Antibiotic resistance is a major problem in Salmonella enterica serovar Typhi, the causative agent of typhoid. Multidrug-resistant (MDR) isolates are prevalent in parts of Asia and Africa and are often associated with the dominant H58 haplotype. Reduced susceptibility to fluoroquinolones is also widespread, and sporadic cases of resistance to third-generation cephalosporins or azithromycin have also been reported. Here, we report the first large-scale emergence and spread of a novel S. Typhi clone harboring resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third-generation cephalosporins in Sindh, Pakistan, which we classify as extensively drug resistant (XDR). Over 300 XDR typhoid cases have emerged in Sindh, Pakistan, since November 2016. Additionally, a single case of travel-associated XDR typhoid has recently been identified in the United Kingdom. Whole-genome sequencing of over 80 of the XDR isolates revealed remarkable genetic clonality and sequence conservation, identified a large number of resistance determinants, and showed that these isolates were of haplotype H58. The XDR S. Typhi clone encodes a chromosomally located resistance region and harbors a plasmid encoding additional resistance elements, including the blaCTX-M-15 extended-spectrum β-lactamase, and carrying the qnrS fluoroquinolone resistance gene. This antibiotic resistance-associated IncY plasmid exhibited high sequence identity to plasmids found in other enteric bacteria isolated from widely distributed geographic locations. This study highlights three concerning problems: the receding antibiotic arsenal for typhoid treatment, the ability of S. Typhi to transform from MDR to XDR in a single step by acquisition of a plasmid, and the ability of XDR clones to spread globally.

Background In 2018 Pakistan initiated its national antimicrobial resistance (AMR) surveillance aligned with Global Antimicrobial Surveillance System (GLASS). To complement this surveillance, we conducted a situational analysis of AMR rates among GLASS organisms in the country. Data from published studies and from antibiograms was compared and role of antibiograms as potential contributors to national AMR surveillance explored. Methods AMR rates for GLASS specified pathogen/antimicrobials combination from Pakistan were reviewed. Data sources included published studies (2006–2018) providing AMR rates from Pakistan (n = 54) as well as antibiograms (2011–2018) available on the Pakistan Antimicrobial Resistance Network (PARN) website. Resistance rates were categorized as follows: Very low: 0–10%, Low: 11–30%, Moderate: 30–50% and High: > 50%. Results Published data from hospital and community/laboratory-based studies report resistance rates of > 50% and 30–50% respectively to 3rd generation cephalosporins, fluoroquinolones and cotrimoxazole amongst Klebsiella pneumoniae and Escherichia coli. Carbapenem resistance rates amongst these organisms remained below 30%. High (> 50%) resistance was reported in Acinetobacter species to aminoglycosides and carbapenems among hospitalized patients. The evolution of ceftriaxone resistant Salmonella Typhi and Shigella species is reported. The data showed > 50% to fluoroquinolones amongst Neisseria gonorrhoeae and the spread of methicillin resistant Staphylococcus aureus (< 30%; 2008) to (> 50%; 2010) in hospital settings. Resistance reported in published studies aligned well with antibiogram data. The latter also captured a clear picture of evolution of resistance over the study period. Conclusion Both published studies as well antibiograms suggest high rates of AMR in Pakistan. Antibiogram data demonstrating steady increase in AMR highlight its potential role towards supplementing national AMR surveillance efforts particularly in settings where reach of national surveillance may be limited.

The percentage of extrapulmonary tuberculosis (EPTB) among new and relapse tuberculosis cases in South Asia (Afghanistan, Pakistan, India, and Bangladesh) ranged from 19% to 23% in 2014. While tuberculosis was reportedly more prevalent in males, a higher preponderance of EPTB was observed in females. National tuberculosis control programs are highly focused on pulmonary tuberculosis. This creates gaps in the surveillance, diagnosis, and study of EPTB among females, which is especially pronounced in the South Asian setting. We have reviewed recently published literatures from January 2010 to June 2016 reporting EPTB in females with a view to evaluate the current epidemiology, risk factors, diagnostic modalities, and treatment outcomes. We report significant gaps in the surveillance of EPTB among women in South Asia, emphasizing the need for greater focus on EPTB in females to overcome current surveillance and knowledge gaps.

Background: Limited capacity of laboratories for antimicrobial susceptibility testing (AST) presents a critical diagnostic bottleneck in resource limited countries. This paper aims to identify such gaps and to explore whether laboratory networks could contribute towards improving AST in low resource settings. Methods: A self-assessment tool to assess antimicrobial susceptibility testing capacity was administered as a pre-workshop activity to participants from 30 microbiology laboratories in 3 cities in Pakistan. Data from public and private laboratories was analyzed and capacity of each scored in percentage terms. Laboratories from Karachi were invited to join a support network. A cohort of five laboratories that consented were provided additional training and updates sessions over a period of 15 months. Impact of training activities in these laboratories was evaluated using a point scoring (0-11) tool. Results: Results of self-assessment component identified a number of areas that required strengthening (scores of ≤60%). These included; readiness for AMR surveillance; 38 and 46%, quality assurance; 49 and 55%, and detection of specific organisms; 56 and 60% for public and private laboratories respectively. No significant difference was detected in AST capacity between public and private laboratories [ANOVA; p > 0.05]. Scoring tool used to assess impact of training within the longitudinal cohort showed an increase from a baseline of 1-5.5 (August 2015) to improved post training scores of 7-11 (October 2016) for the 5 laboratories included. Moreover, statistical analysis using paired t-Test Analysis, assuming unequal variance, indicated that the increase in scored noted represents a statistically significant improvement in the components evaluated [p < 0.05]. Conclusion: Strengthening of laboratory capacity for AMR surveillance is important. Our data shows that close mentoring and support can help enhance capacity for antimicrobial sensitivity testing in resource limited settings. Our study further presents a model wherein laboratory networks can be successfully established and used towards improving diagnostic capacity in such settings.

Background Mutations in the Rv0678, pepQ and atpE genes of Mycobacterium tuberculosis (MTB) have been reported to be associated with reduced antimycobacterial susceptibility to bedaquiline (BDQ). Resistance conferring mutations in treatment naïve MTB strains is likely to have implications for BDQ based new drug regimen that aim to shorten treatment duration. We therefore investigated the genetic basis of resistance to BDQ in MTB clinical isolates from BDQ naïve TB patients from Pakistan. In addition, mutations in genes associated with efflux pumps were investigated as an alternate mechanism of resistance. Methods Based on convenience sampling, we studied 48 MTB clinical isolates from BDQ naïve TB patients. These isolates (from our strain bank) included 38 MDR/pre-XDR/XDR (10 BDQ resistant, 8 BDQ intermediate and 20 BDQ susceptible) and 10 pan drug susceptible MTB isolates. All strains were subjected to whole genome sequencing and genomes were analysed to identify variants in Rv0678, pepQ, atpE, Rv1979c, mmpLS and mmpL5 and drug resistance associated efflux pump genes. Results Of the BDQ resistant and intermediate strains 44% (8/18) had variants in Rv0678 including; two reported mutations S63R/G, six previously unreported variants; L40F, R50Q and R107C and three frameshift mutations; G25fs, D64fs and D109fs. Variants in efflux pumps; Rv1273c (G462K), Rv0507c (R426H) and Rv1634c (E198R) were found to be present in drug resistant isolates including BDQ resistant and intermediate isolates. E198R in efflux pump gene Rv1634c was the most frequently occurring variant in BDQ resistant and intermediate isolates (n = 10). Conclusion We found RAVs in Rv0678 to be commonly associated with BDQ resistance. Further confirmation of the role of variants in efflux pump genes in resistance is required so that they may be incorporated in genome-based diagnostics for drug resistant MTB.