Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) infects lung myeloid cells, but the specific Mtb-permissive cells and host mechanisms supporting Mtb persistence during chronic infection are incompletely characterized. We report that after the development of T cell responses, CD11clo monocyte-derived cells harbor more live Mtb than alveolar macrophages (AM), neutrophils, and CD11chi monocyte-derived cells. Transcriptomic and functional studies revealed that the lysosome pathway is underexpressed in this highly permissive subset, characterized by less lysosome content, acidification, and proteolytic activity than AM, along with less nuclear TFEB, a regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in CD11clo monocyte-derived cells but promotes recruitment of monocytes that develop into permissive lung cells, mediated by the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome functions of macrophages in vitro and in vivo, improving control of Mtb infection. Our results suggest that Mtb exploits lysosome-poor lung cells for persistence and targeting lysosome biogenesis is a potential host-directed therapy for tuberculosis.

primerJinn: a tool for rationally designing multiplex PCR primer sets for amplicon sequencing and performing in silico PCR.

BACKGROUND: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers. RESULTS: We developed primerJinn, a tool that designs a set of multiplex primers and allows for the in silico PCR evaluation of primer sets against numerous input genomes. We used primerJinn to create a multiplex PCR for the sequencing of drug resistance-conferring gene regions from Mycobacterium tuberculosis, which were then successfully sequenced. CONCLUSIONS: primerJinn provides a user-friendly, efficient, and accurate method for designing multiplex PCR primers for targeted sequencing and performing in silico PCR. It can be used for various applications in molecular biology and bioinformatics research, including the design of assays for amplifying and sequencing drug-resistance-conferring regions in important pathogens.

Comparative Performance of Genomic Methods for the Detection of Pyrazinamide Resistance and Heteroresistance in Mycobacterium tuberculosis.

Pyrazinamide is an important component of both drug-susceptible and drug-resistant tuberculosis treatment regimens. Although approximately 50% of rifampin-resistant isolates are also resistant to pyrazinamide, pyrazinamide susceptibility testing is not routinely performed due to the challenging nature of the assay. We investigated the diagnostic accuracy of genotypic and phenotypic methods and explored the occurrence of pyrazinamide heteroresistance. We assessed pyrazinamide susceptibility among 358 individuals enrolled in the South African EXIT-RIF cohort using Sanger and targeted deep sequencing (TDS) of the pncA gene, whole-genome sequencing (WGS), and phenotypic drug susceptibility testing. We calculated the diagnostic accuracy of the different methods and investigated the prevalence and clinical impact of pncA heteroresistance. True pyrazinamide susceptibility status was assigned to each isolate using the Köser classification and expert rules. We observed 100% agreement across genotypic methods for detection of pncA fixed mutations; only TDS confidently identified three isolates (0.8%) with minor variants. For the 355 (99.2%) isolates that could be assigned true pyrazinamide status with confidence, phenotypic DST had a sensitivity of 96.5% (95% confidence interval [CI], 93.8 to 99.3%) and specificity of 100% (95% CI, 100 to 100%), both Sanger sequencing and WGS had a sensitivity of 97.1% (95% CI, 94.6 to 99.6%) and specificity of 97.8% (95% CI, 95.7 to 99.9%), and TDS had sensitivity of 98.8% (95% CI, 97.2 to 100%) and specificity of 97.8% (95% CI, 95.7 to 99.9%). We demonstrate high sensitivity and specificity for pyrazinamide susceptibility testing among all assessed genotypic methods. The prevalence of pyrazinamide heteroresistance in Mycobacterium tuberculosis isolates was lower than that identified for other first-line drugs.

A Human Lung Challenge Model to Evaluate the Safety and Immunogenicity of PPD and Live Bacillus Calmette-Guérin.

Rationale: A human model to better understand tuberculosis immunopathogenesis and facilitate vaccine development is urgently needed.Objectives: We evaluated the feasibility, safety, and immunogenicity of live bacillus Calmette-Guérin (BCG) in a lung-oriented controlled human infection model.Methods: We recruited 106 healthy South African participants with varying degrees of tuberculosis susceptibility. Live BCG, sterile PPD, and saline were bronchoscopically instilled into separate lung segments (n = 65). A control group (n = 34) underwent a single bronchoscopy without challenge. The primary outcome was safety. Cellular and antibody immune signatures were identified in BAL before and 3 days after challenge using flow cytometry, ELISA, RNA sequencing, and mass spectrometry.Measurements and Main Results: The frequency of adverse events was low (9.4%; n = 10), similar in the challenge versus control groups (P = 0.8), and all adverse events were mild and managed conservatively in an outpatient setting. The optimal PPD and BCG dose was 0.5 TU and 104 cfu, respectively, based on changes in BAL cellular profiles (P = 0.02) and antibody responses (P = 0.01) at incremental doses before versus after challenge. At 104 versus 103 cfu BCG, there was a significant increase in number of differentially expressed genes (367 vs. 3; P < 0.001) and dysregulated proteins (64 vs. 0; P < 0.001). Immune responses were highly setting specific (in vitro vs. in vivo) and compartment specific (BAL vs. blood) and localized to the challenged lung segments.Conclusions: A lung-oriented mycobacterial controlled human infection model using live BCG and PPD is feasible and safe. These data inform the study of tuberculosis immunopathogenesis and strategies for evaluation and development of tuberculosis vaccine candidates.

A regimen containing bedaquiline and delamanid compared to bedaquiline in patients with drug-resistant tuberculosis.

There are limited data on combining delamanid and bedaquiline in drug-resistant tuberculosis (DR-TB) regimens. Prospective long-term outcome data, including in HIV-infected persons, are unavailable.We prospectively followed up 122 South African patients (52.5% HIV-infected) with DR-TB and poor prognostic features between 2014 and 2018. We examined outcomes and safety in those who received a bedaquiline-based regimen (n=82) compared to those who received a bedaquiline-delamanid combination regimen (n=40).There was no significant difference in 6-month culture conversion (92.5% versus 81.8%; p=0.26) and 18-month favourable outcome rate (63.4% versus 67.5%; p=0.66) in the bedaquiline versus the bedaquiline-delamanid combination group, despite the latter having more advanced drug resistance (3.7% versus 22.5% resistant to at least five drugs; p=0.001) and higher pre-treatment failure rates (12.2% versus 52.5% with pre-treatment multidrug-resistant TB therapy failure; p<0.001). Although the proportion of prolongation of the QT interval corrected using Fridericia's formula was higher in the combination group (>60 ms from baseline (p=0.001) or >450 ms during treatment (p=0.001)), there were no symptomatic cases or drug withdrawals in either group. Results were similar in HIV-infected patients.A bedaquiline-delamanid combination regimen showed comparable long-term safety compared to a bedaquiline-based regimen in patients with DR-TB, irrespective of HIV status. These data inform regimen selection in patients with DR-TB from TB-endemic settings.

A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic.

BACKGROUND: Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. METHODS: We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. RESULTS: Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. CONCLUSION: The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.

Same-Day Tools, Including Xpert Ultra and IRISA-TB, for Rapid Diagnosis of Pleural Tuberculosis: a Prospective Observational Study.

The diagnosis of pleural tuberculosis (TB) is problematic. The comparative performance of newer same-day tools for pleural TB, including Xpert MTB/RIF Ultra (ULTRA), has hitherto not been comprehensively studied. Adenosine deaminase (ADA), IRISA-TB (interferon gamma ultrasensitive rapid immunosuspension assay), Xpert MTB/RIF, and ULTRA performance outcomes were evaluated in pleural fluid samples from 149 patients with suspected pleural TB. The reference standard was culture positivity (fluid, biopsy specimen, or sputum) and/or pleural biopsy histopathology (termed definite TB). Those designated as having non-TB were negative by microbiological testing and were not initiated on anti-TB treatment. To determine the effect of sample concentration, 65 samples underwent pelleting by centrifugation, followed by conventional Xpert MTB/RIF and ULTRA. Of the 149 patients, 49 had definite TB, 16 had probable TB (not definite but treated for TB), and 84 had non-TB. ULTRA sensitivity and specificity (95% confidence intervals [CI]) were similar to those of Xpert MTB/RIF [sensitivity, 37.5% (25.3 to 51.2) versus 28.6% (15.9 to 41.2), respectively; specificity, 98.8% (96.5 to 100) versus 98.8% (96.5 to 100), respectively]. Centrifugation did not significantly improve ULTRA sensitivity (29.5% versus 31.3%, respectively). Adenosine deaminase and IRISA-TB sensitivity were 84.4% (73.9 to 95.0) and 89.8% (81.3 to 98.3), respectively. However, IRISA-TB demonstrated significantly better specificity (96.4% versus 87.5% [P = 0.034]), positive predictive value (93.6% versus 80.9 [P = 0.028]), and positive likelihood ratio (25.1 versus 6.8 [P = 0.032]) than ADA. In summary, Xpert ULTRA has poor sensitivity for the diagnosis of pleural TB. Alternative assays (ADA and IRISA-TB) are significantly more sensitive, with IRISA-TB demonstrating a higher specificity and rule-in value than ADA in this high-TB-burden setting where HIV is endemic.

Long-term bedaquiline-related treatment outcomes in patients with extensively drug-resistant tuberculosis from South Africa.

Optimal treatment regimens for patients with extensively drug-resistant tuberculosis (XDR-TB) remain unclear. Long-term prospective outcome data comparing XDR-TB regimens with and without bedaquiline from an endemic setting are lacking.We prospectively followed-up 272 South African patients (49.3% HIV-infected; median CD4 count 169 cells·µL-1) with newly diagnosed XDR-TB between 2008 and 2017. Outcomes were compared between those who had not received bedaquiline (pre-2013; n=204) and those who had (post-2013; n=68; 80.9% received linezolid in addition).The 24-month favourable outcome rate was substantially better in the bedaquiline versus the non-bedaquiline group (66.2% (45 out of 68) versus 13.2% (27 out of 204); p<0.001). In addition, the bedaquiline group exhibited reduced 24-month rates of treatment failure (5.9% versus 26.0%; p<0.001) and default (1.5% versus 15.2%; p<0.001). However, linezolid was withdrawn in 32.7% (18 out of 55) of patients in the bedaquiline group because of adverse events. Admission weight >50 kg, an increasing number of anti-TB drugs and bedaquiline were independent predictors of survival (the bedaquiline survival effect remained significant in HIV-infected persons, irrespective of CD4 count).XDR-TB patients receiving a backbone of bedaquiline and linezolid had substantially better favourable outcomes compared to those not using these drugs. These data inform the selection of XDR-TB treatment regimens and roll-out of newer drugs in TB-endemic countries.

Xpert MTB/RIF Results in Patients With Previous Tuberculosis: Can We Distinguish True From False Positive Results?

BACKGROUND: Patients with previous tuberculosis may have residual DNA in sputum that confounds nucleic acid amplification tests such as Xpert MTB/RIF. Little is known about the frequency of Xpert-positive, culture-negative ("false positive") results in retreatment patients, whether these are distinguishable from true positives, and whether Xpert's automated filter-based wash step reduces false positivity by removing residual DNA associated with nonintact cells. METHODS: Pretreatment patients (n = 2889) with symptoms of tuberculosis from Cape Town, South Africa, underwent a sputum-based liquid culture and Xpert. We also compared Xpert results from dilutions of intact or heat-lysed and mechanically lysed bacilli. RESULTS: Retreatment cases were more likely to be Xpert false-positive (45/321 Xpert-positive retreatment cases were false-positive) than new cases (40/461) (14% [95% confidence interval {CI}, 10%-18%] vs 8% [95% CI, 6%-12%];P= .018). Fewer years since treatment completion (adjusted odds ratio [aOR], 0.85 [95% CI, .73-.99]), less mycobacterial DNA (aOR, 1.14 [95% CI, 1.03-1.27] per cycle threshold [CT]), and a chest radiograph not suggestive of active tuberculosis (aOR, 0.22 [95% CI, .06-.82]) were associated with false positivity. CThad suboptimal accuracy for false positivity: 46% of Xpert-positives with CT> 30 would be false positive, although 70% of false positives would be missed. CT's predictive ability (area under the curve, 0.83 [95% CI, .76-.90]) was not improved by additional variables. Xpert detected nonviable, nonintact bacilli without a change in CTvs controls. CONCLUSIONS: One in 7 Xpert-positive retreatment patients were culture negative and potentially false positive. False positivity was associated with recent previous tuberculosis, high CT, and a chest radiograph not suggestive of active tuberculosis. Clinicians may consider awaiting confirmatory testing in retreatment patients with CT> 30; however, most false positives fall below this cut-point. Xpert can detect DNA from nonviable, nonintact bacilli.

primerJinn - a tool for rationally designing multiplex PCR primer sets and in silico PCR.

Background: Multiplex PCR amplifies numerous targets in a single tube reaction and is essential in molecular biology and clinical diagnostics. One of its most important applications is in the targeted sequencing of pathogens. Despite this importance, few tools are available for designing multiplex primers. Results: We developed primerJinn, a tool that designs a set of multiplex primers and allows for the in silico PCR evaluation of primer sets against numerous input genomes. We used primerJinn to create a multiplex PCR for the sequencing of drug resistance-conferring gene regions from Mycobacterium tuberculosis, which were then successfully sequenced. Conclusions: primerJinn provides a user-friendly, efficient, and accurate method for designing multiplex PCR primers and performing in silico PCR. It can be used for various applications in molecular biology and bioinformatics research, including the design of assays for amplifying and sequencing drug-resistance-conferring regions in important pathogens.

Circularization of rv0678 for Genotypic Bedaquiline Resistance Testing of Mycobacterium tuberculosis.

Circular DNA offers benefits over linear DNA in diagnostic and field assays, but currently, circular DNA generation is lengthy, inefficient, highly dependent on the length and sequence of DNA, and can result in unwanted chimeras. We present streamlined methods for generating PCR-targeted circular DNA from a 700 bp amplicon of rv0678, the high GC content (65%) gene implicated in Mycobacterium tuberculosis bedaquiline resistance, and demonstrate that these methods work as desired. We employ self-circularization with and without splints, a Gibson cloning-based approach, and novel 2 novel methods for generating pseudocircular DNA. The circular DNA can be used as a template for rolling circle PCR followed by long-read sequencing, allowing for the error correction of sequence data, and improving the confidence in the drug resistance determination and strain identification; and, ultimately, improving patient treatment. IMPORTANCE Antimicrobial resistance is a global health threat, and drug resistant tuberculosis is a principal cause of antimicrobial resistance-related fatality. The long turnaround time and the need for high containment biological laboratories of phenotypic growth-based Mycobacterium tuberculosis drug susceptibility testing often commit patients to months of ineffective treatment, and there is a groundswell of effort in shifting from phenotypic to sequencing-based genotypic assays. Bedaquiline is a key component to newer, all oral, drug resistant, tuberculosis regimens. Thus, we focus our study on demonstrating the circularization of rv0678, the gene that underlies most M. tuberculosis bedaquiline resistance. We present 2 novel methods for generating pseudocircular DNA. These methods greatly reduce the complexity and time needed to generate circular DNA templates for rolling circle amplification and long-read sequencing, allowing for error correction of sequence data, and improving confidence in the drug resistance determination and strain identification.

A simple, single-tube overlapping amplicon-targeted Illumina sequencing assay.

Targeted amplicon sequencing to identify pathogens, resistance-conferring mutations, and strain types is an important tool in diagnosing and treating infections. However, due to the short read limitations of Illumina sequencing, many applications require the splitting of limited clinical samples between two reactions. Here, we outline hairpin Illumina single-tube sequencing PCR (hissPCR) which allows for the generation of overlapping amplicons containing Illumina indexes and adapters in a single tube, effectively extending the Illumina read length while maintaining reagent and sample input requirements.

selSeq: A method for the enrichment of non-polyadenylated RNAs including enhancer and long non-coding RNAs for sequencing.

Non-polyadenylated RNA includes a large subset of crucial regulators of RNA expression and constitutes a substantial portion of the transcriptome, playing essential roles in gene regulation. For example, enhancer RNAs are long non-coding RNAs that perform enhancer-like functions, are bi-directionally transcribed, and usually lack polyA tails. This paper presents a novel method, selSeq, that selectively removes mRNA and pre-mRNA from samples enabling the selective sequencing of crucial regulatory elements, including non-polyadenylated RNAs such as long non-coding RNA, enhancer RNA, and non-canonical mRNA.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) infects cells in multiple lung myeloid cell subsets and causes chronic infection despite innate and adaptive immune responses. However, the mechanisms allowing Mtb to evade elimination are not fully understood. Here, using new methods, we determined that after T cell responses have developed, CD11clo monocyte-derived lung cells termed MNC1 (mononuclear cell subset 1), harbor more live Mtb compared to alveolar macrophages (AM), neutrophils, and less permissive CD11chi MNC2. Bulk RNA sequencing of sorted cells revealed that the lysosome biogenesis pathway is underexpressed in MNC1. Functional assays confirmed that Mtb-permissive MNC1 have less lysosome content, acidification, and proteolytic activity than AM, and less nuclear TFEB, a master regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in MNC1 in vivo. Instead, Mtb recruits MNC1 and MNC2 to the lungs for its spread from AM to these cell subsets as a virulence mechanism that requires the Mtb ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome function of primary macrophages in vitro and MNC1 and MNC2 in vivo, improving control of Mtb infection. Our results indicate that Mtb exploits lysosome-poor monocyte-derived cells for in vivo persistence, suggesting a potential target for host-directed tuberculosis therapy.

Mycobacterium tuberculosis resides in lysosome-poor monocyte-derived lung cells during chronic infection.

Mycobacterium tuberculosis (Mtb) persists in lung myeloid cells during chronic infection. However, the mechanisms allowing Mtb to evade elimination are not fully understood. Here, we determined that in chronic phase, CD11clo monocyte-derived lung cells termed MNC1 (mononuclear cell subset 1), harbor more live Mtb than alveolar macrophages (AM), neutrophils, and less permissive CD11chi MNC2. Transcriptomic and functional studies of sorted cells revealed that the lysosome biogenesis pathway is underexpressed in MNC1, which have less lysosome content, acidification, and proteolytic activity than AM, and less nuclear TFEB, a master regulator of lysosome biogenesis. Mtb infection does not drive lysosome deficiency in MNC1. Instead, Mtb recruits MNC1 and MNC2 to the lungs for its spread from AM to these cells via its ESX-1 secretion system. The c-Abl tyrosine kinase inhibitor nilotinib activates TFEB and enhances lysosome function of primary macrophages and MNC1 and MNC2 in vivo, improving control of Mtb infection. Our results indicate that Mtb exploits lysosome-poor monocyte-derived cells for in vivo persistence, suggesting a potential target for host-directed tuberculosis therapy.

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.

Anaerobe-enriched gut microbiota predicts pro-inflammatory responses in pulmonary tuberculosis.

BACKGROUND: The relationship between tuberculosis (TB), one of the leading infectious causes of death worldwide, and the microbiome, which is critical for health, is poorly understood. METHODS: To identify potential microbiome-host interactions, profiling of the oral, sputum and stool microbiota [n = 58 cases, n = 47 culture-negative symptomatic controls (SCs)] and whole blood transcriptome were done in pre-treatment presumptive pulmonary TB patients. This was a cross-sectional study. Microbiota were also characterised in close contacts of cases (CCCs, n = 73) and close contacts of SCs (CCSCs, n = 82) without active TB. FINDINGS: Cases and SCs each had similar α- and ß-diversities in oral washes and sputum, however, ß-diversity differed in stool (PERMANOVA p = 0•035). Cases were enriched with anaerobes in oral washes, sputum (Paludibacter, Lautropia in both) and stool (Erysipelotrichaceae, Blautia, Anaerostipes) and their stools enriched in microbial genes annotated as amino acid and carbohydrate metabolic pathways. In pairwise comparisons with their CCCs, cases had Megasphaera-enriched oral and sputum microbiota and Bifidobacterium-, Roseburia-, and Dorea-depleted stools. Compared to their CCSCs, SCs had reduced α-diversities and many differential taxa per specimen type. Cases differed transcriptionally from SCs in peripheral blood (PERMANOVA p = 0•001). A co-occurrence network analysis showed stool taxa, Erysipelotrichaceae and Blautia, to negatively co-correlate with enriched "death receptor" and "EIF2 signalling" pathways whereas Anaerostipes positively correlated with enriched "interferon signalling", "Nur77 signalling" and "inflammasome" pathways; all of which are host pathways associated with disease severity. In contrast, none of the taxa enriched in SCs correlated with host pathways. INTERPRETATION: TB-specific microbial relationships were identified in oral washes, induced sputum, and stool from cases before the confounding effects of antibiotics. Specific anaerobes in cases' stool predict upregulation of pro-inflammatory immunological pathways, supporting the gut microbiota's role in TB. FUNDING: European & Developing Countries Clinical Trials Partnership, South African-Medical Research Council, National Institute of Allergy and Infectious Diseases.

Differential RD-1-specific IFN-γ host responses to diverse Mycobacterium tuberculosis strains in HIV-uninfected persons may be explained by genotypic variation in the ESX-1 region.

OBJECTIVES: Between-person variability in T-cell-specific interferon-gamma release assay (IGRA) responses and discordance between IGRA test formats are poorly understood. METHODS: We evaluated the IFN-γ responses (QuantiFERON-TB Gold-In-Tube [QFT-GIT] and TSPOT-TB) stratified according to the Mycobacterium tuberculosis spoligotype of the culture isolate obtained from the same patients with confirmed active tuberculosis (n = 91). We further analysed differences within the RD-1-encoding ESX-1 region between the different strain types using whole genome sequencing. RESULTS: In HIV-uninfected patients, TSPOT.TB and QFT-GIT IFN-γ responses were 5-fold (p < 0.01) and 2-fold higher (p < 0.05) for those infected with family 33 compared to the LAM strain (additionally, TSPOT.TB responses were 5.6-fold [p < 0.05] and 2.6-fold higher [p < 0.05] for the patients infected with the family 33 versus the X strain and Beijing versus the LAM strain, respectively). Multivariate analysis revealed that strain type (determined by spoligotyping) was independently associated with the magnitude of the IGRA response (varied by IGRA test type) and this is likely explained by variability in the ESX-1 region of Mycobacteriumtuberculosis (determined by next-generation sequencing). CONCLUSIONS: These data have implications for the understanding of between-person heterogeneity in IGRA responses, Mycobateriumtuberculosis-specific host immunity, and the discordance between different IGRA test formats.

Bacterial and host determinants of cough aerosol culture positivity in patients with drug-resistant versus drug-susceptible tuberculosis.

A burgeoning epidemic of drug-resistant tuberculosis (TB) threatens to derail global control efforts. Although the mechanisms remain poorly clarified, drug-resistant strains are widely believed to be less infectious than drug-susceptible strains. Consequently, we hypothesized that lower proportions of patients with drug-resistant TB would have culturable Mycobacterium tuberculosis from respirable, cough-generated aerosols compared to patients with drug-susceptible TB, and that multiple factors, including mycobacterial genomic variation, would predict culturable cough aerosol production. We enumerated the colony forming units in aerosols (≤10 µm) from 452 patients with TB (227 with drug resistance), compared clinical characteristics, and performed mycobacterial whole-genome sequencing, dormancy phenotyping and drug-susceptibility analyses on M. tuberculosis from sputum. After considering treatment duration, we found that almost half of the patients with drug-resistant TB were cough aerosol culture-positive. Surprisingly, neither mycobacterial genomic variants, lineage, nor dormancy status predicted cough aerosol culture positivity. However, mycobacterial sputum bacillary load and clinical characteristics, including a lower symptom score and stronger cough, were strongly predictive, thereby supporting targeted transmission-limiting interventions. Effective treatment largely abrogated cough aerosol culture positivity; however, this was not always rapid. These data question current paradigms, inform public health strategies and suggest the need to redirect TB transmission-associated research efforts toward host-pathogen interactions.