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
Authors: Carratala-Castro Lucia , Ssengooba Willy , Kay Alex , Acácio Sozinho , Ehrlich Joanna , DiNardo Andrew R , Shiba Nosisa , Nsubuga Joachim K , Munguambe Shilzia , Saavedra-Cervera Belén , Manjate Patricia , Mulengwa Durbbin , Sibandze Busizwe , Ziyane Mangaliso , Kasule George , Mambuque Edson , Sekadde Moorine Penninah , Wobudeya Eric , Joloba Moses L , Heyckendorf Jan , Lange Christoph , Hermans Sabine , Mandalakas Anna , García-Basteiro Alberto L. , Lopez-Varela Elisa ,   on behalf of Stool4TB Global Partnership, Sanz Sergi , Dlamini Makhosazana , Dlamini Gcinile , Dlamini Nomathemba , Mthethwa Nkulungwane , Kota Nokwanda , Dube Mbongeni , Maphalala Nontobeko , Nkala Babongile , Dlamini Faith , Shabalala Fortunate , Dlamini Sindisiwe , Maphalala Gugu , Dlamini Lindiwe , Dube Sisi , Fonseca Lee Joao , Machele Nércio , Cumbe Miguel , Lima Agostinho , Magul Katia , Tembe Gustavo , Mudumane Benilde Violeta , Cebola Farida , Rungo Jorcelina , Bila Junior Alberto , Gomes Neide , Mwachan Patricia , Nassolo Maria , Katuwal Sujan , Ang Matthew , Vasiliu Anca , Mejía Rojelio , Bacha Jason , Vambe Debrah , Seeger Abigail , Kontsevaya Irina , Musia Collins , Komba Lilian , Minga Lwijisyo , Mwita Lumumba , Bariki Mtafya , Ntinginya Nyanda Elias 
Published: February 2024

Abstract
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.