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

Department of Laboratory Medicine, University of Washington, CHRMC, 4800 Sand Point Way N.E., Virology Office, G-815, 8G-3, Seattle, WA 98105, USA.
Journal of Clinical Virology (Impact Factor: 3.02). 10/2004; 31(2):123-9. DOI: 10.1016/j.jcv.2004.03.018
Source: PubMed


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.

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    • "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. "
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    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.
    BMC Infectious Diseases 07/2014; 14(1):373. DOI:10.1186/1471-2334-14-373 · 2.61 Impact Factor
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    • "Research intent, hospital based procedure (3) Real-Time PCR [48] [49] [50] [51] [52] [53] Real-time amplification of target DNA or cDNA "
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    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.
    Advances in Virology 12/2013; 2013(2):595768. DOI:10.1155/2013/595768
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    • "All NP swabs and stool samples in which HCoVs were established were also tested for the presence of other viruses. In NP swabs respiratory syncytial virus (RSV), influenza viruses A and B (Flu A-B), parainfluenza viruses 1–3 (PIV 1–3), metapneumovirus (hMPV), human bocavirus (HBoVs), adenovirus (AdV) and rhinovirus (hRV) [18-24] were searched for by real-time RT-PCR. Stool samples were tested for the presence of HBoVs and AdV using molecular methods as described previously [22,23], and for other gastroenteric viruses by electron microscopy (EM). "
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    ABSTRACT: Background Human coronaviruses (HCoVs) are a well-known cause of respiratory infections but their role in gastrointestinal infections is unclear. The objective of our study was to assess the significance of HCoVs in the etiology of acute gastroenteritis (AGE) in children <6 years of age. Methods Stool samples and nasopharyngeal (NP) swabs collected from 260 children hospitalized for AGE (160 also had respiratory symptoms) and 157 otherwise healthy control children admitted for elective surgery were tested for the presence of four HCoVs using real time RT-PCR. Registered at (reg. NCT00987519). Results HCoVs were more frequent in patients with AGE than in controls (23/260, 8.8% versus 4/151, 2.6%; odds ratio, OR 3.3; 95% confidence interval, CI 1.3–10.0; P = 0.01). Three of four HCoV-positive members in the control group, asymptomatic when sampled, recalled gastrointestinal or respiratory symptoms within the previous 14 days. In patients with AGE, HCoVs were present in NP samples more often than in stools (22/256, 8.6%, versus 6/260, 2.3%; P = 0.0004). In 5/6 children with HCoVs detected in stools, the viruses were also detected in NP swabs. Patients had a significantly higher probability of HCoV detection in stool (OR 4; 95% CI 1.4–15.3; P = 0.006) and also in stool and/or NP (OR 3.3, 95% CI 1.3–10.0; P = 0.01) than healthy controls. All four HCoVs species were detected in stool and NP samples. Conclusions Although HCoVs were more frequently detected in patients with AGE than in the control group, high prevalence of HCoVs in NP swabs compounded by their low occurrence in stool samples and detection of other viruses in stool samples, indicate that HCoVs probably play only a minor role in causing gastrointestinal illness in children <6 years old.
    Virology Journal 02/2013; 10(1):46. DOI:10.1186/1743-422X-10-46 · 2.18 Impact Factor
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