Tuberculosis (TB) is a global health threat, but can be difficult to diagnose and manage in pediatric patients due to the weak performance and non-quantitative nature of frontline diagnostic assays, which function even worse when used with patients co-infected with human immunodeficiency virus (HIV). There is an unmet need for a rapid, non-sputum-based quantitative test to detect active TB cases and anti-TB treatment responses in clinically diverse pediatric populations. The proposed research will overcome these obstacles by developing a solid-state nanopore biosensor assay that can rapidly diagnose pediatric TB by measuring Mycobacterium tuberculosis (Mtb)-secreted antigens in patient serum samples. My solid-state nanopore system is easy to operate, has low fabrication/instrument costs, and can perform high-throughput and ultra-sensitive measurements on specific Mtb-derived peptides. The robust portability of this platform also allows its use in resource-limited areas that are subject to high TB prevalence. We identified two highly Mtb-specific peptide fragments of the Mtb virulence antigens CFP-10 and ESAT-6, and validated their clinical performance as biomarkers for active TB disease diagnosis using a mass spectrometry-based assay with 201 adult and 123 pediatric patients and controls chosen from highly relevant cohorts (e.g. HIV-positive/negative, pulmonary/extrapulmonary, Mtb culture-positive/negative, latent TB, and nontuberculous mycobacteria infections). Similar robust overall diagnostic sensitivities and specificities obtained using these biomarkers in adults (88.6% / 93.8%) and children (88.2% / 97.2%) significantly outperformed those reported for other frontline tests. Quantification of serum Mtb antigen concentration was also informative in monitoring the response to anti-TB treatment in HIV- positive adults. My recent results show that nanopores can accurately detect CFP-10 and/or ESAT-6 peptides in a pilot cohort of pediatric TB cases, and indicate that statistical analysis of nanopore results for peptide profiling holds significant diagnostic promise. Based on these findings, I propose that the portable solid-state nanopore biosensor to be analyzed in these studies can improve TB diagnosis in children, particularly in resource-limited areas with high TB prevalence. I will leverage this system and the robustness of Mtb antigen-derived peptide biomarkers to: (1) develop a nanopore-based TB diagnostic assay; (2) validate this assay in a pediatric cohort with and without HIV co- infection; and (3) determine whether Mtb antigens concentrations in serum decrease in response to treatment in a pilot pediatric cohort during anti-TB treatment. Given the success of these proof-of-concept studies, the long- term goal of the proposed research program is to build prototype devices for large-scale on-site clinical validation studies in high TB burden regions, and to extend this biosensing platform to the detection of other disease biomarkers. This research program should hasten the translation of a promising biosensing method into a practical clinic-ready point-of-care tool for disease diagnosis.
Diagnosis of tuberculosis in children is extremely challenging due to atypical symptoms and low bacterial levels associated with pediatric disease, as well as difficulty obtaining required diagnostic specimens. In this work, we propose to employ solid-state nanopore technology, which can recognize single detection events to quantify two tuberculosis-specific peptide biomarkers derived from blood samples, to develop a point-of-care diagnostic system for resource-limited areas. Based on our preliminary studies, we are confident that this diagnostic system will benefit the global tuberculosis control effort by improving early diagnosis in children.