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Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness (ARI) in children. Population-based incidence rates and comprehensive clinical characterizations of disease have not been established.
We conducted population-based prospective surveillance for HMPV in two U.S. counties among children <5 years hospitalized with ARI or fever for two years. Nasal/throat swabs were tested for HMPV by real-time RT-PCR and genotyped.
Forty-two of 1104 (3.8%) children tested positive for HMPV. The overall annual rate of HMPV-associated hospitalizations per 1000 children <5 years was 1.2 (95%CI 0.9–1.6). This rate was highest in infants 0–5 months (4.9/1000 [95%CI 2.9–7.2]), followed by children 6–11 months (2.9/1000 [95%CI 1.4–4.7]). The annual rate of HMPV-associated hospitalizations was less than respiratory syncytial virus, but similar to influenza and parainfluenzavirus (PIV) 3 in all age groups. The mean age of children hospitalized with HMPV was 6 months. Bronchiolitis, pneumonia and asthma were the most common diagnoses in children with HMPV. All four HMPV subgroups were detected during both years at both sites. HPMV was most prominent from March through May.
HMPV was detected in 3.8% of children hospitalized with ARI or fever, with a population incidence similar to that of influenza and PIV3.
Human metapneumovirus (HMPV) is a recently identified paramyxovirus that is a major cause of upper (URI) and lower respiratory illness (LRI) in infants and children worldwide [1–7], including among immunocompromised persons and those with other underlying chronic medical conditions [8–15]. HMPV infection appears to be ubiquitous, as virtually all children are seropositive by the age of 5 years [3, 16–18]. Previous estimates of the percent of acute pediatric LRI associated with HMPV range from 5–25% [2, 4, 5, 7, 19–21], with differences likely resulting from various geographic areas studied, seasonal variability in HMPV circulation, methodological differences, and age groups evaluated. Re-infection occurs throughout life, but subsequent infections are more likely to present with upper than lower respiratory symptoms and to be milder in character than primary infection [1, 2].
Despite studies demonstrating the prevalence of HMPV infection, its actual population-wide disease burden is poorly understood. This study had three objectives. First, we sought to establish population-based rates of HMPV-associated infection in hospitalized children <5 years old with acute respiratory symptoms or fever. Second, to compare the burden of HMPV relative to other respiratory viruses. Finally, while earlier studies outlined the clinical characteristics of hMPV on convenience samples, we wished to comprehensively characterize the clinical presentations from a population-wide perspective.
The New Vaccine Surveillance Network (NVSN) was established by the Centers for Disease Control and Prevention (CDC) to determine the burden of acute respiratory illness (ARI) from vaccine-preventable or potentially preventable agents and to monitor the impact of vaccine use on disease rates . Prospective population-based surveillance was conducted among children <5 years of age hospitalized with acute respiratory symptoms or fever from October 1, 2001 through September 30, 2003 in area hospitals that provided care for over 95% of hospitalized children in Davidson County, TN (Nashville) and Monroe County, NY (Rochester). Institutional Review Boards from both study sites, participating hospitals, and CDC approved the study.
The current study used previously published methods to establish the burden of HMPV in hospitalized children [23–25]. Eligible hospitalized children were under 5 years of age and had received an admission diagnosis of fever alone or acute respiratory infection, which was defined as an illness presenting with fever and/or one or more of the following symptoms: cough, earache, nasal congestion, rhinorrhea, sore throat, vomiting after coughing, wheezing, and labored, rapid, or shallow breathing. Children were excluded if they had respiratory symptoms lasting >14 days at the time of admission, neutropenia from chemotherapy, hospitalization elsewhere within the preceding 4 days, or newborns who had been hospitalized since birth. Study nurses enrolled children admitted to surveillance hospitals from Sunday through Thursday, after obtaining written informed consent. Children were eligible for enrollment if they were admitted within 48 hours of an enrollment day. Nasal and throat swabs were obtained from all children on the day of enrollment (thus within 48 hours of admission), combined into a single tube of viral transport medium, and promptly delivered to the research laboratories at the study sites. Demographic and clinical information were collected using a standardized questionnaire as previously described [22, 26]. We performed statistical comparisons between children infected with HMPV alone or other specific viruses alone; children with more than one virus detected were excluded from analysis. Children were considered as virus-negative if their specimens were negative for HMPV, influenza, respiratory syncytial virus (RSV), parainfluenzaviruses (PIV), human rhinoviruses (HRV) and human coronaviruses (HCoV) by subsequently described testing methods. “High risk” conditions were assessed and included: history of asthma, heart disease, sickle cell anemia, cystic fibrosis, diabetes mellitus, and neuromuscular conditions such as seizures, cerebral palsy or muscular dystrophy. “History of asthma” was determined by asking the parents if their child had ever been diagnosed with asthma by any health care provider or by documentation of a previous diagnosis of asthma in the medical record .
Specimens were divided into aliquots and stored at −80°C until processing. RNA was extracted from pooled nasal and throat swab media and tested for HMPV by real-time RT-PCR, as previously described [1, 27]. Samples were also tested for influenza, RSV, PIV, HRV and HCoV by real-time RT-PCR [23–25, 28, 29]. Specimens that tested positive for HMPV were subjected to conventional nested RT-PCR to amplify and sequence cDNA of the F and G genes, as previously described . HMPV subgroup assignment was made based on either the F or G gene, as either reliably identifies viral subgroup . Sequences were edited and aligned with published HMPV sequences obtained from GenBank using the ClustalW algorithm in MacVector version 10.0 (Accelrys). Phylogenetic analyses were performed using MEGA4 . The datasets were resampled with 500 bootstraps, distances were computed using the Maximum Composite Likelihood method, and phylogeny inferred using the Neighbor-Joining method . Analyses using maximum parsimony and minimum evolution methods yielded similar results (data not shown) and so only the Neighbor-Joining analysis is shown.
Laboratory-confirmed HMPV hospitalization rates per 1000 children were calculated as the weighted number of HMPV-positive ARI or fever hospitalizations divided by the number of children in the county population as determined by the 2000 U.S. Census, multiplied by 1000 [22–26, 29]. Rates were calculated by weighting the observed number of enrolled hospitalizations to account for sampling five days per week and eligible patients who were not enrolled. Rates were determined overall and by demographic subsets with 95% confidence intervals calculated using 1000 bootstrap samples.
Kruskal-Wallis or Pearson tests were used as appropriate for contingency table analyses of signs and symptoms associated with HMPV infection alone compared to RSV, influenza, PIV, or HRV alone, or virus-negative children. Influenza included influenza A and B; PIV included PIV types 1, 2 and 3. For comparative analyses, children with coinfections were excluded. Prospectively collected demographic and clinical data were compared among different viral infections and virus-negative children. Age was categorized as one of three groups (<6, 6–23 and >23 months), based on prior studies of respiratory viruses from this population [22, 23, 25, 26, 29]. All analyses were performed using R version 2.6.2.
Of 1298 children admitted to the surveillance hospitals with respiratory symptoms or fever during enrollment days over the two-year period, 1123 (86.5%) were enrolled. Of 175 children not enrolled, 87 (49.7%) had parents/guardians who refused participation; 62 (35.4%) had no parents/guardians available; 16 (9.1%) were discharged before enrollment; and 10 (5.7%) were not enrolled due to missed admission, unavailability of an interpreter, or refusal of the physician to allow participation. Sufficient samples for HMPV testing were available for 1104 of 1123 (98.3%) study children. Thus, a total of 1104 children were included in the study.
Of the 1104 samples tested, 42 (3.8%, 95% Confidence Interval [CI] 2.8–5.1%) were positive for HMPV, with similar rates in both study years (4.2% in 2001–2002 and 3.3% in 2002–2003) and at both study sites. Demographic characteristics of children with HMPV, RSV, influenza, PIV, or HRV alone, or with no virus detected are presented in Table 1. The median age of children testing positive for HMPV alone was 6 months (range 2–13), similar to those with RSV or influenza, while children with PIV or HRV were older. Gender, race, history of prematurity, and insurance status did not differ between groups. Attendance in out-of-home daycare was less common in children with HMPV and influenza when compared to those with RSV, PIV or HRV. Children with HMPV and influenza had slightly higher rates of exposure to household tobacco smoke compared to other groups. Proportions of general “high-risk” status (including asthma) were higher in children with HMPV and HRV, while proportions of underlying asthma alone were similar between children with HMPV, RSV, and no virus identified. Underlying asthma alone was more common in children with HRV and less common in children with influenza.
Population-based rates of HMPV in hospitalized children are presented in Table 2, along with previously published rates for other common viruses in the NVSN cohort during time periods overlapping this study period [23, 25, 29]. The overall annual rate of HMPV-associated hospitalizations was 1.2 (95% CI 0.9–1.6) per 1000 children <5 years of age. Age-specific rates per 1000 children were highest among younger children: 4.9 (95% CI 2.9–7.2) for 0–5 months of age, 2.9 (95% CI 1.4–4.7) for 6–11 months of age, 0.7 (95% CI 0.1–1.4) for 12–23 months of age, and 0.4 (95% CI 0.1–0.7) for 24–59 months of age. The incidence of HMPV was highest in the 0–11 month old age groups, though RSV remained more common than HMPV in these age groups. The incidence of HMPV in other age groups was similar to that of influenza and PIV3.
The number of all samples collected (including HMPV positive and negative) peaked during January, with 55% of all specimens collected between October 1 and February 28 (Figure 1). However, detection of HMPV in samples peaked slightly later in both years, with 80% of all HMPV infections occurring between February 1 and May 31. The proportion of HMPV positive subjects by month varied from zero during the summer months to a peak of 15% during April.
Clinical features of children with HMPV and other viruses are shown in Table 3. The majority of HMPV-infected children had fever, rhinorrhea, cough, poor feeding, or difficulty breathing. Although most of these symptoms were also present in children with other respiratory viruses, fever was less common in children with HMPV than influenza. Conversely, difficulty breathing and wheezing were more common in HMPV-infected children than in those with influenza or with no virus detected. Sore throat was most common in children with HMPV or PIV. Thirty-eight percent of the HMPV infected children required supplemental oxygen and 2% were treated in the intensive care unit, similar to proportions with other viruses. No child with HMPV in this study died, and the mean duration of hospitalization was 2 days, similar for all viruses. Thirteen children with HMPV had co-detection of other viruses: these included six with HRV, four with influenza virus, two with RSV, and one with PIV-3. The most common discharge diagnoses among children with HMPV only were bronchiolitis (38%), pneumonia (24%), asthma (24%), and URI (3%). Bronchiolitis was most common in children with HMPV and RSV, while pneumonia was more common in HMPV-infected children. A discharge diagnosis of asthma was common in all groups but was less frequent in children with influenza (9%) and most frequent in HRV-infected children (46%). Other diagnoses included croup in a substantial number of children with influenza and PIV [25, 29], and febrile illness alone in children with no respiratory virus identified.
HMPV positive specimens were tested by RT-PCR for the F and G genes and 27 (61%) were genotyped; not all specimens were successfully amplified for F or G by RT-PCR due to RNA degradation. All four subgroups of HMPV were detected in both years; overall, A1 was the most prevalent (44.4%), followed by A2 (29.6%), B1 (18.5%) and B2 (7.4%). However, the distribution of subgroups varied between years, with A1 dominant in Year 1 and A2 and B1 detected equally in Year 2 (Figure 2). There were no differences in subgroups by site. No differences in disease severity were noted among the subgroups (data not shown). Phylogenetic analysis showed genetic diversity even within each of these subgroups with analysis of the F gene showing two sublineages of the A2 subgroup (Figure 3A), and analysis of the G sequences showing two sublineages of the B1 subgroup (Figure 3B). Both of these sublineages circulated during the same season and at the same study site and thus were not unique to either site or year.
We used prospective, population-based, laboratory-confirmed active surveillance to define the burden of HMPV in hospitalized young children. The contribution of HMPV to pediatric hospitalization for ARI or fever was substantial with 1.2 hospitalizations per 1000 children <5 years old per year (95% CI, 0.9–1.6) compared with 3.0 per 1000 (95% CI, 2.8–3.4) for RSV, 0.9 per 1000 (95% CI, 0.8–1.1) for influenza virus, and 0.5 per 1000 (95% CI, 0.4–0.6) for PIV3 [23, 25, 29]. The rate of HMPV-associated hospitalization was highest in infants 0–5 months old at 4.9 per 1000 (95% CI, 2.9–7.2), compared to 16.9 per 1000 (95% CI, 15.3–18.5) for RSV, 4.5 per 1000 (95% CI, 3.4–5.5) for influenza virus, and 1.6 per 1000 (95% CI, 1.0–2.2) for PIV3 [23, 25, 29]. These data suggest that HMPV causes hospitalizations for respiratory symptoms and fever in children <5 years old at rates similar to those of influenza and parainfluenzaviruses. Extrapolating from US census data, our data would project approximately 27,000 HMPV hospitalizations in children <5 years of age annually, underscoring the impact of HMPV on children. HMPV is rarely detected in asymptomatic young children [2, 3, 32, 33].
Although there have been reports of HMPV disease in persons with underlying medical conditions such as prematurity or asthma, few of these studies have been population-based and have enrolled this large number of children [8, 11–15, 19, 34, 35]. One-third of the hospitalized children with HMPV in our study had high-risk conditions, suggesting that these children are at higher risk for hospitalization with HMPV infection. However, two-thirds of HMPV hospitalizations occurred in otherwise healthy children. We did not observe an association between HMPV hospitalization and a history of asthma, in contrast to the association between asthma and human rhinoviruses that has previously been reported in this cohort [24, 36]. Consistent with previous reports, HMPV was associated most frequently with the diagnosis of bronchiolitis [2, 5, 7, 19, 21]. The presenting symptoms of HMPV in this cohort generally were not distinct from those children with other viruses, consistent with other reports [1, 2, 5, 7, 19, 21, 37, 38]. However, very few HMPV-infected children (5%) had fever alone without respiratory symptoms.
HMPV was most prominent at both study sites during the late winter and spring months with peak illness rates in March, April and May. Overall, HMPV accounted for up to 15% of all hospitalizations for children less than five years with ARI or fever with a late spring distribution and was more common than influenza or RSV during this time period [23, 25]. However, the lack of distinctive clinical symptoms and the overlap with other community respiratory viruses highlights the potential utility of rapid sensitive diagnostic tests such as RT-PCR to detect HMPV and other respiratory viruses.
We identified all four subgroups of HMPV during each year and at each study site, although the distribution of subgroups varied by year. The extent of antigenic variability between HMPV subgroups is not clear in animals or in humans [30, 39–41], but the presence of multiple HMPV strains in a single season could affect the design of vaccines or prophylactic antibodies if antigenic variability leads to partial immune escape. We did not find that specific subgroups were associated with more severe disease (data not shown); some studies have suggested this phenomenon , while others have not . Correlation between infecting subtype and disease severity has been shown for RSV [44, 45]. A postulated mechanism for alternating circulation of different HMPV subgroups is population immune pressure [46, 47]. Long-term, prospective, population-based surveillance is needed to establish whether different subgroups of HMPV vary in virulence and whether circulation patterns are the result of immune pressure.
Despite the strength of our prospective population-based surveillance system, our study has limitations. First, surveillance was performed for only two years at only two geographically distant sites (representing two US regions, Northeast and South). Greater regional and temporal differences in viral circulation may have been evident if the study included more years or sites, since others have reported substantial year-to-year variability in HMPV prevalence . Second, institutional differences in medical practice might have contributed to variation in HMPV hospitalization rates, and although enrolled and non-enrolled children did not differ in demographic characteristics, unknown biases may have affected our HMPV burden estimates. Coinfections were present in a minority of children, but the clinical importance of these coinfections is not clear. HRV is identified frequently in asymptomatic individuals and thus especially difficult to interpret as a co-detected virus [48–50]. A large number of children in the study did not have a virus identified by the testing used; this could be due to false negatives or the presence of other pathogens not tested for. Nearly all of the HMPV-infected children had ARI symptoms and not fever alone, but we enrolled children with fever alone in addition to children with ARI. Thus, since the population-based incidence was calculated using all hospitalized children including those with fever alone, we may have underestimated the true incidence of HMPV (and other viruses) in children with ARI. Finally, this study only assessed hospitalized children and the impact of HMPV on emergency department and outpatient visits is unknown.
HMPV is a leading cause of hospitalization for ARI in children less than five years of age, with a population-based incidence similar to that of influenza and PIV3. The majority of HMPV-associated hospitalizations occurred in otherwise healthy children. These data suggest a need for preventive and therapeutic strategies for HMPV and highlight the need to consider HMPV as a cause of ARI among hospitalized children, especially in the spring.
Supported by NIH R03 AI-054790 to JVW and a MedImmune research grant to JEC. The project was supported in part through Cooperative Agreements with the Centers for Disease Control and Prevention (Vanderbilt University U38/CCU417958 and U01/IP000022 and University of Rochester U38/CCU217969 and U01/IP000017).
Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the CDC.
We thank the children and their parents who participated in this study and all of the members of the New Vaccine Surveillance Network. NVSN ARI inpatient study collaborators were as follows. Universityof Rochester: Linda Anderson, Charlene Freundlich, Gladys Lathan, Gerry Lofthus, Andrea Marino, Rebecca Martinez, Kenneth Schnabel, Lynne Shelly, Jennifer Carnahan and Christina Albertin; Vanderbilt University: Diane Kent, Ann Clay, Ayesha Khan, Nayleen Whitehead, Amy Podsiad, and Jody Peters; and CDC: Larry Anderson, John Copeland, Marika Iwane, Ben Schwartz, and Fran Walker.
Conflict of interest information:
John V. Williams has served as a consultant for MedImmune and Novartis.
Kathryn M. Edwards receives research funding from sanofi-pasteur, Wyeth, Novartis, and CSL.
Geoffrey A. Weinberg has served as a consultant for MedImmune.
Marie R Griffin has research funding from MedImmune and Merck.
Caroline B. Hall has served as a consultant for and had research support from MedImmune.
James E. Crowe, Jr. has served as a consultant for Anaptys, Immunobiosciences, Mapp, MedImmune and Novartis and has had research support from MedImmune, Mapp, Alnylam, and sanofi Pasteur.
Chiaoyin K. Wang, Chin-Fen Yang, David Silva, Dan Ye, Marla Chu, Alexis Ta, and Richard R. Spaete were employees of MedImmune at the time of this study.
Role of the industry collaborator: MedImmune provided research support to JEC. De-identified specimens were shipped to MedImmune, who performed extraction, real-time RT-PCR partial sequencing. Results of real-time HMPV testing and sequencing and extracted nucleic acids were returned to Vanderbilt. The study was designed, data analyzed and paper written by the Vanderbilt and NVSN authors.