A stool based qPCR for the diagnosis of TB in children and people living with HIV in Uganda, Eswatini and Mozambique (Stool4TB): a protocol for a multicenter diagnostic evaluation.

BACKGROUND: Tuberculosis (TB) is a major cause of mortality worldwide. Children and people living with HIV (PLHIV) have an increased risk of mortality, particularly in the absence of rapid diagnosis. The main challenges of diagnosing TB in these populations are due to the unspecific and paucibacillary disease presentation and the difficulty of obtaining respiratory samples. Thus, novel diagnostic strategies, based on non-respiratory specimens could improve clinical decision making and TB outcomes in high burden TB settings. We propose a multi-country, prospective diagnostic evaluation study with a nested longitudinal cohort evaluation to assess the performance of a new stool-based qPCR, developed by researchers at Baylor College of Medicine (Houston, Texas, USA) for TB bacteriological confirmation with promising results in pilot studies. METHODS: The study will take place in high TB/HIV burden countries (Mozambique, Eswatini and Uganda) where we will enroll, over a period of 30 months, 650 PLHIV (> 15) and 1295 children under 8 years of age (irrespective of HIV status) presenting pressumptive TB. At baseline, all participants will provide clinical history, complete a physical assessment, and undergo thoracic chest X-ray imaging. To obtain bacteriological confirmation, participants will provide respiratory samples (1 for adults, 2 in children) and 1 stool sample for Xpert Ultra MTB/RIF (Cepheid, Sunnyvale, CA, USA). Mycobacterium tuberculosis (M.tb) liquid culture will only be performed in respiratory samples and lateral flow lipoarabinomannan (LF-LAM) in urine following WHO recommendations. Participants will complete 2 months follow-up if they are not diagnosed with TB, and 6 months if they are. For analytical purposes, the participants in the pediatric cohort will be classified into "confirmed tuberculosis", "unconfirmed tuberculosis" and "unlikely tuberculosis". Participants of the adult cohort will be classified as "bacteriologically confirmed TB", "clinically diagnosed TB" or "not TB". We will assess accuracy of the novel qPCR test compared to bacteriological confirmation and Tb diagnosis irrespective of laboratory results. Longitudinal qPCR results will be analyzed to assess its use as treatment response monitoring. DISCUSSION: The proposed stool-based qPCR is an innovation because both the strategy of using a non-sputum based sample and a technique specially designed to detect M.tb DNA in stool. PROTOCOL REGISTRATION DETAILS: ClinicalTrials.gov Identifier: NCT05047315.

Improving tuberculosis case detection through contact risk stratification by Xpert MTB/RIF Ultra and spatial parameters: Evaluation of an innovative active case finding strategy in Mozambique (Xpatial-TB).

Prompt diagnosis is critical for tuberculosis (TB) control, as it enables early treatment which in turn, reduces transmission and improves treatment outcomes. We investigated the impact on TB diagnosis of introducing Xpert Ultra as the frontline diagnostic test, combined with an innovative active-case finding (ACF) strategy (based on Xpert Ultra semi-quantitative results and spatial parameters), in a semi-rural district of Southern Mozambique. From January-December 2018 we recruited incident TB-cases (index cases, ICs) and their household contacts (HCs). Recruitment of close community contacts (CCs) depended on IC´s Xpert Ultra results, and the population density of their area. TB-contacts, either symptomatic or people living with HIV, were asked to provide a spot sputum for lab-testing. Trends on TB case notification were compared to the previous years and to those of two districts in the south of the Maputo province (control area), using an interrupted time series analysis with and without control (CITS/ITS). A total of 1010 TB ICs (37.1% laboratory-confirmed) were recruited; 3165 HCs and 4730 CCs were screened for TB. Eighty-nine additional TB cases were identified through the ACF intervention (52.8% laboratory-confirmed). The intervention increased by 8.2% all forms of TB cases detected in 2018. Xpert Ultra trace positive results accounted for a high proportion of laboratory confirmations in the ACF cohort (51.1% vs 13.7% of those passively diagnosed). The Number Needed to Screen to find a TB case differed widely among HCs (55) and CCs (153). During the intervention period, a reversal of the previous negative trend in lab-confirmed case notifications was observed in the district. However, the CITS model did not show any statistically significant difference compared to the control area. Paediatric population benefited the most from the ACF strategy and HCs screening seemed an effective intervention to find microbiological confirmed cases in early stages of the disease.

Performance of a stool-based quantitative PCR assay for the diagnosis of tuberculosis in adolescents and adults: a multinational, prospective diagnostic accuracy study.

BACKGROUND: Despite increasing availability of rapid molecular tests for the diagnosis of tuberculosis in high-burden settings, many people with tuberculosis are undiagnosed. Reliance on sputum as the primary specimen for tuberculosis diagnostics contributes to this diagnostic gap. We evaluated the diagnostic accuracy and additive yield of a novel stool quantitative PCR (qPCR) assay for the diagnosis of tuberculosis in three countries in Africa with high tuberculosis burdens. METHODS: We undertook a prospective diagnostic accuracy study in Eswatini, Mozambique, and Tanzania from Sept 21, 2020, to Feb 2, 2023, to compare the diagnostic accuracy for tuberculosis of a novel stool qPCR test with the current diagnostic standard for Mycobacterium tuberculosis DNA detection from sputum and stool, Xpert-MTB/RIF Ultra (Xpert Ultra). Sputum, stool, and urine samples were provided by a cohort of participants, aged 10 years or older, diagnosed with tuberculosis. Participants with tuberculosis (cases) were enrolled within 72 h of treatment initiation for tuberculosis diagnosed clinically or following laboratory confirmation. Participants without tuberculosis (controls) consisted of household contacts of the cases who did not develop tuberculosis during a 6-month follow-up. The performance was compared with a robust composite microbiological reference standard (CMRS). FINDINGS: The cohort of adolescents and adults (n=408) included 268 participants with confirmed or clinical tuberculosis (cases), 147 (55%) of whom were living with HIV, and 140 participants (controls) without tuberculosis. The sensitivity of the novel stool qPCR was 93·7% (95% CI 87·4-97·4) compared with participants with detectable growth on M tuberculosis culture, and 88·1% (81·3-93·0) compared with sputum Xpert Ultra. The stool qPCR had an equivalent sensitivity as sputum Xpert Ultra (94·8%, 89·1-98·1) compared with culture. Compared with the CMRS, the sensitivity of the stool qPCR was higher than the current standard for tuberculosis diagnostics on stool, Xpert Ultra (80·4%, 73·4-86·2 vs 73·5%, 66·0-80·1; p=0·025 on paired comparison). The qPCR also identified 17-21% additional tuberculosis cases compared to sputum Xpert Ultra or sputum culture. In controls without tuberculosis, the specificity of the stool qPCR was 96·9% (92·2-99·1). INTERPRETATION: In this study, a novel qPCR for the diagnosis of tuberculosis from stool specimens had a higher accuracy in adolescents and adults than the current diagnostic PCR gold standard on stool, Xpert-MTB/RIF Ultra, and equivalent sensitivity to Xpert-MTB/RIF Ultra on sputum. FUNDING: National Institutes of Health (NIH) Allergy and Infectious Diseases, and NIH Fogarty International Center.

Fine-grain population structure and transmission patterns of Mycobacterium tuberculosis in southern Mozambique, a high TB/HIV burden area

Genomic studies of the Mycobacterium tuberculosis complex (MTBC) might shed light on the dynamics of its transmis￾sion, especially in high-burden settings, where recent outbreaks are embedded in the complex natural history of the disease. To this end, we conducted a 1 year prospective surveillance-based study in Mozambique. We applied whole￾genome sequencing (WGS) to 295 positive cultures. We fully characterized MTBC isolates by phylogenetics and dating evaluation, and carried out a molecular epidemiology analysis to investigate further associations with pre-defined transmission risk factors. The majority of strains (49.5%, 136/275) belonged to lineage (L) 4; 57.8% of them (159/275) were in genomic transmission clusters (cut-off 5 SNPs), and a strikingly high proportion (45.5%) shared an identical genotype (0 SNP pairwise distance). We found two 'likely endemic' clades, comprising 67 strains, belonging to L1.2, which dated back to the late 19th century and were associated with recent spread among people living with human immunodeficiency virus (PLHIV). We describe for the first time the population structure of MTBC in our region, a high tuberculosis (TB)/HIV burden area. Clustering analysis revealed an unforeseen pattern of spread and high rates of progression to active TB, suggesting weaknesses in TB control activities. The long-term presence of local strains in Mozambique, which were responsible for large transmission among HIV/TB-coinfected patients, calls into question the role of HIV in TB transmission.