Document text
Principal Investigator: Tony Y. Hu
Organization: TULANE UNIVERSITY OF LOUISIANA
Fiscal Year: 2024
Award: $726,852
Funding agency: National Institute of Allergy and Infectious Diseases
ABSTRACT
Every year 10 million people fall ill with tuberculosis (TB) and 1.5 million people die from TB – making it the
leading cause of death from infectious disease – but TB can be difficult to diagnose. Extended M. tuberculosis
(Mtb) cultures are still often used for diagnosis. PCR-based assays (e.g. Xpert MTB/RIF) that detect TB DNA
can provide more rapid results, but require special equipment and, like Mtb culture, exhibit reduced sensitivity
when employed to analyze sputum from individuals with extrapulmonary TB or compromised immune systems.
Blood-based TB assays should detect all forms of TB, but current tests analyze the immune response to Mtb
antigens and cannot distinguish active and latent TB. Sensitive detection of Mtb-derived cell-free DNA (cfDNA)
in the circulation, however, represents a potential new means for enhanced TB diagnosis. Circulating cfDNA is
rapidly degraded after its release and disease-associated cfDNA levels in blood can rapidly change in response
to pathologic changes and physiologic responses. This short half-life can enable “real-time” cfDNA analyses
required for accurate evaluation of the current status of active Mtb infections and rapid granular evaluation of
Mtb treatment responses. However, the limit of detection (LoD) of current PCR-based assays are not sufficient
to reliably detect Mtb cfDNA in blood. We have established a CRISPR-Cas12a-based detection system that can
ultra-sensitively detect trace amount of SARS-CoV-2 RNA in blood to diagnose COVID-19, predict disease
severity, and evaluate infection resolution. We have adapted this approach to develop a blood-based multiplexed
CRISPR-mediated TB diagnosis (CRISPR-TBD) assay that can detect circulating Mtb cfDNA, including SNPs
responsible for drug-resistant TB. Our preliminary data from longitudinal serum samples of patients undergoing
TB treatment provide strong proof-of-principle evidence for the clinical utility of this platform. We have adapted
this approach to a paper strip-based point of care (POC) CRISPR-TBD detection platform suitable for use in
resource-limited regions with high TB burden, without decreasing assay sensitivity. We now propose to: 1)
systematically optimize all CRISPR-TBD paper strip assay steps to improve quantitative detection of Mtb-cfDNA
in POC settings; 2) evaluate the performance of this POC assay to diagnose pulmonary and extrapulmonary TB
and to identify drug-sensitive and -resistant TB cases; 3) quantify Mtb-cfDNA changes in serum during TB
treatment as a measure of treatment efficacy or failure and for early detection of nascent drug resistance; and
4) in-field validate the diagnostic performance of this POC assay in an independent TB patient cohort when
performed in a clinical laboratory in a high endemic TB region and evaluate in parallel the performance our
predictive Mtb-cfDNA model for TB treatment.
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II><Isoniazid resistance><Isoniazid resistant><LAV-HTLV-III><Laboratories><Long-term cohort study><Longitudinal cohort study><Longterm cohort study><Low-resource area><Low-resource community><Low-resource environment><Low-resource region><Low-resource setting><Lung><Lung Respiratory System><Lymphadenopathy-Associated Virus><Lyonization><M tb><M tuberculosis><M tuberculosis antigen><M tuberculosis infection><M. tb><M. tb infection><M. tuberculosis><M. tuberculosis antigen><M. tuberculosis infection><M.tb antigen><M.tb infection><M.tuberculosis infection><MDR Tuberculosis><MDR-TB><MTB infection><Measures><Mediating><Medication><Methods><Modeling><Monitor><Mtb antigen><Multi-Drug Resistant Tuberculosis><MultiDrug Resistance Tuberculosis><Multidrug-Resistant Tuberculosis><Mycobacterium tuberculosis><Mycobacterium tuberculosis (MTB) infection><Mycobacterium tuberculosis antigens><Mycobacterium tuberculosis infection><NIH><National Institutes of Health><Non-Polyadenylated 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