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PCR-based molecular assays have become standard diagnostic procedures for the identification and quantification of human rhinoviruses (HRVs) and other respiratory pathogens in most, if not all, clinical microbiology laboratories. Molecular assays are significantly more sensitive than traditional culture-based and serological methods. This advantage has led to the recognition that HRV infections are common causes for not only upper airway symptoms but also more severe lower respiratory illnesses. In addition, molecular assays improve turnaround time, can be performed by technicians with ordinary skills, and can easily be automated. This chapter describes two highly sensitive and specific PCR-based methods for identifying and quantifying HRVs. The first is a two-step PCR method for the detection and typing of HRV. The second is a pan-HRV real-time quantitative (q) PCR method for measuring viral loads in respiratory samples.

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BACKGROUND: 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. METHODS: 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 (n = 38, January to May) and rainy (n = 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. RESULTS: Wheezing children had a higher [chi(2 )= 5.561, p = 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) (n = 18, 25.7% vs. n = 7, 8.8%; p = 0.005), respiratory syncytial virus B (RSV B) (n = 2, 2.9% vs. n = 4, 5.0%), and enterovirus (n = 1, 1.4% vs. n = 2, 2.5%). Strong odds for rhinoviral infection were observed among nebulised children compared with stable asthmatics (p = 0.005, OR = 3.6, 95% CI = 1.4 - 9.3,). RV was prevalent throughout the year (Dry, n = 6, 15.8%; Rainy, n = 19, 17.0%) and without seasonal association [chi(2 )= 0.028, p = 0.867]. However it was the most frequently detected virus [Dry = 6/10, (60.0%); Rainy = 19/28, (67.9%)] in both seasons. CONCLUSION: 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.

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BACKGROUND: 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. METHODS: 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 (n=38, January to May) and rainy (n=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. RESULTS: Wheezing children had a higher [chi square =5.561, p=0.018] prevalence of respiratory viruses compared with stabilized asthmatics (34.3 per cent (24) versus (vs.) 17.5 per cent (14)). Acute asthmatics were thrice as likely to be infected with a respiratory virus (OR = 2.5, 95 per cent CI = 1.2 - 5.3). The predominant pathogens detected in acute versus stable asthmatics were the rhinovirus (RV) (n=18, 25.7 per cent vs. n=7, 8.8 per cent; p=0.005), respiratory syncytial virus B (RSV B) (n=2, 2.9 per cent vs. n=4, 5.0 per cent), and enterovirus (n=1, 1.4 per cent vs. n=2, 2.5 per cent). Strong odds for rhinovirus infection were observed among nebulised children compared with stable asthmatics (p=0.005, OR = 3.6, 95 per cent CI = 1.4 - 9.3,). RV was prevalent throughout the year (Dry, n=6, 15.8 per cent; Rainy, n=19, 17.0 per cent) and without seasonal association [chi square =0.028, p=0.867]...

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BACKGROUND: 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. METHODS: 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 (n = 38, January to May) and rainy (n = 112, June to December) seasons. Amultitarget, 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. RESULTS: Wheezing children had a higher [c2 = 5.561, p = 0.018] prevalence of respiratory viruses compared with stabilized asthmatics (34.3% (24) versus (vs.) 17.5% (14)).... CONCLUSION: 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.

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BACKGROUND: 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. METHODS AND FINDINGS: 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. CONCLUSIONS: 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.

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Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.

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