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PubMed2026

Assessment of differentially culturable tubercle bacteria assays for the detection of tuberculosis infection in asymptomatic household contacts and the implications for intra-household transmission: a longitudinal cohort study.

BACKGROUND: Conventional culture methods for tuberculosis diagnosis miss differentially culturable tubercle bacteria (DCTB), which grow only in liquid assays supplemented with growth-enhancing factors. This limitation, combined with inadequate contact tracing and screening, often fails to identify asymptomatic individuals, with live bacilli detectable by enhanced culture methods. This shortfall results in undiagnosed reservoirs of bacteria, potentially fuelling ongoing transmission. In this study, we aimed to investigate whether DCTB assays provide greater sensitivity by detecting more Mycobacterium tuberculosis infections than conventional culture and whether this enhanced detection improves the resolution of intrahousehold transmission mapping. In addition, we sought to evaluate whether DCTB populations can progress to conventional culture positivity, thereby highlighting their clinical and epidemiological relevance. METHODS: In this prospective observational longitudinal cohort study, drug-susceptible or rifampicin-resistant tuberculosis index participants aged 12 years or older, were recruited from primary healthcare clinics from two South African districts. Inclusion criteria were informed consent, Xpert MTB/RIF Ultra-positive results, tuberculosis symptoms (>2 weeks), provision of baseline samples, at least one consenting household contact, and documented HIV status. Household contacts of the index patients and control households were also recruited. Sputum specimens were collected at baseline and 2, 4, 8, 12, and 16 months from the index participants and household contacts. Samples were analysed by conventional mycobacterial growth indicator tube (MGIT) culture, and colony-forming unit assays to identify viable bacteria. Enhanced culture to detect DCTB involved serial dilution of sputum in liquid culture, supplemented with M tuberculosis culture filtrate as a source of growth stimulatory factors. Whole-genome sequencing (WGS) of cultured isolates was performed to trace household transmission. FINDINGS: Between June 1, 2020, and Feb 6, 2024, 293 index participants (183 [62%] male), 701 household contacts (453 [65%] female), and 122 control participants (67 [55%] female) were enrolled. At baseline, 249 (85%) of 293 index participants and 110 (16%) of 701 household contact sputum samples were positive for M tuberculosis by MGIT conventional culture. For baseline MGIT-negative specimens, DCTB assays detected M tuberculosis in an additional 21 (7%) of 293 index participants and 26 (4%) of 701 household contacts. Over 16 months of follow-up, DCTB assays identified 61 (8·7%) of 701 additional tuberculosis-positive household contacts not detected by conventional culture. WGS-guided transmission mapping using conventional culture identified transmission in 16 (15%) of 104 households, whereas DCTB assays detected an additional 19 (18%) of 104 transmission events. No evidence of intrahousehold transmission was found in the remaining 69 (66%) of 104 tuberculosis-positive households. Over the 16-month follow-up period, conventional culture identified 233 positive household contacts, of which 195 (84%) were asymptomatic. DCTB assays detected an additional 94 cases of M tuberculosis positivity in household contacts, of which 79 (84%) were asymptomatic. In control households, tuberculosis prevalence at baseline was two (2%) of 122, with an additional three (3%) of 122 identified during follow-up. INTERPRETATION: DCTB assays provide substantial value by detecting asymptomatic individuals missed by conventional culture, revealing a potentially important reservoir of subclinical infection, which could sustain transmission. In addition, DCTB detection uncovers transmission linkages missed by conventional culture, providing a more comprehensive understanding of M tuberculosis transmission dynamics and highlighting the need to incorporate enhanced culture methods into diagnostic and surveillance strategies, to strengthen early case identification and tuberculosis control efforts. FUNDING: National Institutes of Health.

Humans↗
PubMed2025

Diagnostic performance of the Sanity 2.0 assay to detect resistance to rifampicin, isoniazid, and fluoroquinolones in tuberculosis.

UNLABELLED: Effective tuberculosis (TB) management relies on prompt diagnosis of Mycobacterium tuberculosis complex (MTBC) and associated drug resistance. The Sanity 2.0 assay is a high-resolution melting assay designed for direct respiratory sample testing, enabling simultaneous detection of MTBC and resistance to rifampicin (RIF), isoniazid (INH), and fluoroquinolones (FQ) in a single step. This study evaluated its diagnostic performance in two registered multicenter trials among bacteriologically confirmed TB patients. Diagnostic performance was evaluated for MTBC detection, as well as for the identification of resistance to RIF, INH, and FQ, using phenotypic drug susceptibility testing, whole-genome sequencing, and a composite reference standard. Agreement analyses were conducted between the Sanity 2.0 assay and Xpert MTB/RIF and Xpert MTB/XDR. Among 611 patients, the Sanity 2.0 assay detected MTBC in 563 patients, exhibiting a sensitivity of 92.1% (95% CI: 89.7-94.0). For detecting resistance to RIF, INH, and FQ, sensitivities exceeded 90%, with specificities of 95.8% (95% CI: 88.5-98.6), 100.0% (95% CI: 96.4-100.0), and 97.8% (95% CI: 93.8-99.3) against the composite reference standard, respectively. The agreement with Xpert MTB/RIF for RIF detection was 98.6% (95% CI: 96.9-99.3). For INH and FQ resistance, the agreement with Xpert MTB/XDR was 92.0% (95% CI: 88.5-94.5) and 94.3% (95% CI: 91.2-96.3), respectively. The Sanity 2.0 assay is a rapid and user-friendly platform capable of detecting both MTBC and key drug resistance. It demonstrated good diagnostic performance and could potentially be an effective alternative to guide individualized anti-TB treatment, especially in resource-limited settings. IMPORTANCE: Rapid and accurate detection of both Mycobacterium tuberculosis complex (MTBC) and key drug resistance is critical to improving tuberculosis treatment outcomes and reducing transmission. However, current molecular diagnostic workflows often require sequential testing, which can delay the initiation of effective and individualized therapy. We evaluated the Sanity 2.0 assay, an integrated high-resolution melting test that simultaneously detects MTBC and resistance to rifampicin, isoniazid, and fluoroquinolone resistance directly from respiratory samples in about 2-3 hours. The assay demonstrated excellent performance, with MTBC detection sensitivity of 92.1% and drug resistance sensitivities exceeding 90% and specificities over 95% against a composite reference standard, as well as strong concordance with World Health Organization-endorsed molecular assays. Implementation of the Sanity 2.0 assay could streamline TB diagnostic workflows; enable rapid, single-step resistance profiling; and facilitate timely, individualized treatment-particularly in resource-limited settings where rapid and comprehensive resistance testing remains a critical unmet need.

Humans↗
PubMed2025

Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.

UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings. IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.

Isoniazid↗
PubMed2025

Discrepancies in isoniazid susceptibility profiles: Bactec MGIT 960-resistant but GenoType MTBDRplus-susceptible Mycobacterium tuberculosis strains in Hunan, China.

UNLABELLED: Discordant drug susceptibility testing (DST) results between the Bactec MGIT 960 system (MGIT) and the GenoType MTBDRplus assay (MTBDRplus) for isoniazid (INH) complicate clinical decision-making. In this study, we performed minimum inhibitory concentration (MIC) assays and whole-genome sequencing (WGS) on 53 Mycobacterium tuberculosis strains identified as INH-resistant by MGIT but INH-susceptible by MTBDRplus. The variants conferring INH resistance were evaluated by the WHO mutation catalogue. Our results showed that only five strains carried variants classified as "associated with resistance" (Group 1/2), including katG Trp39STOP, katG Ser315Asn, inhA -154G>A, and inhA Ser94Ala. In addition, 44 strains carried 70 variants classified as "Group 3: Uncertain significance" across nine genes, including katG, ahpC, inhA, Rv0010c, Rv1129c, Rv2752c, mshA, dnaA, and Rv1258c. The remaining four strains carried no variants (Groups 1-3) linked to INH resistance. No significant difference in the prevalence of high-level INH resistance was observed between lineage 2 and lineage 4 strains (χ² = 0.232, P = 0.630). Our findings indicate that the variants classified as "uncertain significance" may be the main genetic determinants causing discordant results, highlighting their associations with INH resistance that need to be further investigated. IMPORTANCE: This study addresses a critical challenge in drug susceptibility testing (DST): the discrepancies in DST results for isoniazid (INH) between the Bactec MGIT 960 system and the GenoType MTBDRplus assay. These discordant results significantly complicate treatment decisions, potentially leading to suboptimal patient outcomes. Using MIC assays and WGS on 53 clinical Mycobacterium tuberculosis strains, we provide valuable insights into the genetic basis of INH resistance. Our findings showed that only a small fraction of strains carried variants definitively linked to INH resistance, while a larger number harbored variants of uncertain significance across multiple genes, underscoring the complexity of INH resistance mechanisms. This study highlights the urgent need to refine our understanding of these "Group 3: uncertain significance" variants, as they appear to be a primary driver of the discrepancies. Additionally, this study emphasizes the importance of integrating advanced sequencing tools into DST to improve the accuracy of INH resistance detection.

Isoniazid↗
PubMed2026

Comparative in vitro antimicrobial susceptibility profiles of clofazimine and pyrifazimine against clinical isolates of Mycobacterium tuberculosis in southwest China.

UNLABELLED: Clofazimine (CFZ) is a key drug used to treat drug-resistant tuberculosis (DR-TB), while pyrifazimine (TBI-166) is an improved riminophenazine derivative with better pharmacokinetics. However, there is a lack of data on its susceptibility and resistance in regions with a high disease burden, such as southwestern China. We compared the in vitro antimicrobial activities of CFZ and TBI-166 against 249 DR-TB clinical isolates (99 multidrug-resistant TB [MDR-TB] and 150 pre-extensively drug-resistant TB [pre-XDR-TB] isolates) from southwestern China. TBI-166 exhibited a concentration-dependent biphasic antimicrobial pattern compared to CFZ. TBI-166 showed significantly greater potency at low concentrations (MIC&#x2085;&#x2080; = 0.031 &#xb5;g/mL TBI-166 vs 0.25 &#xb5;g/mL for CFZ; P < 0.001), but attenuated inhibition at high concentrations (MIC&#x2089;&#x2080; > 4 &#xb5;g/mL vs 1 &#xb5;g/mL for CFZ; P < 0.001). However, at high concentrations, the antibacterial effect of TBI-166 is weaker than that of CFZ (40% of TBI-166-resistant isolates have MIC > 4 &#xb5;g/mL, compared to 6.67% of CFZ-resistant isolates, P < 0.001). Epidemiological cutoff values (ECOFFs) were 1.0 &#xb5;g/mL for CFZ and 0.25 &#xb5;g/mL for TBI-166. Based on these in vitro ECOFFs, the resistance rate to TBI-166 (20.1%, 50/249) was significantly higher than that to CFZ (6.0%, 15/249, P < 0.0001). Whole-genome sequencing revealed that mutations in Rv0678 were prevalent in dual-resistant isolates (10/14) and TBI-166 monoresistant isolates (5/40), while Rv1979c mutations were less frequent, and no pepQ mutations were detected. These mutations differed from known hotspots, suggesting potential novel resistance mechanisms. IMPORTANCE: Drug-resistant tuberculosis (DR-TB) remains a major global health challenge, and optimizing treatments for high-burden regions like southwest China is crucial. This study is the first to detail the differential in vitro activities of clofazimine (CFZ) and the novel TBI-166 against clinical DR-TB isolates from southwest China, alongside their resistance-associated genetic profiles. Findings show TBI-166 has enhanced low-concentration potency but higher resistance rates, plus novel mutations in Rv0678 and Rv1979c linked to resistance. These insights will help refine clinical regimens for DR-TB and strengthen regional resistance surveillance, both of which are essential for controlling the spread of DR-TB in southwest China and informing treatment and surveillance strategies in other similar high-burden areas globally.

Clofazimine↗
PubMed2026

First-line drug-resistant tuberculosis among children under 15 years in Ethiopia: insights from phenotypic and whole-genome sequencing approaches.

BACKGROUND: Childhood drug-resistant tuberculosis is often underdiagnosed and inadequately characterized due to the paucibacillary nature of the disease. This study aimed to assess resistance to first-line anti-tuberculosis drugs in children using phenotypic drug susceptibility testing and whole-genome sequencing. METHODS: A retrospective-prospective study was conducted on culture-confirmed childhood tuberculosis cases in Ethiopia (2017&#x2013;2023). Phenotypic drug susceptibility testing was performed on 110 Mycobacterium tuberculosis complex isolates. Whole-genome sequencing was completed for 85 of these isolates, which were analyzed using the TB-Profiler and MTBSeq pipelines. We assessed the sensitivity, specificity, predictive values, and kappa agreement of whole-genome sequencing compared with phenotypic drug susceptibility testing. RESULTS: Phenotypic resistance to at least one first-line anti-TB drug was observed in 26/110 (23.6%) of the examined isolates, with isoniazid resistance being the most frequent, 23/110 (20.9%), followed by rifampicin resistance, 18/110 (16.4%). TB-Profiler showed almost perfect agreement with phenotypic drug susceptibility testing for rifampicin (sensitivity 94.4%, kappa&#x2009;=&#x2009;0.96) and isoniazid (sensitivity 91.3%, kappa&#x2009;=&#x2009;0.91), whereas MTBSeq showed slightly lower performance. Both pipelines demonstrated moderate to weak agreement with phenotypic drug susceptibility testing for detecting resistance to ethambutol, pyrazinamide, and streptomycin. The most frequently observed resistance mutations among phenotypically resistant isolates were rpoB (Ser450Leu), katG (S315Thr), embB (Met306Ile), and pncA (C-11&#xa0;A&#x2009;>&#x2009;G) for rifampicin, isoniazid, ethambutol, and pyrazinamide, respectively. Discrepancies between genotypic and phenotypic drug susceptibility testing were observed across all first-line anti-TB drug-resistant isolates, particularly for ethambutol and pyrazinamide. CONCLUSION: We found a high prevalence of isoniazid resistance, along with rifampicin resistance, underscoring the need for early detection in vulnerable groups. Whole-genome sequencing showed good accuracy for these drugs, with TB-Profiler performing best. CLINICAL TRIAL NUMBER: Not applicable.

Humans↗
PubMed2026

Genetic diversity and drug resistance profiles of Mycobacterium tuberculosis among Ethiopian children as determined by whole-genome sequencing.

UNLABELLED: Ethiopia ranks 30th among the tuberculosis (TB) burden countries, with children representing a significant yet understudied population group. This study aims to investigate the genetic diversity and drug-resistant profile among Ethiopian children. We included children under 15 years of age diagnosed with culture-confirmed pulmonary TB/drug-resistant TB between January 2017 and June 2023. Phenotypic drug susceptibility testing and whole-genome sequencing were conducted for 85 Mycobacterium tuberculosis (MTB) isolates. Demographic data were combined with genomic information. Lineage 4 was the most dominant (77.6%), while lineage 2 was less common (1%). Within lineage 4, several sub-lineages were identified, with lineage 4.2.2.2 being notably the most predominant (48%). Most of these cases were from Oromia (58%), including the hotspot areas for lineage 4 that were identified at a 99% confidence level. Among 17 MDR/pre-XDR-TB isolates, lineages 3 and 4.2.2.2 were the dominantly observed lineages/sub-lineages, with proportions of 29% and 65%, respectively. Of the 85 cases, 30.5% were drug-resistant TB to at least one of the five first-line anti-TB drugs tested by phenotypic drug susceptibility testing. Of these 26 drug-resistant TB cases, 23 were concordant with whole-genome sequencing characterization. The most frequent resistance mutations to rifampicin were found in the rpoB gene, specifically p.Ser450Leu (88%), followed by isoniazid in the katG gene, p.Ser315Thr (86%). Multidrug-resistant TB was strongly associated with MTB lineages (P = 0.007). This study identified high genetic diversity of M. tuberculosis and related drug-resistance mutations, with a strong concordance between whole-genome sequencing-based predictions and phenotypic drug susceptibility testing. IMPORTANCE: Our findings revealed a high genetic diversity of Mycobacterium tuberculosis among Ethiopian children, with the most common lineage being lineage 4, specifically lineage 4.2.2.2, in which a higher frequency of multidrug-resistant tuberculosis (TB) was observed. Additionally, we identified regional hotspots, suggesting ongoing community transmission. Moreover, whole-genome sequencing demonstrated high concordance with phenotypic drug susceptibility testing and identified mutation genes associated with first- and second-line anti-TB drugs, highlighting its usefulness in providing comprehensive results for resistance detection in children. Thus, it is essential for integrating genomic surveillance into childhood TB and drug resistance control.

Humans↗
PubMed2026

MIC-based tuberculosis drug susceptibility testing using Sensititre MYCOTB: a diagnostic accuracy meta-analysis.

Accurate drug susceptibility testing (DST) is crucial for designing effective regimens for multidrug-resistant (MDR) and pre-extensively drug-resistant tuberculosis (pre-XDR TB). Sensititre MYCOTB enables simultaneous determination of minimum inhibitory concentrations (MICs) for multiple drugs, but its diagnostic performance varies across studies. This meta-analysis evaluated the diagnostic performance of Sensititre MYCOTB for key MDR and pre-XDR TB drugs. The protocol was registered in PROSPERO (CRD420251230599). PubMed, Cochrane, Google Scholar, Scopus, ONOS, Web of Science, ScienceDirect, and registries were systematically searched for studies published between 2010 and 2025. Studies comparing the Sensititre MYCOTB with reference DST for Mycobacterium tuberculosis complex (MTBC) were included. Bias assessment and pooled diagnostic accuracy estimates were generated. Fourteen studies, including 1,728 isolates, were analyzed. Rifampicin and isoniazid demonstrated high sensitivity (0.976 [95% CI: 0.94-0.99] and 0.977 [95% CI: 0.95-0.99]) and specificity (0.958 [95% CI: 0.84-0.98] and 0.957 [95% CI: 0.83-0.99], respectively) with low heterogeneity. Amikacin, kanamycin, and ofloxacin demonstrate good diagnostic accuracy, with high specificity (>0.98 [95% CI]). Moderate diagnostic accuracy was observed for ethambutol, streptomycin, ethionamide, and rifabutin. Cycloserine, moxifloxacin, and para-aminosalicylic acid showed inconsistent performance despite excellent specificity (>0.97 [95% CI]). Sensitivity analysis partially improved pooled sensitivity for moxifloxacin 0.801 (95% CI: 0.585-0.924) and para-aminosalicylic acid 0.76 (95% CI: 0.518-0.894), whereas cycloserine remained at 0.436 (95% CI: 0.190-0.725), although heterogeneity persisted. Sensititre MYCOTB DST demonstrates high diagnostic accuracy for MDR-TB and pre-XDR-TB drugs, while caution is required with cycloserine, moxifloxacin, and para-aminosalicylic acid. These findings support the integration of MIC-based testing into clinical decision-making.

Microbial Sensitivity Tests↗
PubMed2026

Draft genome sequences of non-endemic Mycobacterium tuberculosis Indo-Oceanic and West African lineages recovered in Panama.

We report the draft genome sequences of three Mycobacterium tuberculosis isolates recovered from Panamanian residents belonging to the Indo-Oceanic (Lineage 1) and West African (Lineage 5) lineages. These isolates exhibit various drug-resistance profiles, including rifampicin-resistant and multidrug-resistant phenotypes. This highlights the changing molecular epidemiology of tuberculosis in Central America.

DNA sequencing↗
PubMed1992

Mycobacterium tuberculosis in children with human immunodeficiency virus type 1 infection.

A retrospective study was conducted at the Childrens Hospital Center at Jackson Memorial Hospital in Miami, FL, to evaluate the natural history of Mycobacterium tuberculosis infection in nine children with vertically acquired human immunodeficiency virus type 1 infection. The patients' ages ranged from 6 months to 7 years (median age, 42 months). Common presenting symptoms included prolonged fever, cough and anorexia. Only one patient had a positive tuberculin test. Five patients evidenced only pulmonary disease, three patients had pulmonary and extrapulmonary disease and one patient developed extrapulmonary tuberculosis (mastoiditis) and pulmonary interstitial disease that could not be attributed to mycobacterial infection because of lack of information. Organisms isolated before January, 1989, were susceptible to isoniazid and rifampin whereas isolates from three patients cultured after that time were resistant to multiple antituberculosis drugs. The median survival time after M. tuberculosis diagnosis for all children was 20 months. Our study suggests that children with human immunodeficiency virus type 1 infection who have tuberculosis have an increased risk for extrapulmonary disease. A high index of suspicion for the diagnosis of M. tuberculosis should be maintained in human immunodeficiency virus type 1-infected children with prolonged fever and respiratory symptoms. In areas of high endemicity of multidrug-resistant organisms, therapy with a broader panel of drugs may need to be instituted until susceptibility testing becomes available.

AIDS-Related Opportunistic Infections↗
PubMed1992

Attacking today's tuberculosis problem: a multifaceted, coordinated effort.

Tuberculosis is making a comeback in communities across the nation. Increased rates of the disease, particularly with those having HIV/AIDS, have sounded the alarm that quick and decisive action is needed to halt the spread of TB. Multidrug-resistant TB is becoming a primary concern with public health officials. Specific plans and efforts, instigated by the Centers for Disease Control, have outlined the appropriate steps local public health workers, the medical community, and civic and community organizations should take in order to eliminate TB by the year 2010. With the creation of the Task Force on Drug Resistant Tuberculosis, Louisiana has a vehicle with which to combat its growing TB problem.

Acquired Immunodeficiency Syndrome↗
PubMed2025

Advancing the fight against tuberculosis: integrating innovation and public health in diagnosis, treatment, vaccine development, and implementation science.

Tuberculosis (TB) remains one of the leading causes of infectious disease mortality worldwide, increasingly complicated by the emergence of drug-resistant strains and limitations in existing diagnostic and therapeutic strategies. Despite decades of global efforts, the disease continues to impose a significant burden, particularly in low- and middle-income countries (LMICs) where health system weaknesses hinder progress. This comprehensive review explores recent advancements in TB diagnostics, antimicrobial resistance (AMR surveillance), treatment strategies, and vaccine development. It critically evaluates cutting-edge technologies including CRISPR-based diagnostics, whole-genome sequencing, and digital adherence tools, alongside therapeutic innovations such as shorter multidrug-resistant TB regimens and host-directed therapies. Special emphasis is placed on the translational gap-highlighting barriers to real-world implementation such as cost, infrastructure, and policy fragmentation. While innovations like the Xpert MTB/RIF Ultra, BPaLM regimen, and next-generation vaccines such as M72/AS01E represent pivotal progress, their deployment remains uneven. Implementation science, cost-effectiveness analyses, and health equity considerations are vital to scaling up these tools. Moreover, the expansion of the TB vaccine pipeline and integration of AI in diagnostics signal a transformative period in TB control. Eliminating TB demands more than biomedical breakthroughs-it requires a unified strategy that aligns innovation with access, equity, and sustainability. By bridging science with implementation, and integrating diagnostics, treatment, and prevention within robust health systems, the global community can accelerate the path toward ending TB.

diagnostic innovation↗
PubMed2026

Genetic diversity of clinical Mycobacterium bovis BCG isolates from an immunocompromised patient with BCG infection.

BACKGROUND: The Bacillus Calmette-Gu&#xe9;rin (BCG) vaccine is widely administered to prevent severe tuberculosis but can cause serious adverse events, including disseminated BCGosis, in immunocompromised individuals. However, studies investigating the in vivo genetic adaptation and microevolution of this live-attenuated vaccine during prolonged infection remain limited. METHODS: Two clinical Mycobacterium bovis BCG isolates (BCG01 and BCG02) and a lot-matched vaccine strain (VAC) underwent whole-genome sequencing. Phenotypic drug susceptibility testing was performed on the clinical isolates. Genomic relatedness was assessed using SNP-distance clustering and maximum-likelihood phylogeny against global reference strains. Comparative variant analysis was performed to identify mutations specific to BCG01 and BCG02 relative to VAC, and genotypic drug resistance was assessed using TB-Profiler. RESULTS: Phylogenomic analyses and SNP distance confirmed that both clinical isolates were derived from the BCG Tokyo 172 vaccine strain. BCG02 exhibited twice the mutational burden of BCG01, acquiring mutations in genes associated with cell wall biosynthesis (mas, ppsA), regulatory adaptation (pknK, dnaA), and surface antigens (pecA, PE/PPE). Crucially, whereas BCG01 remained susceptible to first-line drugs, BCG02 acquired a canonical rpoB Ser450Leu mutation (100% frequency) and a heteroresistant inhA Ile194Thr mutation (13% frequency), resulting in multidrug-resistant (MDR) BCGosis. CONCLUSION: Although structurally stable, the BCG Tokyo 172 vaccine strain can undergo rapid, clinically significant microevolution and clonal selection within immunocompromised hosts. The in vivo acquisition of multidrug resistance underscores the critical need for pre-vaccination immune screening and comprehensive laboratory monitoring of BCG-associated adverse events.

BCGosis↗
PubMed2026

Patterns of Drug Resistance, Drug Resistance Conferring Mutations and Genomic DNA Methylation Revealed in Mycobacterium tuberculosis From South Africa.

Tuberculosis remains a major public health threat globally, with drug-resistant strains undermining treatment efficacy. We analyzed 126 Mycobacterium tuberculosis (M. tuberculosis) isolates with diverse drug resistance spectra and selected 35 for whole genome sequencing (WGS) using Illumina NextSeq, SMRT PacBio Onso and SMRT PacBio Revio sequencing platforms. The study aimed to characterize drug resistance profiles, compare short- and long-read sequencing performance, identify lineages among South African isolates, detect known drug resistance mutations and their lineage-specific patterns, and utilize long-read SMRT platforms for epigenetic profiling. Multiple drug resistance mutations were identified, some lineage-specific, and notably, East-African-Indian (EAI) Lineage 1 isolates often considered less pathogenic, showed significant potential for multidrug-resistance development, including higher fluoroquinolone resistance as compared to other lineages. Three DNA motifs with methylated adenines, namely CACGCaG, CtCCaG and GaTNNNNRtAC, were detected, with methylation patterns varying by lineage and strain due to mutations in the corresponding methyltransferases (MTases). A particularly notable finding was the stable maintenance of a genetic heterogeneity in the mamB MTase, performing methylation at CACGCaG motifs. These results highlight the combined role of genetic and epigenetic variation in M. tuberculosis adaptive evolution and underscore the value of integrating long-read sequencing into TB surveillance and research.

Mycobacterium tuberculosis↗
PubMed2026

Genomic insights into low-level rifampicin resistance mediated by borderline rpoB mutations in Mycobacterium tuberculosis: prevalence and phylogeny in Northeast China.

The emergence of low-level rifampicin (RIF) resistance in Mycobacterium tuberculosis poses a challenge to tuberculosis (TB) control, as it often leads to discordance between genotypic resistance detected by molecular assays (e.g., Xpert MTB/RIF) and phenotypic susceptibility in conventional drug susceptibility testing (DST). In this study, we performed whole-genome sequencing (WGS) on 17 clinical isolates from Changchun, Northeast China, which exhibited such discordance. All isolates harbored functional borderline mutations in the rpoB RRDR region, predominantly Leu452Pro and Leu430Pro (29% each), followed by His445Asn (18%). RIF minimum inhibitory concentration (MIC) values ranged from &#x2264;0.25 to 1.0 mg/L, confirming low-level resistance. Notably, 53% (9/17) of the isolates were co-resistant to fluoroquinolones and 24% (4/17) to isoniazid (INH). According to WHO classification, 59% (10/17) were pre-extensively drug-resistant TB (Pre-XDR-TB) or multidrug-resistant TB (MDR-TB). Phylogenetic analysis revealed that 94% (16/17) belonged to the East Asian Beijing lineage (Lineage 2.2.1), with no evidence of recent local transmission. These findings underscore the complexity of low-level RIF resistance and its frequent association with broader drug resistance in a dominant lineage, highlighting the need for integrating MIC and WGS into diagnostic algorithms to guide appropriate treatment and surveillance.IMPORTANCEThe accurate detection of RIF resistance is critical for the management of TB, yet standard phenotypic methods often fail to identify strains with low-level resistance conferred by borderline rpoB mutations. This study provides the first genomic characterization of such discordant isolates in Northeast China, revealing a high prevalence of co-resistance to other key drugs and a strong association with the locally dominant Beijing lineage. The findings emphasize that reliance on phenotypic DST alone may lead to underestimation of drug resistance and inappropriate treatment, potentially contributing to the emergence and spread of Pre-XDR-TB and MDR-TB. Incorporating MIC determination and WGS into routine diagnostics could enhance detection, inform tailored therapy, and improve surveillance of these clinically significant strains.

Mycobacterium tuberculosis↗