Seda Yerlikaya

Background: Translation of tuberculosis (TB) biomarker and diagnostic research into tools that improve patient and public health outcomes has been slow, partly because no internationally agreed framework exists defining the use cases that new biomarkers and tests should address. We aimed to identify, validate, and prioritise use cases for TB biomarkers and tests across Mycobacterium tuberculosis ( Mtb ) infection, TB disease, TB treatment, and post-TB care, through an international consensus process. Methods and Findings: We conducted a scoping review of the literature, guidelines, and target product profiles (24 documents; 69 candidate use cases consolidated to 13), followed by a hybrid RAND/UCLA modified Delphi consensus process involving 185 identified interest-holders, including clinicians, researchers, diagnostic developers, industry, funders, civil society, national TB programmes, and policymakers (including WHO representatives). Interest-holders completed an online survey rating agreement with each use case, and attended a consensus meeting to discuss use cases with less than 80% agreement, followed by a further validation meeting. Eleven use cases were retained across four TB care pathway stages: three for Mtb infection, four for disease detection, three for treatment optimisation, and one for post-TB care. Highest-priority use cases were detection of drug-resistant TB, prediction of progression from infection to disease, identification of current Mtb infection, and improved diagnosis of active TB disease. Conclusions: This consensus exercise provides the first comprehensive, prioritised framework of use cases for TB biomarkers and tests, spanning the full care pathway. These eleven priority use cases can guide investment, focus biomarker discovery, and inform future target product profiles, funding calls, and policy development. Applying the framework to the current biomarker pipeline is a key next step to address gaps between innovation and priority needs.

Sample preparation remains a barrier for decentralized, swab-based molecular testing of tuberculosis (TB). Extraction-free workflows offer a simpler alternative, but systematic benchmarking against standard methods is lacking. We evaluated five novel lysis devices, BLINK Shaker Prototype, nPOC-BB, SPS-1, Truelyse, and Thermolyse, against a heat- and bead-beating reference method using contrived M. tuberculosis-spiked tongue and sputum swabs. The primary outcome was lysis efficiency, measured as the relative DNA recovery compared with the reference workflow. Secondary outcomes included nuclease inactivation, biosafety, and usability. In the reference buffer, lysis efficiencies ranged from 51-63% to 95-154% on tongue swabs and 12-54% to 280-644% on sputum swabs across the five devices. In proprietary buffers, performance varied more widely, with lysis efficiencies of 2-4% to 64-80% on tongue swabs and 1% to 94-398% on sputum swabs. Complete biosafety inactivation was achieved by three devices; two showed residual growth (<0.02%). Lysis efficiency of several devices met or exceeded the reference, supporting the feasibility of extraction-free workflows for TB diagnosis, with further optimization of buffer compatibility and biosafety profiles expected to enhance performance.

Background: Biomarkers have been widely proposed as having potential to improve tuberculosis (TB) detection, prediction of progression, and treatment monitoring, but evidence is fragmented and it is unclear which biomarkers have the strongest evidence base. We undertook an umbrella review to synthesise this evidence.Methods: This umbrella review (PROSPERO CRD420251230142) evaluated systematic reviews of biomarkers for three broad TB use-cases: detection of TB disease, prediction of progression to TB disease, and TB treatment monitoring and optimisation. We searched MEDLINE, Web of Science, Global Index Medicus and Cochrane Library for systematic reviews of host- or pathogen-derived biomarkers from January 2018 and appraised review quality using AMSTAR-2. For each biomarker class and use case we summarised the technology readiness, strength of evidence, diagnostic accuracy, extent of independent validation, operational characteristics, and unmet need addressed. We synthesised evidence for each biomarker class qualitatively and quantitatively, against a pre-specified biomarker prioritisation framework.Findings: Forty-five systematic reviews met inclusion criteria: 39 on TB detection, 7 on progression and 6 on treatment monitoring. Nine classes of host- or pathogen-based biomarkers were identified, most commonly host protein, host transcriptomic and host miRNA. For TB detection, evidence was strongest for host protein, host transcriptomic, and pathogen antigen classes, while the immune phenotype, exhaled breath and pathogen DNA/RNA classes were earlier in development, lacking independent validation. Across use cases, estimates of diagnostic accuracy varied widely and were often lower in higher quality studies with clinically relevant populations. In high-quality studies no biomarkers consistently met WHO target product profile thresholds. For TB progression, the strongest evidence was for host transcriptomic, which predicted the short-term risk of TB progression but showed modest performance over longer follow-up intervals. For TB treatment monitoring, evidence to predict clinical outcomes is limited across all classes.Interpretation: Many systematic reviews have been published describing numerous TB biomarkers, especially for the TB detection use case. Robust validation of candidate biomarkers in clinically relevant populations is lacking for many classes. Further prospective head-to-head validation studies are needed to determine which biomarkers have meaningful potential to improve individual and public health outcomes.

Improved diagnostic tools for tuberculosis that are suitable for use in peripheral health centers are essential for reducing the persistent gap between estimated and notified cases. The diagnostic accuracy and usability of the MiniDock MTB test for detecting pulmonary tuberculosis is unknown. We conducted a prospective, cross-sectional study at outpatient centers in India, Nigeria, the Philippines, South Africa, Uganda, Vietnam, and Zambia. Patients 12 years of age or older with presumptive pulmonary tuberculosis were enrolled between September 12, 2024, and March 31, 2025. Assessment with MiniDock MTB was performed with sputum swabs and tongue swabs. Diagnostic accuracy was evaluated against a sputum-culture-based reference and as compared with sputum-smear microscopy and Xpert MTB/RIF Ultra assay. Usability was assessed with a system usability scale and direct observation. A total of 1380 participants were enrolled; 255 (18.5%) had human immunodeficiency virus infection and 226 (16.4%) had culture-confirmed tuberculosis. MiniDock MTB sensitivity was 85.7% (95% confidence interval [CI], 80.4 to 90.0) with sputum and 79.6% (95% CI, 73.8 to 84.7) with tongue swabs; specificity was greater than 97.5% for both. Results of sputum tests with MiniDock MTB closely matched those with Xpert MTB/RIF Ultra for sensitivity (difference, -2.8 percentage points; 95% CI, -6.0 to 0.5). MiniDock MTB had greater sensitivity than smear microscopy for tests of sputum (difference, 24.3 percentage points; 95% CI, 17.9 to 30.7) and tongue swabs (difference, 18.3 percentage points; 95% CI, 12.0 to 24.7). The test showed diagnostic accuracy that was consistent with World Health Organization (WHO) accuracy targets for near-point-of-care tuberculosis diagnostics (≥85% sensitivity for sputum and ≥75% for nonsputum and ≥98% specificity for both). The median score on the system usability scale (range, 0 to 100, with higher scores indicating better perceived usability) was 75 (interquartile range, 65 to 80), which indicated good usability. No adverse events related to the index test were reported. MiniDock MTB met WHO targets for diagnostic accuracy and usability for tuberculosis detection across diverse clinical settings. (Funded by the National Institutes of Health and others; Rapid Research in Diagnostics Development for TB Network and Assessing Diagnostics at Point-of-Care for Tuberculosis ClinicalTrials.gov numbers, NCT04923958 and NCT05941052.).

Background: Accurate nonsputum tests are essential for improving tuberculosis (TB) detection. We report the first evaluation of the Xpert TB/LTBI assay (research-use-only; Cepheid, USA), which detects 9 Mycobacterium tuberculosis antigen-stimulated mRNA targets from blood. Methods: We enrolled individuals ≥12 years with presumptive TB from clinics in Uganda and Vietnam. All participants underwent sputum- (liquid culture; Xpert MTB/RIF Ultra) and blood-based (Xpert TB/LTBI; QuantiFERON-TB Gold Plus [QFT-Plus]) testing. Participants were classified as having active TB (positive sputum results), latent TB infection (LTBI; positive QFT-Plus with negative sputum results), or no TB infection. Diagnostic accuracy for active TB (primary analysis) and of a 2-step algorithm that first predicted TB infection (active TB or LTBI) and then distinguished active TB from LTBI was assessed using logistic regression and receiver operating characteristic analysis. Results: Among 214 participants included, 56.5% were male, 9.3% were living with HIV, 29.0% had active TB, and 31.8% had LTBI. Xpert TB/LTBI area under the curve (AUC) was 0.92 (95% CI: .88–.96) for identifying active TB, and sensitivity was 93.5% (95% CI: 84.6–97.5) and specificity 76.3% (95% CI: 69.0–82.4) at a cut-point that achieved ≥90% sensitivity. In the 2-step algorithm, Xpert TB/LTBI had high accuracy for identifying TB infection in the first step, but accuracy was lower for distinguishing active TB from LTBI in the second step. Conclusions: Xpert TB/LTBI exceeded World Health Organization (WHO)-recommended minimum accuracy targets for a TB screening test. However, further refinement is needed to improve its ability to distinguish active TB from LTBI.

Background Swab-based molecular platforms that enable testing of both sputum (via swabs swirled in sputum) and tongue swabs are emerging as a promising option for more accessible and lower cost molecular testing for tuberculosis (TB). We conducted a multi-country evaluation of two novel swab-based molecular tests: Molbio Truenat MTB Ultima (MTB Ultima) and Pluslife MiniDock MTB Test (MiniDock MTB). Methods Consecutive people ≥12 years old with presumptive TB were enrolled at outpatient health centers in India, Uganda, and Vietnam. We collected two tongue swabs and prepared two sputum swabs for MTB Ultima and MiniDock MTB testing, then evaluated the diagnostic accuracy of MTB Ultima and MiniDock MTB using both swab types against a sputum liquid culture-based microbiological reference standard (MRS). The diagnostic accuracy of swab-based molecular tests was also compared to sputum Xpert MTB/RIF Ultra (Xpert Ultra) and auramine smear microscopy. Findings From January to September 2024, 1,050 participants were included in the tongue swab MTB Ultima evaluation, 197 in the sputum swab MTB Ultima evaluation, and 322 in the MiniDock MTB evaluations. In comparison to sputum Xpert Ultra, sensitivity was similar for both sputum swab MTB Ultima (93.6% vs. 100.0%, difference -6.4%, [95% CI: -15.5, 2.7], p=0.25) and MiniDock MTB (91.0% vs. 94.0%, difference –3.0% [95% CI: -8.6, 2.6], p=0.50). In comparison to sputum smear microscopy, sensitivity was higher for both tongue swab MTB Ultima (77.9% vs. 59.1%, difference 18.8% [95% CI: 10.8, 26.8], p98%) for both tests with sputum swabs and tongue swabs. Interpretation MTB Ultima and MiniDock MTB have similar accuracy to current sputum-based molecular tests with sputum swabs and meet minimum accuracy thresholds for a non-sputum, near point-of-care molecular test with tongue swabs. These tests offer strong potential to make universal molecular testing for TB a reality.

Rationale: C-reactive protein (CRP)-based tuberculosis (TB) screening is recommended for people with HIV. However, its performance among people without HIV and in diverse settings is unknown. Objectives: In a multicountry study, we aimed to determine whether CRP meets the minimum accuracy targets (sensitivity ⩾ 90%, specificity ⩾ 70%) for an effective TB screening test. Methods: Consecutive outpatient adults with cough ⩾2 weeks from five TB endemic countries in Africa and Asia had baseline blood collected for point-of-care CRP testing and HIV and diabetes screening. Sputum samples were collected for Xpert MTB/RIF Ultra (Xpert) testing and culture. CRP sensitivity and specificity (5 mg/L cut-point) was determined in reference to sputum test results and compared by country, sex, and HIV and diabetes status. Variables affecting CRP performance were identified using a multivariate receiver operating curve regression model. Measurements and Main Results: Among 2,904 participants, of whom 613 (21%) had microbiologically confirmed TB, CRP sensitivity was 84% (95% confidence interval [CI], 81–87%) and specificity was 61% (95% CI, 59–63%). CRP accuracy varied geographically, with higher sensitivity in African countries (⩾91%) than Asian countries (64–82%). Sensitivity was higher among men than women (86% vs. 78%; difference, +8%; 95% CI, 1–15%) and specificity was lower among people with HIV than people without HIV (64% vs. 45%; difference, +19%; 95% CI, 13–25%). Receiver operating curve regression identified country and measures of TB disease severity as predictors of CRP performance. Conclusions: Overall, CRP did not achieve the minimum accuracy targets, and its performance varied by setting and in some subgroups, likely reflecting population differences in mycobacterial load.

Background: Xpert MTB/XDR (Cepheid) is recommended by the World Health Organization for drug susceptibility testing in patients with tuberculosis, with potential for rapid detection of isoniazid and fluoroquinolone resistance. However, diagnostic accuracy and clinical utility in a programmatic setting are unknown. Methods: We evaluated the accuracy and clinical utility of Xpert MTB/XDR in patients with rifampicin-resistant pulmonary tuberculosis during programmatic implementation in Georgia between July 2022 and August 2024, using phenotypic drug susceptibility testing (DST) as a reference standard. Results: An overall 140 patients were tested with Xpert MTB/XDR and phenotypic DST, and 94.9% and 33.8% had isoniazid and fluoroquinolone resistance by phenotypic DST, respectively. Xpert MTB/XDR showed 99.2% sensitivity (95% CI, 95.5%–100%) and 100% specificity (95% CI, 54.1%–100%) for isoniazid resistance. Sensitivity and specificity for fluoroquinolone resistance were 88.4% (95% CI, 74.9%–96.1%) and 100% (95% CI, 95.6%–100%). When indeterminate/invalid Xpert MTB/XDR results were included, 17.4% (8/46) and 6.2% (8/129) of patients with phenotypic fluoroquinolone and isoniazid resistance were missed. Median turnaround time for Xpert MTB/XDR was 1 day (IQR, 1–3) and median time to treatment was 4 days (IQR, 1–7). Phenotypic DST results took a median 43 days (IQR, 29–63) longer than Xpert MTB/XDR results. Finally, 95% (115/121; 95% CI, 89.5%–98.2%) of patients had fluoroquinolones appropriately prescribed based on Xpert MTB/XDR results. Conclusions: Programmatic data confirm the high accuracy of Xpert MTB/XDR, despite being below the World Health Organization target product profile targets for fluoroquinolones, with significantly faster time to results than phenotypic DST.

Rapid and accurate point-of-care (POC) tuberculosis (TB) diagnostics are crucial to bridge the TB diagnostic gap. Leveraging recent advancements in COVID-19 diagnostics, we explored adapting commercially available POC SARS-CoV-2 tests for TB diagnosis in line with the World Health Organization (WHO) target product profiles (TPPs). A scoping review was conducted following PRISMA-ScR guidelines to systematically map POC antigen and molecular SARS-CoV-2 diagnostic tests potentially meeting the TPPs for TB diagnostic tests for peripheral settings. Data were gathered from PubMed/MEDLINE, bioRxiv, medRxiv, publicly accessible in vitro diagnostic test databases, and developer websites up to 23 November 2022. Data on developer attributes, operational characteristics, pricing, clinical performance, and regulatory status were charted using standardized data extraction forms and evaluated with a standardized scorecard. A narrative synthesis of the data is presented. Our search yielded 2003 reports, with 408 meeting eligibility criteria. Among these, we identified 66 commercialized devices: 22 near-POC antigen tests, 1 POC molecular test, 31 near-POC molecular tests, and 12 low-complexity molecular tests potentially adaptable for TB. The highest-scoring SARS-CoV-2 diagnostic tests were the near-POC antigen platform LumiraDx (Roche, Basel, Switzerland), the POC molecular test Lucira Check-It (Pfizer, New York, NY, USA), the near-POC molecular test Visby (Visby, San Jose, CA, USA), and the low-complexity molecular platform Idylla (Biocartis, Lausanne, Switzerland). We highlight a diverse landscape of commercially available diagnostic tests suitable for potential adaptation to peripheral TB testing. This work aims to bolster global TB initiatives by fostering stakeholder collaboration, leveraging SARS-CoV-2 diagnostic technologies for TB, and uncovering new commercial avenues to tackle longstanding challenges in TB diagnosis.