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Departments of Pediatrics, Microbiology and Immunology, Medicine
Preventive Medicine, Vanderbilt University Medical Center, Nashville, Tennessee
Division of Clinical Immunology, Johns Hopkins University School of Medicine, Baltimore, Maryland
We determined the prevalence of human metapneumovirus (hMPV) infection in adults with asthma who were prospectively enrolled after hospitalization for an acute asthma exacerbation. Nasal wash specimens collected at admission and 3 months after discharge were tested for hMPV by real-time reverse-transcription polymerase chain reaction assays. hMPV was detected in 7 (6.9%) of 101 subjects at hospitalization and in 1 (1.3%) of 75 subjects at follow-up (odds ratio, 7 [95% confidence interval, 0.9312]; P = .03). None of the patients with hMPV infection at hospitalization tested positive at follow-up, strongly suggesting that hMPV plays a direct etiologic role in acute asthma exacerbations.
Human metapneumovirus (hMPV) is a recently identified paramyxovirus that causes respiratory disease in adults and children worldwide [15]. Limited data have suggested that there is an association between hMPV infection and asthma in children [57], and studies have shown that other respiratory virusessuch as respiratory syncytial virus (RSV), influenza virus, and rhinovirusesare associated with acute asthma exacerbations in adults [811]. However, there have been no published studies of hMPV infection in adults with asthma to date. In the present study, we prospectively recruited adults who were hospitalized with an acute asthma exacerbation for evaluation at both hospitalization and a follow-up visit 3 months after discharge. Thus, our study offered a unique opportunity to test for an association between hMPV infection and asthma at 2 time points in the context of subjects who were acutely ill or convalescent.
Subjects, materials, and methods.
The present investigation was a prospective observational study of 101 individuals 18 years old who were hospitalized for an acute asthma exacerbation at the Vanderbilt University Medical Center from December 1999 to December 2003. The subjects were initially identified by a presumptive admission diagnosis of an asthma exacerbation. Charts on these subjects were then reviewed, to both confirm the diagnosis of asthma and assure that the hospitalization was for asthma. Subjects were excluded if they had a life expectancy of <6 months, congestive heart failure or other active chronic pulmonary disease, or previous enrollment in the study. All eligible subjects were approached for study inclusion on weekdays. A study nurse completed a standardized form that recorded clinical symptoms, medical history, and smoking history. Chronic asthma severity was measured by use of the Johns Hopkins asthma severity score, for which lower scores indicate milder disease [12]. Nasal wash and serum specimens were collected from all subjects, who underwent a physical examination and spirometric testing (forced expiratory volume in 1 s [FEV1]). Subjects were followed during their hospitalization and were seen as outpatients at a follow-up visit at the Vanderbilt University Medical Center General Clinical Research Center 3 months after discharge from the hospital for their acute asthma exacerbation, at which time they underwent a similar evaluation, which included repeated nasal wash specimen collection and spirometric testing. The Vanderbilt University Institutional Review Board approved the present study, and all subjects signed an informed consent form.
Nasal wash specimens were aliquoted and then snap-frozen at -80°C. RNA was extracted by use of phenol-chloroform (TRIzol; Invitrogen) and was reverse transcribed into cDNA by use of Superscript II (Invitrogen) and random primers. Polymerase chain reaction (PCR) was performed with primers specific for the polymerase gene, which is highly conserved among hMPV isolates [1, 5]. hMPV was also tested for by a real-time PCR assay on a Smart Cycler (Cepheid), with nucleocapsid-specific primers and probe [13] and the QuantiTect PCR Kit (Qiagen). The probe used black hole quencher3 as the 3 quencher. These primers and probe have been shown to detect all 4 genetic lineages of hMPV [13], and our lower limit of detection was 50 viral-genome copies/reaction. Specimens were considered to be positive only if they were positive by both PCR assays, which included negative controls. Products were gel purified and cloned into a commercial plasmid vector, and the nucleotide sequence was determined by use of the ABI 3730xl automatic sequencer at the Vanderbilt DNA Sequencing Core. Sequences were aligned to each other and to published hMPV sequences by use of MacVector (version 7.2; Accelrys). Phylogenetic analysis was performed by use of PHYLIP (version 3.6; available at http://evolution.genetics.washington.edu/phylip.html; J. Felsenstein, University of Washington, Seattle); the maximum-likelihood method was used.
Rhinovirus was tested for by a nested PCR. The first-round primers amplified a 380-bp region in the 5 untranslated region of picornaviruses, which includes rhinoviruses. The nested primers amplified a 202-bp rhinovirus-specific region (primers and PCR conditions are available on request). The size of the rhinovirus PCR products was verified by agarose gel electrophoresis. Controls in each PCR run included reactions without cDNA and with cDNA prepared from rhinovirus 16 RNA.
Total serum IgE level was measured in baseline serum specimens by a monoclonal antibodybased ELISA as described elsewhere [14], except that 3,3,5,5-tetramethylbenzidine was used as the substrate; the reaction was stopped by use of 2.5 mol/L H2SO4, and optical-density readings were measured at 450 nm. IgE reference standards were obtained from Pharmacia.
Subjects were stratified by hMPV infection status. Fisher's exact test was used to assess the significance of differences in categorical variable distribution, and Student's t test was used to assess the significance of differences for continuous variables. For non-Gaussian data, continuous variables were compared by nonparametric testing (the Mann-Whitney U test), and categorical variables were compared by Fisher's exact test. Subjects served as their own controls at the 3-month follow-up visits, and a matched-pair analysis was used to test for an association between hMPV infection status and asthma exacerbations requiring hospitalization. It was assumed that hMPV infection status would not be negatively associated with asthma exacerbations, and, thus, the 1-sided McNemar's exact conditional test was used. Because of small numbers, we present an exact confidence interval (CI) for a Mantel-Haenszel common odds ratio (OR). Analyses were performed by use of SAS (version 9.1; SAS Institute).
Results.
The characteristics of the 101 subjects are presented in table 1. The mean ± SD age was 41.9 ± 11.7 years; 21 (21%) subjects were men and 80 (79%) were women; 52 (51%) subjects were white; and the median length of hospitalization was 2.0 days (range, 19 days). Of the 101 subjects, 75 (74%) returned for a follow-up visit. The subjects who returned for follow-up did not differ from those who did not return with respect to age, sex, race, asthma severity score, length of hospitalization, and insurance type (data not shown). The subjects who did not return for follow-up were more likely to be a current smoker (P = .003) but were less likely to require daily 2-agonists (P = .05), to use inhaled corticosteroids (P = .05), and to have a college education (P = .02) (data not shown).
hMPV was detected in 7 (6.9%) of the 101 subjects who were hospitalized for an acute asthma exacerbation and in 1 (1.3%) of the 75 subjects who returned for a follow-up visit. All of the subjects who tested positive for hMPV at hospitalization tested negative at the follow-up visit. The difference in the rate of hMPV positivity between the patients who returned for a follow-up visit (n = 75) and those who did not (n = 26) was not statistically significant (P = .18). All 7 subjects who tested positive for hMPV at hospitalization returned for a follow-up visit. In a matched-pair analysis, the OR for hMPV infection associated with acute asthma exacerbation was found to be 7 (95% CI, 0.9312; P = .03). By use of a symptom index for viral symptoms (which was based on a composite of symptoms that are distinct from asthma symptoms), it was determined that 79 (78%) of the 101 subjects had a symptom index suggestive of an upper respiratory tract infection associated with their acute asthma exacerbation. Of those who were positive for hMPV, 86% had a positive index [15]. Twenty-one (21%) of the 101 subjects tested positive for rhinovirus at hospitalization, and no coinfections with hMPV and rhinovirus were detected.
Seventy-nine percent of the entire cohort were women, and 57% of the subjects who tested positive for hMPV at hospitalization were women; however, this difference was not statistically significant. The hospitalized subjects with hMPV infection did not differ significantly from the hospitalized subjects without hMPV infection with respect to age, race, asthma severity score, length of hospitalization, asthma medication use (inhaled corticosteroids and 2-agonists), and total serum IgE levels (table 1) or with respect to atopy, as assessed by skin-prick testing (data not shown). The differences in mean FEV1 values between hospitalization and follow-up did not differ between the hMPV-infected and the hMPV-uninfected subjects. A greater percentage of the hMPV-infected subjects had a history of daily 2-agonist use, inhaled corticosteroid use, prior intubation, and elevated total serum IgE levels, although none of these differences were statistically significant. The single hMPV isolate detected in a subject at a follow-up visit was in a male subject with no symptomatic complaints, although the presence of nasal discharge was noted on physical examination.
Six of the 8 hMPV infections were identified between the months of January and May, and 6 were detected in 1 of the 4 study years. Two major genetic subgroups of hMPV (A and B) have been described [1, 2, 4, 5], and the isolates recovered in the present study all fell into genogroup A, as determined by phylogenetic analysis (data not shown).
Discussion.
In this prospective study of adult patients with asthma, we found that 6.9% of all hospitalizations for acute asthma exacerbations were associated with hMPV infection. In contrast, at follow-up, hMPV was detected in only 1 subject (1.3%), who was asymptomatic but had clinical rhinitis. These data strongly support the concept that hMPV plays an etiologic role in acute asthma exacerbations and show that asymptomatic infection in our population was uncommon, which is consistent with the findings of previous studies showing that asymptomatic hMPV infections in children and adults are rare [1, 5]. The hMPV-infected subjects in our study also appeared to have more-severe or less-well-controlled asthma than did the uninfected subjects, as was evidenced by higher rates of daily 2-agonist use, inhaled corticosteroid use, elevated total serum IgE levels, and prior intubation. Several studies have suggested that hMPV infection may cause more-severe disease in children and adults with chronic conditions, although selection bias and the difficulty of diagnosing asthma in very young children make this association uncertain [13]. In addition to children, patients with underlying medical conditions, including asthma, likely should be a targeted population for vaccine development.
Of 8 hMPV infections, we identified 6 in 1 of the 4 study years. Other studies have noted variability in the amount of disease attributable to hMPV infection in different years [1, 3]; in one study, hMPV infection accounted for anywhere between 0% and 31% of acute respiratory illnesses in a given season [5]. All of the strains identified in the present study belonged to genogroup A, a finding that is consistent with those of studies showing that, in a given season, viruses from one subgroup may be predominant in a community even if both are present [24].
Previous studies have described significant associations between other respiratory viruses and asthma in adults [811]. A prospective, 4-year study of 1029 adults and children hospitalized with acute respiratory disease, including asthma, detected evidence of viral infection in 45% of the subjects, predominantly with RSV, parainfluenza virus, and influenza virus [9]. A case-control study of inpatient and outpatient adults with asthma that tested for respiratory viruses by PCR detected a virus in 26% of inpatients with asthma, 18% of outpatients with asthma, and 8% of inpatient controls [10]. A study of 46 hospitalized adults with asthma used PCR to test for respiratory viruses at admission and 46 weeks after discharge [11]; this study identified a virus (predominantly picornavirus, adenovirus, and influenza virus) in 48% of adults with asthma at admission, and picornavirus was detected in only 7% of convalescent specimens. In the present study, the majority of acute asthma exacerbations appeared to be attributable to viral illness, with 78% of the subjects having a symptom index suggestive of an upper respiratory tract infection at presentation, a finding similar to that of another study that used PCR to detect an array of viral pathogens that cause upper and lower respiratory tract illness [15]. Additionally, we detected rhinovirus in 21% of our adult subjects hospitalized for an acute asthma exacerbation with no overlap among those with hMPV infection, a finding similar to those of other studies of rhinovirus detection in adults with asthma exacerbations requiring hospitalization [16, 17]. Given the similar prevalences of symptoms of upper respiratory tract infection and rhinovirus detection, the results of the present study are likely generalizable to other populations of adults with asthma.
In this study, no characteristics clearly identified any subgroup of hospitalized adults with asthma who were at higher risk for hMPV infection. The hMPV-infected subjects with asthma were clinically similar to the nonhMPV-infected subjects with regard to rates of fever, length of hospitalization, asthma severity score, and mean FEV1 values. The lack of statistically significant differences may have been due to the relatively small numbers of subjects and limited power. Another potential limitation is that only 74% of the cohort returned for a follow-up visit. Although there were no significant demographic differences between those who did and those who did not return, there was the potential for underestimating the prevalence of hMPV infection in the convalescent group. The strengths of this study include the recruitment of subjects over a 4-year period and the evaluation and testing of patients at 2 time points.
Several studies have investigated the possibility of a link between hMPV infection and asthma in children [57], but an association between infection with this newly described virus and severe asthma exacerbations in adults has not been established. In the present prospective investigation of adults hospitalized for an acute asthma exacerbation, we found a significant association between hMPV infection and acute exacerbations. Our data, collected at 2 time points, provide strong evidence that hMPV plays an etiologic role in acute asthma exacerbations. The characteristics of the hospitalized hMPV-infected subjects suggest that patients with more-severe underlying disease may be at greater risk. Further studies are needed to explore the mechanisms of the interaction between hMPV infection and asthma, which may help us to further understand the well-established link between acute asthma exacerbations and viral illness.
Acknowledgment
We acknowledge and thank Tebeb Gebretsadik (Department of Biostatistics, Vanderbilt University School of Medicine), for her assistance with and oversight of the biostatistical analyses.
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