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

> **NIH NIH R44** · SALUS DISCOVERY, LLC · 2024 · $295,893

## Abstract

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.

## Key facts

- **NIH application ID:** 10918778
- **Project number:** 1R44AI179588-01A1
- **Recipient organization:** SALUS DISCOVERY, LLC
- **Principal Investigator:** Jay Wyatt Warrick
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $295,893
- **Award type:** 1
- **Project period:** 2024-06-25 → 2025-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10918778

## Citation

> US National Institutes of Health, RePORTER application 10918778, 3STEP: A swab-seal-read assay for tuberculosis and RIF resistance enabled by autonomous extraction technology (1R44AI179588-01A1). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10918778. Licensed CC0.

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