As part of a randomized clinical trial evaluating the efficacy of systemic corticosteroid in the treatment of acute expiratory wheezing in children, we investigated the viral etiology of the infections. From September 1, 2000, through May 31, 2002, a total of 293 children participated in the study in the Department of Pediatrics, Turku University Hospital. Study breaks occurred from June to July 2001 and during Christmas week 2001. Inclusion criteria were the following: age from 3 months to 16 years, hospitalization for acute expiratory wheezing, and written informed consent from the parents. Exclusion criteria were the following: chronic diseases other than asthma or allergy, systemic glucocorticoid treatment within 4 weeks before the study, severe wheezing necessitating intensive-care unit treatment, and previous participation in this study. The study protocol was approved by the Ethics Committee of the Turku University Hospital.

Acute expiratory wheezing was called bronchiolitis when it occurred in children <3 years of age. When it recurred >2 times in persons of any age or occurred in persons >3 years of age, the diagnosis of asthma was used (7). To some extent, bronchiolitis and asthma are expressions of the same pathologic process, and no rigid criteria separate these illnessess. All patients were examined by one of the two study physicians (T.J. and P.L.).

On patient's admission, a nasopharyngeal aspirate sample was taken through a nostril by inserting a disposable catheter connected to a mucus extractor to a depth of 5 to 7 cm and retracting it slowly while applying gentle suction with an electric suction device. All specimens were obtained without inserting any solution into the nostrils. Disposable plastic gloves were used, and all surfaces were wiped with disinfectant to prevent contamination. Immediately after the secretion was suctioned, two sterile cotton swabs were dipped in the aspirate. The swabs were then placed in vials containing 2 mL of viral transport medium (5% tryptose phosphate broth, 0.5% bovine serum albumin, and antimicrobial agents in phosphate-buffered saline) for virus culture and PCR assays. The rest of the mucus was used for virus antigen detection. The specimens were transported to the laboratory on the same day at room temperature. The tubes for RSV and HMPV PCR assays were frozen at –70°C before processing. Blood samples were collected on patient's admission and 2–3 weeks after discharge from the hospital.

Viral antigens for adenovirus; influenza A and B viruses; parainfluenza virus types 1, 2, and 3; and RSV were detected by time-resolved fluoroimmunoassay (8). Immunoglobulin (Ig) G antibodies to the same viruses were measured from paired serum samples by enzyme immunoassays as described earlier (9–11). Purified heat-treated coxsackievirus A9, coxsackievirus B3, echovirus 11, and poliovirus 1 were used as an antigen mixture in enterovirus IgG assays and purified heat-treated coxsackievirus A16, coxsackievirus B3, and echovirus 11 in IgM assays (12). Virus culture was performed according to routine protocols in A549, HeLa, and LLC-Mk2 cell lines and human foreskin fibroblasts (13). The supernatants of cell cultures exhibiting a cytopathogenic effect were further studied by antigen detection for adenovirus; influenza A and B viruses; parainfluenza virus types 1, 2, and 3; and RSV or by reverse transcription (RT)-PCR for enteroviruses and rhinovirus. Nucleic acids for RT-PCR were isolated from the nasopharyngeal samples with a commercial kit (High Pure Viral Nucleic Acid Kit, Roche Diagnostics, Mannheim, Germany) according to the manufacturer's instructions. RT-PCR was used to detect enteroviruses and rhinovirus, coronavirus, RSV, and HMPV, as described previously (6,14,15). A case was defined as virus positive if at least one of the tests used was positive for virus. The rates of HMPV, respiratory picornaviruses, and RSV detected during the first study season 2000–2001 have been published (16).

The chi-square test was used for intergroup comparisons of differrent age groups in specific virus groups. The results were analyzed by using SAS software (version 8.2, SAS Institute, Cary, NC).

From September 2000 through May 2002, a total of 661 children were hospitalized for acute expiratory wheezing (Figure 1). Of the 661 patients, 341 did not meet the study criteria: 87 had already participated in the study, 79 were <3 months of age, 55 were not enrolled during study breaks, 48 had had systemic glucocorticoid treatment within 4 weeks, 24 did not need hospitalization, 17 had a chronic disease, 12 had guardians with language difficulties, 11 needed treatment in our intensive care unit, 3 had guardians who were not present, 2 were exposed to varicella, 2 patients' cases were not reported to the study physician, and 1 child was not eligible because of social reasons. The remaining 320 were eligible, but the parents of 27 (8%) children did not give their consent for participation in the study. Eventually, 293 children participated in the study.

The median age of the 293 study patients was 1.6 years (range 3 months–15.2 years). Seventy-six (26%) children were <12 months of age, 152 (52%) children were 12–35 months old, and 65 (22%) children were >3 years. In 179 children, the clinical diagnosis was bronchiolitis and in 114, acute asthma. Of the children with asthma, 49 were <3 years of age, 53% were boys, 38% experienced atopy, and 41% had parents who smoked.

A potential causative viral agent was detected in 88% of the cases (Table 1). RSV (27%), enteroviruses (25%), and rhinovirus (24%) were the most common causative agents, resulting in 31%, 28%, and 28% of 258 virus-positive cases, respectively. The viruses in samples identified by the primary picornavirus PCR test but not identifiable in the liquid-hybridization assay were named rhino/enteroviruses (16%). According to our sequence data, these amplicons have shown >90% homology to human rhinoviruses. The remaining eight viruses studied accounted for 18% of the cases, and none of these viruses was detected in >5% of all cases.

Mixed viral infections were found in 57 (19%) cases and were usually associated with respiratory picornaviruses. Coinfection with enteroviruses and RSV was the most common mixed infection (19%), followed by rhinovirus and RSV (14%), rhino/enterovirus and RSV (11%), and enteroviruses and rhinovirus (9%). Of 12 HMPV infections, 5 were associated with other respiratory viruses.

Most of the viruses (84%), respiratory picornaviruses especially, were detected by using PCR (Table 1). Rhinovirus was cultivated in 25 (38%) of 65 specimens with PCR-positive results, enteroviruses in 14 (24%) of 59 specimens with PCR-positive results, and rhino/enterovirus in 1 (2%) of 46 specimens with PCR-positive results. To compare different methods of detecting RSV infection, we selected the patients whose samples were studied with four methods (n = 257). The recovery rate of RSV by IgG serologic testing was 22%; by virus antigen detection, 21%; by virus culture, 20%; and by PCR, 18%.

Typical of the situation in Finland, a minor RSV epidemic occurred during the spring of 2000, followed by a major epidemic during the winter of 2001 to 2002 (Figure 2). Enterovirus outbreaks were seen during the fall in both 2000 and 2001. Rhinovirus outbreaks occurred during fall and spring of both years. An HMPV epidemic was seen during the winter of 2001. HMPV was detected in 30% of the study children during the 3-month epidemic. During the peak 3 epidemic months of respiratory picornaviruses, from September to November 2000, they accounted for 82% of all cases, and only 5% had other viral causes. During the peak 3 epidemic months of RSV, from November 2001 through January 2002, RSV accounted for 65% of all cases, and other viruses were found in 20%. Influenza A virus epidemics occured in the community from the beginning of October 2000 to the end of March 2001 and from October 2001 to May 2002 (data not shown), but influenza A virus caused only three cases of acute expiratory wheezing for which the patient had to be hospitalized.

RSV (54%), respiratory picornaviruses (42%), and HMPV (11%) were the most common viruses in infants (Figure 3). Respiratory picornaviruses were detected in 65% and RSV in 22% of the cases in children ages 12–35 months. In children aged >3 years, respiratory picornaviruses (82%) were found most frequently.

Comparisons between age groups showed that RSV (p < 0.001) and HMPV (p = 0.0030) infected infants significantly more often than children in other age groups, adenovirus infected children ages 1–2 years significantly more often (p = 0.022), and enteroviruses infected children ages >3 years more often (p = 0.0018; Figure 3). No other significant differences were found.