Whole-genome sequencing for prediction of Mycobacterium tuberculosis drug susceptibility and resistance: a retrospective cohort study

Abstract

SummaryBackgroundDiagnosing drug-resistance remains an obstacle to the elimination of tuberculosis. Phenotypic drug-susceptibility testing is slow and expensive, and commercial genotypic assays screen only common resistance-determining mutations. We used whole-genome sequencing to characterise common and rare mutations predicting drug resistance, or consistency with susceptibility, for all first-line and second-line drugs for tuberculosis.MethodsBetween Sept 1, 2010, and Dec 1, 2013, we sequenced a training set of 2099 Mycobacterium tuberculosis genomes. For 23 candidate genes identified from the drug-resistance scientific literature, we algorithmically characterised genetic mutations as not conferring resistance (benign), resistance determinants, or uncharacterised. We then assessed the ability of these characterisations to predict phenotypic drug-susceptibility testing for an independent validation set of 1552 genomes. We sought mutations under similar selection pressure to those characterised as resistance determinants outside candidate genes to account for residual phenotypic resistance.FindingsWe characterised 120 training-set mutations as resistance determining, and 772 as benign. With these mutations, we could predict 89·2% of the validation-set phenotypes with a mean 92·3% sensitivity (95% CI 90·7–93·7) and 98·4% specificity (98·1–98·7). 10·8% of validation-set phenotypes could not be predicted because uncharacterised mutations were present. With an in-silico comparison, characterised resistance determinants had higher sensitivity than the mutations from three line-probe assays (85·1% vs 81·6%). No additional resistance determinants were identified among mutations under selection pressure in non-candidate genes.InterpretationA broad catalogue of genetic mutations enable data from whole-genome sequencing to be used clinically to predict drug resistance, drug susceptibility, or to identify drug phenotypes that cannot yet be genetically predicted. This approach could be integrated into routine diagnostic workflows, phasing out phenotypic drug-susceptibility testing while reporting drug resistance early.

The epidemiology, pathogenesis, transmission, diagnosis, and management of multidrug-resistant, extensively drug-resistant, and incurable tuberculosis

“…145,146 However, before wholegenome sequencing information can be used in routine clinical practice, several important issues need to be resolved. Whole-genome sequencing is complicated by the need for culture before DNA extraction, 147 incomplete knowledge of all resistance-conferring mutations, 59,148 and the need for a validated pipeline to accurately predict resistance.…”

Clinical implications of the global multidrug-resistant tuberculosis epidemic

“…24 Studies have already illustrated a role for such technology in identifying drug resistance mutations in TB. 25,26 NGS enables sequencing of entire coding regions and allows polymorphisms involved in drug resistance in TB to be further characterised. However, the discordance between phenotypic tests and NGS indicates that some mutations may not have an impact on drug susceptibility, so it is important to correlate new mutations with clinical response to drugs before determining what the effect of a particular mutation may be.…”

“…However, the discordance between phenotypic tests and NGS indicates that some mutations may not have an impact on drug susceptibility, so it is important to correlate new mutations with clinical response to drugs before determining what the effect of a particular mutation may be. 25 …”

Current status and opportunities for therapeutic drug monitoring in the treatment of tuberculosis

“…Whereas in the last two decades especially reverse line blot assays [69][70][71] were applied to detect or exclude the presence of particular resistance mutations in the genome of Mtb, nowadays whole genome sequencing (WGS) provides the possibility to detect all mutations previously associated with resistance. [72] However, the general problem faced at the moment is that the predictive value of resistance mutations is not always clear, especially when they are rarely encountered. [73] For the frequently observed mutations tested in reverse line blot assays, such as the ones associated with rifampicin and isoniazid, the positive-and negative-predictive value is high and this merits direct clinical use of these test results to steer the treatment.…”

“…[72,81] It is conceivable that within a few years a large part of the phenotypic resistance testing could be avoided. Ideally also the correlation between the presence of particular resistance mutations and the level of resistance against all anti-TB drugs will be established, so the selection of the treatment regimen can be based solely on DNA analysis.…”