(T1430-03-17) High-Throughput, Multiplexed CRISPR-Cas13a Assay for Simultaneous Detection of Tuberculosis, HIV, Malaria, and TB Drug-Resistance Markers Using CARMEN

Purpose: Tuberculosis (TB), HIV, and malaria (THM) remain among the top infectious disease killers globally. Tuberculosis (TB) leads them as a killer, causing 1.25 million deaths in 2023, with 30% of HIV-related deaths attributed to TB [1]. In 2022, around 167,000 people died from TB-HIV coinfection [2]. Additionally, 263 million malaria cases and 597,000 deaths occurred worldwide in 2023 [3]. The emergence of drug-resistant TB strains and treatment-challenging HIV and malaria co-infections worsens outcomes, highlighting an urgent need for screening methods that simultaneously detect TB, HIV, and malaria (collectively termed THM), and associated drug-resistance markers in resource-limited settings. However, current diagnostic methods for TB, HIV, and malaria are often performed separately [4]. Here, we present the performance of CRISPR-Cas13-based detection assays that target Mycobacterium tuberculosis (Mtb), Human immunodeficiency virus (HIV), and Plasmodium pathogens. CRISPR-based diagnostics utilize a CRISPR effector protein (e.g., Cas13) and a CRISPR RNA (crRNA) to recognize target sequences with high specificity, triggering collateral cleavage and enzyme activation [5]. These reactions are arrayed for highly multiplexed nucleic acid detection using PolyU-FAM dyes and fluorescence-based readouts via plate readers. This concept enabled the development of Combinatorial Arrayed Reactions for Multiplexed Evaluation of Nucleic acids (CARMEN), which miniaturized Cas13 detection reactions for scalable, highly multiplexed nucleic acid detection - up to 4,608 reactions in a single microwell array [6]. We further tested CARMEN and our CRISPR assays across diverse human sample matrices, namely serum, whole blood, and dried blood spots. Our panel successfully performed in all matrices with high specificity and sensitivity down to 10 copies/µL in a pooled, multiplexed PCR amplified target. We hope to use these assays to monitor infection transmission at a grand scale and improve our understanding of tuberculosis and malaria disease courses, co-infections, and outcomes in the developing world.

Methods: Using published sequence databases and surveillance data, we designed and optimized a 10-target CRISPR-Cas13a panel, including crRNAs for Mycobacterium tuberculosis, HIV-1, HIV-2, four Plasmodium species (P. falciparum, P. vivax, P. ovale, P. malariae), two isoniazid resistance-associated TB mutations (inhA, katG), and a human internal control (RNAseP). Each crRNA was validated using synthetic transcripts to assess specificity, sensitivity, and limit of detection (LOD). The assay workflow included a 2-hour PCR reaction of the target for amplification followed by a 3-hour assay run at 37°C using the CRISPR Cas13a mastermix composed of T7 polymerase, rNTPs, LwaCas13a, Cleavage buffer, RNase inhibitor, and Poly-U FAM. For multiplex evaluation, we utilized the CARMEN platform, which generates thousands of nanoliter-scale CRISPR reactions via microfluidic droplet pairing. The BioMark HD system was employed to barcode, load, quantify, and image CARMEN reactions. To simulate clinical performance, we evaluated the panel using contrived healthy human sample matrices—serum, whole blood and dried blood spots (DBS) from healthy donors spiked with known quantities of synthetic targets ranging from 10,000 (or e⁴) copies/ μL to 10 (or e¹) copies/μL. The DNA/RNA target was then extracted using the Zymo Quick DNA/RNA MagBead Kit. This extracted target DNA/RNA was then subjected to a multiplexed PCR with pooled primer pairs for all the assays. The amplified target and the assay reagents were further loaded into the BioMark HD microfluidic chip and placed into the instrument for reactions to occur for up to 180 minutes with images captured every 5 minutes.

Results: All ten crRNAs exhibited high specificity with minimal off-target activity (Figure 1). Singleplex reactions achieved an LOD of 1 copy/μL. CARMEN-enabled multiplexing showed comparable performance with an LOD of 1 copy/μL for all the crRNAs, allowing parallel evaluation of over 4,500 crRNA–target combinations in a single run. Notably, simultaneous detection of both infection and drug-resistance loci was achieved in a single reaction per sample. The assay also preserved performance across all tested matrices, with high signal-to-noise ratios and consistent detection across pathogen and resistance targets. Across all matrices, the assays maintained consistent sensitivity with serum at 10 copies/µL for all the assays (exception of 100 copies/µL for the inhA detection), Whole blood ranging from 10-100 copies/µL for various assays and Dried blood spot with 10 copies/µL for all the assays (exception of 100 copies/µL for the inhA assay) with distinguishable fluorescence signal (Figure 2, 3). Of these matrices, the best assay performance was observed with Dried Blood spots, yielding the most robust and rapid detection kinetics. Additionally, the detection of inhA and katG mutations was successful even in complex matrices like blood, indicating strong potential for field-deployable TB drug resistance monitoring.

Conclusion: We report the development of a CRISPR-Cas13a-based assay using CARMEN for scalable, multiplexed detection of TB, HIV, malaria, and associated TB drug-resistance mutations. The platform enables robust detection from multiple sample matrices and supports high-throughput, massively parallel, sensitive, and human matrix-compatible detection from a single test deployment in centralized clinical and surveillance settings. This approach offers a promising avenue for comprehensive infectious disease diagnostics, especially in co-infection-prone and resource-limited environments.

References: 1. World Health Organization. (2025, March 14). Tuberculosis.
2. Shen Y. (2024). Mycobacterium tuberculosis and HIV Co-Infection: A Public Health Problem That Requires Ongoing Attention. Viruses, 16(9), 1375.
3. PATH. (2025, April 25). The big push to end malaria: Progress, challenges, and the road ahead.
4. Makam, P., & Matsa, R. (2021). “Big Three” infectious diseases: tuberculosis, malaria and HIV/AIDS. Current Topics in Medicinal Chemistry, 21(31), 2779-2799.
5. Kaminski, M. M., Abudayyeh, O. O., Gootenberg, J. S., Zhang, F., & Collins, J. J. (2021). CRISPR-based diagnostics. Nature biomedical engineering, 5(7), 643-656.
6. Ackerman, C. M., Myhrvold, C., Thakku, S. G., Freije, C. A., Metsky, H. C., Yang, D. K., ... & Sabeti, P. C. (2020). Massively multiplexed nucleic acid detection with Cas13. Nature, 582(7811), 277-282.

Acknowledgements: We acknowledge the support of The Bioengineering Department at the University of California Los Angeles. We are grateful to The Bill and Melinda Gates Foundation for funding our research work.


Figure 1. Specificity assessment of the THM synthetic panel detection. Heatmaps illustrate the fluorescence intensity of synthetic DNA fragments at 1e4 (10,000) copies/uL at 1-hour post-reaction initiation.


Figure 2. Heatmaps illustrating multiplexed assay performance for the Tuberculosis-HIV-Malaria (THM) panel in healthy human serum (n=40). Data shows fluorescence intensity of genomic DNA for Plasmodium falciparum (Pf), Plasmodium vivax (Pv), Plasmodium ovale (Po), Plasmodium malariae (Pm), RNaseP, TB 16s, inhA, katG, and genomic RNA for HIV1, and HIV2 at 10-10,000 copies per uL.


Figure 3. Heatmaps illustrating multiplexed assay performance for the Tuberculosis-HIV-Malaria (THM) panel in Dried Blood spots elution of blood from healthy human individuals (n = 40). Data shows fluorescence intensity of genomic DNA for Plasmodium falciparum (Pf), Plasmodium vivax (Pv), Plasmodium ovale (Po), Plasmodium malariae (Pm), RNaseP, TB 16s, inhA, katG, and genomic RNA for HIV1, and HIV2 at 10-10,000 copies per uL.