Research Projects

1. Rationale of the Research Tuberculosis (TB) is a major global health burden, particularly among children, where early and accurate diagnosis remains a challenge. Pediatric TB is often paucibacillary, with nonspecific clinical symptoms and difficulty in collecting sputum or gastric lavage samples, which limits the utility of conventional microbiological diagnostic methods. Saliva is a non-invasive, easily accessible biofluid that has shown promise in diagnostic applications. Emerging evidence suggests that Mycobacterium tuberculosis (M.tb) DNA can be found as cell-free DNA (cfDNA) in body fluids such as plasma and urine. However, the presence and diagnostic utility of M.tb cfDNA in saliva has never been explored in pediatric pulmonary TB patients. This study proposes to harness salivary cfDNA as a diagnostic biomarker source for pediatric pulmonary TB and to develop a novel, rapid, and field-deployable diagnostic assay. 2. Scientific Objectives 1. Profiling of M.tb-specific cfDNA signatures in saliva samples from pediatric pulmonary TB subjects by whole genome sequencing. 2. Validation of the selectedM.tb-specific salivary cfDNA sequences in pediatric pulmonary TB patients using digital PCR. 3. Development of rapid lateral flow format targeting selected M.tb-specific salivary cfDNA signatures using an integrated recombinase polymerase amplification and CRISPR-Cas12a(RPA/CRISPR-Cas12a) molecular assay 4. Validation of the developed lateral flowRPA/CRISPR-Cas12a assay in paediatric tuberculosis suspects. 3. Hypothesis Saliva from pediatric TB patients contains short cfDNA fragments of M.tb origin that can be selectively amplified and detected using molecular techniques such as RPA and CRISPR-Cas12a-based diagnostics. These fragments are distinct from those in non-TB individuals and can serve as reliable biomarkers for disease detection. 4. Main experiments to be carried out This prospective case-control study will recruit pediatric pulmonary TB suspects (1–15 years) after consent/assent. Unstimulated saliva samples will be collected and grouped as confirmed TB, probable TB and non-tuberculosis based on clinical details, radiological assessment and microbiological analysis. Salivary cfDNA will be extracted using Qiagen's Circulating Nucleic Acid Kit and initially screened using qPCR (IS6110/MPT64) and/or GeneXpert Ultra. Positive samples will undergo whole-genome sequencing to identify unique M.tb-specific cfDNA fragments. These sequences will be validated using digital PCR (dPCR) in all study groups. Validated targets will be used to develop a rapid diagnostic assay based on Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a, integrated into a lateral flow strip. Optimization steps will include reaction temperature, enzyme-to-probe ratios, and template concentration. Finally, the developed assay will be validated using blinded samples (n=150) and results compared against clinical reference standards (CRS) and culture to assess sensitivity and specificity. 5. Significance to the Field This project will pioneer the use of saliva-derived cfDNA for pediatric TB diagnosis and introduce a novel diagnostic platform combining next-generation sequencing, CRISPR-based detection, and lateral flow strip technology. If successful, the assay will offer a non-invasive, rapid, and cost-effective diagnostic tool that can be implemented in resource-limited, high-burden settings. This will significantly improve early detection and treatment outcomes for pediatric TB and contribute to global TB control efforts.