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Genotypic detection of rifampicin resistance in Mycobacterium tuberculosis: analysis of mutations from high- and low-incidence areas using denaturing high-performance liquid chromatography

Abstract number: R2504

Evans J., Parveen A., Smith E.G., Li X., Chan E., Chan R., Hawkey P.

Objectives: With the emergence of MDR- and XDR-TB, there is a need for a rapid method of detecting rifampicin resistance in M. tuberculosis that can be applied to all isolates in a geographic area. Denaturing high performance liquid chromatography (dHPLC) is a rapid screening method that can analyse PCR amplicons up to 700 bp in length. The aim of this study was to evaluate dHPLC analysis of the rpoB gene in M. tuberculosis using a extensive collection of 16 distinct mutations in 10 different codons from Hong Kong and the UK and a collection of 84 consecutive clinical isolates.

Methods: DNA from 52 rifampicin resistant M. tuberculosis isolates from the UK and Hong Kong identified from 1996–2005 was extracted using a sonication and heating method from positive liquid cultures incubated at 37C. Each mutation was defined by DNA sequence analysis using capillary electrophoresis. In order to evaluate clinical utility, 84 consecutive clinical isolates identified as M. tuberculosis by the Hain LifeScience GenoType MTBC assay were analysed by dHPLC. Phenotypic drug sensitivity testing was undertaken using the BD MGIT liquid culture system. A 400 bp product was amplified from each DNA extract, hybridised with a known sensitive control (H37Rv), and analysed on a WAVE system at 67.0C. Mutations were detected if two or more peaks were detected on a chromatogram with one peak indicating no mutation was detected. Sensitivity, specificity, PPV and NPV were calculated.

Results: 45/52 (88.2%) rifampicin resistant isolates with defined DNA mutations were detected by dHPLC at 67.0 °C. 83/84 (98.8%) consecutive DNA extracts were amplified with the amplification failure bring phenotypically rifampicin sensitive. Two isolates were phenotypically rifampicin resistant and dHPLC detected a mutation in the rpoB amplicon for both these isolates (S531L and S531W). dHPLC detected a mutation in 1/82 phenotypically rifampicin sensitive isolates (M482T, a non-cluster I/II mutation). In a combined analysis of all isolates, detection of mutations in the rpoB gene by dHPLC analysis exhibited sensitivity of 88.7%, specificity of 98.8%, PPV of 97.9%, and NPV of 93.0%.

Conclusion: This study of dHPLC analysis of an expanded collection of mutations from high and low areas of incidence and clinical strains in an area of low incidence shows that dHPLC analysis is sensitive and specific and could be implemented in a routine clinical service alongside routine MIRU-VNTR DNA fingerprinting on a WAVE system.

Session Details

Date: 19/04/2008
Time: 00:00-00:00
Session name: 18th European Congress of Clinical Microbiology and Infectious Diseases
Subject:
Location: Barcelona, Spain
Presentation type:
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