Quantification of Circulating Antigens for Pediatric TB Diagnosis andTreatment Monitoring



 DESCRIPTION (provided by applicant): Conventional protocols for adult and pediatric tuberculosis (TB) diagnosis and treatment monitoring rely heavily on time-consuming bacterial culture or unquantifiable DNA detection methods for the presence of small numbers of bacteria. For pediatric TB, diagnosis and treatment are particularly difficult because current clinical protocols demand much from these young patients. Although they comprise a small percentage of the health care caseload, children who are co-infected with human immunodeficiency virus (HIV) and TB represent one of the most vulnerable groups with one of the highest mortality rates. We choose this challenging cohort (provided by a NIAID-sponsored clinical trial) to push the boundaries of what our technology platform can do. In order to address the current limitations in clinical management of pediatric TB, we have developed a rapid and quantitative diagnostic and monitoring platform for active TB wherein porous silicon nanodisks (referred to as "pSiND"), loaded with target biomarkers, are used for identification of disease signatures by a bench-top mass spectrometry (MS). Using this new biomarkers detection modality, pSiND-MS, we could detect and quantify two TB-specific blood-born biomarkers (CFP-10 and ESAT-6) at extremely low concentrations (1.0 fmol), which serve as signs of bacterial infection in advance of physiological manifestations observable by conventional protocols. Characteristics of ESAT-6 and CFP-10, i.e., antigens secreted by actively proliferating Mycobacterium tuberculosis (Mtb), make them ideal biomarkers for active TB diagnosis and candidates for TB vaccine development. We used this approach to distinguish adult patients with active TB from those with latent tuberculosis infection, or healthy volunteers. Also, based on multiplex detection and quantification by pSiND- MS, we differentiated pediatric TB-infected patients (n=36) from non-TB children (n=35) according to the peptidic patterns of CFP-10 and ESAT-6. Our categorization of patient samples matched clinical designations at 100% specificity and 94.4% sensitivity. Just as important, we could render accurate diagnoses within one hour of sample-to-answer processing rather than wait the typical 4-6 weeks. In this proposal, we aim to: 1) design and develop the scale-up nanodisk microfabrication protocol with FDA compliant in cGMP facilities; 2) conduct an extensive clinical validation of pSiND-MS using samples from a large cohort of children with HIV/TB; 3) determine effectiveness of our approach for rapid evaluation of treatment efficacy; and 4) optimize development and clinical validation of a portable pSiDN-miniMS system for identification and quantification of CFP-10 and ESAT-6. The pSiND-MS technology platform has an added advantage in that high-throughput and accurate mass spectrometry has become a virtually essential technology for clinical diagnosis in many parts of the world. The "miniaturized" and easy-to-use MS system at a shoebox size for point-of-care applications is aimed at serving patients in resource-limited areas. Achievement of all of our aims will significantly improve clinical management strategies for global TB control.