Background Pakistan is fifth among high burden countries for tuberculosis. A steady increase is seen in extrapulmonary tuberculosis (EPTB), which now accounts for 20% of all notified TB cases. There is very limited information on the epidemiology of EPTB. This study was performed with the aim to describe the demographic characteristics, clinical manifestations and treatment outcomes of EPTB patients in Pakistan. Method We performed descriptive analysis on routinely collected data for cohorts of TB patients registered nationwide in 2016 at health facilities selected using stratified convenient sampling. Findings Altogether 54092 TB including 15790 (29.2%) EPTB cases were registered in 2016 at 50 study sites. The median age was 24 years for EPTB and 30 years for PTB patients. The crude prevalence of EPTB in females was 30.5% (95%CI; 29.9-31.0) compared to 27.9% (95%CI; 27.3-28.4) in males. The likelihood of having EPTB (OR), was 1.1 times greater for females, 2.0 times for children, and 3.3 times for residents of provinces in the NorthWest .

We report on the first six cases of acquired resistance to bedaquiline in Pakistan. Seventy sequential isolates from 30 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing and by the detection of mutations in relevant genes. We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

Pakistan’s national tuberculosis control programme (NTP) is among the many programmes worldwide that value the importance of subnational tuberculosis (TB) burden estimates to support disease control efforts, but do not have reliable estimates. A hackathon was thus organised to solicit the development and comparison of several models for small area estimation of TB. The TB hackathon was launched in April 2019. Participating teams were requested to produce district-level estimates of bacteriologically positive TB prevalence among adults (over 15 years of age) for 2018. The NTP provided case-based data from their 2010–2011 TB prevalence survey, along with data relating to TB screening, testing and treatment for the period between 2010–2011 and 2018. Five teams submitted district-level TB prevalence estimates, methodological details and programming code. Although the geographical distribution of TB prevalence varied considerably across models, we identified several districts with consistently low notification-to-prevalence ratios. The hackathon highlighted the challenges of generating granular spatiotemporal TB prevalence forecasts based on a cross-sectional prevalence survey data and other data sources. Nevertheless, it provided a range of approaches to subnational disease modelling. The NTP’s use and plans for these outputs shows that, limitations notwithstanding, they can be valuable for programme planning.

Aims and objectives: In the present study, we report on the first seven cases of acquired bedaquiline and clofazimine resistance identified in Pakistan. The study was conducted within the framework of a retrospective surveillance project to monitor the acquisition of resistance to bedaquiline implemented by the National TB Reference Laboratory in Pakistan and TB Supranational Reference Laboratory. Methods: Seventy three sequential isolates from 31 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing in 7H11 medium, and for all isolates showing an increased MIC compared to the baseline isolate, further MIC testing for bedaquiline and clofazimine was conducted using the Bactec MGIT960 (BD, Franklin Lakes, NJ, USA). The H37Rv (ATCC 27294) strain was used as a susceptible control in MIC testing. Bedaquiline dry powder was supplied by Janssen-Pharmaceutica (Beerse, Belgium). WGS was carried out with the Illumina Nextera-XT DNA sample preparation kit to prepare paired-end libraries of 150-bp read length to sequence on an Illumina NextSeq platform. Data analysis and single nucleotide polymorphism (SNP) calling were performed using the MTBseq-Pipeline on low-frequency detection mode. Genes associated with resistance to bedaquiline and/or clofazimine (atpE, Rv0678, pepQ, and Rv1979c) were screened for mutations. Results: We studied 73 isolates from patients (n=31; 8 MDR, 16 pre-XDR, and 7 XDR) enrolled in bedaquiline-containing regimens. The sequential isolates tested included two strains from 22 patients, three from 7 patients, and four from 2 patients. All baseline strains included in the study were sensitive to bedaquiline. Seven patients developed an increase in bedaquiline MICs in 7H11 medium (range, 0.125 to >0.5mg·liter−1) during therapy, and six of them became resistant to bedaquiline according to the current critical concentration proposed for the drug. In all seven patients we detected genetic variants in Rv0678 that were not present at baseline isolates. Moreover in five out of seven patients the treatment was ultimately failed. Conclusions: We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

20We report on the first six cases of acquired resistance to bedaquiline in Pakistan. Seventy sequential 21 culture isolates from 30 drug-resistant tuberculosis patients on bedaquiline-containing regimen were 22 tested for minimum inhibitory concentration (MIC) and whole genome sequencing. An increase in MICs 23 associated with cross-resistance to clofazimine and appearance of specific mutations was documented in 24 six cases. The study underlines that appropriate monitoring is mandatory for the introduction of new drugs. 25 26 27 93 strains collected from two patients reported as "failure" at the end of treatment (patient-4 MIC 0.25 94 mg·L −1 , eight fold higher than baseline and patient-5 MIC 0.125 mg·L −1 , four fold higher than baseline) 95 would have been categorised as susceptible. This finding indicates that monitoring MICs during treatment 96 could be a better predictor for failure than single testing at critical concentration. The genetic basis of 97 4 resistance to bedaquiline is still the subject of much uncertainty. WGS analysis in different studies showed 98 that bedaquiline-clofazimine cross resistance arises through mutations in Rv0678 (6,7,8) and pepQ (9). In 99 this study, we show that the increase of MIC to bedaquiline and clofazimine could be explained by 100 mutations emerging during therapy in Rv0678. As reported in table 1 we observed four different mutations 101 and three of these were previously reported as associated 102 to bedaquiline resistance in MTB clinical strains (8). We show that the same mutations are associated to 103 Clofazimine resistance. As a result, regimens that contain both drugs might have to be reconsidered when 104 these mutations are identified, to reduce the risk of failure for the patients and transmission of such strains 105 in the community. Altogether, these data show that resistance to bedaquiline emerges during treatment 106 and emphasize the importance of using MIC and whenever possible molecular based surveillance in 107 national programmes implementing bedaquiline for the treatment of MDR/XDR-TB to monitor the 108 emergence of resistance. Moreover, a classification of mutations associated to bedaquiline and clofazimine 109 resistance is crucial to lead future development of tools for fast detection of resistance. Introducing drugs 110 without proper diagnostics to monitor drug resistance may lead to amplification of hardly treatable cases. 111 112 Ethics approval 113 Ethical clearance was approved by the Institutional review board for HIV, TB and Malaria programme, 114 Pakistan.