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JAC Advance Access published online on December 18, 2008
Journal of Antimicrobial Chemotherapy, doi:10.1093/jac/dkn506
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Original research

Application of denaturing HPLC to rapidly identify rifampicin-resistant Mycobacterium tuberculosis in low- and high-prevalence areas

Jason T. Evans1,*, Abida Parveen1, Grace E. Smith1, Li Xu1, Edward W. C. Chan2, Raphael C. Y. Chan2 and Peter M. Hawkey1,3

1 HPA West Midlands Laboratory, Heart of England NHS Foundation Trust, Bordesley Green East, Birmingham B9 5SS, UK 2 Department of Microbiology, The Chinese University of Hong Kong, Shatin, NT, Hong Kong SAR, People's Republic of China 3 Division of Immunity and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

Received 4 August 2008; returned 7 September 2008; revised 4 November 2008; accepted 20 November 2008

* Corresponding author. Tel: +44-121-424-0250; Fax: +44-121-772-6229; E-mail: jason.evans{at}heartofengland.nhs.uk

Objectives: Since the emergence of multidrug-resistant and extensively drug-resistant tuberculosis, there has been a call for a rapid assay to detect rifampicin-resistant strains that can be implemented into a routine service to analyse all strains in a specific geographical location. Denaturing HPLC (dHPLC) is a rapid screening test that can detect mutations in PCR amplicons. The aim of this study was to evaluate the dHPLC analysis of rifampicin-resistant Mycobacterium tuberculosis isolates using an extensive strain collection from Hong Kong and the UK and a collection of 84 consecutive clinical isolates.

Methods: DNA from 51 rifampicin-resistant M. tuberculosis strains from the UK and Hong Kong identified from 1996 to 2005 was extracted and each mutation was defined by capillary electrophoresis. A 400 bp PCR product was amplified from each strain, heteroduplexed with a known susceptible control (H37Rv) and analysed by dHPLC at 67.0°C.

Results: Forty-five out of 51 (88.2%) rifampicin-resistant strains with known DNA mutations were detected by dHPLC. Two out of 84 clinical isolates were phenotypically rifampicin-resistant and dHPLC detected a mutation in the rpoB amplicon for both these isolates. dHPLC detected a mutation in 1 out of 82 phenotypically rifampicin-susceptible isolates (M482T, a non-cluster I/II mutation). In a combined analysis of all strains and isolates, mutation detection by dHPLC analysis exhibited 88.2% sensitivity and 98.8% specificity.

Conclusions: This study shows that dHPLC analysis is sensitive and specific and could be implemented in a routine clinical service.

Key Words: antitubercular agents , microbial drug resistance , polymerase chain reaction


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