Genetic diversity of multidrug-resistant Mycobacterium tuberculosis isolates in Punjab, Pakistan.

Pakistan ranks 5th among the world's highest tuberculosis (TB) burden countries and 6th among the countries with the highest prevalence of drug-resistant TB. However, insufficient data are available on the genetic structure of M. tuberculosis strains circulating in this country. The objective of this study was to explore the genetic diversity of multidrug-resistant M. tuberculosis isolates from Punjab, Pakistan with a combination of spoligotyping and 24-loci MIRU-VNTR typing. Among a total of 127 MDR isolates studies, 53 spoligotypes were obtained, split into 14 clusters (n = 88, 69.3%, 2-29 isolates per cluster) and 39 (30.7%) unique patterns. At the phylogenetic level, the most prevalent sublineage was CAS1_DELHI (n = 53, 41.7%), mostly represented by ST 1942 (n = 29, 22.8%), followed by T1 (n = 14, 11%) and Beijing (n = 10, 7.8%). The remaining nine sublineages (CAS, MANU2, EAI5, T2, LAM10_CAM, H1, X1, H4 and CAS2) involved altogether 24 (18.9%) isolates. Twenty-six (20.5%) isolates could not be assigned to any specific clade. MIRU-VNTR typing identified 123 (96.8%), 97 (76.4%) and 65 (51.2%) unique types with a tolerance of 0, 1, and 2 locus differences between the patterns. Upon combined spoligotyping and MIRU-VNTR typing analysis, 123 (96.8%), 108 (85%), and 91 (71.7%) unique types were identified if a tolerance of 0, 1, and 2 locus differences in the MIRU-VNTR patterns was assumed, respectively. Based on the clustering results, the transmission rate for MDR-TB cases under the study was calculated at 3.2%, 15%, and 28.3%. Overall, three clades, namely CAS1_DELHI, T1, and Beijing accounted for the majority of MDR-TB cases in Pakistan. Up to a third of the cases were clustered upon combined spoligotyping and MIRU-VNTR typing, suggesting a moderate level of active transmission.

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

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.

The activity of silver nanoparticles against microalgae of the Prototheca genus.

AIM: To investigate the in vitro activity of silver NPs (AgNPs) against pathogenic microalgae of the Prototheca genus. MATERIALS & METHODS: The antialgal potential of AgNPs against Prototheca species of both clinical and environmental origin was assessed from minimum inhibitory (algistatic) and algicidal concentrations. The in vitro cytotoxicity of AgNPs against bovine mammary epithelial cell line was evaluated by means of the standard MTT assay. RESULTS: AgNPs showed a strong killing activity toward Prototheca algae, as the minimal algicidal concentration (MAC) values matched perfectly the corresponding minimum inhibitory concentration (MIC) values for all species (MAC = MIC, 1-4 mg/l), except P. stagnora (MIC > 8 mg/l). The concentrations inhibitory to pathogenic Prototheca spp. (MIC, 1-4 mg/l) were below the concentrations at which any toxicity in epithelial cells could be observed (CC20 > 6 mg/l). CONCLUSION: The study emphasizes the potential of AgNPs as a new therapeutic tool for the management of Prototheca infections.