The goals of this study were to determine whether rhinovirus colds were associated with distinct patterns of viral replication or neutrophilic airway inflammation in subjects with asthma, and to determine whether these same predictors correlated with exacerbations. Three-fourths of the colds in this study were due to rhinoviruses, and HRV exposure was the primary agent associated with exacerbation in the following 5 days. This rate is at the higher end of those reported in other studies (5
), and may, in part, reflect recruitment strategies designed to enroll subjects at the first sign of a cold and before the exacerbation occurs. Data from the present study provide several novel insights. Foremost, the rate of exacerbations was 4.4-fold higher for group A minor HRV compared with all other groups combined, and may be the first study in adults with asthma to examine differences in HRV group pathogenicity. Published studies in children suggest that HRV-C may be more likely to cause severe exacerbations requiring hospitalization (34
). HRV-C is more difficult to grow in culture, and does not appear to bind ICAM-1 or the LDL-R (49
). Although we did not perform broad surveillance of adults for a complete profile of HRV strains in the community for each cold season, HRV group A has been shown to be more likely to have an increased symptom burden in general populations of adults (50
). Collectively, these data suggest that there may be an age dependence to the intensity of the immune response to HRV, or that prior exposure to HRV-C may provide greater protection against subsequent exposure to this group in adults, in comparison with other HRV groups. Other potential mechanisms for varying HRV group pathogenicity that warrant further study include baseline differences in lower airway epithelial cell expression levels of ICAM-1, LDL-R, and the receptor for HRV-C, inconsistent effects of ICS on the expression of these receptors, and/or potential divergent signaling pathways, leading to production of chemokines and/or antiviral factors (52
The quantitative assessment of HRV copy number and associated lower airway inflammation in patients with asthma relative to control subjects without atopy or asthma has also not been previously shown during natural infections. We demonstrate that the HRV copy number in acute sputum samples from patients with asthma are not different when grouped according to exacerbation status; however, patients with HRV-associated exacerbations had greater than fivefold more neutrophils in their acute sputum samples than subjects with HRV colds, but no exacerbation. These data can be interpreted in several ways. First, it is likely that the variability in measuring HRV RNA copy number in a sputum sample is greater than that associated with neutrophil enumeration. In this sense, the present study may be underpowered to detect differences in lower airway HRV copy number that would be predicted if patients with asthma indeed have a relative deficiency in antiviral factors (22
). Consistent with this notion is our observation that select patients with exacerbations have a sputum-to-peak nasal lavage HRV ratio greater than 1, suggestive of lower airway replication. In this light, it was interesting that post hoc
analysis showed a trend toward increased sputum HRV copy number in patients previously taking ICS. Previous ICS use may be a marker for attenuated antiviral host factor production, and, similarly, administration of oral steroids has been shown to increase the nasal HRV titer during inoculation experiments, such that the increased burden may be an effect of the medicine (55
). Second, patients with exacerbations could have more robust recruitment and/or delayed clearance of neutrophils and other inflammatory cells from the lower airway in the setting of a similar viral challenge. With respect to the latter possibility, defects in alveolar macrophage phagocytosis have been associated with severe asthma phenotypes that are more exacerbation prone (56
). Third, given that the control subjects who are HRV infected and nonatopic in the present study had similar sputum neutrophil counts to those from the patients with asthma, there are likely to be additional features of the asthmatic lower airway that contribute to the exacerbation phenotype. Collectively, these observations will need validation in a larger cohort, with the goal of identifying biomarkers related to the risk of exacerbations during natural infections.
Clearly, the timing of respiratory sample collection is critical in a natural infection and, as such, our study design had a number of strengths and some limitations that should be considered in interpreting these findings. To fully characterize the effects of the acute illness, all participants completed four study visits within the first 14 days after the onset of cold symptoms. That the cold symptom scores in all participants continued to rise during the early visits, the peak nasal lavage HRV copy number occurred on the third day of cold symptoms (similar to that observed in inoculation experiments (26
)), and lung function at the first visit was not different between patients with asthma with or without exacerbation, all suggest that our recruiting efforts enrolled a pre-exacerbation population. Nonetheless, it is possible that some of the acute samples were collected after peak inflammation. The ability to test multiple samples of respiratory secretions with sensitive and comprehensive PCR-based viral diagnostics (58
) adds to the validity of identifying viral illnesses. Furthermore, HRV-positive samples were confirmed by sequencing and molecular typing, which allowed for analysis of group-specific effects. In contrast, previous work by others identified female sex, race, peak expiratory flow variability, lack of use of ICS, and a history of treatment with oral corticosteroid, such as prednisone, as independent risk factors for the incidence and severity of asthma exacerbations (1
). These factors were not significant in the present study, and may reflect a limitation of our sample size and mild baseline disease impairment. Of note, 36 of the initial 84 patients with asthma (42.9%) had required prednisone and/or advanced medical care during episodes before the current study, demonstrating that our recruiting strategy captured an at-risk population. Our entry criteria required an FEV1
≥ 70% predicted at the first visit, and an absence of baseline use of high-dose ICS or prednisone to ensure safety during the complex visit structure and procedural burden. As such, our findings may not apply to those with more severe asthma, although this population will clearly be of interest in terms of the potential interactions between corticosteroids and viral burden. Use of recently adapted nasal sample collection methods at home (47
) may allow us to enroll patients in future protocols with a greater spectrum of disease severity and fewer dedicated study visits.
In conclusion, we show that HRV infection is the most frequent exposure in adults causing seasonal asthma exacerbation in the setting of concomitant upper respiratory symptoms, and that minor group A HRV has the highest rate. Relative to participants with asthma experiencing an HRV cold only, subjects with exacerbations have higher sputum neutrophil counts and an increased chance of having more HRV detected in the sputum compared with the nasal samples. Ongoing work in several laboratories is focused on the identity of rhinovirus species or sequence elements that confer the most pathogenic risk. Additional study is also warranted regarding the mechanisms by which allergic inflammation influences the antiviral response and/or sensitizes the lower airway to injury associated with neutrophilic influx.