Conventional phenotypic drug susceptibility testing (DST) methods for
Mycobacterium tuberculosis
are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci:
rpoB
,
katG
,
embB
,
rrs
,
gyrA
, and the promoter regions of
inhA
and
eis
. The molecular method was evaluated on a panel of 290 clinical isolates of
M. tuberculosis
. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant
M. tuberculosis
as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant
M. tuberculosis
. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in
M. tuberculosis
.
Our findings are consistent with the belief that a mutation at position 1401 in rrs leads to resistance to capreomycin, amikacin and kanamycin. The data also show that tlyA is not a sensitive genetic marker for capreomycin resistance in clinical isolates of M. tuberculosis, as mutations in this gene are infrequent and not all mutations in tlyA lead to capreomycin resistance.
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