Pyrosequencing for rapid detection of Mycobacterium tuberculosis resistance to rifampin, isoniazid, and fluoroquinolones

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.

FIG. 1.

Diagrammatic representation of RDR and corresponding positions of PCR and sequencing primers. The numbers underneath the codons signify the codon number and hot-spot regions. F, forward; R, reverse; R1, first rpoB sequencing primer; R2, second rpoB sequencing primer; K1, katG sequencing primer; G1, gyrA sequencing primer.

FIG. 2.

Pyrograms illustrating representative sequences of each RDR. In each instance, the upper panel shows the sequence for the reference ATCC strain, and the lower panel shows the sequence for a resistant isolate. (A) Rifampin RDR with a mutation in codon 533. (B) Ofloxacin RDR with a mutation in codon 94. Also note the polymorphism in codon 95 of the ofloxacin-resistant isolate. (C) Isoniazid RDR with a mutation in codon 315.