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Objective/Background: Tuberculosis (TB) is a leading cause of death worldwide, with new threats of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. Pakistan is the fifth highest among high-burden TB countries and the fourth highest among high-burden drug-resistant-TB countries. Pakistan is the sixth most populous country in the world, and Pakistani youth is the highest population group in Pakistan and second in the world. This study was aimed at assessing the understanding, awareness, and mindset of university students toward TB, MDR TB, and XDR TB in Lahore. Methods: A cross-sectional questionnaire-based study was performed on 1137 individuals from three major public-sector universities in Lahore, Pakistan. Information regarding their knowledge and attitude toward MDR and XDR TB was gathered using a structured questionnaire. Data collected was analyzed using SPSS version 20. Results: Male (531) and female (606) students were asked about different aspects of MDR and XDR TB. Although 80.47% students had good knowledge about simple TB, a very small fraction had awareness and appropriate knowledge about MDR/XDR-TB. Considering TB as a stigma, only 9.3% students disclosed that they had household TB contact. Only 25% students knew about XDR TB. Conclusion: Our results indicated that a small fraction of people knew the exact definition and treatment duration of MDR TB and XDR TB in our society. There is a need to increase the awareness and knowledge status of university students about MDR and XDR TB.

The current study was undertaken to characterize the RRDR rpoB gene mutations among the rifampicin-resistant Mycobacterium tuberculosis (MTB) isolates from Pakistan. Rifampicin mutation patterns were analyzed by using PCR followed by rpoB gene sequencing. Among the 1080 referred TB cases, 63 (6%) were resistant against at least one first-line TB drug. Out of these 63 resistant isolates, 24 isolates (38%) were found to be resistant to isoniazid and rifampicin. Sequence analysis of multidrug-resistant tuberculosis (MDR-TB) isolates detected a single mutation in the RRDR region of the rpoB gene at codon 531, 516, 512, 528 and 533; however, 5 MDR-TB isolates lack any mutation in the RRDR region. A double mutation was observed in 1 MDR-TB isolate at codon 512 and 516 which are reported for the first time from Pakistan. Moreover, in 1 isolate a novel silent mutation was observed at codon 528. Further studies about these mutations may be helpful in the development of diagnostic tools for the detection of MTB in a high TB endemic area like Pakistan.

Background: GenoType MTBDRplus is a molecular assay for detection of Mycobacterium tuberculosis resistance to isoniazid (INH) and rifampicin (RMP), the two major anti-tuberculosis (TB) drugs. Identification of INH resistance is largely based on the occurrence of mutations in the katG gene, coding for the catalase-peroxidase, or in the promoter region of the inhA gene, coding for the NADH-dependent enoyl-ACP reductase. For testing the RMP resistance, mutations in the rpoB gene, coding for the RNA polymerase β subunit, particularly in the RMP resistance determining region (RRDR) of the gene are investigated. The GenoType MTBDRplus assay has been validated in several countries. The aim of this study was to evaluate the ability of the assay to detect INH and RMP resistance among strains of M. tuberculosis, isolated from Pakistani TB patients, and phenotypically identified as multidrug-resistant (MDR), that is resistant to both INH and RMP. Material/methods: The study included a collection of 100 MDR M. tuberculosis strains isolated from as many Pakistani TB patients over a 9-month period (i.e. between January and September 2014). Drug susceptibility testing was performed using the standard 1% proportion method on Löwenstein-Jensen medium, with INH and RMP critical concentrations of 0.2 mg/L and 40 mg/L, respectively. Genomic DNA was extracted by the cetyl-trimethyl ammonium bromide (CTAB) method. The GenoType MTBDRplus assay (Hain Lifescience, Germany) was done following the manufacturer's instructions. Results: In the katG gene, with MTBDRplus assay, a specific mutation associated with INH resistance (i.e. G944C transition, conferring Ser315Thr amino acid change) was detected in 66 (66%) of the strains. Thirty-four (34%) strains did not carry the katG mutation detected by the assay. Mutations in the mabA-inhA promoter region were detected in 10 (10%) strains (C-15T – in 10 strains, and T-8C – in 2 strains). Seventy-seven (77%) strains tested harboured a mutation in rpoB gene. Mutations in the rpoB gene were of four types: C1349T, A1304T, C1333G, and TC1324CA found in 63 (63%), 11 (11%), 8 (8%), and one (1%) strain, respectively. Of the 100 strains designated as MDR by the proportion method, GenoType MTBDRplus confirmed this phenotype in only 62 strains. The results of GenoType MTBDRplus and the conventional drug susceptibility method were consistent in 70% (70/100) for INH, and 77% (77/100) for RMP. Conclusions: As evidenced in this study, the major concern with the GenoType MTBDRplus assay were false negative results. In comparison to conventional drug susceptibility testing, the assay was unable to detect 30 (30/100; 30%) strains resistant to INH and 23 (23/100; 23%) strains resistant to RMP. The GenoType MTBDRplus failed to identify 38 MDR (38/100; 38%) strains. Resistance in those strains probably originate from mutations in other codons and/or genes than those covered by the test. For detecting INH and RMP resistance in TB cases, especially in high TB incidence countries, such as Pakistan, molecular approaches should still be a complement rather than areplacement to conventional drug susceptibility testing.