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

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.