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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., (© 2024. The Author(s).)

Genomic studies of the Mycobacterium tuberculosis complex (MTBC) might shed light on the dynamics of its transmission, 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.