Ziyaad Waja

Background/Objectives: Access to accurate tuberculosis (TB) diagnostics remains limited, particularly in high-burden settings. The Pluslife MTB assay is among the first molecular tests specifically designed for swab-based detection of Mycobacterium tuberculosis complex (MTBC) and offers near point-of-care use. Methods: We conducted a prospective diagnostic accuracy study in South Africa to evaluate the performance of the Pluslife assay on tongue swabs (TSs) and sputum swabs (SSs) among symptomatic and asymptomatic adults. Results were compared against liquid culture as the reference standard and Xpert MTB/RIF Ultra (Xpert Ultra) as a comparator. Operational characteristics and ease-of-use were assessed through structured observation and Likert-scale scoring by testing personnel. Results: Of the 256 participants enrolled, 92 [36%] were people with HIV (PHIV) and 217 were included in the final analysis. Culture confirmed TB in 41/217 (19%). Pluslife on SSs demonstrated a sensitivity of 85% (95% CI: 70.8–94.4), compared to 83% (95% CI: 67.9–92.8), for Xpert Ultra on sputum and detected one additional case (paired analysis, p = 1.000). Sensitivity on TSs was lower (63%, 95% CI: 46.9–77.9), particularly among PHIV. Specificity exceeded 97% across specimen types. Concordance on TSs between Pluslife and Xpert Ultra increased with higher bacterial loads. Operational evaluation showed short hands-on time and high ease-of-use scores, though limitations were noted for patient identifier recording and troubleshooting on the Pluslife MiniDock device. Conclusions: The Pluslife MTB assay on SSs demonstrated good diagnostic accuracy and favorable usability in this study. Tongue swabs remain feasible but less reliable, supporting sputum as the preferred first specimen for TB diagnosis.

Background: Adjunctive host-directed therapies (HDT) may improve treatment outcomes in tuberculosis (TB). Affordable repurposed drugs can be quickly implemented in clinical practice; however, predictive biomarkers are essential for determining their potential effectiveness. Methods: 72 patients (35.2% people with HIV-1) from South Africa with drug-sensitive pulmonary TB, recruited to the SMA-TB clinical trial of adjunctive acetylsalicylic acid (ASA) and ibuprofen (IBU), were included. CD4+ T-cell subsets and Mtb-specific CD4+ T-cell responses were analyzed by flow cytometry at baseline, week 2, week 8 and week 24 and tested as predictors for microbiological, clinical and radiological outcomes. Results: Most TB patients experienced a fast sputum culture conversion and good clinical and radiological outcomes by week 24, regardless of intervention or HIV coinfection. During TB therapy, fractions of Mtb-specific polyfunctional CD4+ T cells, Th1 and Th1/Th17 subsets, and CD153 expression increased, whereas Ki67+ HLA-DR + CD4+ T cells declined. No statistically significant differences were identified between the ASA or IBU groups and placebo for any defined outcome. Still, higher baseline fractions of polyfunctional and CD153-expressing CD4+ T cells were associated with fast sputum culture conversion in the ASA and placebo arms. Conclusions: CD4+ T-cell subsets show promise as markers of disease severity and potential predictors of microbiological, clinical, and radiological outcomes in treated TB. However, the present findings are insufficient to support the routine use of ASA or IBU in adjunctive HDT regimens, and larger, targeted studies are warranted.

Background: As novel strategies are needed to improve tuberculosis (TB) outcomes and shorten treatment; we evaluated whether host-directed therapy with non-steroidal anti-inflammatory drugs (NSAIDs) could enhance treatment response when added to standard therapy. Methods: We conducted a phase 2b, double-blind, placebo-controlled, randomized trial in South Africa and Georgia. Adults with microbiologically confirmed pulmonary TB were randomized to receive standard therapy plus acetylsalicylic acid (ASA) (300 mg), ibuprofen (IBU) (400 mg), or placebo twice daily for 4 weeks and once daily for 4 weeks. Primary end points were time to a 67% reduction in the clinical symptom/sign-based TB score (TBS) and time to stable sputum culture conversion (SCC). Safety and clinical, radiological and microbiological assessments were performed up to 24 weeks after treatment completion. Results: Of 221 randomized participants, 204 were included in the analysis. No significant differences were observed in the primary end points; median time to 67% TBS reduction was 4.5 weeks (ASA) and 5.0 weeks (IBU and placebo), and median time to stable SCC was 8 weeks in all groups. Both NSAIDs were associated with earlier cavity resolution at week 8, sustained at week 24 for ASA. IBU was associated with greater clinical improvement in multidrug-resistant-TB. Adverse event rates were similar across arms. Conclusions: Adjunctive ASA and IBU did not shorten the time to clinical or microbiologic response but were associated with earlier cavity resolution and improved TBS in specific subgroups, with similar safety to standard therapy alone. Funded by the European Union’s Horizon 2020; ClinicalTrials.gov number, NCT04575519 .

Africa bears the largest burden of morbidity and mortality from hypertension (HT) and related cardiovascular diseases. Thiazide diuretics, including hydrochlorothiazide (HCTZ), are the most prescribed hypertension medicines globally due to their cost effectiveness and minimal side effects. However, studies investigating the genetic variation in HCTZ‐related genes and their effect on drug response have been mainly conducted in European, American, and Asian populations. This study aimed to examine genetic variation in HCTZ response‐related genes among newly diagnosed hypertensives from Soweto, South Africa, irrespective of the treatment approach, and to assess its potential impact on medication response. Using targeted NGS on 364 newly diagnosed hypertensives with at least one follow‐up visit, we identified 1258 unique variants in 16 key HCTZ response genes. Among these, 89 were novel variants, of which 12 were protein‐altering, including 8 predicted to be deleterious. Association analyses with 882 independent variants revealed that carriers of the intronic variant rs1025450259 in GNB3 had significantly greater reduction in BP compared to other patients on heterogeneous treatment strategies, but no significant associations were detected in the subset of individuals treated with HCTZ monotherapy only ( n = 103). For variants previously associated with HCTZ response, we did not replicate prior associations (e.g., rs2611316 in ALDH1A2 ) but highlight important allele frequency differences across global populations. Despite the limitations, the study outcomes support the need for larger, controlled trials to assess genetic influences on HT treatment outcomes, advancing personalized medicine for African populations. This research further underscores the importance of pharmacogenetic studies in continental African populations.

Background: Differences in absolute neutrophil count (ANC) between African and non-African populations have been reported. Neutropenia has also been reported during rifamycin use. ANC values in current tables that assign severity grades to clinical or laboratory events (National Cancer Institute Common Terminology Criteria for Adverse Events [CTCAE] and the National Institute of Allergy and Infectious Diseases Division of AIDS [DAIDS]) might not consistently be applicable among different populations. Methods: During 2 international tuberculosis treatment trials, 337 participants received 10 mg/kg of rifampin, and 3 groups of 361, 81, and 81 participants received 10, 15, and 20 mg/kg of rifapentine, respectively, for 2 months of daily treatment. ANC was measured at baseline and after 2, 4, 6, 8, and 12 weeks of treatment. CTCAE versions 2.0–4.0 and DAIDS version 2.0 define neutropenia grade 3 as ANC 500–999 cells/μL and ANC 400–599 cells/μL, respectively. We analyzed factors associated with neutropenia. Results: Baseline median ANC was lower in Black African participants than in Black non-African participants (P = .004). CTCAE-defined grade 3 or higher neutropenia occurred in 32 (28 in Africa) participants. Proportions did not differ between rifapentine doses. Only 4 events would have been reported by DAIDS criteria. All neutropenia events resolved spontaneously without interrupting treatment. Enrollment in an African country was an independent factor associated with the development of neutropenia. Conclusions: Differences in baseline median ANC can result in misclassification of adverse events or inappropriate clinical management. Development and use of population-specific normal values of ANC could improve guidance for research and clinical management.

Background: HIV is associated with an increased risk of aggressive lymphomas. Lymphadenopathy is common at the time of presentation; therefore, we set out to understand the time to lymphoma diagnosis in people with HIV (PWH) undergoing fine needle aspiration (FNA). Methods: A prospective, observational cohort of PWH, age ≥ 18 years, undergoing FNA in Soweto, South Africa was established between September 2021 and December 2022. Participants were followed up for up to 8 months and provided consent for a medical record review. Results: One hundred and forty-six participants were enrolled, including 76 females (52%) with a median age of 40 years and a median CD4 count of 216 cells/μL. TB was the most common diagnosis (n = 62; 42%), followed by lymphoma (n = 21; 14%), of whom 10 (48%) died either prior to diagnosis or initiating chemotherapy. An additional 2 participants (10%) were still awaiting a biopsy confirmation at 8 months. One participant’s FNA was suggestive of both lymphoma and TB. The median healthcare provider interval, the time from presentation to diagnosis, was 85 days. Conclusions: While TB was the most common diagnosis among PWH undergoing FNA, lymphoma was the leading cause of death. As most deaths occurred prior to chemotherapy, interventions to expedite a lymphoma diagnosis in this high-risk population are needed.

Growing evidence points to the presence of differentially culturable tubercle bacteria (DCTB) in clinical specimens from individuals with active tuberculosis (TB) disease. These bacteria are unable to grow on solid media but can resuscitate in liquid media. Given the epidemiological success of certain clinical genotype families of Mycobacterium tuberculosis, we hypothesize that different strains may have distinct mechanisms of adaptation and tolerance. We used an in vitro carbon starvation model to determine the propensity of strains from lineages 2 and 4 that included the Beijing and LAM families respectively, to generate DCTB. Beijing strains were associated with a greater propensity to produce DCTB compared to LAM strains. Furthermore, LAM strains required culture filtrate (CF) for resuscitation whilst starved Beijing strains were not dependent on CF. Moreover, Beijing strains showed improved resuscitation with cognate CF, suggesting the presence of unique growth stimulatory molecules in this family. Analysis of starved Beijing and LAM strains showed longer cells, which with resuscitation were restored to a shorter length. Cell wall staining with fluorescent D-amino acids identified strain-specific incorporation patterns, indicating that cell surface remodeling during resuscitation was distinct between clinical strains. Collectively, our data demonstrate that M. tuberculosis clinical strains from different genotype lineages have differential propensities to generate DCTB, which may have implications for TB treatment success.

Rationale: We developed a standardized method, possible poor treatment response (PPTR), to help ascertain efficacy endpoints in Study S31/A5349 (NCT 02410772), an open-label trial comparing two 4-month rifapentine-based regimens with a standard 6-month regimen for the treatment of pulmonary tuberculosis (TB). Objectives: We describe the use of the PPTR process and evaluate whether the goals of minimizing bias in efficacy endpoint assessment and attainment of relevant data to determine outcomes for all participants were achieved. Methods: A PPTR event was defined as the occurrence of one or more prespecified triggers. Each PPTR required initiation of a standardized evaluation process that included obtaining multiple sputum samples for microbiology. Measurements and Main Results: Among 2,343 participants with culture-confirmed drug-susceptible TB, 454 individuals (19.4%) had a total of 534 individual PPTR events, of which 76.6% were microbiological (positive smear or culture at or after 17 wk). At least one PPTR event was experienced by 92.4% (133 of 144) of participants with TB-related unfavorable outcome and between 13.8% and 14.7% of participants with favorable and not-assessable outcomes. A total of 75% of participants with TB-related unfavorable outcomes had microbiological confirmation of failure to achieve a disease-free cure. Conclusions: Standardized methodologies, such as our PPTR approach, could facilitate unbiased efficacy outcome determinations, improve discrimination between outcomes that are related and unrelated to regimen efficacy, and enhance the ability to conduct pooled analyses of contemporary trials.