Therapeutic Failure and Acquired Bedaquiline and Delamanid Resistance in Treatment of Drug-Resistant TB.

New classes of antitubercular drugs, diarylquinolines and nitroimidazoles, have been associated with improved outcomes in the treatment of drug-resistant tuberculosis, but that success is threatened by emerging drug resistance. We report a case of bedaquiline and delamanid resistance in a 55-year-old woman in South Africa with extensively drug-resistant tuberculosis and known HIV.

Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

BACKGROUND: Repeated culture reduces within-sample Mycobacterium tuberculosis genetic diversity due to selection of clones suited to growth in culture and/or random loss of lineages, but it is not known to what extent omitting the culture step altogether alters genetic diversity. We compared M. tuberculosis whole genome sequences generated from 33 paired clinical samples using two methods. In one method DNA was extracted directly from sputum then enriched with custom-designed SureSelect (Agilent) oligonucleotide baits and in the other it was extracted from mycobacterial growth indicator tube (MGIT) culture. RESULTS: DNA directly sequenced from sputum showed significantly more within-sample diversity than that from MGIT culture (median 5.0 vs 4.5 heterozygous alleles per sample, p = 0.04). Resistance associated variants present as HAs occurred in four patients, and in two cases may provide a genotypic explanation for phenotypic resistance. CONCLUSIONS: Culture-free M. tuberculosis whole genome sequencing detects more within-sample diversity than a leading culture-based method and may allow detection of mycobacteria that are not actively replicating.

Correction to: Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture.

He authors reported that one of the authors' names was typeset incorrectly in the authorship list.

Opportunities and limitations of genomics for diagnosing bedaquiline-resistant tuberculosis: a systematic review and individual isolate meta-analysis.

BACKGROUND: Clinical bedaquiline resistance predominantly involves mutations in mmpR5 (Rv0678). However, mmpR5 resistance-associated variants (RAVs) have a variable relationship with phenotypic Mycobacterium tuberculosis resistance. We did a systematic review to assess the maximal sensitivity of sequencing bedaquiline resistance-associated genes and evaluate the association between RAVs and phenotypic resistance, using traditional and machine-based learning techniques. METHODS: We screened public databases for articles published from database inception until Oct 31, 2022. Eligible studies performed sequencing of at least mmpR5 and atpE on clinically sourced M tuberculosis isolates and measured bedaquiline minimum inhibitory concentrations (MICs). A bias risk scoring tool was used to identify bias. Individual genetic mutations and corresponding MICs were aggregated, and odds ratios calculated to determine association of mutations with resistance. Machine-based learning methods were used to define test characteristics of parsimonious sets of diagnostic RAVs, and mmpR5 mutations were mapped to the protein structure to highlight mechanisms of resistance. This study was registered in the PROSPERO database (CRD42022346547). FINDINGS: 18 eligible studies were identified, comprising 975 M tuberculosis isolates containing at least one potential RAV (mutation in mmpR5, atpE, atpB, or pepQ), with 201 (20·6%) showing phenotypic bedaquiline resistance. 84 (29·5%) of 285 resistant isolates had no candidate gene mutation. Sensitivity and positive predictive value of taking an any mutation approach was 69% and 14%, respectively. 13 mutations, all in mmpR5, had a significant association with a resistant MIC (adjusted p<0·05). Gradient-boosted machine classifier models for predicting intermediate or resistant and resistant phenotypes both had receiver operator characteristic c statistic of 0·73 (95% CI 0·70-0·76). Frameshift mutations clustered in the α1 helix DNA-binding domain, and substitutions in the α2 and α3 helix hinge region and in the α4 helix-binding domain. INTERPRETATION: Sequencing candidate genes is insufficiently sensitive to diagnose clinical bedaquiline resistance, but where identified, some mutations should be assumed to be associated with resistance. Genomic tools are most likely to be effective in combination with rapid phenotypic diagnostics. This study was limited by selective sampling in contributing studies and only considering single genetic loci as causative of resistance. FUNDING: Francis Crick Institute and National Institute of Allergy and Infectious Diseases at the National Institutes of Health.

Detection of a historic reservoir of bedaquiline/clofazimine resistance-associated variants in Mycobacterium tuberculosis.

BACKGROUND: Drug resistance in tuberculosis (TB) poses a major ongoing challenge to public health. The recent inclusion of bedaquiline into TB drug regimens has improved treatment outcomes, but this advance is threatened by the emergence of strains of Mycobacterium tuberculosis (Mtb) resistant to bedaquiline. Clinical bedaquiline resistance is most frequently conferred by off-target resistance-associated variants (RAVs) in the mmpR5 gene (Rv0678), the regulator of an efflux pump, which can also confer cross-resistance to clofazimine, another TB drug. METHODS: We compiled a dataset of 3682 Mtb genomes, including 180 carrying variants in mmpR5, and its immediate background (i.e. mmpR5 promoter and adjacent mmpL5 gene), that have been associated to borderline (henceforth intermediate) or confirmed resistance to bedaquiline. We characterised the occurrence of all nonsynonymous mutations in mmpR5 in this dataset and estimated, using time-resolved phylogenetic methods, the age of their emergence. RESULTS: We identified eight cases where RAVs were present in the genomes of strains collected prior to the use of bedaquiline in TB treatment regimes. Phylogenetic reconstruction points to multiple emergence events and circulation of RAVs in mmpR5, some estimated to predate the introduction of bedaquiline. However, epistatic interactions can complicate bedaquiline drug-susceptibility prediction from genetic sequence data. Indeed, in one clade, Ile67fs (a RAV when considered in isolation) was estimated to have emerged prior to the antibiotic era, together with a resistance reverting mmpL5 mutation. CONCLUSIONS: The presence of a pre-existing reservoir of Mtb strains carrying bedaquiline RAVs prior to its clinical use augments the need for rapid drug susceptibility testing and individualised regimen selection to safeguard the use of bedaquiline in TB care and control.

Management of atrial fibrillation in chronic kidney disease: double trouble.

Chronic kidney disease (CKD) has a very well-established link with cardiovascular disease. Below stage 3 CKD (glomerular filtration rate <60 mL/min), there is a progressive increase in both total mortality and cardiovascular-specific mortality as kidney function declines; indeed, it is more likely for a patient with CKD stage 3 to die of cardiovascular disease than to progress to CKD stage 4 and beyond. Arrhythmia is particularly common in patients with CKD. Depending on the study and measurement used, the prevalence of patients with CKD with chronic atrial fibrillation (AF) is quoted at 7% to 18%, rising to 12% to 25% for those older than 70 years. These rates are up to 2 to 3 times higher than in the general population. Of all patients with AF, 10% to 15% will have CKD. However, not all standard rate and rhythm methods are suitable for this population and those that are tend to be less effective. Meanwhile, anticoagulation has long been a thorny subject, with much conflicting evidence around the balance between bleeding and stroke risk. To help clarify this, we first highlight the challenges of performing evidence-based medicine in the patient with renal disease, and then review recent and emerging research to suggest an approach to the management of patients with renal disease who have AF. We also review the potential role of the different new oral anticoagulant drugs in CKD.

Opportunities and limitations of genomics for diagnosing bedaquiline-resistant tuberculosis: an individual isolate metaanalysis.

Background: Clinical bedaquiline resistance predominantly involves mutations in mmpR5 (Rv0678). However, mmpR5 resistance-associated variants (RAVs) have a variable relationship with phenotypic M. tuberculosis resistance. We performed a systematic review to (1) assess the maximal sensitivity of sequencing bedaquiline resistance-associated genes and (2) evaluate the association between RAVs and phenotypic resistance, using traditional and machine-based learning techniques. Methods: We screened public databases for articles published until October 2022. Eligible studies performed sequencing of at least mmpR5 and atpE on clinically-sourced M. tuberculosis isolates and measured bedaquiline minimum inhibitory concentrations (MICs). We performed genetic analysis for identification of phenotypic resistance and determined the association of RAVs with resistance. Machine-based learning methods were employed to define test characteristics of optimised sets of RAVs, and mmpR5 mutations were mapped to the protein structure to highlight mechanisms of resistance. Results: Eighteen eligible studies were identified, comprising 975 M. tuberculosis isolates containing ≥1 potential RAV (mutation in mmpR5, atpE, atpB or pepQ), with 201 (20.6%) demonstrating phenotypic bedaquiline resistance. 84/285 (29.5%) resistant isolates had no candidate gene mutation. Sensitivity and positive predictive value of taking an 'any mutation' approach was 69% and 14% respectively. Thirteen mutations, all in mmpR5, had a significant association with a resistant MIC (adjusted p<0.05). Gradient-boosted machine classifier models for predicting intermediate/resistant and resistant phenotypes both had receiver operator characteristic c-statistics of 0.73. Frameshift mutations clustered in the alpha 1 helix DNA binding domain, and substitutions in the alpha 2 and 3 helix hinge region and in the alpha 4 helix binding domain. Discussion: Sequencing candidate genes is insufficiently sensitive to diagnose clinical bedaquiline resistance, but where identified a limited number of mutations should be assumed to be associated with resistance. Genomic tools are most likely to be effective in combination with rapid phenotypic diagnostics.

Rab11 function in Trypanosoma brucei: identification of conserved and novel interaction partners.

The Ras-like GTPase Rab11 is implicated in multiple aspects of intracellular transport, including maintenance of plasma membrane composition and cytokinesis. In metazoans, these functions are mediated in part via coiled-coil Rab11-interacting proteins (FIPs) acting as Rab11 effectors. Additional interaction between Rab11 and the exocyst subunit Sec15 connects Rab11 with exocytosis. We find that FIPs are metazoan specific, suggesting that other factors mediate Rab11 functions in nonmetazoans. We examined Rab11 interactions in Trypanosoma brucei, where endocytosis is well studied and the role of Rab11 in recycling well documented. TbSec15 and TbRab11 interact, demonstrating evolutionary conservation. By yeast two-hybrid screening, we identified additional Rab11 interaction partners. Tb927.5.1640 (designated RBP74) interacted with both Rab11 and Rab5. RBP74 shares a coiled-coil architecture with metazoan FIPs but is unrelated by sequence and appears to play a role in coordinating endocytosis and recycling. A second coiled-coil protein, Tb09.211.4830 (TbAZI1), orthologous to AZI1 in Homo sapiens, interacts exclusively with Rab11. AZI1 is restricted to taxa with motile cilia/flagella. These data suggest that Rab11 functions are mediated by evolutionarily conserved (i.e., AZI1 and Sec15) and potentially lineage-specific (RBP74) interactions essential for the integration of the endomembrane system.

Evolution of Mycobacterium tuberculosis drug resistance in the genomic era.

Mycobacterium tuberculosis has acquired drug resistance to all drugs that have been used against it, including those only recently introduced into clinical practice. Compared to other bacteria, it has a well conserved genome due to its role as an obligate human pathogen that has adapted to a niche over five to ten thousand years. These features facilitate reconstruction and dating of M. tuberculosis phylogenies, giving key insights into how resistance has been acquired and spread globally. Resistance to each new drug has occurred within five to ten years of clinical use and has occurred even more rapidly with recently introduced drugs. In most cases, resistance-conferring mutations come with a fitness cost, but this can be overcome by compensatory mutations which restore fitness to that of wild-type bacteria. It is likely that M. tuberculosis acquires drug resistance while maintaining limited genomic variability due the generation of low frequency within-host variation, combined with ongoing purifying selection causing loss of variants without a clear fitness advantage. However, variants that do confer an advantage, such as drug resistance, can increase in prevalence amongst all bacteria within a host and become the dominant clone. These resistant strains can then be transmitted leading to primary drug resistant infection in a new host. As many countries move towards genomic methods for diagnosis of M. tuberculosis infection and drug resistance, it is important to be aware of the implications for the evolution of resistance. Currently, understanding of resistance-conferring mutations is incomplete, and some targeted genetic diagnostics create their own selective pressures. We discuss an example where a rifampicin resistance-conferring mutation which was not routinely covered by standard testing became dominant. Finally, resistance to new drugs such as bedaquiline and delamanid is caused by individually rare mutations occurring across a large mutational genomic target that have been detected over a short time, and do not provide statistical power for genotype-phenotype correlation - in contrast to longer-established drugs that form the backbone of drug-sensitive antituberculosis therapy. Therefore, we need a different approach to identify resistance-conferring mutations of new drugs before their resistance becomes widespread, abrogating their usefulness.

Bedaquiline Adherence Measured by Electronic Dose Monitoring Predicts Clinical Outcomes in the Treatment of Patients With Multidrug-Resistant Tuberculosis and HIV/AIDS.

BACKGROUND: Novel regimens have revolutionized multidrug-resistant tuberculosis (MDR-TB) treatment; however, medication adherence remains challenging and poorly characterized. We hypothesized that bedaquiline adherence, measured using electronic dose monitoring, would predict MDR-TB treatment outcomes. SETTING: This is a prospective cohort study conducted in KwaZulu-Natal, South Africa. METHODS: Adults with MDR-TB and HIV initiating bedaquiline and on antiretroviral therapy (ART) were eligible. Separate electronic dose monitoring devices measured bedaquiline and ART adherence through 6 months, calculated as observed versus expected doses. Whole-genome sequencing was performed to identify bedaquiline resistance-associated variants. RESULTS: From November 2016 through February 2018, 199 participants with MDR-TB and HIV were enrolled and followed up through treatment completion (median 17.2 months interquartile range 12.2-19.6). The median bedaquiline adherence was higher than ART adherence (97 vs. 89%, P < 0.001) but correlated (r2 = 0.68, P < 0.001). High bedaquiline adherence (≥90%) compared with lower adherence was associated with improved end of treatment successful outcome (83.4% vs. 46.3%, P < 0.001), decreased mortality (11.0% vs. 29.6% P = 0.004), and improved retention in care through end of treatment (94.5% vs. 79.6% P = 0.002). Modeling identified a highly significant but linear association between bedaquiline adherence and outcome. On multivariable analysis, bedaquiline adherence was independently associated with mortality and outcome. Bedaquiline resistance-associated variants were seen in 12% (7/57) of sequenced isolates (7% baseline, 5% emergent) with only 28.6% experiencing successful treatment outcome. CONCLUSIONS: Bedaquiline adherence through 6 months independently predicted end of MDR-TB treatment outcome, but a specific bedaquiline adherence threshold was not identified. Interventions to optimize bedaquiline adherence are urgently needed to improve MDR-TB HIV treatment outcomes.

Novel use of endoluminal repair as prophylaxis of aortic rupture secondary to radiotherapy for lung cancer.

Non-small-cell lung cancer (NSCLC) invading the aorta is staged as T(4). Only 9% of T(4) tumors are resected; the alternative is chemoradiotherapy, but for peri-aortic NSCLC, radiation damage to the aortic wall can induce fatal rupture. We report the case of a 76 year-old man with a 3-cm left lower lobe NSCLC clearly invading the aortic wall. A thoracic stent graft was inserted prophylactically to prevent aortic rupture. He then received 64 Gy radiotherapy in 32 fractions, resulting in tumor shrinkage. Prophylactic aortic endografting, a less invasive treatment than open surgery, may enable high dose irradiation of the aortic wall.

Population-level emergence of bedaquiline and clofazimine resistance-associated variants among patients with drug-resistant tuberculosis in southern Africa: a phenotypic and phylogenetic analysis.

BACKGROUND: Bedaquiline and clofazimine are important drugs in the treatment of drug-resistant tuberculosis and are commonly used across southern Africa, although drug susceptibility testing is not routinely performed. In this study, we did a genotypic and phenotypic analysis of drug-resistant Mycobacterium tuberculosis isolates from cohort studies in hospitals in KwaZulu-Natal, South Africa, to identify resistance-associated variants (RAVs) and assess the extent of clofazimine and bedaquiline cross-resistance. We also used a comprehensive dataset of whole-genome sequences to investigate the phylogenetic and geographical distribution of bedaquiline and clofazimine RAVs in southern Africa. METHODS: In this study, we included M tuberculosis isolates reported from the PRAXIS study of patients with drug-resistant tuberculosis treated with bedaquiline (King Dinuzulu Hospital, Durban) and three other cohort studies of drug-resistant tuberculosis in other KwaZulu-Natal hospitals, and sequential isolates from six persistently culture-positive patients with extensively drug-resistant tuberculosis at the KwaZulu-Natal provincial referral laboratory. Samples were collected between 2013 and 2019. Microbiological cultures were done as part of all parent studies. We sequenced whole genomes of included isolates and measured bedaquiline and clofazimine minimum inhibitory concentrations (MICs) for isolates identified as carrying any Rv0678 variant or previously published atpE, pepQ, and Rv1979c RAVs, which were the subject of the phenotypic study. We combined all whole-genome sequences of M tuberculosis obtained in this study with publicly available sequence data from other tuberculosis studies in southern Africa (defined as the countries of the Southern African Development Community), including isolates with Rv0678 variants identified by screening public genomic databases. We used this extended dataset to reconstruct phylogenetic relationships across lineage 2 and 4 M tuberculosis isolates. FINDINGS: We sequenced the whole genome of 648 isolates from 385 patients with drug-resistant tuberculosis recruited into cohort studies in KwaZulu-Natal, and 28 isolates from six patients from the KwaZulu-Natal referral laboratory. We identified 30 isolates with Rv0678 RAVs from 16 (4%) of 391 patients. We did not identify any atpE, pepQ, or Rv1979c RAVs. MICs were measured for 21 isolates with Rv0678 RAVs. MICs were above the critical concentration for bedaquiline resistance in nine (43%) of 21 isolates, in the intermediate category in nine (43%) isolates, and within the wild-type range in three (14%) isolates. Clofazimine MICs in genetically wild-type isolates ranged from 0·12-0·5 µg/mL, and in isolates with RAVs from 0·25-4·0 µg/mL. Phylogenetic analysis of the extended dataset including M tuberculosis isolates from southern Africa resolved multiple emergences of Rv0678 variants in lineages 2 and 4, documented two likely nosocomial transmission events, and identified the spread of a possibly bedaquiline and clofazimine cross-resistant clone in eSwatini. We also identified four patients with pepQ frameshift mutations that may confer resistance. INTERPRETATION: Bedaquiline and clofazimine cross-resistance in southern Africa is emerging repeatedly, with evidence of onward transmission largely due to Rv0678 mutations in M tuberculosis. Roll-out of bedaquiline and clofazimine treatment in the setting of limited drug susceptibility testing could allow further spread of resistance. Designing strong regimens would help reduce the emergence of resistance. Drug susceptibility testing is required to identify where resistance does emerge. FUNDING: Wellcome Trust, National Institute of Allergy and Infectious Diseases and National Center for Advancing Translational Sciences of the National Institutes of Health.

Dynamics of within-host Mycobacterium tuberculosis diversity and heteroresistance during treatment.

BACKGROUND: Studying within-host genetic diversity of Mycobacterium tuberculosis (Mtb) in patients during treatment may identify adaptations to antibiotic and immune pressure. Understanding the significance of genetic heteroresistance, and more specifically heterozygous resistance-associated variants (RAVs), is clinically important given increasing use of rapid molecular tests and whole genome sequencing (WGS). METHODS: We analyse data from six studies in KwaZulu-Natal, South Africa. Most patients (>75%) had baseline rifampicin resistance. Sputum was collected for culture at baseline and at between two and nine intervals until month six. Positive cultures underwent WGS. Mixed infections and reinfections were excluded from analysis. FINDINGS: Baseline Mtb overall genetic diversity (at treatment initiation or major change to regimen) was associated with cavitary disease, not taking antiretroviral therapy if HIV infected, infection with lineage 2 strains and absence of second-line drug resistance on univariate analyses. Baseline genetic diversity was not associated with six-month outcome. Genetic diversity increased from baseline to weeks one and two before returning to previous levels. Baseline genetic heteroresistance was most common for bedaquiline (6/10 [60%] of isolates with RAVs) and fluoroquinolones (9/62 [13%]). Most patients with heterozygous RAVs on WGS with sequential isolates available demonstrated RAV persistence or fixation (17/20, 85%). New RAVs emerged in 9/286 (3%) patients during treatment. We could detect low-frequency RAVs preceding emergent resistance in only one case, although validation of deep sequencing to detect rare variants is required. INTERPRETATION: In this study of single-strain Mtb infections, baseline within-host bacterial genetic diversity did not predict outcome but may reveal adaptations to host and drug pressures. Predicting emergent resistance from low-frequency RAVs requires further work to separate transient from consequential mutations. FUNDING: Wellcome Trust, NIH/NIAID.

Rapid identification of a Mycobacterium tuberculosis full genetic drug resistance profile through whole genome sequencing directly from sputum.

INTRODUCTION: Resistance to second-line tuberculosis drugs is common, but slow to diagnose with phenotypic drug sensitivity testing. Rapid molecular tests speed up diagnosis, but can only detect limited mutations. Whole genome sequencing (WGS) of culture isolates can generate a complete genetic drug resistance profile, but is delayed by the initial culture step. In the case presented here, successful WGS directly from sputum was achieved using targeted enrichment. CASE REPORT: A 29-year-old Nigerian woman was diagnosed with tuberculosis. Xpert MTB/RIF and Hain line probe assays identified rpoB and inhA mutations consistent with rifampicin and intermediate isoniazid resistance, and a further possible mutation conferring fluoroquinolone resistance. WGS directly from sputum identified a further inhA mutation consistent with high-level isoniazid resistance and confirmed the absence of fluoroquinolone resistance. Isoniazid was stopped, and the patient has completed 18 months of a fluoroquinolone-based regimen without relapse. DISCUSSION: Compared to rapid molecular tests (which can only examine a limited number of mutations) and WGS of culture isolates (which requires a culture step), WGS directly from sputum can quickly generate a complete genetic drug resistance profile. In this case, WGS altered the clinical management of drug-resistant tuberculosis and demonstrated potential for guiding individualized drug treatment where second-line drug resistance is common.