Fluoroquinolone resistance detection in Mycobacterium tuberculosis with locked nucleic acid probe real-time PCR.
METHODS
Pham Ngoc Thach Hospital for Tuberculosis and Lung Diseases, Ho Chi Minh City, Vietnam.


OBJECTIVE
Fluoroquinolones (FQs) are increasingly used in the treatment of tuberculosis (TB) and are the second-line drugs of choice for treatment of multidrug-resistant TB. We aimed to set up a polymerase chain reaction (PCR) based assay to detect the most common FQ-resistance-associated mutations in gyrase A (gyrA) of Mycobacterium tuberculosis.


METHODS
A total of 42 FQ-resistant and 40 FQ-susceptible isolates were collected in 2005-2006 and sequenced in gyrA. Using sequencing results as gold standard, a real-time PCR using three locked nucleic acid probes (LNA-PCR) was designed to detect mutations at positions 90, 91 and 94 (97% of gyrA FQ-resistance-associated mutations) and evaluated.


RESULTS
Sequencing of 42 FQ-resistant isolates revealed no gyrA mutations in 10 isolates, 20 isolates had a single mutation and 12 isolates showed double peaks at resistance-associated alleles, suggesting a heterogeneous population. With LNA-PCR, all wild-type and 19/20 mutant isolates were correctly identified. Eleven of 12 heterogeneous isolates were correctly identified as resistant mutants. Overall, 71% ([19 + 11]/42) of phenotypically FQ-resistant isolates were detected. Specificity was 100% on 40 FQ-susceptible isolates.


CONCLUSIONS
This assay provides a simple and rapid means to reliably detect FQ-resistance-associated gyrA mutations in M. tuberculosis.
van Doorn
H R
HR
Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. rvandoorn@oucru.org
An
D D
DD
de Jong
M D
MD
Lan
N T N
NT
Hoa
D V
DV
Quy
H T
HT
Chau
N V V
NV
Duy
P M
PM
Tho
D Q
DQ
Chinh
N T
NT
Farrar
J J
JJ
Caws
M
M
D000995:Antitubercular Agents / Q000494:pharmacology*; D027081:DNA Gyrase / Q000235:genetics*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D024841:Fluoroquinolones / Q000494:pharmacology*; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D018088:Tuberculosis, Multidrug-Resistant / Q000235:genetics*
D000995:Antitubercular Agents; D024841:Fluoroquinolones; D009841:Oligonucleotides; C477371:locked nucleic acid; D027081:DNA Gyrase