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Abstract Background Fluoroquinolones (FQs) are potential drugs that inhibit DNA synthesis and are used in the treatment of multidrug-resistant tuberculosis (TB) and short-term anti-TB regimens. In recent years, a high proportion of FQ resistance has been observed in Mycobacterium tuberculosis isolates. The development of FQ resistance in multidrug-resistant TB negatively impacts patient treatment outcome and is a serious threat to control of TB. Methods The study included a total of 562 samples from patients with pulmonary TB that had been on anti-tuberculosis therapy. MTBDRsl assays were performed for the molecular detection of mutations. Sequence analysis was performed for the characterization and mutational profiling of FQ-resistant isolates. Results FQ resistance was observed in 104 samples (18.5%), most of which were previously treated and treatment failure cases. A total of 102 isolates had mutations in DNA gyrase subunit A (gyrA), while mutations in gyrB were observed in only two isolates. Mutational analysis revealed that the mutations mostly alter codons 94 (replacing aspartic acid with glycine, D94G) and 90 (replacing alanine with valine, A90V). In MDR and treatment failure cases, resistance to FQs was most commonly associated with the D94G mutation. In contract, a high proportion of A90V mutations were observed in isolates that were newly diagnosed. Conclusion The findings suggest that genotypic assays for FQ resistance should be carried out at the time of initial diagnosis, before starting treatment, in order to rule out mutations that impact the potential use of FQs in treatment and to control drug resistance.

A high percentage of camel handlers in Saudi Arabia are seropositive for Middle East respiratory syndrome coronavirus. We found that 12/100 camel handlers and their family members in Pakistan, a country with extensive camel MERS-CoV infection, were seropositive, indicating that MERS-CoV infection of these populations extends beyond the Arabian Peninsula.

The human pathogen Acinetobacter baumannii has emerged as a frequent cause of hospital-acquired infections, but infection of animals has rarely been observed. Here we analyzed an outbreak of epidemic pneumonia killing hundreds of sheep on a farm in Pakistan and identified A. baumannii as the infecting agent. A pure culture of strain AbPK1 isolated from lungs of sick animals was inoculated into healthy sheep, which subsequently developed similar disease symptoms. Bacteria re-isolated from the infected animals were shown to be identical to the inoculum, fulfilling Koch’s postulates. Comparison of the AbPK1 genome against 2283 A. baumannii genomes from the NCBI database revealed that AbPK1 carries genes for unusual surface structures, including a unique composition of iron acquisition genes, genes for O-antigen synthesis and sialic acid-specific acetylases of cell-surface carbohydrates that could enable immune evasion. Several of these unusual and otherwise rarely present genes were also identified in genomes of phylogenetically unrelated A. baumannii isolates from combat-wounded US military from Afghanistan indicating a common gene pool in this geographical region. Based on core genome MLST this virulent isolate represents a newly emerging lineage of Global Clone 2, suggesting a human source for this disease outbreak. The observed epidemic, direct transmission from sheep to sheep, which is highly unusual for A. baumannii, has important consequences for human and animal health. First, direct animal-to-animal transmission facilitates fast spread of pathogen and disease in the flock. Second, it may establish a stable ecological niche and subsequent spread in a new host. And third, it constitutes a serious risk of transmission of this hyper-virulent clone from sheep back to humans, which may result in emergence of contagious disease amongst humans.

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.

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:Drug resistant tuberculosis (DR-TB) is a major public health problem in developing countries such as Pakistan. OBJECTIVE:The current study was conducted to assess the frequency of drug resistant tuberculosis including multi drug resistance (MDR- TB) as well as risk factors for development of DR-TB, in Punjab, Pakistan. METHODOLOGY:Drug susceptibility testing (DST) was performed, using proportion method, for 2367 culture positive Mycobacterium tuberculosis (MTB) cases that were enrolled from January 2012 to December 2013 in the province of Punjab, Pakistan, against first-line anti-tuberculosis drugs. The data was analyzed using statistical software; SPSS version 18. RESULTS:Out of 2367 isolates, 273 (11.5%) were resistant to at least one anti-TB drug, while 221 (9.3%) showed MDR- TB. Risk factors for development of MDR-TB were early age (ranges between 10-25 years) and previously treated TB patients. CONCLUSION:DR-TB is a considerable problem in Pakistan. Major risk factors are previous history of TB treatment and younger age group. It emphasizes the need for effective TB control Program in the country.

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.