Evaluating culture-free targeted next-generation sequencing for diagnosing drug-resistant tuberculosis: a multicentre clinical study of two end-to-end commercial workflows

Abstract

Background: Drug-resistant tuberculosis remains a major obstacle in ending the global tuberculosis epidemic. Deployment of molecular tools for comprehensive drug resistance profiling is imperative for successful detection and characterisation of tuberculosis drug resistance. We aimed to assess the diagnostic accuracy of a new class of molecular diagnostics for drug-resistant tuberculosis.

Methods: We conducted a prospective, cross-sectional, multicentre clinical evaluation of the performance of two targeted next-generation sequencing (tNGS) assays for drug-resistant tuberculosis at reference laboratories in three countries (Georgia, India, and South Africa) to assess diagnostic accuracy and index test failure rates. Eligible participants were aged 18 years or older, with molecularly confirmed pulmonary tuberculosis, and at risk for rifampicin-resistant tuberculosis. Sensitivity and specificity for both tNGS index tests (GenoScreen Deeplex Myc-TB and Oxford Nanopore Technologies [ONT] Tuberculosis Drug Resistance Test) were calculated for rifampicin, isoniazid, fluoroquinolones (moxifloxacin, levofloxacin), second line-injectables (amikacin, kanamycin, capreomycin), pyrazinamide, bedaquiline, linezolid, clofazimine, ethambutol, and streptomycin against a composite reference standard of phenotypic drug susceptibility testing and whole-genome sequencing.

Findings: Between April 1, 2021, and June 30, 2022, 832 individuals were invited to participate in the study, of whom 720 were included in the final analysis (212, 376, and 132 participants in Georgia, India, and South Africa, respectively). Of 720 clinical sediment samples evaluated, 658 (91%) and 684 (95%) produced complete or partial results on the GenoScreen and ONT tNGS workflows, respectively, with 593 (96%) and 603 (98%) of 616 smear-positive samples producing tNGS sequence data. Both workflows had sensitivities and specificities of more than 95% for rifampicin and isoniazid, and high accuracy for fluoroquinolones (sensitivity approximately ≥94%) and second line-injectables (sensitivity 80%) compared with the composite reference standard. Importantly, these assays also detected mutations associated with resistance to critical new and repurposed drugs (bedaquiline, linezolid) not currently detectable by any other WHO-recommended rapid diagnostics on the market. We note that the current format of assays have low sensitivity (≤50%) for linezolid and more work on mutations associated with drug resistance is needed.

Interpretation: This multicentre evaluation demonstrates that culture-free tNGS can provide accurate sequencing results for detection and characterisation of drug resistance from Mycobacterium tuberculosis clinical sediment samples for timely, comprehensive profiling of drug-resistant tuberculosis.

Funding: Unitaid.

Figure 1:. Study profile

pDST=phenotypic drug susceptibility testing. WGS=whole-genome sequencing. *Indicators of and risk factors for drug resistance were: (1) a positive rifampicin resistance result by GeneXpert MTB/RIF or GeneXpert MTB/RIF Ultra; or (2) not responding to tuberculosis treatment with positive sputum smear or culture after ≥3 months of standard tuberculosis treatment; or (3) previously diagnosed with rifampicin-resistant or multidrug-resistant tuberculosis and did not respond to tuberculosis treatment with positive sputum smear or culture after ≥3 months of a standard multidrug-resistant tuberculosis regimen; or (4) previously received >1 month of treatment for a prior tuberculosis episode; or (5) close contact with an individual with known drug-resistant tuberculosis.

Figure 2:. Sequencing success by bacterial load proxies (AFB smear status, study GeneXpert semiquantitative result category, and HIV status)

Total numbers of samples in each category are indicated in parentheses on the y-axis. Blue bars indicate the number of samples for each targeted next-generation sequencing (tNGS) solution with complete resistance profiles (ie, all tNGS targets were sequenced and reported). Purple bars indicate the number of samples for each tNGS solution with partial resistance profiles (ie, some tNGS targets failed to sequence). Grey bars indicate the number of samples with no sequence data generated (ie, no tNGS results were reported). AFB=acid-fast bacilli. GS=GenoScreen Deeplex Myc-TB. ONT=Oxford Nanopore Technologies Tuberculosis Drug Resistance Test.

Figure 3:. Sensitivity and specificity of GenoScreen (A) and ONT (B) targeted next-generation sequencing workflows for detection of drug resistance compared with a composite reference of MGIT phenotypic drug susceptibility testing and whole-genome sequencing

Sensitivity is shown in the forest plot diagrams, while both sensitivity and specificity are shown in the adjacent tables. Bedaquiline and clofazimine do not have a composite reference as there were no resistance-associated mutations identified in the 2021 WHO mutation catalogue; for these drugs, only MGIT phenotypic drug susceptibility testing was used as a reference. The dotted line represents 50% sensitivity. TP=true positive. FP=false positive. FN=false negative. TN=true negative. ONT=Oxford Nanopore Technologies. MGIT=Mycobacteria Growth Indicator Tube.