Studies based on polymerase chain reaction (PCR) for the detection of respiratory viruses have established that human rhinoviruses (HRV) of species A, B, and C are the most frequent agents of acute respiratory infections (ARI) in all age groups.1 Moreover, these agents are also associated with up to 70% of virus-related wheezing exacerbations.2 The most recently identified HRV species C has been detected in association with bronchiolitis, wheezing, and asthma exacerbations requiring hospitalization.3 However, most studies focusing on HRV-C have been hospital-based, or restricted to inpatients, a selection that may have narrowed the perception of the clinical spectrum that may be caused by HRV-C. We have assessed the frequencies of HRV species in children seen as outpatients at a primary care facility in Sao Paulo, southeast Brazil.
PATIENTS AND METHODS
This study was done with stored nasal aspirates collected from March to December 2008 as part of a previous prospective study of ARI in children of less than 12 years of age (median, 4 years) without any underlying disease (eg, asthma), in the city of São Paulo. Children were seen as outpatients at a primary care facility by a pediatrician who, upon a clinical diagnosis of ARI, filled out a form with clinical and epidemiologic information, and obtained an informed consent from parents or guardians. The follow-up of all patients was accompanied by the physician in subsequent office visits, or by telephone, with data entered on the form, until the resolution of respiratory episode. The median time from the onset of symptoms to the collection of nasal washings was 3 days (1–12 days).
Viral RNA was extracted using QIAamp Viral RNA extraction Kit (Qiagen, Hilden, Germany), according to manufacturer's instructions. Amplifications of HRV 5′ untranslated region and VP4/VP2 gene were performed by the reverse transcriptase PCR assay as described previously,4 with minor modifications. Positive (HRV type 39 stock) and negative controls were included in all tested sample batches. The assay was standardized to detect HRV RNA equivalent to 10−3.25 TCID50 of HRV 39.
DNA sequencing was done with BigDye Terminator Cycle Sequencing Ready Reaction Kit, according to the manufacturer's instructions (Applied Biosystems, Foster City, CA), using the same primers as in the PCR. Phylogenetic relationships were assessed by maximum likelihood by the PhyML software.5 Trees were replicated 100 times to provide bootstrap support for clades. The analyses were performed using the software Topali v2.5,6 comparing with HRV sequences available in GenBank (National Center for Biotechnology Information).
Positive samples for different HRV species were distributed in 2 categories, according to the presence of wheezing. χ2 (statistical package for social sciences [SPSS] v11.5, SPSS Inc., Chicago, IL) and Fisher exact test (GraphPad InStat v3.06 software) were applied when appropriate. Data on sample positivity for respiratory syncytial virus (RSV) and adenovirus (AdV) by immunofluorescence (SimulFluor Respiratory Screen Kit, Chemicon, US) and for human bocavirus (HBoV) by PCR7 were reviewed to assess the frequency of coinfections.
Samples from 120 patients (1 sample per patient) were analyzed. Although most patients had symptoms of upper ARI, fever was present in 50.8% (61/120) and wheezing in 47.5% (57/120) of them. Rhinovirus RNA was detected in 46.7% of the patients (56/120), and the median age of HRV-positive patients was 3 years (ranging from 1 month to 10 years). Of the HRV positive samples, wheezing was present in 51.8% (29/56), dyspnea in 41.1% (21/56), fever in 41.1%, and all 3 symptoms simultaneously in 33.9% (19/56). Coinfection with other respiratory viruses was found in 14.3% (8/56) of the samples: 7.3% (4/56) with RSV, 5.3% (3/56) with HBoV, and 1.8% (1/56) with AdV.
Partial genome sequencing revealed that of the 56 HRVs detected, 12 (21.4%) were HRV A, 6 (10.7%) were HRV B, and 33 (58.9%) were of HRV C. Therefore, HRV C was detected in 27.5% (33/120) of the study patients.
Five HRVs could not be identified by the partial genome sequencing performed (1 from a wheezing child and 4 from nonwheezing children). Of the 28 wheezing children from whom HRV was sequenced, 60.7% (17/28) were infected with HRV C, 28.5% (8/28) with HRV A, and 10.7% (3/28) with HRV B. In the wheezers group, 13.8% of patients (4/29) were coinfected with RSV and 6.9% (2/29) with HBoV. Of the 23 HRVs obtained from nonwheezing patients, 69.5% (16/23) were HRV C, 17.4% (4/23) were HRV A, and 13% (3/23) were HRV B. Coinfections with HBoV and AdV were detected with the same frequency, 3.7% (1/27), in the nonwheezing group. Differences in frequencies of the 3 HRV species between patients with and without wheezing were not significant. When patients were stratified into 8 age groups, HRV C was the predominant species in all age groups between 6 months and 5 years of age (Fig. 1). Association between HRV species frequency and age was performed in both, wheezer and nonwheezer groups. There was no significant association of wheezing with any of the 3 HRV species in any of the age groups.
In this outpatient study, HRV C was the most frequently detected HRV species in association with ARI in children, including the very young. All ARI episodes in this study had a favorable outcome, none of the patients required hospitalization and no difference was detected between wheezers and nonwheezers regarding the frequencies of HRV species. These results are in contrast with those from previously published studies, in which HRV C infections were frequent in hospitalized children with respiratory diseases and were associated with asthma, recurrent wheezing, and bronchiolitis.8 However, most studies incriminating HRV C as a frequent cause of severe lower respiratory tract ARI and asthma have been hospital-based, in which very young children may have been overrepresented, thereby introducing a possible selection bias.9–11 In this regard, Calvo et al (2010)8 described that HRV C infections were not associated with more severe disease than HRV A infections in hospitalized patients. Assessment of the full range of clinical manifestations of HRV species in children must include not only patients at different levels of health care, but also in the households. Therefore, HRV species determination in samples obtained in community-based studies should be done to address this issue.
1. Gern JE. The ABCs of rhinoviruses, wheezing
, and asthma. J Virol
2. Friedlander SL, Busse WW. The role of rhinovirus
in asthma exacerbations. J Allergy Clin Immunol
3. Miller EK, Edwards KM, Weinberg GA, et al. A novel group of rhinoviruses is associated with asthma hospitalizations. J Allergy Clin Immunol
4. Savolainen C, Mulders MN, Hovi T. Phylogenetic analysis of rhinovirus
isolates collected during successive epidemic seasons. Virus Res
5. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol
6. Milne I, Wright F, Rowe G, et al. TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments. Bioinformatics
7. Allander T, Tammi MT, Eriksson M, et al. Cloning of a human parvovirus by molecular screening of respiratory tract samples. Proc Natl Acad Sci U S A
8. Calvo C, Casas I, García-García ML, et al. Role of rhinovirus
C respiratory infections in sick and healthy children in Spain. Pediatr Infect Dis J
9. Calvo C, Garcia ML, Pozo F, et al. Role of rhinovirus
C in apparently life-threatening events in infants, Spain. Emerg Infect Dis
10. Piotrowska Z, Vázquez M, Shapiro ED, et al. Rhinoviruses are a major cause of wheezing
and hospitalization in children less than 2 years of age. Pediatr Infect Dis J
11. Arden KE, Mackay M. Newly identified human rhinoviruses: molecular methods heat up the cold viruses. Rev Med Virol