JCM Accepts, published online ahead of print on 21 October 2009

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J. Clin. Microbiol. doi:10.1128/JCM.01229-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Pyrosequencing for Rapid Detection of Mycobacterium tuberculosis Resistance to Rifampin, Isoniazid and Fluoroquinolones

Lulette Tricia C. Bravo, Marion J. Tuohy, Concepcion Ang, Raul V. Destura, Myrna Mendoza, Gary W. Procop, Steven M. Gordon, Geraldine S. Hall, and Nabin K. Shrestha^*

Cleveland Clinic, Cleveland, OH, USA; University of the Philippines-Philippine General Hospital, Manila, Philippines

^* To whom correspondence should be addressed. Email: shrestn{at}ccf.org.

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.