Angelique Kany Kany Luabeya

The WHO has recommended tongue swabs (TS) as alternative samples for microbiological diagnosis of tuberculosis. We evaluated the effects of oral hygiene and food/drink intake on TS performance in South Africa. Food/drink intake prior to sampling marginally decreased Mycobacterium tuberculosis DNA signal strength, but neither behaviour decreased diagnostic sensitivity.

Mycobacterium tuberculosis (MTB) disease is a major global health threat with most tuberculosis (TB) cases occurring in low-and middle-income countries (LMIC) with limited healthcare infrastructure. Near-point-of-care testing which can be deployed at peripheral clinical settings is needed to start treatment earlier and thereby improve treatment outcomes. Here we report the development and preliminary characterization of an MTB detection assay that utilizes tongue swab or sputum specimens for The DASH® Rapid PCR System which employs cartridge-based automated sequence specific capture sample prep combined with dual target qPCR multicopy MTB insertion sequences IS 6110 and IS 1081 amplification and detection. MTB is resistant to conventional bacterial lysis techniques; therefore, we evaluated two pre-cartridge lysing techniques, mechanical lysis and sonication, and selected sonication for all subsequent studies. The DASH MTB assay demonstrated a limit of detection of 2.5 MTB cells/swab with no detection of 10 non-tuberculosis Mycobacterium strains. Clinical testing of 100 (49 positive and 51 negative) de-identified blinded sputa from South African symptomatic clinic attendees yielded an overall test sensitivity of 96% (100% for smear positive samples and 88% for smear negative samples) and specificity of 88% when compared to sputum culture. In a separate study of 110 tongue swab specimens (70 positive and 40 negative) from South African symptomatic clinic attendees, the sensitivity was 91% and the specificity was 100%. We further demonstrated that the test is compatible with peripheral LMIC settings via external battery operation and cartridge stability at 45°C for up to one year. Importance: Tuberculosis (TB) is the single most deadly infectious disease with 1.23 million deaths in 2024. Near-point-of-care testing which can be deployed at peripheral settings that lack laboratory infrastructure to deliver prompt and accurate diagnosis is needed to start treatment earlier and thereby improve treatment outcomes. In this study, we have developed an automated test to detect Mycobacterium tuberculosis (MTB), the cause of TB, from sputum and tongue swab specimens. Its high sensitivity and specificity, rapid time to result, and compatibility with environments that lack air conditioning and consistent electricity make this assay suitable for diverse clinical settings.

Tongue swabs (TS) are novel non-sputum specimens for molecular diagnosis of tuberculosis (TB). Analysis of swabs by using Cepheid Xpert MTB/RIF Ultra® (Xpert Ultra) is less sensitive than the use of certain manual qPCR methods but is desirable given the widespread use and familiarity of Xpert Ultra. This study evaluated an easy-to-use protocol for analyzing TS by Xpert Ultra. TS samples from symptomatic South African patients were previously collected and tested by Xpert Ultra using a protocol that requires a heating block (termed Heat+TE). Replicate, paired samples from the same patients were tested by a newly reported method that does not require heating (termed 2:1 SR). The diagnostic accuracy of the two methods was compared. Because the sensitivity of TS testing is lowest in patients with low sputum bacillary loads, the resolving power of this paired comparison was maximized by prioritizing samples from such participants (N=108), 88 of whom were TB-positive by sputum microbiological reference standard (MRS) and 20 were TB-negative. Within this sample set, the Heat+TE method was 42.5% (38/88; 95CI 33-54%) sensitive and the 2:1 SR method was 52.3% (46/88; 95CI 41-63%) sensitive relative to sputum MRS. Both methods were 100% (20/20; 95% CI 83-100%) specific. A secondary analysis compared the sensitivities of single-sample versus dual-sample testing within a cohort (N=241) that included both high- and low-bacillary load participants. Testing of a single sample was 75.5% (182/241; 95CI 70-81%) sensitive relative to sputum MRS, whereas dual sampling was 81.3% (196/241; 95CI 76-86%) sensitive.

Background New safe and effective TB vaccine strategies to replace infant BCG vaccination are needed urgently. We evaluated the safety, reactogenicity and immunogenicity of three doses of the live-attenuated Mycobacterium tuberculosis (Mtb) vaccine candidate, MTBVAC, in comparison to BCG in South African newborns. Methods Healthy infants, HIV-unexposed, BCG-naïve newborns without history of close TB contact were randomly allocated into three sequential cohorts to receive a single intradermal dose of BCG (SSI, 2.5×10⁵ CFU) or MTBVAC (2.5×10⁴; 2.5×10⁵ CFU; or 2.5×106 CFU). Results 228 pregnant women consented, and 99 newborns were enrolled. Seventy-eight infants across all 3 cohorts had local reactions, all rated mild, except one grade 2 erythema. Induration, swelling, and erythema was more common with increased MTBVAC dosage. Induration and swelling were more common in MTBVAC 2.5x106 than in BCG and reactogenicity in MTBVAC 2.5x105 was same as BCG. Twelve infants experienced 14 vaccine-unrelated SAEs including one death due to bronchopneumonia. Eight infants commenced TB treatment for unconfirmed pulmonary TB (BCG n=4 and MTBVAC 2.5x104 CFU n=4) and one for unconfirmed TB meningitis (BCG). MTBVAC was highly immunogenic at all 3 doses, inducing predominantly Th1-cytokine-expressing CD4 T-cells, which peaked at day 56 and waned thereafter. The 2.5×105 and 2.5×106 CFU MTBVAC doses were more immunogenic than BCG, inducing very similar response magnitudes and phenotypes. Vaccination with any MTBVAC dose resulted in QFT conversion in most infants at Day 56, but these responses waned and reverted to QFT-negative in more than half by the end of the 1-year follow-up period. Conclusion MTBVAC appeared safe and well tolerated and immunogenic at doses between 2.5×10⁴ CFU and 2.5×106 CFU in South Africans newborns. The 2.5×105 CFU MTBVAC dose was selected for the ongoing phase 3 trial in a high TB prevalence setting.

Oral swabs are emerging as a non-invasive sample type for diagnosing infectious diseases including Ebola, tuberculosis (TB), and COVID-19. To assure proper sample collection, sample adequacy controls (SACs) are needed that detect substances indicative of samples collected within the oral cavity. This study evaluated two candidate SACs for this purpose. One detected representative oral microbiota ( Streptococcus species DNA) and the other, human cells (human mitochondrial DNA, mtDNA). Quantitative PCR (qPCR) assays for the two target cell types were applied to buccal swabs (representing samples collected within the oral cavity) and hand swabs (representing improperly collected samples) obtained from 51 healthy U.S. volunteers. Quantification cycle (Cq) cutoffs that maximized Youden’s index were established for each assay. The streptococcal target at a Cq cutoff of ≤34.9 had 99.0% sensitivity and specificity for oral swab samples, whereas human mtDNA perfectly distinguished between hand and mouth swabs with a Cq cutoff of 31.3. The human mtDNA test was then applied to buccal, tongue, and gum swabs that had previously been collected from TB patients and controls in South Africa, along with “air swabs” collected as negative controls (total N = 292 swabs from 71 subjects). Of these swabs, 287/292 (98%) exhibited the expected Cq values. In a paired analysis the three oral sites yielded indistinguishable amounts of human mtDNA, however PurFlock™ swabs collected slightly more human mtDNA than did OmniSwabs™ (p = 0.012). The results indicate that quantification of human mtDNA cannot distinguish swabs collected from different sites within the mouth. However, it can reliably distinguish oral swabs from swabs that were not used orally., which makes it a useful SAC for oral swab-based diagnosis.

SETTING South Africa. OBJECTIVE 1) To measure changes in the adolescent prevalence of latent tuberculous infection (LTBI) between 2005 and 2015, and 2) to evaluate medium-term impact of TB control measures on LTBI prevalence. DESIGN We compared baseline data from a cohort study (2005–2007) and a vaccine trial (2014–2015) which enrolled adolescents from the same eight South African high schools. LTBI was defined based on QuantiFERON^®-TB Gold In-Tube test positivity. RESULTS We analysed data from 4880 adolescents between 2005 and 2007, and 1968 adolescents between 2014 and 2015, when the average LTBI prevalence was respectively 43.8% (95%CI 28.4–59.1) vs. 48.5% (95%CI 41.1–55.8). Age-specific LTBI prevalence increased between the ages 12 and 18 years by 13% only in lower socio-economic quintile schools, where the average LTBI prevalence was unchanged between the two periods (54% vs. 53%). In the highest socio-economic quintile schools, LTBI prevalence did not increase with age; however, the average LTBI prevalence increased from 20% to 38% between the two periods. CONCLUSION Adolescent LTBI prevalence remained high and constant over a decade, suggesting that Mycobacterium tuberculosis transmission to children was not impacted in the medium term by effective TB control efforts. Trends in adolescent LTBI prevalence should be interpreted in the context of the sociodemographic factors that affect the risk of transmission before and during adolescence.

Diagnostic tests for tuberculosis (TB) usually require collection of sputum, a viscous material derived from human airways. Sputum can be difficult and hazardous to collect and challenging to process in the laboratory. Oral swabs have been proposed as alternative sample types that are noninvasive and easy to collect. This study evaluated the biological feasibility of oral swab analysis (OSA) for the diagnosis of TB. Swabs were tested from South African adult subjects, including sputum GeneXpert MTB/RIF (GeneXpert)-confirmed TB patients (n = 138), sputum GeneXpert-negative but culture-positive TB patients (n = 10), ill non-TB patients (n = 37), and QuantiFERON-negative controls (n = 34). Swabs were analyzed by using a manual, nonnested quantitative PCR (qPCR) targeting IS6110. Two swab brands and three sites within the oral cavity were compared. Tongue swabbing yielded significantly stronger signals than cheek or gum swabbing. A flocked swab performed better than a more expensive paper swab. In a two-phase study, tongue swabs (two per subject) exhibited a combined sensitivity of 92.8% relative to sputum GeneXpert. Relative to all laboratory-diagnosed TB, the diagnostic yields of sputum GeneXpert (1 sample per subject) and OSA (2 samples per subject) were identical at 49/59 (83.1%) each. The specificity of the OSA was 91.5%. An analysis of “air swabs” suggested that most false-positive results were due to contamination of manual PCRs. With the development of appropriate automated methods, oral swabs could facilitate TB diagnosis in clinical settings and patient populations that are limited by the physical or logistical challenges of sputum collection.

Rationale: Global tuberculosis (TB) control requires effective vaccines in TB-endemic countries, where most adults are infected with Mycobacterium tuberculosis (M.tb). Objectives: We sought to define optimal dose and schedule of H56:IC31, an experimental TB vaccine comprising Ag85B, ESAT-6, and Rv2660c, for M.tb-infected and M.tb-uninfected adults. Methods: We enrolled 98 healthy, HIV-uninfected, bacillus Calmette-Guérin–vaccinated, South African adults. M.tb infection was defined by QuantiFERON-TB (QFT) assay. QFT-negative participants received two vaccinations of different concentrations of H56 in 500 nmol of IC31 to enable dose selection for further vaccine development. Subsequently, QFT-positive and QFT-negative participants were randomized to receive two or three vaccinations to compare potential schedules. Participants were followed for safety and immunogenicity for 292 days. Measurements and Main Results: H56:IC31 showed acceptable reactogenicity profiles irrespective of dose, number of vaccinations, or M.tb infection. No vaccine-related severe or serious adverse events were observed. The three H56 concentrations tested induced equivalent frequencies and functional profiles of antigen-specific CD4 T cells. ESAT-6 was only immunogenic in QFT-negative participants who received three vaccinations. Conclusions: Two or three H56:IC31 vaccinations at the lowest dose induced durable antigen-specific CD4 T-cell responses with acceptable safety and tolerability profiles in M.tb-infected and M.tb-uninfected adults. Additional studies should validate applicability of vaccine doses and regimens to both QFT-positive and QFT-negative individuals. Clinical trial registered with www.clinicaltrials.gov (NCT01865487).

Objective: Paediatric laboratory reference intervals used in Africa and Asia may be derived from historical intervals of predominantly Caucasian infants in Europe or North America. These intervals may therefore not be compatible with the range of normality for developing country populations. We aimed to compare haematology and biochemistry parameters in healthy South African infants with local laboratory reference intervals.Methods: We compared the baseline haematology and biochemistry results of 634 (316 male and 318 female) HIV‐unexposed infants, aged 3–6 months, living in a rural area of the Western Cape Province, South Africa, against laboratory reference intervals supplied by the South African National Health Laboratory Services (NHLS). We calculated the percentage of observed values out of bound (in terms of lower and upper limits) compared to laboratory reference intervals.Results: Of the 634 healthy infants screened, 316 (49.84%) were male and 318 (50.16%) female. A majority (91.05%) had platelet counts above the laboratory reference interval upper limit (350 × 10⁹cells/l), while over half, 54.85% and 56.98% had mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values below the lower limits of 77.0–105.0 fl and 26.0–34.0 pg, respectively. A small proportion were outside the reference limits for haematocrit, namely 15.71% below and 7.14% above the normal limits of 0.31–0.38 l/l. For male and female infants, 33.65% and 18.04% of alkaline phosphatase (ALP) values and 7.01% and 14.56% of alanine transaminase (ALT) values were above the upper limits, respectively. For male infants, 10.83% of gamma‐glutamyl transferase (GGT) values, and for female infants, 31.11% of GGT values were below the lower limits of 12 U/l for males and 15 U/l for females. We observed no significant deviations (>10% out of bound) from NHLS reference intervals in the remaining haematology and biochemistry parameters measured.Conclusions: Haematology and biochemistry parameters in apparently healthy South African infants deviate frequently from national laboratory reference intervals, including abnormalities consistent with subclinical hypochromic microcytic anaemia. It is important that clinical laboratory reference intervals for children are derived locally, rather than being adopted from Caucasian norms in developed countries, because clinical trials of vaccines, drugs and diagnostics are increasingly conducted in sub‐Saharan Africa.