Abstract
The goal of this study was to apply temperature-mediated heteroduplex analysis using denaturing high-performance liquid chromatography to identify pyrazinamide (PZA) resistance in
Mycobacterium tuberculosis
isolates and simultaneously differentiate between
M. tuberculosis
and
Mycobacterium bovis
. Features that contributed to an optimal assay included the use of two different reference probes for the
pncA
gene targets from wild-type
M. tuberculosis
and wild-type
M. bovis
, optimization of the column temperature, increasing the starting concentration of the elution buffer, and reducing the rate of elution buffer increase (slope). A total of 69 strains were studied, including 48 wild-type
M. tuberculosis
strains (13 were PZA-resistant strains) and 21
M. bovis
strains (8 were BCG strains). In all isolates tested, wild-type
M. tuberculosis
generated a single-peak pattern when mixed with the
M. tuberculosis
probe and a double-peak pattern with the
M. bovis
probe. In contrast, all
M. bovis
isolates generated a double-peak pattern when mixed with the
M. tuberculosis
probe and a single-peak pattern with the
M. bovis
probe. PZA-resistant mutant
M. tuberculosis
isolates generated characteristic patterns that were easily distinguishable from both wild-type
M. tuberculosis
and
M. bovis
isolates. Chromatographic patterns generated by the two reference probes allowed the rapid detection of PZA resistance with the simultaneous ability to distinguish between
M. tuberculosis
and
M. bovis
. This approach may allow the detection of drug resistance-associated mutations, with potential application to clinical and epidemiological aspects of tuberculosis control.