Affiliation:
1. Cleveland Clinic, Cleveland, Ohio
2. University of the Philippines—Philippine General Hospital, Manila, Philippines
Abstract
ABSTRACT
After isoniazid and rifampin (rifampicin), the next pivotal drug class in
Mycobacterium tuberculosis
treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of the
rpoB
,
katG
, and
gyrA
genes occur with frequencies of 97%, 50%, and 85% among
M. tuberculosis
isolates resistant to rifampin, isoniazid, and fluoroquinolones, respectively. Sequences are highly conserved, and certain mutations correlate well with phenotypic resistance. We developed a pyrosequencing assay to determine
M. tuberculosis
genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102
M. tuberculosis
clinical isolates from the Philippines for susceptibility to rifampin, isoniazid, and ofloxacin by using the conventional submerged-disk proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified by using PCR primers directed toward the RDR of the
rpoB
,
katG
, and
gyrA
genes, and pyrosequencing was performed on the extracts. The
M. tuberculosis
H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivities and specificities of pyrosequencing were 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100% for the detection of resistance to rifampin, isoniazid, and ofloxacin, respectively. Pyrosequencing is thus a rapid and accurate method for detecting
M. tuberculosis
resistance to these three drugs.
Publisher
American Society for Microbiology
Cited by
55 articles.
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