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Nucleic acid sequence based amplification and molecular beacon detection for the real-time identification of respiratory syncytial virus in paediatric respiratory specimens

Abstract number: p1657

Manji  R., Zhang  F., Ginocchio  C.

Background: 

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infection in infants and young children, with bronchiolitis and pneumonia being the major clinical manifestations. The rapid diagnosis of RSV infections is of central importance for individual patient management (rational use of antibiotics and antiviral agents), hospital infection control and monitoring epidemiological disease patterns. This study included a technical validation and a retrospective clinical evaluation of a real time NASBA assay for the detection of RSV A and RSV B in paediatric respiratory samples.

Methods: 

Samples tested included: dilution panels of in vitro transcribed RNA, local RSV isolates, isolates of common respiratory pathogens, and frozen respiratory specimens (nasopharyngeal aspirates, washes or swabs) from 231 children (age range: 5 d to 2 yr) who were evaluated in the paediatric emergency department for respiratory disease. Nucleic acid (NA) isolation, amplification and detection were performed using the NucliSens EasyQ Basic Kit and NucliSens EasyQ RSV A+B reagents (bioMérieux). Specimen NAs and a RSV 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 RSV specific primer pair. Included in the reaction were a RSV 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 analyzer (bioMérieux). Results were compared to direct immunofluorescence (DFA) and/or viral culture using R-Mix cells (Diagnostic Hybrids, OH).

Results: 

The limit of detection for RSV was 2 RNA copies/rxn and the 100% detection rate was 5 copies/rxn. The assay was 100% specific for RSV with no cross reactivity to other respiratory pathogens. The NASBA assay detected 10% more positive specimens than DFA and 44% more positive samples than VC. The NPVs of the assays were: NASBA 94.6%, DFA 90.8% and VC 78.0%.

Conclusions: 

The NucliSens EasyQ RSV assay demonstrated superior sensitivity to both DFA and viral culture for the detection of RSV A and B from respiratory specimens. The assay was easy to use, required minimal hands on time (1 hr) and a faster time to results as compared to rapid culture (4 hr vs. 24–72 hr).

Session Details

Date: 01/08/2007
Time: 00:00-00:00
Session name: XXIst ISTH Congress
Subject:
Location: Oxford, UK
Presentation type:
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