Detection of Rhinoviruses by Tissue Culture and Two Independent Amplification Techniques, Nucleic Acid Sequence-Based Amplification and Reverse Transcription-PCR, in Children with Acute Respiratory Infections during a Winter Season

Loens, Katherine; Goossens, Herman; Laat, C. de; Foolen, H.; Oudshoorn, P.; Pattyn, S. R.; Sillekens, Peter; Ieven, Margareta

doi:10.1128/jcm.44.1.166-171.2006

Cited by 61 publications

(55 citation statements)

References 48 publications

(42 reference statements)

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“…HRV are responsible for a substantial proportion of upper respiratory illnesses (19). Because infectious HRV are difficult to recover from clinical samples, it has been postulated that HRV may be responsible for other respiratory illnesses currently of unknown etiology (1,8,18,24). The fact that less than 1 50% tissue culture infectious dose of HRV caused an experimental human rhinovirus infection (5) suggests that tissue culture may not efficiently detect HRV.…”

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confidence: 99%

Comparison of Results of Detection of Rhinovirus by PCR and Viral Culture in Human Nasal Wash Specimens from Subjects with and without Clinical Symptoms of Respiratory Illness

et al. 2007

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Human rhinoviruses (HRV) cause acute upper respiratory illness. The frequency of HRV-associated illnesses appears greater when PCR assays are used to detect rhinoviruses. The present study performed PCR-based detection of HRV upon entry of subjects into respiratory syncytial virus and parainfluenza type 3 vaccine trials when subjects were symptom-free and upon subsequent development of clinical symptoms of respiratory illness during the trial. The background of HRV PCR positivity in symptom-free individuals (30/139 [22%]) was only slightly lower than in those with respiratory illness (28/77 [36%]). For subjects with multiple samples, it was estimated that HRV was detectable by PCR for approximately 100 days before, during, and after clinical symptoms were documented. PCR is a remarkably more sensitive method of detecting HRV than is tissue culture. The presence of HRV RNA may not always reflect an association with infectious virus production. The limited association of HRV RNA with illness suggests caution in assigning causality of HRV PCR positivity to clinical symptoms of respiratory illness.Over 100 serotypes of human rhinoviruses (HRV), members of the family Picornaviridae, have been identified through community surveillance of respiratory illness (19). HRV has traditionally been identified by growth in tissue culture, as characterized by cytopathic effect. HRV are differentiated from enteroviruses by the capacity to grow at 33°C and acid lability. HRV are responsible for a substantial proportion of upper respiratory illnesses (19). Because infectious HRV are difficult to recover from clinical samples, it has been postulated that HRV may be responsible for other respiratory illnesses currently of unknown etiology (1,8,18,24). The fact that less than 1 50% tissue culture infectious dose of HRV caused an experimental human rhinovirus infection (5) suggests that tissue culture may not efficiently detect HRV. Recent reports suggest that PCR detection extends the scope of HRV illness to include lower respiratory tract illness (10) and establishes a strong association between rhinovirus and exacerbations of asthma (12,26). HRV has been shown to replicate in cells of both the upper (14, 27) and lower (21) respiratory tracts and can grow at the higher temperature of the lower respiratory tract (22).However, more limited studies have detected HRV by PCR in 12 to 20% of asymptomatic children and in a relatively high percentage of adenoid and tonsillar tissues, suggesting that identification of HRV by PCR may not necessarily confirm it as the etiologic agent (16,20,23,25). Identification of rhinovirus infection by PCR may not be accompanied by a serologic response, an unusual occurrence with most symptomatic respiratory viral infections (4).Clinical studies evaluating the safety of live, attenuated strains of respiratory syncytial virus (RSV) (17, 29) and parainfluenza virus type 3 (PIV3) (3) have recently been carried out with adults and children as young as 4 weeks of age. Nasal wash samples were collected from sympt...

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Comparison of Results of Detection of Rhinovirus by PCR and Viral Culture in Human Nasal Wash Specimens from Subjects with and without Clinical Symptoms of Respiratory Illness

et al. 2007

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“…These assays typically target the 5Ј noncoding region (5ЈNCR) of the viral genome that contains highly conserved sequences suitable for molecular assay development. However, most of these assays require postamplification processing of the amplicon by gel electrophoresis, probe hybridization, sequencing, or restriction analysis to confirm and differentiate HRVs from HEVs (1,3,4,5,14,24,31,34,41). More recently, real-time RT-PCR assays have been described for HRVs/HEVs (8,9,25,38,44) that offer potentially rapid, sensitive, and quantitative results and that are less prone to amplicon contamination.…”

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Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Lu¹,

Holloway

Dare³

et al. 2008

J Clin Microbiol

257

229

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Human rhinoviruses (HRVs) are important contributors to respiratory disease, but their healthcare burden remains unclear, primarily because of the lack of sensitive, accurate, and convenient means of determining their causal role. To address this, we developed and clinically validated the sensitivity and specificity of a real-time reverse transcription-PCR (RT-PCR) assay targeting the viral 5 noncoding region defined by sequences obtained from all 100 currently recognized HRV prototype strains and 85 recently circulating field isolates. The assay successfully amplified all HRVs tested and could reproducibly detect 50 HRV RNA transcript copies, with a dynamic range of over 7 logs. In contrast, a quantified RNA transcript of human enterovirus 68 (HEV68) that showed the greatest sequence homology to the HRV primers and probe set was not detected below a concentration of 5 ؋ 10 5 copies per reaction. Nucleic acid extracts of 111 coded respiratory specimens that were culture positive for HRV or HEV were tested with the HRV real-time RT-PCR assay and by two independent laboratories that used different in-house HRV/HEV RT-PCR assays. Eighty-seven HRVculture-positive specimens were correctly identified by the real-time RT-PCR assay, and 4 of the 24 HEVpositive samples were positive for HRV. HRV-specific sequences subsequently were identified in these four specimens, suggesting HRV/HEV coinfection in these patients. The assay was successfully applied in an investigation of a coincidental outbreak of HRV respiratory illness among laboratory staff.

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“…The 5= NCR of picornaviruses, due to its high rate of conservation, is an optimal target for highly sensitive diagnostic assays (4,(13)(14)(15)(16)(17)(18)(19). We have compared several combinations of primers corresponding to different conserved stretches in the 5= NCR of HRVs and HEVs, and we obtained the most sensitive RT-PCR assay with the primers for the sites utilized here (30).…”

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confidence: 99%

“…Both HRVs and HEVs have conserved 5= noncoding regions (NCRs) and a few nearly identical sequence motifs, allowing the design of universal primers for their amplification in RT-PCR assays. For differentiation between HRVs and HEVs, however, these assays require a nested format, post-PCR hybridization, agarose gel electrophoresis, or sequencing (13)(14)(15)(16)(17)(18)(19), making them prone to contamination or cumbersome for diagnostic use. Recently, RT-PCR assays using real-time amplification have been developed for specific detection of HRVs and/or HEVs (20)(21)(22).…”

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confidence: 99%

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

et al. 2013

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Detection of Rhinoviruses by Tissue Culture and Two Independent Amplification Techniques, Nucleic Acid Sequence-Based Amplification and Reverse Transcription-PCR, in Children with Acute Respiratory Infections during a Winter Season

Cited by 61 publications

References 48 publications

Comparison of Results of Detection of Rhinovirus by PCR and Viral Culture in Human Nasal Wash Specimens from Subjects with and without Clinical Symptoms of Respiratory Illness

Comparison of Results of Detection of Rhinovirus by PCR and Viral Culture in Human Nasal Wash Specimens from Subjects with and without Clinical Symptoms of Respiratory Illness

Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

Simultaneous Detection and Differentiation of Human Rhino- and Enteroviruses in Clinical Specimens by Real-Time PCR with Locked Nucleic Acid Probes

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