Detecting active tuberculosis disease: a blood-based Mycobacterium tuberculosis antigen detection asssay

Project Summary/Abstract HIV infection directly impacts both Mycobacterium tuberculosis pathogenesis and disease severity. Timely and accurate diagnosis of active tuberculosis (TB) disease is critical for people living with HIV (PLWH) as TB disease accounts for one third of all HIV-related deaths. Yet, currently there are no diagnostic solutions for active TB disease that are rapid, inexpensive, and highly sensitive; and solutions that are available are designed primarily as sputum-based solutions and optimized to detect pulmonary disease. This exacerbates the diagnostic gap for PLWH, as PLWH are more likely to present with paucibacillary and extrapulmonary forms of the disease. Novel point-of-care, rapid and accurate diagnostic solutions that have been optimized and tested among PLWH are critically needed to reduce treatment delays and improve treatment outcomes. Our central hypothesis is that the identification of the M. tuberculosis specific antigen, 10-kDa culture filtrate protein or CFP-10, in circulating body fluids, specifically blood, is indicative of actively replicating bacteria, and the detection of CFP-10 could potentially be used to diagnose both pulmonary and extrapulmonary disease. Historically direct detection of M. tuberculosis specific antigens has been hampered by the expense and complexity of detecting ultra-low concentrations of circulating antigens in biofluids. However, recent technological advancements in chip-based electrochemical immunosensors have resulted in increased sensitivity, options for miniaturization, and improved operational simplicity allowing for the detection of ultra-low concentrations of target antigens in micro-volume clinical specimens. Using seed funding from SD-CFAR our team developed a prototype Mycobacteria tuberculosis antigen detection assay that successfully detected the M. tuberculosis specific antigen CFP-10 in a limited number of clinical samples from patients with culture confirmed TB. While the initial success of this prototype assay was very promising, more data is needed prior to any further clinical validation studies. In this proposed study we aim to 1) determine the analytical performance of the prototype assay by conducting a series of laboratory- based experiments using contrived samples to standardize the assay, assess its sensitivity and performance parameters (dynamic range, precision, and repeatability), determine the specificity of the assay in the presence of non-tuberculosis mycobacterium, and evaluate its storage stability: and 2) we will conduct a limited clinical performance study of the prototype assay using 144 previously collected (72 from PLWH) and well characterized clinical samples from the R2D2 network (with a pre-selected TB positivity of 50%). The low cost and relatively simple approach of the prototype assay make it ideal for use at the clinic level as a low-tech, point-of-care diagnostic. If ultimately successful, this assay has the potential to fundamentally change global TB diagnostics.