Application of denaturing HPLC to rapidly identify rifampicin-resistant Mycobacterium tuberculosis in low- and high-prevalence areas.
OBJECTIVE
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.
Evans
Jason T
JT
HPA West Midlands Laboratory, Heart of England NHS Foundation Trust, Bordesley Green East, Birmingham B9 5SS, UK. jason.evans@heartofengland.nhs.uk
Parveen
Abida
A
Smith
Grace E
GE
Xu
Li
L
Chan
Edward W C
EW
Chan
Raphael C Y
RC
Hawkey
Peter M
PM
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D019075:Electrophoresis, Capillary; D006723:Hong Kong; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity; D014376:Tuberculosis / Q000382:microbiology; D006113:United Kingdom
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin