3STEP: A swab-seal-read assay for tuberculosis and RIF resistance enabled by autonomous extraction technology

ABSTRACT In 2021 there were 10.6 million new cases of Mycobacterium tuberculosis (TB) and 1.6 million TB-related deaths, making TB the second leading infectious cause of death worldwide behind only COVID-19. Standard TB diagnostic methods are insufficient in that they either 1) utilize challenging sample types and are often invasive (e.g., sputum, gastric aspiration, lymph node biopsy), 2) lack sufficient sensitivity and/or specificity (e.g., culture, reliance on clinical observation alone), or 3) cannot be widely implemented in community point-of-care (POC) settings. As a result, there remains a significant unmet need for affordable POC technologies that can be used at local or temporary clinics, urgent care facilities, pharmacies, and school health clinics with easier to acquire samples (e.g., urine or oral swabs). In addition, TB is notoriously difficult to lyse adding another challenge to the creation of a POC solution. Salus Discovery has developed 3STEP, a new LAMP-based POC NAT for TB that directly overcomes the above mentioned challenges. 3STEP provides lab-quality, PCR sensitivity (<10 CFU/swab analytical sensitivity) with the speed and simplicity of a lateral flow assay (LFA), (i.e., a simple tongue swab, 3 user steps, no liquid transfers, and results within ~30 minutes). 3STEP also addresses challenges of spectral multiplexing in LAMP reactions by enabling spatial multiplexing for integrated detection of drug-resistant infections. Importantly, our novel stabilized interface technology (SIFT) used to create 3STEP enables both an ultra-cheap disposable and an affordable isothermal instrument design. In this SBIR Fast Track Phase I/Phase II proposal we will continue developing 3STEP for the detection of TB and rifampin resistant (RR) TB from tongue swabs by: In Aim 1 (Phase I), optimizing 3STEP for TB oral swabs using contrived samples, including designing and implementing RR testing into the spatial multiplexing capabilities of 3STEP; In Aim 2 (Phase I), prototyping and testing a POC lysis module; In Aim 3 (Phase I), assessing clinical sensitivity in a small pilot study using bio-banked samples collected through FIND’s FEND-TB program; In Aim 4 (Phase II), finalizing the 3STEP assay and consumable for an expanded clinical evaluation; And in Aim 5 (Phase II), performing a larger scale clinical evaluation of the multiplexed 3STEP TB/RR assay. Results will be important to subsequent funding efforts for 3STEP commercial development (assay and reader) and to demonstrate field-readiness for participation in TB diagnostic evaluation studies similar to R2D2, DriveDx4TB, SMART4TB, and FEND-TB.