Anti-Epileptic Drugs therapy that was started at birth because of the presence of seizures).

quality of life. This result could be achieved through the use of protocols for early diagnosis (e.g. including EEG and neurological examination follow up) or prevention (e.g. by the continuation or rational discontinuation of Anti-Epileptic Drugs therapy that was started at birth because of the presence of seizures). Furthermore these results could be a starting point for a prospective study that includes a larger sample (considering the limited number of our patients) and a larger number of variables. Indeed our kind of approach, on the basis of more statistically significant data, could be for example very useful for the application of neuroprotection protocols in order to identify asphyxiated patients at risk of permanent encephalopathy[8] which could be prevented through selective cerebral hypothermia[3]. Competing interests The authors declare that they have no competing interests. Authors' contributions AA dealt with the recruitment of case history. FR, MS, FA conceived the study, took part in the study planning and carried out the statistical analysis. All authors drafted the manuscript. All authors read and approved the final manuscript. Supplementary Material Additional file 1 Patients with both symptomatic epilepsy and cerebral palsy at one year of age. Parameters at birth and at one year cut-off. Click here for file Acknowledgements Authors express special thanks to Chief Physician, Medical and Paramedical staff of Child Neuropsychiatry Division of our Department for their help and for logistical support during our research.

Background Very low birth weight infants (VLBWI), i.e. those weighing < 1500 grams, account for a very small percentage of all live births but make a disproportionately large contribution to neonatal mortality and morbidity [1]. In 2002, 53.9% of infants who died in the USA were VLBWI [1]. In Italy, the incidence of VLBWI is about one out of 100 live births (5000 VLBWI per year) [1,2]. Moreover, mortality rates in VLBWI are 100-fold higher than in other infants [1]. Management of VLBWI at the intensive care level is crucial for reducing mortality, morbidity and the risk of long-term complications [3,4]. In the last 20 years, the increased use of prenatal steroids and supplementary surfactant, and wider use of ventilation techniques, has provided major improvements in intensive care of VLBWI [5]. However, only a small number of studies involving large populations of VLBWI are available to date. More such studies may provide information on mortality and treatment approaches, and form the basis for more effective and less expensive therapeutic strategies [6].

ded major improvements in intensive care of VLBWI [5]. However, only a small number of studies involving large populations of VLBWI are available to date. More such studies may provide information on mortality and treatment approaches, and form the basis for more effective and less expensive therapeutic strategies [6]. VLBWI vary substantially in terms of mortality and response to therapy, according to birth weight (BW) and to gestational age (GA) [7]. Moreover, clinical outcomes may vary between different areas as a result of specific treatment strategies employed in individual intensive care units, including emergency transport [6]. Therefore, databases collecting information about VLBWI, classified on the basis of GA and BW, may be important repositories for clinical decision-making. The most notable example of such a database is that of the Vermont Oxford Network (VON) [8,9]. At its foundation in 1990, VON included 36 intensive care units. Currently, VON comprises more than 500 units worldwide, and records mortality, morbidity and treatments for about 40000 VLBWI per year. At the end of each year, the performance for each individual unit is compared with the overall VON results. In Italy, only five intensive care units were included in VON in 2004, and thus little information on VLBWI morbidity and treatments is available [1]. Therefore, therapeutic strategies based on VON data rely upon the experiences of different geographic areas [1]. Such choices may not be completely suitable for individual local units.

five intensive care units were included in VON in 2004, and thus little information on VLBWI morbidity and treatments is available [1]. Therefore, therapeutic strategies based on VON data rely upon the experiences of different geographic areas [1]. Such choices may not be completely suitable for individual local units. S. Chiara hospital is the only neonatal intensive care unit (NICU) in the Province of Trento (Italy). It serves a population of about 460000 people with about 5000 infants per year, of which one in 100 are VLBWI, admitting the totality of the inborn and outborn VLBWI of the province. Its provincial organization is based on the "in-uterus" transport to S. Chiara and emergency transport from referral delivery points for only few cases every year. S. Chiara is a 20-bed unit with eight beds dedicated to intensive care, each providing assisted ventilation and monitoring of vital parameters. NO treatment, radiography, echography and haemogas-analysis devices, and infusion pumps are also available. The working team includes 9 neonatologists and 25 nurses. Operative, surgical, orthopaedic and neuropsychiatric units are present in the paediatric department of S. Chiara. A specialised ophthalmologist is dedicated to diagnosis of retinopathy of premature (ROP). In the Trento area all the patients go directly home from S. Chiara NICU. There is no other centre where they can be transferred to. Even the follow up for the first 2 years of life is made exclusively by S. Chiara NICU of Trento. The S. Chiara neonatal intensive care unit provides emergency transport for the entire Trento area.

nto area all the patients go directly home from S. Chiara NICU. There is no other centre where they can be transferred to. Even the follow up for the first 2 years of life is made exclusively by S. Chiara NICU of Trento. The S. Chiara neonatal intensive care unit provides emergency transport for the entire Trento area. Rather than a comparison among NICUs, this study wants to be a comparison between two areas, the small Trento-area and the big VON-area. The aim of this retrospective analysis is to compare mortality, morbidity and treatment of VLBWI in the S. Chiara intensive care unit during 2000–2005 with those recorded in VON during 2004. In particular, this analysis focuses on infants weighing ≤ 1000 grams. Methods In the Trento area, only 50 VLBWI are recorded each year compared with about 40000 in VON. Therefore, consecutive VLBWI data reported in S. Chiara during a six-year period (2000–2005) were compared with a single year for VON (2004).

Rather than a comparison among NICUs, this study wants to be a comparison between two areas, the small Trento-area and the big VON-area. The aim of this retrospective analysis is to compare mortality, morbidity and treatment of VLBWI in the S. Chiara intensive care unit during 2000–2005 with those recorded in VON during 2004. In particular, this analysis focuses on infants weighing ≤ 1000 grams. Methods In the Trento area, only 50 VLBWI are recorded each year compared with about 40000 in VON. Therefore, consecutive VLBWI data reported in S. Chiara during a six-year period (2000–2005) were compared with a single year for VON (2004). Baseline data (inborn infants, use of prenatal steroids, multiple births, incidence of small for gestational age infants [SGA babies, defined for birth weight below the 10th percentile, in babies of the same gestational age [8]], caesarean sections, congenital anomalies, need for intubation in delivery room) were collected for the two populations. The incidence of respiratory distress syndrome (RDS), defined as radiologically confirmed clinical signs of respiratory distress [5], and of pneumothorax were evaluated, as well as the frequency of chronic lung disease (CLD), defined as the need for oxygen supplementation at 28 days and 36 weeks [1]. In terms of respiratory complications, the frequency of surfactant administration (Curosurf®; Chiesi Farmaceutici, Parma, Italy [200 mg/kg bolus]) was recorded. Surfactant was given as a prophylactic measure in the delivery room to all babies with GA < 29 weeks. In Trento area the policy about ELBWI (22–24 weeks of GA) is based on the active care with resuscitation since the 22nd week. In general the Trento policy for all the VLBWI is based on the early prophylactic administration of surfactant in Trento in delivery room or in the first hours of life; this step is followed by the clinical evaluation that permits to continue with a minimal handling or, if strictly necessary, with high invasivity (catheters, antibiotics, ventilations, etc...). Furthermore, the need for nasal continuous positive airway pressure (NCPAP) and conventional ventilation (Newport and Cub Bear ventilators; Burke & Burke) was observed. Other complications recorded included: patent ductus arteriosus (PDA), as determined by echocardiography (treatment with indomethacin [Liometacen®, Chiesi Farmaceutici, 0.20 mg/kg/day i.v.

nuous positive airway pressure (NCPAP) and conventional ventilation (Newport and Cub Bear ventilators; Burke & Burke) was observed. Other complications recorded included: patent ductus arteriosus (PDA), as determined by echocardiography (treatment with indomethacin [Liometacen®, Chiesi Farmaceutici, 0.20 mg/kg/day i.v. for three days, administered over 20 minutes] was also recorded); necrotising enterocolitis (NEC), defined as gastrointestinal signs and symptoms associated with specific radiological and operative evidence [5]; total, grade III and IV (Papile's classification) intraventricular haemorhage (IVH), diagnosed with ultrasonography [5]; and cystic periventricular leukomalacia (PVL), defined on ultrasound as multiple small cysts located in the external angles of the lateral ventricles, fronto-parietal or occipital peri-ventricular white matter [5]. ROP was scored according to international classification [5]. ROP screening frequency and results, and the need for surgical intervention for this condition were also evaluated. Overall mortality was calculated, as well as the total number of discharged infants and those discharged on human milk. The incidence of each parameter was calculated in both the overall population and sub-groups defined by BW (501–750 grams, 751–1000 grams, 1001–1250 grams, 1251–1500 grams). Incidences were compared using odds ratios (OR) and the respective 95% confidence intervals (95% CI). The differences between the two populations were calculated with the Mantel-Haenzel estimate (MH).

the overall population and sub-groups defined by BW (501–750 grams, 751–1000 grams, 1001–1250 grams, 1251–1500 grams). Incidences were compared using odds ratios (OR) and the respective 95% confidence intervals (95% CI). The differences between the two populations were calculated with the Mantel-Haenzel estimate (MH). Results A total of 250 VLBWI in the Trento area and 38895 in VON were evaluated. Baseline data for the two populations, and BW-defined sub-groups, are summarised in Table 1 (Additional file 1). The mean birth weight in each BW category is similar in the two populations. VON in 2004 did not report the mean gestational age. Table 2 (Additional file 2) lists the prenatal data for the two populations. The frequency of inborn infants was significantly higher in Trento than in VON: 91% vs 84% (MH 8.56; p-value 0.003). In addition, some treatments, such as the administration of prenatal steroids (82% vs 74%; MH 7.47 and p-value 0.006) and caesarean section were significantly more frequent in the Trento area than in VON. The frequencies of congenital anomalies, multiple births were similar between Trento and VON. SGA infants were less frequent in Trento.

eatments, such as the administration of prenatal steroids (82% vs 74%; MH 7.47 and p-value 0.006) and caesarean section were significantly more frequent in the Trento area than in VON. The frequencies of congenital anomalies, multiple births were similar between Trento and VON. SGA infants were less frequent in Trento. Table 3 (Additional file 3) lists the incidence of respiratory complications and related treatments in the two populations. In Trento the overall need for intubation was significantly less frequent in the sub-group with BW 1251–1500 grams, but significantly more VLBWI with BW ≤ 1000 grams were given surfactant prophylaxis compared with VON. Compared with VON, significantly fewer VLBWI in every Trento weight group developed RDS, and therefore needed specific treatment (MH 18.55; p-value 0.00001), with the use of conventional ventilation and nasal CPAP (never before mechanical ventilation and never with high flow nasal cannula) significantly less frequent in Trento. Only in the sub-group with BW 501–750 grams the frequency was similar in the two populations. Greater use of surfactant prophylaxis and lower incidence of RDS in Trento compared with VON were particularly evident in the 501–1000 grams group. The same trend was found for CLD at 36 weeks: 5% vs 36% in VON (MH 102.5; p-value 0.00000). In Trento postnatal steroids for CLD are not used. The overall frequency of pneumothorax was similar in the two populations.

wer incidence of RDS in Trento compared with VON were particularly evident in the 501–1000 grams group. The same trend was found for CLD at 36 weeks: 5% vs 36% in VON (MH 102.5; p-value 0.00000). In Trento postnatal steroids for CLD are not used. The overall frequency of pneumothorax was similar in the two populations. Table 4 (Additional file 4) summarises the incidence of other complications and of indomethacin administration. In Trento, PDA (14% vs 37%; MH 54.51; p-value 0.00000) and subsequent use of indomethacin (12% vs 34%; MH 55.65; p-value 0.00000) were significantly less frequent than in VON. This difference was greatest in the two lowest BW groups, as was also the case with respiratory complications. In Trento, NEC (2% vs 6%; MH 8.57; p-value 0.003) was significantly less frequent than in VON. Moreover, even if all the IVHs were significantly less frequent in Trento than in VON, the analysis of IVH-grade did not show any difference. There were no significant differences in the incidence of PVL. Table 5 (Additional file 5) lists the frequencies of other parameters analysed, including mortality. Screening for ROP was significantly more common in Trento than in VON (83% vs 67%; MH 29.5; p-value 0.000001). The overall incidence of ROP was significantly lower in the S. Chiara intensive care unit, in all the BW sub-groups. No significant difference was evident in the incidence of severe grade of ROP.

ng mortality. Screening for ROP was significantly more common in Trento than in VON (83% vs 67%; MH 29.5; p-value 0.000001). The overall incidence of ROP was significantly lower in the S. Chiara intensive care unit, in all the BW sub-groups. No significant difference was evident in the incidence of severe grade of ROP. Overall mortality was comparable in the two populations (10% and 14% in Trento and VON, respectively). Furthermore, in Trento, significantly more infants were discharged on human milk than in VON, in both the overall population (MH 139.7; p-value 0.00000) and in BW sub-groups [Figure 1]. Figure 1 Percentage of very low birth weight infants discharged on human milk in Trento area (2000–2005) and VON (2004). Discussion This analysis reports the complications and treatments in the S. Chiara hospital (Trento, Italy) neonatal intensive care unit during 2000–2005. As the only intensive care centre in Trento (an area comprising 6200 km2), and serving the entire population (500000 inhabitants, with 5000 newborns per year), S. Chiara data might be an important source of epidemiological information. Data recorded in S. Chiara were compared with the entire VON database (S. Chiara is part of this network), therefore comparing a small area (2000–2005) with a global group, represented by VON (2004). In this way, it is possible to critically evaluate therapeutic strategies used in S. Chiara, in a global management of a perinatal area.

S. Chiara were compared with the entire VON database (S. Chiara is part of this network), therefore comparing a small area (2000–2005) with a global group, represented by VON (2004). In this way, it is possible to critically evaluate therapeutic strategies used in S. Chiara, in a global management of a perinatal area. The two populations had similar rates of congenital anomalies, SGA infants, and multiple births; some intervention strategies were significantly more common in Trento than in VON. For instance, caesarean section, which is a common choice in Italy, especially in recent years [10]. In Trento, the use of prenatal steroids and the use of supplementary surfactant prophylaxis is significantly more common than in VON. The surfactant strategy is based on a multicentre retrospective European study that suggested that prophylactic surfactant administered within 15 minutes of delivery is associated with a significantly greater reduction in RDS than rescue therapy (administration after 15 minutes of life) [11]. Moreover, a head-to-head comparison of surfactant prophylaxis versus rescue therapy confirmed the same finding, suggesting that prophylaxis is associated with a reduction in mortality [12].

ry is associated with a significantly greater reduction in RDS than rescue therapy (administration after 15 minutes of life) [11]. Moreover, a head-to-head comparison of surfactant prophylaxis versus rescue therapy confirmed the same finding, suggesting that prophylaxis is associated with a reduction in mortality [12]. The use of prenatal steroids with the greater use of surfactant prophylaxis may be associated with the lower incidence of RDS reported in Trento. Effective management of this condition could reduce the risk of CLD, as suggested by the present analysis and by another study [13]. Surfactant prophylaxis, administered within the first few minutes of birth in the delivery room, may also result in fewer infants requiring assisted ventilation, thus reducing invasive procedures without worsening clinical outcomes [11]. The reduction in frequency of respiratory complications, as CLD, in Trento compared with VON is particularly evident in the two lowest BW groups (501–750 grams and 751–1000 grams), which are characterised by a particularly high risk. In these groups, surfactant prophylaxis was more common, further suggesting that this treatment may be associated with important improvements in clinical outcomes and better management of neonatal respiration, in terms of peak inspiration volume, intermittent positive pressure ventilation and fraction of inhaled oxygen. These results are consistent with the multicentre study by Bevilacqua et al, in which surfactant prophylaxis was associated with a lower incidence of RDS compared with rescue therapy [11]. It is noteworthy that VLBWI receiving surfactant prophylaxis had a lower BW than those treated with rescue therapy [11].

ed oxygen. These results are consistent with the multicentre study by Bevilacqua et al, in which surfactant prophylaxis was associated with a lower incidence of RDS compared with rescue therapy [11]. It is noteworthy that VLBWI receiving surfactant prophylaxis had a lower BW than those treated with rescue therapy [11]. In Trento, the overall rates of PDA, NEC and IVH were lower than in VON, occasionally reaching statistical significance. As observed for respiratory complications, the difference was greatest in the lowest BW groups. Overall, these data are comparable with those reported in the Bevilacqua study [11]. It may be that RDS is less common in Trento area because of several factors: more prenatal steroids; surfactant in delivery room; low amount of infused liquids, thanks to the fact that in Trento the enteral feeding with bank human milk and mother milk is preferred. This management, mainly in the first week of life, gives as a consequence high weight loss (about 20%), less PDA, less RDS and less CLD, but also less indomethacin treatment, less catheters and less antibiotics (with lower risk of complications). In Trento it is usually used only one dose of surfactant (in delivery room or in NICU) with extubation as soon as possible, also in delivery room. It is preferred NCPAP instead of mechanical ventilation when the baby permits it.

methacin treatment, less catheters and less antibiotics (with lower risk of complications). In Trento it is usually used only one dose of surfactant (in delivery room or in NICU) with extubation as soon as possible, also in delivery room. It is preferred NCPAP instead of mechanical ventilation when the baby permits it. Even the lower risk of NEC in Trento compared with VON may be associated with specific therapeutic strategies of the S. Chiara intensive care unit, such as the very early administration of human milk (from the second hour from birth, when possible). In fact, the percentage of discharged infants receiving human milk was significantly higher in Trento than in VON (83% vs. 44%, respectively). Since 1993, the S. Chiara unit has employed the early exclusive enteral feeding (EEEF) protocol that is widely used in Scandinavian countries [14]. This protocol, which is targeted to specific VLBWI conditions, is based on the exclusive administration of human milk, either from the mother or from a donor, to VLBWI weighing 750–1250 grams and with GA > 26 weeks. The EEEF protocol may be suitable for VLBWI undergoing CPAP, but cannot be suggested for those treated with mechanical ventilation or presenting with asphyxia, metabolic acidosis, hypotension, sepsis or persistent hypoglycaemia. During 2000–2005 in S. Chiara, the EEEF protocol was initiated in 51.4% of VLBWI weighing 750–1250 grams and with a GA > 26 weeks. Among these infants, only one out of five (10.3% of the overall population) required further nutritional support, while the majority (41.0% of the total) did not.

persistent hypoglycaemia. During 2000–2005 in S. Chiara, the EEEF protocol was initiated in 51.4% of VLBWI weighing 750–1250 grams and with a GA > 26 weeks. Among these infants, only one out of five (10.3% of the overall population) required further nutritional support, while the majority (41.0% of the total) did not. Several studies have confirmed the importance of VLBWI feeding with human milk. The high content of oligosaccharides in human milk may improve the development of immune system and prevent onset of NEC [15]. The administration of human milk to VLBWI is recommended by American Academy of Pediatrics and Canadian Pediatrics Society because of its excellent energetic properties and for its important effects on neural, cognitive and psychological development [16,17]. In particular, feeding with human milk is of great importance in VLBWI weighing less than 1000 grams, because it is associated with significant improvements in survival and clinical outcomes [18]. In Trento, significantly more VLBWI in both the overall population (MH 139.7; p-value 0.00000) and in BW sub-groups were discharged on human milk, compared with VON. Screening for ROP was about 2.5 times more common in Trento than in VON. This strategy allows more rapid diagnosis of the potential presence of ROP. The frequency of this condition (overall and severe grades) was lower in Trento than in VON. An early diagnosis of ROP is of particular importance to prevent the risk of short- and long-term damage to vision [19].

common in Trento than in VON. This strategy allows more rapid diagnosis of the potential presence of ROP. The frequency of this condition (overall and severe grades) was lower in Trento than in VON. An early diagnosis of ROP is of particular importance to prevent the risk of short- and long-term damage to vision [19]. Mortality was comparable in the two populations, although a trend towards lower mortality in Trento was evident, especially in infants with BW ≤ 750 grams. It is possible that the therapeutic strategies adopted in Trento, such as surfactant prophylaxis and widespread use of human milk, may be associated with a reduction in complications and, as a consequence, with a lower mortality in this high-risk class of VLBWI.

o was evident, especially in infants with BW ≤ 750 grams. It is possible that the therapeutic strategies adopted in Trento, such as surfactant prophylaxis and widespread use of human milk, may be associated with a reduction in complications and, as a consequence, with a lower mortality in this high-risk class of VLBWI. Conclusion The comparison between VLBWI data collected during 2000–2005 in the S. Chiara intensive care unit of Trento and those derived from the VON database shows that the frequency of the most important complications associated with intensive treatment was, in most cases, lower in the Trento area. This difference may be due, at least in part, to a greater use of prenatal steroids, to a less aggressive surfactant approach, based on prophylactic treatment instead of rescue therapy, and greater feeding with human milk using the EEEF protocol, which may be associated with better development and lower incidence of NEC and other complications. It is noteworthy that surfactant prophylaxis is associated with reduced need for ventilation assistance. Other factors, such as more extensive screening for ROP, might contribute to decrease time and cost associated with intensive care and to limit short- and long-term consequences. These differences are particularly evident in lower BW infants, which present a higher risk. In conclusion, this study suggests that a less aggressive therapeutic strategy, mostly based on prevention and on global management, may be associated with an improvement in clinical outcomes in preterm infants.

Conclusion The comparison between VLBWI data collected during 2000–2005 in the S. Chiara intensive care unit of Trento and those derived from the VON database shows that the frequency of the most important complications associated with intensive treatment was, in most cases, lower in the Trento area. This difference may be due, at least in part, to a greater use of prenatal steroids, to a less aggressive surfactant approach, based on prophylactic treatment instead of rescue therapy, and greater feeding with human milk using the EEEF protocol, which may be associated with better development and lower incidence of NEC and other complications. It is noteworthy that surfactant prophylaxis is associated with reduced need for ventilation assistance. Other factors, such as more extensive screening for ROP, might contribute to decrease time and cost associated with intensive care and to limit short- and long-term consequences. These differences are particularly evident in lower BW infants, which present a higher risk. In conclusion, this study suggests that a less aggressive therapeutic strategy, mostly based on prevention and on global management, may be associated with an improvement in clinical outcomes in preterm infants. Competing interests The authors declare that they have no competing interests.

Conclusion The comparison between VLBWI data collected during 2000–2005 in the S. Chiara intensive care unit of Trento and those derived from the VON database shows that the frequency of the most important complications associated with intensive treatment was, in most cases, lower in the Trento area. This difference may be due, at least in part, to a greater use of prenatal steroids, to a less aggressive surfactant approach, based on prophylactic treatment instead of rescue therapy, and greater feeding with human milk using the EEEF protocol, which may be associated with better development and lower incidence of NEC and other complications. It is noteworthy that surfactant prophylaxis is associated with reduced need for ventilation assistance. Other factors, such as more extensive screening for ROP, might contribute to decrease time and cost associated with intensive care and to limit short- and long-term consequences. These differences are particularly evident in lower BW infants, which present a higher risk. In conclusion, this study suggests that a less aggressive therapeutic strategy, mostly based on prevention and on global management, may be associated with an improvement in clinical outcomes in preterm infants. Competing interests The authors declare that they have no competing interests. Authors' contributions GDN had primary responsibility for protocol development, patients enrollment, data analysis and writing the text. MB and RM were responsible for patients evaluation schedule and Oxford Data Base maintenance. FP, AP, and AV contributed to the protocol development and execution of the study.

Competing interests The authors declare that they have no competing interests. Authors' contributions GDN had primary responsibility for protocol development, patients enrollment, data analysis and writing the text. MB and RM were responsible for patients evaluation schedule and Oxford Data Base maintenance. FP, AP, and AV contributed to the protocol development and execution of the study. Supplementary Material Additional file 1 Table 1. Baseline data for the Trento and VON populations. Click here for file Additional file 2 Table 2. Prenatal data for the Trento and VON populations. Click here for file Additional file 3 Table 3. Incidence of respiratory complications and related treatments in Trento and VON. Click here for file Additional file 4 Table 4. Incidence of non-respiratory complications and indomethacin administration in Trento and VON. Click here for file Additional file 5 Table 5. Incidence of ROP and mortality in Trento and VON. Click here for file Acknowledgements The authors thank the neonatologist Silvia Graziani, Elena Franco and Elisabetta Chiodin for their continuing support and Ms Liliana Iseppi for administrative assistance.

Introduction Feeding intolerance is a recurrent problem in the clinical care of preterm infants and occur mainly in the first week of life, suggesting the presence of a maturation pattern of gastrointestinal tract [1]. It is known that functional maturation of the gastrointestinal tract is quite different over time with respect to its anatomical development [2-4]. Adequate levels of some digestive enzymes are reached only at the end of gestation and lactase activity at 34 weeks gestation is only 30% of the level of full-term newborns [3]. To date there is little data available about the development of the motility function and of the mucosal barrier in newborns during early days of life. Gastrointestinal motility can be recorded as a measure of gastric electrical activity, of the wall movements, and of gastric emptying time. A reliable method for recording gastric motility is cutaneous electrogastrography (EGG) [5-7]; electrogastrographic studies in newborns have demonstrated the absence of normal slow waves at birth and a maturation process modulated by enteral feedings [8-11]. Gastric emptying (GE) can be assessed by ultrasonography which is considered a non-invasive technique particularly suitable for young patients [12].

[5-7]; electrogastrographic studies in newborns have demonstrated the absence of normal slow waves at birth and a maturation process modulated by enteral feedings [8-11]. Gastric emptying (GE) can be assessed by ultrasonography which is considered a non-invasive technique particularly suitable for young patients [12]. The functional integrity of the mucosal barrier of the intestine partly depends on the close interaction of adjacent mucosal cells. The most reliable in vivo method to study this functional integrity is the sugar absorption test (SAT), which has been performed on adults [13] and newborns, both preterm [14] and term ones [15]. Some of the key events involving permeability actually take place in the neonatal period, when the barrier is leakier. Coordinated motor function in the gastrointestinal tract plays a crucial role in the intestinal transportation, absorption and maintenance of the enteric bacterial ecology [16]. In particular, delayed intestinal transit time may contribute to increased mucosal permeability, and even to facilitated bacterial translocation [17]. The aim of the study was to investigate gastric motility and intestinal permeability to verify if a maturation pattern exists in preterm newborns during the first month of life.

The functional integrity of the mucosal barrier of the intestine partly depends on the close interaction of adjacent mucosal cells. The most reliable in vivo method to study this functional integrity is the sugar absorption test (SAT), which has been performed on adults [13] and newborns, both preterm [14] and term ones [15]. Some of the key events involving permeability actually take place in the neonatal period, when the barrier is leakier. Coordinated motor function in the gastrointestinal tract plays a crucial role in the intestinal transportation, absorption and maintenance of the enteric bacterial ecology [16]. In particular, delayed intestinal transit time may contribute to increased mucosal permeability, and even to facilitated bacterial translocation [17]. The aim of the study was to investigate gastric motility and intestinal permeability to verify if a maturation pattern exists in preterm newborns during the first month of life. Methods Infants and protocol The study was performed at the Neonatology Section of the Department of Pediatrics at the University of Bari. Healthy preterm newborns, born at a gestational age of 28–36 weeks, a birth weight > 1800 g, normal Apgar score, and a post natal age < = 24 h, were eligible to participate in the study. Newborns with: a) respiratory distress, b) congenital malformation, c) inborn errors of metabolism, or d) proven sepsis or infection, were not included. From an initial group of 38 preterm newborns, 18 entirely bottle-fed infants completed the study. The others were excluded for various reasons: a change in milk formula (4 newborns); an infectious disease (1 newborn); withdrawal from the study (7 newborns); inability to perform the scheduled SAT due to early transfer to another hospital (1 newborn) and/or failure to collect urine within the scheduled collection day (7 newborns). All the newborns enrolled reached the total amount of enteral feeding within the first week of life. All the preterm newborns were exclusively bottle-fed with the same preterm standard formula throughout the intervention period. The daily formula intake was approximately 30 ml/kg/day at baseline and 180 ml/kg/day at the end on the study.

rolled reached the total amount of enteral feeding within the first week of life. All the preterm newborns were exclusively bottle-fed with the same preterm standard formula throughout the intervention period. The daily formula intake was approximately 30 ml/kg/day at baseline and 180 ml/kg/day at the end on the study. Gastric electrical activity, gastric emptying time and intestinal permeability were recorded on days 3, 7, 15, and 30 after birth in order to evaluate the time changes in motility and permeability. The range of the data collection period was rigorously narrow (± 1 day). From birth until the end of the study, episodes of regurgitation, vomiting, number of evacuations, the time of complete emission of meconium, and the daily amount of feedings, were recorded. Written informed consent was obtained from the parents, and the study was approved by our local institutional ethics committee.

. From birth until the end of the study, episodes of regurgitation, vomiting, number of evacuations, the time of complete emission of meconium, and the daily amount of feedings, were recorded. Written informed consent was obtained from the parents, and the study was approved by our local institutional ethics committee. Assessment of gastric electrical activity The EGG recordings were performed using portable equipment before and 120 min after meal, following a fasting period of 4 hours. Two silver-silver chloride bipolar electrodes (Clear Trace, ConMed, Utica, NY USA) were placed on the cleaned abdominal surface overlying the antro-pyloric axis to obtain the best signal-noise ratio. The reference electrode was placed to form an equilateral triangle [18]. Electrogastrography was performed using a portable EGG recorder (UPS 2020, Medical Management Systems, MMS, The Netherlands). The recordings and analysis of the EGG parameters (dominant frequency and normal slow wave percentage, power ratio) were previously described in different papers [11,19]

uilateral triangle [18]. Electrogastrography was performed using a portable EGG recorder (UPS 2020, Medical Management Systems, MMS, The Netherlands). The recordings and analysis of the EGG parameters (dominant frequency and normal slow wave percentage, power ratio) were previously described in different papers [11,19] Assessment of gastric emptying The ultrasound gastric emptying examinations were always performed by the same investigator using a real-time apparatus (Image Point HX, Hewlett Packard Company, Palo Alto, CA, USA) equipped with a 3.5 MHz linear probe. The ultrasound examination and the measure of the antral area were performed according to the procedure reported in a previous work [11]. The EGG and GE were simultaneously recorded to avoid differences due to the rapid changes in physiological parameters. During the same EGG recording session, antral measurements were made before the test meal, and at regular 30-min intervals up to 180 min after the meal. In each patient, the half emptying time (T1/2) was calculated [11,12,20].

taneously recorded to avoid differences due to the rapid changes in physiological parameters. During the same EGG recording session, antral measurements were made before the test meal, and at regular 30-min intervals up to 180 min after the meal. In each patient, the half emptying time (T1/2) was calculated [11,12,20]. Assessment of intestinal permeability The SAT was performed after oral ingestion, by suckling, of a solution containing 5.0 g of lactulose and 2.0 g of mannitol (Sigma Aldrich s.r.l., Milano, Italy) per 100 ml water (375 mosml/l) at a dose of 2 ml/kg of body weight. The newborns fasted two hours before and after the oral administration of the solution. All the urine passed in the subsequent five hours was collected in an adhesive urine bag (Sincrolag s.r.l. Italy). The complete urine volume was measured and stored at -80°C until analysis. Urinary concentration of lactulose and mannitol were determined by ion exchange chromatography with pulse amperometric detection [21]. Lactulose is a disaccharide that crosses the intestinal epithelium by passive diffusion through the paracellular tight junctions. Mannitol is a monosaccharide that crosses the intestinal epithelium mainly by transcellular passive diffusion through aqueous pores [22]. The evidence of an exclusively transcellular permeation of monosaccharides is still controversial. Mannitol is used for osmotic shrinkage of membrane vesicles, which would not be possible if permeation across cell membranes were unrestricted, and many experimental physiologists use it as an extracellular fluid volume marker suggesting a paracellular route of permeation for this probe [23].

es is still controversial. Mannitol is used for osmotic shrinkage of membrane vesicles, which would not be possible if permeation across cell membranes were unrestricted, and many experimental physiologists use it as an extracellular fluid volume marker suggesting a paracellular route of permeation for this probe [23]. The urinary excretion percentage of lactulose and mannitol are markers for paracellular and transcellular diffusion respectively. To correct for non-mucosal factors that may affect the intestinal uptake of these saccharides, including rate of gastric emptying, intestinal transit time and renal clearance, the urinary percentage of lactulose and mannitol were expressed as the L/M ratio. Data analysis The data were first analyzed using simple descriptive statistics of centrality and dispersion. Clinical parameters are expressed as median and range and physiological data are expressed as mean ± SEM. However, because of the sample size and absence of a normal distribution of the data, only non-parametric statistical analysis tests were performed. The overall effect over time of EGG, GE and SAT parameters was determined by a repeated measures analysis (Friedman Friedman Repeated Measures Anova). Because of missing data at some points, multiple comparisons were non available and differences among the recording points of EGG, GE and SAT parameters were made using the Wilcoxon signed rank test. All the differences were considered significant at a 5% level. The software package used for the statistical analysis was STATA (STATA ver 4.0 Statistical Software, Stata Corporation).

e non available and differences among the recording points of EGG, GE and SAT parameters were made using the Wilcoxon signed rank test. All the differences were considered significant at a 5% level. The software package used for the statistical analysis was STATA (STATA ver 4.0 Statistical Software, Stata Corporation). Results Anthropometric and clinical parameters These parameters are reported in Table 1. It clearly shows that a homogeneous group was collected. None of the newborns presented significant regurgitation and/or vomiting from birth until the last day of examination and all passed the meconium within the second day of life. All newborns reached the total amount of enteral feeding (140 ml/kg/day) within the seven days. Table 1 Baseline anthropometric and clinical data of the newborns which completed the study (n = 18) Gestational age 34 [2] Birth weight (g) 2140 [305] Apgar score 8 [1] Male/Female 8/10 Vaginal/caesarian delivery 7/11 Data are expressed as Medians and range or numbers Electrogastrographic and Gastric emptying data Figure 1(a, b, c) shows the pattern over time of the percentage of normal slow waves recorded before and after meal, and the pattern of power ratio (Power ratio: Friedman Repeated Measures p = 0.18; Wilcoxon signed rank test, day 3 vs day 7 p = 0.02). Figure 2 plots the ultrasound T1/2 over time (Friedman Repeated Measures Analysis p = 0.69; Wilcoxon signed rank test day 3 vs day 7 p = 0.08). Both power ratio and gastric emptying time did show a slightly difference comparing day 3 and day 7, without a significant improvement over time.

est, day 3 vs day 7 p = 0.02). Figure 2 plots the ultrasound T1/2 over time (Friedman Repeated Measures Analysis p = 0.69; Wilcoxon signed rank test day 3 vs day 7 p = 0.08). Both power ratio and gastric emptying time did show a slightly difference comparing day 3 and day 7, without a significant improvement over time. Figure 1 Gastric electrical activity are reported as percentage of gastric slow waves (SW) at baseline (a), after meal (b), and power ratio (PR) (c). Repeated measurements analysis did not demonstrate any improvement in power ratio over time. Only a difference at day 7 respect to day 3 is evident. Data are means ± SEM. Figure 2 Gastric emptying time is reported as the half emptying time (T1/2). Repeated measurements analysis did not show any improvement in T1/2 over time. A slightly difference at day 7 respect to day 3 is only evident. Data are means ± SEM. Intestinal permeability data Measurement of lactulose excretion demonstrated an evident reduction at day 7 and the subsequent recording days (Figure 3a). On the other hand, measurement of mannitol excretion demonstrated a fluctuation over time and an increase on day 7 without reaching a significant difference (Figure 3b). The L/M ratio showed a deep decline between day 3 and day 7, then the ratio became constantly low (Friedman Repeated Measures Analysis p = 0.004; Wilcoxon signed rank test: day 3 vs day 7 p < 0.05, day 3 vs day 15 p < 0.05, day 3 vs day 30 p < 0.05 (Figure 3c).

y 7 without reaching a significant difference (Figure 3b). The L/M ratio showed a deep decline between day 3 and day 7, then the ratio became constantly low (Friedman Repeated Measures Analysis p = 0.004; Wilcoxon signed rank test: day 3 vs day 7 p < 0.05, day 3 vs day 15 p < 0.05, day 3 vs day 30 p < 0.05 (Figure 3c). Figure 3 Intestinal permeability pattern as determined by urinary excretion of orally administered lactulose (a), mannitol (b), and L/M ratio (c) respectively. L/M ratio persistently and significantly reduces after day 3 (see text). Data are means ± SEM. Discussion In preterm newborns the gastric electrical activity is quite stable with slight differences in power ratio and emptying at given recording days. On the contrary, intestinal permeability showed a persistent improvement over the first week of postnatal life. A few studies have investigated the gastric motility and intestinal permeability in preterm newborns. We studied the gastric electrical activity, gastric emptying and intestinal permeability in a time series in order to account for the effect of the different physiological variables over time. The pattern of slow wave percentage in the normal neonates showed a stable 3 cpm activity over time. During the first month of life the slow wave percentage was usually reported to be about 38% [24] from birth to 4 weeks, whilst according to others the slow wave percentage was about 50% [25]. Our data from premature newborns showed a higher percentage of normal slow wave, probably as a result of our broad interval in the normal EGG frequency ranges.

he slow wave percentage was usually reported to be about 38% [24] from birth to 4 weeks, whilst according to others the slow wave percentage was about 50% [25]. Our data from premature newborns showed a higher percentage of normal slow wave, probably as a result of our broad interval in the normal EGG frequency ranges. Intestinal immaturity is limited largely to infants of less than 34 weeks gestation but may extend to older gestational ages. Intestinal immaturity could explain poor gastroduodenal coordination and excessive quiescence in motor activity reported in very immature infants as poor gastric emptying, duodenogastric reflux and gastroduodenal hypomotility [26,27]. Our group of healthy newborns were of about 34 weeks gestation and showed a normal EGG parameters and gastric emptying time, even if subtle differences between the recording days were found. These findings confirm that gastric development is complete in late preterm infants [28-30].

gastroduodenal hypomotility [26,27]. Our group of healthy newborns were of about 34 weeks gestation and showed a normal EGG parameters and gastric emptying time, even if subtle differences between the recording days were found. These findings confirm that gastric development is complete in late preterm infants [28-30]. Different sugar-absorption tests for measuring intestinal permeability for sugars have been studied in a variety of gastrointestinal diseases. In vivo mannitol is absorbed via the transcellular pathway and serves as a marker of transcellular uptake [22,23] while lactulose is only slightly absorbed, but exclusively across the intestinal membrane through the intercellular junctions, and serves as a marker for mucosal integrity [31]. In our study L/M ratio was sharply reduced at day 7, then it remained stable. The clinical significance of an increased intestinal permeability is still under investigation. Although alterations in intestinal permeability could cause bacterial translocation and septic complications, no evidence is reported in humans to support this assumption [32,33]. A close relationship between luminal factors and permeability was demonstrated only for IgA, ovoalbumin, and bacterial peptides [34-36]. Overall, the human neonate shows a developmental pattern of sugar intestinal permeability that resembles gut closure observed in other mammals; intestinal permeability decreases faster in breast-fed newborns than in those fed with adapted or hydrolysed formula [37,38]. However, both decreased and increased permeability during the first months of life have been reported [14,39,40]. The reasons for such discrepancies lie in the differences in study design such as gestational age, clinical condition, feeding regiments and postnatal age at the time of the studies. Our data are similar to that of Van Elburg, actually preterm newborns permeability is higher during the first 2 days of life than up to 6 days later, independently of birth weight and gestational age [41]. Our data showed a slight increase in mannitol permeability in day 7 and a dramatic reduction of L/M ratio between day 3 and day 7 related to reduced lactulose permeability. Even if the relationship between feeding and intestinal maturation was not studied in our paper, some authors have demonstrated that the starting of enteral feeding induces an increase in intestinal barrier function [42].

dramatic reduction of L/M ratio between day 3 and day 7 related to reduced lactulose permeability. Even if the relationship between feeding and intestinal maturation was not studied in our paper, some authors have demonstrated that the starting of enteral feeding induces an increase in intestinal barrier function [42]. The fact that adult patients fed with total parenteral nutrition showed an impaired intestinal permeability confirms the link between enteral nutrition and permeability [43]. In conclusion, healthy late preterm newborns showed mature EGG and gastric emptying and a rapid improvement in intestinal permeability. The role of enteral nutrients is not merely linked to nourishing the developing intestine of the premature infants but may represent a kick off point. Optimization of nutrition in preterm infants could have major implications for health and outcome. Abbreviations EGG: Cutaneous electrogastrography; GE: Gastric emptying; SAT: Sugar absorption test; DF: Dominant frequency; SW: Slow waves; PR: Power ratio; FFT: Fast Fourier transform; T1/2: Half emptying time. Competing interests The authors declare that they have no competing interests.

In conclusion, healthy late preterm newborns showed mature EGG and gastric emptying and a rapid improvement in intestinal permeability. The role of enteral nutrients is not merely linked to nourishing the developing intestine of the premature infants but may represent a kick off point. Optimization of nutrition in preterm infants could have major implications for health and outcome. Abbreviations EGG: Cutaneous electrogastrography; GE: Gastric emptying; SAT: Sugar absorption test; DF: Dominant frequency; SW: Slow waves; PR: Power ratio; FFT: Fast Fourier transform; T1/2: Half emptying time. Competing interests The authors declare that they have no competing interests. Authors' contributions GR conceived of the study, carried out the recording of the gastric electrical activity and drafted the manuscript. FI conceived of the study, and carried out the permeability tests and polish the manuscript. RF participated in the design of the study. OM carried out the permeability tests and gastric emptying studies. GS participated in the design of the study. MB performed the statistical analysis. LC participated in the coordination of the study. RF participated in the coordination of the study and polish the manuscript.

Background During the last fifty years there has been a profound change in the approach to children who are hospitalized whether for short or long periods of time [1-9]. In fact, to humanize the management of children in hospitals has become a serious concern of civil society and one of the main goals of public and private health centers, health care providers and governments [10]. The concept of family-centered care has developed initially in the economically advantaged countries. It was the result of an increased social awareness, which focused particularly on the importance of meeting the psychosocial and developmental needs of children, with an emphasis on the role of families in promoting the health and well-being of their children [1-9]. Family-centered care in pediatrics (Appendix 1) is based on the understanding that the family is the child's primary source of strength and support and that the child's and family's perspectives and information are important in clinical decision making [11]. It is now a consolidated notion for family-centered trained professionals, that health care experiences can enhance parents' confidence in their roles, and eventually increase the competence of children and young adults to take responsibility for their own health care. Mostly in anticipation of the transition to adult service systems [12,13]. In particular, family-centered care has been promoted during the last fifteen years as the philosophies, principles and practices that put the family at the heart or center of services; the family has been identified as the driving force [14].

. Mostly in anticipation of the transition to adult service systems [12,13]. In particular, family-centered care has been promoted during the last fifteen years as the philosophies, principles and practices that put the family at the heart or center of services; the family has been identified as the driving force [14]. In parallel, the notion of family pediatrics (family-oriented care) developed among pediatricians as a concept not in contrast but in harmony although different from family-centered care. As recently emphasized by the report of the American Academy of Pediatrics (AAP) Task Force on the Family, family pediatrics aims at extending the responsibilities of the pediatrician to include screening, assessment, and referral of parents for physical, emotional, social problems or health risk behaviors that can adversely affect the health and emotional or social well-being of their child [11]. This paper, will debate the concepts of family-centered care and family pediatrics with the aim to emphasize the importance of the beneficial interfacing between the two conceptions. Furthermore, the unprecedented, rapid transformations of socioeconomic, demographic and ethnic characteristics of western societies and their cultural repositioning in the global context, has brought to public attention other important aspects and notions related to family-centered care and family pediatrics. Including cultural diversity and cultural competence, which will also be discussed in the paper.

ethnic characteristics of western societies and their cultural repositioning in the global context, has brought to public attention other important aspects and notions related to family-centered care and family pediatrics. Including cultural diversity and cultural competence, which will also be discussed in the paper. Public policies established by federal, state, and local governing and administrative bodies clearly have a great impact on the health, safety, and welfare of children and their families and on the ability of pediatricians to serve them. The organization of pediatric care show profound differences and divergent problems throughout the world, particularly between economically advantaged and challenged Counties. However, significant differences exist also among the economically developed Countries. For instance, in the US although the concepts of family-oriented and family-centered care emerged and developed to become a consolidated part of its health systems since over fifty years, the AAP observes that the US stands apart from most similarly developed countries in having no coherent set of public policies that would create a social context supportive of families, traditional or otherwise [11]. In Europe, a recent study of the Union of National European Paediatric Societies and Associations (UNEPSA) emphasized that the diversity of pediatric care in different countries in Europe is extreme and the differences are difficult to be analyzed and harmonized [15]. In a further study [16], UNEPSA also investigated the status of primary pediatric care in Europe. Data from the UNEPSA study were obtained form 34/48 European (EU and non-EU) countries, including Turkey, Switzerland, and Israel, which covered over 85% of the European population by serving approximately 158 million children who were younger than 15 years. Twelve of the 34 countries had a system for primary care for children, 6 had a general practitioner/family doctor system, and 16 had a combined system for the care of children who are younger 15. Three countries (Bulgaria, Croatia, and Czech Republic) were reported to have a public health reform in progress, including a shift from the pediatric system to the combined system. Three countries (8%) did not have any pediatricians working at the primary level of care. Fourteen countries (41%) reported having community pediatricians.

Bulgaria, Croatia, and Czech Republic) were reported to have a public health reform in progress, including a shift from the pediatric system to the combined system. Three countries (8%) did not have any pediatricians working at the primary level of care. Fourteen countries (41%) reported having community pediatricians. A most interesting case seems to be that regarding Italy, where family-pediatrics and family-pediatricians may be considered respectively equivalent to the Italian Public Health Institution "Pediatria di Famiglia" and to the professional figure of "Pediatra di Famiglia" also called "Pediatra di Libera Scelta", which in Italy is part of the public health system. Furthermore, most recently the Italian Ministry of Health, the major health care provider in that Country, made an effort to explore the possibility to establish the pediatric "Casa della Salute", which was designed to be an institution somehow comparable to the Medical Home typical of the Anglo-Saxon medical system. The paper aims at emphasizing the importance of positively interfacing family-centered care and family-oriented care concepts in pediatrics, also suggesting that most favorable environments to practice an effective family-centered care in pediatrics should be provided by health care institutions.

A most interesting case seems to be that regarding Italy, where family-pediatrics and family-pediatricians may be considered respectively equivalent to the Italian Public Health Institution "Pediatria di Famiglia" and to the professional figure of "Pediatra di Famiglia" also called "Pediatra di Libera Scelta", which in Italy is part of the public health system. Furthermore, most recently the Italian Ministry of Health, the major health care provider in that Country, made an effort to explore the possibility to establish the pediatric "Casa della Salute", which was designed to be an institution somehow comparable to the Medical Home typical of the Anglo-Saxon medical system. The paper aims at emphasizing the importance of positively interfacing family-centered care and family-oriented care concepts in pediatrics, also suggesting that most favorable environments to practice an effective family-centered care in pediatrics should be provided by health care institutions. Discussion From the concept of Medical Home to Family-centered care Historically, family-centered care has evolved from the concept of Medical Home, which is not a building, house, or hospital, but rather an approach to providing comprehensive primary care. A Medical home is defined as a primary care that is accessible, continuous, comprehensive, family centered, coordinated, compassionate, and culturally effective. In a medical home, a pediatric clinician works in partnership with the family/patient to assure that all of the medical and non-medical needs of the patient are met [13]. Through this partnership, the pediatric clinician can help the family/patient access and coordinate specialty care, educational services, out-of-home care, family support, and other public and private community services that are important to the overall health of the child/youth and family. As a matter of fact, for many years family-centered care had the characteristic of an effective medical home [12] and much of the early work focused on hospitals. For instance, as a multitude of studies progressively emphasized the impact of separating hospitalized children from their families, many institutions adopted policies that welcomed family members to be with their child around the clock and also encouraged their presence during medical procedures.

focused on hospitals. For instance, as a multitude of studies progressively emphasized the impact of separating hospitalized children from their families, many institutions adopted policies that welcomed family members to be with their child around the clock and also encouraged their presence during medical procedures. In Europe, such new concepts and policies gradually became accepted, although only few research studies and extensive reports, frequently of humanistic rather than scientific type, were produced on the topic. Conversely, in the Anglo-Saxon Countries family-center care has long been a matter of public attention and scientific investigation. In the New World, family-centered care was given further bust by consumer-led movements of the 1960s and 1970s and by professionals in education, health, and child development, which prompted US Federal legislation of the late 1980s and 1990s, much of it targeted at children with special needs, to provide additional validation of the importance of family-centered principles [13].

by consumer-led movements of the 1960s and 1970s and by professionals in education, health, and child development, which prompted US Federal legislation of the late 1980s and 1990s, much of it targeted at children with special needs, to provide additional validation of the importance of family-centered principles [13]. Family-centered care concepts merging in family pediatrics Nowadays, attention for family-centered care continues to grow in economically advantaged Countries and its momentum is further supported by a growing body of research and by prestigious organizations. For instance, the American Academy of Pediatrics (AAP) has incorporated some of the principles of family-centered care into its policy statements [14] and the US Institute of Medicine in its 2001 report Crossing the Quality Chasm: A New Health System for the 21st Century, emphasized the need to ensure the involvement of patients in their own health care decisions, to better inform patients of treatment options, and to improve patients' and families' access to information [17].

Institute of Medicine in its 2001 report Crossing the Quality Chasm: A New Health System for the 21st Century, emphasized the need to ensure the involvement of patients in their own health care decisions, to better inform patients of treatment options, and to improve patients' and families' access to information [17]. The embedding of family-centered care concepts in family-oriented care in pediatrics has produced a positive new approach to health care which shapes health care policies, programs, facility design, and day-to-day interactions among patients, families, physicians, and other health care professionals [18]. However, the practice of family-centered care by health care professionals could be productive only if the community of health care professionals recognized the vital role that families play in ensuring the health and well-being of children and family members of all ages. In that view, it is critical that health practitioners acknowledge that emotional, social, and developmental support are integral components of health care. Furthermore, each child and family's innate strengths should be respected and the health care experience must be considered as an opportunity to build on these strengths and support families in their care-giving and decision-making roles [19].

l, social, and developmental support are integral components of health care. Furthermore, each child and family's innate strengths should be respected and the health care experience must be considered as an opportunity to build on these strengths and support families in their care-giving and decision-making roles [19]. Core principles of family-centered care Under many aspects, family-centered care can be considered as an extension of patient-focused care, a concept that acquired general attention in the early 1990s [8]. The fundamental premise of patient-focused care was to abandon the traditional approach in which care was based on what worked well from an organizational perspective, promoting the concept that delivery of care should be based on the needs of the patient. Family-centered care simply takes patient-focused care to the next step and expands the concern to include in the loop of care those persons who are important in a patient's life [13].

orked well from an organizational perspective, promoting the concept that delivery of care should be based on the needs of the patient. Family-centered care simply takes patient-focused care to the next step and expands the concern to include in the loop of care those persons who are important in a patient's life [13]. It is now a consolidated convincement in pediatrics that acceptance and correct practice of family-centered approaches will produce better health outcomes and wiser allocation of resources as well as greater patient and family satisfaction. However, much confusion remains over what family-centered critical care actually is [8]. Many clinicians incorrectly associate family-centered care with open visiting. This misconception originates, in part, from the common implementation of policies for flexible visiting hours in units that are attempting to provide more family-oriented care. Given that family-centered care is a philosophical approach to care that recognizes the needs of patients' family members as well as the important role that family members play during a patient's illness, it is therefore not defined by a singular intervention. In fact, no single intervention and not even a group of interventions will ensure a family-focused environment. For instance, it would be wrong to believe that simply allowing a family member to be at a patient's bedside 24 hours a day would mean that the staff met the family's needs. In fact, having a family member present in a situation in which staff members are not equipped to meet the family's needs could ultimately have unfavorable consequences. Family members may be more stressed if they feel ignored by a nurse, neglected by physicians or are made to believe that they are somehow in the way or interfering with the patient's care [8].

a situation in which staff members are not equipped to meet the family's needs could ultimately have unfavorable consequences. Family members may be more stressed if they feel ignored by a nurse, neglected by physicians or are made to believe that they are somehow in the way or interfering with the patient's care [8]. An important step toward the establishing of family-centered care concepts has been made in 2003 by the American Academy of Pediatrics, which issued a policy statement indicating nine key principles, that should guide the practice of family-centered care in pediatrics (Appendix 2). The AAP statement indicates that family-centered care is grounded in collaboration among patients, families, physicians, nurses, and other professionals for the planning, delivery, and evaluation of health care as well as in the education of health care professionals. This partnership proposed by the AAP statement implies that practice of family-centered care in pediatrics should pursue a number of unavoidable tasks, including the support of youth as they transition to adulthood, acknowledgment of family as the constant in a child's life and the honor of cultural diversity and family traditions. Such tasks are reported in Appendix 3.

es that practice of family-centered care in pediatrics should pursue a number of unavoidable tasks, including the support of youth as they transition to adulthood, acknowledgment of family as the constant in a child's life and the honor of cultural diversity and family traditions. Such tasks are reported in Appendix 3. The 2003 AAP statement has been undoubtedly a significant step toward the correct management of the needs of hospitalized children. However, in spite of the widespread attention and growing knowledge regarding family-centered care, there is still confusion over this concept and its practice may cause frustrations for many staff members who think that family-centered care may not be in the best interest of either patients or health personnel. In general, adult patients who are able should always be asked to what extent they want their family to participate in care. Family's involvement is presented as a choice and lets patients know that family members are welcome should the patients so decide [8]. Patients may, in fact, not want any visitors or any information given out to family members. Of course, in pediatrics the practice of these concepts is facilitated by the nature of the patients and their dependence by families or guardians. However, it is also important that care for the needs of children and family's involvement are not misrepresented as opportunities for the staff to alleviate their job or to charge families with duties or responsibilities that do not pertain to them.

nature of the patients and their dependence by families or guardians. However, it is also important that care for the needs of children and family's involvement are not misrepresented as opportunities for the staff to alleviate their job or to charge families with duties or responsibilities that do not pertain to them. On the other hand it is equally important and helpful for staff members to see that the essence of family-centered care is consistent with patient-centered care. To such regard, a great concern often raised by staff members is that family-centered care demands that staff relinquish all structures within the unit that allow some form of order in this otherwise chaotic environment. This concern should be considered absolutely not the case. For example, during a critical illness, patients' families will benefit from guidance and structure to help them deal with the situation. It is crucial that staff members fully understand what family-centered care is and is not, to avoid any room for disruptive discussions, as well as they must be reassured by knowing that boundaries and limitations are still in place and that the expertise of staff members remains a critical factor in ensuring the success of family-centered care [8,13].

ully understand what family-centered care is and is not, to avoid any room for disruptive discussions, as well as they must be reassured by knowing that boundaries and limitations are still in place and that the expertise of staff members remains a critical factor in ensuring the success of family-centered care [8,13]. The important concept that must be reiteratively stressed is that the needs of the children are always the priority, even in a family-centered environment. Research indicates that it is important to the hospitalized child's family members to be assured that he/she is receiving the best possible care [20]. Interventions such as having family members present during procedures and resuscitations help to reassure family members that everything possible is being done for the patient [21]. Understanding and meeting children's needs in hospitals should always be the priority for both the patient's family and health care providers. Finally, family-centered way of practice requires that outdated rules and regulations that were imposed for the benefit of the organization rather than children or their families should be reviewed and reconsidered. Structures and policies that provide for the support and safety of patients and their family members are generally welcomed by family members and help staff members to carry out their responsibilities in a timely and efficient manner [8].

ation rather than children or their families should be reviewed and reconsidered. Structures and policies that provide for the support and safety of patients and their family members are generally welcomed by family members and help staff members to carry out their responsibilities in a timely and efficient manner [8]. Importance of Cultural Diversity and Cultural Competence for family-centered care in the context of socioeconomic, demographic and ethnic changes Cultural Diversity is generally defined by coexistence of numerous distinct ethnic, racial, religious, or cultural groups within one social unit, organization, or population. As a source of exchange, innovation and creativity, cultural diversity is as necessary for humankind as biodiversity is for nature. In this sense, it is the common heritage of humanity and should be recognized and affirmed for the benefit of present and future generations. This approach to cultural diversity should be taken by economically advantaged Countries in confronting socioeconomic, demographic and ethnical changes within their civil societies. In our increasingly diverse societies, Cultural Diversity is essential to ensure harmonious interaction among people and groups with plural, varied and dynamic cultural identities as well as their willingness to live together [22-24].

ronting socioeconomic, demographic and ethnical changes within their civil societies. In our increasingly diverse societies, Cultural Diversity is essential to ensure harmonious interaction among people and groups with plural, varied and dynamic cultural identities as well as their willingness to live together [22-24]. Cultural competence is defined as a set of values, behaviors, attitudes, and practices within a system, organization, program or among individuals which enables them to work effectively cross culturally (Appendix 4). Further, it refers to the capability to respect the beliefs, language, inter-personal styles and behaviors of individuals and families receiving services, as well as staff who are providing such services. At a systems, organizational, or program level, cultural competence requires a comprehensive and coordinated plan that includes interventions at all the levels from policy-making to the individual, and is a dynamic, ongoing, process that requires a long-term commitment. An important component of cultural competence is linguistic competence, the capacity of an organization and its personnel to communicate effectively, and convey information in a manner that is easily understood by diverse audiences including persons of limited local language proficiency, those who are not literate or have low literacy skills, and individuals with disabilities.

competence, the capacity of an organization and its personnel to communicate effectively, and convey information in a manner that is easily understood by diverse audiences including persons of limited local language proficiency, those who are not literate or have low literacy skills, and individuals with disabilities. Cultural Competence and Cultural Diversity are profoundly interconnected to the concept and practice of family-centered care. In fact, Cultural Competence is necessary in providing care to culturally diverse families. Family-centered care values the strengths, cultures, traditions and expertise that everyone brings to a respectful family/professional partnership, where families feel they can be decision makers with providers at different levels, in the care of their own children and as advocates for systems and policies supportive of children and youth with special health care needs. It requires culturally competent attitudes and practices in order to develop and cultivate those partnerships and to have the knowledge and skills that will enable the health care practitioners to be "family-centered" with the many diverse families that exist and they interact with. Various and untraditional strategies can also be adopted to support health system in providing proper care to sick children, including those who are hospitalized. For instance, building relationships with community cultural brokers, is an approach that can help health care institutions and health care professionals in understanding rules and behaviors of different communities [25-29].

Group 3 comprised 291 children. Mean PCT level was 0.42 (± 0.18) ng/ml. Nine children had PCT levels > 0.5 ng/ml (values ranges from 0.59 to 18.64 ng/ml, median 0.86 ng/ml) [Figure 2]. Among them, 3 had transient acute synovitis, 3 had lower respiratory tract infection, 1 gingivitis, 1 tonsillitis and 1 was discharged with no diagnosis but recovered without antibiotic treatment. Seventy three children had fever (6 in group 1, 23 in group 2 and 44 in group 3) and 12 of them had PCT values > 0.5 ng/ml (2 in group 1, 4 in group 2 and 6 in group 3). In this study, the specificity (Sp) of the PCT as a marker of bacterial infection (comparing Group 1 and Group 3) was 96.9% [95% CI, 94.2-98.6], the sensitivity (Se) 25% [95% CI, 3.2-65.1], the positive predictive value (PPV) 18.2% [95% CI, 2.3-51.8] and the negative predictive value (NPV) 97.9% [95% CI, 95.5-99.2] [Table 1]. When comparing Groups 1 and 2 versus Group 3 the Sp was 96.9% [95% CI, 94.2-98.6], the Se 12.5% [95% CI, 4.7-25.2], the PPV 40% [95% CI, 16.3-67.7] and the NPV 87% [95% CI, 82.9-90.5]. When considering only children with fever, the Sp was 86.3% [95% CI, 82.3-90.2], the Se 20.7% [95% CI, 3.8-43.2], the PPV 50% [95% CI, 18.9-65.4] and the NPV 62.3% [95% CI, 31.2-82.3]. After stratification by suspected/confirmed osteomyelitis versus non-infected children, the Se was 20% [95% CI, 3.7-41.6], the Sp 96.9% [95% CI, 94.2-98.6], the PPV 30.7% [95% CI, 13.7-48.9] and the NPV 94.6% [95% CI, 91.2-97.5]. For the suspected/confirmed septic arthritis versus non-infected children, the Se was 7.1% [95% CI, 4.8-19.5], the Sp 96.9% [95% CI, 94.2-98.6], the PPV 18.1% [95% CI, 4.1-38.6] and the NPV 91.5% [95% CI, 87.4-94.3]. The receiver operating characteristic (ROC) curves comparing Group 1 versus Group 3 and Group 1 and 2 versus Group 3 are plotted in Figure 3 and 4. The area under the curves (AUC) is 0.59 [95% CI, 0.45-0.79] for proven infection versus no infection and the AUC for proven or suspected infection versus no infection is 0.54 [95% CI, 0.50-0.61].

health system in providing proper care to sick children, including those who are hospitalized. For instance, building relationships with community cultural brokers, is an approach that can help health care institutions and health care professionals in understanding rules and behaviors of different communities [25-29]. In brief, knowledge of cultural diversity is vital at all levels of health system and knowledge about cultures and their impact on interaction with health care is essential for health care professionals, whether they are practicing in a clinical setting, education, research or administration. Knowledge and skills related to the important concept of cultural diversity and acquaintance with cultural competence are factors that must be considered strategic to strengthen and broaden health care delivery systems. Summary In conclusion, the social, political and most in general cultural changes in today's globalized world, has accelerated various innovative approaches in different sectors of society, including the health system. Although Family-centered care applies to patients of all ages, and it may be practiced in any health care setting, this unique approach is of particular importance in the management of hospitalized children, given the role that hospitalization may play as a time of potential crisis for the child and family [11].

ystem. Although Family-centered care applies to patients of all ages, and it may be practiced in any health care setting, this unique approach is of particular importance in the management of hospitalized children, given the role that hospitalization may play as a time of potential crisis for the child and family [11]. Family-centered care should be integrated in the management of children in teaching and non teaching hospitals; and education and training in family-centered care should be provided to all trainees, students, and residents as well as staff members [30]. In accordance with the American Academy of Pediatrics'recommendations [13], core concepts of family-centered care should be incorporated into all aspects of pediatricians' professional practice, whether it is private practice or in hospitals (Appendix 5). Whatever the context, pediatricians should unequivocally convey respect for parents' or guardians' unique insight into and understanding of their child's behavior and needs, should actively seek out their observations, and should appropriately incorporate family preferences into the care plan. Decisions on a patient's plan of care should be made only after such consultation has been made.

for parents' or guardians' unique insight into and understanding of their child's behavior and needs, should actively seek out their observations, and should appropriately incorporate family preferences into the care plan. Decisions on a patient's plan of care should be made only after such consultation has been made. Finally, the most favorable environments to practice an effective family-centered care in pediatrics must be provided by health care institutions. They should design their facilities to promote the philosophy of family-centered care and in hiring of staff, developing job descriptions, and designing performance-appraisal processes, they should make explicit the expectation of collaboration with patients and families and other family-centered behaviors. Competing interests DECLARATION: All the authors declare that they have no competing interests as they have been described in the "manuscript sections for Debate Articles" of the IJP. Authors' contributions All the Authors equally contributed to the article. In particular, they have equally made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; furthermore, they have been involved in drafting the manuscript or revising it critically for important intellectual content; Finally, all authors read and approved the final manuscript.

have equally made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; furthermore, they have been involved in drafting the manuscript or revising it critically for important intellectual content; Finally, all authors read and approved the final manuscript. Appendix 1 Family-Centered Care: Definition and its Importance in Pediatrics • Family-centered care. An innovative approach to the planning, delivery, and evaluation of health care that is grounded in mutually beneficial partnerships among health care patients, families, and providers. It incorporates the patient, the health care provider, and the family in all aspects of care. • Heart of Pediatric family-centered care. The belief that health care providers and the family are partners, working together to best meet the needs of the child. Parents and family members provide the child's primary strength and support. Their information and insights can enhance the professional staff's technical knowledge, improve care and help design better programs and friendlier systems 13 Neff JM et al. Pediatrics – 2003 Appendix 2 Core Principles of Family-Centered Care 1. Respecting each child and his or her family 2. Honoring racial, ethnic, cultural, and socioeconomic diversity and its effect on the family's experience and perception of care 3. Recognizing and building on the strengths of each child and family, even in difficult and challenging situations 4. Supporting and facilitating choice for the child and family about approaches to care and support

characteristic (ROC) curves comparing Group 1 versus Group 3 and Group 1 and 2 versus Group 3 are plotted in Figure 3 and 4. The area under the curves (AUC) is 0.59 [95% CI, 0.45-0.79] for proven infection versus no infection and the AUC for proven or suspected infection versus no infection is 0.54 [95% CI, 0.50-0.61]. Table 1 Sensitivity, Specificity, Positive Predictive Value and Negative Predictive Value of PCT with the [95% CI] Groups Sensitivity Specificity PPV NPV 1 vs 3 25 [3.2-65.1] 96.9 [94.2-98.6] 18.2 [2.3-51.8] 97.9 [95.5-99.2] 1+2 vs 3 12.5 [4.7-25.2] 96.9 [94.2-98.6] 40 [16.3-67.7] 87 [82.9-90.5] 1+2 vs 3 (fever) 20.7 [3.8-43.2] 86.3 [82.3-90.2] 50 [18.9-65.4] 62.3 [31.2-82.3] 1+2 vs 3 (osteomyelitis) 20 [3.7-41.6] 96.9 [94.2-98.6] 30.7 [13.7-48.9] 94.6 [91.2-97.5] 1+2 vs 3 (septic arthritis) 7.1 [4.8-19.5] 96.9 [94.2-98.6] 18.1 [4.138.6] 91.5 [87.4-94.3] Figure 3 ROC curve for PCT, proven infection versus no infection. Figure 4 ROC curve for PCT, proven/suspected infection versus no infection.

2. Honoring racial, ethnic, cultural, and socioeconomic diversity and its effect on the family's experience and perception of care 3. Recognizing and building on the strengths of each child and family, even in difficult and challenging situations 4. Supporting and facilitating choice for the child and family about approaches to care and support 5. Ensuring flexibility in organizational policies, procedures, and provider practices so services can be tailored to the needs, beliefs, and cultural values of each child and family 6. Sharing honest and unbiased information with families on an ongoing basis and in ways they find useful and affirming 7. Providing and/or ensuring formal and informal support (eg, family-to-family support) for the child and parent(s) and/or guardian(s) during pregnancy, childbirth, infancy, childhood, adolescence, and young adulthood 8. Collaborating with families at all levels of health care, in the care of the individual child and in professional education, policy making, and program development 9. Empowering each child and family to discover their own strengths, build confidence, and make choices and decisions about their health 11 American Academy of Pediatrics – 2003 Appendix 3 Tasks of Family-Centered Care in Pediatrics Family-centered care 1. Acknowledges the family as the constant in a child's life. 2. Builds on family strengths. 3. Supports the child in learning about and participating in his/her care and decision-making. 4. Honors cultural diversity and family traditions. 5. Recognizes the importance of community-based services. 6. Promotes an individual and developmental approach.

Appendix 3 Tasks of Family-Centered Care in Pediatrics Family-centered care 1. Acknowledges the family as the constant in a child's life. 2. Builds on family strengths. 3. Supports the child in learning about and participating in his/her care and decision-making. 4. Honors cultural diversity and family traditions. 5. Recognizes the importance of community-based services. 6. Promotes an individual and developmental approach. 7. Encourages family-to-family and peer support. 8. Supports youth as they transition to adulthood. 9. Develops policies, practices, and systems that are family-friendly and family-centered in all settings. 10. Celebrates successes. Sources • 31 National Center for Family-Centered Care: Family-Centered Care for Children with Special Health Care Needs. Bethesda, MD: Association for the Care of Children's Health; 1989 • 32 Bishop et al.: Family/Professional Collaboration for Children with Special Health Care Needs and their Families. Burlington, VT: University of Vermont, Department of Social Work; 1993. • 33 Bishop et al: Family-Centered Care Projects 1 and 2 (2002–2004). Algodones, NM: Algodones Associates; 2004 Appendix 4 Principles of Cultural Competence An organization should 1) Value diversity in families, staff, providers and communities; 2) Have the capacity for cultural self-assessment; 3) Be conscious of the dynamics inherent when cultures interact, e.g. families and providers; 4) Institutionalize cultural knowledge; and 5) Develop adaptations to service delivery and partnership building reflecting an understanding of cultural diversity. An individual should 1) Examine one's own attitude and values;

2) Have the capacity for cultural self-assessment; 3) Be conscious of the dynamics inherent when cultures interact, e.g. families and providers; 4) Institutionalize cultural knowledge; and 5) Develop adaptations to service delivery and partnership building reflecting an understanding of cultural diversity. An individual should 1) Examine one's own attitude and values; 2) Acquire the values, knowledge, and skills for working in cross cultural situations; and 3) Remember that every one has a culture. Sources • 34 Maternal and Child Health Bureau (MCHB): Guidance and Performance Measures for Discretionary Grants, Health Resources and Services Administration. Washington, DC: U.S. Department of Health and Human Services; 2004. • 23 Cross T, Bazron B, Dennis K, Isaacs M: Towards a culturally competent system of care. Washington, DC: Georgetown University Child Development Center, CASSP Technical Assistance Center; 1989 • 35 Goode J: Definition of Linguistic Competence. Washington, DC: National Center for Cultural Competence; 2004.

• 34 Maternal and Child Health Bureau (MCHB): Guidance and Performance Measures for Discretionary Grants, Health Resources and Services Administration. Washington, DC: U.S. Department of Health and Human Services; 2004. • 23 Cross T, Bazron B, Dennis K, Isaacs M: Towards a culturally competent system of care. Washington, DC: Georgetown University Child Development Center, CASSP Technical Assistance Center; 1989 • 35 Goode J: Definition of Linguistic Competence. Washington, DC: National Center for Cultural Competence; 2004. Appendix 5 Strategies for an Effective Family-Centered Care in Hospital and Private Practice • Conduct attending physician rounds (ie, patient presentations and rounds discussions) in the patients' rooms with the family present should be standard practice. This will facilitate the exchange of information between the family and other members of the child's health care team and encourage the involvement of the family in the decisions that are commonly made during rounds. In teaching hospitals, a lasting impression will be made on students and house staff when they are encouraged in this process by their attending physician. • Invite parents and guardians to be present with their child during medical procedures and offered support before, during, and after the procedure. Working with families in decision making and information sharing in all practice settings should always take into account the older child's and young adult's capacity for independent decision making and right to privacy and confidentiality.

medical procedures and offered support before, during, and after the procedure. Working with families in decision making and information sharing in all practice settings should always take into account the older child's and young adult's capacity for independent decision making and right to privacy and confidentiality. • Promote the active participation of all children in the management and direction of their own health care, beginning at an early age and continuing into adult health care. During their work in collaboration with families and other health care professionals, pediatricians should also examine systems of care, individual interactions with patients and families, and patient flow and should modify these as needed to improve the patient's and family's experience of care. • Share information with children and families in ways that are useful and affirming in every health care encounter • Encourage and facilitate family-to-family support and networking, particularly with families of similar cultural and linguistic backgrounds or families who have children with the same type of medical condition. • Invite the families to collaborate in pediatric research programs. Families should have a voice at all levels in shaping the research agenda, in determining how children and families participate in research, and in deciding how research findings will be shared with children and families

• Encourage and facilitate family-to-family support and networking, particularly with families of similar cultural and linguistic backgrounds or families who have children with the same type of medical condition. • Invite the families to collaborate in pediatric research programs. Families should have a voice at all levels in shaping the research agenda, in determining how children and families participate in research, and in deciding how research findings will be shared with children and families • Create opportunities for children and families to serve as advisors in family advisory councils, committees, and task forces dealing with operational issues in hospitals, clinics, and office-based practices; as participants in quality improvement initiatives; as educators of staff and professionals in training; and as leaders or co-leaders of peer support programs. 11 American Academy of Pediatrics – 2003 13 Neff JM et al. Pediatrics – 2003 Acknowledgements The Authors would like to acknowledge the non-profit scientific association "Giordano Emilio Ghirardi", Foggia, Italy and the non-profit Family Association "Associazione Italiana Celiachia" AIC-Puglia, Italy, for their support and collaboration with the Institute of Paediatrics of the University of Foggia. Their positive cooperation and support has been useful to further establish the concept of Family centred care in the geographical area served by the University Hospital of Foggia.

Introduction Almost all children are infected by Respiratory Syncytial Virus (RSV) at least once by 2 years of age, and approximately 1–2% of infants will require hospitalization for RSV-associated Lower Respiratory Tract Infections (LRTI) [1-3]. Infection rates vary from 50–70% during the first year of life, to 100% during the second-third year of age. Immunity, however, is not complete, and reinfection is common [1-4]. Evidence of RSV infection has been found in every geographic area studied. In countries with a temperate climate, outbreaks generally occur during the autumn and last until spring, with the number of infections peaking in January-March. In tropical areas, epidemics generally coincide with the rainy season [5-7]. Although palivizumab can reduce the rate of hospitalization due to RSV-LRTI, it is costly to administer, therefore local data need to be evaluated to formulate guidelines and justify its use, especially in countries with economic difficulties [8]. In Italy, RSV outbreaks begin in the late winter and last until spring, reaching a peak in March. Recent climate changes have influenced the regional trend of the country, altering the start and peak of the RSV epidemic season [9,10]. Objective the Authors conducted this study to evaluate the epidemiological and clinical patterns of RSV infection in infants hospitalized for LRTI in in Palermo, South Italy, Sicily. Methods Patients A prospective surveillance study was performed from October 1, 2005 to April 30, 2006. The study population consisted of children admitted to the "G. Di Cristina", Children Hospital of Palermo.

Objective the Authors conducted this study to evaluate the epidemiological and clinical patterns of RSV infection in infants hospitalized for LRTI in in Palermo, South Italy, Sicily. Methods Patients A prospective surveillance study was performed from October 1, 2005 to April 30, 2006. The study population consisted of children admitted to the "G. Di Cristina", Children Hospital of Palermo. Consent for enrolment was sought from parents by study personnel. All children less than 2 years of age who were hospitalized with symptoms suggesting LRTI were enrolled in the study. LRTI were categorized on the basis of clinical and roentgenographic findings, according to the criteria proposed by Ruuskanen and Ogra [11]. The disease was diagnosed: a) as wheezy bronchitis, when an acute illness characterized by cough, rhonchi, and expiratory wheezing was detected; b) as bronchiolitis, when wheezing dyspnea, tachypnea and CXR hyperinflation of the lung with or without areas of collapse were present; c) as pneumonia when radiographic findings of lung parenchymal involvement, with interstitial-alveolar infiltrates and/or consolidation, were found. The radiologist and the clinicians allocating patients to these categories were blinded as to RSV status.

ation of the lung with or without areas of collapse were present; c) as pneumonia when radiographic findings of lung parenchymal involvement, with interstitial-alveolar infiltrates and/or consolidation, were found. The radiologist and the clinicians allocating patients to these categories were blinded as to RSV status. Demographical, clinical and microbiological data were prospectively collected and entered into an Access Database (Microsoft). The following parameters were recorded: demographic characteristics, breast-feeding, history of underlying diseases, history of admissions for respiratory problems, start and duration of symptoms, need for intensive care, oxygen supplementation, results of microbiology tests and chest radiograph. We also evaluated the incidence of LRTI in all newborns hospitalized in the N.I.C.U. of the 'Dipartimento Materno Infantile-IMI' and the 'V. Cervello' Hospital in Palermo during the same period. Informed consent was obtained from a parent or guardian. The study was approved by the Ethics Committee of the ARNAS Hospital of Palermo, Italy. Oximetry Oxygen saturation (SaO2) and pulse rate were determined upon admission and then daily, using a portable Nellcor oximeter (Nellcor, Inc., Hayward, CA). Data were always collected for at least 5 min (except for longer periods of observation when measurements were not stable or not considered reproducible), with the child awake and quiet in room air and, when needed, in oxygen. Oxygen was administered when SaO2 was <90% and amounts of oxygen supplementation were recorded.

ta were always collected for at least 5 min (except for longer periods of observation when measurements were not stable or not considered reproducible), with the child awake and quiet in room air and, when needed, in oxygen. Oxygen was administered when SaO2 was <90% and amounts of oxygen supplementation were recorded. Admission in Intensive Care Unit (ICU) was decided when the cardio-respiratory or the general conditions of the patients required intensive care. Virologic Studies Nasopharyngeal swabs and throat specimens were collected at the time of LRTI and tested for a panel of respiratory viruses by immunofluorescent test (PathoDx® Respiratory Virus Panel Kit, Immunofluorescent test for 7 respiratory viruses: influenza A; influenza B; adenovirus; parainfluenza 1, 2, 3; and RSV) in shell vial pre-CPE assays, and conventional tube culture confirmation (CE, Remel, Lenexa, KS 66215. USA), or for RSV alone by enzymatic diagnostic test (Now® RSV Test, Binax) according to medical request-record. Statistical analysis Analysis was based on comparison of demographic data between RSV + and RSV - infants. Between groups comparison was performed by means of chi-square test for categorical variables and by Student's t-test for continuous variables collected in the study. Data were processed with a SPSS-pc statistical program. A significance level of p < 0.05 was used throughout the study.

Statistical analysis Analysis was based on comparison of demographic data between RSV + and RSV - infants. Between groups comparison was performed by means of chi-square test for categorical variables and by Student's t-test for continuous variables collected in the study. Data were processed with a SPSS-pc statistical program. A significance level of p < 0.05 was used throughout the study. Results Of the 1219 children referred to the Emergency Unit of the 'G. Di Cristina' Children's Hospital between November 2005 and May 2006, 705 (58%) were hospitalized with suspected LRTI. The trend of LRTI hospitalization started in November 2005, lasting until May 2006 with an epidemic peak in March (figure 1). Of these 705 children, n°35 were over 2 years old and n°25 had a history of two or more previous hospitalizations and were carried out from the study. Figure 1 Seasonal trend of RSV infection in 335 enrolled infants. Of the remaining 645 infants, only 335 showed viral investigation and were enrolled in the study.

Results Of the 1219 children referred to the Emergency Unit of the 'G. Di Cristina' Children's Hospital between November 2005 and May 2006, 705 (58%) were hospitalized with suspected LRTI. The trend of LRTI hospitalization started in November 2005, lasting until May 2006 with an epidemic peak in March (figure 1). Of these 705 children, n°35 were over 2 years old and n°25 had a history of two or more previous hospitalizations and were carried out from the study. Figure 1 Seasonal trend of RSV infection in 335 enrolled infants. Of the remaining 645 infants, only 335 showed viral investigation and were enrolled in the study. The retrospective interview of the pediatricians on because the viral investigation did not perform in the excluded patients has showed that in 60% of the cases the patients have been hospitalized in the weekend when viral analysis was not routinely available, in 10% of the cases the infants were early tested, in 3% of the cases the parents have refused the investigation and in the remaining 27% of cases the clinical, instrumental and epidemiological investigations were considered suitable for the management of LRTI. The percentage of exclusion showed an equally distribution with the trend of hospitalization.

e early tested, in 3% of the cases the parents have refused the investigation and in the remaining 27% of cases the clinical, instrumental and epidemiological investigations were considered suitable for the management of LRTI. The percentage of exclusion showed an equally distribution with the trend of hospitalization. RSV was identified in 178/335 patients (53%), parainfluenza type 3 virus in 14, influenza A virus in 10 and adenovirus in 2 cases. No combined virus infection was observed. The epidemic peak of the LRTI occurred in March, with a prevalence of RSV positivity of 63% raising up to 80% in April, while no RSV positivity was seen in October, November or December (figure 1). The characteristic of 335 studied infants are shown in table 1. Table 1 Demographic and clinical data of the 335 RSV studied infants in the season of November 2005–April 2006

RSV was identified in 178/335 patients (53%), parainfluenza type 3 virus in 14, influenza A virus in 10 and adenovirus in 2 cases. No combined virus infection was observed. The epidemic peak of the LRTI occurred in March, with a prevalence of RSV positivity of 63% raising up to 80% in April, while no RSV positivity was seen in October, November or December (figure 1). The characteristic of 335 studied infants are shown in table 1. Table 1 Demographic and clinical data of the 335 RSV studied infants in the season of November 2005–April 2006 RSV Positive (n = 178) RSV Negative (n = 157) P %Male (n = 214) 59 41 > 0.05 %Female (n = 121) 73 17 > 0.05 Age (months) 3 ± 1.4 5 ± 7.1 <0.05 %Age at time of hospitalization < 3 months (n = 132) 63 37 > 0.05 %Age at time of hospitalization < 6 months (n = 220) 59 41 <0.05 %Age at time of hospitalization > 6 months (n = 115) 43 57 > 0.05 Gestational age (weeks) 38 ± 1,7 38 ± 1,9 > 0.05 %Gestational age >36 weeks (n = 315) 45 55 > 0.05 Birth weight (gr.) 2986 ± 680 3023 ± 609 > 0.05 Birth weight group > 2500 g (n = 291) 44.3 55.7 > 0.05 Previous Hospitalization for LRTI (n = 97) 37 63 > 0.05 Breastfeeding (n = 187) 58 129 <0.05 Days of hospitalization 6.4 ± 3.5 5.6 ± 3.1 <0.05 Days of oxygen therapy (n = 235) 4.9 ± 3.3 4.1 ± 2.3 <0.05 Data are shown as percentage or means ± SD. The value of p refers to statistical significance of differences between the two populations; Independently from the RSV positivity, when evaluating the age distribution, 132 (39%) of them were less than 3 months old and 220 (65%) were less than 6 months old.

RSV Positive (n = 178) RSV Negative (n = 157) P %Male (n = 214) 59 41 > 0.05 %Female (n = 121) 73 17 > 0.05 Age (months) 3 ± 1.4 5 ± 7.1 <0.05 %Age at time of hospitalization < 3 months (n = 132) 63 37 > 0.05 %Age at time of hospitalization < 6 months (n = 220) 59 41 <0.05 %Age at time of hospitalization > 6 months (n = 115) 43 57 > 0.05 Gestational age (weeks) 38 ± 1,7 38 ± 1,9 > 0.05 %Gestational age >36 weeks (n = 315) 45 55 > 0.05 Birth weight (gr.) 2986 ± 680 3023 ± 609 > 0.05 Birth weight group > 2500 g (n = 291) 44.3 55.7 > 0.05 Previous Hospitalization for LRTI (n = 97) 37 63 > 0.05 Breastfeeding (n = 187) 58 129 <0.05 Days of hospitalization 6.4 ± 3.5 5.6 ± 3.1 <0.05 Days of oxygen therapy (n = 235) 4.9 ± 3.3 4.1 ± 2.3 <0.05 Data are shown as percentage or means ± SD. The value of p refers to statistical significance of differences between the two populations; Independently from the RSV positivity, when evaluating the age distribution, 132 (39%) of them were less than 3 months old and 220 (65%) were less than 6 months old. Four (1%) infants were born before 32 weeks gestation, 16 (5%) were born between 33–35 weeks and the remaining 315 patients (94%) were born after the 36th week. All infants born before 32 weeks gestation were RSV-negative and received palivizumab prophylaxis. None of the RSV positive children received it. The distribution of gestational age (GA) showed that in our study population it was <36 weeks in 6% of the subjects versus 4.0% in general population [12,13].

the 36th week. All infants born before 32 weeks gestation were RSV-negative and received palivizumab prophylaxis. None of the RSV positive children received it. The distribution of gestational age (GA) showed that in our study population it was <36 weeks in 6% of the subjects versus 4.0% in general population [12,13]. Evaluating the distribution of birth weights, 4 infants weighed under 1.500 g, 10 between 1.500–2.000 g, and 30 between 2000 and 2.500 g and the remaining 291 infants over 2.500 at birth. 187 of the 335 children (55,8%) were breast-fed for more than 3 months. Of the 335 enrolled children, 224 (67%) had diagnosis of bronchiolitis, 35 (10%) had pneumonia, and 76 had wheezy bronchitis (23%); respectively, 55% of the patients with bronchiolitis, 16% of those with pneumonia, and 29% of those with wheezy bronchitis showed RSV infection. One infant was affected by bronchopulmonary dysplasia and 4 were affected by congenital cardiopathies. During the studied period, three cases of RSV was identified in 3 preterm newborns (2 patients born at 28 weeks gestation and 1 born in the 34th week) hospitalized from birth in the neonatal intensive care unit (NICU). The RSV respiratory diseases occurred before discharge when they stayed in post-intensive area.

One infant was affected by bronchopulmonary dysplasia and 4 were affected by congenital cardiopathies. During the studied period, three cases of RSV was identified in 3 preterm newborns (2 patients born at 28 weeks gestation and 1 born in the 34th week) hospitalized from birth in the neonatal intensive care unit (NICU). The RSV respiratory diseases occurred before discharge when they stayed in post-intensive area. The age at time of admission was lower in RSV + rather than in RSV-negative (RSV.) infants (p = 0.00). The likelihood to be RSV+, rather than RSV-negative (RSV-), was higher for infants < 6 months (p = 0.00) and lower in infants with a history of breast-feeding (p = 0.02) (table 1). Other demographic and clinical characteristics were evaluated but were not significantly different between RSV+ and RSV- infants. Comparing severity of disease, a tendency toward longer hospitalization and a longer oxygen therapy were detected in RSV+ infants when compared with RSV-infants. (Table 1). Furthermore, 10 RSV + infants were admitted to the Pediatric Intensive Care Unit, on average for 11.3 days versus no one infant in the RSV - patients. Only 1 patient required assisted ventilation. Discussion Previous Italian surveillance study of acute respiratory infections (ARI) in hospitalized infants had showed that respiratory syncytial virus is the prevalent etiological infectious agent [10]. The incidence of ARI tracked closely with the RSV season, with the majority of infections occurring from late winter through early spring.

lian surveillance study of acute respiratory infections (ARI) in hospitalized infants had showed that respiratory syncytial virus is the prevalent etiological infectious agent [10]. The incidence of ARI tracked closely with the RSV season, with the majority of infections occurring from late winter through early spring. RSV is a significant cause of morbidity leading to the hospitalization of children in Palermo, particularly infants under 2 years of age. The seasonal distribution of our patients shows that RSV infections peak in late spring, in contrast to Northern Italy. This pattern is similar to that reported for tropical countries, where RSV infection also occurs in the summer [5,14]. Efforts to prevent this infection are based on case management, vaccination and the identification of risk factors. Furthermore, in our report, we attempted to delineate clinical pattern of RSV + hospitalized infants in a population of south Italian children who experience extremely high rates of hospitalization for LRTI. Our results show that age at time of admission for LRTI was lower for infants RSV + rather than RSV- (3 months versus 5 months) and the age 0–6 months increased the risk of RSV hospitalization.

or) characterised by a high sensitivity (0.06 ng/ml) allowing a good discrimination of values around the threshold of 0.5 ng/ml. By comparison, PCT determined by PCT- Q-test, an immunochromatographic semi-quantitative assay, is not sensitive enough to discriminate values around 0.5 ng/ml as positive or negative values. There are several limitations to our study. The low incidence of positive bacteriological findings for the children considered as infected may be a bias. Nevertheless, bacteriologic diagnosis of orthopedic infections is known to be difficult and bacteriological samples are positive in a few numbers of cases [3,4]. On the other hand, this study was conducted in an urban teaching institution (tertiary pediatric care hospital), which has different referral patterns and serves a different population of patients than other institutions. We diagnosed 48 children as infected during this one year study, while Butbul-Aviel included 23 infected children during a two years study. Also, because there is no true gold standard for the diagnosis of skeletal infection except for positive bacteriological cultures, we applied an accepted standard supported by the literature: a combination of clinical condition, bacteriological findings, laboratory markers, completed by an imaging study when needed.

ospitalized infants in a population of south Italian children who experience extremely high rates of hospitalization for LRTI. Our results show that age at time of admission for LRTI was lower for infants RSV + rather than RSV- (3 months versus 5 months) and the age 0–6 months increased the risk of RSV hospitalization. This is not surprising as a chronological age of 3 months or less at the onset of the RSV season is a known risk factor for severe RSV-induced LRTI [12-14]. In addition to the small size of the conducting airways and incomplete development of the lung structure, these infants are less likely to have RSV-neutralizing maternal antibodies than infants born after the peak of the RSV season [15]. Lung growth and a more efficient immune response may explain why a chronological age of > 6 months at the time of hospitalization is associated with a lower likelihood of being hospitalized for a LRTI due to RSV [14-16]. In our report, the study of LRTI population revalues the protective effects of breast-feeding to RSV infection, recently underlined by some studies. It is not clear how breast-feeding reduces the risk of infection, although immunomodulatory constituents of human milk seem to be protective [17]. Although recent immunological studies have tried to define the mucosal and/or systemic mechanisms of protection against respiratory infections during the early months of life, more studies are required to identify which elements modify the evolution of disease. Therefore the protective role of breast-feeding must be carefully considered [17,18].

al studies have tried to define the mucosal and/or systemic mechanisms of protection against respiratory infections during the early months of life, more studies are required to identify which elements modify the evolution of disease. Therefore the protective role of breast-feeding must be carefully considered [17,18]. The statistical significant association between RSV infection and days of hospitalization and oxygen therapy confirms that this infection is cause of severity illness and that prompt recognition of the diagnosis that include early treatment with oxygen therapy helped to improve the clinical state of the children [10,19-21]. In particular, hospital stay greater than 5 days is considered one of relevant data for score range of illness severity [21,22]. In accordance with this observation is the remark that in our study all infants hospitalized in ICU were RSV+. Nosocomial RSV infection were observed in NICU during the studied season. These findings showed the relevant role of viral infection to determine severe respiratory illness in hospitalized patients. Several studies conducted in both community and healthcare settings have also demonstrated that promoting hand-washing and good hygiene practices to be adopted when coughing can help reduce the transmission of acute respiratory infection (ARI) [22]

ction to determine severe respiratory illness in hospitalized patients. Several studies conducted in both community and healthcare settings have also demonstrated that promoting hand-washing and good hygiene practices to be adopted when coughing can help reduce the transmission of acute respiratory infection (ARI) [22] Due to the low number of children with specific characteristics, other risk factors such as presence of chronic lung diseases or of congenital heart disease, congenital or acquired immunodeficiency, haematological malignancies, bone-marrow or organ transplants, and cystic fibrosis were not analyzed. There were two major limitations in our study. Firstly, no clinical score was used in our hospital for children with LRTI when the children were admitted to the Paediatric Infectious Disease Unit and clinical data collected. Secondly, this study reports the results of only one tertiary care medical center in Palermo. A comprehensive study including local clinics, regional hospitals and medical centers would provide more details of RSV infections in Sicily. In conclusion, RSV is the most important viral pathogen in infants and children under the age of 2 years. The seasonal trend of RSV infections in Palermo is "atypical", with peaks in the late winter and spring. Furthermore, the data provided by the study on the expected onset and end of the RSV season indicates that palivizumab prophylaxis start should be discussed keeping in mind the substantial variability in community RSV season timing. Competing interests The authors declare that they have no competing interests.

In conclusion, RSV is the most important viral pathogen in infants and children under the age of 2 years. The seasonal trend of RSV infections in Palermo is "atypical", with peaks in the late winter and spring. Furthermore, the data provided by the study on the expected onset and end of the RSV season indicates that palivizumab prophylaxis start should be discussed keeping in mind the substantial variability in community RSV season timing. Competing interests The authors declare that they have no competing interests. Authors' contributions PD conceived of the study, participated in its design and coordination and drafted the manuscript. AR conceived of the study and participated in its design and coordination. LS carried out the virologic studies. AG collected demographical, clinical and microbiological data of the children hospitalized in the Division of Infectious Diseases of the "G. Di Cristina" Children's Hospital of Palermo. AP collected demographical, clinical and microbiological data of the children hospitalized in the Division of Infectious Diseases of the "G. Di Cristina" Children's Hospital of Palermo. FF collected demographical, clinical and microbiological data of the children referred to Emergency Unit of the "G. Di Cristina" Children's Hospital of Palermo. PD participated in the coordination of the study. MC collected demographical, clinical and microbiological data of the children hospitalized in the Cystic Fibrosis Center of the "G. Di Cristina" Children Hospital of Palermo. DP performed the statistical analysis. DM collected demographical, clinical and microbiological data of the children referred to Emergency Unit of the "G. Di Cristina" Children's Hospital of Palermo. GC conceived of the study and participated in its design and coordination. He also collected demographical, clinical and microbiological data of the children hospitalized in the N.I.C.U. of the "Dipartimento Materno Infantile-IMI" and the "V. Cervello" Hospital of Palermo. All authors read and approved the final manuscript.

Introduction Sir, Infectious agents are believed to play a role in the pathogenesis of some autoimmune diseases. Among the most investigated micro-organisms are Streptococci and Herpes viruses [1-3]. An abnormal T cell response to low levels of some Staphylococcus aureus superantigens was demonstrated in vitro in Behçet's disease (BD) [4]. Similar correlations have also been reported in vivo. The relationship between nasal Staphylococcus aureus carriage and Wegener granulomatosis (WG) has been demonstrated [5]. Moreover, exacerbation of the clinical signs of BD due to Staphylococcal gingival infection has been described. Not only were oral ulcers aggravated, but also genital ulcers and skin lesions worsened. Elimination of the bacterial infection resulted in complete recovery [6]. In the following report, we describe three paediatric cases that tested positive for nasal Staphylococcus aureus concomitantly with the reactivation of their autoimmune ocular inflammation.

ated, but also genital ulcers and skin lesions worsened. Elimination of the bacterial infection resulted in complete recovery [6]. In the following report, we describe three paediatric cases that tested positive for nasal Staphylococcus aureus concomitantly with the reactivation of their autoimmune ocular inflammation. Case Report Three boys, aged between 8 and 11 years, were referred to our Service for severe ocular inflammation with progressive visual impairment. Clinical, haematological and instrumental evaluations allowed to exclude an infectious origin of the inflammation, which was eventually diagnosed as bilateral idiopathic pars planitis in two cases, and unilateral posterior uveitis with papillitis in one case. Systemic immune-modulating treatment resulted in satisfactory control of the ocular disease, with visual recovery in 15-30 days. Nasal swab specimens obtained pre-treatment, while ocular inflammation was still active, tested negative for bacterial colonisation in all cases. The three young patients experienced a relapse of their ocular inflammation after approximately 45, 150 and 180 days, respectively. Nasal swab culture was repeated at the time of reactivation and tested positive for Staphylococcus aureus in all cases, in the absence of any respiratory symptoms. Immune-modulating treatment was temporally increased and associated with systemic antimicrobial therapy (lasting 7-10 days). Complete quiescence of the ocular inflammation was achieved after three weeks. At this time nasal swabs tested negative in 2/3 boys.

reus in all cases, in the absence of any respiratory symptoms. Immune-modulating treatment was temporally increased and associated with systemic antimicrobial therapy (lasting 7-10 days). Complete quiescence of the ocular inflammation was achieved after three weeks. At this time nasal swabs tested negative in 2/3 boys. Discussion Several immunological mechanisms have been proposed to explain the relationship between some infectious agents and an active status of autoimmune diseases. In WG, genetic polymorphism of the receptor for the Fc fragment of immunoglobulins G (FcγR) may decrease Staphylococcus aureus clearance, thus promoting chronic carriage and resulting in preferential binding of FcγR to a subset of IgG-ANCA isotypes characterised by marked immunogenicity [7]. Furthermore, as significant sequence homology exists between the mammalian and microbial Heat Shock Proteins (50% homology), it has been suggested that bacterial HSP-responsive T cells stimulate autoreactive T cells by cross-reactivity mechanisms [1]. In fact, both antistreptococcal and antiretinal HSP60 antibodies were raised in the serum samples of patients with BD and uveitis [8]. Increased anti-HSP65 antibody responses were also present in the cerebrospinal fluid of patients with neuro-BD with parenchimal involvement [9].

ells by cross-reactivity mechanisms [1]. In fact, both antistreptococcal and antiretinal HSP60 antibodies were raised in the serum samples of patients with BD and uveitis [8]. Increased anti-HSP65 antibody responses were also present in the cerebrospinal fluid of patients with neuro-BD with parenchimal involvement [9]. We describe a small paediatric case series in which an extraocular Staphylococcus aureus infection may have acted as trigger for reactivation of autoimmune ocular inflammation. In these cases, nasal swabs proved useful to identify and treat the nasal bacterial carrier status of the young patients. A recent series stated that the mean rate of nasal Staphylococcus aureus colonisation in childhood is 36%, showing an age-related parabolic distribution with peak incidence at the age 11 years [10]. This corresponds to the age range in our patients. A larger number of cases, however, is necessary to evaluate if the elimination of nasal staphylococcal colonisation, even if asymptomatic, may help control the autoimmune ocular inflammation.

Background Stump appendicitis is a delayed complication of appendectomy. It is rare and few cases reported in the paediatric literature. The authors report on another case in a child and focus on the diagnostic peculiarities of this entity. Case A 9-year-old boy was admitted with a chief complaint of right lower quadrant pain of 24 hours duration associated to bilious vomiting and fever. His surgical history reveals an open appendectomy performed three years ago. On physical examination, he was not in distress and has a fever of 39°C. Abdominal palpation demonstrated tenderness in the right lower quadrant and guarding over the appendectomy scar. The remainder of the abdomen was soft and nontender. However, no abdominal masses were appreciated and the rectal and scrotal examinations were normal. The white blood cell count was 23.500 cells/mm3 with 87% neutrophils. Plain abdominal radiograph (figure 1) and ultrasonography (figure 2) revealed fecalith localized in the right iliac fossa. The diagnosis of stump appendicitis was advocated and confirmed at laparotomy. Through the same Mac Burney's approach, a gangrenous and perforated appendiceal stump of 35 mm was found and completely removed (figure 3). The post-operative course was uneventful after 18 months follow-up period. Figure 1 Plain abdominal radiograph demonstrating fecalith (arrow). Figure 2 Ultrasonography revealing fecalith (1) into appendiceal stump (2). Figure 3 Pathologic specimen showing gangrenous and perforated appendiceal stump.

Case A 9-year-old boy was admitted with a chief complaint of right lower quadrant pain of 24 hours duration associated to bilious vomiting and fever. His surgical history reveals an open appendectomy performed three years ago. On physical examination, he was not in distress and has a fever of 39°C. Abdominal palpation demonstrated tenderness in the right lower quadrant and guarding over the appendectomy scar. The remainder of the abdomen was soft and nontender. However, no abdominal masses were appreciated and the rectal and scrotal examinations were normal. The white blood cell count was 23.500 cells/mm3 with 87% neutrophils. Plain abdominal radiograph (figure 1) and ultrasonography (figure 2) revealed fecalith localized in the right iliac fossa. The diagnosis of stump appendicitis was advocated and confirmed at laparotomy. Through the same Mac Burney's approach, a gangrenous and perforated appendiceal stump of 35 mm was found and completely removed (figure 3). The post-operative course was uneventful after 18 months follow-up period. Figure 1 Plain abdominal radiograph demonstrating fecalith (arrow). Figure 2 Ultrasonography revealing fecalith (1) into appendiceal stump (2). Figure 3 Pathologic specimen showing gangrenous and perforated appendiceal stump. Discussion Stump appendicitis is the re-inflammation of the residual appendiceal tissue after an appendectomy [1-3]. It represents a rare delayed complication of appendectomy which is unknown by most clinicians [1-7]. Its frequency is under-estimated and under-reported [4,5,7,8]. Some factors have been suggested for the development of this condition. An appendicael stump that is left too long represents the most advocated etiologic factor [1-5,7-12]. Our patient had a relatively long stump. Inadequate identification of the appendicael base, because of severe local inflammation, retrocecal or sub-serous appendix, has been also suggested [1,4,5,10]. Moreover, the incidence of this complication seems to increase until the introduction of laparoscopic approach, probably due to absence of tactile feedback [1,4,13]. The age of the patients ranges from 11 to 72 years with [1,2,4,14]. Only three cases are reported in the paediatric literature [1,2,14]. The time of onset ranges from 2 weeks to decades after appendectomy [1,2,5,8-10,14-16]. The recognition of stump appendicitis can be challenging and are often delayed, leading to serious complications [1,4,8,16]. Thus, early diagnosis is necessary and should be considered when evaluating any patient with recurrent right lower quadrant abdominal pain and a history of appendectomy [16]. Clinically, patients present with signs and symptoms similar to appendicitis or acute abdomen [6]. The presence of an appendectomy scar does not absolutely rule out the possibility of stump appendicitis. Physician should keep in mind a possible incomplete appendiceal resection to prevent delayed diagnosis and treatment. As in our case, a high clinical suspicion and the presence of fecalith may help to diagnose the disease. Ultrasonography and CT scan of the abdomen constitute the modalities of choice for confirming the diagnosis [2,4,5,9,11,14]. Laparoscopy seems to be better than conventional laparotomy. It permits to perform a global ispection of abdominal cavity and an easier adhesiolysis [10,11,14]. Treatment is based on complete removal of the appendix [2,8].

of the abdomen constitute the modalities of choice for confirming the diagnosis [2,4,5,9,11,14]. Laparoscopy seems to be better than conventional laparotomy. It permits to perform a global ispection of abdominal cavity and an easier adhesiolysis [10,11,14]. Treatment is based on complete removal of the appendix [2,8]. Conclusion Stump appendicitis is a real entity. Its diagnosis frequently missed or delayed, should be considered in any patient with right lower quadrant pain even if there is a history of appendectomy. Complete removal of the appendix is imperative and is the only mean to prevent the occurrence of this complication. Consent Written informed consent was obtained from the patient for publication of this case report and accompanying images Competing interests The authors declare that they have no competing interests. Authors' contributions MG drafted and conceived the manuscript and FF SS RJ MH participated in its design

1+2 vs 3 (fever) 20.7 [3.8-43.2] 86.3 [82.3-90.2] 50 [18.9-65.4] 62.3 [31.2-82.3] 1+2 vs 3 (osteomyelitis) 20 [3.7-41.6] 96.9 [94.2-98.6] 30.7 [13.7-48.9] 94.6 [91.2-97.5] 1+2 vs 3 (septic arthritis) 7.1 [4.8-19.5] 96.9 [94.2-98.6] 18.1 [4.138.6] 91.5 [87.4-94.3] Figure 3 ROC curve for PCT, proven infection versus no infection. Figure 4 ROC curve for PCT, proven/suspected infection versus no infection. Discussion Early identification of skeletal infection is still a challenge for the clinicians, especially in the Emergency Department. Many trials found that PCT has a good specificity and a good positive predictive value for systemic bacterial infection, especially meningitis, pyelonephritis, pulmonary and neonates infections. In previously published reports, the cut off level beyond which a bacterial infection is considered as definite, ranges between 0.5 and 1 ng/ml [19,20]. In our series, with a PCT cut off level of 0.5 ng/ml, the Group 1 and 3 did not differ with a sensitivity of 25% (and only 12.5% if we consider all the infected children- Group 1 and 2 versus Group 3) and a specificity of 96.9%. Nine patients had false positive results (PCT level > 0.5 ng/ml in Group 3) and only two had positive results when infection was confirmed. Moreover, considering the ROC curves, there is no PCT cut-off level which allows to identify bone and joint infection. Our experience in the determination of the PCT levels in the initial workup of all patients admitted in the hospital with suspected bone or joint infection shows a low sensitivity (PCT level was negative in 75% of proven infection). This does not place it above clinical judgment for the correct discrimination of patients with skeletal infection. Butbul-Aviel [18] evaluated PCT values in 44 children admitted to the hospital for fever, limping and suspicion of osteomyelitis or septic arthritis. They found a higher sensitivity (58.3%) only for osteomyelitis, but the same than us for septic arthritis (27.2%). In our series, if we consider only osteomyelitis, the sensitivity is 16.6%. Their results are not confirmed by our study which may be because of our greater number of inclusions. Also we included all the patients cared for non traumatic decreased active motion of a skeletal segment, with or without fever.

Today anthracyclines are among the most powerful drugs used for the treatment of oncologic diseases both in childhood and adulthood. Nevertheless their major antineoplastic efficacy can be seriously impaired by collateral toxic effects causing profound alterations in cardiac muscle. These effects can be associated to acute clinical manifestations, occurring within 24 hours from the beginning of treatment, such as hyperkinetic arrhythmias and/or reversible heart failure (myocarditis-pericarditis syndrome); subacute manifestations, occurring after weeks or months (up to 30 months), leading rapidly to progressive heart failure and 60% mortality; chronic manifestations, occurring 4-20 years after the treatment, with progressive irreversible cardiac insufficiency [1]. The most interesting aspects are connected to late chronic cardiotoxicity that is particularly insidious. It has a long term asymptomatic course or presents slight electrocardiographic and/or echocardiographic anomalies that later evolve into chronic cardiomiopathy, dilated type in adulthood and restrictive-dilated in childhood, that is refractory to medical treatment [2]. Another peculiar feature of chronic anthracycline cardiotoxicity is that it is strictly linked to drug cumulative dose. Indeed, the incidence of anthracycline - induced cardiomyopathy (AIC) and heart failure increases from 7% of cases for total doses of 550 mg/m2/bs, to 15% for 600 mg/m2/bs and 30-40% for 700 mg/m2/bs [3].

her peculiar feature of chronic anthracycline cardiotoxicity is that it is strictly linked to drug cumulative dose. Indeed, the incidence of anthracycline - induced cardiomyopathy (AIC) and heart failure increases from 7% of cases for total doses of 550 mg/m2/bs, to 15% for 600 mg/m2/bs and 30-40% for 700 mg/m2/bs [3]. Pathological studies on experimental animal models and human endomyocardial biopsies have shown that AIC is characterized by histological alterations consisting in multiple areas of interstitial fibrosis associated with the presence of cardiomyocytes with vacuolar degeneration or compensatory hypertrophy. Necrotic cardiomyocytes with histiocytic infiltration, and stromal oedema with myocardial fibers dissociation can also be observed. Electron microscopy revealed that the damage caused by anthracyclines to cardiomyocytes appears as loss of myofibrils, distention of sarcoplasmic reticulum, mitochondrial swelling, increased lysosomal number and disorganization of nuclear chromatine [4-6]. In order to explain these alterations, numerous pathogenetic mechanisms have been proposed [6], and three seem to be the most important: free radical release secondary to the binding of anthracyclines to intracellular iron, interaction with nuclear and mitochondrial DNA, and gene activation with biochemical transduction signals inducing apoptosis [7,8].

ns, numerous pathogenetic mechanisms have been proposed [6], and three seem to be the most important: free radical release secondary to the binding of anthracyclines to intracellular iron, interaction with nuclear and mitochondrial DNA, and gene activation with biochemical transduction signals inducing apoptosis [7,8]. Free radicals cardiac toxicity can be caused by direct damage of the mitochondrial respiratory chain with consequent decrease in energy production, due to phosphorilative processes impairment, and reduction of cardiomyocytes following the release of pro-apoptotic factors. Both effects lead to altered systolic function [7] (Figure 1). Further harmful actions of free radicals are associated with membrane lipid peroxidation and cytoskeleton protein oxidation. These events cause the dysfunction of membrane and sarcotubular ATP-ases systems with consequent intracellular calcium increase, and altered sarcomeric motility impairing the relaxing ability of cardiomyocytes that induces deficient diastolic function [9] (Figure 1). Initially, the loss of contractile elements is compensated by the hypertrophy of surviving cardiomyiocytes, thus masking the alteration of systolic function. On the other hand, cardiac cells have a low content of antioxidant systems and can be easily damaged by oxidative stress. Figure 1 Role of free radicals in the pathogenesis of anthracycline cardiomyopathy.

Free radicals cardiac toxicity can be caused by direct damage of the mitochondrial respiratory chain with consequent decrease in energy production, due to phosphorilative processes impairment, and reduction of cardiomyocytes following the release of pro-apoptotic factors. Both effects lead to altered systolic function [7] (Figure 1). Further harmful actions of free radicals are associated with membrane lipid peroxidation and cytoskeleton protein oxidation. These events cause the dysfunction of membrane and sarcotubular ATP-ases systems with consequent intracellular calcium increase, and altered sarcomeric motility impairing the relaxing ability of cardiomyocytes that induces deficient diastolic function [9] (Figure 1). Initially, the loss of contractile elements is compensated by the hypertrophy of surviving cardiomyiocytes, thus masking the alteration of systolic function. On the other hand, cardiac cells have a low content of antioxidant systems and can be easily damaged by oxidative stress. Figure 1 Role of free radicals in the pathogenesis of anthracycline cardiomyopathy. Furthermore, interferences with nuclear DNA can inhibit protein synthesis and cardiac tissues growth and down-regulate contractile, sarcotubular and cytosolic proteins. Moreover, these interferences can determine the re-expression of genes that are active during the embrio-fetal period when they code the synthesis of both pro-apoptotic factors and enzymatic and functionally immature muscular proteins. Conversely, interferences with mitochondrial DNA mainly affect the mitochondrial respiratory chain function that can be seriously impaired by the inhibition of cardiolipin, a phospholipid which plays a crucial role in the regulation of cardiac energetic processes. Alterations of the subunits of mitochondrial respiratory complexes can also cause the release of cytochrome c, which can determine cardiomyocytes apoptosis by activating caspases and metalloproteinases enzymatic system. [10,11] (Figure 2). All these processes involving both nuclear and mitochondrial DNA may be linked to anthracycline alcoholic metabolites, and their negative effects on cellular energetic metabolism, protein synthesis and myocardial tissues development can explain the different clinical evolution of AIC in adulthood and in childhood [12]. In adults the loss of cardiomyocytes induced by apoptosis, together with the inhibition of compensatory hypertrophy and with the energetic deficit, can cause ventricular dilatation resulting from the thinning of ventricular walls and the reduction of contractile force, leading to the development of dilated cardiomyopathy. In children the dilatation of ventricular cavities can be associated with a restrictive hemodynamic status following reduced cardiac dimensions caused by the slower development of myocardial mass. This reduces ventricular compliance and thus determines restrictive-dilated cardiomyopathy [13,14] (Figure 3). The subsequent evolution of these two types of AIC is characterized by inexorable progressive deterioration of cardiac function leading to severe and refractory heart failure with fatal exitus.

yocardial mass. This reduces ventricular compliance and thus determines restrictive-dilated cardiomyopathy [13,14] (Figure 3). The subsequent evolution of these two types of AIC is characterized by inexorable progressive deterioration of cardiac function leading to severe and refractory heart failure with fatal exitus. Figure 2 Pathogenetic mechanisms of anthracycline cardiomyopathy. Figure 3 Different haemodynamic evolution of anthracycline cardiomyopathy in relation to patient's age.

yocardial mass. This reduces ventricular compliance and thus determines restrictive-dilated cardiomyopathy [13,14] (Figure 3). The subsequent evolution of these two types of AIC is characterized by inexorable progressive deterioration of cardiac function leading to severe and refractory heart failure with fatal exitus. Figure 2 Pathogenetic mechanisms of anthracycline cardiomyopathy. Figure 3 Different haemodynamic evolution of anthracycline cardiomyopathy in relation to patient's age. At this point, the mechanisms responsible for this negative evolution of AIC can overlap others that progressivley exacerbate all dilated chronic cardiomyopathies (regardless of their aetiology) and are strictly influenced by the neuro-hormonal response triggered by the chronic cardiac contractility deficit. This response activates the adrenergic and renin-angiotensin-aldosterone system and the release of catecholamines, angiotensin and aldosterone. Excess of circulating amines down-regulates cardiac adrenergic receptors and reduces the inotropic response to adrenergic signals thus further impairing contractile deficit. Peripheral vasoconstriction and hydro-saline retention are induced respectively by angiotensin and aldosterone, and increase pre and the post-load thus jeopardizing cardiac performance and giving rise to a vicious circle with progressive cardiac dysfunction [15,16] (Figure 4). Moreover, at the genesis of this dysfunction, besides the above-mentioned negative haemodynamic effects, modifications of the ultrastructure of cardiac muscle induced by the same adrenergic amines, aldosterone and angiotensin may be involved. Recent studies on molecular cardiology showed that these substances can be released inside the cardiac muscle after "cardiac mechano-receptors stimulation" due to bio-mechanical stress induced by volume and/or pressure overload secondary to heart failure [17]. This intra-myocardial neuro-hormonal response is far greater than that of the circulatory district and, like catecholamines, angiotensin and aldosterone, includes cardiomyocytes release of endothelin, cytokines and peptides growth factors (Figure 5). These molecules, acting in autocrine and paracrine fashion in the same cardiomyiocytes and in the surrounding tissues, determine profound modifications of ultrastructural cardiac architecture with functional alterations at the level of the finest subcellular mechanisms.

cytokines and peptides growth factors (Figure 5). These molecules, acting in autocrine and paracrine fashion in the same cardiomyiocytes and in the surrounding tissues, determine profound modifications of ultrastructural cardiac architecture with functional alterations at the level of the finest subcellular mechanisms. These ultrastructural changes constitute the basis of the so called "myocardial remodelling" a biological process, peculiar to the natural history of chronic heart failure, characterized by several cellular and molecular events consisting of hypertrophic and apoptotic processes of myocardial cells, mesenchymal fibrotic and inflammatory reactions and of cytoskeleton and cellular matrix alterations making the myocardium more vulnerable [18] (Figure 5). Instead of decreasing cardiac work and oxygen request through the reduction of the afterload, the same compensatory hypertrophy of myocardial fibres induced by biomechanical stress seems to further damage cardiac performance. In remodelling myocardial muscle growth stimuli also activate biochemical signals that promote myocardiocytes apoptosis leading to pathologic hypertrophy with a negative effect on myocardial activity. This event is different from what normally happens in physiological hypertrophy of subjects doing physical activity, in whom biochemical signals that stimulate the myocardiocytes hypertrophy are associated with signals that promote their surviving [19] (Figure 6). An important aspect on myocardial remodelling is, above all, the modification of heart genetic expression that is characterized by the reactivation of fetal genetic program under the stimulation of some of the biochemical mediators released by myocardial fibres (adrenergic amines, endothelin, growth factors etc). Two events characterize these genetic modifications: a) re-expression of genes that were particularly active in fetal heart, such as the gene of the beta-myosin (molecule with low ATP-ase activity), some proto-oncogenes that induce apoptosis, such as C-Jun and C-Fos genes and the genes encoding for the α1 subunit of Na-K ATP-ase that can cause contractile dysfunction and instability of the membrane potential; b) suppression of genes that are active in adult heart, such as those regulating the sarcotubular ATP-ase, β1- adrenergic receptors and the lipid beta oxidation, with negative consequences on cardiac diastolic function and on the energetic metabolism.

e contractile dysfunction and instability of the membrane potential; b) suppression of genes that are active in adult heart, such as those regulating the sarcotubular ATP-ase, β1- adrenergic receptors and the lipid beta oxidation, with negative consequences on cardiac diastolic function and on the energetic metabolism. All these harmful events, connected to the cardiac remodelling, cause precarious heart function, and thus explain the fatal progressive evolution of chronic heart failure associated to AIC [20]. Figure 4 Factors determining progression of cardiac insufficiency in anthracycline cardiomyopathy. Figure 5 Effects of intramyocardial neuro-hormonal response to biomechanic stress. Figure 6 Different to growth stimuli in myocardial remodelling.

All these harmful events, connected to the cardiac remodelling, cause precarious heart function, and thus explain the fatal progressive evolution of chronic heart failure associated to AIC [20]. Figure 4 Factors determining progression of cardiac insufficiency in anthracycline cardiomyopathy. Figure 5 Effects of intramyocardial neuro-hormonal response to biomechanic stress. Figure 6 Different to growth stimuli in myocardial remodelling. Prevention is particularly important in children, who, thanks to modern treatments, can survive leukemia and other tumoral diseases for several decades. The various approaches proposed are not always completely efficacious and include: cumulative dose under 450 mg/m2bs, use of anthracycline analogouses (epirubicin, idarubicin, mitoxantrone), alternative methods of administration (continuous slow infusion instead of rapid bolus, or liposome encapsulated anthracyclines) and, above all, use of antioxidants [3,6]. Although classic molecules such as tocopherol, ascorbic acid and acetylcysteine have displayed encouraging results against acute anthracycline toxicity, they have not demonstrated clear clinical benefits in chronic cardiomyopathty [6]. More recent studies reported that probucol, a lipid-lowering drug, that also exerts an antioxidant effect and promotes the activities of endogenous antioxidants, was effective in preventing anthracycline cardiomyopathty and heart failure in animal experiments, but further clinical trials are required [6]. To date the most promising agent is dexrarozane, an iron-chelator capable of preventing the formation of extremely reactive hydroxyl radicals catalyzed by the anthracycline-iron complex [21]. Clinical trials conducted in children have demonstrated that this drug has an effective cardioprotective action and reduces the cardiac side-effects of anthracyclines for up to 5 years after chemotherapy [22]. Longer follow-up are required to determine the long-term cardioprotective effects of dexrazozane.

omplex [21]. Clinical trials conducted in children have demonstrated that this drug has an effective cardioprotective action and reduces the cardiac side-effects of anthracyclines for up to 5 years after chemotherapy [22]. Longer follow-up are required to determine the long-term cardioprotective effects of dexrazozane. The effectiveness of conventional therapy of chronic heart failure traditionally based on the use of digitalis, vasodilators, diuretics and beta-blockers is debated. Even if these drugs can transitorily improve the hemodynamic status of subjects with AIC, they are not able to prevent cardiac insufficiency progressing toward more severe forms requiring cardiac transplantation [23].

ure traditionally based on the use of digitalis, vasodilators, diuretics and beta-blockers is debated. Even if these drugs can transitorily improve the hemodynamic status of subjects with AIC, they are not able to prevent cardiac insufficiency progressing toward more severe forms requiring cardiac transplantation [23]. However, recent researches seem to offer new perspectives for pathogenetic treatment of AIC aimed at blocking the molecular mechanisms responsible for apoptotic, inflammatory and fibrotic phenomena connected to neuro-hormonal response causing heart remodelling, this being the key pathogenetic factor involved in the progression of chronic heart failure, regardless of its ethiology. Various treatments include the use of direct antagonists of angiotensin (losartan) and endothelin (bosentan), and of natriuretic peptides, physiologic antagonists of renine-angiotensine-aldosterone system. But the most promising seem to be based on the use of anticytokinic substances (monoclonal antibodies, soluble receptors) in particular those targeting tumour necrosis factor (TNF), or on the use of other substances that stimulate cardiomyocytes survival, such as growth factors (GH, IGF-1) and cardiotrophin, or those avoiding their apoptosis, such as caspases and metalloproteinases inhibitors [24-27]. These studies are still fragmentary, and the sometimes conflicting results need to be confirmed by larger clinical trials. Regarding AIC in particular, recent attention has been focused on some substances, such as Kinin B1 receptors (KB1R) antagonists and erythropoietin (Epo), that are able to module the function of AKT system which potentiates the biochemical signals connected to the survival of cardiomyiocytes at subcellular level and inhibits the mechanisms that stimulate apoptosis. Experimental studies in animals have shown that anthracyclines are capable of inducing the over expression of KB1R in cardiomyocytes, and this overexpression inhibiting the AKT pathway determines the appearance of apoptotic and inflammatory phenomena in the cardiac tissues. These negative effects in mice could be prevented by deleting the KB1R gene or by stimulating kinin B2 receptors that have a protective effect on cardiac muscle [28] (Figure 7). Regarding Epo, it has been shown that this molecule can directly stimulate the AKT biochemical system inside myocardial cells, hence promote the release of antiapoptotic, antioxidant and anti-inflammatory factors [29] (Figure 8).

lating kinin B2 receptors that have a protective effect on cardiac muscle [28] (Figure 7). Regarding Epo, it has been shown that this molecule can directly stimulate the AKT biochemical system inside myocardial cells, hence promote the release of antiapoptotic, antioxidant and anti-inflammatory factors [29] (Figure 8). The results of these researches suggest that pharmacological antagonists of KB1R and Epo might be beneficial in AIC. Nevertheless, these potential therapeutic strategies have to be proven in further studies and has to be evaluated whether pharmacological KB1R antagonists and Epo can prevent the development of AIC or might even be curative when administered after the onset of the disease. Figure 7 Potential therapeutic use of B1 and B2 kinin receptors (B1R, B2R) modulators to prevent anthracycline-induced myocardial damage. Figure 8 Possible mechanism by which erythropoietin is cardioprotective. On the basis of these considerations, it is likely that in the near future the better knowledge of the subtle biochemical mechanisms regulating the function and survival of cardiac cells and the emerging perspectives of a "molecular ventricular assistance" connected to the developing gene therapy of chronic heart failure may allow a more rational preventive and therapeutic approach to cardiac insufficiency associated to dilated cardiomyopathies and therefore revolutionize also the prognosis of AIC [30,31]. Competing interests The author declares that they have no competing interests.

On the basis of these considerations, it is likely that in the near future the better knowledge of the subtle biochemical mechanisms regulating the function and survival of cardiac cells and the emerging perspectives of a "molecular ventricular assistance" connected to the developing gene therapy of chronic heart failure may allow a more rational preventive and therapeutic approach to cardiac insufficiency associated to dilated cardiomyopathies and therefore revolutionize also the prognosis of AIC [30,31]. Competing interests The author declares that they have no competing interests. Acknowledgements The author would like to thank G. Vitaliti and N. Bonanno for their technical collaboration.

itis (27.2%). In our series, if we consider only osteomyelitis, the sensitivity is 16.6%. Their results are not confirmed by our study which may be because of our greater number of inclusions. Also we included all the patients cared for non traumatic decreased active motion of a skeletal segment, with or without fever. Another difference with Butbul-Aviel's study is the test we used. We used a quantitative method (PCT sensitive Kryptor) characterised by a high sensitivity (0.06 ng/ml) allowing a good discrimination of values around the threshold of 0.5 ng/ml. By comparison, PCT determined by PCT- Q-test, an immunochromatographic semi-quantitative assay, is not sensitive enough to discriminate values around 0.5 ng/ml as positive or negative values.

Introduction Bone and joint infections may occur at any age but they are more common in children and present a diagnostic challenge in the emergency department. Delays in diagnosis can lead to disabling sequelae. Most children diagnosed early recover completely with proper management [1].

Introduction Bone and joint infections may occur at any age but they are more common in children and present a diagnostic challenge in the emergency department. Delays in diagnosis can lead to disabling sequelae. Most children diagnosed early recover completely with proper management [1]. No specific laboratory test exists for the diagnosis of bone and joint infections, with the exception of isolation of an organism from the bone or synovial fluid which is considered the gold standard, although its sensitivity ranges from 30% to 90% [2,3]. When obtained, up to 40% of blood cultures are positive, helping to identify a pathogen agent. Laboratory markers, such as elevated white blood cell count (WBC) and C-reactiv protein (CRP) levels may be helpful but are not specific [4]. Procalcitonin (PCT) serum level is very low in healthy patients (< 0.1 ng/ml) and increases rapidly in response to bacterial endotoxins [5,6]. PCT plasma concentrations are raised in severe bacterial infections (bacterial meningitis, septic shock, bacteremia and pyelonephritis), but remain fairly low in viral infections and non-specific inflammatory diseases (with a cut-off level of 0.5 ng/ml) [7-14]. In adults also, PCT is an accurate marker for bacterial infection when differentiating bacterial infection from non-infective causes of inflammation or viral infection [15-18]. Martinot, in a prospective study comparing 11 bacterial arthritis with 18 rheumatoid arthritis and 13 crystal induced arthritis, found that serum PCT is a poorly sensitive (55%) but specific (94%) marker of bacterial infection [16]. To our knowledge, there is only one study in children which tested PCT as a diagnostic aid in skeletal infections [18]. In children admitted for fever and limping, Butbul-Aviel found for PCT a sensitivity of 43.5% and a specificity of 100%. They concluded that PCT is an important informative marker in the diagnosis of osteomyelitis but not in septic arthritis. The aim of our study was to evaluate the sensitivity, specificity and predictive values of PCT for identifying bone and joint infection in children admitted in the emergency department for suspected osteomyelitis or septic arthritis.

tant informative marker in the diagnosis of osteomyelitis but not in septic arthritis. The aim of our study was to evaluate the sensitivity, specificity and predictive values of PCT for identifying bone and joint infection in children admitted in the emergency department for suspected osteomyelitis or septic arthritis. Materials and methods This study was a prospective trial in which cases were collected consecutively. It was carried out between November 2004 and November 2005 in the Emergency Department of an academic tertiary care hospital in Paris, France. All the children presented in the pediatric emergency department for non traumatic decreased active motion of a skeletal segment, with or without fever (rectal temperature higher than 38°C), were prospectively studied. Children who had previously received antibiotics and newborns were excluded. All the investigators enrolling patients are emergency physicians, board certified, specialty trained in pediatrics. The infection was suspected on clinical conditions, bacteriological findings, and laboratory markers completed by imaging study. Clinical conditions were defined as pain during passive movements, failure to move an extremity, pain, tenderness, warmth, and erythema of the involved bone, limping or decreased active motion in the involved joint, with or without fever.

ons, bacteriological findings, and laboratory markers completed by imaging study. Clinical conditions were defined as pain during passive movements, failure to move an extremity, pain, tenderness, warmth, and erythema of the involved bone, limping or decreased active motion in the involved joint, with or without fever. Laboratory markers (PCT, WBC, and CRP) were obtained on admission, before initiation of intravenous antibiotic, when the infection was suspected. PCT was measured by an automatic quantitative method (PCT Sensitive Kryptor, BRAHMS France SAS). This assay has an improved functional assay sensitivity of 0.06 ng/ml and results ranges from 0.06 to 50 ng/ml. Values of PCT levels > 0.5 ng/ml were considered as abnormal. The results of the PCT values were obtained 24 hours after the admission, so this did not interfere with the medical decision. Blood cultures were collected only in children with fever. Bone aspiration was performed in children suspected for osteomyelitis with a periosteal abscess on ultrasound. Joint fluid aspirations were collected in children with articular effusion, suspected on clinical and ultrasound findings, when temperature was higher than 38°C. All bacterial cultures were performed before antibiotic treatment. A plain radiography of the affected area was performed in all patients. In all cases of children with suspected osteomyelitis, skeletal scintigraphy with 99m Tc-methylene diphosphonate was obtained. Ultrasound was performed in all children with suspected articular effusion.

Blood cultures were collected only in children with fever. Bone aspiration was performed in children suspected for osteomyelitis with a periosteal abscess on ultrasound. Joint fluid aspirations were collected in children with articular effusion, suspected on clinical and ultrasound findings, when temperature was higher than 38°C. All bacterial cultures were performed before antibiotic treatment. A plain radiography of the affected area was performed in all patients. In all cases of children with suspected osteomyelitis, skeletal scintigraphy with 99m Tc-methylene diphosphonate was obtained. Ultrasound was performed in all children with suspected articular effusion. Patients were assigned to three groups according to the degree of suspected infection: Group 1 Confirmed infection: this group consisted of patients which had one positive bacteriological culture (blood, bone aspiration or joint fluid aspiration). Group 2 Presumed infection: this group consisted of children with positive laboratory findings (neutrophilia > 10000/mm3 and/or CRP > 20 mg/l) and either purulent bone or joint fluid aspirations or positive scintigraphy but negative cultures. Purulent aspirate was defined as cloudy macroscopic liquid and presence of deteriorated polymorphic neutrophils.

oup consisted of children with positive laboratory findings (neutrophilia > 10000/mm3 and/or CRP > 20 mg/l) and either purulent bone or joint fluid aspirations or positive scintigraphy but negative cultures. Purulent aspirate was defined as cloudy macroscopic liquid and presence of deteriorated polymorphic neutrophils. Group 3 Non infected patients: these children had to fulfil three conditions: negative laboratory findings (all blood tests including CRP values and cultures), no X-ray or ultrasound abnormalities. Children with hip effusion, without fever and without biologic inflammatory syndrome, were classified as "transient monoarticular synovitis" and were included in group 3. All children in group 3 recovered within one week without any antibiotic treatment. Recovery was assessed one week later during an orthopedic specialist visit (blinded for PCT results). Results Three hundred sixty children were admitted between November 2004 and November 2005 in the pediatric emergency department for suspected osteomyelitis or septic arthritis. Twenty-one of them were excluded (lost blood samples for PCT or insufficient blood volume) [Figure 1]. Three hundred thirty nine patients, aged 1 month old to 14 years old (median 3 years, mean age 4 years), were included in the analysis (6 patients had 2 blood samples). One hundred and eighteen were girls and 221 boys. Figure 1 trial profile.

Results Three hundred sixty children were admitted between November 2004 and November 2005 in the pediatric emergency department for suspected osteomyelitis or septic arthritis. Twenty-one of them were excluded (lost blood samples for PCT or insufficient blood volume) [Figure 1]. Three hundred thirty nine patients, aged 1 month old to 14 years old (median 3 years, mean age 4 years), were included in the analysis (6 patients had 2 blood samples). One hundred and eighteen were girls and 221 boys. Figure 1 trial profile. Group 1 comprised 8 patients with documented bacterial infection. Mean PCT level was 4.99 (± 2.3) ng/ml. Two of them (25%) had PCT levels > 0.5 ng/ml [Figure 2]. One had a multifocal osteomyelitis (PCT = 37.26 ng/ml) after varicella and one had septic arthritis of the knee (PCT = 1.01 ng/ml). The other patients had septic arthritis in 5 cases and one had osteomyelitis. There were one positive blood culture (Streptococcus), two positive bone aspirations (one Staphylococcus and one Salmonella), and five positive joint fluid aspirations (4 Kingella Kingae and 1 Streptococcus). Figure 2 distribution of PCT values ≥ 0.5 (in ng/ml) in the three groups. Group 2 comprised 40 children which were presumed infected. Mean PCT level was 0.48 (± 0.12) ng/ml. Four (10%) had PCT levels > 0.5 ng/ml (values ranges from 0.62 to 6.94 ng/ml, median 0.75 ng/ml) [Figure 2]. They all had presumed osteomyelitis with positive scintigraphy but negative bone aspiration culture. Fourteen children with presumed osteomyelitis and 22 with presumed septic arthritis had PCT levels < 0.5 ng/ml.

our (10%) had PCT levels > 0.5 ng/ml (values ranges from 0.62 to 6.94 ng/ml, median 0.75 ng/ml) [Figure 2]. They all had presumed osteomyelitis with positive scintigraphy but negative bone aspiration culture. Fourteen children with presumed osteomyelitis and 22 with presumed septic arthritis had PCT levels < 0.5 ng/ml. Group 3 comprised 291 children. Mean PCT level was 0.42 (± 0.18) ng/ml. Nine children had PCT levels > 0.5 ng/ml (values ranges from 0.59 to 18.64 ng/ml, median 0.86 ng/ml) [Figure 2]. Among them, 3 had transient acute synovitis, 3 had lower respiratory tract infection, 1 gingivitis, 1 tonsillitis and 1 was discharged with no diagnosis but recovered without antibiotic treatment. Seventy three children had fever (6 in group 1, 23 in group 2 and 44 in group 3) and 12 of them had PCT values > 0.5 ng/ml (2 in group 1, 4 in group 2 and 6 in group 3).

y. Also, because there is no true gold standard for the diagnosis of skeletal infection except for positive bacteriological cultures, we applied an accepted standard supported by the literature: a combination of clinical condition, bacteriological findings, laboratory markers, completed by an imaging study when needed. Staphylococcus aureus was described in large cohorts as the most common identifiable causative organism accounting for more than 50% of isolated organism in acute hematogenous osteomyelitis and 30% in septic arthritis [4,21]. The Staphylococcus aureus is under-represented in our case series (50% of the osteomyelitis and none of the septic arthritis) probably because of the little number (eight) of positive bacteriological findings. Laboratory measurements should only be performed if they help making decisions about patients and/or are useful to follow the patient's evolution. Despite abundant literature on procalcitonin and its diagnostic accuracy in severe bacterial infections (bacterial meningitis, septic shock, bacteremia and pyelonephritis), in our series we found a low sensitivity (PCT level was positive in 25% of proven infection). Considering the low value of sensitivity, PCT cannot be used as a screening test for identifying skeletal infections in children. Larger studies are needed to evaluate still more the place of PCT measurements in the diagnosis of osteomyelitis and septic arthritis. Competing interests The authors declare that they have no competing interests.

Laboratory measurements should only be performed if they help making decisions about patients and/or are useful to follow the patient's evolution. Despite abundant literature on procalcitonin and its diagnostic accuracy in severe bacterial infections (bacterial meningitis, septic shock, bacteremia and pyelonephritis), in our series we found a low sensitivity (PCT level was positive in 25% of proven infection). Considering the low value of sensitivity, PCT cannot be used as a screening test for identifying skeletal infections in children. Larger studies are needed to evaluate still more the place of PCT measurements in the diagnosis of osteomyelitis and septic arthritis. Competing interests The authors declare that they have no competing interests. Authors' contributions SF and BC drafted the manuscript. CH and BL carried out the immunoassays. SF, BC, GC, SP and CG participated in the sequence alignment. JPJ performed the statistical analysis. SF, CG, GC conceived of the study, and participated in its design and coordination. All the authors read and approved the final manuscript. Acknowledgements We thank Jean-Philippe JAÏS, from the Biostatistical Department, for his help in the statistical analysis.

Introduction Many multifactorial diseases may have a homogeneous clinical presentation but still be heterogeneous from a molecular standpoint and the concept that every patient's disease is somehow unique is gaining ground, in which case a customized, individually-targeted therapy would be desirable [1]. For the development of such a personalized therapeutic approach to be feasible, diseases need to be defined and characterized at molecular level [1]. Recent advances in high-throughput technologies (e.g. mass spectrometry and NMR-based spectroscopy) have enabled the identification of biomarker profiles that characterize disease sub-phenotypes, providing the basis for the development of new, targeted drugs, potentially enabling us to provide "the right therapy for the right patient" [1]. It is on such technologies that the so-called "-omic" sciences rely, e.g. genomics, transcriptomics, proteomics, metabolomics, where these "-omic" terms have been formulated to define approaches capable of identifying groups of biomarkers characteristic of a particular disease within the set of genes, mRNAs, proteins and metabolites of a given organism [2,3].

lled "-omic" sciences rely, e.g. genomics, transcriptomics, proteomics, metabolomics, where these "-omic" terms have been formulated to define approaches capable of identifying groups of biomarkers characteristic of a particular disease within the set of genes, mRNAs, proteins and metabolites of a given organism [2,3]. Metabolomics is defined as the analysis and interpretation of the global metabolic data expressing the multiparametric metabolic response of living systems to genetic modification, pathophysiological stimuli and environmental influences [4,5]. Metabolomics takes a non-selective approach and, by means of a comprehensive overview of the metabolites, enables the "metabolic fingerprint" of a sample to be obtained [6]. Metabolomics is the latest of the -omic sciences and it is considered the one that comes closest to expressing phenotype, giving us the chance to look at genotype-phenotype, as well as genotype-envirotype relationships. In fact, although the metabolic profile can be seen as the ultimate expression of the information contained in the genetic code, it is also influenced by several factors unrelated to the genome, such as interactions with commensal microorganisms, nutritional factors, environmental agents, and exposure to drugs or toxic substances [3,6].

the metabolic profile can be seen as the ultimate expression of the information contained in the genetic code, it is also influenced by several factors unrelated to the genome, such as interactions with commensal microorganisms, nutritional factors, environmental agents, and exposure to drugs or toxic substances [3,6]. From a clinical standpoint, the metabolomic analysis has two major potential applications. The first concerns the early diagnosis and characterization of disease phenotypes. Metabolomic analysis can detect a pattern of metabolites that discriminate between groups of subjects, enabling the metabolic characterization of a disease, or of a disease phenotype. This is an exploratory process, since unexpected or even unknown metabolites may turn out to be important in this discrimination, paving the way to the formulation of new pathophysiological hypotheses [2,4]. The second potential clinical application concerns the identification of individual metabolomic characteristics able to predict drug effectiveness and/or toxicity - an approach called pharmacometabolomics, which appears to be a promising branch of metabolomics for screening human populations, implying the concrete possibility of a genuinely customized approach to treatment [1]. The concept of pharmacometabolomics has been first introduced by a seminal study conducted on mice demonstrating that the hepatotoxic effects of the analgesic paracetamol can be predicted on the basis of the pre-treatment metabolomic urinary profiles [7].

possibility of a genuinely customized approach to treatment [1]. The concept of pharmacometabolomics has been first introduced by a seminal study conducted on mice demonstrating that the hepatotoxic effects of the analgesic paracetamol can be predicted on the basis of the pre-treatment metabolomic urinary profiles [7]. Methods used in metabolomic analysis The methods used in metabolomic analysis are generally based on mass spectrometry or NMR-based spectroscopy, since these techniques can handle complex biological samples with a high sensitivity, selectivity and throughput [8]. Mass spectrometry, usually combined with chromatographic separation methods, enables the molecules in a sample to be separated on the basis of their mass-to-charge ratio and their representation in a spectrum [9,10]. 1H-NMR spectroscopy enables the detection of almost all proton-containing metabolites in a sample, different molecules producing different signals in the NMR spectrum [11]. Some of the advantages of this technique are that it is non-selective and fast, and usually demands no sample preparation [11]. It is also non-destructive and can be applied to the analysis of tissue samples that will subsequently remain available for further diagnostic analyses [11]. A recent development is represented by the combination of NMR and MS data, which may improve the identification of unknown metabolites [12].

1H-NMR spectroscopy enables the detection of almost all proton-containing metabolites in a sample, different molecules producing different signals in the NMR spectrum [11]. Some of the advantages of this technique are that it is non-selective and fast, and usually demands no sample preparation [11]. It is also non-destructive and can be applied to the analysis of tissue samples that will subsequently remain available for further diagnostic analyses [11]. A recent development is represented by the combination of NMR and MS data, which may improve the identification of unknown metabolites [12]. Both NMR and MS are powerful spectroscopic methods for generating multivariate datasets: NMR and MS spectra are highly complex and the biological information they contain can only be extracted by applying bioinformatic tools, such as pattern recognition methods. These are computer-based procedures that can be classified as unsupervised or supervised [13]. The unsupervised methods reduce the complexity of the data contained in the spectra and represent them by means of plots that the human eye can interpret. This approach helps to identify any intrinsic sample clustering, to see whether different groups of individuals (e.g. healthy vs ill) can be discriminated by the characteristics of their spectra [13]. The supervised methods use a training set of samples (of known classification) to create a mathematical model that is then used to test an independent dataset: unlike the unsupervised methods, they enable us to predict which group a new sample belongs to on the strength of the characteristics of its spectra [13].

[13]. The supervised methods use a training set of samples (of known classification) to create a mathematical model that is then used to test an independent dataset: unlike the unsupervised methods, they enable us to predict which group a new sample belongs to on the strength of the characteristics of its spectra [13]. Once a metabolic pattern typical of a given condition has been characterized, the analysis may go on to identify single biomarkers relevant to sample clustering. Fundamental support for molecular identification comes from various on-line databases, the most comprehensive of which is the Human Metabolomic Database [14]. Pediatric clinical studies Metabolomic analysis can be applied to the study of biological fluids collected in non-invasive or minimally-invasive ways (e.g. urine, exhaled breath condensate, blood). Being highly informative and suitable for use on non-invasively collected samples too, the metabolomic approach seems particularly promising in the field of pediatric medicine [15]. A number of recently-published studies have applied the metabolomic approach to the pediatric population. Some of these studies evaluated how physiological variables, such as age or diet, can affect children's metabolomic profiles.

Pediatric clinical studies Metabolomic analysis can be applied to the study of biological fluids collected in non-invasive or minimally-invasive ways (e.g. urine, exhaled breath condensate, blood). Being highly informative and suitable for use on non-invasively collected samples too, the metabolomic approach seems particularly promising in the field of pediatric medicine [15]. A number of recently-published studies have applied the metabolomic approach to the pediatric population. Some of these studies evaluated how physiological variables, such as age or diet, can affect children's metabolomic profiles. Using an NMR-based metabolomic analysis, Gu et al demonstrated the effect of age on the urinary metabolite profile in pre-adolescent children [16]. Likewise, a recent Italian study evaluated urine samples in genetically homogeneous healthy populations, demonstrating that the excretion of most amino acids is age-dependent [17]. Understanding the age-related characteristics of the metabolomic profile can facilitate the interpretation of its pathological modifications [16]. To study the effects of diet, Bertram et al [18] compared the metabolomic urinary profiles of two groups of children on a diet rich in milk versus meat proteins, demonstrating that the samples could be successfully discriminated according to the children's diet, with a significantly greater urinary excretion of creatine in the children on a diet rich in meat. Other pediatric studies have focused on the metabolomic profiles of different biofluids in the presence of pathological processes.

To study the effects of diet, Bertram et al [18] compared the metabolomic urinary profiles of two groups of children on a diet rich in milk versus meat proteins, demonstrating that the samples could be successfully discriminated according to the children's diet, with a significantly greater urinary excretion of creatine in the children on a diet rich in meat. Other pediatric studies have focused on the metabolomic profiles of different biofluids in the presence of pathological processes. In one publication, metabolomics was used to study disorders due to inborn errors of metabolism [19]. Mass spectrometry is commonly used in the diagnosis of inborn errors of metabolism to seek specific metabolites (targeted analysis), but this study was the first to use untargeted metabolomic analyses on plasma samples. Investigating disorders of proprionate metabolism (methylmalonic acidemia [MMA] and propionic acidemia [PA]), the authors found that the most important metabolite for discriminating between the healthy and the ill was propionyl carnitine, which is indeed the target compound for screening newborn for MMA and PA by tandem-MS. This result validates the role of untargeted metabolomic analysis in identifying biomarkers of disease. In addition, untargeted metabolomic analysis showed that many other compounds are important in differentiating both between healthy and ill, and between cases of MMA and PA [19].

newborn for MMA and PA by tandem-MS. This result validates the role of untargeted metabolomic analysis in identifying biomarkers of disease. In addition, untargeted metabolomic analysis showed that many other compounds are important in differentiating both between healthy and ill, and between cases of MMA and PA [19]. A Japanese group described a characteristic metabolomic profile in the cerebrospinal fluid of children with influenza-associated encephalopathy, suggesting that it might be possible to identify specific biomarkers useful for the early diagnosis of this disease [20]. In a study by our group [21], metabolomics was applied to analyzing exhaled breath condensate (breathomics): this biofluid is collected non-invasively by cooling the exhaled air and its composition is believed to reflect that of the airway lining fluid [21]. NMR-based metabolomic analyses of exhaled breath condensate could clearly discriminate between asthmatic and healthy children, with 95% success rate in their classification. Many authors believe that asthma should no longer be considered a single disease and that efforts should be made to identify the different biochemical and inflammatory profiles behind asthma symptoms in order to treat them with specifically-targeted therapies [22]. The metabolomic approach may have a role in characterizing these asthma sub-phenotypes.

should no longer be considered a single disease and that efforts should be made to identify the different biochemical and inflammatory profiles behind asthma symptoms in order to treat them with specifically-targeted therapies [22]. The metabolomic approach may have a role in characterizing these asthma sub-phenotypes. Still in the field of pediatric pulmonology, it has recently been demonstrated that metabolomic analysis of bronchoalveolar fluid can distinguish between different degrees of inflammation in children with cystic fibrosis, with the promise of identifying new biomarkers of inflammation [23]. Metabolomic analysis has also proved useful in identifying early urinary biomarkers of acute kidney injury after cardiopulmonary bypass surgery [24]: using ultra-performance liquid chromatography (UPLC)/MS based metabolomic analyses on urine samples collected 4 and again 12 hours after surgery, the authors were able to identify the children that would develop acute kidney injury over the next 3 days. From a clinical standpoint, finding early metabolic biomarkers may improve our understanding of the pathophysiological mechanisms involved in acute kidney injury and enable a timely diagnosis of this disease after pediatric cardiac surgery.

ntify the children that would develop acute kidney injury over the next 3 days. From a clinical standpoint, finding early metabolic biomarkers may improve our understanding of the pathophysiological mechanisms involved in acute kidney injury and enable a timely diagnosis of this disease after pediatric cardiac surgery. Finally, an interesting longitudinal study applying the metabolomic analysis to serum samples recently demonstrated that a metabolic dysregulation precedes the onset of the autoimmunity associated with type-1 diabetes and the following progression to clinical diabetes [25]. This study has potential therapeutic implications too: based on their findings, the authors suggest that an immunomodulatory therapy might be more useful than immunosuppression in the prevention of the disease [25]. Conclusion Metabolomics is a novel approach that promises to enable the detection of states of disease, to stratify patients based on biochemical profiles and to monitor disease progression. Metabolomic analysis may also be able to orient the choice of therapy, identify responders and predict toxicity (pharmacometabolomics), paving the way to a customized therapy. There are many potential applications for metabolomics in pediatric medicine, inasmuch as it is a highly informative technique that can also be used on non-invasively collected samples.

Introduction Allergen specific immunotherapy (SIT) is the practice of administering gradually increasing doses of the specific causative allergen to reduce the clinical reactivity of allergic subjects, and is the only treatment targeting the causes of hypersensitivity and not only the symptoms, as done by drugs [1]. The traditional, subcutaneous immunotherapy (SCIT) was burdened by the problem of systemic reactions which may be sometimes severe and - though very rarely - even fatal [2]. This was the background to develop non injections routes for SIT and particularly sublingual immunotherapy (SLIT), that emerged as a real treatment option for respiratory allergy [3]. A number of studies was conducted to evaluate efficacy and safety of SLIT, the first meta-analysis - including 22 placebo-controlled trials - concluded for positive results in both issues, but the number of studies on children was too low to draw definite conclusions [4]. Since then, many other studies became available and make possible to analyze SLIT in children in its well defined aspects as well as in sides still requiring more solid data.

olled trials - concluded for positive results in both issues, but the number of studies on children was too low to draw definite conclusions [4]. Since then, many other studies became available and make possible to analyze SLIT in children in its well defined aspects as well as in sides still requiring more solid data. Efficacy of Slit in Children The clinically efficacy of SLIT, as of SIT in general, is evaluated by the decrease in symptom scores of rhinitis and asthma and in consumption of symptomatic drugs. Many placebo-controlled studies are conducted on small patient populations and cannot achieve a reliable statistical significance, but their combined evaluation by the tool of meta-analysis is considered an adequate method to obtain more robust data [5]. The results obtained by the Cochrane Collaboration method [6] are expressed as standardized mean difference (SMD) and allow to compare the effect of SLIT on actively and placebo treated patients. Also systematic reviews, that is, literature analysis without using the Cochrane method, are available.

ore robust data [5]. The results obtained by the Cochrane Collaboration method [6] are expressed as standardized mean difference (SMD) and allow to compare the effect of SLIT on actively and placebo treated patients. Also systematic reviews, that is, literature analysis without using the Cochrane method, are available. The progressive increase in number of SLIT studies addressing the pediatric population made possible to perform specific meta-analyses and systematic reviews. The first systematic review included the studies up to June 2003, which were evaluated qualitatively, and concluded for low to moderate efficacy of SLIT only in children with house dust mite induced mild to moderate asthma [7]. A meta-analysis by Olaguibel et al including 7 randomized controlled studies conducted on children aged up to 14 years was substantially in agreement, since it found that SLIT was significantly effective on asthma symptoms (SMD -1.42, p = 0.01) and on drug consumption (SMD -1.01, p = 0.06), while the improvement did not reach the significance for nasal and conjunctival symptoms [8].

led studies conducted on children aged up to 14 years was substantially in agreement, since it found that SLIT was significantly effective on asthma symptoms (SMD -1.42, p = 0.01) and on drug consumption (SMD -1.01, p = 0.06), while the improvement did not reach the significance for nasal and conjunctival symptoms [8]. A further meta-analysis on SLIT in children was published in 2006 [9]: in this case the evaluation concerned the efficacy on allergic rhinitis including 10 randomized controlled studies, with an overall number of 484 patients (245 actively and 239 placebo treated). A significant reduction of both symptoms (SMD - 0.56, p = 0.02) and medication (- 0.76, p = 0.03) was observed. A notable aspect was provided from the sub-analysis addressing the length of treatment and the kind of allergen administered, which demonstrated a higher efficacy for durations longer than 18 months and for pollen allergens compared to house dust mites. The same group performed a meta-analysis on the efficacy of SLIT in allergic asthma, analyzing 9 studies on pediatric patients which included a total number of 441 patients, 232 actively treated and 209 placebo-treated [10]. A significant reduction was found in both symptoms scores (SMD - 1.14, p = 0.02) and drug use (SMD -1.63, p = 0.007).

ed a meta-analysis on the efficacy of SLIT in allergic asthma, analyzing 9 studies on pediatric patients which included a total number of 441 patients, 232 actively treated and 209 placebo-treated [10]. A significant reduction was found in both symptoms scores (SMD - 1.14, p = 0.02) and drug use (SMD -1.63, p = 0.007). A systematic review in the same year by Roder et al evaluating any form of immunotherapy in children concluded for no evidence of effectiveness in the subgroup of 11 studies on SLIT, but the review was based on analysis of each single study and not on pooling all data together [11]. The authors justified such approach with the relevant heterogeneity of the available studies.

ating any form of immunotherapy in children concluded for no evidence of effectiveness in the subgroup of 11 studies on SLIT, but the review was based on analysis of each single study and not on pooling all data together [11]. The authors justified such approach with the relevant heterogeneity of the available studies. Actually, heterogeneity, which is mainly due to different scoring systems in the various studies. is a limit of meta-analysis. However, a data source alternative to meta-analysis are studies conducted on large numbers of patients that provide adequate statistical power. The recent preparations for SLIT in orosoluble tablet of grass pollen extract were evaluated on large populations, including 253 children treated by a one grass (Phleum pratense) extract [12], and 278 children treated with a 5-grass pollen extract [13]. These studies showed a highly significant improvement in symptom and rescue medications scores in actively treated compared with placebo treated patients during the grass pollen season. Thus, the criticism on the efficacy of SLIT in children does not seem to have ground. An updated and balanced review on this issue by Larenas-Linnemann was recently published [14]. The author after accurate analysis of all the available studies concluded that "evidence of effect is confirmed for SLIT in children with allergic rhinitis or asthma caused by pollen exposure", while there is yet room for investigations on long-term effects and preventive action of SLIT, as well as on optimal dosing for dust mites. Indeed, the dosing is a pivotal factor, and the dose-dependence of efficacy in children treated with pollen extract was clearly demonstrated both clinically [15] and immunologically [16]. Dose-response studies in mite allergic children are warranted. Another important observation concerns the capacity of SLIT to prevent the development of asthma in children with seasonal rhinitis treated with grass pollen extract compared with subjects treated with standard symptomatic drugs [17].

immunologically [16]. Dose-response studies in mite allergic children are warranted. Another important observation concerns the capacity of SLIT to prevent the development of asthma in children with seasonal rhinitis treated with grass pollen extract compared with subjects treated with standard symptomatic drugs [17]. Safety of Slit in Children All systematic revisions and meta-analysis found that the most common adverse events to SLIT, regardless the age, are local reactions in the oropharynx - with itching, tingling and swelling in the mouth - followed by local gastrointestinal reactions - with nausea, vomiting or diarrhea - and that systemic reactions such as asthma, rhinitis, or urticaria, are quite rare [4,18-20]. An increased risk of systemic reactions is apparent in subjects undergoing SLIT because of previous systemic reactions to SCIT [21,22]. In particular, one of the cases of anaphylaxis concerned a pediatric patient, who had had urticaria to previous SCIT treatment and developed an anaphylactic reaction after the very first dose of a grass pollen tablet formulation with no updosing phase [21].

g SLIT because of previous systemic reactions to SCIT [21,22]. In particular, one of the cases of anaphylaxis concerned a pediatric patient, who had had urticaria to previous SCIT treatment and developed an anaphylactic reaction after the very first dose of a grass pollen tablet formulation with no updosing phase [21]. Some studies addressed specific safety issues in children. SLIT was well tolerated using ultra-rush schedules - that reach the maintenance dose in a few hours [23] - and also starting the treatment during the pollen season [24]. Two studies demonstrated that SLIT is safe also in children younger than 5 years (that is the age limit indicated for SCIT), as assessed by comparable rate and kind of adverse effects in patients aged less or more than 5 years [25,26]. A further observation regarded children treated with one or multiple allergen extracts, who showed comparable rates of side effects, more than 90% being mild and self-resolving [27]. Concerning SLIT with house dust mites extracts, a recent study (including both adults and children) reported a comparable safety and tolerability in patients treated continuously or intermittently, i.e. 2-month treatment alternate to-2 month suspension [28]. Issues Influencing the Feasibility of Slit As stated above, SLIT has clear evidence of efficacy and safety. Still, there are aspects influencing its clinical results, such as the compliance, or its prescription, such as the cost-effectiveness.

Concerning SLIT with house dust mites extracts, a recent study (including both adults and children) reported a comparable safety and tolerability in patients treated continuously or intermittently, i.e. 2-month treatment alternate to-2 month suspension [28]. Issues Influencing the Feasibility of Slit As stated above, SLIT has clear evidence of efficacy and safety. Still, there are aspects influencing its clinical results, such as the compliance, or its prescription, such as the cost-effectiveness. Compliance to SLIT According to established definitions, compliance is "The extent to which a patient's behavior matches the prescriber's advice" and adherence is "The extent to which the patient's behavior matches agreed recommendations from the prescriber" [29], and both of them are essential for the clinical outcome of a medical treatment. A number of studies conducted on SCIT showed that the major cause of noncompliance was the inconvenience, related to injections and particularly to their frequency, and the cost of the treatment [30]. SLIT has different compliance issues than SCIT, because it is administered at home by patients themselves and thus it is not affected by most causes reported for non-compliance to allergen injections, having instead compliance problems similar to drug treatment. Some studies not specifically designed for compliance (for instance safety and tolerability analyses) reported that treatment withdrawal is frequently caused by repeated local reactions in the mouth or at gastrointestinal level [4,18]. Concerning specific compliance and adherence studies, the available data indicate quite satisfactory results.

ally designed for compliance (for instance safety and tolerability analyses) reported that treatment withdrawal is frequently caused by repeated local reactions in the mouth or at gastrointestinal level [4,18]. Concerning specific compliance and adherence studies, the available data indicate quite satisfactory results. In a study on children treated with SLIT by an allergen extract in monodoses, parents were interviewed by unscheduled phone calls at the third and sixth month of SLIT and asked to count at once the remaining doses; a compliance rate higher than 75% was found in 85% of children at the third month and in 84% of children at the sixth month; the major cause of withdrawal (5.6% of cases) was the cost of treatment, while side effects accounted for 1.4% of stopping [31]. In a study comparing compliance to SLIT, SCIT and local nasal immunotherapy in children, data on SLIT concerned 806 patients, 173 of whom (21.4%) were noncompliant, with a highly significant difference (p < 0.0001) for a better compliance in hospital setting (90.5%) compared to private office setting (61.2%); the most common reason of withdrawal was the cost of treatment, reported globally in 36.4% of cases, followed by inconvenience, feeling of inefficacy, and side effects [32].

with a highly significant difference (p < 0.0001) for a better compliance in hospital setting (90.5%) compared to private office setting (61.2%); the most common reason of withdrawal was the cost of treatment, reported globally in 36.4% of cases, followed by inconvenience, feeling of inefficacy, and side effects [32]. Cost-effectiveness of SLIT Many studies are available, recently reviewed, showing that SLIT provides economic advantage compared with drug treatment by bringing a better clinical outcome at a reduced cost or an extra benefit at a very acceptable extra cost [33]. Concerning children, fhe first published study dealt with the evaluation of cost effectiveness of SLIT, with the high dose suggested in the ARIA document [34], in subjects with allergic rhinitis and asthma. From records of pediatric patients seen for respiratory allergy, who had 1-year data prior to receive SLIT and 3-year data on high dose SLIT, outcome measures (the number of exacerbations, visits, absence from nursery or school) were analyzed. Moreover, direct costs (Euro spent on drugs, specialists visits, and SLIT) and indirect costs (costs resulting from children school and parental work loss) were considered. A second analysis compared a sub-group of children with allergic asthma, using a control group for costs, based on records of patients not treated with SLIT, extracted from a network-database of pediatricians. An overall number of 135 children were analyzed, 46 with perennial and 89 with seasonal allergy, with comparable gender and age distribution. A substantial reduction was found in all outcome measures during SLIT compared with the previous period. The average annual cost/patient was Euro 2672 before SLIT initiation and Euro 629/year during SLIT. Similar results were found for allergen subgroups. The asthma analysis involved 41 children with SLIT and 35 controls, and also showed a substantial reduction in outcomes, though the direct cost per patient over the 4 years follow-up was € 1182 for SLIT-treated children and € 1100 for controls [35]. A study conducted in France estimated that in children treated with SLIT for house dust mite or pollen allergies, the incremental costs per asthma case avoided over a 7-year period, compared with standard symptomatic treatment, were 3938 Euro for dust mite and 824 Euro for pollen allergy; of note, there was an economic advantage of SLIT also versus SCIT for pollen allergy, since the incremental cost for the latter was 1708 Euro [36].

incremental costs per asthma case avoided over a 7-year period, compared with standard symptomatic treatment, were 3938 Euro for dust mite and 824 Euro for pollen allergy; of note, there was an economic advantage of SLIT also versus SCIT for pollen allergy, since the incremental cost for the latter was 1708 Euro [36]. Conclusion SLIT was successfully introduced in Europe mainly on safety grounds and in some countries, including Italy, is currently more frequently employed than SCIT. The analysis of the abundant literature supports the use of SLIT in children with rhinitis and asthma caused by sensitization to seasonal allergens, while further studies are needed to demonstrate a full effectiveness in sensitization to perennial allergens. Favourable data obtained from studies on compliance and cost-effectiveness make SLIT a feasible treatment for treatment with respiratory allergy. Competing interests The authors declare that they have no competing interests. Authors' contributions All author participated equally in reviewing the literaure, discussing data and drafting the manuscript. All authors read and approved the final manuscript.

Background Postnatal depression (PD) is the most common disorder following childbirth and a social problem for public welfare: ten to fifteen women out of one hundred suffer from this disorder [1]. PD is a severe condition that has been described as "a thief who steals maternity"; up to 50% of the cases are diagnosed, and approximately 49% of women who seek help feel desperately depressed [2]. If untreated, a large number of these mothers continue to be depressed until the end of the first and the second postnatal years.

severe condition that has been described as "a thief who steals maternity"; up to 50% of the cases are diagnosed, and approximately 49% of women who seek help feel desperately depressed [2]. If untreated, a large number of these mothers continue to be depressed until the end of the first and the second postnatal years. Women who suffer from PD are exposed to an increased risk of future depression, relapses, thoughts of abusing their children, and face difficulties in the child-mother relationship [3]. Maternal Depression (MD) may have a strong negative impact on the social, cognitive and behavioral development of children, including an increased rate of behavioral problems at school [4,5]. The obstetrician should be the first to identify those mothers who risk developing depression, but this can be quite difficult as symptoms often appear after the routine 4 to 6 week postnatal examination. Moreover, mothers with PD often do not recognize the symptoms of depression. This is a result of the difficulty in identifying the following signs as symptoms of PD: weight loss, irritability, crying fits and fatigue (often considered as the physiological adaptation to life with a newborn child). This is the reason why many women do not receive an immediate diagnosis or an appropriate treatment program [6]. Pediatricians may be the only medical workers that are routinely met by mothers during the first twelve months of the baby's life [7]. A self- report rating scale routinely administered in pediatrics could be a useful tool to recognize the risk of PD. The Edinburgh Postnatal Depression Scale is the most widely used screening scale for PD [8]. It has been validated in Holland, Australia, Portugal, Sweden, Italy, Spain, United Arab Emirates, France and India [6,8-15].

rating scale routinely administered in pediatrics could be a useful tool to recognize the risk of PD. The Edinburgh Postnatal Depression Scale is the most widely used screening scale for PD [8]. It has been validated in Holland, Australia, Portugal, Sweden, Italy, Spain, United Arab Emirates, France and India [6,8-15]. The EPDS has already been administered in a pediatrics setting at the University Rochester Medical Centre (NY), although the impact of this screening instrument on the visit time was not assessed. However, the length of the visit is an important factor to take into consideration when working in a busy pediatric clinic. As a matter of fact, if we extend visit times, we risk reducing the total number of daily visits.

Centre (NY), although the impact of this screening instrument on the visit time was not assessed. However, the length of the visit is an important factor to take into consideration when working in a busy pediatric clinic. As a matter of fact, if we extend visit times, we risk reducing the total number of daily visits. In this research project we focused on PD risk in mothers who have recently given birth. Many studies highlighted that the most significant risk factors for PD are: young maternal age, absence of a social support, immigration, and lack of a supporting spouse [16]. In addition, we also took into account what the outcome would be if new fathers were evaluated as well. To date, only a few studies have investigated PD risk in fathers, and no study has been conducted in Italy. Men who have manual or working class occupations and low social integration are more likely to become depressed [9]. In this study we will refer to parents at both low and high risk of suffering from depression on the basis of the EPDS score. In fact, a high EPDS score does not mean postnatal depression but only a high risk of suffering from PD. A psychiatrist was involved to confirm the diagnosis of PD. The aims of our study were: 1) to check the feasibility of assessing the risk of PD in parents using the EPDS; 2) to provide correlations between PD risk and socio-demographic information, both in mothers and in fathers; 3) to check correlations between high EPDS scoring fathers in couples with EPDS positive mothers and high EPDS scoring fathers in couples with EPDS negative mothers.

ng the risk of PD in parents using the EPDS; 2) to provide correlations between PD risk and socio-demographic information, both in mothers and in fathers; 3) to check correlations between high EPDS scoring fathers in couples with EPDS positive mothers and high EPDS scoring fathers in couples with EPDS negative mothers. Methods Setting The study was conducted at the Pediatric Clinic of the Policlinico A. Gemelli, Catholic University Hospital, Rome. This clinic admits 4000 patients a year, and it is a teaching site for pediatric residents and medical students. Participants Our team included: a senior pediatrician, two pediatric residents, two psychologists, and two psychiatrists. The EPDS was proposed to all parents, regardless of age and nationality, at the first postnatal check-up within the first year of baby's birth. Unmarried women were also included. Postnatal check-up examinations were carried out by the senior pediatrician with the aid of pediatric residents. Research Tools EPDS is a paper-and-pencil self-reporting questionnaire composed of 10 questions and a 0-3 point scale. The cut-off score is 9 for women and 7 for men [11,17,18].

Participants Our team included: a senior pediatrician, two pediatric residents, two psychologists, and two psychiatrists. The EPDS was proposed to all parents, regardless of age and nationality, at the first postnatal check-up within the first year of baby's birth. Unmarried women were also included. Postnatal check-up examinations were carried out by the senior pediatrician with the aid of pediatric residents. Research Tools EPDS is a paper-and-pencil self-reporting questionnaire composed of 10 questions and a 0-3 point scale. The cut-off score is 9 for women and 7 for men [11,17,18]. Procedure EPDS was proposed by a pediatrician, before clinical examination. The informed consent was obtained by explaining the meaning of the EPDS and we asked participants to complete questionnaires, without any help, and to answer according to their feelings during the previous seven days. In the case of high scores, the EPDS was repeated after five weeks, especially when we visited babies in the first 15 days of life, as, after delivery, many women experience 'baby blues'. Baby blues is considered a normal stage of early motherhood, usually disappearing some days after delivery. The EPDS was translated and validated into several languages (Italian, French, Spanish, English, Arabic, and Punjabi) for parents who did not understand Italian. We translated the EPDS into Singhalese and used it, although this version was not officially validated.

Procedure EPDS was proposed by a pediatrician, before clinical examination. The informed consent was obtained by explaining the meaning of the EPDS and we asked participants to complete questionnaires, without any help, and to answer according to their feelings during the previous seven days. In the case of high scores, the EPDS was repeated after five weeks, especially when we visited babies in the first 15 days of life, as, after delivery, many women experience 'baby blues'. Baby blues is considered a normal stage of early motherhood, usually disappearing some days after delivery. The EPDS was translated and validated into several languages (Italian, French, Spanish, English, Arabic, and Punjabi) for parents who did not understand Italian. We translated the EPDS into Singhalese and used it, although this version was not officially validated. Whenever our test results were rated as 'high', we told mothers or fathers or sometimes both parents, to undergo a psychologist-psychiatrist examination. Without an appropriate psychiatric evaluation, a high EPDS score does not mean PD. Sometimes, in the self-reporting test, people report anxiety, mood instability, depressed mood, and other transient emotional disturbances which disappear within a few hours or days. A PD diagnosis requires a woman to be experiencing dysphoric mood and several other symptoms such as appetite, sleep or psychomotor disturbances, excessive feeling of guilt, fatigue and suicidal thoughts for a minimum of two weeks.

, and other transient emotional disturbances which disappear within a few hours or days. A PD diagnosis requires a woman to be experiencing dysphoric mood and several other symptoms such as appetite, sleep or psychomotor disturbances, excessive feeling of guilt, fatigue and suicidal thoughts for a minimum of two weeks. Study Population This cross-sectional study started in January 2005 and ended in November of the same year. 1130 infants were examined at the first postnatal check-up (median 17 days, range 15-20 days). We proposed the EPDS to 1628 parents; 1621 subjects, 1122 mothers of the 1127 (99.6%) and 499 fathers of the 501 (99.6%) completed the EPDS and were included in this study. Five mothers with previous depression symptoms and two fathers who did not complete the EPDS were excluded. The male group was smaller than the female one, due to the fact that fathers were investigated only from August onwards and mothers often attended the clinic alone. We excluded mothers with a history of depression since we intended to check only symptoms of PD, which is a perinatal pathology, not to be confused with depression. Statistical Analysis To evaluate the feasibility of assessing postnatal depression symptoms, we calculated by how many minutes (average and range) the completion of the questionnaire extended the visit time, and the percentage of compilers.

We excluded mothers with a history of depression since we intended to check only symptoms of PD, which is a perinatal pathology, not to be confused with depression. Statistical Analysis To evaluate the feasibility of assessing postnatal depression symptoms, we calculated by how many minutes (average and range) the completion of the questionnaire extended the visit time, and the percentage of compilers. The prevalence of the risk of PD was calculated as the percentage of mothers who scored ≥ 10 and as the percentage of fathers who scored ≥ 8. The characteristics of subjects at high PD risk and at low PD risk were compared using the Chi-squared test for each following characteristic: maternal and paternal age, marital status, employment, educational level, nationality, nursing, number of pregnancies, gestational age, delivery, mother's and father's pathologies, baby's weight at birth, gender, infant hospitalization and pathologies, and the season of the interview. Crude odds ratios (OR) were also calculated for all variables and adjusted OR were also calculated for variables where the univariate analysis showed a statistically significant association (p-value for Chi squared test < 0.05). Adjusted OR were calculated with the construction of a multivariate logistic regression model using the backward elimination method. The fit of the model was assessed using the Hosmer-Lemenshow test.