Evaluating a New Stool Based qPCR for Diagnosis of Tuberculosis in Children and People Living With HIV

Tuberculosis (TB) continues to be a leading cause of morbidity and mortality among children and people living with HIV (PLHIV). Despite significant progress in TB diagnostics, improvement of childhood TB diagnosis continues to be a major challenge and is a key pillar of the WHO "End TB Strategy". TB laboratory confirmation is particularly challenging in children and PLHIV given the difficulty in obtaining sputum samples, and the pauci-bacillary nature of disease. In consequence, bacteriological confirmation of pulmonary TB in young children and immunosuppressed PLHIV remains disappointingly low. Inability to bacteriologically confirm TB, results in both i) under diagnosis which leads to worse outcomes including increased mortality and ii) over or under diagnosis, and poor resource allocation. Given the limitations of currently available sputum-based diagnostic tests in these vulnerable populations, there is a need to develop new tools and identify easy to collect non-respiratory specimens which, combined, could improve bacteriological confirmation. Preliminary data suggest that a new platform, an innovative stool homogenization and DNA isolation method, adds value to existing sputum-based diagnostics by increasing the rates of bacteriological confirmation, and could also be useful as a monitoring tool for treatment response. This platform has the potential to be adapted to a point of care (POC) diagnostic and thus easily implemented in resource-constrained basic health care centres.

Objective: The aim of this project is to evaluate the diagnostic performance of the stool bead-based real-time quantitative PCR (qPCR) platform for TB diagnosis in children and PLHIV. This will be evaluated in the high TB and HIV burden settings of Mozambique, Eswatini and Uganda, under the hypothesis that it will narrow the large TB case detection gap by improving TB confirmation rates in children and PLHIV, while proving feasible and acceptable.

Primary objective:

• Evaluate the diagnostic accuracy of a stool-based real-time quantitative PCR for detecting DNA of Mycobacterium tuberculosis (MTB qPCR), compared to a composite reference standard (sputum and stool Xpert Ultra, sputum culture and urine TB-LAM).

Secondary objectives:

• Evaluate the diagnostic accuracy of the stool-based Mtb qPCR to individual sputum and stool Xpert Ultra, urine LAM, sputum smear and sputum culture (MGIT) results using a consensus clinical case definition as the reference standard.
• Evaluate the usefulness of the quantitative stool-based MTB qPCR platform as a tool to monitor treatment response for children and PLHIV.
• To create a biorepository of well characterized pediatric samples at each of the study sites to support the future development and evaluation of novel biomarker research.

Methodology: This is a prospective diagnostic evaluation study with a nested longitudinal cohort evaluation. During a 30-month recruitment period, people with presumptive TB will consecutively enroll being part of two study groups: Children less than 8 years of age, irrespective of HIV status (N=1295) and adults living with HIV, irrespective of immunological status (N=650). Sixty extra people will take part as healthy (asymptomatic) controls. After clinical, radiological and bacteriological evaluation, TB cases will be treated according to national guidelines. Participants diagnosed with TB will be followed up for 6 months since treatment initiation in order to assess treatment response and outcomes. A clinical case definition will be established as a reference standard and defined as any participant in whom a decision is made to start ATT (TB cases will be classified as confirmed or unconfirmed). Specifically, children will be classified into 3 distinct pediatric endpoint categories (confirmed TB, unconfirmed TB and Unlikely TB) based on bacteriological, radiological and clinical criteria, following international consensus guidelines.