Genetic Determinants of Drug Resistance in Mycobacterium tuberculosis and Their Diagnostic Value.

Farhat MR1,2, Sultana R3, Iartchouk O4, Bozeman S5, Galagan J6, Sisk P7, Stolte C8, Nebenzahl-Guimaraes H9, Jacobson K10, Sloutsky A11, Kaur D12, Posey J13, Kreiswirth BN14, Kurepina N15, Rigouts L16, Streicher EM17, Victor TC18, Warren RM19, van Soolingen D20, Murray M21,22.

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Abstract

BACKGROUND:

The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of anti-tuberculosis drugs.

OBJECTIVES:

To determine the optimal molecular approach needed, we sought to create a comprehensive catalogue of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance.

METHODS:

We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug resistance loci in M.tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1397 clinical M.tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line anti-tuberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool.

RESULTS:

The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity, but was lower for other drugs. The number of mutations needed to diagnose resistance is large and for the 13 drugs studied it was 238 across 18 genetic loci.

CONCLUSION:

These data suggest that a comprehensive M.tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole genome sequencing, encode resistance to second-line TB drugs.