Molecular screening of new patients suspected for TB could help in the effective control of TB in Pakistan as it is a high TB burden country. It will be informative to understand the prevalence of multi drug resistance for a better drug regimen management in this geographical area. The Rifampicin resistance determining region (RRDR) sequencing was used to identify mutations associated with drug resistance in DNA extracts from 130 known multidrug resistant (MDR) cultured strains and compared with mutations observed in DNA extracts directly from 86 sputum samples from consecutive newly diagnosed cases in Lahore, Pakistan. These newly diagnosed samples were positive for smear microscopy, chest X-ray and presumed sensitive to first line drugs. In the known MDR group the most frequent mutations conferring resistance were found in rpoB531 (n = 51, 39.2%). In the newly diagnosed tuberculosis group with no history of MDR, mutations in rpoB531 were seen in 10 of the samples (11.6%). Collectively, all mutations in the RRDR region studied were observed in 80 (61.5%) of known MDR cases and in 14 (16.3%) of the newly diagnosed cases. Using the RRDR as a surrogate marker for MDR, sequences for the newly diagnosed (presumed sensitive) group indicate much higher levels of MDR than the 3.9% WHO 2015 global estimate and suggests that molecular screening directly from sputum is urgently required to effectively address the detection and treatment gaps to combat MDR in this high burden country.

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Background: Multi drug resistant tuberculosis(MDR-TB) is more difficult to diagnose and treat, leading to high mortality. Accurate and rapid detection of MDR-TB is critical for timely initiation of treatment. Evaluating new drug resistance diagnostic tools such as Genotype MTBDRplus VER 2.0 assay offer opportunity to scale up drug susceptibility testing(DST) capacity in Ethiopia.Methods & Materials: A cross sectional study was conducted from December to August, 2015 on presumptive MDR-TB patients. Analysis of 72 smear positive and 197 smear negative sputum samples was done with Genotype MTBDRplus VER 2.0 assay and compared with the reference, BACTEC MGIT 960 culture and DST. Sensitivity, specificity, PPV and NPV of the MTBDRplus VER 2.0 assay was calculated, comparing the results with the reference method and results was interpreted based on 95% confidence interval, statistical significant was taken at p-value <0.05.Results: The sensitivity, specificity, PPV and NPV of Genotype MTBDRplus VER 2.0 assay were 96.4, 100, 100 and 96.9%, respectively for the detection of MDR-TB from direct smear positive sputum samples. Only 14(54%) samples had valid results with LPA among the 26 smear negative culture positive samples. The remaining 8(30.6%) and 4(15.4%) were invalid and negative with LPA, respectively. The sensitivity and specificity of Genotype MTBDRplus VER 2.0 assay was 100% for the detection of MDR-TB among 14 direct smear negative and culture positive sputum samples. The most common mutations associated with RMP and INH resistance was S531L and S315TL, respectively. A single rare mutation (C15T/A16G) was also detected in this study. Conclusion:The diagnostic performance of Genotype MTBDRplus VER 2.0 assay in direct smear positive sputum sample was highly sensitive and specific for early detection of MDR-TB. However, the diagnostic performance of Genotype MTBDRplus VER 2.0 assay in direct smear negative sputum sample was low and showed high level of invalid results so it is unlikely to implement Genotype MTBDRplus VER 2.0 assay for the detection of MDR-TB in direct smear negative sample in our routine settings until the method is optimized. Hence, large scale further studies are needed in direct smear negative samples.

Nepal is geographically located between India and China, a region containing significant Tuberculosis (TB) and Multi-Drug Resistance (MDR-TB) burdens. However, limited information is available on the phylogenetic diversity of Mycobacterium tuberculosis (Mtb) in Nepal. To gain further insight into the diversity of Mtb in Nepal, consecutive clinical samples from 176 newly diagnosed pulmonary tuberculosis patients were collected from two hospitals in Nepal. Insertion Site IS6110 Fluorescent Amplified Fragment Length Polymorphism (FAFLP) PCR and rpoB sequence analysis were carried out on genomic DNA extracts of cultured strains to assign them to accepted genetic lineages and identify MDR-TB. In this study, the IS6110 based characterisation showed a prevalence of 36.36% Central Asian Strain (CAS), 18.75% Beijing, 7.95% Haarlem, 3.97% X, 2.2% each of Latin American Mediterranean (LAM), T-Uganda and T, 1.7% S and 24.4% were unassigned. Further, 3.9% of total M. tuberculosis isolates were of rifampicin resistant genotypes thus indicating that the prevalence of MDR could be higher than the country wide prevalence of MDR among new TB cases (2.2%) as reported by the national drug resistance survey carried out in 2011/2012.