Denaturing HPLC for high-throughput screening of rifampicin-resistant Mycobacterium tuberculosis isolates.
OBJECTIVE
To evaluate the use of denaturation high-performance liquid chromatography (dHPLC) as a rapid method to detect rifampicin (RMP) resistance based on mutations in the rpoB gene in a high-volume laboratory setting.


METHODS
A total of 132 RMP-resistant Mycobacterium tuberculosis strains with different rpoB mutation were used to optimise the running condition of dHPLC as a pilot study. A blind correlation study was subsequently done between dHPLC and in vitro RMP susceptibility tests on 3167 M. tuberculosis strains in a high-throughput clinical setting.


RESULTS
In the pilot study, rpoB mutation could be detected on 116/132 (87.9%) RMP-resistant strains by dHPLC. In the second phase of the study, 84/3107 (2.7%) clinical M. tuberculosis isolates were RMP-resistant. The sensitivity and specificity of dHPLC in the prediction of RMP resistance were 70/84 (83.3%) and 70/77 (91.0%), respectively. The specificity became 100% when 511 Leu to Pro mutation was excluded from the RMP resistance-related genetic changes.


CONCLUSIONS
In the detection of RMP resistance in a high-throughput laboratory setting, dHPLC has been demonstrated to be rapid, simple, workable, automatable and inexpensive in terms of running costs and the labour involved.
Yip
C W
CW
Department of Health, Tuberculosis Reference Laboratory, Centre for Health Protection, Kowloon, Hong Kong SAR, China.
Leung
K L
KL
Wong
D
D
Cheung
D T L
DT
Chu
M Y
MY
Tang
H S
HS
Kam
K M
KM
D001426:Bacterial Proteins / Q000235:genetics; D002851:Chromatography, High Pressure Liquid; D012321:DNA-Directed RNA Polymerases; D024881:Drug Resistance, Bacterial; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification*; D012293:Rifampin / Q000494:pharmacology*; D013997:Time Factors; D014397:Tuberculosis, Pulmonary / Q000382:microbiology*
D001426:Bacterial Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin