Isoniazid resistance profile and associated levofloxacin and pyrazinamide resistance in rifampicin resistant and sensitive isolates/from pulmonary and extrapulmonary tuberculosis patients in Pakistan: A laboratory based surveillance study 2015-19.

BACKGROUND: Pakistan is among top five high burden countries for tuberculosis and drug resistant TB. Among rifampicin sensitive new pulmonary TB (PTB), prevalence of isoniazid resistance is 8.3% (95%CI: 7.0-10.7) and resistance to fluoroquinolone is higher (11·1%, 95%CI: 7·8-14·3) than isoniazid resistance. METHOD: Five year retrospective data (2015-2019) of drug susceptibility testing (DST) for Mycobacterium tuberculosis isolates, performed using recommended phenotypic (pDST) and/or genotypic (gDST) methods was analyzed stratified by rifampicin results for isoniazid resistance profiles and associated levofloxacin and pyrazinamide resistance. FINDINGS: DST data was analyzed from 11045 TB patients. Isolates were tested using pDST (87%), gDST (92%) and both methods (79.5%). For both rifampicin and isoniazid, a significant difference (P < .001) was noted between resistance detected by pDST and gDST. Among isolates, tested by both methods (8787), 49% were resistant to rifampicin and 51.7% to isoniazid with discordance in resistant results of 15.8% for each, with 13.2% (570) of rifampicin resistance reported sensitive by pDST and 14.2% (660) of isoniazid resistance missed by gDST. Estimated isoniazid resistance among rifampicin sensitive new PTB, extrapulmonary TB and previously treated PTB was 9.8% (95%CI: 8.7-11.1), 6.8% (95%CI: 5.4-8.5) and 14.6% (95%CI: 11.8-17.9) respectively. Significant differences were reported between the genotypic profile of isoniazid resistance associated with rifampicin-resistant and sensitive isolates including detectable mutations (87% vs 71.6%), frequency of inhA (7.6% and 30.2%) and katG mutations (76.1% vs 41.2%) respectively. Among rifampicin resistant and sensitive isolates, a significantly higher level of resistance to levofloxacin and pyrazinamide was seen associated with isoniazid resistance. CONCLUSION: There are risks and many challenges in implementing WHO recommended treatment for isoniazid resistant tuberculosis. The laboratory based surveillance can complement random surveys in country specific planning for TB diagnostics and appropriate treatment regimens.

Extrapulmonary tuberculosis in Pakistan- A nation-wide multicenter retrospective study.

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 North-West. The most common forms of EPTB were pleural (29.6%), lymphatic (22.7%) and abdominal TB (21.0%). Pleural TB was the most common clinical manifestation in adults (34.2%) and abdominal TB in children (38.4%). An increase in the prevalence of pleural and osteoarticular and decline in lymphatic and abdominal TB was observed with advancing age. Diversity in demography and clinical manifestations were noted between provinces. The treatment success rate for all type EPTB was significantly high compared to bacteriology confirmed PTB with the exception of EPTB affecting CNS with a high mortality rate. CONCLUSIONS: The study provides an insight into demography, clinical manifestations and treatment outcomes of EPTB. Further studies are needed to explain significant diversities observed between provinces, specific risk factors and challenges concerning EPTB management.

Acquisition of Cross-Resistance to Bedaquiline and Clofazimine following Treatment for Tuberculosis in Pakistan.

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.

Genetic sequencing for surveillance of drug resistance in tuberculosis in highly endemic countries: a multi-country population-based surveillance study.

BACKGROUND: In many countries, regular monitoring of the emergence of resistance to anti-tuberculosis drugs is hampered by the limitations of phenotypic testing for drug susceptibility. We therefore evaluated the use of genetic sequencing for surveillance of drug resistance in tuberculosis. METHODS: Population-level surveys were done in hospitals and clinics in seven countries (Azerbaijan, Bangladesh, Belarus, Pakistan, Philippines, South Africa, and Ukraine) to evaluate the use of genetic sequencing to estimate the resistance of Mycobacterium tuberculosis isolates to rifampicin, isoniazid, ofloxacin, moxifloxacin, pyrazinamide, kanamycin, amikacin, and capreomycin. For each drug, we assessed the accuracy of genetic sequencing by a comparison of the adjusted prevalence of resistance, measured by genetic sequencing, with the true prevalence of resistance, determined by phenotypic testing. FINDINGS: Isolates were taken from 7094 patients with tuberculosis who were enrolled in the study between November, 2009, and May, 2014. In all tuberculosis cases, the overall pooled sensitivity values for predicting resistance by genetic sequencing were 91% (95% CI 87-94) for rpoB (rifampicin resistance), 86% (74-93) for katG, inhA, and fabG promoter combined (isoniazid resistance), 54% (39-68) for pncA (pyrazinamide resistance), 85% (77-91) for gyrA and gyrB combined (ofloxacin resistance), and 88% (81-92) for gyrA and gyrB combined (moxifloxacin resistance). For nearly all drugs and in most settings, there was a large overlap in the estimated prevalence of drug resistance by genetic sequencing and the estimated prevalence by phenotypic testing. INTERPRETATION: Genetic sequencing can be a valuable tool for surveillance of drug resistance, providing new opportunities to monitor drug resistance in tuberculosis in resource-poor countries. Before its widespread adoption for surveillance purposes, there is a need to standardise DNA extraction methods, recording and reporting nomenclature, and data interpretation. FUNDING: Bill & Melinda Gates Foundation, United States Agency for International Development, Global Alliance for Tuberculosis Drug Development.