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

Abstract

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 degrees 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.

• Authors: Jason T Evans, Abida Parveen, Grace E Smith, Li Xu, Edward W C Chan, Raphael C Y Chan, Peter M Hawkey
• Journal: The Journal of antimicrobial chemotherapy (J Antimicrob Chemother) Vol. 63 Issue 2 Pg. 295-301 (Feb 2009) ISSN: 1460-2091 [Electronic] England
• PMID: 19095682 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antitubercular Agents
• Bacterial Proteins
• DNA, Bacterial
• rpoB protein, Mycobacterium tuberculosis
• DNA-Directed RNA Polymerases
• Rifampin

Topics

• Antitubercular Agents (pharmacology)
• Bacterial Proteins (genetics)
• Chromatography, High Pressure Liquid
• DNA, Bacterial (genetics)
• DNA-Directed RNA Polymerases
• Drug Resistance, Bacterial
• Electrophoresis, Capillary
• Hong Kong
• Humans
• Microbial Sensitivity Tests (methods)
• Mutation, Missense
• Mycobacterium tuberculosis (drug effects, genetics, isolation & purification)
• Nucleic Acid Denaturation
• Polymerase Chain Reaction
• Rifampin (pharmacology)
• Sensitivity and Specificity
• Tuberculosis (microbiology)
• United Kingdom