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

Abstract

Rationale: 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 antituberculosis drugs.

Objectives: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog 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 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis 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.

Measurements and main 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.

Conclusions: 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 tuberculosis drugs.

Keywords: molecular diagnostics; multidrug-resistant tuberculosis; sensitivity and specificity.

Figure 1.

Geographic and genetic diversity of the isolates. (A) Isolate principal genetic group (PGG) (26) by country of origin. The digits represent the number of isolates collected from each country. Isolate numbers less than 10 are not displayed. (B) Neighbor-joining phylogenetic tree of Mycobacterium tuberculosis isolates. All red tones belong to the Europe–Africa–Americas lineage 4 (taupe, Haarlem; magenta, X/low-copy clade; orange, T; maroon, Latin American–Mediterranean). Blue, East Asia lineage 2 (e.g., Beijing); purple, East Africa and India lineage 3 (e.g., Central Asian Strain). S = sensitive.

Figure 2.

Diagnostic performance of serially pared predictive models by drug. Sensitivity is plotted in black, specificity in red. Dashed lines represent ±1 SD. Vertical lines represent the minimal set of predictive mutations chosen (in an automated step-down fashion) beyond which the sensitivity drops >1 SD from the mean sensitivity for the full model. AMK = amikacin; CAP = capreomycin; CIP = ciprofloxacin; EMB = ethambutol; ETH = ethionamide; INH = isoniazid; KAN = kanamycin; LEVO = levofloxacin; OFLX = ofloxacin; PAS = paraaminosalicylic acid; PZA = pyrazinamide; RIF = rifampicin; STR = streptomycin.