New Manual Quantitative Polymerase Chain Reaction Assay Validated on Tongue Swabs Collected and Processed in Uganda Shows Sensitivity That Rivals Sputum-based Molecular Tuberculosis Diagnostics.

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.

Yield and Efficiency of Novel Intensified Tuberculosis Case-Finding Algorithms for People Living with HIV.

RATIONALE: The recommended tuberculosis (TB) intensified case finding (ICF) algorithm for people living with HIV (symptom-based screening followed by Xpert MTB/RIF [Xpert] testing) is insufficiently sensitive and results in unnecessary Xpert testing. OBJECTIVES: To evaluate whether novel ICF algorithms combining C-reactive protein (CRP)-based screening with urine Determine TB-LAM (TB-LAM), sputum Xpert, and/or sputum culture could improve ICF yield and efficiency. METHODS: We compared the yield and efficiency of novel ICF algorithms inclusive of point-of-care CRP-based TB screening and confirmatory testing with urine TB-LAM (if CD4 count ≤100 cells/µl), sputum Xpert, and/or a single sputum culture among consecutive people living with HIV with CD4 counts less than or equal to 350 cells/µl initiating antiretroviral therapy in Uganda. MEASUREMENTS AND MAIN RESULTS: Of 1,245 people living with HIV, 203 (16%) had culture-confirmed TB including 101 (49%) patients with CD4 counts less than or equal to 100 cells/µl. Compared with the current ICF algorithm, point-of-care CRP-based TB screening followed by Xpert testing had similar yield (56% [95% confidence interval, 49-63] vs. 59% [95% confidence interval, 51-65]) but consumed less than half as many Xpert assays per TB case detected (9 vs. 4). Addition of TB-LAM did not significantly increase diagnostic yield relative to the current ICF algorithm but provided same-day diagnosis for 26% of TB patients with advanced HIV. Addition of a single culture to TB-LAM and Xpert substantially improved ICF yield, identifying 78% of all TB cases. CONCLUSIONS: Point-of-care CRP-based screening can improve ICF efficiency among people living with HIV. Addition of TB-LAM and a single culture to Xpert confirmatory testing could enable HIV programs to increase the speed of TB diagnosis and ICF yield.

A Novel, 5-Transcript, Whole-blood Gene-expression Signature for Tuberculosis Screening Among People Living With Human Immunodeficiency Virus.

BACKGROUND: Gene-expression profiles have been reported to distinguish between patients with and without active tuberculosis (TB), but no prior study has been conducted in the context of TB screening. METHODS: We included all the patients (n = 40) with culture-confirmed TB and time-matched controls (n = 80) enrolled between July 2013 and April 2015 in a TB screening study among people living with human immunodeficiency virus (PLHIV) in Kampala, Uganda. We randomly split the patients into training (n = 80) and test (n = 40) datasets. We used the training dataset to derive candidate signatures that consisted of 1 to 5 differentially-expressed transcripts (P ≤ .10) and compared the performance of our candidate signatures with 4 published TB gene-expression signatures, both on the independent test dataset and in 2 external datasets. RESULTS: We identified a novel, 5-transcript signature that met the accuracy thresholds recommended for a TB screening test. On the independent test dataset, our signature had an area under the curve (AUC) of 0.87 (95% confidence interval [CI] 0.72-0.98), with sensitivity of 94% and specificity of 75%. None of the 4 published TB signatures achieved desired accuracy thresholds. Our novel signature performed well in external datasets from both high (AUC 0.81, 95% CI 0.74-0.88) and low (0.81, 95% CI 0.77-0.85) TB burden settings. CONCLUSIONS: We identified the first gene-expression signature for TB screening. Our signature has the potential to be translated into a point-of-care test to facilitate systematic TB screening among PLHIV and other high-risk populations.

New manual qPCR assay validated on tongue swabs collected and processed in Uganda shows sensitivity that rivals sputum-based molecular TB diagnostics.

Background: Reliance on sputum-based testing is a key barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce and sputum processing increases the complexity and cost of molecular assays. 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 two health centers in Kampala, Uganda. We collected routine demographic and clinical information, sputum for routine TB testing (one Xpert MTB/RIF Ultra® and two liquid cultures), and up to four tongue swabs for same-day qPCR. We evaluated tongue swab qPCR diagnostic accuracy in reference to sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared two swab types (Copan FLOQSWAB® and Steripack® spun polyester swabs). Results: Among 397 participants, 43.1% were female, median age was 33 years, 23.5% were living with HIV (PLHIV) and 32.3% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% [96.2-99.1] of participants. Tongue swab qPCR sensitivity was 91.0% [84.6-94.9] and specificity 98.9% [96.2-99.8] vs. microbiological reference standard (MRS). A single tongue swab recovered a seven-log range of TB copies, with a decreasing recovery trend among four serial swabs. We found no difference between swab types. Conclusions: Tongue swabs show promise as an alternative to sputum for TB diagnosis, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

Transaminitis prevalence among HIV-infected adults eligible for tuberculosis preventive therapy.

OBJECTIVE: To assess the prevalence of severe transaminitis precluding tuberculosis (TB) preventive therapy (TPT) initiation for people with HIV (PWH) in a high TB/HIV burden setting. DESIGN/METHODS: We conducted a secondary analysis of data from a prospective cohort study of PWH with pre-antiretroviral therapy (ART) CD4 + counts 350 cells/µl or less undergoing systematic TB screening from two HIV clinics in Uganda. For this analysis, we excluded patients with culture-confirmed TB and patients without aspartate transaminase (AST) or alanine transaminase (ALT) levels measured within three months of enrollment. We compared the proportion of patients with any transaminitis (AST or ALT greater than one times the upper limit of normal ULN) and severe transaminitis (AST or ALT >3 times ULN) for patients screening negative for TB by symptoms and for those screening negative by C-reactive protein (CRP). We also assessed the proportion of patients with transaminitis by self-reported alcohol consumption. RESULTS: Among 313 participants [158 (50%) women, median age 34 years (IQR 27-40)], 75 (24%) had any transaminitis and six (2%) had severe transaminitis. Of 32 of 313 (10%) who screened negative for TB by symptoms, none had severe transaminitis. In contrast, six-times more PWH screened negative for TB by CRP (194 of 313; 62%), of whom only four (2.1%) had severe transaminitis. Differences in the proportion with any and severe transaminitis according to alcohol consumption were not statistically significant. CONCLUSION: Prevalence of severe transaminitis was low among PWH without culture-confirmed TB in this setting, and is therefore, unlikely to be a major barrier to scaling-up TPT.

Impact of hematocrit on point-of-care C-reactive protein-based tuberculosis screening among people living with HIV initiating antiretroviral therapy in Uganda.

Point-of-care C-reactive protein (POC CRP) testing is a potential tuberculosis (TB) screening tool for people living with HIV (PLHIV). Unlike lab-based assays, POC assays do not routinely adjust CRP levels for hematocrit, potentially resulting in TB screening status misclassification. We compared the diagnostic accuracy of unadjusted and hematocrit-adjusted POC CRP for culture-confirmed TB among PLHIV with CD4 cell-count ≤350 cells/uL initiating antiretroviral therapy (ART) in Uganda. We prospectively enrolled consecutive adults, measured POC CRP (Boditech; normal <8 mg/L), collected two spot sputum specimens for comprehensive TB testing, and extracted pre-ART hematocrit from clinic records. Of the 605 PLHIV included, hematocrit-adjusted POC CRP had similar sensitivity (80% vs 81%, difference +1% [95% CI -3 to +5], P= 0.56) and specificity (71% vs 71%, difference 0% [95% CI -1 to +1], P= 0.56) for culture-confirmed TB, relative to unadjusted POC CRP. When used for TB screening, POC CRP may not require adjustment for hematocrit. However, larger studies may be required if differences close to the clinically meaningful threshold are to be detected.

Field evaluation of a prototype tuberculosis lipoarabinomannan lateral flow assay on HIV-positive and HIV-negative patients.

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.

Characterization of oral swab samples for diagnosis of pulmonary tuberculosis.

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.

Evaluation of multi-antigen serological screening for active tuberculosis among people living with HIV.

Better triage tests for screening tuberculosis (TB) disease are needed for people living with HIV (PLHIV). We performed the first evaluation of a previously-validated 8-antigen serological panel to screen PLHIV for pulmonary TB in Kampala, Uganda. We selected a random 1:1 sample with and without TB (defined by sputum culture) from a cohort of PLHIV initiating antiretroviral therapy. We used a multiplex microbead immunoassay and an ensemble machine learning classifier to determine the area under the receiver operating characteristic curve (AUC) for Ag85A, Ag85B, Ag85C, Rv0934-P38, Rv3881, Rv3841-BfrB, Rv3873, and Rv2878c. We then assessed the performance with the addition of four TB-specific antigens ESAT-6, CFP-10, Rv1980-MPT64, and Rv2031-HSPX, and every antigen combination. Of 262 participants (median CD4 cell-count 152 cells/µL [IQR 65-279]), 138 (53%) had culture-confirmed TB. The 8-antigen panel had an AUC of 0.53 (95% CI 0.40-0.66), and the additional 4 antigens did not improve performance (AUC 0.51, 95% CI 0.39-0.64). When sensitivity was restricted to ≥90% for the 8- and 12-antigen panel, specificity was 2.2% (95% CI 0-17.7%) and 8.1% (95% CI 0-23.9%), respectively. A three-antigen combination (Rv0934-P38, Ag85A, and Rv2031-HSPX) outperformed both panels, with an AUC of 0.60 (95% CI 0.48-0.73), 90% sensitivity (95% CI 78.2-96.7%) and 29.7% specificity (95% CI 15.9-47%). The multi-antigen panels did not achieve the target accuracy for a TB triage test among PLHIV. We identified a new combination that improved performance for TB screening in an HIV-positive sample compared to an existing serological panel in Uganda, and suggests an approach to identify novel antigen combinations specifically for screening TB in PLHIV.

Point-of-care C-reactive protein and risk of early mortality among adults initiating antiretroviral therapy.

OBJECTIVES: In resource-limited settings, mortality in the initial months following antiretroviral therapy (ART) initiation remains unacceptably high. Novel tools to identify patients at highest risk of poor outcomes are needed. We evaluated whether elevated C-reactive protein (CRP) concentrations predict poor outcomes among people living with HIV (PLWH) initiating ART. METHODS: We enrolled and followed for 3-months consecutive PLWH with pre-ART CD4 T-cell counts 350 cells/µl or less initiating ART from two HIV clinics in Uganda. Pre-ART CRP concentrations were measured from capillary blood using a point-of-care (POC) assay. After excluding patients with prevalent tuberculosis - the leading cause of HIV death - we measured 3-month mortality rates using Kaplan-Meier curves, used Cox regression to compare differences in survival, and used logistic regression to compare differences in the odds of opportunistic infections, between patients with and without elevated POC CRP (≥8 mg/l). RESULTS: Of 1293 patients included [median CD4 T-cell count 181 (interquartile range 82-278)], 23 (1.8%) died within 3 months, including 19 of 355 (5.4%) with elevated POC CRP and four of 938 (0.4%) with nonelevated POC CRP. Eighty-six (6.7%) patients were diagnosed with opportunistic infections, including 39 of 355 (11.0%) with elevated POC CRP and 47 of 938 (5.0%) with nonelevated POC CRP. Elevated POC CRP was associated with mortality (adjusted hazard ratio 10.87, 95% confidence interval 3.64-32.47) and opportunistic infection (adjusted odds ratio 1.95, 95% confidence interval 1.23-3.07). CONCLUSION: Among PLWH with advanced HIV, elevated pre-ART POC CRP concentrations are associated with early mortality and opportunistic infections. Pre-ART POC CRP testing may reduce mortality by identifying patients at high risk for poor outcomes.

Brief Report: Yield and Efficiency of Intensified Tuberculosis Case-Finding Algorithms in 2 High-Risk HIV Subgroups in Uganda.

BACKGROUND: Tuberculosis (TB) risk varies among different HIV subgroups, potentially impacting intensified case finding (ICF) performance. We evaluated the performance of the current ICF algorithm [symptom screening, followed by Xpert MTB/RIF (Xpert) testing] in 2 HIV subgroups and evaluated whether ICF performance could be improved if TB screening was based on C-reactive protein (CRP) concentrations. METHODS: We enrolled consecutive adults with CD4 counts ≤350 cells/µL initiating antiretroviral therapy and performed symptom screening, CRP testing using a low-cost point-of-care (POC) assay, and collected sputum for Xpert testing. We compared the yield and efficiency of the current ICF algorithm to POC CRP-based ICF among patients new to HIV care and patients engaged in care. RESULTS: Of 1794 patients, 126/1315 (10%) new patients and 21/479 (4%) engaged patients had Xpert-positive TB. The current ICF algorithm detected ≥98% of all TB cases in both subgroups but required ≥85% of all patients to undergo Xpert testing. POC CRP-based ICF halved the proportion of patients in both subgroups requiring Xpert testing relative to the current ICF algorithm and had lower yield among patients engaged in care [81% vs. 100%, difference -19% (95% confidence interval: -41 to 3)]. Among patients new to care, POC CRP-based ICF had similar yield as the current ICF algorithm [93% vs. 98%, difference -6% (95% confidence interval: -11 to 0)]. CONCLUSIONS: Among patients new to care, POC CRP-based screening can improve ICF efficiency without compromising ICF yield, whereas symptom-based screening may be necessary to maximize ICF yield among patients engaged in care.

Point-of-care C-reactive protein-based tuberculosis screening for people living with HIV: a diagnostic accuracy study.

BACKGROUND: Symptom-based screening for tuberculosis is recommended for all people living with HIV. This recommendation results in unnecessary Xpert MTB/RIF testing in many individuals living in tuberculosis-endemic areas and thus poor implementation of intensified case finding and tuberculosis preventive therapy. Novel approaches to tuberculosis screening are needed to help achieve global targets for tuberculosis elimination. We assessed the performance of C-reactive protein (CRP) measured with a point-of-care assay as a screening tool for active pulmonary tuberculosis. METHODS: For this prospective study, we enrolled adults (aged ≥18 years) living with HIV with CD4 cell count less than or equal to 350 cells per µL who were initiating antiretroviral therapy (ART) from two HIV/AIDS clinics in Uganda. CRP concentrations were measured at study entry with a point-of-care assay using whole blood obtained by fingerprick (concentration ≥10 mg/L defined as screen positive for tuberculosis). Sputum samples were collected for Xpert MTB/RIF testing and culture. We calculated the sensitivity and specificity of point-of-care CRP and WHO symptom-based screening in reference to culture results. We repeated the sensitivity analysis with Xpert MTB/RIF as the reference standard. FINDINGS: Between July 8, 2013, and Dec 15, 2015, 1237 HIV-infected adults were enrolled and underwent point-of-care CRP testing. 60 (5%) patients with incomplete or contaminated cultures were excluded from the analysis. Of the remaining 1177 patients (median CD4 count 165 cells per µL [IQR 75-271]), 163 (14%) had culture-confirmed tuberculosis. Point-of-care CRP testing had 89% sensitivity (145 of 163, 95% CI 83-93) and 72% specificity (731 of 1014, 95% CI 69-75) for culture-confirmed tuberculosis. Compared with WHO symptom-based screening, point-of-care CRP testing had lower sensitivity (difference -7%, 95% CI -12 to -2; p=0·002) but substantially higher specificity (difference 58%, 95% CI 55 to 61; p<0·0001). When Xpert MTB/RIF results were used as the reference standard, sensitivity of point-of-care CRP and WHO symptom-based screening were similar (94% [79 of 84] vs 99% [83 of 84], respectively; difference -5%, 95% CI -12 to 2; p=0·10). INTERPRETATION: The performance characteristics of CRP support its use as a tuberculosis screening test for people living with HIV with CD4 count less than or equal to 350 cells per µL who are initiating ART. HIV/AIDS programmes should consider point-of-care CRP-based tuberculosis screening to improve the efficiency of intensified case finding and increase uptake of tuberculosis preventive therapy. FUNDING: National Institutes of Health; President's Emergency Plan for AIDS Relief; University of California, San Francisco, Nina Ireland Program for Lung Health.