Real-world comparison of two molecular methods for detection of respiratory viruses

Background: Molecular polymerase chain reaction (PCR) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies. Molecular assays have generally been evaluated by comparing them to conventional direct fluorescent antibody (DFA) or viral culture techniques, with few published direct comparisons between molecular methods or between institutions. We sought to perform a real-world comparison of two molecular respiratory viral diagnostic methods between two experienced respiratory virus research laboratories.Methods: We tested nasal and throat swab specimens obtained from 225 infants with respiratory illness for 11 common respiratory viruses using both a multiplex assay (Respiratory MultiCode-PLx Assay [RMA]) and individual real-time RT-PCR (RT-rtPCR).Results: Both assays detected viruses in more than 70% of specimens, but there was discordance. The RMA assay detected significantly more human metapneumovirus (HMPV) and respiratory syncytial virus (RSV), while RT-rtPCR detected significantly more influenza A. We speculated that primer differences accounted for these discrepancies and redesigned the primers and probes for influenza A in the RMA assay, and for HMPV and RSV in the RT-rtPCR assay. The tests were then repeated and again compared. The new primers led to improved detection of HMPV and RSV by RT-rtPCR assay, but the RMA assay remained similar in terms of influenza detection.Conclusions: Given the absence of a gold standard, clinical and research laboratories should regularly correlate the results of molecular assays with other PCR based assays, other laboratories, and with standard virologic methods to ensure consistency and accuracy

Detection of Pathogenic Bacteria During Rhinovirus Infection is Associated with Increased Respiratory Symptoms and Exacerbations of Asthma

Background Detection of either viral or bacterial pathogens is associated with wheezing in children, however the influence of both bacteria and virus on illness symptoms has not been described. Objective We evaluated bacterial detection during peak RV season in children with and without asthma to determine if an association exists between bacterial infection and the severity of RV illnesses. Methods 308 children (166 with asthma, 142 without asthma) ages 4–12 years provided five consecutive weekly nasal samples during September, and scored cold and asthma symptoms daily. Viral diagnostics and quantitative PCR for Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis were performed on all nasal samples. Results Detection rates were 53%, 17% and 11% for H. influenzae, S. pneumoniae and M. catarrhalis, respectively, with detection of RV increasing the risk of detecting bacteria within the same sample (OR 2.0, 95% CI 1.4–2.7, p<0.0001) or the following week (OR 1.6 (1.1–2.4), p=0.02). In the absence of RV, S. pneumoniae was associated with increased cold symptoms (mean 2.7 (95% CI 2.0–3.5) vs. 1.8 (1.5–2.2), p=0.006) and moderate asthma exacerbations (18% (12%–27%) vs. 9.2% (6.7%–12%), p=0.006). In the presence of RV, S. pneumoniae was associated with increased moderate asthma exacerbations (22% (16%–29%) vs. 15% (11%–20%), p=0.01). Furthermore, M. catarrhalis detected alongside RV increased the likelihood of experiencing cold and/or asthma symptoms compared to isolated detection of RV (OR 2.0 (1.0–4.1), p=0.04). Regardless of RV status, H. influenzae was not associated with respiratory symptoms. Conclusion RV infection enhances detection of specific bacterial pathogens in children with and without asthma. Furthermore, these findings suggest that M. catarrhalis and S. pneumoniae contribute to the severity of respiratory illnesses, including exacerbations of asthma

Inhaled corticosteroid use is associated with increased circulating tregulatory cells in children with asthma

BACKGROUND: T regulatory (Treg) cells are important in balancing immune responses and dysregulation of Treg cells has been implicated in the pathogenesis of multiple disease states including asthma. In this study, our primary aim was to determine Treg cell frequency in the peripheral blood of children with and without asthma. The secondary aim was to explore the association between Treg cell frequency with allergen sensitization, disease severity and medication use. METHODS: Peripheral blood mononuclear cells from healthy control subjects (N = 93) and asthmatic children of varying disease severity (N = 66) were characterized by multi-parameter flow cytometry. RESULTS: Our findings demonstrate that children with asthma had a significantly increased frequency of Treg cells compared to children without asthma. Using a multivariate model, increased Treg cell frequency in children with asthma was most directly associated with inhaled corticosteroid use, and not asthma severity, allergic sensitization, or atopic status of the asthma. CONCLUSION: We conclude that low dose, local airway administration of corticosteroids is sufficient to impact the frequency of Treg cells in the peripheral blood. These data highlight the importance of considering medication exposure when studying Treg cells and suggest inhaled corticosteroid use in asthmatics may improve disease control through increased Treg cell frequency

Distribution and seasonality of rhinovirus and other respiratory viruses in a cross-section of asthmatic children in Trinidad, West Indies

<p>Abstract</p> <p>Background</p> <p>Childhood asthma in the Caribbean is advancing in prevalence and morbidity. Though viral respiratory tract infections are reported triggers for exacerbations, information on these infections with asthma is sparse in Caribbean territories. We examined the distribution of respiratory viruses and their association with seasons in acute and stable asthmatic children in Trinidad.</p> <p>Methods</p> <p>In a cross-sectional study of 70 wheezing children attending the emergency department for nebulisation and 80 stable control subjects (2 to 16 yr of age) in the asthma clinic, nasal specimens were collected during the dry (<it>n </it>= 38, January to May) and rainy (<it>n </it>= 112, June to December) seasons. A multitarget, sensitive, specific high-throughput Respiratory MultiCode assay tested for respiratory-virus sequences for eight distinct groups: human rhinovirus, respiratory syncytial virus, parainfluenza virus, influenza virus, metapneumovirus, adenovirus, coronavirus, and enterovirus.</p> <p>Results</p> <p>Wheezing children had a higher [χ²= 5.561, <it>p </it>= 0.018] prevalence of respiratory viruses compared with stabilized asthmatics (34.3% (24) versus (vs.) 17.5% (14)). Acute asthmatics were thrice as likely to be infected with a respiratory virus (OR = 2.5, 95% CI = 1.2 – 5.3). The predominant pathogens detected in acute versus stable asthmatics were the rhinovirus (RV) (<it>n </it>= 18, 25.7% vs. <it>n </it>= 7, 8.8%; <it>p </it>= 0.005), respiratory syncytial virus B (RSV B) (<it>n </it>= 2, 2.9% vs. <it>n </it>= 4, 5.0%), and enterovirus (<it>n </it>= 1, 1.4% vs. <it>n </it>= 2, 2.5%). Strong odds for rhinoviral infection were observed among nebulised children compared with stable asthmatics (<it>p </it>= 0.005, OR = 3.6, 95% CI = 1.4 – 9.3,). RV was prevalent throughout the year (Dry, <it>n </it>= 6, 15.8%; Rainy, <it>n </it>= 19, 17.0%) and without seasonal association [χ²= 0.028, <it>p </it>= 0.867]. However it was the most frequently detected virus [Dry = 6/10, (60.0%); Rainy = 19/28, (67.9%)] in both seasons.</p> <p>Conclusion</p> <p>Emergent wheezing illnesses during childhood can be linked to infection with rhinovirus in Trinidad's tropical environment. Viral-induced exacerbations of asthma are independent of seasons in this tropical climate. Further clinical and virology investigations are recommended on the role of infections with the rhinovirus in Caribbean childhood wheeze.</p

A Diverse Group of Previously Unrecognized Human Rhinoviruses Are Common Causes of Respiratory Illnesses in Infants

Human rhinoviruses (HRVs) are the most prevalent human pathogens, and consist of 101 serotypes that are classified into groups A and B according to sequence variations. HRV infections cause a wide spectrum of clinical outcomes ranging from asymptomatic infection to severe lower respiratory symptoms. Defining the role of specific strains in various HRV illnesses has been difficult because traditional serology, which requires viral culture and neutralization tests using 101 serotype-specific antisera, is insensitive and laborious.To directly type HRVs in nasal secretions of infants with frequent respiratory illnesses, we developed a sensitive molecular typing assay based on phylogenetic comparisons of a 260-bp variable sequence in the 5' noncoding region with homologous sequences of the 101 known serotypes. Nasal samples from 26 infants were first tested with a multiplex PCR assay for respiratory viruses, and HRV was the most common virus found (108 of 181 samples). Typing was completed for 101 samples and 103 HRVs were identified. Surprisingly, 54 (52.4%) HRVs did not match any of the known serotypes and had 12-35% nucleotide divergence from the nearest reference HRVs. Of these novel viruses, 9 strains (17 HRVs) segregated from HRVA, HRVB and human enterovirus into a distinct genetic group ("C"). None of these new strains could be cultured in traditional cell lines.By molecular analysis, over 50% of HRV detected in sick infants were previously unrecognized strains, including 9 strains that may represent a new HRV group. These findings indicate that the number of HRV strains is considerably larger than the 101 serotypes identified with traditional diagnostic techniques, and provide evidence of a new HRV group

Rhinovirus illnesses during infancy predict subsequent childhood wheezing

Background: The contribution of viral respiratory infections during infancy to the development of subsequent wheezing and/ or allergic diseases in early childhood is not established. Objective: To evaluate these relationships prospectively from birth to 3 years of age in 285 children genetically at high risk for developing allergic respiratory diseases. Methods: By using nasal lavage, the relationship of timing, severity, and etiology of viral respiratory infections during infancy to wheezing in the 3rd year of life was evaluated. In addition, genetic and environmental factors that could modify risk of infections and wheezing prevalence were analyzed. Results: Risk factors for 3rd year wheezing were passive smoke exposure (odds ratio [OR] 5 2.1), older siblings (OR 5 2.5), allergic sensitization to foods at age 1 year (OR 5 2.0), any moderate to severe respiratory illness without wheezing during infancy (OR 5 3.6), and at least 1 wheezing illness with respiratory syncytial virus (RSV; OR 5 3.0), rhinovirus (OR 5 10) and/or non-rhinovirus/RSV pathogens (OR 5 3.9) during infancy. When viral etiology was considered, 1st-year wheezing illnesses caused by rhinovirus infection were the strongest predictor of subsequent 3rd year wheezing (OR 5 6.6; P &lt; .0001). Moreover, 63% of infants who wheezed during rhinovirus seasons continued to wheeze in the 3rd year of life, compared with only 20% of all other infants (OR 5 6.6; P &lt; .0001). Conclusion: In this population of children at increased risk of developing allergies and asthma, the most significant risk factor for the development of preschool childhood wheezing is the occurrence of symptomatic rhinovirus illnesses during infancy that are clinically and prognostically informative based on their seasonal nature. (J Allergy Clin Immunol 2005;116:571-7.

Enhanced Neutralizing Antibody Responses to Rhinovirus C and Age-Dependent Patterns of Infection

Knowledge of prevalent RV types, antibody responses, and populations at risk based on age and genetics may guide the development of vaccines or other novel therapies against this important respiratory pathogen.Longitudinal data from the Childhood Origins of ASThma (COAST) birth cohort study were analyzed to determine relationships between age and RV-C infections. Neutralizing antibodies specific for rhinovirus A (RV-A) and RV-C (3 types each) were determined using a novel polymerase chain reaction-based assay. We pooled data from 14 study cohorts in the United States, Finland, and Australia and used mixed-effects logistic regression to identify factors related to the proportion of RV-C versus RV-A detection.In COAST, RV-A and RV-C infections were similarly common in infancy, while RV-C was detected much less often than RV-A during both respiratory illnesses and scheduled surveillance visits (pRhinovirus C (RV-C) can cause asymptomatic infection and respiratory illnesses ranging from the common cold to severe wheezing.To identify how age and other individual-level factors are associated with susceptibility to RV-C illnesses.</div

Real-world comparison of two molecular methods for detection of respiratory viruses

Background: Molecular polymerase chain reaction (PCR) based assays are increasingly used to diagnose viral respiratory infections and conduct epidemiology studies. Molecular assays have generally been evaluated by comparing them to conventional direct fluorescent antibody (DFA) or viral culture techniques, with few published direct comparisons between molecular methods or between institutions. We sought to perform a real-world comparison of two molecular respiratory viral diagnostic methods between two experienced respiratory virus research laboratories.Methods: We tested nasal and throat swab specimens obtained from 225 infants with respiratory illness for 11 common respiratory viruses using both a multiplex assay (Respiratory MultiCode-PLx Assay [RMA]) and individual real-time RT-PCR (RT-rtPCR).Results: Both assays detected viruses in more than 70% of specimens, but there was discordance. The RMA assay detected significantly more human metapneumovirus (HMPV) and respiratory syncytial virus (RSV), while RT-rtPCR detected significantly more influenza A. We speculated that primer differences accounted for these discrepancies and redesigned the primers and probes for influenza A in the RMA assay, and for HMPV and RSV in the RT-rtPCR assay. The tests were then repeated and again compared. The new primers led to improved detection of HMPV and RSV by RT-rtPCR assay, but the RMA assay remained similar in terms of influenza detection.Conclusions: Given the absence of a gold standard, clinical and research laboratories should regularly correlate the results of molecular assays with other PCR based assays, other laboratories, and with standard virologic methods to ensure consistency and accuracy