Alfred Andama

Rationale: Sputum-based testing using Xpert MTB/RIF Ultra (Xpert) is the most common molecular testing method for diagnosing tuberculosis (TB). Objectives: To evaluate whether sputum quality influences Xpert positivity and diagnostic accuracy. Methods: We screened consecutive people for presumptive TB in India, the Philippines, Vietnam, Nigeria, South Africa, Uganda, and Zambia as part of the R2D2 TB Network and ADAPT studies. Participants provided 2-3 sputum samples for Xpert and culture reference testing. The quality of the first sputum sample was graded following standardized procedures by trained research staff and used for Xpert testing. We performed logistic regression to evaluate whether sputum grade was independently associated with Xpert positivity, and calculated sensitivity and specificity of Xpert against a culture-based microbiological reference standard (MRS). Measurements and Main Results: Among 1,855 participants, 798 (43%) were female, 348 (19%) were living with HIV (PLHIV), and 1795 (97%) had a cough of ≥2 weeks. Overall, 313 (17%) had a positive Xpert result. Most sputum samples were salivary (83%). Xpert positivity was lowest among salivary samples (16.1%) and highest among purulent samples (31.2%). After adjusting for demographic and clinical variables, there was no significant association between any sputum grade and Xpert positivity. Xpert sensitivity (salivary: 89%, mucoid: 91%, mucopurulent: 87%, purulent: 100%) and specificity (>98%) were high across sputum grades. Conclusions: Sputum quality was not independently associated with Xpert positivity and Xpert sensitivity was high across all sputum grades. These findings support molecular testing of all sputum samples for TB diagnosis regardless of macroscopic appearance. Scientific Knowledge on the Subject: Xpert MTB/RIF Ultra (Xpert Ultra) is a WHO-recommended initial diagnostic test for pulmonary tuberculosis. Historical laboratory practice, derived from the smear microscopy era, has emphasized sputum macroscopic quality, with many programs recommending rejection of salivary sputum as inadequate. Prior studies evaluating sputum quality and Xpert performance yielded mixed results, and were limited by small sample sizes, single-country designs, heterogeneous sputum grading systems, and predominant use of the first-generation Xpert MTB/RIF assay. What This Study Adds to the Field: In this large and geographically diverse evaluation of 1,855 participants across seven high-burden countries, macroscopic sputum quality was not independently associated with Xpert Ultra positivity or diagnostic accuracy. Xpert Ultra demonstrated high sensitivity and specificity across all sputum grades, including salivary specimens, which comprised over 80% of samples. These findings, generated by using a standardized sputum grading protocol and a culture-based microbiological reference standard, provide robust evidence that sputum quality-based rejection criteria are unnecessary for Xpert Ultra testing. Revising laboratory guidelines to accept all expectorated sputum regardless of appearance could improve case detection and reduce diagnostic delays in high-burden settings.

Background: Tongue swabs are a promising alternative specimen for tuberculosis (TB) diagnosis. Although test specificity exceeds 98%, sensitivity is lower than sputum-based molecular testing. We investigated whether the use of tongue swabs could increase sample availability, resulting in similar diagnostic yield. Methods: In this cross-sectional study (July 2024–January 2025), we screened consecutive people with presumptive TB at health centers in the Philippines, Vietnam, Uganda, and Zambia. Participants were asked to provide tongue swabs and referred for routine sputum collection. Tongue swabs were tested in research laboratories using the MiniDock MTB Test (Guangzhou Pluslife Biotech Co., Ltd., China); sputum was tested using WHO-recommended molecular testing per national guidelines. We compared diagnostic yield, defined as proportion of positive test results among all participants, between tongue swab- and sputum-based molecular testing with a prespecified 3.0% non-inferiority margin. Results: Of 1639 participants, 851 (51.9%) were female, 415 (25.3%) were living with HIV, and 132 (8.1%) were children <5 years. All provided tongue swabs, but only 1389 (84.7%) produced sputum. Diagnostic yield was 3.8% (63/1639) for tongue swabs and 4.1% (68/1639) for sputum-based (68/1639, 4.1%) molecular testing. The difference (0.3%, 95% CI −0.6 to +1.2) was within the prespecified non-inferiority margin. Results were consistent across countries and key subgroups (age, sex, and HIV status). Conclusions: Tongue swab-based molecular testing with MiniDock MTB achieved non-inferior diagnostic yield compared with sputum-based molecular testing. These findings support scale-up of swab-based platforms as a cost-efficient alternative, particularly where sputum collection is challenging or smear microscopy remains the primary diagnostic method.

Rationale: Computer-aided detection algorithms for automated chest X-ray reading have been endorsed by the World Health Organization for tuberculosis triage, but independent, multi-country assessment of current products is needed to guide implementation. Objectives: We included chest X-rays from adults who presented to outpatient facilities with at least 2 weeks of cough in India, Madagascar, the Philippines, South Africa, Tanzania, Uganda, and Vietnam. Methods: We calculated and compared the accuracy overall and by country and key groups for 7 computer-aided detection algorithms: CAD4TB, qXR, INSIGHT CXR, DrAid, Genki, InferRead, and Radify. We determined if any computer-aided detection product could achieve the minimum target accuracy for a tuberculosis triage test (≥ 90% sensitivity and ≥ 70% specificity). Results: Of 3901 individuals included, the median age was 41 years (IQR, 29-54 years), 12.9% were people living with HIV, 8.2% were living with diabetes, and 21.2% had a prior history of tuberculosis. Specificity ranged from 30.9% to 73.5% at 90% sensitivity. CAD4TB achieved the highest specificity at 90% sensitivity (73.5% specific [95% CI, 71.9%-75.1%]), although qXR and INSIGHT CXR also achieved the target 70% specificity. There was heterogeneity by country and subgroup that improved with population-specific thresholds, except for people living with HIV, 50 years and older, or with a history of tuberculosis. Conclusions: Multiple computer-aided detection algorithms achieved the minimum target accuracy for a tuberculosis triage test among symptomatic individuals with cough. Further efforts are needed to integrate computer-aided detection into routine tuberculosis case detection programs in high-burden communities.

Point-of-care (POC) tests are needed to improve tuberculosis (TB) detection, but how TB-affected persons value POC tests is unknown. Simulations using discrete choice experiment-based data from 1149 TB-affected participants across 5 countries demonstrated that POC TB tests were highly preferred, even with modestly lower accuracy compared to standard facility-based testing.

Background: Tongue swabs are a promising alternative specimen for tuberculosis (TB) diagnosis, with high specificity (>98%) but lower sensitivity than sputum-based molecular tests. We investigated whether increased sample availability could offset lower sensitivity, resulting in similar diagnostic yield. Methods: From September 2024-January 2025, we screened consecutive people with presumptive TB at health centers in the Philippines, Vietnam, Uganda, and Zambia. Enrolled participants were asked to provide tongue swabs and referred for routine sputum collection. Tongue swabs were tested in research laboratories using the MiniDock MTB Test (Guangzhou Pluslife Biotech Co., Ltd., China); sputum was tested using WHO-recommended molecular testing per national guidelines. We compared diagnostic yield, defined as positive test results among those seeking testing, between tongue swab- and sputum-based molecular testing with a pre-specified non-inferiority margin of ±3%. Findings: Of 1,639 participants, 851 (51·9%) were female, 415 (25·3%) were living with HIV and 132 (8·1%) were children Interpretation: TB diagnostic yield from tongue swabs was non-inferior to sputum-based molecular testing. These data support scale-up of swab-based molecular platforms as a lower-cost alternative to sputum-based tests, particularly in settings where sputum collection is challenging, or smear microscopy remains standard. Funding: Supported by the Bill and Melinda Gates Foundation.

Background. Community-acquired pneumonia (CAP) remains an important cause of morbidity and mortality in people with HIV (PWH), and antimicrobial resistance (AMR) leads to poor treatment outcomes. Better tests are required to overcome the low sensitivity of sputum Gram stain and culture for pneumonia diagnosis. Molecular diagnostic tests rapidly detect respiratory pathogens and markers of AMR, but few studies have examined their role in PWH. Objectives. To investigate the additional yield of the Biofire FilmArray Pneumonia Panel plus (FilmArrayPN-PCR), an automated nested multiplex polymerase chain reaction system, over culture for diagnosis of CAP, and determine clinical predictors of AMR in PWH. Methods. We enrolled adult PWH hospitalised with cough <2 months in a prospective cohort in Kampala, Uganda. Participants provided expectorated sputum samples for testing by FilmArrayPN-PCR and culture. We performed drug susceptibility testing of cultured sputum isolates and detection of genetic markers of AMR on sputum by FilmArrayPN-PCR. Results. The 107 participants enrolled had a median (interquartile range) age of 40 (31 - 46) years, 50.5% (n=54/107) were female, and 74.8% (n=80/107) had recent antibiotic use. The median duration of cough was 3 (1 - 4) weeks. FilmArrayPN-PCR increased the detection of respiratory pathogens by 64.5% (95% confidence interval (CI) 54.8 - 73.1; p<0.001) and detected AMR in 25.2% (n=27/107). Baseline room air oxygen saturation <92% (adjusted odds ratio (aOR) 9.20; 95% CI 2.52 - 33.57; p=0.001) and prior antibiotic use (aOR 4.14; 95% CI 1.04 - 16.51; p=0.04) were independent predictors of AMR. Conclusion. FilmArrayPN-PCR increased the diagnostic yield of pathogens, and a low baseline oxygen saturation (<92%) and prior antibiotic use were associated with an increased risk of AMR in hospitalised PWH with CAP.

Background: Children with non-severe TB may benefit from short-course treatment, but point-of-care tools are needed to stratify disease severity. We prospectively evaluated the Cepheid Xpert MTB-Host Response (HR) prototype cartridge for distinguishing TB severity in children with pulmonary TB (PTB) in The Gambia and Uganda. Methods: We included children <15 with microbiologically confirmed or clinically diagnosed unconfirmed PTB. Severity was defined using the World Health Organization (WHO) guidelines for a four-month, drug-susceptible regimen. Capillary or venous blood was tested with the HR cartridge for PCR-based detection of 3 mRNA genes and calculation of a TB score from cycle thresholds. We generated receiver operating characteristic curves with the TB score to classify severe TB and assessed if Xpert-HR could achieve the WHO target accuracy for treatment optimization (≥90% sensitivity, ≥70% specificity). Results: Among 106 children, the median age was 4 years (IQR 1-7), 56.6% were female, and 13.2% were living with HIV. In all children with PTB, Xpert-HR achieved an AUC of 0.67 (95% CI 0.55-0.78), with 89.3% sensitivity (95% CI 71.8-97.7) and 29.5% specificity (95% CI 19.7-40.9, cutoff ≤ −0.60). By confirmation status, Xpert-HR approached the target accuracy in children with Confirmed TB, with 62.5% specificity (95% CI 24.5-91.5) at 91.7% sensitivity (95% CI 61.5-99.8, cut-off ≤ −1.349). Among children with Unconfirmed TB, specificity was lower (24.3%, 95% CI 14.8-36.0) at 93.8% sensitivity (95% CI 69.8-99.8, cutoff ≤ −0.450). Target accuracy was almost achieved in children 5-9 regardless of confirmation status (100% sensitivity [95% CI 71.5-100], 66.7% specificity [95% CI 43.0-85.4], cutoff ≤ −1.35), but specificity (28.2%, 95% CI 18.6-39.5) was lower for children < 5 (92.9% sensitivity, 95% CI 76.5-99.1, cutoff ≤ −0.550). Conclusions: Xpert-HR approached the target accuracy to stratify PTB severity in older children and those with Confirmed TB but had lower specificity in children with Unconfirmed TB. Child-specific signatures may be needed to improve performance in younger children with paucibacillary disease.

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.

Background: Accessible, accurate screening tests are necessary to advance tuberculosis case finding and early detection in high-burden countries. Methods: We prospectively screened adults with ≥2 weeks of cough at primary health centers in the Philippines, Vietnam, South Africa, Uganda, and India. Participants underwent chest radiography, Cepheid Xpert TB Host Response (Xpert HR) testing, and point-of-care C-reactive protein (CRP) testing (Boditech). Chest radiographs were processed using CAD4TB v7, a computer-aided detection (CAD) algorithm. We assessed diagnostic accuracy against a microbiologic reference standard (sputum Xpert Ultra, culture). Optimal cutoff points were chosen to maximize specificity at 90% sensitivity. Two-test screening algorithms were considered, using (1) sequential negative serial screening (with positive defined as positive on either test) and (2) sequential positive serial screening (with positive defined as positive on both tests). Results: Between July 2021 and August 2022, a total of 1392 participants with presumptive tuberculosis had valid index tests and reference standard results, and 303 (22%) had confirmed tuberculosis. In head-to-head comparisons, CAD4TB v7 showed the highest specificity at 90% sensitivity (70.3% vs 65.1% for Xpert HR [95% confidence interval for absolute difference in specificity, 1.6%–8.9%] and vs 49.7% for CRP [17.0%–24.3%]). Three 2-test screening algorithms met World Health Organization target product profile minimum accuracy thresholds and had higher accuracy than any test alone. At 90% sensitivity, the specificity was 79.6% for Xpert HR–CAD4TB (sequential negative), 75.9% for CRP-CAD4TB (sequential negative), and 73.7% for Xpert HR–CAD4TB (sequential positive). Conclusions: CAD4TB achieves target product profile targets and outperforms Xpert HR and CRP. Combining screening tests further increased accuracy. Clinical Trials Registration. NCT04923958

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.