Diagnosis of infection is typically based on direct detection of the pathogen and/or its products in bodily fluids and tissues. This approach has limited sensitivity and speed of detection. We propose to develop a novel, host-based diagnostic assay platform for blood-based assays combining: (1) the precision of antigen-specific immune responses, (2) the versatility and robustness of nucleic acid detection by fluorescence in situ hybridization (FISH), and (3) the diagnostic and prognostic power of single-cell analysis by flow cytometry. Our preliminary work shows that stimulation of peripheral blood T lymphocytes ex vivo induces expression of mRNA for key cytokines (IL-2, IFN-? and TNF-?) that is detected by hybridization with gene-specific FISH probes and single- and multi-parameter flow cytometry. We now propose to move this platform through preclinical product development for diagnosis of tuberculosis (TB) in partnership with Oxford Immunotec, a world leader in FDA-approved, host-based TB diagnostics. TB is the selected pathology because: (i) it is a global health problem;(ii) M. tuberculosis infection is diagnosed by detecting antigen- specific T cell responses;and (iii) i has a complex clinical presentation ranging from asymptomatic, latent infection (LTBI) to active pulmonary tuberculosis (PTB). Application of our FISH-Flow platform to TB diagnosis is expected to yield a test with superior diagnostic accuracy and speed than the existing tests. Moreover, the platform has the unparalleled potential to distinguish between LTBI and PTB through its multi-parameter capacity, since stages/progression of infection are associated with different cytokine profiles. We will optimize key assay parameters and verify the resulting standardized assay for protocol robustness and reproducibility, assay duration, and initial assessment of diagnostic accuracy in comparison with an existing FDA-approved test. Future kit manufacturability will also be explored. The first outcome will be a simple and accurate single- cytokine assay for LTBI diagnosis that equals or exceeds the clinical accuracy of the existing LTBI diagnostics and surpasses them in speed and ease of execution. The second outcome will be the evaluation of the three- cytokine readout for distinguishing LTBI from PTB. Established collaborations at the PI site and at Oxford Immunotec with clinical sites in regions with low and high TB prevalence will provide blood samples from donors in all TB diagnostic groups. We have also shown that the FISH-Flow protocol is amenable to adaption to microfluidics-based, automated platforms. A third outcome will be automation of all assay steps upstream of flow-cytometry detection (from sample preparation to nuclei acid staining) to obtain a semi-automated device operating with standard flow cytometry. This work, conducted by our bioengineer partner who is experienced in commercializing diagnostic devices, should eventually lead to a hand-held, fully automated device with sample-in-answer-out capability. The FISH-Flow platform in its manual and automated versions will be applicable to detection of receptor-mediated responses in many infectious and non-infectious pathologies.

Public Health Relevance

Fast, affordable and reliable diagnosis of an infection with the bacteria that cause tuberculosis before a person shows symptoms is critical to eliminating tuberculosis. This collaborative proposal establishes a partnership toward development of a next-generation diagnostic test for tuberculosis between academic investigators and a medical diagnostics company that has already developed FDA-approved tuberculosis diagnostics. The plan is designed to refine and validate a promising diagnostic procedure (assay) that detects the responses of human immune cells to tuberculosis infection, resulting in both manual and semi-automated versions of the assay that will determine whether a person is infected with the bacteria and the stage of that infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI104615-01
Application #
8474448
Study Section
Special Emphasis Panel (ZAI1-LG-M (J4))
Program Officer
Jacobs, Gail G
Project Start
2013-08-15
Project End
2018-07-31
Budget Start
2013-08-15
Budget End
2014-07-31
Support Year
1
Fiscal Year
2013
Total Cost
$1,354,623
Indirect Cost
$362,240
Name
Rutgers University
Department
Public Health & Prev Medicine
Type
Schools of Medicine
DUNS #
078795851
City
Newark
State
NJ
Country
United States
Zip Code
07103
Guerrini, Valentina; Prideaux, Brendan; Blanc, Landry et al. (2018) Storage lipid studies in tuberculosis reveal that foam cell biogenesis is disease-specific. PLoS Pathog 14:e1007223
Ravi, Janani; Anantharaman, Vivek; Aravind, L et al. (2018) Variations on a theme: evolution of the phage-shock-protein system in Actinobacteria. Antonie Van Leeuwenhoek 111:753-760
Arrigucci, Riccardo; Bushkin, Yuri; Radford, Felix et al. (2017) FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Protoc 12:1245-1260
Manganelli, Riccardo; Gennaro, Maria Laura (2017) Protecting from Envelope Stress: Variations on the Phage-Shock-Protein Theme. Trends Microbiol 25:205-216
Radford, Felix; Tyagi, Sanjay; Gennaro, Maria Laura et al. (2016) Flow Cytometric Characterization of Antigen-Specific T Cells Based on RNA and Its Advantages in Detecting Infections and Immunological Disorders. Crit Rev Immunol 36:359-378
Vir, Pooja; Arrigucci, Riccardo; Lakehal, Karim et al. (2015) Single-Cell Cytokine Gene Expression in Peripheral Blood Cells Correlates with Latent Tuberculosis Status. PLoS One 10:e0144904
Bushkin, Yuri; Radford, Felix; Pine, Richard et al. (2015) Profiling T cell activation using single-molecule fluorescence in situ hybridization and flow cytometry. J Immunol 194:836-41
Shi, Lanbo; Salamon, Hugh; Eugenin, Eliseo A et al. (2015) Infection with Mycobacterium tuberculosis induces the Warburg effect in mouse lungs. Sci Rep 5:18176