Usefulness of MTBDRplus assay for rapid genotypic detection of Mycobacterium tuberculosis resistant to isoniazid and rifampicin directly in clinical specimens

Abstract number: R2503

Lacoma A., García N., Prat C., Blanco S., Haba L., Fuenzalida L., Maldonado J., Ruiz Manzano J., Domínguez J.

Objectives: To evaluate the usefulness of MTBDRplus assay for detecting resistance of Mycobacterium tuberculosis against isoniazid (INH) and rifampicin (RIF) in smear-negative and smear-positive clinical specimens.

Methods: Sixty-five clinical samples (53 sputa, 3 bronchoalveolar lavages, 5 bronchial aspirates, 3 pleural effusions and 1 lymph node) were recovered from 28 patients. All the samples were processed by conventional mycobacterial procedures and after strain isolation on solid culture, the antibiotic susceptibility was assessed by BACTEC460TB. According to the Ziehl-Neelsen staining 39 samples were smear-negative (19 INHs/RIFs, 4 INHr/RIFs and 3 INHr/RIFr) and 26 were smear-positive (12 INHs/RIFs, 1 INHr/RIFs and 26 INHr/RIFr). The assay, which consisted of DNA isolation, multiplex PCR with biotinylated primers and reverse hybridisation on nitrocellulose strips, was carried out following the manufacturer instructions. MTBDRplus combines control, wild type and mutation probes, which allow the detection of the most common resistance mediating mutations in rpoB, katG and inhA.

Results: Valid test results were obtained for 51 samples (78.4%). The 14 invalid results corresponded to Ziehl-Neelsen negative samples. The concordance between MTBDRplus susceptibility pattern and BACTEC460TB result for the samples with valid result was 98% (50/51) for RIF and 96.2% (49/51) for INH. In relation to RIF, the discordant sample was resistant according to the MTBDRplus, because of the absence of one of the wild-type bands, but susceptible according to BACTEC460TB. The two discordant samples for INH, belonging to the same patient, were RIFr/INHr according to BACTEC but the MTBDRplus INH pattern was susceptible. We detected 30 rpoB mutations for the 29 RIF resistant samples that consisted of: 17 D526Y (56.7%), 9 D516V (30%) and 4 S531L (13.3%). Mutations in katG and inhA for the 30 INH resistant samples were: katG S315T in 11 cases (39.3%) and both katG S315T and inhA-15 C-T in 17 cases (60.7%).

Conclusions: MTBDRplus assay is an easy to perform test that allows the rapid detection of RIF and INH resistant M. tuberculosis directly from clinical sample with a high sensitivity, being appropriated for the use in the routine work flow.