Comparative Analysis of Whole Genome Sequencing

To examine the potential analytical advantage of whole genome sequencing comparison was made with three commercial tests: (1) the Xpert MTB/RIF (Cepheid Inc., USA) which targets the rpoB gene for RMP resistance; (2) the LPA MTBDRplus for MDR-TB (Hain Lifescience, Germany) which targets rpoB, katG and inhA for resistance to RMP and INH; and (3) the LPA MTBDRsl (Hain Lifescience, Germany) which targets gyrA, rrs and embB for resistance to the fluoroquinolones (FLQ), aminoglycosides and ethambutol, respectively. In silico versions were developed based on the polymorphisms used by these assays and their performance compared to the whole genome mutation library. In particular, in silico analysis of the six datasets was performed and analytical sensitivities and specificities of the inferred resistance relative to the reported phenotype were compared (Figure 2, Additional file 1: Figures S3 and S4). KvarQ [35 (link)], a new tool that directly scans fastq files of bacterial genome sequences for known genetic polymorphisms, was run across all 792 samples using the MTBC test suite and default parameters. Sensitivity and specificity achieved by this method using phenotypic DST results as the reference standard were calculated.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.