In a case study published in the journal Mycobacteria, a team of microbiologists and infectious disease specialists from the Hospices Civils de Lyon (France) describe the management of a young patient hospitalized for smear-positive pulmonary tuberculosis. On day 4, the Xpert® MTB/RIF Ultra assay, followed by a second rapid test (MDR/MTB ELITe MGB®), both detected rifampicin resistance, indicating the need for an intensive MDR-TB treatment regimen. Indeed, at the time of diagnosis (end of 2024), the 6-month short-course treatment with bedaquiline, pretomanid, linezolid and moxifloxacin was not available in France.

For confirming this indication, the team chose to rapidly apply targeted next-generation sequencing on a patient’s sputum sample using Deeplex® Myc-TB, capable of directly identifying resistance mutations to 15 anti-tuberculosis drugs in a single test. The result, available within just a few days, identified the P454L mutation in the rpoB gene, which is located just outside the rifampicin resistance-determining region (RRDR) normally targeted by Xpert assays. This mutation is classified by the WHO as of uncertain significance (group 3, no clear evidence of association with resistance). No resistance mutation was detected in any of the targets of the 14 other anti-TB drugs.

This information proved decisive: in consultation between clinicians and microbiologists, the patient was treated as a drug-susceptible tuberculosis case, with a bridging strategy including levofloxacin while awaiting phenotypic confirmation. Eighteen days later, whole genome sequencing performed after culturing confirmed the P454L mutation in the rpoB gene in isolation and the absence of resistance mutation in any other gene targets.  By day 31, phenotypic drug susceptibility testing using MGIT and determination of the minimal inhibitory concentration (0.125 mg/L) using broth microdilution confirmed full susceptibility to rifampicin. Treatment was simplified by discontinuating levofloxacin, and the patient was cured within six months, with no relapse observed after three months’ follow-up.

This case of false-positive rifampicin resistance detected by indirect, real-time PCR tests highlights the clinical value of culture-free direct sequencing with the Deeplex® Myc-TB assay: by precisely identifying the nature and gene position of the mutation, the use of this approach prevented an unnecessary prolonged and toxic MDR-TB treatment, while ensuring a multidisciplinary evidence-based decision.