Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus

J, Klemenc; Ali, Syed Asad; Johnson, Marion; Tollefson, Sharon J.; Talbot, H. Keipp; Hartert, Tina V.; Edwards, Kathryn M.; Williams, John V.

doi:10.1016/j.jcv.2012.05.005

Cited by 50 publications

(34 citation statements)

References 46 publications

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“…Viral testing was performed in the research lab, and the results were not available to clinicians; thus clinical diagnoses were assigned by the patients’ health care providers independent of viral detection. A total of 215 subjects enrolled at our site tested positive for HMPV by reverse-transcriptase polymerase chain reaction (RT-PCR) [20]. We queried the NVSN database to identify subjects who had chest radiographs obtained; of these 215 HMPV-positive children, 77 (36%) underwent chest radiography at the discretion of the treating clinician.…”

Section: Methodsmentioning

confidence: 99%

Chest radiographic features of human metapneumovirus infection in pediatric patients

et al. 2017

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Background Human metapneumovirus (HMPV) was identified in 2001 and is a common cause of acute respiratory illness in young children. The radiologic characteristics of laboratory-confirmed HMPV acute respiratory illness in young children have not been systematically assessed. Objective We systematically evaluated the radiographic characteristics of acute respiratory illness associated with HMPV in a prospective cohort of pediatric patients. Materials and methods We included chest radiographs from children <5 years old with acute respiratory illness who were enrolled in the prospective New Vaccine Surveillance Network (NVSN) study from 2003 to 2009 and were diagnosed with HMPV by reverse transcription-polymerase chain reaction (RT-PCR). Of 215 HMPV-positive subjects enrolled at our tertiary care children’s hospital, 68 had chest radiographs obtained by the treating clinician that were available for review. Two fellowship-trained pediatric radiologists, independently and then in consensus, retrospectively evaluated these chest radiographs for their radiographic features. Results Parahilar opacities were the most commonly observed abnormality, occurring in 87% of children with HMPV. Hyperinflation also occurred frequently (69%). Atelectasis (40%) and consolidation (18%) appeared less frequently. Pleural effusion and pneumothorax were not seen on any radiographs. Conclusion The clinical presentations of HMPV include bronchiolitis, croup and pneumonia. Dominant chest radiographic abnormalities include parahilar opacities and hyperinflation, with occasional consolidation. Recognition of the imaging patterns seen with common viral illnesses like respiratory syncytial virus (RSV) and HMPV might facilitate diagnosis and limit unnecessary antibiotic treatment.

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Section: Methodsmentioning

confidence: 99%

Chest radiographic features of human metapneumovirus infection in pediatric patients

et al. 2017

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“…Both NS and NP samples were analyzed at Vanderbilt for identification of influenza, human metapneumovirus (MPV), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza virus 3 (PIV3), and adenovirus (ADV) using nucleic acid extraction and real-time RT-PCR methods previously described. 5-8 To maximize the efficiency of the comparisons, NP samples were tested for the targets identified in the paired NS, and for completeness, a random sample of NP paired to NS that had tested negative for all study viruses were also tested for all targets. For both NS and NP, samples with a minimum volume of 200 μl were included, and the samples were considered positive if the RT-PCR CT value was <40.…”

Section: Methodsmentioning

confidence: 99%

Concordance between RT-PCR-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing

Grijalva

Griffin

Edwards

et al. 2014

Journal of Clinical Virology

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Background Epidemiologic studies of respiratory infections frequently rely on separate sample collections for the detection of bacteria and viruses. The requirement for two specimens presents cost, logistical, and acceptability challenges. Objectives To determine the agreement in detection of respiratory viruses using RT-PCR between two different types of samples collected on the same day: nasal swabs preserved in viral transport medium (NS) and nasopharyngeal swabs preserved in skim milk-tryptone-glucose-glycerol [STGG] medium (NP), the current standard for pneumococcal colonization studies. Study design Paired NS and NP samples were collected between May 2009 and September 2011 as part of the RESPIRA-PERU study, a large prospective cohort of Andean children <3 years of age. NS samples used polyester swabs and viral transport medium whereas NP samples used rayon wire-handled swabs and STGG medium. Samples were tested for influenza, human metapneumovirus (MPV), respiratory syncytial virus (RSV), human rhinovirus (HRV), parainfluenza virus 3 (PIV3) and adenovirus (ADV) using real-time RT-PCR. We calculated the agreement, and compared cycle thresholds (CT) between NP and NS samples. Results Among 226 paired NP-NS samples, we observed very high agreement with a Kappa statistic ranging from 0.71 for ADV to 0.97 for MPV. CT values were similar for both strategies. Conclusions NP samples preserved in STGG provide a simple and reliable strategy for identification of both pneumococcus and respiratory viruses. This single specimen collection strategy could be used for epidemiologic studies, especially in resource-limited settings. Furthermore, archived NP-STGG specimens from previous studies could be reliably tested by RT-PCR for viruses.

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“…Nucleic acid amplification, such as RT-PCR, is the most commonly used diagnostic method [11]; sever al commercial methods include hMPV probes among their multiple respiratory pathogen diagnostic arrays [12]. Due to the slow in vitro growth and the unsteady cytopathic effect that is produced, viral cultures have low sensitivity, which might explain the delay in the discovery of this virus.…”

Section: Introductionmentioning

confidence: 99%

Human Metapneumovirus: Laboratory Methods for Isolation, Propagation, and Plaque Titration

et al. 2018

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The human metapneumovirus (hMPV) is an important viral agent associated with severe infections of the upper and lower airways, especially in young children and immunosuppressed subjects. Nevertheless, in vitro studies of hMPV are very difficult due to the little knowledge we have on its laboratory manipulation. Objective: The aim of this study was to isolate and propagate hMPV from patients, and to establish a method to quantify the virus by plaque assay. Method: As part of a Latin American respiratory virus surveillance study, 12 nasal secretion samples – hMPV-positive by direct fluorescence – were inoculated on LLC-MK2 cells to isolate the virus. The supernatants were re-inoculated and the cytopathic effect and syncytium formation were evaluated daily; the infection was confirmed by immunofluorescence and RT-PCR. A protocol to titrate the harvested virus was established inoculating serial dilutions on LLC-MK2 cells, and agarose was then added as an overlay. After different time periods, the monolayers were fixed and stained with Naphthol blue/black or crystal violet and finally the viral titer was obtained. Results: Eight out of 12 hMPV-positive respiratory samples were positive for the isolation and confirmed by RT-PCR and immunofluorescence, but the cytopathic effect and syncytium formation were observed only in 5 cultures. One out of 8 viral isolates was used for propagation and plaque assay standardization. We found that incubation for 7 days in the semisolid overlay yielded plaques with appropriate size and shape to be counted, although crystal violet staining showed slightly larger plaques than those seen with Naphthol blue/black staining. Conclusions: The isolation and propagation from patient-derived hMPV and the standardization of a practical, reliable, and inexpensive method of detection and quantification of hMPV were carried out, without the additional use of antibodies that had not been reported previously. These results offer some important insights for future studies of cellular and molecular biology of hMPV.

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Real-time reverse transcriptase PCR assay for improved detection of human metapneumovirus

Cited by 50 publications

References 46 publications

Chest radiographic features of human metapneumovirus infection in pediatric patients

Chest radiographic features of human metapneumovirus infection in pediatric patients

Concordance between RT-PCR-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing

Human Metapneumovirus: Laboratory Methods for Isolation, Propagation, and Plaque Titration

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