Respiratory virus infections are the most important trigger of respiratory illnesses in young childhood. Human rhinovirus (HRV) and respiratory syncytial virus (RSV) are reported as the most frequent causing agents of respiratory illness in selected populations of hospitalized children and in children at high risk for atopic diseases.1–4 Data on the occurrence and clinical impact of respiratory pathogens during respiratory tract infections in unselected infants are scarce. Most of these studies have used rather insensitive conventional methods for virus detection. The development and use of polymerase chain reaction (PCR) methods have markedly increased the identification of viral pathogens.5,6
Because of the scarcity of data on the prevalence of respiratory pathogens in young children, prospective data on the clinical impact of respiratory pathogens during respiratory tract infections are almost lacking. For a long time, it has been suggested that some respiratory pathogens cause more severe respiratory illness than others. RSV is most often mentioned to cause severe respiratory tract illness in young children, resulting in prolonged postviral wheezing.7,8 HRV, on the contrary, was thought to be limited to the upper respiratory tract, causing only mild symptoms.9 During the last decade the perspective on HRVs has changed, as HRVs seem to be able to infect the lower respiratory tract as well.10 Moreover, some symptomatic HRV infections could play an important role in recurrent wheezing later on in life.11
In view of this, we questioned whether there is a difference in the clinical impact between the various respiratory pathogens. In this prospective population based birth cohort study, we described the occurrence and clinical impact of respiratory pathogens in infants whose parents reported respiratory infection episodes during the first year of life.
The study was done as part of the ongoing wheezing illnesses study leidsche rijn, a prospective ongoing population-based birth cohort study on determinants of wheezing illnesses. Study design and rationale of wheezing illnesses study leidsche rijn were described elsewhere.12 Briefly, healthy infants were enrolled in this study at the age of 2 to 3 weeks, before any respiratory symptoms had occurred and followed until they reached the age of 1 year. Exclusion criteria were gestational age <36 weeks, major congenital abnormalities and neonatal respiratory disease. Inclusion of infants that participated in this part of the study was done from October 2003 to September 2006. In total, 450 parents were asked to participate in this part of the study. Hundred thirty-seven parents did not return a sample and 8 infants were lost to follow-up. The study was approved by the local medical ethics committee (UMC Utrecht) and the parents gave written informed consent.
During the first year of life all parents filled in a daily questionnaire with regard to respiratory symptoms in their baby. The respiratory symptoms considered were: cough, wheeze (a whistling noise coming from the chest and not the nose), with or without fever (temperature above 38°C). To relate respiratory virus positivity to specific respiratory symptoms parents were asked to take nose and throat swabs at the second day of a reported episode with respiratory symptoms. Parents were instructed by research physicians on how to recognize the various respiratory symptoms. A symptomatic episode was defined by the presence of cough and/or wheeze, with or without fever, for a period of 2 days or longer. The end of a symptomatic episode was defined as having a period of at least 2 days without any respiratory symptoms.
Primary care visits and physicians-diagnoses during the first year of life were recorded according to the International Classification system of Primary Care (ICPC).13 We defined visits to the physician as the occurrence of a “respiratory ICPC,” ie, dyspnea (R02), wheezing (R03), cough (R05), acute upper tract infection (R74), acute bronchi(oli)tis (R78), pneumonia (R81), asthma like symptoms (R96), or other less prevalent respiratory ICPC’s (breath problems [R04], sneeze [R07], other symptoms of the nose [R08], symptoms of the throat [R21], abnormal sputum [R25], concern about respiratory illness [R27], acute laryngitis [R77], influenza [R88], other infection of the airways [R83], and other respiratory diseases [R99]).
Respiratory pathogens were detected from the respiratory samples by PCR. After receiving precise instruction at the beginning of the study, parents collected the samples by rubbing one of the nostrils and posterior oropharynx using separate cotton-tipped swabs. After sampling, the 2 swabs were collected into a single vial containing gelatine, lactoalbumine, yeast viral transport (GLY) medium with pimaricin 0.1 mg/mL as viral transport medium and sent to our laboratory via mail. Samples were stored at −20°C until analysis. Sampling of respiratory pathogens by the parents using nose and throat swabs has been shown to be feasible and reliable. Both the sampling frequency and the viral recovery rate in parental samples are at least as efficient compared with sampling by a dedicated research nurse.14
The respiratory pathogens HRV and enterovirus, human metapneumovirus (hMPV), human coronaviruses OC43 and 229E, and Chlamydophila pneumoniae and Mycoplasma pneumoniae were analyzed as described.15
The PCR for adenovirus was performed on conventional PCR (PE 9700) and analyzed by gel electrophoresis.16 The real-time PCR for coronavirus NL63, influenzavirus A and B, RSV A end B was performed using the Lightcycler 2.0 format with Lightcycler Taqman Mastermix (Roche).16 Sensitivity and specificity are state of the art as monitored by Quality Control Molecular Diagnostics (www.qcmd.org) (QCMD) panels.
The independent T test was used to compare the number of reported episodes between children with or without siblings. Kruskal-Wallis test was used to compare the number of reported episodes between children born in different seasons.
Logistic regression was used to investigate the clinical impact of different respiratory pathogens. An infection with a single respiratory pathogen was compared with a single infection with all other pathogens. The clinical impact was assessed using 5 different parameters; duration of the reported respiratory episode, percentage of cough and percentage of wheeze during each episode, the presence or absence of fever, and physician visits related to the respiratory episode. Information of physician visits was available for 95% of all single infections. Results are presented as odds ratios (OR) with their 95% confidence interval (CI) and P value. A P < 0.05 was considered significant. All analyses were performed using SPSS, 2001, version 15.0 (SPSS Institute, Inc, Chicago, IL).
Three hundred five infants participated in this study (152 girls and 153 boys). Of these infants we had a mean documented observed period of 11.5 months (96% follow-up; range: 1–12). These infants reported a median of 5 episodes of respiratory illness per infant/y (range: 1–35; Table 1). Figure 1 (Supplemental Digital Content 1, https://links.lww.com/A954) shows the number of episodes reported by the parents reported throughout the year and the number of samples collected each month. There was no significant difference in number of reported episodes in children with or without siblings (Independent T test, P = 0.23) or children born in the winter compared with other seasons (Kruskal-Wallis test, P = 0.88).
In total, 668 samples were collected. The first sample was generally taken during the first episode at a median age of 4.5 months (interquartile range: 3.0–7.0). Table 2 shows the PCR results of all collected samples. In the majority of the samples 1 or more viruses were detected (85%). HRV was by far the most common pathogen, it was found in 72% of the samples followed by RSV in 11% and coronaviruses in 8% of the samples. Coronavirus OC43 was the most common type of the coronaviruses followed by NL63 and 229E (80%, 18%, and 2%, respectively). For RSV, the types A and B were equally often found (53% and 47%, respectively). The majority of the influenzavirus infections were caused by influenzavirus type A (83% and 17%, for influenza virus type A and B, respectively). Multiple respiratory pathogens were found in 15% of the samples. M. pneumoniae and C. pneumoniae were predominantly found as a coinfection and not as a single pathogen. Adenovirus was exclusively found as a coinfection.
The seasonal distribution of the pathogens is shown in Figure 2. HRV infections occurred throughout the year. RSV infections occurred mainly during the winter months (from November to January). The influenza virus showed a peak incidence in the beginning of the year (late winter) until April.
The clinical impact of the various respiratory pathogens was studied in episodes with single pathogen detections (468 samples). The contribution of days with cough or wheeze within each episode was calculated to correct for the varying duration of the episodes. Further, the presence or absence of fever was reported during each respiratory episode. Details of the respiratory episodes, including duration, type of symptoms, and health care usage, are provided (Table 3). The median duration of symptoms during a pathogen- positive episode was 9.5 days. Cough was almost always present during the entire episode for all respiratory pathogens. Single hMPV infections were observed in only 6 cases. Episodes with hMPV infections were associated with the longest duration and the highest proportion of wheeze. Wheeze was also frequently reported in infections with RSV. Influenza virus was associated with a shorter duration of the episode, yet fever was present during all episodes. The duration of an episode with multiple viral infections was shorter compared with episodes with a single virus infection.
The number of physician visits for respiratory symptoms were recorded during the first year of life. Follow-up data on physician visits were available of 289 (95%) infants; the other (5%) infants had a physician outside the research district. In total, 777 visits were made for respiratory symptoms (range: 0-25 visits), with a median of 1 visit per infant/y. A hundred one visits to the physician with respiratory complaints could be linked to a sampled episode (13% of the physician visits). In 88% (89 of 101) of the samples, which was linked to physician visits, 1 or more viruses were detected. In Table 3, we compared the number of physician visits for the different pathogens. Physician visits occurred most often for infections with RSV, hMPV, and enterovirus. The number of physician visits in episodes with multiple virus infections was comparable to episodes with a single virus.
Finally, we compared the clinical impact of infections with HRV to infections with RSV (Table 4, Supplemental Digital Content 2, https://links.lww.com/A955). The duration of infections with RSV was shorter compared with infections with HRV, this difference was statistically significant (OR: 0.95, 95% CI: 0.91–1.00, P = 0.03). There was no statistically significant difference in the proportion of cough and wheeze during episodes with a RSV and HRV infection. Fever was reported more frequently in episodes with RSV than in episodes with HRV, this difference was borderline significant (OR: 1.96; CI: 0.94–4.09, P = 0.07). More physician visits were linked to episodes with RSV infection than episodes with HRV infection (OR: 2.18; CI: 0.96–4.99, P = 0.06).
This study shows that respiratory viruses and atypical bacteria are frequently found in infants with lower respiratory tract symptoms during the first year of life. HRV was the most frequent detected respiratory pathogen followed by RSV and coronavirus. We observed that infections with RSV were more frequently associated with wheeze, fever, and physician visits compared with infections with HRV. Nevertheless, the high prevalence and duration of HRV infections results in a higher burden of disease compared with RSV infections.
Some methodologic aspects need to be considered. In this large prospective study, viral samples were only taken during respiratory episodes. Recent literature shows that respiratory viruses are also frequently found in children without respiratory symptoms.15,18 Further, molecular detection techniques have increased viral pathogen identification, but it is uncertain whether is represents true infections. A longitudinal study with regular viral sampling regardless of respiratory symptoms could give a more detailed picture of the occurrence of respiratory pathogens in the first year of life. However, besides description of the prevalence of different viruses, the focus of the present study was to compare the relative pathogenicity of different viruses. Although we have no data on the absolute pathogenicity of viruses, the study is useful to compare different viruses.
In the present study, parents sampled their infants during respiratory episodes. In general, the first sample was taken during the first reported episode at a median age of 4.5 months. In a recently published Swiss birth cohort,19 the first acute respiratory tract illness was reported at a median age of 6 months and respiratory samples were collected by research nurses. Even though parent-sampling led to earlier sampling in this study, a proportion of parents did not sample at all. These results show that the method of sampling by either parents or research nurse is never complete and respiratory episodes are missed. Systematic sampling could be essential to further unravel the importance of respiratory pathogens during episodes with symptoms and in episodes without symptoms.
The most common respiratory pathogens we found were HRV, coronavirus, and RSV. HRV was by far the most prevalent virus in this study, as it was identified in nearly three-quarter of the samples. Besides, the contribution of HRV as single virus infection was even higher (85%). Overall, this study confirms the high occurrence of HRVs in respiratory tract infections during the first year of life.3,4 Interestingly, C. pneumoniae, M. pneumoniae, and adenovirus were predominantly found as a coinfection and not as a single pathogen. This result might indicate that the role of these pathogens in young infants with respiratory symptoms is limited.20,21
The clinical impact of the respiratory pathogens was derived from the duration, type of symptoms, and health care utilization. Influenza virus was associated with the shortest duration of illness. All influenza-positive episodes were presented with fever. In the literature, fever is a well known symptom of influenzavirus but usually the duration is also thought to be extended.22 Infections with hMPV resulted in the longest duration of symptoms and the highest proportion of wheeze. Further, a high proportion of the hMPV infections, although the numbers were small, led to a physician visit. These findings were comparable to other studies for the impact of hMPV in children.22,23 Cough was not a good discriminatory symptom as it was reported during the complete episode regardless of type of pathogen. The results show that the pattern of response to a pathogen was too diverse to draw conclusions for the clinical impact of single pathogens.
Until recently, HRVs have been thought to cause only mild symptoms in contrast to RSV, which is seen as a virus causing more severe illness.24 In some studies RSV was detected in up to 80% of the bronchiolitis cases,25 which could be suggestive for a high pathogenicity of this virus. We compared the clinical impact of HRV infections to infections with RSV and observed some differences. HRV infections were associated with a longer period of illness. Infections with RSV, on the other hand, were more often associated with wheeze, fever, and physician visits. This might indicate that infections with RSV cause more severe illness compared with infections with HRV. However, the occurrence of single-HRV infections is almost 13 times greater than to RSV infections. In addition, the absolute number of physician visits made during episodes with a HRV infection is nearly 7 times greater than to RSV infections. So, even though RSV infections might lead to more severe respiratory illness, due to the presenting symptoms, the occurrence, and the duration of HRV infections lead to a higher burden of disease. These results are in line with the growing evidence of the importance of HRV infections in respiratory illnesses. Although HRVs also seem to be associated with recurrent wheezing later on in life,11 more follow-up studies are needed to further investigate the long-term effects of rhinovirus infections early in life.
The authors thank all the parents and children who participated, Khodeza Koppenol-Moeliker and Rolien Bekkema for their dedicated assistance, and Myriam Olling-de Kok for secretarial support and also thank Bianca Zwan and Toyba Yimam for the laboratory assistance (National Institute of Public Health and the Environment, Bilthoven, The Netherlands).
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