Simultaneous detection of Mycobacterium tuberculosis complex and rifampin/isoniazid resistance with sanity 2.0 MTBC-MDR: a moderate-complexity automated NAAT.

OBJECTIVE

To compare the performance of Sanity 2.0 with that of Xpert MTB/RIF and phenotypic drug susceptibility testing (DST) for the detection of Mycobacterium tuberculosis complex (MTBC) and resistance to rifampicin (RIF) and isoniazid (INH).

METHODS

Acid-fast staining positive sputum samples, collected from February to July in 2024, were used for solid culture, DST, Xpert MTB/RIF, and Sanity 2.0 assay.

RESULTS

The limit of detection of the Sanity 2.0 assay was 3 CFU/mL for MTB detection, and 30 CFU/mL for RIF and INH resistance mutations. Compared to Xpert MTB/RIF, complete agreement was observed for the detection of MTB with sensitivity of 99.3% (296/298), as well as for RIF resistance. Using DST as the reference method, Sanity 2.0 exhibited a sensitivity of 96.55% and a specificity of 99.25% for the detection of RIF resistance. For INH resistance, Sanity 2.0 demonstrated a sensitivity of 78.95% and a specificity of 99.22%. Among three cases with discordant rifampicin resistance profiles between Sanity 2.0 and DST, one Sanity 2.0-sensitive/phenotypically-resistant case was confirmed as sensitive by sequencing, and two Sanity 2.0-resistant/phenotypically-sensitive cases carried a mutation in rpoB at codon 531 (TCG→TTG) and 526 (CAC→AGC), respectively. Among two INH-resistant cases by Sanity 2.0 that were sensitive by DST, one isolate carried a mutation in the ahpC promoter region (–15 C > T), and the other had a katG mutation at codon 315 (AGC→ACC). All eight cases that were INH-sensitive by Sanity 2.0 but resistant by DST were confirmed to be sensitive by sequencing.

CONCLUSION

Sanity 2.0 represents a rapid and automated molecular assay with high sensitivity for MTBC and RIF/INH resistance detection, showing comparable performance to established assays and making it well-suited for decentralized healthcare settings as a complementary diagnostic tool.