# Rapid Detection of TB from Blood using Cell-Free DNA and CRISPR

> **NIH NIH R01** · UNIVERSITY OF CALIFORNIA, SAN DIEGO · 2023 · $795,192

## Abstract

Project Summary
While >90% of TB cases are curable, treatment is dependent on diagnostics based exclusively on the detection
of Mycobacterium tuberculosis (Mtb) in patient sputum, despite the limitations of sputum as a diagnostic
sample. Sputum is difficult to process, almost impossible to obtain from patients with paucibacillary TB, such as
people living with HIV (PLWH) and children and is not diagnostic for patients with extrapulmonary TB. A low-
cost, rapid test using blood instead of sputum could transform TB diagnosis for ALL patients and
reduce the current diagnostic gap between patients with and without HIV co-infection. Most active TB
disease (ATB) is still diagnosed with sputum smear microscopy, despite its low sensitivity and specificity. This
lack of diagnostic rigor contributes to the ongoing underdiagnosis of ~3 million TB cases/year, resulting in
continued transmission and poor patient outcomes, especially among PLWH in whom mortality is highest. Our
long-term goal is to transform TB diagnosis with a blood test that accurately distinguishes patients with ATB
from latent TB infection (LTBI), and other pulmonary disorders regardless of co-infection with HIV. The
objective of this study is to demonstrate the analytical and clinical performance of a novel assay for rapid
diagnosis and treatment monitoring of ATB that exploits the precise molecular affinity of CRISPR and
promiscuous nuclease activity of Cas12a to detect ultra-low concentrations of Mtb cell free DNA (cfDNA)
circulating in blood. Preliminary analytical studies indicate assay detection limits of <1fg/µL of Mtb cfDNA in
blood and no cross-reactivity with non-tuberculosis mycobacteria (NTM) DNA. Preliminary clinical studies
including adults and children with presumptive TB, as well as samples from a diagnostically challenging cohort
of symptomatic immunocompromised children living with HIV, demonstrated high diagnostic sensitivity and
specificity in a pooled adult and pediatric group (>90%), and high diagnostic sensitivity (85%) in children. We
will achieve our proposed objective through the following specific aims: AIM 1: Validate a lateral flow version
of the CRISPR-TB assay for low resource settings. Our current working assay requires a PCR step and
fluorescence reader. Preliminary studies indicate we can significantly simplify this workflow using isothermal
amplification and a novel lateral flow (LF) strip for detection of Mtb cfDNA. We will expand on our pilot studies
to validate the existing LF assay using contrived and clinical samples. AIM 2: Define the dynamics of the Mtb
cfDNA signal for treatment monitoring. Preliminary studies indicate the Mtb cfDNA signal decreases to
extinction after treatment initiation but can have non-linear dynamics. We will define the variability and
trajectory of the Mtb cfDNA signal in biobanked serial samples from 34 patients treated for drug resistant TB
over a year, and 50 prospectively collected patients with drug susceptible TB treat...

## Key facts

- **NIH application ID:** 10620065
- **Project number:** 1R01AI174964-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN DIEGO
- **Principal Investigator:** Richard S Garfein
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $795,192
- **Award type:** 1
- **Project period:** 2023-07-17 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10620065

## Citation

> US National Institutes of Health, RePORTER application 10620065, Rapid Detection of TB from Blood using Cell-Free DNA and CRISPR (1R01AI174964-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10620065. Licensed CC0.

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