Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid and Fluoroquinolones. Journal of clinical microbiology [J Clin Microbiol] Journal article

Title	Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid and Fluoroquinolones.
Author(s)	Bravo LT, Tuohy MJ, Ang C, Destura RV, Mendoza M, Procop GW, Gordon SM, Hall GS, Shrestha NK
Institution	Cleveland Clinic, Cleveland, OH, USA; University of the Philippines-Philippine General Hospital, Manila, Philippines.
Source	J Clin Microbiol 2009 Oct 21.
Abstract	After isoniazid and rifampin, the next pivotal drug class in M. tuberculosis (MTB) 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 MTB 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 MTB genotypic resistance to rifampin, isoniazid, and fluoroquinolones. We characterized 102 MTB clinical isolates from the Philippines for susceptibility to rifampin, isoniazid and ofloxacin using the conventional disc submerged proportion method and validated our pyrosequencing assay using these isolates. DNA was extracted and amplified using PCR primers directed towards the RDR of the rpoB, katG and gyrA genes, and pyrosequencing performed on the extracts. The MTB H37Rv strain (ATCC 25618) was used as the reference strain. The sensitivity and specificity of pyrosequencing was 96.7% and 97.3%, 63.8% and 100%, and 70.0% and 100%, respectively, for the detection of resistance to rifampin, isoniazid and ofloxacin. Pyrosequencing is thus a rapid and accurate method for detecting MTB resistance to these three drugs.
Language	ENG
Pub Type(s)	JOURNAL ARTICLE
PubMed ID	19846642

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