Field evaluation of nanopore targeted next-generation sequencing to predict drug-resistant tuberculosis from native sputum in South Africa and Zambia

Rapid and comprehensive drug susceptibility testing (DST) is essential for diagnosing and treating drug-resistant tuberculosis effectively, and next-generation sequencing can be an effective genotypic DST method. We implemented and evaluated the performance of a nanopore targeted sequencing assay, called the Tuberculosis Drug Resistance Test (TBDR, Oxford Nanopore Diagnostics, Ltd., United Kingdom), which predicts drug resistance to 16 TB drugs, at a South African reference laboratory and a district diagnostic laboratory in Zambia. We compared the sequencing success rates between unprocessed and decontaminated sputum samples and determined the diagnostic accuracy against local DST (Xpert MTB/RIF Ultra, Xpert MTB/XDR, and BD BACTEC MGIT phenotypic DST). We prospectively sequenced 236 samples and have 148 samples with sequencing results from unprocessed and decontaminated sputum. We obtained successful sequencing results from 66.4% (94/148) unprocessed sputum samples and 75% (111/148) decontaminated samples. Sequencing success rates at the two sites differed, with 50.7% (36/71) successful sequencing results from unprocessed sputum in Zambia and 75.3% (58/77) in South Africa. Samples with "low" bacterial load, measured by Xpert MTB/RIF Ultra, tended to produce fewer successful sequencing results. TBDR sequencing predicted resistances in 48 samples, detecting resistance for rifampicin (n = 41) and isoniazid (n = 20), as well as 10 second-line drugs (n = 15). Sensitivity was variable compared to phenotypic DST, ranging from 33 (ethionamide) to 94% (rifampicin), while specificity remained above 90% for all drugs, except clofazimine. The TBDR assay can provide rapid, comprehensive genotypic DST. Technical and operational challenges need to be addressed for its broader implementation in high tuberculosis-burden settings. IMPORTANCE This study illustrates the use of the Tuberculosis Drug Resistance Test (TBDR, Oxford Nanopore Diagnostics, Ltd., United Kingdom) as a rapid drug susceptibility testing (DST) approach for diagnosing drug-resistant TB in the high TB-burden countries of South Africa and Zambia. The TBDR assay predicts resistance to 16 TB drugs, including first- and second-line treatments. By implementing the TBDR assay in a national reference laboratory in South Africa and a district diagnostic laboratory in Zambia, we demonstrate how this technology can provide faster diagnostic results (days) compared to traditional phenotypic DST methods (similar to 2 months), with adequate sensitivity. Missed resistances compared to phenotypic DST indicate that technical improvements are needed. Successful sequencing from unprocessed and decontaminated sputum samples at different sites suggests feasibility in diverse settings, though operational challenges remain. Implementing this rapid, comprehensive DST approach could enhance drug-resistant tuberculosis diagnosis and treatment, ultimately improving patient outcomes and helping to combat tuberculosis in high-burden regions.