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.

D.5. Primary Outcomes, Statistical Power, and Sample Size The primary outcome for Aim 1 is cfDNA levels for a) children with confirmed and unconfirmed TB ("TB cases"), b) children with unlikely TB (UTB), and c) well children (WC). Based on preliminary data, the mean cfDNA levels (and standard deviations) for these groups were 4.2 (4.0), 2.0 (3.2), and 1.1 (0.03), respectively. We hypothesize that TB children will have significantly higher serum concentrations of cfDNA than the UTB and WC groups. Statistical power and sample size: With a two-sided type I error is set at 0.05, statistical power set at 80%, 42 subjects per group are required to detect a significant difference between the TB and UTB children (based on the formulae by Hulley et al. ). Comparisons between the TB and WC group requires 13 children per comparison group. Based on data from current studies and local Ministry of Health (see D.7. below) we anticipate 135 TB cases per year (55 in 2 mo-6 yrs.; 80 in 7-14 yrs.), or approximately 200 TB cases per 18-month study period (for test population and validation population). We also anticipate 80 unlikely TB cases per year (40 in 2 mo-6 yrs.: 40 in 7-14 yrs.), or approximately 105 unlikely TB cases per 18-month study period (18 months each for test population and validation population recruitment). Subjects will be stratified into two age groups (2 months-6 years, 7-14 years). With 42 required TB cases per age group, our proposed sample exceeds the minimum requirements by 50% or more per age group. The enrollment targets here will provide flexibility in meeting recruitment goals and non-response rates.

D.6. Procedures and specimen collection. For hospitalized patients with suspected tuberculosis, enrollment, clinical evaluation, and sample collection are performed in the same room; for outpatients the process of clinical evaluation, gastric aspirate sampling will be performed in the consulting room of Pneumology clinic at the pediatric hospital and blood sampling will be collected in the clinical laboratory phlebotomy room. Follow-up specimen collection will be done through community clinics where patients receive their weekly allotment of TB medicines, with follow-ups at 1- and 2-weeks post treatment initiation. A 2-month follow-up visit in conjunction with a routine physician visit is scheduled, to allow for a later follow-up cfDNA biomarker specimen.

All participants under evaluation for TB will have procedures at the time of enrollment including:

* Medical history and clinical evaluation required to accurately assess each criterion that contributes to their subsequent assignment to TB outcome groups, based on the 2015 revised consensus criteria. * Quantiferon-TB Gold In-Tube testing to detect immunologic evidence of tuberculosis (including latent infection \[LTBI\]) will be performed in our laboratory at Universidad Catolica, based on standard protocols * HIV serology unless known to be HIV+, by Abbott Alere Determine HIV 1/2 (See D.8 Data analysis for analytical adjustments by HIV serostatus) * COVID-19 PCR or antigen test-- suspect TB patients only. (If positive these subjects are excluded) * Chest radiograph-- suspect TB patients only; not performed on well controls. AP and lateral views. Digital images processed in DICOM will be transmitted to Dr. Zimic's lab for analysis. * Lung ultrasound-- suspect TB patients only; Not performed on well controls. Ultrasound examination is performed based on a standard protocol supervised by Dr. Fentress, imaging the chest in perpendicular planes in the midclavicular line anteriorly and posteriorly from apices to diaphragm, and in the midaxillary line from axilla to diaphragm, for a total of 12 views per participant. , , * Specimen collection for TB microbiology and PCR-- suspect TB patients only; not performed on well controls. All subjects with a work-up for suspected TB will have at least three sputum-equivalent specimens collected for TB microbiology and PCR analysis by Xpert MTB/RIF. Acceptable specimens include (1) gastric aspirates, (2) expectorated sputum (for older subjects who can produce sputum), or (3) string test specimens, conducted by our standard protocol. Specimens will be collected in the early morning prior to eating, and specimen collection times will be separated by at least 24 hours. In cases where parents or attending physicians only agree to two specimens, a third specimen will not be collected. Anti-TB treatment will be initiated when indicated by the physician following collection of the last specimen for TB microbiology. * Microbiologic analysis for TB isolation and detection will be performed on specimens collected from suspect TB patients, including (1) Ziehl-Neelsen AFB smear, (2) TB culture, and (3) and TB PCR detection by Xpert MTB/RIF. TB cultures will use the MODS technique that we developed in Peru 8, 10 * Serum for cfDNA collected by venipuncture. We ship all serum cfDNA samples to Tulane by World Courier, on dry ice, and we aliquot all samples so half will be shipped and the other half stored at -80 C.

Participation by well control subjects will end after enrollment specimen collection and evaluations. TB treatment will be initiated for all subjects with presumptive TB disease, based on the attending physician's clinical opinion and based on Bolivian national TB treatment standards. Study participants with compatible disease and on anti-TB therapy will have the follow-up procedures listed at the times specified:

* Clinical evaluation (1 week, 2 weeks, and 2 months post enrollment and start of treatment) * Serum for cfDNA and whole blood for CBC (1 week, 2 weeks, and 2 months post enrollment) * For subjects on antituberculous therapy and not demonstrating improvement-repeat evaluation (per clinician decision at 2 weeks or 2 months post enrollment). Minimum 2 samples for smear, culture, and Xpert MTB/RIF (Gastric aspirates \[by intubation/string test\] or sputum)

TB culture techniques. Specimens collected will be cultured for M. tuberculosis using the Microscopic-Observation Drug-Susceptibility (MODS) Method.8 Standard volumes of each decontaminated specimen will be inoculated into modified Middlebrook 7H9 media and cultured in a sterile 24-well plate. Plates are placed in a plastic resealable bag, incubated at 37°C, and examined every other day for up to 30 days by inverted light microscopy. Presumptive TB isolates with cording are reported as positive and confirmed by IS 6110 PCR.

CRISPR-TBD assays: The circulating cfDNA will be extracted with Quick-cfDNA Serum \& Plasma at the Hu lab at Tulane. CRISPR-TBDB requires an PCR-based target amplification prior to CRISPR-mediated fluorescent signal production, following procedures recently published by Huang et. al. 23 The CRISPR-TBDB data will be evaluated in silico analysis using SnapGene software (version 5.0.8) and by triplicate CRISPR-TBDB assays.

Chest radiograph and lung ultrasound studies and data processing. See Specific Aim #3 below.

D.7. Study group assignment. We will employ an observational study design with 5 study groups of children ages 2 months to 14 years, as detailed below. These are A) confirmed pulmonary TB, B) unconfirmed pulmonary TB, C) unlikely TB, D) well age-matched controls (which will be divided into 2 groups based on presence or absence of LTBI). Most children evaluated for TB and started on treatment as determined by the attending (non-study) physician will fall into groups A (approximately 10%) and B (60-65%), based on recent data. Groups A and B together are referred to here as "TB cases". Approximately 25-30% of subjects who meet entry criteria based

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