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Demonstration of targeted next-generation sequencing using the Oxford Nanopore Technologies tuberculosis assay for the detection of drug resistant tuberculosis in Kyrgyzstan

A Kulzhabaeva(1,3) A Iskakova(1,2) G Saparova(2) F Tilekova(2) B Myrzaliev(3,4) M Ahmatov(1,3) A Duishekeeva(1,3) A Soorombaeva(1) A Toktogonova(2) M Sydykova(2) A Slyzkyi(4) A Kadyrov(2) G Kalmambetova(2) E Tiemersma(4) K Kremer(4)

1:KNCV Tuberculosis Foundation Kyrgyzstan office, Bishkek, Kyrgyzstan; 2:National Center for Phthisiology, Bishkek, Kyrgyzstan; 3:Kyrgyz State Medical Academy, Bishkek, Kyrgyzstan; 4:KNCV Tuberculosis Foundation, The Hague, The Netherlands

We present the first results of an ongoing study that validates the Oxford Nanopore Technologies (ONT) targeted next-generation sequencing (tNGS) tuberculosis (TB) assay and compares its use to standard of care (SOC) drug susceptibility testing (DST) in Kyrgyzstan.

For validation, 82 smear-positive sputum samples were subjected to both the ONT tNGS TB assay (OND-CUST-KIT on MinION MK1B) and the Deeplex Myc-TB kit (GenoScreen, on MiSeq (Illumina)). 6/82 samples had indeterminate results. Among the remaining 76 samples, concordance was 100% for isoniazid/rifampicin/amikacin/kanamycin; 98.6% for fluoroquinolones/capreomycin/linezolid, 96.0% for bedaquiline/clofazimine/ethambutol/streptomycin; 93.4% for pyrazinamide, and 92.1% for ethionamide. High concordance was observed between the two assays for high-confidence-graded mutations.

For the comparative study that will enrol 782 Xpert-TB-positive patients, sputum samples of participants from Kyrgyzstan, aged >=18 years with signs and symptoms of pulmonary TB, are being collected for testing with the ONT assay and SOC-DST (i.e. Xpert, line probe assay and phenotypic DST). From January-April 2024, 776 patients were enrolled, of which 140 (18%) had Xpert MTB/RIF detected. Of these, 75 were sequenced and 21 also had SOC-DST-results. SOC-DST revealed isoniazid but not rifampicin resistance in nine (43%) patients and rifampicin plus isoniazid resistance in ten (48%) patients. Generally, the ONT tNGS assay had results concordant with SOC-DST, but identified additional resistance, including to levofloxacin and moxifloxacin, in 18 patients.

This study shows the usefulness of ONT tNGS for the diagnosis of TB in Kyrgyzstan and highlights the potential of incorporating tNGS into routine TB diagnostic algorithms for accurate and timely treatment decisions.

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