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Bedaquiline Microheteroresistance A patient in South Africa was treated with a multidrug regimen for multidrug-resistant tuberculosis; the regimen included bedaquiline, to which the isolate was sus...

Background Antigen-detecting rapid diagnostic tests (Ag-RDTs) for the detection of SARS-CoV-2 offer new opportunities for testing in the context of the COVID-19 pandemic. Nasopharyngeal swabs (NPS) are the reference sample type, but oropharyngeal swabs (OPS) may be a more acceptable sample type in some patients. Methods We conducted a prospective study in a single screening center to assess the diagnostic performance of the Panbio™ COVID-19 Ag Rapid Test (Abbott) on OPS compared with reverse-transcription quantitative PCR (RT-qPCR) using NPS during the second pandemic wave in Switzerland. Results 402 outpatients were enrolled in a COVID-19 screening center, of whom 168 (41.8%) had a positive RT-qPCR test. The oropharyngeal Ag-RDT clinical sensitivity compared to nasopharyngeal RT-qPCR was 81% (95%CI: 74.2–86.6). Two false positives were noted out of the 234 RT-qPCR negative individuals, which resulted in a clinical specificity of 99.1% (95%CI: 96.9–99.9) for the Ag-RDT. For cycle threshold values ≤ 26.7 (≥ 1E6 SARS-CoV-2 genomes copies/mL, a presumed cut-off for infectious virus), 96.3% sensitivity (95%CI: 90.7–99.0%) was obtained with the Ag-RDT using OPS. Interpretation Based on our findings, the diagnostic performance of the Panbio™ Covid-19 RDT with OPS samples, if taken by a trained person and high requirements regarding quality of the specimen, meet the criteria required by the WHO for Ag-RDTs (sensitivity ≥80% and specificity ≥97%) in a high incidence setting in symptomatic individuals.

Aims & objectives: Potentially transformative new tuberculosis (TB) drugs like bedaquiline are undergoing roll-out, however, this is largely in the absence of programmatic drug susceptibility testing (DST). We lack information on how susceptibility changes during treatment in patients on bedaquiline-containing regimens, especially in at risk patients who have complex treatment histories, are in programmatic rather than trial environments, and have a delayed treatment response (defined here as sustained culture-positivity). Methods: Serial isolates from 51 patients with drug resistant (DR-)TB who were culture-positive after ≥4 months of a programmatically administered bedaquiline-containing regimen, were collected. Bedaquiline phenotypic DST in MGIT 960 (1μg/ml), targeted deep sequencing (Rv0678, atpE, pepQ) and whole genome sequencing was done on paired isolates (pre-bedaquiline initiation, post-four-month). Results: 24/51 (47%) patients were phenotypically and genotypically resistant (39% acquired resistance). Excluding one patient with an unknown history, prior clofazimine exposure was associated with bedaquiline-resistance [21/24 (88%) bedaquiline resistant cases had prior clofazimine vs. 12/26 (46%) susceptibles; p=0.002]. Diverse combinations of single nucleotide polymorphisms (SNPs) and indels were in the Rv0678 promoter region and the Rv0678 and atpE genes. Examples of newly described resistance associated variants (RAVs) include Rv0678 -8 T/G and atpE 223 C/T. RAVs were not in defined hotspots and sometimes occurred concurrently with atpE RAVs. Discussion: The rate of bedaquiline resistance acquisition in this population is alarmingly high and associated with prior clofazimine exposure. The diverse RAVs pose challenges to molecular test development. This study highlights the existence of a potentially infectious pool of bedaquiline-resistant patients present under programmatic conditions in a resource-constrained setting and illustrates the danger of starting patients with complex histories on a novel drug without routinely available DST.

Resistance to first-line anti-tuberculosis (TB) drugs continues to threaten global TB control with over 500 000 cases of rifampicin resistant (RR)-TB being reported in 2018. Treatment of RR-TB requires the use of toxic drugs often leading to adverse events, poor adherence and poor treatment outcomes. In order to address the toxicity of treatment and to improve treatment outcomes the World Health Organization has promoted the use of injection free regimens which include new and repurposed drugs: bedaquiline (BDQ), linezolid (LZD) and clofazimine (CFZ). In 2017, South Africa rolled out the inclusion of BDQ in the treatment of all pre-XDR- and XDR-TB cases as well as patients experiencing adverse events from kanamycin. In late 2018, South Africa included BDQ into three injection free regimens for treating RR-TB. This was done before routine drug susceptibility testing for BDQ could be implemented. Resistance to BDQ occurs primarily through mutations in Rv0678 (mmpR), pepQ and atpE. Understanding which mutations confer resistance and why they arise in a patient will be critical for early diagnosis as well as for preventing the emergence of resistance thereby ensuring successful treatment. Collation of the mutations identified in Rv0678 shows that BDQ resistance is largely driven by small insertions/deletions which disrupt the function of the repressor thereby leading to over expression of the efflux pump mmpL5 responsible for extruding BDQ from the cell. Understanding the association between gene mutation and phenotypic resistance remains critical for the development of new molecular diagnostics to rapidly diagnose resistance thereby affording the clinician the opportunity to adjust the regimen. Analysis of serial isolates collected from patients receiving BDQ (Access program pre-2017) has shown the emergence of mutations in Rv0678, atpE and pepQ during treatment in patients who remain culture positive after three months of treatment. Using targeted deep sequencing it is now possible to track the emergence of resistant sub-populations at the sub-phenotypic level (i.e., micro-heteroresistance) thereby affording the opportunity to study the association of the emergence of resistance with drug regimens, MIC distributions, PK and PD data and treatment outcomes. Our analysis showed that mutations conferring resistance occurred soon after the introduction of BDQ into the regimen and that clones harbouring these mutations were subsequently selected. Most surprising was the observation that different resistant sub-populations emerged during the period after the cessation of BDQ in the regimen. This demonstrates the continuing selective pressure as a result of the long half-life of BDQ. Although the origin of the distinct resistant clones is unknown, we hypothesize that resistance evolved independently in spatially separated lesions subsequently rupturing into the airways. A surprising observation was that concomitant emergence of different variants of the rpoC gene suggesting a compensatory mechanism to overcome the fitness cost of Rv0678 mutations.

Most cases of multidrug-resistant (MDR) tuberculosis (TB) are never diagnosed (328,300 of the ∼490,000 cases in 2016 were missed). The Xpert MTB/RIF assay detects resistance only to rifampin, despite ∼20% of rifampin-resistant cases being susceptible to isoniazid (a critical first-line drug). Consequently, many countries require further testing with the GenoType MTBDRplus assay. However, MTBDRplus is not recommended for use on smear-negative specimens, and thus, many specimens require culture-based drug susceptibility testing. Furthermore, MTBDRplus requires specialized expertise, lengthy hands-on time, and significant laboratory infrastructure and interpretation is not automated. To address these gaps, we evaluated the accuracy of the FluoroType MTBDR (FluoroType) assay. Sputa from 244 smear-positive and 204 smear-negative patients with presumptive TB (Xpert MTB positive, n = 343) were tested. Culture and MTBDRplus on isolates served as reference standards (for active TB and MDR-TB, respectively). Sanger sequencing and MTBDRplus, both of which were performed on sputa, were used to resolve discrepancies. The sensitivity of FluoroType for the detection of M. tuberculosis complex was 98% (95% confidence interval [CI], 95 to 99%) and 92% (95% CI, 84 to 96%) for smear-positive and smear-negative specimens, respectively (232/237 versus 90/98 specimens; P < 0.009). The sensitivity and specificity for smear-negative specimens were 100% and 97%, respectively, for rifampin resistance; 100% and 98%, respectively, for isoniazid resistance; and 100% and 100%, respectively, for MDR-TB. FluoroType identified 98%, 97%, and 97% of the rpoB, katG, and inhA promoter mutations, respectively. FluoroType has excellent sensitivity with sputa equivalent to that of MTBDRplus with the isolates and can provide rapid drug susceptibility testing for rifampin and isoniazid. In addition, the capacity of FluoroType to simultaneously identify virtually all mutations in the rpoB, katG, and inhA promoter may be useful for individualized treatment regimens.

Xpert MTB/RIF (Xpert) is a widely-used test for tuberculosis (TB) and rifampicin-resistance. Second-line drug susceptibility testing (DST), which is recommended by policymakers, typically requires additional specimen collection that delays effective treatment initiation. We examined whether cartridge extract (CE) from used Xpert TB-positive cartridges was, without downstream DNA extraction or purification, suitable for both genotypic DST (MTBDRplus, MTBDRsl), which may permit patients to rapidly receive a XDR-TB diagnosis from a single specimen, and spoligotyping, which could facilitate routine genotyping. To determine the limit-of-detection and diagnostic accuracy, CEs from dilution series of drug-susceptible and -resistant bacilli were tested (MTBDRplus, MTBDRsl). Xpert TB-positive patient sputa CEs (n = 85) were tested (56 Xpert-rifampicin-susceptible, MTBDRplus and MTBDRsl; 29 Xpert-rifampicin-resistant, MTBDRsl). Spoligotyping was done on CEs from dilution series and patient sputa (n = 10). MTBDRplus had high non-valid result rates. MTBDRsl on CEs from dilutions ≥103CFU/ml (CT ≤ 24, >“low” Xpert semiquantitation category) was accurate, had low indeterminate rates and, on CE from sputa, highly concordant with MTBDRsl isolate results. CE spoligotyping results from dilutions ≥103CFU/ml and sputa were correct. MTBDRsl and spoligotyping on CE are thus highly feasible. These findings reduce the need for additional specimen collection and culture, for which capacity is limited in high-burden countries, and have implications for diagnostic laboratories and TB molecular epidemiology.

Recurrent tuberculosis (TB) after successful TB treatment occurs due to endogenous reactivation (relapse) or exogenous reinfection. We revisited the conclusions of relapse in a high TB incidence setting that were drawn on the basis of IS6110 restriction fragment length polymorphism (RFLP) analysis in a large retrospective cohort study in suburban Cape Town, South Africa. Using whole genome sequencing (WGS), we undertook pair-wise genome comparison of Mycobacterium tuberculosis strains cultured from diagnostic sputum samples collected at the index and recurrent TB episode for 25 recurrent TB cases who had been classified as relapse based on identical DNA fingerprint patterns in the earlier study. We found that paired strain genome sequences were identical or showed minimal variant differences in 22 of 25 recurrent TB cases, consistent with relapse. One showed 20 variant differences, suggestive of exogenous reinfection. Two of the 25 had mixed infections, each with the index episode strain detected as the dominant strain at recurrence in one of these patients, the minority strain harboured drug-resistance conferring mutations (rpoB, katG). In conclusion, our study highlights the additional value of WGS for investigating recurrent TB in settings with high infection pressure and closely related circulating strains, where the extent of re- and mixed infection may be underestimated.

Tuberculosis (TB) remains an important public health problem with an estimated 10.4 million new cases diagnosed in 2015 (1). Of these, approximately 4.3 million cases went undiagnosed or unreported, posing a major hurdle to the eradication of TB (1). The control of TB is further threatened by the HIV epidemic and the emergence and spread of multidrug-resistant (MDR) TB, defined as resistance to rifampicin and isoniazid. In 2015, there were an estimated 480,000 new cases of MDR-TB and an additional 100,000 people were diagnosed with rifampicin-resistant TB. These cases result in continued transmission in communities and health care settings due to the detection and treatment gap, with only 1 million cases (30% of the 3.4 million bacteriologically confirmed cases or 10% of all cases globally) having had a drug susceptibility test for rifampicin in 2015. The WHO has therefore included early diagnosis of TB and universal drug susceptibility testing as one of its core priorities for global TB control (2).