Emergence of bedaquiline resistance in a high tuberculosis burden country.

RATIONALE: Bedaquiline has been classified as a group A drug for the treatment of multidrug-resistant tuberculosis (MDR-TB) by the World Health Organization; however, globally emerging resistance threatens the effectivity of novel MDR-TB treatment regimens. OBJECTIVES: We analysed pre-existing and emerging bedaquiline resistance in bedaquiline-based MDR-TB therapies, and risk factors associated with treatment failure and death. METHODS: In a cross-sectional cohort study, we employed patient data, whole-genome sequencing (WGS) and phenotyping of Mycobacterium tuberculosis complex (MTBC) isolates. We could retrieve baseline isolates from 30.5% (62 out of 203) of all MDR-TB patients who received bedaquiline between 2016 and 2018 in the Republic of Moldova. This includes 26 patients for whom we could also retrieve a follow-up isolate. MEASUREMENTS AND MAIN RESULTS: At baseline, all MTBC isolates were susceptible to bedaquiline. Among 26 patients with available baseline and follow-up isolates, four (15.3%) patients harboured strains which acquired bedaquiline resistance under therapy, while one (3.8%) patient was re-infected with a second bedaquiline-resistant strain. Treatment failure and death were associated with cavitary disease (p=0.011), and any additional drug prescribed in the bedaquiline-containing regimen with WGS-predicted resistance at baseline (OR 1.92 per unit increase, 95% CI 1.15-3.21; p=0.012). CONCLUSIONS: MDR-TB treatments based on bedaquiline require a functional background regimen to achieve high cure rates and to prevent the evolution of bedaquiline resistance. Novel MDR-TB therapies with bedaquiline require timely and comprehensive drug resistance monitoring.

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid.

OBJECTIVES: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

Design of Multidrug-Resistant Tuberculosis Treatment Regimens Based on DNA Sequencing.

BACKGROUND: Comprehensive and reliable drug susceptibility testing (DST) is urgently needed to provide adequate treatment regimens for patients with multidrug-resistant/rifampicin-resistant tuberculosis (MDR/RR-TB). We determined whether next-generation sequencing (NGS) analysis of Mycobacterium tuberculosis complex isolates and genes implicated in drug resistance can guide the design of effective MDR/RR-TB treatment regimens. METHODS: NGS-based genomic DST predictions of M. tuberculosis complex isolates from MDR/RR-TB patients admitted to a TB reference center in Germany between 1 January 2015 and 30 April 2019 were compared with phenotypic DST results of mycobacteria growth indicator tubes (MGIT). Standardized treatment algorithms were applied to design individualized therapies based on either genomic or phenotypic DST results, and discrepancies were further evaluated by determination of minimal inhibitory drug concentrations (MICs) using Sensititre MYCOTBI and UKMYC microtiter plates. RESULTS: In 70 patients with MDR/RR-TB, agreement among 1048 pairwise comparisons of genomic and phenotypic DST was 86.3%; 76 (7.2%) results were discordant, and 68 (6.5%) could not be evaluated due to the presence of polymorphisms with yet unknown implications for drug resistance. Importantly, 549 of 561 (97.9%) predictions of drug susceptibility were phenotypically confirmed in MGIT, and 27 of 64 (42.2%) false-positive results were linked to previously described mutations mediating a low or moderate MIC increase. Virtually all drugs (99.0%) used in combination therapies that were inferred from genomic DST were confirmed to be susceptible by phenotypic DST. CONCLUSIONS: NGS-based genomic DST can reliably guide the design of effective MDR/RR-TB treatment regimens.

Secretome characterization of clinical isolates from the Mycobacterium abscessus complex provides insight into antigenic differences.

BACKGROUND: Mycobacterium abscessus (MAB) is a widely disseminated pathogenic non-tuberculous mycobacterium (NTM). Like with the M. tuberculosis complex (MTBC), excreted / secreted (ES) proteins play an essential role for its virulence and survival inside the host. Here, we used a robust bioinformatics pipeline to predict the secretome of the M. abscessus ATCC 19977 reference strain and 15 clinical isolates belonging to all three MAB subspecies, M. abscessus subsp. abscessus, M. abscessus subsp. bolletii, and M. abscessus subsp. massiliense. RESULTS: We found that ~ 18% of the proteins encoded in the MAB genomes were predicted as secreted and that the three MAB subspecies shared > 85% of the predicted secretomes. MAB isolates with a rough (R) colony morphotype showed larger predicted secretomes than isolates with a smooth (S) morphotype. Additionally, proteins exclusive to the secretomes of MAB R variants had higher antigenic densities than those exclusive to S variants, independent of the subspecies. For all investigated isolates, ES proteins had a significantly higher antigenic density than non-ES proteins. We identified 337 MAB ES proteins with homologues in previously investigated M. tuberculosis secretomes. Among these, 222 have previous experimental support of secretion, and some proteins showed homology with protein drug targets reported in the DrugBank database. The predicted MAB secretomes showed a higher abundance of proteins related to quorum-sensing and Mce domains as compared to MTBC indicating the importance of these pathways for MAB pathogenicity and virulence. Comparison of the predicted secretome of M. abscessus ATCC 19977 with the list of essential genes revealed that 99 secreted proteins corresponded to essential proteins required for in vitro growth. CONCLUSIONS: This study represents the first systematic prediction and in silico characterization of the MAB secretome. Our study demonstrates that bioinformatics strategies can help to broadly explore mycobacterial secretomes including those of clinical isolates and to tailor subsequent, complex and time-consuming experimental approaches accordingly. This approach can support systematic investigation exploring candidate proteins for new vaccines and diagnostic markers to distinguish between colonization and infection. All predicted secretomes were deposited in the Secret-AAR web-server ( http://microbiomics.ibt.unam.mx/tools/aar/index.php ).

Microevolution of Mycobacterium tuberculosis Subpopulations and Heteroresistance in a Patient Receiving 27 Years of Tuberculosis Treatment in Germany.

Preexisting and newly emerging resistant pathogen subpopulations (heteroresistance) are potential risk factors for treatment failure of multi/extensively drug resistant (MDR/XDR) tuberculosis (TB). Intrapatient evolutionary dynamics of Mycobacterium tuberculosis complex (Mtbc) strains and their implications on treatment outcomes are still not completely understood. To elucidate how Mtbc strains escape therapy, we analyzed 13 serial isolates from a German patient by whole-genome sequencing. Sequencing data were compared with phenotypic drug susceptibility profiles and the patient's collective 27-year treatment history to further elucidate factors fostering intrapatient resistance evolution. The patient endured five distinct TB episodes, ending in resistance to 16 drugs and a nearly untreatable XDR-TB infection. The first isolate obtained, during the patient's 5th TB episode, presented fixed resistance mutations to 7 anti-TB drugs, including isoniazid, rifampin, streptomycin, pyrazinamide, prothionamide, para-aminosalicylic acid, and cycloserine-terizidone. Over the next 13 years, a dynamic evolution with coexisting, heterogeneous subpopulations was observed in 6 out of 13 sequential bacterial isolates. The emergence of drug-resistant subpopulations coincided with frequent changes in treatment regimens, which often included two or fewer active compounds. This evolutionary arms race between competing subpopulations ultimately resulted in the fixation of a single XDR variant. Our data demonstrate the complex intrapatient microevolution of Mtbc subpopulations during failing MDR/XDR-TB treatment. Designing effective treatment regimens based on rapid detection of (hetero) resistance is key to avoid resistance development and treatment failure.

Two Pandemics, One Challenge-Leveraging Molecular Test Capacity of Tuberculosis Laboratories for Rapid COVID-19 Case-Finding.

In many settings, the ongoing coronavirus disease (COVID-19) pandemic coincides with other major public health threats, in particular tuberculosis. Using tuberculosis (TB) molecular diagnostic infrastructure, which has substantially expanded worldwide in recent years, for COVID-19 case-finding might be warranted. We analyze the potential of using TB diagnostic and research infrastructures for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. We focused on quality control by adapting the 12 Quality System Essentials framework to the COVID-19 and TB context. We conclude that diagnostic infrastructures for TB can in principle be leveraged to scale-up SARS-CoV-2 testing, in particular in resource-poor settings. TB research infrastructures also can support sequencing of SARS-CoV-2 to study virus evolution and diversity globally. However, fundamental principles of quality management must be followed for both TB and SARS-CoV-2 testing to ensure valid results and to minimize biosafety hazards, and the continuity of TB diagnostic services must be guaranteed at all times.

Mycobacterium tuberculosis Complex Lineage 3 as Causative Agent of Pulmonary Tuberculosis, Eastern Sudan1.

Pathogen-based factors associated with tuberculosis (TB) in eastern Sudan are not well defined. We investigated genetic diversity, drug resistance, and possible transmission clusters of Mycobacterium tuberculosis complex (MTBC) strains by using a genomic epidemiology approach. We collected 383 sputum specimens at 3 hospitals in 2014 and 2016 from patients with symptoms suggestive of TB; of these, 171 grew MTBC strains. Whole-genome sequencing could be performed on 166 MTBC strains; phylogenetic classification revealed that most (73.4%; n = 122) belonged to lineage 3 (L3). Genome-based cluster analysis showed that 76 strains (45.9%) were grouped into 29 molecular clusters, comprising 2-8 strains/patients. Of the strains investigated, 9.0% (15/166) were multidrug resistant (MDR); 10 MDR MTBC strains were linked to 1 large MDR transmission network. Our findings indicate that L3 strains are the main causative agent of TB in eastern Sudan; MDR TB is caused mainly by transmission of MDR L3 strains.

Population Structure of Mycobacterium bovis in Germany: a Long-Term Study Using Whole-Genome Sequencing Combined with Conventional Molecular Typing Methods.

Mycobacterium bovis is the primary cause of bovine tuberculosis (bTB) and infects a wide range of domestic animal and wildlife species and humans. In Germany, bTB still emerges sporadically in cattle herds, free-ranging wildlife, diverse captive animal species, and humans. In order to understand the underlying population structure and estimate the population size fluctuation through time, we analyzed 131 M. bovis strains from animals (n = 38) and humans (n = 93) in Germany from 1999 to 2017 by whole-genome sequencing (WGS), mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, and spoligotyping. Based on WGS data analysis, 122 out of the 131 M. bovis strains were classified into 13 major clades, of which 6 contained strains from both human and animal cases and 7 only strains from human cases. Bayesian analyses suggest that the M. bovis population went through two sharp anticlimaxes, one in the middle of the 18th century and another one in the 1950s. WGS-based cluster analysis grouped 46 strains into 13 clusters ranging in size from 2 to 11 members and involving strains from distinct host types, e.g., only cattle and also mixed hosts. Animal strains of four clusters were obtained over a 9-year span, pointing toward autochthonous persistent bTB infection cycles. As expected, WGS had a higher discriminatory power than spoligotyping and MIRU-VNTR typing. In conclusion, our data confirm that WGS and suitable bioinformatics constitute the method of choice to implement prospective molecular epidemiological surveillance of M. bovis The population of M. bovis in Germany is diverse, with subtle, but existing, interactions between different host groups.

Antimicrobial Susceptibility and Phylogenetic Relations in a German Cohort Infected with Mycobacterium abscessus.

Mycobacterium abscessus is a highly antibiotic-resistant opportunistic pathogen causing clinically challenging infections in patients with preexisting lung diseases or under immunosuppression. Hence, reliable antibiotic susceptibility data are required for effective treatment. Aims of this study were to investigate (i) the congruence of genotypic and phenotypic antimicrobial susceptibility testing, (ii) the relationship between resistance profile and clinical course, and (iii) the phylogenetic relations of M. abscessus in a German patient cohort. A total of 39 isolates from 29 patients infected or colonized with M. abscessus underwent genotypic and phenotypic drug susceptibility testing. Clinical data were correlated with susceptibility data. Phylogenetic analysis was performed by means of whole-genome sequencing (WGS) and single-nucleotide polymorphism (SNP) analysis. Macrolide resistance was mainly mediated by functional Erm(41) methyltransferases (T28 sequevars) in M. abscessus subsp. abscessus (n = 25) and M. abscessus subsp. bolletii (n = 2). It was significantly associated with impaired culture conversion (P = 0.02). According to the core SNP phylogeny, we identified three clusters of closely related isolates with SNP distances below 25. Representatives of all circulating global clones (Absc. 1, Absc. 2, and Mass. 1) were identified in our cohort. However, we could not determine evidence for in-hospital interhuman transmission from clinical data. In our patient cohort, we identified three M. abscessus clusters with closely related isolates and representatives of the previously described international clusters but no human-to-human in-hospital transmission. Macrolide and aminoglycoside susceptibility data are critical for therapeutic decision-making in M. abscessus infections.

What Is Resistance? Impact of Phenotypic versus Molecular Drug Resistance Testing on Therapy for Multi- and Extensively Drug-Resistant Tuberculosis.

Rapid and accurate drug susceptibility testing (DST) is essential for the treatment of multi- and extensively drug-resistant tuberculosis (M/XDR-TB). We compared the utility of genotypic DST assays with phenotypic DST (pDST) using Bactec 960 MGIT or Löwenstein-Jensen to construct M/XDR-TB treatment regimens for a cohort of 25 consecutive M/XDR-TB patients and 15 possible anti-TB drugs. Genotypic DST results from Cepheid GeneXpert MTB/RIF (Xpert) and line probe assays (LPAs; Hain GenoType MTBDRplus 2.0 and MTBDRsl 2.0) and whole-genome sequencing (WGS) were translated into individual algorithm-derived treatment regimens for each patient. We further analyzed if discrepancies between the various methods were due to flaws in the genotypic or phenotypic test using MIC results. Compared with pDST, the average agreement in the number of drugs prescribed in genotypic regimens ranged from just 49% (95% confidence interval [CI], 39 to 59%) for Xpert and 63% (95% CI, 56 to 70%) for LPAs to 93% (95% CI, 88 to 98%) for WGS. Only the WGS regimens did not contain any drugs to which pDST showed resistance. Importantly, MIC testing revealed that pDST likely underestimated the true rate of resistance for key drugs (rifampin, levofloxacin, moxifloxacin, and kanamycin) because critical concentrations (CCs) were too high. WGS can be used to rule in resistance even in M/XDR strains with complex resistance patterns, but pDST for some drugs is still needed to confirm susceptibility and construct the final regimens. Some CCs for pDST need to be reexamined to avoid systematic false-susceptible results in low-level resistant isolates.

Validation of the FluoroType MTBDR Assay for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates.

For Mycobacterium tuberculosis complex (MTBC), the rapid and accurate diagnosis of drug resistance is crucial to ensure early initiation of appropriate therapy. Recently, a new molecular diagnostic test, the FluoroType MTBDR, aimed at detecting rifampin and isoniazid resistance has become available. This study aimed to evaluate the FluoroType MTBDR in comparison to phenotypic drug susceptibility testing (DST) using M. tuberculosis complex isolates. MTBC isolates underwent phenotypic DST and were tested using the FluoroType MTBDR and Genotype MTBDRplus. Sanger sequencing of the key regions of rpoB, katG, inhA, and aphC was performed for isolates with discordant phenotypic and molecular results. Furthermore, isolates with specific wild-type bands missing in the Genotype MTBDRplus, indicating the presence of a mutation, were investigated by Sanger sequencing. Specificity and sensitivity, defined as the proportions of isolates correctly determined as susceptible and resistant by the FluoroType MTBDR compared to phenotypic DST, were calculated. A total of 180 culture isolates were included; phenotypic DST showed 85 isolates susceptible to isoniazid and rifampin, 7 with isoniazid monoresistance, 7 with rifampin monoresistance, and 81 with multidrug resistance. The specificity of the FluoroType MTBDR was 100% (95% confidence interval [CI], 96.0 to 100%) for both rifampin and isoniazid. The sensitivity was 91.7% (95% CI, 83.6 to 96.6%) for isoniazid and 98.9% (95% CI, 93.8 to 100.0%) for rifampin. The FluoroType MTBDR has a high sensitivity and specificity for the detection of rifampin and isoniazid resistance when using culture isolates.

Rapid Microarray-Based Detection of Rifampin, Isoniazid, and Fluoroquinolone Resistance in Mycobacterium tuberculosis by Use of a Single Cartridge.

The rapid and robust identification of mutations in Mycobacterium tuberculosis complex (MTBC) strains mediating multidrug-resistant (MDR) and extensively drug-resistant (XDR) phenotypes is crucial to combating the MDR tuberculosis (TB) epidemic. Currently available molecular anti-TB drug susceptibility tests either are restricted to a single target or drug (i.e., the Xpert MTB/RIF test) or present a risk of cross-contamination due to the design limitations of the open platform (i.e., line probe assays). With a good understanding of the technical and commercial boundaries, we designed a test cartridge based on an oligonucleotide array into which dried reagents are introduced and which has the ability to identify MTBC strains resistant to isoniazid, rifampin, and the fluoroquinolones. The melting curve assay interrogates 43 different mutations in the rifampin resistance-determining region (RRDR) of rpoB, rpoB codon 572, katG codon 315, the inhA promoter region, and the quinolone resistance-determining region (QRDR) of gyrA in a closed cartridge system within 90 min. Assay performance was evaluated with 265 clinical MTBC isolates, including MDR/XDR, non-MDR, and fully susceptible isolates, from a drug resistance survey performed in Swaziland in 2009 and 2010. In 99.5% of the cases, the results were consistent with data previously acquired utilizing Sanger sequencing. The assay, which uses a closed cartridge system in combination with a battery-powered Alere q analyzer and which has the potential to extend the current gene target panel, could serve as a rapid and robust point-of-care test in settings lacking a comprehensive molecular laboratory infrastructure to differentiate TB patients infected with MDR and non-MDR strains and to assist clinicians with their early treatment decisions.

Some Synonymous and Nonsynonymous gyrA Mutations in Mycobacterium tuberculosis Lead to Systematic False-Positive Fluoroquinolone Resistance Results with the Hain GenoType MTBDRsl Assays.

In this study, using the Hain GenoType MTBDRsl assays (versions 1 and 2), we found that some nonsynonymous and synonymous mutations in gyrA in Mycobacterium tuberculosis result in systematic false-resistance results to fluoroquinolones by preventing the binding of wild-type probes. Moreover, such mutations can prevent the binding of mutant probes designed for the identification of specific resistance mutations. Although these mutations are likely rare globally, they occur in approximately 7% of multidrug-resistant tuberculosis strains in some settings.

Role of Alanine Racemase Mutations in Mycobacterium tuberculosis d-Cycloserine Resistance.

A screening of more than 1,500 drug-resistant strains of Mycobacterium tuberculosis revealed evolutionary patterns characteristic of positive selection for three alanine racemase (Alr) mutations. We investigated these mutations using molecular modeling, in vitro MIC testing, as well as direct measurements of enzymatic activity, which demonstrated that these mutations likely confer resistance to d-cycloserine.

LOC-SERS: A Promising Closed System for the Identification of Mycobacteria.

A closed droplet based lab-on-a-chip (LOC) device has been developed for the differentiation of six species of mycobacteria, i.e., both Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM), using surface-enhanced Raman spectroscopy (SERS). The combination of a fast and simple bead-beating module for the disruption of the bacterial cell with the LOC-SERS device enables the application of an easy and reliable system for bacteria discrimination. Without extraction or further treatment of the sample, the obtained SERS spectra are dominated by the cell-wall component mycolic acid. For the differentiation, a robust data set was recorded using a droplet based LOC-SERS device. Thus, more than 2100 individual SERS spectra of the bacteria suspension were obtained in 1 h. The differentiation of bacteria using LOC-SERS provides helpful information for physicians to define the conditions for the treatment of individual patients.

Occurrence of rpoB mutations in isoniazid-resistant but rifampin-susceptible Mycobacterium tuberculosis isolates from Germany.

Four out of 143 phenotypically isoniazid-resistant but rifampin-susceptible Mycobacterium tuberculosis strains that were isolated from patients in Germany in 2011 had mutations in the rifampin resistance-determining region of rpoB. After performing drug susceptibility testing (DST) with two methods, the proportion method on Löwenstein-Jensen medium and using the Bactec 960 Mycobacteria Growth Indicator Tube system, we conclude that the two methods are equally reliable for phenotypic DST and MIC determination.

Dual-centre evaluation of the FluoroType MTBDR version 2 assay for detection of Mycobacterium tuberculosis complex and resistance-conferring mutations in pulmonary and extrapulmonary samples from Denmark, Germany and Sierra Leone.

OBJECTIVES: We evaluated the ability of FluoroType MTBDR version 2 (FTv2; Hain Lifescience), a second-step real-time PCR assay, to simultaneously detect Mycobacterium tuberculosis complex (MTBC) DNA and mutations conferring resistance to rifampicin (RIF) and isoniazid (INH), in pulmonary and extrapulmonary samples from patients and compared them with corresponding cultures. METHODS: FTv2 MTBC was evaluated on 1815 and 432 samples from Denmark (DK) and Germany (DE), respectively. RIF and INH resistance mutations were assessed in the German samples and 110 samples from Sierra Leone and subsequently compared to phenotypic antimicrobial susceptibility testing and a composite reference DNA (CRD) based on the GenoType MTBDR line-probe assay and Sanger sequencing or whole-genome sequencing. RESULTS: Of the 584 (557 smear-negative) Danish and 277 (85 smear-negative) German sputum samples, 42 (16) and 246 (54) were culture positive, and 44 (18) and 222 (35) were FTv2 positive, providing an FTv2 sensitivity and specificity of 0.86 (0.63) and 0.98 (DK), 0.90 (0.65) and 1.00 (DE), respectively. The count, sensitivities, and specificities for all pulmonary samples were 1434, 0.79, and 0.99 (DK) and 347, 0.86, and 1.00 (DE), respectively; for extrapulmonary samples, 381, 0.33, 0.99 (DK) and 83, 0.50, and 1.00 (DE). The valid count, sensitivity, and specificity compared with CRD for detecting resistance mutations were RIF 355, 0.99, 0.96, and INH 340, 1.00, and 0.98, respectively. DISCUSSION: FTv2 reliably detects MTBC DNA in pulmonary and extrapulmonary samples and detects resistance mutations for INH and RIF resistance in inhA promoter, katG, and rpoB genes.