A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus

Ha, Lien Anh; Doorn, H. Rogier van; Bryant, Juliet E.; Nghiem, My Ngoc; Van, Vinh Chau Nguyen; Vo, Cong Khanh; Nguyen, Minh Dung; Tran, Thi Hong; Farrar, Jeremy; Jong, Menno D. de

doi:10.1016/j.jviromet.2011.11.012

Cited by 42 publications

(42 citation statements)

References 36 publications

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“…Viral genomic loads for RSV positive samples were determined at the laboratory of P.A.P by Q-PCR. Threshold cycle (CT) values were determined, thereby providing a semiquantitative measure of viral genomic load, as previously described [45]. …”

Section: Methodsmentioning

confidence: 99%

Genomic Loads and Genotypes of Respiratory Syncytial Virus: Viral Factors during Lower Respiratory Tract Infection in Chilean Hospitalized Infants

Espinosa

Martín

Torres

et al. 2017

IJMS

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Abstract:The clinical impact of viral factors (types and viral loads) during respiratory syncytial virus (RSV) infection is still controversial, especially regarding newly described genotypes. In this study, infants with RSV bronchiolitis were recruited to describe the association of these viral factors with severity of infection. RSV antigenic types, genotypes, and viral loads were determined from hospitalized patients at Hospital Roberto del Río, Santiago, Chile. Cases were characterized by demographic and clinical information, including days of lower respiratory symptoms and severity. A total of 86 patients were included: 49 moderate and 37 severe cases. During 2013, RSV-A was dominant (86%). RSV-B predominated in 2014 (92%). Phylogenetic analyses revealed circulation of GA2, Buenos Aires (BA), and Ontario (ON) genotypes. No association was observed between severity of infection and RSV group (p = 0.69) or genotype (p = 0.87). After a clinical categorization of duration of illness, higher RSV genomic loads were detected in infants evaluated earlier in their disease (p < 0.001) and also in infants evaluated later, but coursing a more severe infection (p = 0.04). Although types and genotypes did not associate with severity in our children, higher RSV genomic loads and delayed viral clearance in severe patients define a group that might benefit from new antiviral therapies.

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Section: Methodsmentioning

confidence: 99%

Genomic Loads and Genotypes of Respiratory Syncytial Virus: Viral Factors during Lower Respiratory Tract Infection in Chilean Hospitalized Infants

Espinosa

Martín

Torres

et al. 2017

IJMS

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“…Viral RNA was extracted from 100 μl of nasopharyngeal specimens using the MagNA Pure 96 nucleic acid extraction system (Roche Diagnostics, Basel, Switzerland) following the manufacturer's instructions. Viral RNA was eluted in 60 μl elution buffer and 5 μl viral RNA was used in the diagnostic and subtyping real‐time RT‐PCR for RSV A or B infection as described previously . The remaining viral RNA was stored at −80°C until further analyses.…”

Section: Methodsmentioning

confidence: 99%

Characterization of hospital and community‐acquired respiratory syncytial virus in children with severe lower respiratory tract infections in Ho Chi Minh City, Vietnam, 2010

Tuấn

Thanh

Hai

et al. 2015

Influenza Resp Viruses

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BackgroundHuman respiratory syncytial virus (RSV) is an important community and nosocomial pathogen in developed countries but data regarding the importance of RSV in developing countries are relatively scarce.MethodsDuring a 1-year surveillance study in 2010, we took serial samples from children admitted to the Emergency Unit of the Respiratory Ward of Children's Hospital 1 in Ho Chi Minh City, Vietnam. RSV was detected within 72 hours of admission to the ward in 26% (376/1439; RSV A: n = 320; RSV B: n = 54; and RSV A and B: n = 2). Among those negative in the first 72 hours after admission, 6·6% (25/377) acquired nosocomial RSV infection during hospitalization (RSV A: n = 22; and RSV B: n = 3).ResultsChildren with nosocomial RSV infection were younger (P = 0·001) and had a longer duration of hospitalization (P < 0·001). The rate of incomplete recovery among children with nosocomial RSV infection was significantly higher than among those without (P < 0·001). Phylogenetic analysis of partial G gene sequences obtained from 79% (316/401) of positive specimens revealed the co-circulation of multiple genotypes with RSV A NA1 being predominant (A NA1: n = 275; A GA5: n = 5; B BA3: n = 3; B BA9: n = 26; and B BA10: n = 7). The RSV A GA5 and RSV B BA3 genotypes have not been reported from Vietnam, previously.ConclusionBesides emphasizing the importance of RSV as a cause of respiratory infection leading to hospitalization in young children and as a nosocomial pathogen, data from this study extend our knowledge on the genetic diversity of RSV circulating in Vietnam.

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“…Commercial (Do et al, 2012). In this study, we designed and validated two one-step TaqMan RT-qPCR assays to efficiently detect, quantify and subtype RSV.…”

Section: Qcmd Resultsmentioning

confidence: 99%

“…Different RSV genes have been targeted in these assays, such as the matrix, the polymerase (Kuypers et al, 2004), or the fusion protein genes (Mentel et al, 2003;van Elden et al, 2003), but the N gene is the most widely used due to its high conservation and the number of N sequences available in Genbank. Several assays were designed to subtype RSV, but most were validated more than 10 years ago (Dewhurst-Maridor et al, 2004;Do et al, 2012;Gueudin et al, 2003;Hu et al, 2003). In order to include Table 5 Validation of subtyping efficiency.…”

Section: Discussionmentioning

confidence: 99%

“…Real-time quantitative PCR (RT-qPCR) is the gold standard for both accurate RSV diagnosis and quantification and several assays have been developed during the last decades (Chi et al, 2007;Choudhary et al, 2013;Dewhurst-Maridor et al, 2004;Do et al, 2012;Gueudin et al, 2003;Hu et al, 2003;Kuypers, http://dx.doi.org/10.1016/j.jviromet.2016.05.004 0166-0934/© 2016 Elsevier B.V. All rights reserved. Mentel et al, 2003;van Elden et al, 2003), although these have to be adapted constantly to take into account genetic variations among circulating strains.…”

Section: Introductionmentioning

confidence: 99%

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A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

Essaidi-Laziosi

Lyon

Mamin

et al. 2016

Journal of Virological Methods

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a b s t r a c tHuman respiratory syncytial virus (RSV) is a major health problem and the main cause of hospitalization due to bronchiolitis. RSV is divided into two antigenic subgroups, RSV-A and -B that co-circulate worldwide. Rapid and sensitive detection is desirable for proper patient handling while assessment of viral load may help to evaluate disease severity and progression. Finally RSV subtyping is needed to determine the prevalence and pathogenicity of each RSV subgroup, as well as their sensitivity to treatment. In this study, we took into account the most recent circulating RSV variants and designed two quantitative TaqMan one-step RT-PCR assays to detect and quantify both RSV subgroups separately. Standard dilutions of transcripts of positive and negative polarities were included in the assay validation to assess potential differences in sensitivity on negative-sense genomes and positive-sense RNAs. In addition, RSV detection in respiratory specimens of different types and sampled in different populations was compared to commercially available RSV diagnostic tools. Altogether, the RSV-A and -B assays revealed sensitive and quantitative over a wide range of viral loads, with a slight improved sensitivity of the RSV-B assay on positive sense transcripts, and allowed accurate RSV subtyping. We thus provide a useful tool for both RSV diagnostics and research.

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A sensitive real-time PCR for detection and subgrouping of human respiratory syncytial virus

Cited by 42 publications

References 36 publications

Genomic Loads and Genotypes of Respiratory Syncytial Virus: Viral Factors during Lower Respiratory Tract Infection in Chilean Hospitalized Infants

Genomic Loads and Genotypes of Respiratory Syncytial Virus: Viral Factors during Lower Respiratory Tract Infection in Chilean Hospitalized Infants

Characterization of hospital and community‐acquired respiratory syncytial virus in children with severe lower respiratory tract infections in Ho Chi Minh City, Vietnam, 2010

A new real-time RT-qPCR assay for the detection, subtyping and quantification of human respiratory syncytial viruses positive- and negative-sense RNAs

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