Genotype mtbdrplus

The drug susceptibility pattern of the Mycobacterium tuberculosis complex was determined using the Line Probe Assay (LPA) test. The test is based on the DNA-strip technology and permits the molecular genetics identification of the Mycobacterium tuberculosis complex and its resistance to rifampicin and/or isoniazid. The manufacturer’s instruction demonstrated that the identification of rifampicin resistance is enabled by the detection of the most significant mutations of the rpoB gene (coding for the β-sub-unit of the RNA polymerase). For the detection of a high level isoniazid resistance, the katG gene (coding for the catalase peroxidase) was examined, and for the detection of a low level isoniazid resistance the promoter region of the inhA gene (coding for the NADH enoyl ACP reductase) was examined. Line probe Assay was accomplished following the manufacturer’s instructions (GenoType MTBDRplus, Hain Life science GmbH, 2009, Germany).

Throughout the study period, gDST for RMP, INH and FQ was performed by Line Probe assay(LPA), using GenoType MTBDRplus and MTBDRsl version 2.0 (Hain Lifescience, Nehren, Germany). MGIT 960 automated system (BD, Sparks, MD, USA) was used to perform pDST at recommended critical concentrations for RMP (1.0ug/ml), INH (0.1ug/ml), PZA(100ug/ml) and ofloxacin (2ug/ml) during 2015–17 and LFX (1.0ug/ml) during 2018–19 [16 ].
All clinical samples were processed for culture and pDST was performed for all confirmed Mtb isolates. LPA was introduced in late 2015 and gDST was increasingly performed directly on clinical samples from known RMP resistant patients. Culture isolates were used for gDST for patients having RMP resistance with invalid results on direct testing, known RMP sensitive or with unknown RMP status. Sequencing was not performed as facilities were not established.
All recommended quality control measures for DST were followed during study period including, testing of known sensitive and resistant control strains with each batch of DST [17 ] and regular participation in annual external quality assessments conducted by WHO collaborating center (Supra National TB reference laboratory, ITM, Antwerp, Belgium) with successful certification for first and second line DST.

Genomic DNA was extracted from 0.5 ml of OM-S decontaminated sediment using the commercially available kit GenoLyse (Hain Lifescience, Nehren, Germany). Only the samples from TB confirmed cases (i.e. culture positive for MTB) were processed for LPA (GenoType MTBDRplus; Hain Lifescience) testing. DNA amplification and hybridization were performed according to manufacturer’s instructions.

All isolates were freshly subcultured on liquid medium incubated in MGIT 960, before being tested. The Genotype MTBDRplus and MTBDRsl (Hain Lifescience, GmbH, Germany) were performed according to the manufacturer’s instructions. These are Line Probe Assays (LPAs) that are based on DNA strip technology. Genotype MTBDRsl was performed by an experienced operator, who received standardized training on the method and it has been done, routinely, in the past 4 years in our Lab. The test itself has internal quality control and the final results were double checked by two blinded experienced operators.

The evaluation of the 24-well plate method in detecting MDR isolates was also compared with the GenoType MTBDRplus. For aminoglycosides and fluoroquinolones the phenotypic results from the 24 well assay was compared to the HAIN MTBDRsl assay through the detection of mutations in the gyrA and rrs genes, respectively. Essentially, the MTBDRplus and MTBDRsl assays share the same procedure (GenoType® MTBDRplus, GenoType® MTBDRsk Hain Life science GmbH, Germany). The MTBDRplus test is based on PCR amplification of specific regions of RIF and INH resistance conferring genes, rpoB, katG and inhA followed by reverse hybridization of amplicons to specific probes pre-blotted to membrane strips and colorimetric detection of the key mutations associated with RIF and INH resistance [20 (link)].