Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing: From Sequence Data to Resistance Profiles.

Whole genome sequencing of Mycobacterium tuberculosis complex (MTBC) isolates has been shown to provide accurate predictions for resistance and susceptibility for many first- and second-line anti-tuberculosis drugs. However, bioinformatic pipelines and mutation catalogs to predict antimicrobial resistances in MTBC isolates are often customized and detailed protocols are difficult to access. Here, we provide a step-by-step workflow for the processing and interpretation of short-read sequencing data and give an overview of available analysis pipelines.

Use of Whole Genome Sequencing for Mycobacterium tuberculosis Complex Antimicrobial Susceptibility Testing.

Whole genome sequencing (WGS) is becoming an important diagnostic tool for antimicrobial susceptibility testing of Mycobacterium tuberculosis complex (MTBC) isolates in many countries. WGS protocols usually start with the preparation of a DNA-library: the critical first step in the process. A DNA-library represents the genomic content of a DNA sample and consists of unique short DNA fragments. Although available DNA-library protocols come with manufacturer instructions, details of the entire process, including quality controls, instrument parameters, and run evaluations, often need to be developed and customized by each laboratory to implement WGS technology effectively. Here, we provide a detailed workflow for a DNA-library preparation based on an adapted Illumina protocol optimized for the reduction of reagent costs.

First detection of a Mycobacterium tuberculosis XDR clinical isolate harbouring an RpoB I491F mutation in a Ukrainian patient treated in Germany, October 2023.

This report documents the case of a Ukrainian patient infected with an extensively drug-resistant (XDR) lineage 2 Mycobacterium tuberculosis strain harbouring the rifampicin resistance mutation RpoB I491F. This mutation is not detected by routine molecular WHO-recommended rapid diagnostics, complicating the detection and treatment of these strains. The occurrence of such mutations underscores the need for enhanced diagnostic techniques and tailored treatment regimens, especially in eastern Europe where lineage 2 strains and XDR-tuberculosis are prevalent.

Clinical and genomic features of Mycobacterium avium complex: a multi-national European study.

BACKGROUND: The Mycobacterium avium complex (MAC) comprises the most frequent non-tuberculous mycobacteria (NTM) in Central Europe and currently includes twelve species. M. avium (MAV), M. intracellulare subsp. intracellulare (MINT), and M. intracellulare subsp. chimaera (MCH) are clinically most relevant. However, the population structure and genomic landscape of MAC linked with potential pathobiological differences remain little investigated. METHODS: Whole genome sequencing (WGS) was performed on a multi-national set of MAC isolates from Germany, France, and Switzerland. Phylogenetic analysis was conducted, as well as plasmids, resistance, and virulence genes predicted from WGS data. Data was set into a global context with publicly available sequences. Finally, detailed clinical characteristics were associated with genomic data in a subset of the cohort. RESULTS: Overall, 610 isolates from 465 patients were included. The majority could be assigned to MAV (n = 386), MCH (n = 111), and MINT (n = 77). We demonstrate clustering with less than 12 SNPs distance of isolates obtained from different patients in all major MAC species and the identification of trans-European or even trans-continental clusters when set into relation with 1307 public sequences. However, none of our MCH isolates clustered closely with the heater-cooler unit outbreak strain Zuerich-1. Known plasmids were detected in MAV (325/1076, 30.2%), MINT (62/327, 19.0%), and almost all MCH-isolates (457/463, 98.7%). Predicted resistance to aminoglycosides or macrolides was rare. Overall, there was no direct link between phylogenomic grouping and clinical manifestations, but MCH and MINT were rarely found in patients with extra-pulmonary disease (OR 0.12 95% CI 0.04-0.28, p < 0.001 and OR 0.11 95% CI 0.02-0.4, p = 0.004, respectively) and MCH was negatively associated with fulfillment of the ATS criteria when isolated from respiratory samples (OR 0.28 95% CI 0.09-0.7, p = 0.011). With 14 out of 43 patients with available serial isolates, co-infections or co-colonizations with different strains or even species of the MAC were frequent (32.6%). CONCLUSIONS: This study demonstrates clustering and the presence of plasmids in a large proportion of MAC isolates in Europe and in a global context. Future studies need to urgently define potential ways of transmission of MAC isolates and the potential involvement of plasmids in virulence.

Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

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.

Genomic diversity and clinical relevance of Mycobacterium simiae.

Introduction: Mycobacterium simiae is a slow-growing non-tuberculous mycobacterium that can cause non-tuberculous mycobacterium (NTM) pulmonary disease and extrapulmonary infections. Until now, detailed genomic and clinical characteristics, as well as possible transmission routes of this rare pathogen remain largely unknown. Methods: We conducted whole genome sequencing of available M. simiae isolates collected at a tertiary care centre in Central Germany from 2006 to 2020 and set them into context with publicly available M. simiae complex sequences through phylogenetic analysis. Resistance, virulence and stress genes, as well as known Mycobacteriaceae plasmid sequences were detected in whole genome raw reads. Clinical data and course were retrieved and correlated with genomic data. Results: We included 33 M. simiae sensu stricto isolates from seven patients. M. simiae showed low clinical relevance with only two patients fulfilling American Thoracic Society (ATS) criteria in our cohort and three receiving NTM-effective therapy. The bacterial populations were highly stable over time periods of up to 14 years, and no instances of mixed or re-infections with other strains of M. simiae were observed. Clustering with <12 single nucleotide polymorphisms distance was evident among isolates from different patients; however, proof for human-to-human transmission could not be established from epidemiological data. Conclusion: Overall, the available sequence data for M. simiae complex was significantly extended and new insights into its pathogenomic traits were obtained. We demonstrate high longitudinal genomic stability within single patients. Although we cannot exclude human-to-human transmission, we consider it unlikely in the light of available epidemiological data.

Prison as a driver of recent transmissions of multidrug-resistant tuberculosis in Callao, Peru: a cross-sectional study.

Background: We sought to identify resistance patterns and key drivers of recent multidrug-resistant tuberculosis (MDR-TB) transmission in a TB-prevalent area in Peru. Methods: Cross-sectional study including MDR Mycobacterium tuberculosis complex (Mtbc) strains identified in Callao-Peru between April 2017 and February 2019. Mtbc DNA was extracted for whole genome sequencing which was used for phylogenetic inference, clustering, and resistance mutation analyses. Clusters indicative of recent transmission were defined based on a strain-to-strain distance of ≤5 (D5) single nucleotide polymorphisms (SNPs). Epidemiologic factors linked to MDR-TB clustering were analyzed using Poisson regression. Findings: 171 unique MDR-Mtbc strains were included; 22 (13%) had additional fluoroquinolone resistance and were classified as pre-XDR. Six strains (3.5%) harboured bedaquiline (BDQ) resistance mutations and were classified as MDR + BDQ. 158 (92%) Mtbc strains belonged to lineage 4 and 13 (8%) to lineage 2. Using a cluster threshold of ≤5 SNPs, 98 (57%) strains were grouped in one of the 17 D5 clusters indicative of recent transmission, ranging in size from 2 to the largest cluster formed by 53 4.3.3 strains (group_1). Lineage 4.3.3 strains showed the overall highest cluster rate (43%). In multivariate analyses, current or previous imprisonment was independently associated with being part of any MDR-TB transmission clusters (adjusted prevalence ratio [aPR], 1.45; 95% CI, 1.09-1.92). Interpretation: Pre-XDR-TB emerged in more than 10% of the MDR-TB strains investigated. Transmission of 4.3.3 Mtbc strains especially of the dominant group_1 clone is a major driver of the MDR-TB epidemic in Callao. Current or previous imprisonment was linked to recent MDR-TB transmissions, indicating an important role of prisons in driving the MDR-TB epidemic. Funding: This work was supported in part by the ERANet-LAC Network of the European Union, Latin America and the Caribbean Countries on Joint Innovation and Research Activities, and FONDECYT. Additional support was received from Leibniz Science Campus Evolutionary Medicine of the Lung, the Deutsche Forschungsgemeinschaft (German Research Foundation, under Germany's Excellence Strategy-EXC 2167 Precision Medicine in Inflammation), and the Research Training Group 2501 TransEvo.

Bedaquiline Resistance after Effective Treatment of Multidrug-Resistant Tuberculosis, Namibia.

Bedaquiline is currently a key drug for treating multidrug-resistant or rifampin-resistant tuberculosis. We report and discuss the unusual development of resistance to bedaquiline in a teenager in Namibia, despite an optimal background regimen and adherence. The report highlights the risk for bedaquiline resistance development and the need for rapid drug-resistance testing.

Refined understanding of the impact of the Mycobacterium tuberculosis complex diversity on the intrinsic susceptibility to pretomanid.

Previous work reported unprecedented differences in the intrinsic in vitro susceptibility of the Mycobacterium tuberculosis complex (MTBC) to pretomanid (Pa) using the Mycobacteria Growth Indicator Tube (MGIT) system. We tested 125 phylogenetically diverse strains from all known MTBC lineages (1-9) without known Pa resistance mutations and four strains with known resistance mutations as controls. This confirmed that MTBC, unlike most bacteria-antimicrobial combinations, displayed substantial differences in the intrinsic susceptibility relative to the technical variation of Pa MIC testing. This was also the case for the Middlebrook 7H11 (7H11) medium, demonstrating that these differences were not specific to MGIT. Notably, lineage 1 was confirmed to have intrinsically elevated MICs compared with lineages 2, 3, 4, and 7 (L2-4/7), underlining the urgent need for WHO to publish its decision of whether lineage 1 should be deemed treatable by BPaL(M), the now preferred all-oral regimen for treating rifampin-resistant tuberculosis. Lineages 5 and 6, which are most frequent in West Africa, responded differently to Pa, with lineage 5 being more similar to L2-4/7 and lineage 6 being more susceptible. More data are needed to determine whether 7H11 MICs are systematically lower than those in MGIT. IMPORTANCE: This study confirmed that the Mycobacterium tuberculosis complex lineage 1, responsible for 28% of global tuberculosis cases, is less susceptible to pretomanid (Pa). It also refined the understanding of the intrinsic susceptibilities of lineages 5 and 6, most frequent in West Africa, and lineages 8 and 9. Regulators must review whether these in vitro differences affect the clinical efficacy of the WHO-recommended BPaL(M) regimen and set breakpoints for antimicrobial susceptibility testing accordingly. Notably, regulators should provide detailed justifications for their decisions to facilitate public scrutiny.

Enhanced active case finding of drug-resistant tuberculosis in Namibia: a protocol for the hotspots, hospitals, and households (H3TB) study.

INTRODUCTION: Namibia is a high tuberculosis (TB)-burden country with an estimated incidence of 460/100 000 (around 12 000 cases) per year. Approximately 4.5% of new cases and 7.9% of previously treated TB cases are multidrug resistant (MDR) and 47% of patients with MDR-TB are HIV coinfected. Published data suggest a clustering of MDR-TB transmission in specific areas. Identifying transmission clusters is key to implementing high-yield and cost-effective interventions. This includes knowing the yield of finding TB cases in high-transmission zones (eg, community hotspots, hospitals or households) to deliver community-based interventions. We aim to identify such transmission zones for enhanced case finding and evaluate the effectiveness of this approach. METHODS AND ANALYSIS: H3TB is an observational cross-sectional study evaluating MDR-TB active case finding strategies. Sputum samples from MDR-TB cases in three regions of Namibia will be evaluated by whole genome sequencing (WGS) in addition to routine sputum investigations (Xpert MTB/RIF, culture and drug susceptibility testing). We will collect information on household contacts, use of community spaces and geographical map intersections between participants, synthesising these data to identify transmission hotspots. We will look at the feasibility, acceptability, yield and cost of case finding strategies in these hotspots, and in households of patients with MDR-TB and visitors of hospitalised patients with MDR-TB. A compartmental transmission dynamic model will be constructed to evaluate the impact and cost-effectiveness of the strategies if scaled. ETHICS AND DISSEMINATION: Ethics approval was obtained. Participants will give informed consent. H3TB will capitalise on a partnership with the Ministry of Health and Social Services to follow up individuals diagnosed with MDR-TB and integrate WGS data with innovative contact network mapping, to allow enhanced case finding. Study data will contribute towards a systems approach to TB control. Equally important, it will serve as a role model for similar studies in other high-incidence settings.

Molecular determinants of multidrug-resistant tuberculosis in Sierra Leone.

Multidrug-resistant tuberculosis (MDR-TB) management has become a serious global health challenge. Understanding its epidemic determinants on the regional level is crucial for developing effective control measures. We used whole genome sequencing data of 238 of Mycobacterium tuberculosis complex (MTBC) strains to determine drug resistance profiles, phylogeny, and transmission dynamics of MDR/rifampicin-resistant (RR) MTBC strains from Sierra Leone. Forty-two strains were classified as RR, 196 as MDR, 5 were resistant to bedaquiline (BDQ) and clofazimine (CFZ), but none was found to be resistant to fluoroquinolones. Sixty-one (26%) strains were resistant to all first-line drugs, three of which had additional resistance to BDQ/CFZ. The strains were classified into six major MTBC lineages (L), with strains of L4 being the most prevalent, 62% (n = 147), followed by L6 (Mycobacterium africanum) strains, (21%, n = 50). The overall clustering rate (using ≤d12 single-nucleotide polymorphism threshold) was 44%, stratified into 31 clusters ranging from 2 to 16 strains. The largest cluster (n = 16) was formed by sublineage 2.2.1 Beijing Ancestral 3 strains, which developed MDR several times. Meanwhile, 10 of the L6 strains had a primary MDR transmission. We observed a high diversity of drug resistance mutations, including borderline resistance mutations to isoniazid and rifampicin, and mutations were not detected by commercial assays. In conclusion, one in five strains investigated was resistant to all first-line drugs, three of which had evidence of BDQ/CFZ resistance. Implementation of interventions such as rapid diagnostics that prevent further resistance development and stop MDR-TB transmission chains in the country is urgently needed. IMPORTANCE: A substantial proportion of MDR-TB strains in Sierra Leone were resistant against all first line drugs; however this makes the all-oral-six-month BPaLM regimen or other 6-9 months all oral regimens still viable, mainly because there was no FQ resistance.Resistance to BDQ was detected, as well as RR, due to mutations outside of the hotspot region. While the prevalence of those resistances was low, it is still cause for concern and needs to be closely monitored.

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance.

OBJECTIVES: Heteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has been linked with poor treatment outcomes. We compared the analytical sensitivity of molecular methods, such as GeneXpert and whole genome sequencing (WGS) in detecting heteroresistance when compared with the 'gold standard' phenotypic assay: the agar proportion method (APM). METHODS: Using two rounds of proficiency surveys with defined monoresistant BCG strains and mixtures of susceptible/resistant M. tb, we determined the limit of detection (LOD) of known resistance associated mutations. RESULTS: The LOD for rifampin-R (RIF-R) detection was 1% using APM, 60% using GeneXpert MTB/RIF, 10% using GeneXpert MTB/RIF Ultra and 10% using WGS. While WGS could detect mutations beyond those associated with RIF resistance, the LOD for these other mutations was also 10%. Additionally, we observed instances where laboratories did not report resistance in the majority population, yet the mutations were present in the raw sequence data. CONCLUSION: The gold standard APM detects minority resistant populations at a lower proportion than molecular tests. Mycobacterium bovis BCG strains with defined resistance and extracted DNA from M. tb provided concordant results and can serve in quality control of laboratories offering molecular testing for resistance. Further research is required to determine whether the higher LOD of molecular tests is associated with negative treatment outcomes.

Emergence of bedaquiline-resistant tuberculosis and of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains with rpoB Ile491Phe mutation not detected by Xpert MTB/RIF in Mozambique: a retrospective observational study.

BACKGROUND: In 2021, an estimated 4800 people developed rifampicin-resistant tuberculosis in Mozambique, 75% of which went undiagnosed. Detailed molecular data on rifampicin-resistant and multidrug-resistant (MDR) tuberculosis are not available. Here, we aimed at gaining precise data on the determinants of rifampicin-resistant and MDR tuberculosis in Mozambique. METHODS: In this retrospective observational study, we performed whole-genome sequencing of 704 rifampicin-resistant Mycobacterium tuberculosis complex (Mtbc) strains submitted to the National Tuberculosis Reference Laboratory (NTRL) in Maputo, Mozambique, between 2015 and 2021. Phylogenetic strain classification, genomic resistance prediction, and cluster analysis were performed. FINDINGS: Between Jan 1, 2015, and July 31, 2021, 2606 Mtbc isolates with an isoniazid or rifampicin resistance were identified in the NTRL biobank, of which, 1483 (56·9%) were from men, 1114 (42·7%) from women, and nine (0·4%) were unknown. Genome-based drug-resistant prediction classified 704 Mtbc strains as rifampicin resistant. 628 (89%) of the 704 Mtbc strains were classified MDR; of those, 146 (23%) were pre-extensively drug resistant (pre-XDR; additional fluoroquinolone resistance), and 24 (4%) extensively drug resistant (XDR; combined fluoroquinolone and bedaquiline resistance). Overall, 61 (9%) of 704 strains revealed resistance to bedaquiline: five (7%) of 76 rifampicin resistant plus bedaquiline resistant, 32 (7%) of 458 MDR plus bedaquiline resistant, and 24 (100%) of 24 XDR. Prevalence of bedaquiline resistance increased from 3% in 2016 to 14% in 2021. The cluster rate (12 single-nucleotide polymorphism threshold) was 42% for rifampicin-resistant strains, 78% for MDR strains, 94% for pre-XDR strains, and 96% for XDR Mtbc strains. 31 (4%) of 704 Mtbc strains, belonging to a diagnostic escape outbreak strain previously described in Eswatini (group_56), had an rpoB Ile491Phe mutation which is not detected by Xpert MTB/RIF (no other rpoB mutation). Of these, 23 (74%) showed additional resistance to bedaquiline, 13 (42%) had bedaquiline and fluoroquinolone resistance, and two (6%) were bedaquiline, fluoroquinolone, and delamanid resistant. INTERPRETATION: Pre-XDR resistance is highly prevalent among MDR Mtbc strains in Mozambique and so is bedaquiline resistance; and the frequency of bedaquiline resistance quadrupled over time and was found even in Mtbc strains without fluoroquinolone resistance. Importantly, strains with Ile491Phe mutation were frequent, accounting for 31% (n=10) of MDR plus bedaquiline-resistant strains and 54% (n=13) of XDR Mtbc strains. Given the current diagnostic algorithms and treatment regimens, both the emergence of rifampicin resistance due to Ile491Phe and bedaquiline resistance might jeopardise MDR tuberculosis prevention and care unless sequencing-based technology is rolled out. The potential cross border spread of diagnostic escape strains needs further investigation. FUNDING: The German Ministry of Health through the Seq_MDRTB-Net project, the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy Precision Medicine in Inflammation and the Research Training Group 2501 TransEvo, the Leibniz Science Campus Evolutionary Medicine of the Lung, and the German Ministry of Education and Research via the German Center for Infection Research.

The effect of M. tuberculosis lineage on clinical phenotype.

Six lineages of Mycobacterium tuberculosis sensu stricto (which excludes M. africanum) are described. Single-country or small observational data suggest differences in clinical phenotype between lineages. We present strain lineage and clinical phenotype data from 12,246 patients from 3 low-incidence and 5 high-incidence countries. We used multivariable logistic regression to explore the effect of lineage on site of disease and on cavities on chest radiography, given pulmonary TB; multivariable multinomial logistic regression to investigate types of extra-pulmonary TB, given lineage; and accelerated failure time and Cox proportional-hazards models to explore the effect of lineage on time to smear and culture-conversion. Mediation analyses quantified the direct effects of lineage on outcomes. Pulmonary disease was more likely among patients with lineage(L) 2, L3 or L4, than L1 (adjusted odds ratio (aOR) 1.79, (95% confidence interval 1.49-2.15), p<0.001; aOR = 1.40(1.09-1.79), p = 0.007; aOR = 2.04(1.65-2.53), p<0.001, respectively). Among patients with pulmonary TB, those with L1 had greater risk of cavities on chest radiography versus those with L2 (aOR = 0.69(0.57-0.83), p<0.001) and L4 strains (aOR = 0.73(0.59-0.90), p = 0.002). L1 strains were more likely to cause osteomyelitis among patients with extra-pulmonary TB, versus L2-4 (p = 0.033, p = 0.008 and p = 0.049 respectively). Patients with L1 strains showed shorter time-to-sputum smear conversion than for L2. Causal mediation analysis showed the effect of lineage in each case was largely direct. The pattern of clinical phenotypes seen with L1 strains differed from modern lineages (L2-4). This has implications for clinical management and could influence clinical trial selection strategies.

Two-stage tuberculosis diagnostics: combining centrifugal microfluidics to detect TB infection and Inh and Rif resistance at the point of care with subsequent antibiotic resistance profiling by targeted NGS.

Globally, tuberculosis (TB) remains the deadliest bacterial infectious disease, and spreading antibiotic resistances is the biggest challenge for combatting the disease. Rapid and comprehensive diagnostics including drug susceptibility testing (DST) would assure early treatment, reduction of morbidity and the interruption of transmission chains. To date, rapid genetic resistance testing addresses only one to four drug groups while complete DST is done phenotypically and takes several weeks. To overcome these limitations, we developed a two-stage workflow for rapid TB diagnostics including DST from a single sputum sample that can be completed within three days. The first stage is qPCR detection of M. tuberculosis complex (MTBC) including antibiotic resistance testing against the first-line antibiotics, isoniazid (Inh) and rifampicin (Rif). The test is automated by centrifugal microfluidics and designed for point of care (PoC). Furthermore, enriched MTBC DNA is provided in a detachable sample tube to enable the second stage: if the PCR detects MTBC and resistance to either Inh or Rif, the MTBC DNA is shipped to specialized facilities and analyzed by targeted next generation sequencing (tNGS) to assess the complete resistance profile. Proof-of-concept testing of the PoC test revealed an analytical sensitivity of 44.2 CFU ml-1, a diagnostic sensitivity of 96%, and a diagnostic specificity of 100% for MTBC detection. Coupled tNGS successfully provided resistance profiles, demonstrated for samples from 17 patients. To the best of our knowledge, the presented combination of PoC qPCR with tNGS allows for the fastest comprehensive TB diagnostics comprising decentralized pathogen detection with subsequent resistance profiling in a facility specialized in tNGS.

Tuberculosis Variant with Rifampin Resistance Undetectable by Xpert MTB/RIF, Botswana.

GeneXpert MTB/RIF, a tool widely used for diagnosing tuberculosis, has limitations for detecting rifampin resistance in certain variants. We report transmission of a pre-extensively drug-resistant variant in Botswana that went undetected by GeneXpert. The public health impact of misdiagnosis emphasizes the need for comprehensive molecular testing to identify resistance and guide treatment.