Flow Direct: Next Generation Urine LFA for TB Diagnosis in Point-of-Care Settings

ABSTRACT Mycobacterium tuberculosis (TB) is a global threat to human health and accounts for 20% of adult deaths in low- income countries (~1.6 million deaths/year), yet only ~23% of new TB patients receive a WHO recommended nucleic acid test, while 37% receive tests with significantly lower sensitivity (e.g., smear microscopy), and ~40%

cases do not have access to testing. The critical gap in TB diagnostics, and the weakest link in global TB care, is access to high-quality TB testing. In order to address this, the WHO has developed target product profiles for the highest-priority TB diagnostic technologies and key among them is an affordable (<$2) urine-based antigen

test (e.g., lateral flow assay or LFA) for point-of-care (POC) testing in low resource settings. An accurate urine- based antigen test that can provide both simplicity (add-wait-read operation) and low cost (<$2) has the potential to close the immense gap in access to quality TB testing. However, the only recommended and commercially available urine LFA for TB is the Determine TB LAM (DTB- LAM) antigen test from Abbott. While high specificity enables use of the DTB-LAM test as a diagnostic, its limited sensitivity has restricted its recommended use to severely immunocompromised populations (e.g., HIV+) (~10% of all TB patients). More expensive LFA technologies like the FujiFilm TB LAM LFA with silver amplification (~$8/test) and the ultra-sensitive electrochemiluminescence ELISA (ECL) ($10k instrument) platforms can offer improved limits of detection (~50 fold lower than the DTB-LAM LFA) and generally improve clinical sensitivity without compromising specificity, but they have similarly fallen short with immunocompetent patients. To overcome this unmet need, Salus has recently developed the new Flow Direct assay, an affordable (<$2) LFA-based, next-generation POC immunoassay that, similar to Abbott’s DTB-LAM and FujiFilm’s TB LAM assays, targets LAM. However, Flow Direct reaches beyond Abbott’s and FujulFilm’s capabilities by providing: of on-device removal of inhibitory urine matrix components, a visual readout, multiplexing with multiple Abs, and a similar (or better) LOD than the ultrasensitive ECL laboratory instrument (i.e., < 10 pg/mL of LAM). Briefly, through removal of inhibitory urine matrix components, Salus has now demonstrated the ability to detect LAM in both HIV- AND HIV+ samples that falsely test negative by ultra-sensitive ECL, meaning Flow Direct has the potential to be a single LFA-based test that can accurately diagnose TB in the vast majority of patients. Here we propose to demonstrate the value of Flow Direct for TB urine LAM by completing the following Aims: In Aim 1 (Phase I), screening, optimizing, and benchmarking membrane-based matrix removal from TB- spiked samples; In Aim 2 (Phase I), performing verification of on-device matrix removal from spiked TB+ and TB- samples; in Aim 3 (Phase II), finalizing the flow assay and device manufacturing for clinical evaluations; and in Aim 4 (Phase II), evaluating the Flow device both in bio-banked clinical samples and in-country. Results will be important to subsequent funding efforts for Flow Direct commercial development.