key: cord-349279-wbb7h2zu authors: Walker, Gregory J.; Stelzer‐Braid, Sacha; Shorter, Caroline; Honeywill, Claire; Wynn, Matthew; Willenborg, Christiana; Barnes, Phillipa; Kang, Janice; Pierse, Nevil; Crane, Julian; Howden‐Chapman, Philippa; Rawlinson, William D. title: Viruses associated with acute respiratory infection in a community‐based cohort of healthy New Zealand children date: 2019-05-07 journal: J Med Virol DOI: 10.1002/jmv.25493 sha: doc_id: 349279 cord_uid: wbb7h2zu Acute respiratory infections (ARIs) are a major cause of morbidity among children. Respiratory viruses are commonly detected in both symptomatic and asymptomatic periods. The rates of infection and community epidemiology of respiratory viruses in healthy children needs further definition to assist interpretation of molecular diagnostic assays in this population. Children otherwise healthy aged 1 to 8 years were prospectively enrolled in the study during two consecutive winters, when ARIs peak in New Zealand. Parents completed a daily symptom diary for 8 weeks, during which time they collected a nasal swab from the child for each clinical ARI episode. A further nasal swab was collected by research staff during a clinic visit at the conclusion of the study. All samples were tested for 15 respiratory viruses commonly causing ARI using molecular multiplex polymerase chain reaction assays. There were 575 ARIs identified from 301 children completing the study, at a rate of 1.04 per child‐month. Swabs collected during an ARI were positive for a respiratory virus in 76.8% (307 of 400), compared with 37.3% (79 of 212) of swabs collected during asymptomatic periods. The most common viruses detected were human rhinovirus, coronavirus, parainfluenza viruses, influenzavirus, respiratory syncytial virus, and human metapneumovirus. All of these were significantly more likely to be detected during ARIs than asymptomatic periods. Parent‐administered surveillance is a useful mechanism for understanding infectious disease in healthy children in the community. Interpretation of molecular diagnostic assays for viruses must be informed by understanding of local rates of asymptomatic infection by such viruses. metapneumovirus (HMPV), human enterovirus (EV), and adenovirus (ADV), are the most common viral aetiological agents associated with ARIs. 8 Although most respiratory viral infections are mild, they can potentially cause life-threatening illness, particularly in high-risk groups such neonates, the elderly, and those with chronic respiratory disease. Advances in molecular diagnosis have made it possible to easily and quickly identify viruses in people with ARIs, with quantitative polymerase chain reaction (PCR) now considered by most to be the reference standard for detection of respiratory viruses over traditional methods such as viral culture. 9 However, the clinical significance of virus detection is not always clear, as respiratory viruses are also detected in asymptomatic subjects. 10, 11 Further understanding of the role of viral detection in ARIs may improve clinical decision-making around infection control and therapy. Direct comparisons between studies of respiratory virus epidemiology can be difficult for a number of reasons. Climatic and social factors affect the survival and transmission of respiratory viruses, as well as host mobility and susceptibility. 12, 13 This results in many viruses having characteristic seasonal patterns that vary by geographical location. Reported epidemiology is also affected by study design. Only one-fifth of ARIs result in a health care visit, 14 were recruited for the study. This was made up of 138 children from a previous study, 17 while the remaining 167 participants replied to advertising via staff email for the local district health boards, via posters and through referral from family and friends. There were 50 of 372 participants deemed ineligible for various reasons including that they slept in more than one bedroom, the bedroom was already heated, or they were going away for more than 2 weeks during the study period. Before commencement of the study 17 of 372 participants withdrew, while 4 of 305 participants commenced the program but were later excluded after failing to comply with study conditions. The remaining cohort for analysis was 301 children, PCR, respectively, as previously described. 18 Sequences were compared to the NCBI nonredundant nucleotide (nr/nt) database using the Basic Local Alignment Search Tool (BLAST) on the NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and genotype assigned according to the closest identifying sequence. Details of primers used in detection of respiratory viruses are included in the supplementary material (Table S1 ). Any period in which participants had a multi-day cumulative symptom score ≥4, with a single day symptom score ≥2 was defined as an ARI episode. The end of an ARI episode was marked by three consecutive symptom-free days. A symptom score correction (daily baseline score of −1) was applied to participants who were considered to have background rhinitis. This included children who had (i) rhinitis symptoms (runny nose or sneezing) for ≥49 of the 56 study days, (ii) no more than 7 days with no symptoms, or (iii) 7 or more days of only rhinitis symptoms. In cases where multiple swabs were taken during a single ARI episode, only the first swab taken was included in analysis. The exception to this was if a new virus was detected in subsequent swabs, or if swabs were taken >30 days apart. Risk ratio (RR) was calculated as the proportion of samples positive for a virus with ARI symptoms divided by the proportion of samples negative for a respiratory virus with ARI symptoms. There were 16 856 child-days of symptom observed and a total of 612 nasal swabs collected from the 301 participants. There were 575 ARIs identified (Table 1) . Nasal swabs were received from 400 of 575 of these episodes (69.6%). In addition, 212 swabs were taken when children were asymptomatic, most of which were performed during the clinical visit by researchers at the conclusion of the study. Due to inclusion of children from a previous study of wheezing, those with asthma were highly represented within our cohort (33.6%). Overall, children with asthma displayed a small increase in the rate of ARIs per child-month (1.13 vs 1.00) compared with nonasthmatics (Table S2 ), but this increase was not significant (P = 0.057). Of the 400 swabs taken during an ARI, 307 (76.8%) were positive for a virus. During an ARI, detection of respiratory viruses in nasal swabs was highest amongst the youngest age group (1 year old), and decreased to age 4 ( Table 1 ). During ARIs, multiple viruses were codetected in 37 (9.3%) swabs. The most commonly detected viruses in samples collected during ARI were HRV (52.8%), HCoV (11.0%), parainfluenza virus (PIF) (6.0%), IFV (4.5%), RSV (3.8%), and HMPV (3.5%). These were all significantly more likely to be associated with ARI episodes than asymptomatic detection (Table 2 ). AdV was detected with similar frequency in both ARI (3.3%) and asymptomatic samples (3.8%), while detection of HBoV was relatively low in both groups. Detection of any virus and codetection of viruses were both significantly associated with swabs collected during ARI episodes. Sequencing was performed on 193 of a possible 284 picornavirus positive samples (68.0%). Rhinovirus strains HRV-A and HRV-C were significantly associated with ARI episodes (Table 2) . Overall, detection of individual viruses was similar between asthmatic and nonasthmatic children in swabs taken during an ARI (Table S3) . HRV-B (P = 0.042) and RSV (P = 0.034) were significantly over represented in asthmatics during ARIs. ARIs were most frequent in May (1.39), which was also the month in which the least data was collected. This was followed by June (1.22) August (1.21), and July (1.07). The rate of ARIs steadily decreased following the winter season ( Figure 1 ). HRV was the predominant virus detected in each month, and was most prevalent in late-winter and spring (Figure 1 ). HCoV and RSV had peaks during early winter, while AdV peaked in spring. IFV and HMPV were most prevalent in July, but were also detected in other months. PIF was detected consistently throughout study months, although not in May. There is a need for improved population data regarding respiratory viruses in children, particularly in the Southern Hemisphere. This is the first study assessing the epidemiology of an extended range of respiratory viruses in a community cohort of healthy children in New Zealand. Infection with respiratory viruses was common in both symptomatic and asymptomatic children, and this study demonstrated These relatively high frequencies were true of children with asthma (1.12) and those without asthma (1.00). The proportion of ARIs associated with a virus-positive swab in this population declined with age. Children 1 year of age were positive for a respiratory virus in 88.9% of samples taken during an ARI, compared to 69.8% in participants aged greater than or equal to 5 years age. A similarly designed household surveillance study showed that weekly detections of viruses in young (aged <5) and older (aged 5-17) children were significantly more frequent than in adults. 20 In the current study swabs were taken at the onset of illness, when viral load reportedly peaks for some respiratory viruses. 21 It is possible that this method is more likely to detect viruses associated with ARI than other studies obtaining weekly swabs. Sample sizes of each virus detected here were too small to compare individual viruses between age groups. However, a multi-country community surveillance study has previously shown that with the exception of influenza, prevalence of individual respiratory viruses was highest in younger children. 3 The rates of ARI per child-month were similar between age groups, suggesting that other factors such as development of allergy may play a more significant role than age in acute respiratory health. The study was conducted during the winter/spring seasons of 2 consecutive years. Consistent with previous reports in the Southern Hemisphere RSV peaked in this pediatric population in early winter. 4, 22 Interestingly, we found a similar pattern for detection of HCoV. It has recently been reported in China that while overall HCoV infection is most prevalent in winter, HCoV subtypes display different epidemic patterns. 23 As only a small number of HCoVs were detected (n = 50), we did not distinguish between subtypes when reporting our results. Influenza was detected from June to October and peaked in July. A limitation was that no data were collected for the summer and autumn periods, capturing only the times of highest respiratory virus activity. This may account for the low detection of AdV, HBoV, and EV in comparison to other year-round epidemiological studies. 24 A yearround analyses may also allow for investigation of phenomena such as the February back to school HRV peaks. 25 Our study differs from a previous NZ-based analysis of viruses in hospitalized children with LRTI, 22 where relatively high rates of RSV (39%) and HMPV (7%) were detected. In our community-based study, incidence of these viruses was considerably lower. Previous groups have reported that RSV and HMPV rarely occur in asymptomatic subjects, 11, 26, 27 suggesting that detection of these viruses is almost always of clinical relevance. IFV may be present in people with few or no symptoms. However, the duration and copy number of viral shedding is considerably lower in these cases, 28 and further research is needed to understand the contribution of asymptomatic detection to community transmission. In the present study, we found these viruses in very few samples of asymptomatic subjects, and demonstrated these three viruses (and others) to be significantly associated with ARIs. Respiratory viruses were detected in a large proportion of swabs taken during asymptomatic periods (37.3%), with the vast majority of these detections caused by HRV. This is unsurprising as HRVs were the most frequently detected virus overall, and have been commonly detected in asymptomatic children before. 11 It has previously been unclear whether subtypes of HRV have varying clinical impact on their hosts, with studies of healthy, 5 and asthmatic 18 children reporting no differences in symptom risk between HRV subtypes. Our data, along with another recent study 19 suggest that HRV-A and HRV-C subtypes are significantly more likely to cause symptoms related to ARI. In our study HRV-B, AdV, and HBoV were detected similarly in samples from both ARI episodes and asymptomatic periods. While their corresponding risk ratios are not considered significant, the number of detections of these viruses is relatively small, and a larger analysis would be required to rule out the clinical significance of detecting HRV-B, AdV and HBoV in ARIs. The effect of viral coinfection on respiratory disease severity in children has not been well established. A number of studies have found increased hospitalizations, longer length of stay in hospital, and higher mortality when two or more respiratory viruses were detected. [29] [30] [31] However, others report no association between viral coinfection and clinical outcomes 32,33 including a systematic review of 43 such publications. 34 In this study we found a codetection was observed in 9.3% of total virus detections during ARIs, which is similar to rates reported previously. 4 We also codetected viruses in a small number of swabs collected during asymptomatic periods, and show that codetections are significantly more likely to be associated with ARIs. 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