Open AccessPyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid, and Fluoroquinolones
Author(s) -
Lulette Tricia C. Bravo,
Marion J. Tuohy,
Concepcion Ang,
Raul V. Destura,
Myrna Mendoza,
Gary W. Procop,
Steven M. Gordon,
Geraldine S. Hall,
Nabin K. Shrestha
Publication year - 2009
Publication title -
journal of clinical microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.349
H-Index - 255
eISSN - 1070-633X
pISSN - 0095-1137
DOI - 10.1128/jcm.01229-09
Subject(s) - rpob , isoniazid , mycobacterium tuberculosis , rifampicin , microbiology and biotechnology , tuberculosis , pyrosequencing , ofloxacin , biology , drug resistance , antibacterial agent , virology , antibiotics , ciprofloxacin , genetics , gene , medicine , pathology
After isoniazid and rifampin (rifampicin), the next pivotal drug class inMycobacterium tuberculosis treatment is the fluoroquinolone class. Mutations in resistance-determining regions (RDR) of therpoB ,katG , andgyrA genes occur with frequencies of 97%, 50%, and 85% amongM. 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 determineM. tuberculosis genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102M. 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 therpoB ,katG , andgyrA genes, and pyrosequencing was performed on the extracts. TheM. 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 detectingM. tuberculosis resistance to these three drugs.