Rapid determination of anti-tuberculosis drug resistance from whole-genome sequences

Abstract

Mycobacterium tuberculosis drug resistance (DR) challenges effective tuberculosis disease control. Current molecular tests examine limited numbers of mutations, and although whole genome sequencing approaches could fully characterise DR, data complexity has restricted their clinical application. A library (1,325 mutations) predictive of DR for 15 anti-tuberculosis drugs was compiled and validated for 11 of them using genomic-phenotypic data from 792 strains. A rapid online 'TB-Profiler' tool was developed to report DR and strain-type profiles directly from raw sequences. Using our DR mutation library, in silico diagnostic accuracy was superior to some commercial diagnostics and alternative databases. The library will facilitate sequence-based drug-susceptibility testing.

Figure 1

Polymorphism in the curated library used for predicting multi-drug resistant TB (MDR-TB) and extensive-drug resistant TB (XDR-TB). (A) Polymorphisms associated with MDR-TB. (B) Polymorphisms associated with XDR-TB. Colour-coded bars in the Circos plot represent genes described to be involved in drug resistance (from Table 1). On top of each of these bars a grey histogram shows the mutation density (calculated as the number of polymorphic sites within windows of 20 bp) derived from the curated list of DR-associated mutations. These grey areas highlight the presence of DR-associated regions in candidate genes, which in some cases span the whole gene (for example, katG) or are confined to a certain region of the gene (for example, rpoB). Vertical black lines indicate the frequency of mutations (that is, the number of times the mutation has been observed) in phenotypically resistance isolates. Internal black lines show co-occurring mutations both within and between genes. The thickness of these lines is proportional to the frequency of the mutations appearing together.

Figure 2

Inferred analytical accuracies of the whole genome mutation library and three commercial molecular tests for resistance. In silico analysis of published sequence data using mutation libraries derived from XpertMTB/RIF (Cepheid Inc., USA) (purple), MTBDRsl (red) and MTBDRplus (orange) (Hain Life Sciences, Germany), and the curated whole genome library (blue). For each library in silico inferred resistance phenotypes were compared to reported phenotypes obtained from conventional drug susceptibility testing. Errors bars correspond to 95% confidence intervals. Abbreviations: AMK, amikacin; CAP, capreomycin; EMB, ethambutol; ETH, ethionamide; INH, Isoniazid; KAN, kanamycin; MDR, multi-drug resistance; MOX, moxifloxacin; OFX, ofloxacin; PZA, pyrazinamide; RMP, rifampicin; STR, streptomycin; XDR, extensive drug resistance.