Background: Molecular diagnosis using the Xpert MTB/RIF Ultra assay enables rapid detection of Mycobacterium tuberculosis and rifampicin resistance. Objectives: This study evaluated the diagnostic performance and cost efficiency of a two-sample pooled testing approach compared with standard individual testing. Materials and methods: A cross-sectional diagnostic study was conducted among 3,504 presumptive TB patients. Sputum specimens were tested by Xpert Ultra individually, with those results serving as the reference standard, and in two-sample pools. The pooling protocol utilized 2.0 mL from each specimen to maximize load volume. Deconvolution, which required retesting both individual specimens, was mandatory for all positive and invalid pooled results. Performance metrics, RIF resistance concordance, and cost-effectiveness modeled on cartridge consumption and direct cost 550 Thai Baht per unit were compared. Results: The pooled method showed excellent concordance with the individual method, yielding 240 concordant positives and 3,249 concordant negatives, with no statistically significant difference in MTB detection (McNemar χ²=0.267, p=0.605). The pooled approach achieved a sensitivity of 96.39% (95% CI, 93.25-98.33) and a specificity of 99.82% (95% CI, 99.60-99.93). The assay maintained reliable detection of RIF resistance, indicating that pooling did not compromise molecular accuracy. In practice, 1,752 pooled runs were performed, with 105 (5.7%) error results and 246 positive pools requiring deconvolution. Including repeats and deconvolution, total cartridge use was 2,349 compared with 3,504 for individual testing, corresponding to an actual cost reduction of 32.96% (approx. 180,000 Thai Baht saved per 1,000 tests). Conclusion: Two-sample pooled Xpert Ultra testing demonstrated high diagnostic accuracy and doubled analytical throughput. Although deconvolution limited cost savings to approximately 33%, the strategy proved highly cost-effective and operationally feasible. This method offers a practical, scalable approach for optimizing molecular TB diagnostics and resource utilization, especially in low- to moderate-prevalence settings.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, remains the deadliest human pathogen. Treatment is hampered by drug resistance and the persistence of slow-growing or non-replicating populations. Rifampicin, a cornerstone of first-line therapy, inhibits transcription during promoter escape, but resistance mutations undermine efficacy and drive resistance spread. We revisited the transcription cycle as an antibacterial target by characterizing AAP-SO2, an RNA polymerase inhibitor with whole-cell activity against Mtb. AAP-SO2 slows the nucleotide addition cycle, disrupting elongation and termination. Rifampicin-resistant mutations impose fitness costs by perturbing the balance of these steps, creating exploitable weaknesses. Inhibition of transcription with AAP-SO2 reduced the evolution of rifampicin resistance and was especially effective against the most common resistant mutant. Combination treatment with rifampicin and AAP-SO2 synergistically killed non-replicating Mtb in an ex vivo rabbit granuloma model. These findings show that exploiting functional vulnerabilities of the transcription cycle can counter rifampicin resistance and improve clearance of recalcitrant Mtb populations.

Abdominal tuberculosis (TB) poses diagnostic difficulties due to its vague symptoms and low bacterial load. Culture, the gold standard, is limited by a prolonged turnaround time of up to eight weeks. Xpert MTB/RIF Ultra (Xpert Ultra), a rapid, automated molecular test, can detect Mycobacterium tuberculosis and rifampicin resistance in under two hours. This study evaluated the diagnostic accuracy of Xpert Ultra for abdominal TB using both culture and a composite reference standard (CRS), and assessed its agreement in rifampicin resistance detection with phenotypic and genotypic drug susceptibility testing (DST). A prospective observational study was conducted from September 2019 to March 2021 at a tertiary care centre in North India. Adults with clinical and radiological features of abdominal TB were enrolled. Relevant abdominal samples were collected and tested using smear, culture (BACTEC MGIT 960), Xpert Ultra, histopathology, and clinical response. Rifampicin resistance was confirmed using MGIT 960 and GenoType MTBDRplus. Of 176 eligible patients, 144 were enrolled, yielding 152 abdominal samples: lymph node aspirates (52%), biopsies (42.8%), pus/aspirates (3.3%), and ascitic/omental fluids (2%). Xpert Ultra showed 84% diagnostic accuracy against CRS and 75% against culture. Rifampicin resistance detection showed 100% concordance with both phenotypic and genotypic DST. Xpert Ultra offers high diagnostic accuracy and excellent concordance in rifampicin resistance detection for abdominal TB. Its speed and reliability make it a valuable diagnostic tool in high-burden settings.

Introduction: Tuberculosis (TB), primarily caused by Mycobacterium tuberculosis, remains a major global health threat, further intensified by the emergence of drug-resistant strains. Rapid molecular diagnostic tools such as the GeneXpert MTB/RIF assay have significantly improved early detection of TB and identification of rifampicin resistance, offering faster and more accurate results than conventional methods. Aim: To compare smear microscopy with the GeneXpert MTB/ RIF assay for the rapid detection of Mycobacterium tuberculosis and simultaneous detection of rifampicin resistance. Materials and Methods: A cross-sectional study was conducted over one year (September 2022 to September 2023) at Geetanjali Medical College and Hospital, Udaipur, Rajasthan, India. A total of 314 clinical specimens (233 pulmonary and 81 extrapulmonary) were tested using both Ziehl–Neelsen (ZN) staining and the GeneXpert MTB/RIF assay. Specimens were processed according to standard protocols for both diagnostic methods. Demographic details such as age and gender were recorded. Statistical analysis was performed using the Chi-square test, with a p-value <0.001 was considered statistically significant. Results: Among the 314 samples, males constituted the majority (204; 65%), and the most affected age group was 51- 70 years (133; 42.36%). Using GeneXpert, 87 (27.7%) samples tested positive for M. tuberculosis, whereas smear microscopy detected only 60 (19.1%). Among pulmonary samples (n=233), GeneXpert detected MTB in 81 (35%) cases, while smear microscopy detected 58 (25%). Among extrapulmonary samples (n=81), GeneXpert detected MTB in 6 (7%) cases, whereas smear microscopy detected only 1 (1%). Of the total samples, 59 (19%) were positive by both methods, 28 (9%) were positive only by GeneXpert, and 1 (0.3%) sample was positive only by smear. GeneXpert demonstrated a 6% higher diagnostic yield for extrapulmonary TB compared with smear microscopy. Rifampicin resistance was identified in 10 (11%) of the 87 GeneXpert-positive samples, indicating the presence of potential multidrug-resistant TB. Conclusion: The GeneXpert MTB/RIF assay is more sensitive than smear microscopy for detecting MTB in both pulmonary and extrapulmonary samples. It enables rapid diagnosis and simultaneous detection of rifampicin resistance, which is essential for the timely initiation of appropriate therapy.

Xpert MTB/RIF probe reactivity to non-tuberculosis mycobacteria cultured in MGIT broth samples.

A 3-month clofazimine-rifapentine-containing regimen for drug-susceptible tuberculosis versus standard of care (Clo-Fast): a randomised, open-label, phase 2c clinical trial.

High rates of acquired resistance to fluoroquinolones, bedaquiline, and linezolid in patients failing treatment against drug-resistant tuberculosis in the Republic of Moldova.

Bacillus velezensis SF-10 modulates the rhizosphere core microbiome by stimulating the probiotic community to control tomato wilt disease.

This study aimed to evaluate the efficacy of a dry extract solution of Gratiola officinalis L. against reference and clinical strains of M. tuberculosis, including multidrug-resistant (MDR) strains. Material and Methods — The extract of Gratiola officinalis L. was prepared using the authors’ original technique. Its effects were assessed in vitro against a reference strain of M. tuberculosis that was clinically sensitive, a clinical MDR M. tuberculosis strain (with nucleotide substitutions in codon 531 of the rpoB gene [rifampicin resistance] and codon 315 of the katG gene [isoniazid resistance]), and additional sensitive and reference strains. Results — Minimum inhibitory concentrations (MICs) of the Gratiola officinalis L. extract were determined. At 14 mg/mL, the extract exerted a complete bactericidal effect against the M. tuberculosis reference strain. At 26.6 mg/mL, growth of the clinically sensitive M. tuberculosis strain (sensitive to isoniazid, rifampicin, ethambutol, and streptomycin) was fully suppressed. At 53.1 mg/mL, growth of the MDR M. tuberculosis strain (resistant to rifampicin, isoniazid, streptomycin, and ethambutol) was fully suppressed. Conclusion — The Gratiola officinalis L. extract demonstrated high bacteriostatic and bactericidal activity against Mycobacterium tuberculosis. This activity was evident against a reference strain (assessed via colony growth dynamics) and, using the serial dilution method, against a sensitive clinical strain and an MDR strain resistant to isoniazid and rifampicin.

Utility of the Urine GeneXpert MTB/RIF Assay for the Diagnosis of Mycobacterium Tuberculosis and Assessing Rifampicin Resistance in Sputum-scarce HIV Infected Individuals: A Scoping Review

Drug-resistant tuberculosis (DR-TB) continues to pose a threat to public health worldwide. Rifampicin (RIF) resistance is mostly caused by mutations in the rpoB gene, which codes for the β -subunit of RNA polymerase and is also an important surrogate marker for multidrug-resistant tuberculosis (MDR). This study aimed to detect the rpoB gene mutations associated with RIF resistance and identify the risk factors for MDR/ RIF resistance patterns in individuals infected with pulmonary TB. A facility-based cross-sectional study was conducted at selected TB treatment center hospitals (Felegehiwot, Debre-tabor, University of Gondar, Debark, and Metema hospitals) from June to December 2023 in the Northwestern Amhara regional state of Ethiopia. A total of 206 pulmonary TB patient's sputum samples were included. The study participants' Socio-demographics and clinical and behavioral characteristics were collected through semi-structured questionnaires. Then all GeneXpert® MTB/RI-positive sputum specimens of bacterial isolates were culturedin a conventional egg-based solid Lowenstein-Jensen (LJ) medium. MTB Genomic DNA was extracted using GenoLyze Kit. The allele-specific Amplification Refractory Mutation System Polymerase Chain Reaction (ARMS PCR) approach was employed on whole DNA samples from 206 Culture positive isolates using three distinct codon-specific primers (D516V, H526Y, and S531L). An isolate is classified as RR-TB if it carries any mutation in the rpoB gene. Most Single nucleotide polymorphism (SNP) mutations were observed on rpoB S531L 19 (9.2%). Of 206 confirmed clinical isolates, 21 (10.2%) were RIF Resistant, while the remaining 185 (89.8%) were RIF susceptible. Before TB treatment history (AOR = 4.27, CI 1.29-14.20, p = 0.02), and Window opening practice of patients (AOR = 6.17, CI 1.22-31.29, p = 0.03) were significantly associated with RR-TB development. The prevalence of RR (RIF Resistant) -TB among TB-confirmed cases was 21 (10.2%). This implies that RR-TB is a serious health problem in the study population. The S531L was the most common mutation conferring resistance to RIF. Not applicable.

The World Health Organization (WHO) recommends testing stool with Xpert MTB/RIF Ultra (Xpert-Ultra) as an initial diagnostic test for detection of tuberculosis (TB) and rifampicin resistance in children. The option of testing stool on the Truenat platform could benefit children and their caregivers in remote areas where GeneXpert is not available. We report the results of research to validate a protocol for processing and testing stool on Truenat using an adapted Simple One Step (SOS) stool method in routine settings in Nigeria. Stool specimens from children with presumptive TB were tested with Truenat MTB Plus (Truenat) and Xpert-Ultra using a comparative cross-sectional study design. A total of 510 children were enrolled and submitted a stool specimen. Of these, 482 (94.5%) had valid results on both platforms, 28 (5.8%) with MTB detected and 454 MTB not detected. Of the 28 with MTB detected, eight were detected on both platforms, seven on Truenat only, and 13 on Xpert-Ultra only. The concordance rate between Truenat and Xpert-Ultra was high at 95.8%. Significantly more non-determinate (error/invalid) results were observed with Truenat compared to Xpert-Ultra (4.7% versus 1.2% respectively; P < 0.001). Truenat can accurately detect MTB using 100 mg of stool with an adapted SOS stool protocol. Further optimizing extraction methods could reduce the frequency of non-determinate results. These results hold promise for expansion of childhood TB diagnosis services to hard-to-reach areas with limited infrastructure that are suitable for Truenat placement. Global scale-up of these alternative testing approaches will be necessary to close the gap in childhood TB case finding.IMPORTANCEFollowing WHO recommendations, high TB burden countries have commenced testing stool on GeneXpert to improve TB case finding among children and others who may not easily produce sputum. In previous work, we adapted the Simple One Step (SOS) stool method for GeneXpert for the Truenat platform. In the present study, we validated the revised processing method under routine conditions in health care facilities in Nigeria. We tested stool from 510 children with presumptive TB on both the GeneXpert and Truenat platforms across a variety of health care settings. Concordance between Truenat and Xpert MTB detection was high at 95.8%, confirming stool can be tested on Truenat in routine services. Our study demonstrates that TB diagnostic testing for children can be provided in peripheral health facilities located in remote geographic areas with limited infrastructure where Truenat can be placed. Global scale-up will help further close the gap in childhood TB case finding.

We report the discovery of 4'-thiothymidine (4'sT), a sulfur-containing nucleoside antibiotic produced by the nematode symbiont Photorhabdus asymbiotica. 4'sT exhibits antibacterial activity against Escherichia coli, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. The production of 4'sT was increased in rifampicin-resistant mutants. Heterologous expression of the radical SAM enzyme-containing gene cluster confirmed its biosynthetic origin. Resistant mutants mapped to tdk, which encodes thymidine kinase in the thymidine salvage pathway. Mechanistic studies revealed that 4'sT is a prodrug activated via the thymidine salvage pathway and incorporated into DNA, where it inhibits replication and triggers the SOS response. Clinical E. coli isolates were resistant, but inhibition of de novo thymidylate synthesis with trimethoprim rendered them highly susceptible, indicating that strain-selective activity is governed by differences in thymidine metabolism. These findings highlight the role of metabolic context in determining antibiotic selectivity and demonstrate the utility of rifampicin resistance for assessing silent biosynthetic gene clusters to discover new antimicrobial compounds.IMPORTANCEIntroducing novel antibiotics is essential to counter the spread of drug-resistant pathogens. Here, we report the discovery of 4'-thiothymidine (4'sT), a nucleoside antibiotic from the nematode symbiont Photorhabdus asymbiotica, identified through activation of a silent biosynthetic gene cluster. 4'sT features an unusual 4'-thiosugar moiety. We identified its biosynthetic gene cluster, including a radical SAM enzyme presumably involved in sulfur incorporation. 4'sT exhibits strain-selective activity that is governed by differences in thymidine metabolism rather than variations in the molecular target. These findings expand our knowledge of antibiotics with unusual selective activity.

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.

BackgroundTuberculous spondylitis (TS, Pott’s disease) accounts for up to 50% of extrapulmonary TB cases and presents a significant clinical challenge due to destructive spinal involvement, frequent neurological complications and difficulties in detecting Mycobacterium tuberculosis (Mtb) in bone tissue. Molecular diagnostics have improved pathogen detection; however, cases with concomitant HIV andHCV infections remain problematic. These conditions, marked by immunosuppression, systemic inflammation, endothelial dysfunction and altered host-pathogen interactions, are linked to atypical TS progression, rapid disease course and reduced treatment efficacy. Analysing Mtb genome mutations is critical for rational chemotherapy selection, especially when rapid drug susceptibility results are unavailable.ObjectivesTo define the mutation patterns and frequency associated with resistance to isoniazid, rifampicin, fluoroquinolones and aminoglycosides in Mtb isolated from bone tissue of TS patients coinfected with HIV and HCV, and to justify including this group in the high-risk category for MDR TB.MethodsA retrospective and prospective study included 300 patients with a confirmed diagnosis of TS, who were observed from 2016 to 2021 in a specialized clinic. Patients were grouped as follows: 20% without viral infections, 8% infected only with HCV, 6% with HIV alone and 66% with combined HIV+HCV infection. Surgical bone specimens were analysed using microscopy, culture on solid media and molecular-genetic techniques (PCR/PCR reverse hybridization). When detecting MBT DNA in amounts insufficient for identifying mutations, a repeat study was conducted. Among 324 PCR-positive cases, 267 samples contained sufficient DNA for mutation detection. Key genetic loci examined included rpoB, katG, gyrA/gyrB, rrs and eis. Results were correlated with patients’ viral infection status.ResultsPCR showed the highest sensitivity, detecting Mtb DNA in 83% of bone samples, compared to 28% with microscopy and 24% with culture. Resistance-related mutations were common: rpoB mutations (rifampicin resistance) were found in 72.3% of strains, predominantly the S531L substitution (86.5%); katG mutations (isoniazid resistance) in 79.4%, mainly S315T (96.2%). Fluoroquinolone resistance (gyrA/gyrB) was present in 31.6% of samples, aminoglycoside resistance (rrs, eis) in 51.6%. HIV infection increased the risk of MDR Mtb 4.16-fold (95% CI 2.11–7.67) and HCV 2.29-fold (95% CI 1.36–4.86). Patients with dual HIV+HCV infection showed the lowest proportion of drug-susceptible strains and the highest frequency of pre- XDR Mtb. Overall, the likelihood of detecting at least MDR Mtb in this subgroup was nearly four times higher than in patients without viral infections.ConclusionMolecular diagnostics outperform traditional bacteriology in detecting the TS pathogen and its drug resistance mutations. The high frequency of rpoB and katG mutations, combined with changes in gyrA/gyrB, rrs and eis, confirms a significant risk of MDR/pre-XDR Mtb in TS patients with HIV/HCV coinfections. Given the time required for results and low culture sensitivity, personalized therapy based on molecular-genetic analysis of bone specimens is optimal. When such data are unavailable, empirical treatment targeting MDR/pre-XDR Mtb is justified. Molecular diagnostics thus serve both as a rapid detection tool and a key to stratifying patients by MDR Mtb risk for informed personalized or empirical treatment decisions.

Tuberculosis (TB) remains a significant global public health challenge, particularly in low- and middle-income countries. The emergence of rifampicin-resistant tuberculosis further complicates control efforts. Despite national efforts, there are limited data on the prevalence of Mycobacterium tuberculosis and their rifampicin resistance in Ghana, especially at the facility level. This study aimed to determine the prevalence of Mycobacterium tuberculosis and rifampicin resistance among presumptive tuberculosis cases at the Ho Teaching Hospital over a three-year period. The study used a retrospective design and collected secondary data from the Microbiology Laboratory Unit of Ho Teaching Hospital between 2022 and 2024. Data on patient demographics, Mycobacterium tuberculosis detection, and rifampicin resistance were retrieved and analysed using descriptive and inferential statistics. Mycobacterium tuberculosis and rifampicin resistance were identified using the GeneXpert MTB/RIF assay. Statistical analyses were performed using STATA version 15 with statistical significance set at p < 0.05. Out of 2,225 presumptive tuberculosis cases, 203 tested positive for the infection, resulting in an overall prevalence of 9.1% (95% CI: 7.9–10.4). The prevalence was significantly higher among males (12.4%) compared to females (5.7%), and highest among young adults aged 18–24 years (12.8%). Of the 166 Mycobacterium tuberculosis-positive cases tested for rifampicin susceptibility, 3 (1.8%) were resistant. Although rifampicin resistance was more common among females and adults, the differences were not statistically significant. Although the detection rate of rifampicin resistance among newly diagnosed Mycobacterium tuberculosis cases was low (1.8%), it remains a significant public health concern, hence the need for enhancing surveillance systems and prioritising early detection strategies.

Undetected rifampicin-resistant tuberculosis associated with rpoB I491F and V170F mutations in Botswana: Diagnostic implications.

"Comparative study of gastric aspirate and bronchoalveolar lavage smear with CBNAAT examination for diagnosis of pulmonary tuberculosis in sputum smear negative suspected patients".

A retrospective analysis of pulmonary and extra-pulmonary tuberculosis drug resistance in Punjab, Pakistan (2012-2022).

Direct targeted next-generation sequencing for diagnosis of drug-resistant tuberculosis from clinical samples - An update.