Direct thin-layer agar for bedaquiline-susceptibility testing of Mycobacterium tuberculosis at BSL2 level yields high accuracy in 15 days from sputum processing.

Cuella Martin, I.; Runyambo, D.; De Bock, S.; Diels, M.; Niyompano, H.; Hakizayezu, F.; Keysers, J.; de Rijk, W. B.; Habimana, Y. M.; Gahamanyi, N.; Ardizzoni, E.; Muvunyi, C. M.; de Jong, B.; Semuto Ngabonziza, J.-C.; Rigouts, L.

doi:10.1101/2024.11.18.624108

bioRxiv · 10.1101/2024.11.18.624108

Direct thin-layer agar for bedaquiline-susceptibility testing of Mycobacterium tuberculosis at BSL2 level yields high accuracy in 15 days from sputum processing.

Cuella Martin, I.·Runyambo, D.·De Bock, S.·Diels, M.·Niyompano, H.·Hakizayezu, F.·Keysers, J.·de Rijk, W. B.·Habimana, Y. M.·Gahamanyi, N.·Ardizzoni, E.·Muvunyi, C. M.·de Jong, B.·Semuto Ngabonziza, J.-C.·Rigouts, L.

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Abstract

This study evaluated thin-layer agar (TLA) as a faster alternative for both indirect MIC determination of bedaquiline (BDQ) from culture isolates and direct drug-susceptibility testing (DST) from sputum samples. Indirect BDQ-MIC results from TLA were compared to the established 7H11 solid DST. Direct-TLA-DST performance was assessed using 143 baseline sputum samples from rifampicin-resistant TB cases. Direct-TLA tested susceptibility to rifampicin, isoniazid, levofloxacin, and BDQ, with results compared to Lowenstein-Jensen (LJ) and MGIT. For indirect BDQ-MIC determination, TLA demonstrated high accuracy, with MIC values for H37Rv falling within the WHO control range (0.015-0.12g/ml). The incubation conditions (5-10% CO2, standard) and reading time (day 7, 14) significantly affected results, with D7 readings in standard incubator providing optimal accuracy. Direct-TLA-DST detected MTB in 53.8% of samples, compared to 55.9% on LJ and 69.4% in MGIT. Uninterpretable results due to contamination or medium drying were low (4.9%). Median time to result was 15 days for smear-positive and 22 days for smear-negative samples, faster than WHO endorsed methods. Sensitivity was 100% for rifampicin and 87.8% for isoniazid, with a specificity of 100% for all drugs except isoniazid (96.2%). No BDQ nor levofloxacin resistance was detected, thus direct TLA sensitivity could not be assessed. In conclusion, direct-TLA-DST offers a reliable and faster alternative to conventional DST methods for BDQ and other anti-TB drugs. Essentially, this technique can be operated at BioSafety Level 2, allowing decentralization of pDST for managing drug-resistant TB in settings with limited laboratory infrastructure. ImportanceThis paper addresses the critical need for faster direct drug-susceptibility testing (DST) on sputum, especially for bedaquiline (BDQ), which is a key drug in treating drug-resistant TB. Currently, there is a lack of rapid, reliable methods for direct BDQ testing from sputum samples, limiting timely and accurate treatment decisions and monitoring. By demonstrating the potential of thin-layer agar (TLA) for direct BDQ-MIC determination, this study offers a promising solution that could significantly improve patient care.

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Cuella Martin, I., Runyambo, D., De Bock, S., Diels, M., Niyompano, H., Hakizayezu, F., Keysers, J., de Rijk, W. B., Habimana, Y. M., Gahamanyi, N., Ardizzoni, E., Muvunyi, C. M., de Jong, B., Semuto Ngabonziza, J.-C., Rigouts, L.. 2024-11-20. Direct thin-layer agar for bedaquiline-susceptibility testing of Mycobacterium tuberculosis at BSL2 level yields high accuracy in 15 days from sputum processing.. https://doi.org/10.1101/2024.11.18.624108

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