Diagnostic Innovations for Pediatric Tuberculosis in Bolivia

Pediatric subject populations, enrollment, and follow-up: During the first two years of the study, a "test population" will be recruited for 18 months to establish quantitative cfDNA standards and ranges for each clinical outcome group, to assess predictive values for cfDNA levels as a biomarker of clinical outcome. We will characterize the dynamics of cfDNA levels in peripheral blood in two age-based groups of children (2 months-6 years ["younger children"], 7-14 years ["older children"]). A stratified analysis with these age-based subgroups is logical because the diagnostic test yield and clinical presentations are different in these groups.

For Aim 2 during years 3-4 of the study, a "validation population" will be recruited for 18 months to validate quantitative cfDNA standards and ranges for each clinical outcome group. Subject recruitment, informed consent, data collection, and study groups for analysis will be the same as for the "test population" in Specific Aim #1. Subjects in each study group will be stratified into age-based subgroups (2 months-6 years, 7-14 years). Serial levels of cfDNA will be assessed and characterized for children in the confirmed and unconfirmed TB groups, to validate normalization of cfDNA values with effective anti-TB therapy.

Table 1. Project Timeline

Initial enrollment and data/specimen collection will be done prior to initiation of TB treatment in the hospital or clinic setting. Inclusion criteria: Children ages 2 months to 14 years identified through the clinics and hospital wards of the "Dr. Mario Ortiz Suarez" Children's Hospital in Santa Cruz and presenting for evaluation for symptomatic respiratory disease and suspicion of tuberculosis will be eligible for enrollment (inclusion criteria based on Bolivian Ministry of Health guidelines for suspect cases of tuberculosis in children ). After screening for exclusion criteria (prior treatment for TB within the past year, current treatment for prevention of TB, weight < 2.5 kg., or clinical instability, positive COVID-19 diagnostic test) study staff will present the study verbally to the parents and provide a brochure with more detailed information designed for both parents and older. Parental informed consent and pediatric participant assent will be obtained. The study will include collection of specimens for diagnostic testing and clinical data as outlined below, but decisions on treatment for tuberculosis will be made by the attending physician who is not involved in the study. As the study groups for data analysis are determined in part based on the results of diagnostic tests performed and on clinical response to treatment, subjects will not be assigned to study groups on enrollment. Study group assignment (confirmed TB, unconfirmed TB, and unlikely TB) will be determined by the project biostatistician after the subject completes all study activities (see D.5. and D.7. study group assignment for criteria and sample size by group). As the Bolivian guidelines for pediatric TB allow for clinical evaluation of children with respiratory disease and only a few TB-related criteria, this recruitment strategy will enroll a population of suspect TB patients that will allow us to compare outcomes in "TB cases" (confirmed TB and unconfirmed TB) and in ill patients who do not meet NIH case definitions for unconfirmed TB (i.e., the unlikely TB group, see D7). This unlikely TB group will serve as "ill/respiratory disease controls", separate from the "well controls" group. On a weekly basis, well control subjects without respiratory symptoms and age-matched (+ 2 years) to suspected TB cases will also be recruited from community health clinics. Well controls will only have a single set of specimens, and no invasive specimens.

D.4. Bolivia study staff: Recruitment of study participants and specimen collection activities will be managed by a physician study coordinator, supervised by co-investigator Dr. Ramiro Cabrera, pediatric pulmonologist and regional consultant for pediatric tuberculosis in Santa Cruz. Clinical and patient related activities will be further overseen by PIs Richard Oberhelman (pediatric infectious diseases specialist) and Robert Gilman (ID specialist), as well as by ID specialists Jeffrey Tornheim and Lima-based Prisma site director Carlton Evans.