Evaluation of quantitative and type-specific real-time RT-PCR assays for detection of respiratory syncytial virus in respiratory specimens from children

ArticleinJournal of Clinical Virology 31(2):123-9 · October 2004with3 Reads
Impact Factor: 3.02 · DOI: 10.1016/j.jcv.2004.03.018 · Source: PubMed
Abstract

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract morbidity in young children and immunosuppressed patients. To rapidly and accurately quantify and subtype RSV in respiratory samples, we developed and evaluated two real-time RT-PCR assays. A quantitative assay was designed using primers for a consensus region of the matrix protein gene and a subtype-specific assay for RSV-A and RSV-B detection was designed using primers for the polymerase gene. Quantitative RSV RT-PCR results of pediatric nasal wash samples submitted to the University of Washington Virology Laboratory from December 2002, through May 2003, were compared to those of an indirect fluorescent antibody RSV antigen detection assay (FA). Specificity of the RT-PCR assay was high, with no amplification of eleven common respiratory viruses and eight herpes viruses. Among 751 samples, RSV was detected in 267 (35.6%) by FA and in 286 (38.1%) by RT-PCR. Median RSV copy number in nasal wash samples that were positive by both FA and RT-PCR was 2.5 x 10(7) copies/mL versus a median of 3.0 x 10(4) copies/mL for samples positive by RT-PCR only (P < 0.001). The detection and quantity of RSV in respiratory specimens was associated with younger age, but not with gender or hospitalization. Among positive samples from this Seattle cohort, 52% were subtype A and 48% were subtype B. Both subtypes were detected with similar viral loads among all patient groups (stratified by age, gender, and hospitalization), and throughout the specimen collection period. These real-time RT-PCR assays provide a rapid, specific, and highly sensitive alternative for detecting, quantifying, and subtyping RSV in clinical specimens.

    • "...avirus (HBoVs), adenovirus (AdV) and human rhinovirus (hRV) were searched for by real-time RT-PCR [23][24][25][26][27][28][29]. Stool samples from patients with AGE were tested for the presence of norov..."
      All NP swabs and stool samples in which HCoVs were established were also tested for the presence of several other viruses. In NP swabs respiratory syncytial virus (RSV), influenza viruses A and B (Inf A-B), parainfluenza viruses 1–3 (PIV 1–3), human metapneumovirus (hMPV), human bocavirus (HBoVs), adenovirus (AdV) and human rhinovirus (hRV) were searched for by real-time RT-PCR [23][24][25][26][27][28][29]. Stool samples from patients with AGE were tested for the presence of noroviruses of genogroups I and II, human astroviruses, HBoV and AdV using molecular methods as described previously [27, 28, 30, 31], whereas group A rotavirus and adenovirus type 40/41 were detected by antigen-ELISA Premier Rotaclone and Premier Adenoclone (Meridian Bioscience, Cincinnati, OH).
    [Show abstract] [Hide abstract] ABSTRACT: Trial registration: ClinicalTrials.gov NCT00987519.
    Full-text · Article · May 2016 · PLoS ONE
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    • "...cimens with RSV detected were additionally tested for quantitative viral load and subtype [14][15][16]. IRB approval for the study was obtained from the Johns Hopkins University Bloomberg School of Publ..."
      RSV testing was performed using real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) [13]. Specimens with RSV detected were additionally tested for quantitative viral load and subtype [14][15][16]. IRB approval for the study was obtained from the Johns Hopkins University Bloomberg School of Public Health, Seattle Children's Hospital, Cincinnati Children's Hospital, the Institute of Medicine at Tribhuvan University, and the Nepal Health Research Council. Oral consent was obtained from study participants due to low literacy rates in the population.
    [Show abstract] [Hide abstract] ABSTRACT: Background: Respiratory syncytial virus (RSV) is the most important cause of viral pneumonia in children worldwide. A maternal vaccine may protect both the mother and infant from RSV illness. The epidemiology and clinical presentation of RSV in pregnant and postpartum women is not well-described. Methods: Data were collected from a prospective, randomized trial of influenza immunization in pregnant women in rural southern Nepal. Women were enrolled in their second trimester of pregnancy and followed until six months postpartum. Active weekly home-based surveillance for febrile respiratory illness was performed. Mid-nasal swabs collected with episodes of respiratory illness were tested for RSV by real-time polymerase chain reaction. Results: RSV was detected in 14 (0.4%) illness episodes in 3693 women over 3554 person-years of surveillance from 2011-2014. RSV incidence was 3.9/1000 person-years overall, and 11.8/1000 person-years between September and December. Seven (50%) women sought care for RSV illness; none died. Of the 7 (50%) illness episodes during pregnancy, all had live births with 2 (29%) preterm births and a median birthweight of 3060 grams. This compares to 469 (13%) preterm births and a median birthweight of 2790 grams in women without RSV during pregnancy. Of the 7 mothers with postpartum RSV infection, RSV was detected in 4 (57%) of their infants. Conclusions: RSV was an uncommon cause of febrile respiratory illness in mothers during pregnancy in Nepal. These data will inform prevention and therapeutic strategies against RSV in resource-limited settings.
    Full-text · Article · Mar 2016 · PLoS ONE
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    • "...probes specific for the following 17 respiratory viruses were used: AdV [27], PIV 1 to 4 [28], RSV [29] , human metapneumovirus (hMPV) [30], pandemic influenza virus A(H1N1) pdm09, seasonal influenza vir..."
      Each 25-μl reaction mixture contained 5 μl of eluted RNA or DNA and 5 μl of 5× One Step RT-PCR buffer containing 12.5 mM magnesium chloride, 400 μM each deoxynucleoside triphosphate, 40 ng of bovine serum albumin per μl, 0.4 μM primers and 0.2 μM probes, and 1 μl of One Step RT-PCR enzyme mix. Primers and probes specific for the following 17 respiratory viruses were used: AdV [27], PIV 1 to 4 [28], RSV [29] , human metapneumovirus (hMPV) [30], pandemic influenza virus A(H1N1) pdm09, seasonal influenza virus A (H1N1, H3N2) (SIA) [31], seasonal influenza virus B (SIB) [32], human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV- NL63), human coronavirus HKU1 (HCoV-HKU1) [33], rhinovirus (HRV) [34], human parechovirus (HPeV) [35], and enterovirus (EV) [36]. The 7500 Real Time PCR system from Applied Biosystems was used with the following cycling conditions: 30 min at 50°C for reverse transcription, 15 min at 95°C for denaturation, then 45 cycles for 15 s at 95°C, 30 s at 60°C, and 10 s at 40°C.
    [Show abstract] [Hide abstract] ABSTRACT: Background Surveillance of influenza-like illness (ILI) in Central Africa began only recently, and few data are therefore available on the circulation of influenza virus and other respiratory viruses. In Gabon, a Central African country, we established a surveillance network in four major towns in order to analyze cases of ILI among patients who visited health centers between March 2010 and June 2011, and to determine the viral etiology. Methods Nasal swabs were sent for analysis to the Centre International de Recherches Médicales de Franceville, where they were screened for 17 respiratory viruses in a multiplex real-time reverse transcription polymerase chain reaction for all pathogens according the following pairs: adenovirus/parainfluenza virus 4, respiratory syncytial virus/human metapneumovirus, parainfluenza virus 1/parainfluenza virus 2, pandemic influenza virus A/seasonal influenza virus A (H1N1, H3N2)/seasonal influenza virus B, human coronaviruses 229E/OC43, human coronaviruses NL63/HKU1, rhinovirus/human parechovirus, and enterovirus/parainfluenza virus 3. Results We analyzed a total of 1041 specimens, of which 639 (61%) were positive for at least one virus. Three-quarters of the patients were children under five years old. We therefore focused on this age group, in which 68.1% of patients were positive for at least one virus. The most common viruses were adenoviruses (17.5%), followed by parainfluenza viruses (PIVs) 1–4 (16.8%), enteroviruses (EV) (14.7%), respiratory syncytial virus (RSV) (13.5%), and influenza virus (11.9%). The prevalence of some viruses was subject to geographic and seasonal variations. One-third of positive samples contained more than one virus. Conclusions Like most studies in the world, the virus PIVs, EV, RSV, Influenza virus, HRV were predominant among children under five years old in Gabon. An exception is made for adenoviruses which have a high prevalence in our study. However adenoviruses can be detected in asymptomatic persons. These finding gave a better knowledge of the circulation and the seasonality of the viruses involved in ILI in Gabon.
    Full-text · Article · Jul 2014 · BMC Infectious Diseases
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    • "... of disease but evidence is controversial on the link between disease severity and viral load [5,202122. An interaction between detection of other viruses (i.e. ..."
      Children with lower respiratory tract infection shed for longer than those with upper respiratory tract infection (8·4 vs. 1·4 days). Duration of shedding may be related to severity of disease but evidence is controversial on the link between disease severity and viral load [5,202122. An interaction between detection of other viruses (i.e.
    [Show abstract] [Hide abstract] ABSTRACT: SUMMARY RSV is the most important viral cause of pneumonia and bronchiolitis in children worldwide and has been associated with significant disease burden. With the renewed interest in RSV vaccines, we provide realistic estimates on duration, and influencing factors on RSV shedding which are required to better understand the impact of vaccination on the virus transmission dynamics. The data arise from a prospective study of 47 households (493 individuals) in rural Kenya, followed through a 6-month period of an RSV seasonal outbreak. Deep nasopharyngeal swabs were collected twice each week from all household members, irrespective of symptoms, and tested for RSV by multiplex PCR. The RSV G gene was sequenced. A total of 205 RSV infection episodes were detected in 179 individuals from 40 different households. The infection data were interval censored and assuming a random event time between observations, the average duration of virus shedding was 11·2 (95% confidence interval 10·1-12·3) days. The shedding durations were longer than previous estimates (3·9-7·4 days) based on immunofluorescence antigen detection or viral culture, and were shown to be strongly associated with age, severity of infection, and revealed potential interaction with other respiratory viruses. These findings are key to our understanding of the spread of this important virus and are relevant in the design of control programmes.
    Full-text · Article · Jun 2014 · Epidemiology and Infection
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    • "... and N gene, the RSV matrix gene and polymerase gene have also been used to detect RSV in children [53]. The latter method had the ability to quantify and classify RSV with high efficiency and rapidity. ..."
      Several other reports also emphasize the applicability of realtime PCR in detection of RSV in immunocompromised patients [52]. Besides the RSV F and N gene, the RSV matrix gene and polymerase gene have also been used to detect RSV in children [53]. The latter method had the ability to quantify and classify RSV with high efficiency and rapidity.
    [Show abstract] [Hide abstract] ABSTRACT: Human respiratory syncytial virus (RSV) is a common cause of respiratory infection in infants and the elderly, leading to significant morbidity and mortality. The interdisciplinary fields, especially biotechnology and nanotechnology, have facilitated the development of modern detection systems for RSV. Many anti-RSV compounds like fusion inhibitors and RNAi molecules have been successful in laboratory and clinical trials. But, currently, there are no effective drugs for RSV infection even after decades of research. Effective diagnosis can result in effective treatment, but the progress in both of these facets must be concurrent. The development in prevention and treatment measures for RSV is at appreciable pace, but the implementation into clinical practice still seems a challenge. This review attempts to present the promising diverse research approaches and advancements in the area of diagnosis, prevention, and treatment that contribute to RSV management.
    Full-text · Article · Dec 2013 · Advances in Virology
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    • "...and rhinoviruses) and PCR (for adenovirus and bocavirus) using previously described methods25262728293031. Samples were considered positive if the PCR amplification plot crossed the threshold at less t..."
      Swabs were processed in the laboratory as soon as possible after receipt. Specimens were tested for qualitative detection by a panel of 8 single or multiplexed real time reversetranscription (RT)–PCR (for respiratory syncytial virus, influenza virus types A and B, parainfluenza virus types 1–4, human metapneumovirus, human coronaviruses [subtypes OC43, 229E, NL63, and HKU1] and rhinoviruses) and PCR (for adenovirus and bocavirus) using previously described methods25262728293031. Samples were considered positive if the PCR amplification plot crossed the threshold at less than 40 cycles (cycle threshold [C T ] <40).
    [Show abstract] [Hide abstract] ABSTRACT: Understanding the importance of respiratory viruses in children with cystic fibrosis (CF) has been limited because of challenges using clinic- or hospital-based diagnostic testing. We conducted a pilot study to assess feasibility of home self- (or parent-) collection of nasal swabs (NS). Cystic fibrosis patients aged 6-18 years with new respiratory illness participated. In clinic, a deep nasal flocked swab was collected by research staff and compared with an anterior foam NS obtained after instillation of saline spray. At home, up to 2 self-collections of paired foam NS (with and without saline) were collected and mailed for real-time polymerase chain reaction (PCR) testing. Paired swabs were collected from 28 patients: 18 sets in clinic (deep nasal vs saline foam NS) and 43 sets at home (saline vs dry foam NS) with 9 (50%) and 35 (81%) virus detections, respectively. Home-collected NS were obtained closer to illness onset, with a mean difference in symptom days of -2.3 between home and clinic collections (95% confidence interval [CI] -3.5, -1.2; P < .001). Rhinovirus comprised 73% of virus detections; the difference in mean PCR cycle threshold values for rhinovirus between swabs collected at home versus clinic was -3.8 (95% CI -6.8, -0.9; P = .014), indicating significantly higher viral load for home-collected swabs. Home-collected foam NS had a higher positivity rate compared with clinic-collected swabs, likely because collection was closer to illness onset. Home self-collection is feasible and well tolerated for timely respiratory virus diagnosis and provides a novel approach for clinical diagnostics and surveillance of respiratory virus infections among CF patients.
    Preview · Article · Dec 2013
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