Evaluation of Genotype MTBDRplus and MTBDRsl Assays for Rapid Detection of Drug Resistance in Extensively Drug-Resistant Mycobacterium tuberculosis Isolates in Pakistan

Abstract

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries alongside the 6th among countries with the highest burden of drug-resistant TB, including multi-drug resistant (MDR)-TB. Methods for rapid and reliable drug susceptibility testing (DST) are prerequisite for the prompt institution of effective anti-TB treatment. The aim of this study was to evaluate the efficiency of Genotype MTBDRplus and MTBDRsl assays for the detection of MDR and (pre-) extensively drug-resistant (XDR-TB) isolates in Pakistan. The study included 47 pre-XDR and 6 XDR-TB isolates, recovered from 53 patients from Pakistan. Conventional DST was performed using the standard 1% proportion method on the Löwenstein-Jensen medium. For molecular determination of drug resistance, GenoType MTBDRplus and GenoType MTBDRsl assays (Hain Lifescience, Germany) were used. To evaluate discrepancies between conventional and molecular DST results, mutation profiling was performed by amplifying and sequencing seven genetic loci, i.e., katG, inhA, and mabA-inhA promoter, rpoB, gyrA, embB, rrs. The sensitivity of Genotype MTBDRplus was 71.7% for isoniazid (INH) and 79.2% for rifampicin (RIF). Sequence analysis revealed non-synonymous mutations in 93.3 and 27.3% of isolates phenotypically resistant to INH and RIF, respectively, albeit susceptible when tested by GenoType MTBDRplus. GenoType MTBDRsl had a sensitivity of 73.6, 64.7, 20, 25, and 100% for the detection of fluoroquinolones, ethambutol, kanamycin, amikacin, and capreomycin resistance, respectively. Upon sequencing, mutations were detected in 20, 77.8%, and all isolates phenotypically resistant to aminoglycosides, ethambutol, and fluoroquinolones, respectively, yet declared as susceptible with GenoType MTBDRsl. Low sensitivities seriously impede the large-scale application of the Genotype MTBDRplus and MTBDRsl assays. Unless further optimized, the currently available line-probe assays should rather be auxiliary to the conventional, phenotype-based methods in the detection of MDR- and XDR-TB in Pakistan.

Genomic Analysis Identifies Mutations Concerning Drug-Resistance and Beijing Genotype in Multidrug-Resistant Mycobacterium tuberculosis Isolated From China

“…In the present study, the additional mutation loci found in katG except in codon 315 made the mutations in the inhA promoter and oxyR-ahpC less meaningful for predicting INH resistance for that mutation in the latter two regions increased only 2.7% sensitivity by WGS. Although several molecular DSTs for INH resistance testing are recommended by WHO, several reports show that the calculated sensitivity among clinical isolates is far lower 90% ( Li et al, 2015 ; Javed et al, 2016 , 2018 ; World Health Organization, 2016 ; Maningi et al, 2018 ). According to the standard ( World health organization, 2014 ) and the actual situations in clinical practice of new molecular DST assays ( Li et al, 2015 ; Javed et al, 2016 , 2018 ; World Health Organization, 2016 ; Maningi et al, 2018 ), the sensitivity for STR by WGS in the present study was recognized to achieve to an acceptable degree (using rpsL and rrs 530 loop and 912 loop, 83%), and the specificity was excellent (97.8%) while the sensitivity of embB for predicting EMB resistance was excellent (90.9%), but the specificity (65.1%) was far lower than the standard, which requires more than 95% ( World health organization, 2014 ).…”

Evaluation of GenoType MTBDRplus for the detection of drug-resistant Mycobacterium tuberculosis on isolates from Karachi, Pakistan

“…Our study demonstrated a higher sensitivity for detecting RIF and INH resistance via LPA as compared to that reported in another study conducted in Pakistan (RIF: 98.8% vs 79.2%; INH: 90.6% vs 71.7%). [12]…”

“…The reports of the percentage of mutations in the kat G gene range from 31.8%[13] to 96.9%[14] of isolates tested, while the percentage of mutations in the inh A promoter region ranges from 1.5% to 45.9%. [13] The percent of having mutations at both genes ranges from 1.2%[12] to 23.0%. [15] For example, in comparing the percentage of each mutation to those found in similar settings, those identified in Karachi were lower than the 83.0% of kat G mutations[16] and the 21.4% of inhA mutations[6] found in individuals with isolates resistant to INH in India.…”

Comparison of Quantamatrix Multiplexed Assay Platform and GenoType MTBDR Assay Using Smear-Positive Sputum Specimens From Patients With Multidrug- Resistant/Extensively Drug-Resistant Tuberculosis in South Korea

“…Our results showed that the sensitivity for detection of EMB resistance (44.4%) was lower than that for resistance to other drugs. In previous studies, the sensitivity of MTBDR sl for detection of EMB resistance was 64.7% (Javed et al, 2018), 57.5% (Jian et al, 2018), and 62% (Simons et al, 2015). Javed et al (2018) reported that the most frequent mutations were at the embB codons 497 and 406.…”

“…In previous studies, the sensitivity of MTBDR sl for detection of EMB resistance was 64.7% (Javed et al, 2018), 57.5% (Jian et al, 2018), and 62% (Simons et al, 2015). Javed et al (2018) reported that the most frequent mutations were at the embB codons 497 and 406. However, only mutations at codon 306 were determined in our study, suggesting that this may have been the cause of the low sensitivity for EMB resistance that we observed.…”