Neonatal Virus Infection Research Articles

Expert recommendations for the prevention of common respiratory viral infections in neonates

Neonates, particularly preterm infants, are a susceptible population to respiratory viral infections. Currently, aside from influenza, there are no antiviral medications specifically approved for the treatment of respiratory viral infections in neonates; therefore, prevention of these viral infections is particularly crucial for neonates. The Neonatal HealthCare Committee of Chinese Maternal and Child Health Association, based on domestic and international clinical evidence and combined with clinical practice experience, and after thorough discussion by relevant experts, has developed eight expert recommendations. These include preventive strategies against influenza virus, respiratory syncytial virus, and severe acute respiratory syndrome coronavirus 2 infections, intended for reference in clinical practice.

PD-1 signaling in neonates restrains CD8+ T cell function and protects against respiratory viral immunopathology

Respiratory viral infections, including human metapneumovirus (HMPV), remain a leading cause of morbidity and mortality in neonates and infants. However, the mechanisms behind the increased sensitivity to those respiratory viral infections in neonates are poorly understood. Neonates, unlike adults, have several anti-inflammatory mechanisms in the lung, including elevated baseline expression of programmed death ligand 1 (PD-L1), a ligand for the inhibitory receptor programmed cell death protein 1 (PD-1). We thus hypothesized that neonates would rely on PD-1:PD-L1 signaling to restrain antiviral CD8 responses. To test this, we developed a neonatal primary HMPV infection model using wild-type C57BL/6 (B6) and Pdcd1-/- (lacking PD-1) mice. HMPV-infected neonatal mice had increased PD-L1/PD-L2 co-expression on innate immune cells but a similar number of antigen-specific CD8+ T cells and upregulation of PD-1 to that of adult B6 mice. Neonatal CD8+ T cells had reduced interferon-gamma (IFN-γ), granzyme B, and interleukin-2 production compared with B6 adults. Pdcd1-/- neonatal CD8+ T cells had markedly increased production of IFN-γ and granzyme B compared with B6 neonates. Pdcd1-/- neonates had increased acute pathology with HMPV or influenza. Pdcd1-/- neonates infected with HMPV had long-term changes in pulmonary physiology with evidence of immunopathology and a persistent CD8+ T-cell response with increased granzyme B production. Using single-cell ribonucleic acid sequencing from a child lacking PD-1 signaling, a similar activated CD8+ T-cell signature with increased granzyme B expression was observed. These data indicate that PD-1 signaling critically limits CD8+ T-cell effector functions and prevents immunopathology in response to neonatal respiratory viral infections.

Diagnostic Virology-Based Spectrum of Neonatal Viral Infections at a Tertiary Center: Time to Switch Over

Background: Viral infections are important etiologies of neonatal sepsis, especially in premature/immune-compromised babies, and are associated with long-term morbidity and mortality. Timely diagnosis of viral infections using newer diagnostic tests in neonates is a necessary prerequisite for reducing the global burdens of neonatal morbidity and mortality. Objectives: This study aimed to assess the patterns of viral infections in newborns over one year. Methods: A total of 660 newborns were included in this hospital-based, cross-sectional, and observational study based on inclusion/exclusion criteria and after obtaining the required clearance from the institutional ethical committee. All newborns admitted in NICUs for suspected clinical sepsis were included except those with congenital malformation, hypoxic-ischemic encephalopathy (HIE), pre-term for care, and negative consent. Results: This study investigated 660 subjects with suspected sepsis, out of which 560 had non-viral sepsis (group one) and 100 had viral sepsis (group two). A significant difference was observed between the two groups in terms of mean hemoglobin level, mean platelet counts, and mean transaminases level. Cytomegaloviruses (CMV), rubella, and herpes simplex virus (HSV) were found in 9.1%, 4.5%, and 3.8% of the subjects, respectively. Conclusions: It was recommended that all neonatologists and pediatricians involved in neonatal care should suspect a viral agent as a possible cause of sepsis and utilize the diagnostic and treatment facilities with antiviral agents whenever and wherever possible.

Neonatal BCG vaccination is associated with a long-term DNA methylation signature in circulating monocytes.

Trained immunity describes the capacity of innate immune cells to develop heterologous memory in response to certain exogenous exposures. This phenomenon mediates, at least in part, the beneficial off-target effects of the BCG vaccine. Using an in vitro model of trained immunity, we show that BCG exposure induces a persistent change in active histone modifications, DNA methylation, transcription, and adenosine-to-inosine RNA modification in human monocytes. By profiling DNA methylation of circulating monocytes from infants in the MIS BAIR clinical trial, we identify a BCG-associated DNA methylation signature that persisted more than 12 months after neonatal BCG vaccination. Genes associated with this epigenetic signature are involved in viral response pathways, consistent with the reported off-target protection against viral infections in neonates, adults, and the elderly. Our findings indicate that the off-target effects of BCG in infants are accompanied by epigenetic remodeling of circulating monocytes that lasts more than 1 year.

Early life exposure to poly I:C impairs striatal DA-D2 receptor binding, myelination and associated behavioural abilities in rats

Early-life viral infections critically influence the brain development and have been variously reported to cause neuropsychiatric diseases such as Schizophrenia, Parkinson’s diseases, demyelinating diseases, etc. To investigate the alterations in the dopaminergic system, myelination and associated behavioral impairments following neonatal viral infection, the viral immune activation model was created by an intraperitoneal injection of Poly I:C (5 mg/kg bw/ip) to neonatal rat pups on PND-7. The DA-D2 receptor binding was assessed in corpus striatum by using 3H-Spiperone at 3, 6 and 12 weeks of age. MOG immunolabelling was performed to check myelination stature and myelin integrity, while corpus callosum calibre was assessed by Luxol fast blue staining. Relative behavioral tasks i.e., motor activity, motor coordination and neuromuscular strength were assessed by open field, rotarod and grip strength meter respectively at 3, 6 and 12 weeks of age. Following Poly I:C exposure, a significant decrease in DA-D2 receptor binding, reduction in corpus callosum calibre and MOG immunolabelling indicating demyelination and a significant decrease in locomotor activity, neuromuscular strength and motor coordination signify motor deficits and hypokinetic influence of early life viral infection. Thus, the findings suggest that early life poly I:C exposure may cause demyelination and motor deficits by decreasing DA-D2 receptor binding affinity.

Neonatal Brain Injury From SARS-CoV-2: Fact or Fiction?

Neonatal Brain Injury From SARS-CoV-2: Fact or Fiction?

Diminished secretion and function of IL-29 is associated with impaired IFN-α response of neonatal plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are key players in the antiviral immune response and type III IFNs such as IL‐29 appear to play a pivotal role in pDC function. Pronounced susceptibility to viral infections in neonates is partly resulting from diminished antiviral immune mechanisms. Accordingly, the aim of the present study was to investigate the impact of IL‐29 in the altered immune response of neonatal pDCs. PBMCs of adult and term newborns were stimulated with CpG‐ODN2216 in the presence or absence of IL‐29 and assessed for IFN‐α production, downstream‐signaling, and activation marker expression. A significantly lower IL‐29 production after TLR9‐specific stimulation was demonstrated in neonatal pDCs. IL‐29 enhanced the IFN‐α production of pDCs in adults compared to newborns. Newborn pDCs displayed a significantly lower surface expression of IL‐10 and IL‐28Rα receptor resulting in diminished STAT1 and IRF7 activation. Interestingly, concomitant stimulation with CpG‐ODN2216/IL‐29 had no impact on the expression of surface activation and maturation markers of pDCs in neither population. The diminished antiviral immune response of neonatal pDCs is associated with reduced production and cellular responses toward IL‐29. Potential therapeutic agents enhancing the IL‐29 response in neonatal pDCs possibly augment viral protection in newborns.

Clinical Features of Respiratory Syncytial Virus Infection in Neonates: A Single Center Study

Clinical Features of Respiratory Syncytial Virus Infection in Neonates: A Single Center Study

Maternal and infant death after probable vertical transmission of chikungunya virus in Brazil \u2013 case report

BackgroundChikungunya virus infection in neonates is relatively rare and can lead to death.Case presentationWe report the occurrence of the first death of a mother and child after probable vertical transmission of chikungunya virus in Brazil. A 28-year-old pregnant woman with hypertension presented with symptoms compatible with an arboviral disease at 34 weeks’ gestation. She developed preeclampsia with severe respiratory failure which resulted in the emergency cesarean section, and the patient died 12 days after the onset of symptoms. The pre-term newborn weighed 2535 g, with an Apgar score of 4/8. He was referred to the neonatal ICU with neutrophilia and thrombocytopenia, several seizure episodes, and hemorrhagic disorders, which resulted in death. Chikungunya IgM antibody was detected in the cerebrospinal fluid.ConclusionsWe present the first documented maternal and neonatal death in Brazil after probable chikungunya infection during pregnancy.

ENTEROVIRALES AS A REASON OF INTRAUTERINE INFECTIONS OF FETUS AND NEONATAL DISEASES

Introduction. Intrauterine infections remain an urgent issue of modern Perinatology and belong to the group of diseases diagnosis of which is associated with some difficulties. The number of causative agents of intrauterine infections in case of ante-, intra- and perinatal infectionis practically unlimited. However, one of the first places belongs to pathogens of viral infections with enteroviruses being of the most interest, as a transplacental (vertical) way of infection the fetus and neonates was proved. The significance of enteroviruses in the pathology of the fetus and neonate is unknown, since there is no verification ofserotypes of enteroviruses in pregnant women and neonates.The objective is to determine the effect of enteroviruses on the fetus and neonate based on the verification of circulating enteroviruses serotypes, the definition of risk factors of the perinatal anamnesis, clinical course ofintrauterine enteroviral infection.Materials and methods. Clinical and laboratory examination of 834 mothers and their sick neonates with suspected intrauterine infections in the first 3 days of life was conducted. The subject of the study was a group of 224 infants with congenital viral infections. Intrauterineenteroviral infection was verified in 108 cases. General clinical and special methods of research were used. Specific diagnosis of intrauterine enteroviral infection was made using the cultural method, polymerase chain reaction and the modified reaction of complement binding withthe use of poly- and monovalent enteroviral diagnostic sera on the enzyme-immunoassay analyzer. Material for examination of children and mothers was the blood taken from the umbilical and peripheral veins and feces. Statistical processing was carried out with the computation of parametric and nonparametric criteria. The results of the research. In 224 children with congenital viral infections during verification of an etiological pathogen, prenatal enteroviral infections was diagnosed in 108 cases (48.2%), including 54 (50,0%) mature and 54 (50.0%) premature ones. In most casesenteroviruses were verified in association with other intrauterine enteroviral infection pathogens (mixed forms) in 91 (84,3%): in association with respiratory vi ruses in 33 (36,3%), with the causative agents of TORCH-group in 27 (29,7%), with bacterial pathogens in 31 (34,0%) and in the form of monoinfection in 17 (15,7%) neonates. On the basis of laboratory examinations the role of various serologicaltypes of enteroviruses in the structure of congenital viral infections in neonates was identified for the first time, with serotypes of entero 68-71 – in 67 (62,0%), echo – in 32 (29,6%) and Coxsackie B – in 15 (13,9%) predominating.The most severe course of infection was observed in premature infants. The most severe course of intrauterine enteroviral infection with the fatal outcome was observed in neonates in case of association of Coxsackie B6 with bacterial pathogens and influenza virus.Summary. On the basis of specific diagnostic methods, the role of various enteroviruses serotypes in the pathology of the fetus and neonates was identified. The most common perinatal risk factors for the development of intrauterine enteroviral infection in neonates are the presence of acute respiratory viral infections in pregnant women,abortion, premature labor, anemia, placental dysfunction, preeclampsia. The fact that enteroviruses cause visceral forms of lesions (serotypes of Coxacques B and ECHO) as well as subclinical forms of infection (serotypes of entero-68-71) in newborns was proved. Increased frequency (48,2%) of enteroviral isolation in neonate patients withintrauterine enteroviral infections, peculiarities of the severe clinical course motivate early diagnosis in case of visceral and generalized forms of intrauterine infections.

The proposal of Paediatric Virology and its perspectives: An interview with Professor of Paediatrics Maria Theodoridou.

Professor Maria Theodoridou, Emeritus Professor of Paediatrics at the University of Athens, is one of the few paediatricians in Greece, who have experienced almost all the infectious diseases of the second half of the 20th century and their severe consequences, prior to the widespread adoption of immunisations. A milestone during her career was the establishment of a specialised National Reference Unit for the care of paediatric patients with acquired immune deficiency syndrome (AIDS) at the ‘Aghia Sophia’ Children's Hospital in Athens, Greece. According to Professor Theodoridou, training on the prevention, management and treatment of neonatal and paediatric viral infections represents a new educational challenge for both community as well as hospital-based paediatric health professionals. The debate of the potential strategically principal role of Paediatric Virology subspecialists in the primary, secondary and tertiary clinical practice is definitely necessary and needs further discussion and evaluation, she adds. She describes the difficulties that Greece, a country under a long-standing financial crisis, faces for the hospital-based management of paediatric viral infections and refers to the future advances, which are expected in the field of diagnosis and treatment of viral infections in neonates and children. In the context of the 3rd Workshop on Paediatric Virology, which will be held in Athens on October 7th, 2017, Professor Theodoridou will focus on the immigration crisis and vaccination policy.

Toll-Like Receptor (TLR)-2 Mediates Recruitment of Neutrophils During Influenza Infection in a Neonatal Murine Model

Abstract Infants less than six months of age are particularly susceptible to influenza infection due to their impaired cytotoxic T cell response. The neonate relies on innate immunity to contain infection without excessive inflammatory response, by using pattern recognition molecules, like collectins and TLRs. TLR2 has been identified as a bacterial recognition receptor. However, little is known about its role in viral recognition and associated pathogenesis, particularly in the neonate. To address this, 3-day old TLR-2−/− and control C57Bl/6 mice were infected with influenza type A virus strain PR/8/34 (H1N1) and tracked for survival. Bronchoalveolar lavage fluid (BALF) was collected and lungs were harvested at post infection day (PID) 1, 3, and 6. BALF was analyzed by flow cytometry for cellular infiltration in the airways. RNA was isolated and analyzed via real-time PCR for viral loads and various cytokines. There was a striking difference in survival between the TLR-2−/− (61%) and control neonates (25%) (p< 0.01). Surprisingly, TLR-2−/− mice had higher viral loads than controls (p < 0.05) on PID 1, which was coupled with lower neutrophil numbers in the BALF (p < 0.02). No significant differences were noted on PID 3 or 6 for neutrophil counts or viral loads. On PID 3, TLR-2−/− mice had more than a 2-fold lower transcription of CXCL-5, a key chemoattractant for neutrophils. Therefore, the resistance of neonatal TLR-2−/− mice to influenza infection was not due to heightened influenza clearance. Rather, there was impaired neutrophil recruitment, which potentially reduced pulmonary inflammation. Future studies using our neonatal murine model could elucidate the mechanism of TLR-2 and CXCL-5 mediated inflammation during neonatal viral infection.

Cutaneous manifestations of systemic viral diseases in neonates: an update.

Dermatologic findings may be the first signs of a neonatal viral infection. This review provides an update of the diagnostic features and therapies for selected viral illnesses [herpes simplex virus (HSV), varicella zoster virus, enterovirus, and Zika virus] that present with cutaneous manifestations in the neonate. HSV DNA polymerase chain reaction of plasma and cerebrospinal fluid, routinely used in the diagnosis of neonatal HSV, may have expanded utility in assessing prognosis and acyclovir therapeutic efficacy. Maternal antiviral suppressive therapy may alter the clinical appearance of congenital HSV, resulting in delayed diagnosis and treatment. VariZIG, a varicella zoster immune globulin, is a US Food and Drug Administration approved form of prophylaxis for varicella. The Centers for Disease Control and Prevention has expanded the period of VariZIG eligibility for preterm infants, a group particularly susceptible to severe varicella infection. For severe neonatal enterovirus sepsis, the results of a randomized, double-blind, placebo-controlled trial of pleconaril, a viral capsid inhibitor, suggest that this compound is an effective therapy. Human Parechovirus type 3, a strain within a newly formed viral genus, has a similar, and potentially underestimated, clinical presentation to enterovirus sepsis. However, a distinctive erythematous palmoplantar rash may be specific to human Parechovirus type 3 infection. Perinatal Zika virus infection in the neonate may present with a nonspecific macular and papular rash. As this rash is not specific, obtaining a maternal travel history and, if appropriate, requesting additional diagnostic testing are critical for early diagnosis. Neonatal rashes may be harmless and transient, whereas others may reflect the presence of a severe systemic illness. Recognizing key cutaneous features of viral-associated rashes may aid in the prompt and accurate diagnosis and treatment of neonatal viral illnesses.

Viral Infections in Neonates with Suspected Late-Onset Bacterial Sepsis-A Prospective Cohort Study.

Objective The aim of our study was to evaluate the occurrence of viral infections in infants with suspected late-onset bacterial sepsis in a neonatal intensive care unit. Methods In a prospective study, infants with suspected late-onset bacterial sepsis underwent viral testing alongside routine blood culture sampling. Using a multiplex reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay, nasopharyngeal aspirates were analyzed for adenovirus, respiratory syncytial virus (RSV), influenza virus A and B, H1N1 virus, parainfluenza virus 1 to 4, metapneumovirus, coronavirus, and picornavirus. Stools were examined for adenovirus, rotavirus, norovirus, and enterovirus. Results Between August 2010 and March 2014, data of 88 infants with 137 episodes of suspected late-onset bacterial sepsis were analyzed. Six infants were diagnosed with a respiratory viral infection (2 × RSV, 4 × picornavirus). Blood culture–proven bacterial sepsis was detected in 15 infants. Neither viral–bacterial coinfections nor polymerase chain reaction positive stool samples were found. Conclusion Respiratory viruses can be detected in a considerable number of neonates with suspected late-onset bacterial sepsis. In contrast, gastrointestinal viral or enterovirus infections appear uncommon in such cases.

Interferon gamma protects neonatal neural stem/progenitor cells during measles virus infection of the brain.

BackgroundIn the developing brain, self-renewing neural stem/progenitor cells (NSPC) give rise to neuronal and glial lineages. NSPC survival and differentiation can be altered by neurotropic viruses and by the anti-viral immune response. Several neurotropic viruses specifically target and infect NSPCs, in addition to inducing neuronal loss, which makes it difficult to distinguish between effects on NSPCs that are due to direct viral infection or due to the anti-viral immune response.MethodsWe have investigated the impact of anti-viral immunity on NSPCs in measles virus (MV)-infected neonates. A neuron-restricted viral infection model was used, where NSPCs remain uninfected. Thus, an anti-viral immune response was induced without the confounding issue of NSPC infection. Two-transgenic mouse lines were used: CD46+ mice express the human isoform of CD46, the MV entry receptor, under the control of the neuron-specific enolase promoter; CD46+/IFNγ-KO mice lack the key anti-viral cytokine IFNγ. Multi-color flow cytometry and Western Blot analysis were used to quantify effects on NSPC, neuronal, and glial cell number, and quantify effects on IFNγ-mediated signaling and cell markers, respectively.ResultsFlow cytometric analysis revealed that NSPCs were reduced in CD46+/IFNγ-KO mice at 3, 7, and 10 days post-infection (dpi), but were unaffected in CD46+ mice. Early neurons showed the greatest cell loss at 7 dpi in both genotypes, with no effect on mature neurons and glial cells. Thus, IFNγ protected against NSPC loss, but did not protect young neurons. Western Blot analyses on hippocampal explants showed reduced nestin expression in the absence of IFNγ, and reduced doublecortin and βIII-tubulin in both genotypes. Phosphorylation of STAT1 and STAT2 occurred independently of IFNγ in the hippocampus, albeit with distinct regulation of activation.ConclusionsThis is the first study to demonstrate bystander effects of anti-viral immunity on NSPC function. Our results show IFNγ protects the NSPC population during a neonatal viral CNS infection. Significant loss of NSPCs in CD46+/IFNγ-KO neonates suggests that the adaptive immune response is detrimental to NSPCs in the absence of IFNγ. These results reveal the importance and contribution of the anti-viral immune response to neuropathology and may be relevant to other neuroinflammatory conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0571-1) contains supplementary material, which is available to authorized users.

Paediatric Virology: A new paediatric subspecialty? A proposal at the Workshop on Paediatric Virology, Athens, October 10, 2015.

Paediatric Virology: A new paediatric subspecialty? A proposal at the Workshop on Paediatric Virology, Athens, October 10, 2015.

Population-based Incidence and Etiology of Community-acquired Neonatal Viral Infections in Bangladesh: A Community-based and Hospital-based Surveillance Study.

The etiology of >90% of cases of suspected neonatal infection remains unknown. We conducted community-based surveillance in conjunction with hospital-based surveillance in a rural region in Bangladesh from June 2006 to September 2007 to assess the incidence and etiology of community-acquired viral infections among neonates. Community health workers (CHWs) assessed neonates at home on days 0, 2, 5 and 8 after birth and referred cases of suspected illness to the hospital (CHW surveillance). Among neonates with clinically suspected upper respiratory tract infection (URTI), pneumonia, sepsis and/or meningitis, virus identification studies were conducted on nasal wash, cerebrospinal fluid and/or blood specimens. In the hospital-based surveillance, similar screening was conducted among all neonates (referred by CHWs and self-referred) who were admitted to the hospital. CHW surveillance found an incidence rate of 15.6 neonatal viral infections per 1000 live births with 30% of infections identified on the day of birth. Among neonates with suspected sepsis, a viral etiology was identified in 36% of cases, with enterovirus accounting for two-thirds of those infections. Respiratory syncytial virus was the most common etiologic agent among those with viral pneumonia (91%) and URTI (68%). There was a low incidence (1.2%) of influenza in this rural population. Viral infections are commonly associated with acute newborn illness, even in the early neonatal period. The estimated incidence was 5-fold greater than reported previously for bacterial infections. Low-cost preventive measures for neonatal viral infections are urgently needed.

5ICCN_006: Viral infections in neonates with suspected nosocomial bacterial sepsis

Prenatal attachment can be described as the parents’ emotions, perceptions and behaviors that are related to the fetus. This relationship has been described as the most basic form of the human intimacy and represents the earlier internalized representation of the fetus that both parents typically acquire and elaborate during pregnancy. The quality of the relationship between an infant and his or her parent is an important factor influencing the child’s later development, both cognitive and emotional. There is evidence – even though yet unclear – that demographic, perinatal and psychological variables may correlate with attachment. In this perspective, it is essential to recognize the factors influencing attachment of parents towards their fetus and to planning psychosocial interventions in antepartum units or in obstetric clinics, in order to preserve a positive physical and emotional development of the infant and to provide family-centered prenatal care. Particular attention should be paid to women hospitalized for a high-risk pregnancy, since this condition involves a high distress that often results in feelings of anxiety and depression, that can hinder an adequate mother–fetus attachment.© 2014 Elsevier Ireland Ltd. All rights reserved.

Clinical and Laboratory Characteristics of Disseminated Herpes Simplex Virus Infection in Neonates

Disseminated herpes simplex virus (HSV) infection is the most fulminant type of neonatal HSV infection and has the highest mortality. Early diagnosis and treatment are essential for patient survival. We describe the clinical presentation, laboratory characteristics, and outcomes of neonates with disseminated HSV infection at our institution. A retrospective review of electronic medical records from 2006 to 2013 was performed. Only neonates with disseminated HSV infection, confirmed by using polymerase chain reaction or viral culture results, were included. Twenty-two cases were identified; the age range was 1 to 14 days. The majority of patients did not have a maternal history of HSV or a history of maternal fever at delivery. Eleven of the patients were delivered by cesarean delivery, and 3 of these patients did not have prolonged rupture of membranes. Neonatal fever, the most common historical characteristic, was present in only one-half of the patients. Pneumonia and respiratory distress were present in one-half of the patients. Serum aspartate aminotransferase and alanine aminotransferase levels were elevated in most, but not all, patients. The blood HSV polymerase chain reaction was positive in all patients tested. Of the 22 study patients, 16 survived and 6 died. The majority of the patients who died had respiratory disease and a delay in the initiation of acyclovir therapy. Disseminated HSV infection in neonates can be challenging to diagnose and is associated with high mortality. Clinicians must strongly consider this diagnosis, test the blood for HSV polymerase chain reaction, and initiate early treatment in the appropriate clinical scenarios.

Respiratory viral infections are not uncommon in neonatal intensive care units

Full-term and preterm infants admitted to neonatal intensive care units (NICUs) face a high risk of infections, due to the immaturity of their innate and adoptive immune systems, inadequate protection through maternal immunity and the need for repeated invasive procedures (1). In recent years, our knowledge of viral infections in neonates has increased, due to multiplex polymerase chain reaction (PCR)-based techniques. It is now possible to screen for as many as 17 viruses from a single mucus sample (2). However, the role of viral respiratory tract infections in the symptoms of infants admitted to NICUs at birth is still poorly understood. In a recent review, 32 respiratory viral outbreaks in NICUs were reported (3). These were caused by several different respiratory viruses, including respiratory syncytial virus (RSV) (89 patients in 11 outbreaks), enteroviruses (101 patients in 10 outbreaks) and adenovirus (79 patients in six outbreaks). In addition, outbreaks of coronavirus, rhinovirus, influenza A virus and parainfluenza virus infections have been reported (4–7). An epidemic may result in the temporary closure of a NICU. Recently, a NICU was closed for 28 days after adenovirus type 19 caused an outbreak of keratoconjunctivitis, which affected 12 NICU infants, two NICU staff members, two relatives of patients and two members of the ophthalmologic team (8). We report on an observational study on the use of a multiplex PCR in a NICU in Finland. The study was carried out at Turku University Hospital, the only tertiary level NICU in south-west Finland, which serves a population of about 750 000. An average of 600–700 infants are admitted to the NICU annually, resulting in approximately 6000 patient care days per year. About one-third of the infants are preterm, and 50–85 very low birth weight infants are treated annually. Two to four patients are treated in one room. Parents are encouraged to stay with their infant and provided with unlimited access. Visitors with respiratory tract infection symptoms are not allowed into the NICU, and siblings under school age cannot visit if there is an RSV outbreak in the community. Otherwise, healthy siblings are allowed access. During the study period from 1 January 2009 to 30 June 2011, 1589 infants were admitted to the unit and 76 (5%) were evaluated for respiratory viruses. A nasopharyngeal aspirate was taken from infants if they presented with symptoms of respiratory infection, such as rhinorrhea, sneezing, increased bronchial secretions, episodes of bradycardia and/or desaturations, or if they had been exposed to someone with a respiratory infection. We collected and analysed 139 samples from 76 infants for this study. Nasopharyngeal aspirates were collected in sterile tubes and analysed on the same or following day. Nucleic acids were extracted from the aspirates using Nuclisense easyMag extractor (Biomerieux, Boxtel, the Netherlands). Respiratory virus genomes were detected using Seeplex RV12 multiplex PCR assay for adenovirus, influenza A and B viruses, parainfluenza types 1–3 viruses, RSVa, RSVb, rhinovirus, human metapneumovirus, and coronaviruses 229E/NL63 and OC43/HKU1 (Seegene, Seoul, Korea). In addition, specimens were tested in a real-time PCR assay for enteroviruses, rhinovirus and RSV (9). During the two-and-a-half-year study period, no outbreaks of viral respiratory tract infection occurred in the NICU. Of the 139 samples taken from 76 infants, 28 samples (20%) from 15 infants (20%) were positive for one or more viruses. Six babies were positive for rhinovirus, five for parainfluenza type 3 virus, one for parainfluenza type 2