ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis (M.tb), is a leading infectious disease and cause of death worldwide. The growing burden of drug-resistant (DR)-TB is complicating TB treatment. Early diagnosis of TB with drug susceptibility testing (DST) is critical for successful treatment and is the first pillar of the World Health Organization’s (WHO) End TB Strategy. DST is achieved via phenotypic or genotypic methods. Traditionally, phenotypic DST is performed on solid (Löwenstein Jensen) or liquid media (MGIT) in a two-step process: first a culture to identify M.tb growth, and then re-culture of the isolate with the drugs to be tested. In addition to requiring biosafety level II-plus labs, the DST process, if available in low-middle income settings, can take 42 to ~6 months from sample collection to notification of results to the clinical provider resulting in treatment delays, continued transmission, and higher mortality. Conversely, genotypic DST has many advantages, including a reduced time to result (< 2h for GeneXpert) and the possibility of deployment to at or near point of care (POC). However, its widespread use in high TB burden resource-limited settings is hindered by the need for regular power supply and importantly cost. Thus, NIH/NIAID is redirecting attention to innovative and simple phenotypic DST solutions to be deployed at or near POC. The goal of this application is to develop the 1G test into the 2G test, providing higher flexibility to perform DST for 1st and 2nd frontline drugs, including drugs prescribed for DS- and DR-TB regimens such RIPE (DS-TB oral regimen composed of RIF/INH/PZA/Ethambutol), HPMZ (DS-TB 4-month short course oral drug regimen composed of INH/Rifapentine/MFX/PZA) and BPaL [MDR- and pre-XDR oral drug regimen composed of bedaquiline (BDQ), pretomanid (PMD) and linezolid (LNZ)], as well as clofazimine (CFZ) and delamanid (DLM), other WHO recommended oral agents for DR-TB. Because the 2G test is non-proprietary, its cost is expected to be extremely low (< $8) and mainly driven by the cost of drugs. Further, for the 1G test we tested a simple step to digest/decontaminate sputa that does not require equipment, meeting the near to POC test definition. We will optimize this sputum-processing protocol for use with the 2G test. We propose to: Aim 1) Develop and validate the 2G test by defining the stability and critical concentration (CC) for new drugs against known DR-M.tb strains, and optimize appropriate sputum digestion and decontamination protocols for this test; Aim 2) Determine the agreement of the 2G test with current gold standard methods for phenotypic DST for each of the 11 drugs, and Aim 3) Determine the accuracy of the 2G test against reference phenotypic DST protocols using freshly collected sputa in field settings and assess its usability, acceptability, and feasibility. We expect that the novel, simple, affordable and sustainable 2G test will provide a significant improvement when compared to current phenotypic DST reference methods, allowing rapid and tailored treatment for DS-/DR-TB in low- and middle-income countries with high TB burden.

PROJECT NARRATIVE We propose to develop and validate a novel sustainable, easy-to-use phenotypic drug susceptibility testing (DST) test for drug resistant tuberculosis (TB) using non-invasive specimens (i.e. sputum). We will focus on validating and confirming drugs recently approved by WHO to treat drug susceptible (RIPE and HPMZ drug regimens) and resistant (BPaL drug regimen) TB and confirm its efficacy against current phenotypic DST reference tests. This new DST test will be validated further in field settings (Democratic Republic of Congo and the US-Mexico border), where TB is a major health issue and is under-reported due to lack of widely available diagnosis.