Study Participants and Study Protocol

This prospective study was conducted in Turku, Finland, from October 9, 2000, through May 20, 2001. The participating children were recruited before the start of the respiratory season in daycare centers, family daycare, and schools in our area (22). All children <13 years of age were eligible for participation; no exclusion criteria were used. Of 1,458 children initially enrolled, 1,338 were closely monitored throughout the entire follow-up period. The baseline characteristics of participating children are shown in Table 1.

Parents were asked to bring their children to the study clinic for examination by a study physician whenever fever or signs of respiratory tract infection appeared. The study clinic was open every day, and all visits were free. In addition to full clinical examination, chest or sinus radiographs were obtained if pneumonia or sinusitis was suspected on the basis of clinical symptoms. Acute otitis media (AOM) was diagnosed by pneumatic otoscopy, aided by routine use of tympanometry and spectral-gradient acoustic reflectometry. Children without any complications at the first visit were reexamined after 5–7 days, or whenever the parents deemed it necessary.

The parents were provided with a symptom diary in which they recorded daily the child’s symptoms and absences from daycare or school and the parents’ absences from work because of the child’s illness. Only actual days lost were recorded. Thus, days of illness occurring on free weekends or other days off were not recorded as causing absenteeism.

Viral Sampling

At each visit for a new respiratory tract infection, a nasal swab was obtained from a depth of 2–3 cm in the nostril by using a sterile cotton swab that was then inserted into a vial containing viral transport medium (23). The specimens were kept in a refrigerator and transported daily to the laboratory at the Department of Virology, University of Turku, where they were subjected to viral culture for influenza viruses, parainfluenza viruses, respiratory syncytial virus, and adenovirus, and PCR assays for rhinoviruses and enteroviruses. The specimens were then frozen at –70°C until thawed for the purposes of this study.

Detection of hMPV

RNA was extracted from nasal swab specimens by using a High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland) according to the manufacturer’s protocol. hMPV was identified in specimens by using reverse transcription–PCR (RT-PCR). Briefly, hMPV was amplified in a 1-step RT-PCR. The RT-PCR mixture contained RT-PCR buffer (50 mmol/L Tris-HCl, pH 8.4, 50 mmol/L NaCl, 4 mmol/L MgCl₂), 0.5 mmol/L MgCl₂, 2 mmol/L dithiothreitol, 0.6 mmol/L deoxynucleoside triphosphates, 1 μmol/L of each primer, 20 U Maloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA), 4 U Recombinant RNAsin ribonuclease inhibitor (Promega), and 1 U DyNAzyme (Finnzymes, Espoo, Finland). The total volume of the reaction mixture was 50 μL and contained 5 μL of extracted RNA. The RT-PCR for hMPV RNA was conducted at 42°C for 45 min, then at 95°C for 7 min. cDNA amplification consisted of 40 cycles (denaturation at 95°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 1 min) and final extension at 72°C for 10 min. Primers for hMPV were from the L gene (10), and the forward primer was biotinylated.

Biotinylated hMPV RT-PCR products were detected by using a liquid hybridization assay. In this assay, 10 μL of each RT-PCR product was mixed with 50 μL of DELFIA assay buffer (PerkinElmer Finland Oy, Turku, Finland), added to 3 parallel microtitration wells coated with streptavidin (PerkinElmer Finland Oy), and incubated at room temperature for 30 min. Wells were washed at room temperature with Tris-NaCl buffer containing 0.5% Tween 20, denatured for 5 min with 150 μL denaturation buffer (25 mmol/L NaOH and 5 mmol/L EDTA), hybridized with Eu-labeled hMPV-probe (5′-CTG TTA ATA TCC CAC ACC AG-3′) at 40°C for 2 hours. The hybridization was performed with 100 μL of hybridization solution, in which 2 ng Eu-labeled hMPV probe per well was diluted with DELFIA assay buffer that included 0.85 mmol/L NaCl and 0.1% Tween 20. Unspecific hybridizations were removed with hot (50°C) Tris-NaCl buffer containing 0.5% Tween 20. To enhance fluorescence, 200 μL DELFIA Enhancement solution (PerkinElmer Finland Oy) was added per well and incubated for 10 min with shaking. Fluorescence was measured by using a Victor 1420 Multilabel Counter (PerkinElmer Finland Oy).

Definitions

AOM was diagnosed by signs of inflammation of the tympanic membrane, the presence of middle ear effusion, and >1 signs of acute infection. The diagnosis of pneumonia was based on radiologic confirmation of the condition. Both complications were associated with hMPV infection if they were diagnosed <14 days after the clinical visit at which the hMPV-positive specimen was obtained.

Ethics

The study protocol was reviewed and approved by the Ethics Committee of Turku University Hospital. Written informed consent was obtained from the parents of all participating children.

hMPV Outbreak

Figure 1

Figure 1. Number of children with human metapneumovirus infections during each week of the study period, Finland, 2000–2001.

The first case of hMPV infection in the study cohort was identified during the week of December 4, 2000, and the last case was identified during the week of April 23, 2001 (Figure 1). hMPV infections were diagnosed weekly over 14 consecutive weeks from January 8, 2001, through April 15, 2001. Overall, hMPV infection was diagnosed in 47 children (26 boys and 21 girls). The median age of the children was 3.0 years; 81% were <5 years of age.

Incidence Rates of hMPV Infection in Different Age Groups

The incidence of hMPV infection was highest (7.6%) in children <2 years of age at the start of the respiratory season (Table 2). Of a subset of 30 children <1 year of age, 3 (10.0%) acquired hMPV infection during follow-up. The incidence rates of hMPV decreased gradually with age. The overall incidence of hMPV infection in the study cohort of 1,338 children was 3.5% (95% confidence interval [CI] 2.5%–4.5%).

Relative Effect of hMPV among All Respiratory Infections

The relative proportion of hMPV infections among all respiratory infections was greatest in children <2 years of age, in whom hMPV accounted for 1.7% of all respiratory infections during the winter season (Table 3). During the 14-week period of major hMPV circulation, hMPV accounted for 4.2% of all infections in children <2 years of age. The relative effect of hMPV decreased with age. In the entire cohort of 1,338 children, hMPV accounted for 1.3% (95% CI 0.9%–1.7%) of all respiratory infections during the whole respiratory season and 2.7% (95% CI 1.9%–3.5%) of all infections during the 14-week period of continuous hMPV circulation.

Figure 2

Figure 2. Weekly proportions of human metapneumovirus infections among all respiratory infections in the study children, Finland, 2000–2001.

The proportion of hMPV infections among all respiratory infections during each week of the study is shown in Figure 2. The relative effect of hMPV was highest during the week of February 19, during which the virus accounted for 7.1% of all respiratory infections in the study cohort.

Co-infections with hMPV

Another virus was detected concomitantly in 8 (17%) of 47 hMPV-infected children. Three children had enterovirus, 2 had rhinovirus, and 1 each had influenza virus, parainfluenza virus, and a nontypeable picornavirus together with hMPV.

Clinical Characteristics of hMPV Infection

The clinical findings of hMPV infection were analyzed in 39 children in whom hMPV was the only virus detected (Table 4): 97% had a cough, 90% had rhinitis, and 72% had a fever. The median duration of symptomatic illness was 8 days. AOM was the most frequently diagnosed complication; it occurred in 61% of children <3 years of age. Wheezing was observed in 10% of the hMPV-infected children and laryngitis in 8%. A total of 38% of the children were treated with antimicrobial drugs. None of the children was referred to a hospital.

Socioeconomic Effect of hMPV

Overall, 54% of hMPV-infected children were absent from daycare or school for >1 day (Table 4). The mean duration of their absence was 3.3 days. In 38% of cases, a parent missed >1 day of work because of the child’s hMPV illness. The average duration of parental work loss was 2.9 days.

Persistence of hMPV RNA in Nasal Swabs after Acute Illness

In 27 (57%) of the 47 children with hMPV infection, a follow-up nasal swab was obtained during a subsequent respiratory infection. The median interval between the initial hMPV-positive specimen and the subsequent sample was 42 days (range 7–82 days). hMPV could not be detected in any of the subsequent samples after hMPV illness.