Nucleic acid sequence based amplification and molecular beacon detection for the real-time identification of human metapneumovirus in paediatric respiratory specimens
Abstract number: o64
Manji R., Zhang F., Ginocchio C.
Human metapneumovirus (hMPV) may account for about 10% of pediatric upper and/or lower respiratory tract disease in which a respiratory virus, such as RSV, influenza or parainfluenza, could not be detected. Studies have also suggested that hMPV may contribute to increased severity of disease when present with other viral pathogens. Most laboratories cannot routinely diagnose hMPV infections since the virus is slow growing and there are limited reagents for confirmation. The rapid diagnosis of hMPV infections is of central importance for patient management (rational use of antibiotics and antiviral agents), hospital infection control and for understanding the epidemiological disease patterns of hMPV. This study included a technical validation and retrospective clinical evaluation of a real time NASBA assay for the detection of hMPV in pediatric respiratory samples.
Samples tested included: dilution panels of an hMPV viral isolate, isolates of common respiratory pathogens, and frozen respiratory specimens (nasopharyngeal aspirates, washes or swabs) from 232 children (age range: 5 days to 2 years) who presented with respiratory disease. Nucleic acid (NA) isolation, amplification and detection were performed using NucliSens EasyQ Basic Kit and NucliSens EasyQ hMPV reagents (bioMérieux). Specimen nucleic acids and an hMPV specific internal RNA control (IC) were co-extracted using NucliSens Magnetic Extraction Reagents and the NucliSens miniMAG instrument (bioMérieux) and co-amplified using a single hMPV specific primer pair. Included in the reaction were an hMPV specific molecular beacon (5'-FAM) and an IC specific molecular beacon (5'-ROX). Target amplification and continuous monitoring of emitted fluorescence were performed using a NucliSens EasyQ analyser (bioMérieux).
The limit of detection for hMPV was 2.5 TCID50 per reaction and the 90% detection rate was 12.5 TCID50 per reaction. The assay was 100% specific for hMPV, with no cross reactivity detected to a panel of viral and bacterial respiratory pathogens. The overall hMPV prevalence rate was 2.6% and the rate for samples with no other viral pathogen detected was 3.8%. One sample was also positive for RSV.
The NucliSens EasyQ hMPV assay demonstrated excellent sensitivity and specificity. The assay was easy to use, required minimal hands on time (1 hour) and easily provided a same day result. Additional year round studies will provide important epidemiologic data.
|Session name:||XXIst ISTH Congress|
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