Bedaquiline resistance in patients with drug-resistant tuberculosis in Cape Town, South Africa: a retrospective longitudinal cohort study.

BACKGROUND: Bedaquiline is a life-saving tuberculosis drug undergoing global scale-up. People at risk of weak tuberculosis drug regimens are a priority for novel drug access despite the potential source of Mycobacterium tuberculosis-resistant strains. We aimed to characterise bedaquiline resistance in individuals who had sustained culture positivity during bedaquiline-based treatment. METHODS: We did a retrospective longitudinal cohort study of adults (aged ≥18 years) with culture-positive pulmonary tuberculosis who received at least 4 months of a bedaquiline-containing regimen from 12 drug-resistant tuberculosis treatment facilities in Cape Town, South Africa, between Jan 20, 2016, and Nov 20, 2017. Sputum was programmatically collected at baseline (ie, before bedaquiline initiation) and each month to monitor treatment response per the national algorithm. The last available isolate from the sputum collected at or after 4 months of bedaquiline was designated the follow-up isolate. Phenotypic drug susceptibility testing for bedaquiline was done on baseline and follow-up isolates in MGIT960 media (WHO-recommended critical concentration of 1 µg/mL). Targeted deep sequencing for Rv0678, atpE, and pepQ, as well as whole-genome sequencing were also done. FINDINGS: In total, 40 (31%) of 129 patients from an estimated pool were eligible for this study. Overall, three (8%) of 38 patients assessable by phenotypic drug susceptibility testing for bedaquiline had primary resistance, 18 (47%) gained resistance (acquired or reinfection), and 17 (45%) were susceptible at both baseline and follow-up. Several Rv0678 and pepQ single-nucleotide polymorphisms and indels were associated with resistance. Although variants occurred in Rv0676c and Rv1979c, these variants were not associated with resistance. Targeted deep sequencing detected low-level variants undetected by whole-genome sequencing; however, none were in genes without variants already detected by whole-genome sequencing. Patients with baseline fluoroquinolone resistance, clofazimine exposure, and four or less effective drugs were more likely to have bedaquiline-resistant gain. Resistance gain was primarily due to acquisition; however, some reinfection by resistant strains occurred. INTERPRETATION: Bedaquiline-resistance gain, for which we identified risk factors, was common in these programmatically treated patients with sustained culture positivity. Our study highlights risks associated with implementing life-saving new drugs and shows evidence of bedaquiline-resistance transmission. Routine drug susceptibility testing should urgently accompany scale-up of new drugs; however, rapid drug susceptibility testing for bedaquiline remains challenging given the diversity of variants observed. FUNDING: Doris Duke Charitable Foundation, US National Institute of Allergy and Infectious Diseases, South African Medical Research Council, National Research Foundation, Research Foundation Flanders, Stellenbosch University Faculty of Medicine Health Sciences, South African National Research Foundation, Swiss National Science Foundation, and Wellcome Trust.

Genetic variants and their association with phenotypic resistance to bedaquiline in Mycobacterium tuberculosis: a systematic review and individual isolate data analysis.

BACKGROUND: Bedaquiline is a crucial drug for control of rifampicin-resistant tuberculosis. Molecular drug resistance assays could facilitate effective use of bedaquiline and surveillance of drug resistance emergence. To facilitate molecular assay development, we aimed to identify genomic markers of bedaquiline resistance. METHODS: In this systematic review and individual isolate analysis, we searched Europe PubMed Central and Scopus for studies published from the inception of each database until Oct 19, 2020, that assessed genotypic and phenotypic bedaquiline resistance in clinical or non-clinical Mycobacterium tuberculosis isolates. All studies reporting on the assessment of variants in the four genes of interest (Rv0678, atpE, pepQ, and Rv1979c) and phenotypic bedaquiline data in both clinical and non-clinical samples were included. We collated individual isolate data from eligible studies to assess the association between genomic variants with phenotypic bedaquiline resistance, using a standardised method endorsed by WHO. Risk of bias of the extracted data was independently assessed by two authors using the Quality Assessment of Diagnostic Accuracy Studies tool for clinical studies and Systematic Review Center for Laboratory Animal Experimentation tool for animal studies. The primary outcome was to identify mutations associated with resistance in four genes of interest (Rv0678, atpE, pepQ, and Rv1979c); for each genomic variant, the odds ratio (OR), 95% CI, and p value were calculated to identify resistance markers associated with bedaquiline resistance. This study is registered with PROSPERO, CRD42020221498. FINDINGS: Of 1367 studies identified, 41 published between 2007 and 2020 were eligible for inclusion. We extracted data on 1708 isolates: 1569 (91·9%) clinical isolates and 139 (8·1%) non-clinical isolates. We identified 237 unique variants in Rv0678, 14 in atpE, 28 in pepQ, and 11 in Rv1979c. Most clinical isolates with a single variant reported in Rv0678 (229 [79%] of 287 variants), atpE (14 [88%] of 16 variants), pepQ (32 [100%] of 32 variants), or Rv1979c (115 [98%] of 119 variants) were phenotypically susceptible to bedaquiline. Except for the atpE 187G→C (OR ∞, [95% CI 13·28-∞]; p<0·0001) and Rv0678 138_139insG (OR 6·91 [95% CI 1·16-47·38]; p=0·016) variants, phenotypic-genotypic associations were not significant (p≥0·05) for any single variant in Rv0678, atpE, pepQ, and Rv1979c. INTERPRETATION: Absence of clear genotypic-phenotypic associations for bedaquiline complicates the development of molecular drug susceptibility tests. A concerted global effort is urgently needed to assess the genotypic and phenotypic drug susceptibility of M tuberculosis isolates, especially in patients who have received unsuccessful bedaquiline-containing regimens. Treatment regimens should be designed to prevent emergence of bedaquiline resistance and phenotypic drug susceptibility tests should be used to guide and monitor treatment. FUNDING: Research Foundation Flanders, South African Medical Research Council, Department of Science and Innovation - National Research Foundation, National Institute of Health Institute of Allergy and Infectious Diseases, and Doris Duke Charitable Foundation.

Health care seeking patterns of rifampicin-resistant tuberculosis patients in Harare, Zimbabwe: A prospective cohort study.

BACKGROUND: Delays in seeking and accessing treatment for rifampicin-resistant tuberculosis (RR-TB) and multi-drug resistant (MDR-TB) are major impediments to TB control in high-burden, resource-limited settings. METHOD: We prospectively determined health-seeking behavioural patterns and associations with treatment outcomes and costs among 68 RR-TB patients attending conveniently selected facilities in a decentralised system in Harare, Zimbabwe. RESULTS: From initial symptoms to initiation of effective treatment, patients made a median number of three health care visits (IQR 2-4 visits) at a median cost of 13% (IQR 6-31%) of their total annual household income (mean cost, US$410). Cumulatively, RR-TB patients most frequently first visited private facilities, i.e., private pharmacies (30%) and other private health care providers (24%) combined. Median patient delay was 26 days (IQR 14-42 days); median health system delay was 97 days (IQR 30-215 days) and median total delay from symptom onset to initiation of effective treatment was 132 days (IQR 51-287 days). The majority of patients (88%) attributed initial delay in seeking care to "not feeling sick enough." Total delay, total cost and number of health care visits were not associated with treatment or clinical outcomes, though our study was not adequately powered for these determinations. CONCLUSIONS: Despite the public availability of rapid molecular TB tests, patients experienced significant delays and high costs in accessing RR-TB treatment. Active case finding, integration of private health care providers and enhanced service delivery may reduce treatment delay and TB associated costs.

Development and validation of a liquid chromatography-tandem mass spectrometry method for quantifying delamanid and its metabolite in small hair samples.

New all-oral regimens for rifampin-resistant tuberculosis (RR-TB) are being scaled up globally. Measurement of drug concentrations in hair assesses long-term drug exposure. Delamanid (DLM) is likely to be a key component of future RR-TB treatment regimens, but a method to describe its quantification in hair via liquid chromatography-tandem mass spectrometry (LC-MS/MS) has not previously been described. We developed and validated a simple, fast, sensitive, and accurate LC-MS/MS method for quantifying DLM and its metabolite DM-6705 in small hair samples. We pulverized and extracted two milligrams of hair in methanol at 37 °C for two hours, and diluted 1:1 with water. A gradient elution method eluted DLM, DM-6705, and the internal standard OPC 14714 within 3 min, bringing overall analysis time to 5.5 min. The method has limits of detection (LOD) of 0.0003 ng/mg for DLM and 0.003 ng/mg for DM-6705. The established linear dynamic ranges are 0.003-2.1 ng/mg and 0.03-21 ng/mg for DLM and DM-6705, respectively. Eleven of 12 participant hair samples had concentrations within DLM's linear dynamic range, while all 12 samples had concentrations within the quantifiable range for DM-6705. The ranges of concentrations observed in these clinical samples for DLM and DM-6705 were 0.004-0.264 ng/mg hair and 0.412-12.041 ng/mg hair respectively. We demonstrate that while DLM was detected in hair at very low levels, its primary metabolite DM-6705 had levels approximately 100 times higher. Measuring DM-6705 in hair may accurately reflect long-term adherence to DLM-containing regimens for drug-resistant TB.

Presentation and outcome of suspected sepsis in a high-HIV burden, high antiretroviral coverage setting.

OBJECTIVE: To define sepsis syndromes in high-HIV burden settings in the antiretroviral therapy (ART) era. METHODS: We characterized a prospective cohort of adults presenting to a tertiary emergency department in Harare, Zimbabwe with suspected community-acquired sepsis using blood and urine cultures, urine tuberculosis lipoarabinomannan (TB LAM), and serum cryptococcal antigen (CrAg) testing. The primary outcome was 30-day all-cause mortality. RESULTS: Of 142 patients enrolled 68% (n=96/142, 95% confidence interval (CI) [60-75%]) were HIV-positive, 41% (n=39/96, 95% CI [31-50%]) of whom were ART-naïve. Among HIV-positive patients, both opportunistic pathogens (TB LAM-positivity, 36%, 95% CI [24-48%]; CrAg-positivity, 15%, 95% CI [7-23%]) and severe non-AIDS infections (S. pneumoniae urine antigen-positivity 12%, 95% CI [4-20%]; bacteraemia 17% (n=16/96, 95% CI [9-24%]), of which 56% (n=9/16, 95% CI [30-80%]) were gram-negative organisms) were common. Klebsiella pneumoniae recovered from blood and urine was uniformly resistant to ceftriaxone, as were most Escherichia coli isolates. Acknowledging the power limitations of our study, we conclude that relative to HIV-negative patients, HIV-positive patients had modestly higher 30-day mortality (adjusted hazard ratio (HR) 1.88, 95% CI [0.78-4.55]; p=0.16, and 3.59, 95% CI [1.27-10.16], p=0.02) among those with and without viral suppression, respectively. CONCLUSION: Rapid point-of-care assays provide substantial clinically actionable information in the setting of suspected sepsis, even in areas with high ART coverage. Antimicrobial resistance to first-line antibiotics in high burden settings is a growing threat.