Despite acute viral respiratory infections being important causes of morbidity and mortality in infants worldwide, little is known about these infections in the first 4 weeks of life (referred henceforth as the neonatal period). The few studies describing respiratory viruses in neonates are unlikely to be widely generalizable as they are predominantly from neonatal units1,2 or neonates presenting to hospital with respiratory symptoms.3,4 While several community-based birth cohort studies tested for respiratory viruses,5,6 few collected samples before 4 weeks of age and then only tested for a limited number of viruses.7 It is important to understand respiratory virus exposure in this age group as early infection, even if asymptomatic, may influence future respiratory health.8 Hence, we describe the respiratory viruses detected in neonates in a community-based birth cohort.
MATERIALS AND METHODS
The Observational Research in Childhood Infectious Diseases study (clinicaltrials.gov: NCT01304914) is an unselected, community-based birth cohort study in the subtropical city of Brisbane, Australia.9 After initial approaches in antenatal clinics, healthy full-term infants born between September 2010 and October 2012 were enrolled in the study. At enrolment, parents provided their demographic, social and health characteristics, and when available, pregnancy and birth details. Parents collected anterior nasal swab specimens from their infant shortly after birth and weekly thereafter. The specimens were mailed directly to the research laboratory. Parents also completed a daily symptom diary. Post birth, parents provided their own nasal swab specimens and reported respiratory symptoms within the previous 2 weeks.
Swabs were batch-tested for human rhinovirus (HRV), respiratory syncytial virus (RSV), influenza viruses, parainfluenza viruses, adenovirus, human metapneumovirus, human coronaviruses (HCoV), human bocavirus and human polyoma viruses WU and KI using previously validated real-time polymerase chain reaction assays.9 A nested polymerase chain reaction targeting the HRV VP4/VP2 region was used to genotype HRV-positive samples.10
A new virus detection occurred when a new virus species or another HRV genotype was detected. We defined each swab as representing 7 days of study time, with a maximum of 28 days per neonate. The association between potential risk factors (sex, mode of delivery—vaginal versus Caesarean, first-born status, season, parental history of asthma or eczema, household tobacco smoke exposure and breastfeeding) and new virus infections was assessed using Poisson regression. As categories of season and breastfeeding were not mutually exclusive for individual neonates, these 2 variables were analyzed using mixed-effects Poisson regression with neonate included as a random effect. All models were offset using the natural logarithm of study time. Effect estimates are expressed as incident rate ratios (IRR) with 95% confidence intervals (CIs). A failure curve of first virus detections was constructed from life tables. Data were analyzed using Stata v12.0 (StataCorp, College Station, TX).
The Children’s Health Queensland (HREC/10/QRCH/16), Royal Brisbane and Women’s Hospital (HREC/10/QRBW/125), and The University of Queensland (2010000820) Human Research Ethics Committees approved the study.
Viruses Detected and Symptoms in Neonates
Overall, 157/164 enrolled and eligible (74 males, 47.1%) neonates provided 574 nasal swab specimens (range 1–5 per neonate), yielding 552 weeks of data (87.9% of maximum expected). Of the 43 virus-positive swabs from 29 (10 males) neonates (Fig., Supplemental Digital Content 1, http://links.lww.com/INF/C529), new infections were detected in 34 swabs (incidence rate 0.25 virus infections per neonatal period; 95% CI: 0.18, 0.34).The earliest virus detection was at 2 days of age and the age at first detected infection was consistent throughout the neonatal period (Fig., Supplemental Digital Content 2, http://links.lww.com/INF/C530).
HRV was observed in 21/29 (72.4%) neonates with positive detections. Overall, HRV was found in 31 weekly swabs, including from all 4 neonates with viruses detected in the first week of life. HCoV was detected in 4 neonates (5 swabs) and parainfluenza-3 virus in 2 neonates (2 swabs), with 1 neonate each having RSV-A (3 swabs), influenza-B virus (1 swab) and human metapneumovirus (1 swab). No co-detections were identified.
Thirteen neonates had 14 symptomatic episodes associated with virus detection (Fig., Supplemental Digital Content 1, http://links.lww.com/INF/C529). The earliest symptomatic virus detections were in the first week of life, on days 6 and 7 (both HRV-A), and associated symptoms commenced on days 9 and 11, respectively. Eleven showed upper respiratory symptoms of nasal discharge/congestion (4 also had a dry cough), including subject 66 who experienced 2 discrete respiratory episodes (corresponding to 2 different strains of HRV-A). The remaining 2 infants exhibited lower respiratory symptoms: subject 67 had wheezing (HCoV-229E detected on day 13), while subject 161 had wheezing accompanied by fever and a wet cough (HCoV-HKU1 detected on days 14 and 21, and HRV on day 28). Subjects 4, 51, 67, 134, 142 and 161 sought medical advice, with subject 51 diagnosed with otitis media at 22 days of age. None were hospitalized. In contrast, 13 neonates had asymptomatic viral detections. This included subject 71 with influenza-B on day 16, and subject 25 shedding RSV-A between days 9 and 26. Symptom diary data were unavailable for 3 neonates with single HRV detections.
Being the first-born child was associated with a reduced incidence of infection (IRR 0.5; 95% CI: 0.2, 0.9, P = 0.02; Table 1). In contrast, no associations were observed between other risk factors (sex, mode of delivery, season, parental history of asthma/eczema, household tobacco exposure or exclusive breastfeeding) and virus infection as a neonate.
Of the 31 HRV-positive samples (21 neonates), 9 (29.0%) were sequenced, but could not be assigned a specific HRV genotype (unclassified novel VP4/VP2 sequences) based on a GenBank database search (http:http://www.ncbi.nlm.nih.gov), while 4 (12.9%) HRV detections could not be sequenced, usually because of insufficient viral RNA template loads. Of the 18 samples able to be typed, 11 (61.1%) were HRV-A, 2 (11.1%) were HRV-B and 5 (27.8%) were HRV-C. Overall, there were 17 different subtypes among the 27 HRVs able to be sequenced. Apart from HRV-B52 and HRV-C02 (each detected in 2 unrelated neonates), clustering of HRV genotypes was confined to individuals. Repeat detection of homologous genotypes for 1–2 weeks was observed in 4 neonates (subjects 29, 66, 120 and 139). In contrast, 4 subjects (44, 51, 66 and 120) had either different HRV genotypes or different unclassified HRV sequences in subsequent swabs.
Viruses Detected and Symptoms in Parents
A total of 239 (127 mothers) parents submitted swabs. Nine (3 mothers) parents (3.8%, 95% CI: 1.9%, 7.5%) of 8 neonates had respiratory viruses detected, 8 with HRV and 1 with influenza-A virus, with no co-detections. Five parents with HRV reported nasal discharge/congestion, while one other complained of fever, myalgia and lassitude. Of the 8 neonates with infected parents, subject 51 had different HRV genotypes detected on days 18 and 25, subject 100 had a different HRV genotype detected on day 28 and subject 71, whose parents had HRV, became infected with influenza-B. The remaining 5 neonates remained asymptomatic and virus negative. No transmission events between parents and infants were detected.
In a community-based, urban Australian birth cohort of 157 healthy, full-term infants with high exclusive breastfeeding rates and little household tobacco exposure, we detected a mean of 0.25 (95% CI: 0.18, 0.34) new respiratory virus infections per neonatal period. These infections occurred consistently throughout the neonatal period. Being an only child, and presumably less exposed to other children, was associated with a reduced risk of virus detection. HRVs representing diverse genotypes predominated and almost half of all episodes associated with respiratory virus detections were asymptomatic. Virus shedding was transient (5–14 days) and serial HRV detections were as likely to result from new genotype acquisition as continued detection of the original infection. Finally, although numbers were small, there was no evidence of transmission between parents harboring respiratory viruses around the time of delivery and their newborn infants.
In conclusion, respiratory virus infections were common in neonates and many were asymptomatic. HRV predominated and our future studies will assess the association between early HRV exposure and ensuing frequency and severity of respiratory infections, as well as with later respiratory health, mucosal immunity and lung function.
It is possible that the frequent subclinical viral detections we observed were due to some protection against symptomatic illnesses afforded by maternal transplacental and breastmilk antibodies.11 Indeed, neonates who appear healthy may be important sources of virus transmission. Early exposure to HRV could also have implications for infants with a family history of atopy or asthma. High-risk birth cohort studies have found a close relationship between HRV infections in infancy and subsequent asthma development, particularly with HRV-A and C species.12 Recently, a cross-sectional study of the Danish asthma birth cohort (COPSAC2010) detected viruses (predominantly HRV) in 12% of neonates at 4 weeks of age.8 Some of the infected neonates exhibited an exaggerated type-2 mucosal immune response, suggesting early HRV exposure may trigger aberrant immune programing in susceptible individuals and promote subsequent development of asthma and allergic sensitization.8,12 Any preventive intervention will therefore need to begin early.
HRV genotype diversity occurs in neonates, as well as in infants and children.13 Moreover, even during single acute respiratory infection episodes, continued HRV presence in neonates is most likely from sequential infections by different HRV genotypes rather than continued shedding of the original genotype. In HRV-positive infants with sequential detections, we observed a change of genotype 50% of the time. These findings, and those from other reports,5,13 emphasize the importance of nucleotide sequencing when studying HRV infections.
Our study’s strengths include the prospective collection of swabs at birth and then weekly thereafter, allowing respiratory viruses to be detected during the neonatal period. Parent swabs and symptom history also enabled us to examine possible transmission from parent to newborn, which appeared limited, although this observation was constrained by the small number of positive swabs. It is also possible that parents may have missed identifying mild respiratory and nonspecific symptoms in their newborn infants. Finally, and similar to other community-based HRV studies,13 41.9% of sequences from HRV detections either failed to align with HRV reference prototype sequences or could not be genotyped, usually because of low viral RNA template loads. Nevertheless, the Observational Research in Childhood Infectious Diseases study design, with its weekly sampling and HRV sequencing, was able to demonstrate sequential detection by the same and different HRV genotypes.
We acknowledge the generosity and efforts of the families who participated in the study, as well as the efforts of other members of the Observational Research in Childhood Infectious Diseases Study team: Anne Cook, Frances Maguire, Rebecca Holding, Kevin Jacob, Seweryn Bialasiewicz and Jane Gaydon, and the study support data managers are Lynne Grimwood and Patricia Sloots.
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