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BackgroundThe World Health Organization (WHO) has endorsed the next-generation Xpert MTB/RIF Ultra (Ultra) cartridge, and Uganda is currently transitioning from the older generation Xpert MTB/RIF (Xpert) cartridge to Ultra as the initial diagnostic test for pulmonary tuberculosis (TB). We assessed the diagnostic accuracy of Ultra for pulmonary TB among adults in Kampala, Uganda.MethodsWe sampled adults referred for Xpert testing at two hospitals and a health center over a 12-month period. We enrolled adults with positive Xpert and a random 1:1 sample with negative Xpert results. Expectorated sputum was collected for Ultra, and for solid and liquid culture testing for Xpert-negative patients. We measured sensitivity and specificity of Ultra overall and by HIV status, prior history of TB, and hospitalization, in reference to Xpert and culture results. We also assessed how classification of results in the new "trace" category affects Ultra accuracy.ResultsAmong 698 participants included, 211 (30%) were HIV-positive and 336 (48%) had TB. The sensitivity of Ultra was 90.5% (95% CI 86.8-93.4) and specificity was 98.1% (95% CI 96.1-99.2). There were no significant differences in sensitivity and specificity by HIV status, prior history of TB or hospitalization. Xpert and Ultra results were concordant in 670 (96%) participants, with Ultra having a small reduction in specificity (difference 1.9, 95% CI 0.2 to 3.6, p=0.01). When "trace" results were considered positive for all patients, sensitivity increased by 2.1% (95% CI 0.3 to 3.9, p=0.01) without a significant reduction in specificity (- 0.8, 95% CI - 0.3 to 2.0, p=0.08).ConclusionsAfter 1 year of implementation, Ultra had similar performance to Xpert. Considering "trace" results to be positive in all patients increased case detection without significant loss of specificity. Longitudinal studies are needed to compare the benefit of greater diagnoses to the cost of overtreatment.

The performance of urine Xpert MTB/RIF Ultra (Xpert Ultra) for pulmonary TB diagnosis is unknown. HIV-positive and HIV-negative adults were enrolled at two health facilities in Kampala, Uganda. We compared the accuracy of urine Xpert Ultra and Determine TB-LAM in reference to sputum-based testing (positive Xpert MTB/RIF or culture), and assessed incremental yield. Urine Xpert Ultra had low sensitivity (17.2%, 95% CI 12.3-23.2) but high specificity (98.1%, 95% CI 94.4-99.6). Sensitivity reached 50.0% (95% CI 28.2-71.8) among HIV-positive patients with CD4

Background: Sputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain., Methods: From June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester)., Results: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently., Conclusions: Tongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics., Competing Interests: Potential conflicts of interest. A. C. reports participation on the Medaica Advisory Board and stock or stock options for Medaica. All remaining authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Detection of tuberculosis at the point-of-care (POC) is limited by the low sensitivity of current commercially available tests. We describe a diagnostic accuracy field evaluation of a prototype urine Tuberculosis Lipoarabinomannan Lateral Flow Assay (TB-LAM LFA) in both HIV-positive and HIV-negative patients using fresh samples with sensitivity and specificity as the measures of accuracy. This prototype combines a proprietary concentration system with a sensitive LFA. In a prospective study of 292 patients with suspected pulmonary tuberculosis in Uganda, the clinical sensitivity and specificity was compared against a microbiological reference standard including sputum Xpert MTB/RIF Ultra and solid and liquid culture. TB-LAM LFA had an overall sensitivity of 60% (95%CI 51-69%) and specificity of 80% (95%CI 73-85%). When comparing HIV-positive (N = 86) and HIV-negative (N = 206) patients, there was no significant difference in sensitivity (sensitivity difference 8%, 95%CI -11% to +24%, p = 0.4351) or specificity (specificity difference -9%, 95%CI -24% to +4%, p = 0.2051). Compared to the commercially available Alere Determine TB-LAM Ag test, the TB-LAM LFA prototype had improved sensitivity in both HIV-negative (difference 49%, 95%CI 37% to 59%, p<0.0001) and HIV-positive patients with CD4+ T-cell counts >200cells/μL (difference 59%, 95%CI 32% to 75%, p = 0.0009). This report is the first to show improved performance of a urine TB LAM test for HIV-negative patients in a high TB burden setting. We also offer potential assay refinement solutions that may further improve sensitivity and specificity., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: HR, LI, and BH are employees of Biopromic AB. Biopromic AB is developing a point-of-care TB LAM test similar to the one presented. JTC, BDG, AB, BBL, HVH, VMH, SB, BHW, CB, DB, and AS were employees of Global Good Fund, Intellectual Ventures. BH is also an author on patents in the field of TB immunoassays, “Monoclonal antibody, method, kit and use” (WO2016012449) and “Method for removing inhibitory components” (US20190302115). JTC, BDG, and BHW are authors on a patent in the field of analyte concentration devices, “Device for rapid detection of tuberculosis-lipoarabinomannan (TB-LAM) with enhanced sensitivity” (US10830760). AP is an author on patents in the field of mycobacteria detection, “Novel anti-lam and anti-pim6/lam monoclonal antibodies for diagnosis and treatment of mycobacterium tuberculosis infections” (US20190038747) and “Methods for dual detection and differentiation of infection by mycobactierum tuberculosis complex and nontuberculous mycobacteria” (WO2020018806). None of the disclosed competing interest alter our adherence to PLOS ONE policies on sharing data and materials.

Oral swab analysis (OSA) has been shown to detect Mycobacterium tuberculosis (MTB) DNA in patients with pulmonary tuberculosis (TB). In previous analyses, qPCR testing of swab samples collected from tongue dorsa was up to 93% sensitive relative to sputum GeneXpert, when 2 swabs per patient were tested. The present study modified sample collection methods to increase sample biomass and characterized the viability of bacilli present in tongue swabs. A qPCR targeting conserved bacterial ribosomal rRNA gene (rDNA) sequences was used to quantify bacterial biomass in samples. There was no detectable reduction in total bacterial rDNA signal over the course of 10 rapidly repeated tongue samplings, indicating that swabs collect only a small portion of the biomass available for testing. Copan FLOQSwabs collected ~2-fold more biomass than Puritan PurFlock swabs, the best brand used previously (p = 0.006). FLOQSwabs were therefore evaluated in patients with possible TB in Uganda. A FLOQSwab was collected from each patient upon enrollment (Day 1) and, in a subset of sputum GeneXpert Ultra-positive patients, a second swab was collected on the following day (Day 2). Swabs were tested for MTB DNA by manual IS6110-targeted qPCR. Relative to sputum GeneXpert Ultra, single-swab sensitivity was 88% (44/50) on Day 1 and 94.4% (17/18) on Day 2. Specificity was 79.2% (42/53). Among an expanded sample of Ugandan patients, 62% (87/141) had colony-forming bacilli in their tongue dorsum swab samples. These findings will help guide further development of this promising TB screening method., Competing Interests: At the time of study design and evaluation SB, CMB, DB, and AS received salary support from the Global Good Fund. At the time of study design and evaluation GH, KPN, AML, CO, DM, and KJM were employed by Intellectual Ventures Laboratory. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials. A number of the authors are currently affiliated with GH Labs and Roche Molecular Systems, but were not affiliated with them during the study, and these companies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.