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Background The World Health Organization–approved Xpert MTB/XDR test detects Mycobacterium tuberculosis and resistance to isoniazid, fluoroquinolones, ethionamide, and injectable drugs directly in specimens. This pragmatic, laboratory-based study assessed the diagnostic accuracy and feasibility of a reflex testing approach, where Xpert MTB/XDR was performed on residual specimens previously processed for Xpert MTB/RIF Ultra. Methods Routine respiratory specimens, processed for Xpert MTB/RIF Ultra, were stored in sample reagent buffer at 2°C–8°C. If rifampicin resistant, the residual specimen was assessed for adequate volume (≥2 mL) and tested with Xpert MTB/XDR, with storage time recorded. A second specimen was used for routine and reference standard testing (culture and sequencing). Results Specimens (99% sputum) from 763 participants submitted to 2 large routine laboratories were included. Xpert MTB/XDR yielded valid resistance detection results in 639 (84%), compared with 507 (66%) for routine testing (difference [95% CI], 18% [13%–22%]). The median turnaround time for results was 23 hours for Xpert MTB/XDR and 15 days for routine testing. While 748 specimens (98%) were ≥2 mL, only 102 (13%) were stored for ≤4 hours. By the reference standard, 284 of 394 (72%) were isoniazid resistant, and 57 of 380 (15%) were fluroquinolone resistant. The sensitivities of Xpert MTB/XDR were 94% (95% CI, 91%–97%) for isoniazid and 91% (81%–97%) for fluoroquinolone resistance detection. The specificities were 98% (94%–100%) and 100% (98%–100%), respectively. Conclusions Xpert MTB/XDR performed favorably compared with the reference, and the reflex testing approach increased results availability over routine testing, while dramatically decreasing turnaround time from weeks to hours. Laboratory workflow precluded testing within the manufacturer-recommended 4-hour storage time, but longer storage did not appear detrimental. [ABSTRACT FROM AUTHOR]

Several studies have shown increased in vitro cytokine responses to non-related pathogens after Bacillus Calmette-Guérin (BCG) vaccination. A total of 158 infants (80 BCG administered within 7 days of birth; 78 controls) were bled 4 days post-randomization, and at age 3 and 13 months. Geometric mean concentrations of IL-1β, TNF-α, IL-6 (24 h stimulation) and IFN-γ, IL-10, IL-17, IL-22 (96 h stimulation) in response to in vitro stimulation with RPMI, LPS, PHA, Escherichia coli, Streptococcus pneumoniae, Candida albicans and BCG were compared among BCG vaccinated children and controls. BCG vaccination did not affect in vitro cytokine production, except IFN-γ and IL-22 response to BCG. Stratifying for 'age at randomization' we found a potentiating effect of BCG on cytokine production (TNF-α, IL-6, IL-10) in the 4 days post randomization stimulations, among children who were vaccinated at age 2-7 days versus age 0-1 days. BCG vaccination did not potentiate cytokine production to non-BCG antigens. At 4 days post randomization, BCG was associated with higher cytokine production in the later randomized children. [ABSTRACT FROM AUTHOR]

This study explored Mycobacterium tuberculosis (MTB) growth from tongue swabs, both experimentally infected after sampling from healthy controls, or sampled from patients with smear-microscopy confirmed pulmonary tuberculosis (PTB). For both, we evaluated the performance of NALC-NaOH/MGIT960 (MGIT), Kudoh-Ogawa (KO), and cetylpyridinium chloride-Löwenstein-Jensen (CPC/LJ) culture processing methods. Experimentally spiked swabs from 20 participants exhibited 94.4% MTB growth when inoculated within 7 days of CPC exposure, declining significantly after 14-21 days (p<0.00001). KO-processed specimens showed 100% MTB growth, with a non-significant reduction after storage (94.1%; p=0.21), and all spiked swabs yielded growth in MGIT. In the field evaluation on 99 PTB patients, MGIT isolated MTB from 89% of tongue swabs, with an 8% contamination rate, compared to 99% MGIT positivity from sputum. Solid media had lower positivity, 62% for KO and 49% for CPC/LJ, suggesting MGIT as optimal for growing MTB from tongue swabs. Further testing of presumptive PTB patients is recommended., Competing Interests: Declaration of competing interest None of the authors have a competing interest in this research to declare., (Copyright © 2024. Published by Elsevier Inc.)

Background: Truenat MTB Plus (MTB Plus) and MTB Ultima (Ultima) are World Health Organization-endorsed low-complexity tuberculosis (TB) tests, however, performance data are scarce. Methods: Adults (≥18 years; n=498) self-presenting with symptoms to primary care clinics in Cape Town, South Africa (19/02/2016-22/02/2023) provided sputa. We evaluated the accuracy of MTB Plus and Ultima, with Xpert MTB/RIF Ultra (Ultra) as a comparator, vs. a single culture (TB reference standard) or MTBDR plus on an isolate (rifampicin susceptibility reference standard). Results: The proportion of MTB Plus and Ultima unsuccessful results was 20% (95% confidence interval 17, 23) and 14 (11, 16), respectively, with ≥half resolving upon retesting the same eluate. In a three-way analysis, MTB Plus, Ultima and Ultra had sensitivities of 84% (78, 88), 90% (85, 93), and 92% (87, 95), and specificities of 95% (92, 97), 85% (80, 88) and 95% (92, 97) for TB. The proportion of unsuccessful results for MTB-RIF Dx done the same day as DNA extraction was 9% (3, 16; MTB Plus-positives) and 18% (10, 26; Ultima-positives) [if after day-of-extraction, these were 27% (18, 35) and 44% (35, 51)]. Same-day rifampicin susceptibility testing was often unsuccessful in samples with "very low" load [73% (58, 89) MTB Plus, 75% (65, 86) Ultima] but had 100% (40, 100) sensitivity and 99% (96, 100) specificity (for both MTB Plus- or Ultima-positive DNA). Lot variation in unsuccessful and false-positive results was observed. Conclusion: Ultima showed comparable sensitivity to Ultra but specificity, lot variation, and, like MTB-RIF Dx, unsuccessful result rates were suboptimal. Funding: European & Developing Countries Clinical Trials Partnership, and South African Medical Research Council.

Drug susceptibility testing (DST) is essential for effectively starting people on effective tuberculosis (TB) regimens. No accuracy data exists for the new high-throughput LiquidArray MTB-XDR (LA-XDR) test, which detects Mycobacterium tuberculosis complex (MTBC) and susceptibility to the fluoroquinolones, amikacin, ethambutol, and linezolid (the latter two drugs have no rapid molecular DSTs available). We enrolled (n=720) people with presumptive TB who provided two sputa for Xpert MTB/RIF Ultra and culture (MTBC reference standard). Phenotypic DST and Sanger sequencing served as a composite reference standard. Manual FluoroLyse and automated GenoXtract-fleXT (fleXT) DNA extraction methods were compared. For MTBC, LA-XDR using fleXT-extracted or FluoroLyse-extracted DNA had similar sensitivities (85-87%; which improved upon eluate retesting) and specificities (99%). Drug susceptibility sensitivities varied: 94% (86, 98) for fluoroquinolones, 64% (45, 80) for amikacin, and 88% (79, 93) for ethambutol (specificities 97-100%). LA-XDR detected 86% (6/7) phenotypically resistant linezolid isolates. LA-XDR with fleXT had indeterminate proportions of 8% (21/251) for fluoroquinolones, 1% (2/251) for ethambutol, 25% (63/251) for amikacin, and 37% (93/251) for linezolid. In a hypothetical population of 100 smear-negative fluoroquinolones-resistant cases, 24% (24/100) could be missed due to an unsuccessful result (1 fleXT error and, for LA-XDR, 2 invalid results, 15 MTBC-negative, 6 fluoroquinolone-indeterminate, 1 false-susceptible). LA-XDR met the minimum WHO target product profile for a next-generation sputum-based moderate complexity DST with high sensitivity for fluoroquinolones and ethambutol resistance, moderate sensitivity for amikacin resistance, and promise for linezolid resistance, for which more data are needed. Improved MTBC detection would reduce missed resistance., Competing Interests: Disclosure Statement The authors declare no competing interests.

Drug development for tuberculosis is hindered by the methodological limitations in the definitions of patient outcomes, particularly the slow organism growth and difficulty in obtaining suitable and representative samples throughout the treatment. We developed target product profiles for biomarker assays suitable for early-phase and late-phase clinical drug trials by consulting subject-matter experts on the desirable performance and operational characteristics of such assays for monitoring of tuberculosis treatment in drug trials. Minimal and optimal criteria were defined for scope, intended use, pricing, performance, and operational characteristics of the biomarkers. Early-stage trial assays should accurately quantify the number of viable bacilli, whereas late-stage trial assays should match the number, predict relapse-free cure, and replace culture conversion endpoints. The operational criteria reflect the infrastructure and resources available for drug trials. The effective tools should define the sterilising activity of the drug and lower the probability of treatment failure or relapse in people with tuberculosis. The target product profiles outlined in this Review should guide and de-risk the development of biomarker-based assays suitable for phase 2 and 3 clinical drug trials., Competing Interests: Declaration of interests SHG and WS developed the tuberculosis bacterial load assay and are collaborating with LifeArc to bring the assay to market. IK is participating in studies evaluating the feasibility of the TB22 signature developed by her research group as a treatment monitoring tool. JH holds patent shares for the TB22 signature. LJS is an employee of Johnson & Johnson and reports employee stock and stock options from Johnson & Johnson. NHe is an employee of the University Hospital (LMU) and reports grants to the institute from Beckman Coulter, European and Developing Countries Clinical Trials Partnership (EDCTP; EU Horizon 2020), and DZIF for research and evaluation of new tuberculosis diagnostics. NHi reports grants or contract and consulting fees from Otsuka Novel Product Muenich (ONPG) and having detailed information on the LAM sputum biomarker. RvC reports INtegrative omics and aspirin response to define the ThERapeutiC targEts for Pediatric TBM grant (R01AI165721) from the National Institutes of Health and participating in the Data Safety Monitoring Board for three randomised controlled trials focused on tuberculosis meningitis. SBG, MK, MR, and JBT are employees of FIND, the global alliance for diagnostics, a not-for-profit foundation that supports the evaluation of publicly prioritised tuberculosis assays and implementation of WHO-approved (guidance and prequalification) assays using donor grants. FIND has product evaluation agreements with several private-sector companies that design diagnostics for tuberculosis and other diseases. These agreements strictly define FIND’s independence and neutrality with regard to these private-sector companies. SBG and MR are also members of the New Diagnostics Working Group (NDWG) Stop TB Partnership. CL is a patent holder on biomarkers for monitoring of tuberculosis treatment. All other authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Tuberculosis (TB) remains a global public health threat, and the development of rapid and precise diagnostic tools is the key to enabling the early start of treatment, monitoring response to treatment, and preventing the spread of the disease., Objectives: An overview of recent progress in host- and pathogen-based TB diagnostics., Sources: We conducted a PubMed search of recent relevant articles and guidelines on TB screening and diagnosis., Content: An overview of currently used methods and perspectives in the following areas of TB diagnostics is provided: immune-based diagnostics, X-ray, clinical symptoms and scores, cough detection, culture of Mycobacterium tuberculosis and identifying its resistance profile using phenotypic and genotypic methods, including next-generation sequencing, sputum- and non-sputum-based molecular diagnosis of TB and monitoring of response to treatment., Implications: A brief overview of the most relevant advances and changes in international guidelines regarding screening and diagnosing TB is provided in this review. It aims at reviewing all relevant areas of diagnostics, including both pathogen- and host-based methods., (Copyright © 2023 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Introduction: Tuberculosis (TB) is an important cause of morbidity and mortality among people living with HIV (PLHIV). Current WHO-recommended strategies for diagnosing TB among hospitalized PLHIV rely on symptom screening and disease severity to assess eligibility for urine lipoarabinomannan lateral flow (LF-LAM) and molecular testing. Despite these recommendations, autopsy studies show a large burden of undiagnosed TB among admitted PLHIV. The EXULTANT trial aims to assess the impact of an expanded screening strategy using three specimens (sputum, stool, and urine) for TB diagnosis among PLHIV admitted to hospitals in two high HIV and TB burden African countries., Methods: This is a multicenter, pragmatic, individually randomized controlled trial conducted across eleven hospitals in Tanzania and Mozambique. Participants in the intervention arm will be tested with Xpert MTB/RIF Ultra® from expectorated sputum, stool, and urine samples, with additional urine LF-LAM testing in the first 24 h after hospital admission, irrespective of the presence of the symptoms. The control arm will implement the WHO standard of care recommendations. Hospitalized adults (≥ 18 years) with a confirmed HIV-diagnosis, irrespective of antiretroviral (ART) therapy status or presence of TB symptoms will be assessed for eligibility at admission. Patients with a pre-existing TB diagnosis, those receiving anti-tuberculosis therapy or tuberculosis preventive treatment in the 6 months prior to enrolment, and those transferred from other hospitals will not be eligible. Also, participants admitted for traumatic reasons such as acute abdomen, maternal conditions, scheduled surgery, having a positive SARS-CoV2 test will be ineligible. The primary endpoint is the proportion of participants with microbiologically confirmed TB starting treatment within 3 days of enrolment., Discussion: The EXULTANT trial investigates rapid implementation after admission of a new diagnostic algorithm using Xpert MTB/RIF Ultra® in several non-invasive specimens, in addition to LF-LAM, in hospitalized PLHIV regardless of TB symptoms. This enhanced strategy is anticipated to detect frequently missed TB cases in this population and is being evaluated as an implementable and scalable intervention., Trial Registration: Trial reference number: NCT04568967 (ClinicalTrials.gov) registered on 2020-09-29., (© 2024. The Author(s).)

This study evaluated the performance of cobas MTB and cobas MTB-RIF/INH for the diagnosis of tuberculosis and detection of rifampicin (RIF) and isoniazid (INH) resistance. Adults presenting with pulmonary tuberculosis symptoms were recruited in South Africa, Moldova, and India. Performance of cobas MTB was assessed against culture, whereas cobas MTB-RIF/INH was assessed using phenotypic drug susceptibility testing and whole-genome sequencing as composite reference standards. Xpert MTB/RIF (Xpert) or Xpert MTB/RIF Ultra (Ultra) was used as a comparator. The overall sensitivity and specificity of cobas MTB were 95% (95% CI, 93%-96%) and 96% (95% CI, 95%-97%). Among smear-negatives, the sensitivity of cobas MTB was 75% (95% CI, 66%-83%). Among participants tested with both cobas MTB and Xpert, sensitivity was 96% (95% CI, 94%-97%) for cobas MTB and 95% (95% CI, 93%-97%) for Xpert. Among participants tested with both cobas MTB and Ultra, sensitivity was 88% (95% CI, 81%-92%) for cobas MTB and 89% (95% CI, 83%-93%) for Ultra. Sensitivity and specificity of cobas MTB-RIF/INH for RIF and INH detection were 90% (95% CI, 84%-94%) and 100% (95% CI, 99%-100%), and 89% (95% CI, 84%-93%) and 99.5% (95% CI, 98%-100%), respectively. The cobas MTB and cobas MTB-RIF/INH assays exhibited high performance in a diverse population and present a suitable option for molecular detection of tuberculosis and RIF and INH resistance., Competing Interests: Disclosure Statement M.d.V., A.P.-N., A.M., S.O., P.N., and M.R. are employees of FIND. S.G.S., J.M., I.K., and A.P. were employed by FIND at the time of the study. S.S., S.S.C., and K.D. are employees of FIND, India. F.P.M. changed employment from Research Center Borstel (Borstel, Germany) to Roche (Rotkreuz, Switzerland) after the completion of the study in July 2022. (A.M., S.O., P.N., S.S., S.S.C., J.M., I.K., and A.P. are Study Group members.), (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: CD4 measurement is pivotal in the management of advanced HIV disease. VISITECT® CD4 Advanced Disease (AccuBio Limited, Alva, UK; VISITECT) is an instrument-free, point-of-care, semi-quantitative test allowing visual identification of a CD4 ≤200 cells/µl, or >200 cells/µl from finger-prick or venous blood., Methods: As part of a diagnostic accuracy study of FUJIFILM SILVAMP TB LAM (clinicaltrials.gov: NCT04089423), people living with HIV of ≥18 years old were prospectively recruited in seven countries from outpatient departments if a tuberculosis symptom was present, and from inpatient departments. Participants provided venous blood for CD4 measurement using flow cytometry (reference standard) and finger-prick blood for VISITECT (index text), performed at point-of-care. Sensitivity, specificity, and positive and negative predictive values of VISITECT to determine a CD4 ≤200 cells/µl were evaluated., Results: Among 1604 participants, the median flow cytometry CD4 was 367 (IQR 128-626) cells/µl and 521 (32.5%) had a CD4 ≤200 cells/µl. VISITECT sensitivity was 92.7% (483/521, 95% CI 90.1-94.7%) and specificity was 61.4% (665/1083, 95% CI 58.4-64.3%). For participants with a CD4 between 0-100, 101-200, 201-300, 301-500, and >500 cells/µl, VISITECT misclassified 4.5% (95% CI 2.5-7.2%), 12.5 (95% CI 8.0-18.2%), 74.1% (95% CI 67.0-80.5%), 48.0% (95% CI 42.5-53.6%), and 22.6% (95% CI 19.3-26.3%), respectively., Conclusions: VISITECT's sensitivity, but not specificity, met the World Health Organization's minimal sensitivity and specificity threshold of 80% for point-of-care CD4 tests. VISITECT's quality needs to be assessed and its accuracy optimized. VISITECT´s utility as CD4 triage test should be investigated., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)