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

Jason T. Evans*, Abida Parveen, Grace E. Smith, Li Xu, Edward W.C. Chan, Raphael C.Y. Chan, Peter M. Hawkey

*Corresponding author for this work

    Research output: Contribution to journalArticlepeer-review

    4 Citations (Scopus)


    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.

    Original languageEnglish
    Pages (from-to)295-301
    Number of pages7
    JournalJournal of Antimicrobial Chemotherapy
    Issue number2
    Publication statusPublished - 2009

    Bibliographical note

    Funding Information:
    DNA sequence analysis was undertaken at the Functional Genomics and Proteomics Laboratories, Division of Biosciences, University of Birmingham. This facility is funded by grant 6/JIF13209 from the BBSRC. We wish to thank all microbiology laboratories in the Midlands that refer specimens to the Health Protection Agency Midlands Regional Centre for Mycobacteriology, public health teams, health protection units and the staff at the Midlands Regional Centre for Mycobacteriology for isolation and identification.


    • Antitubercular agents
    • Microbial drug resistance
    • Polymerase chain reaction


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