Description:
014 Multiplexed Digital Counting of Single Molecules for Advanced Molecular Diagnosis
Fast-Track proposals will not be accepted.
Direct to Phase II will not be accepted.
Number of anticipated awards: 1
Budget (total costs):
Phase I: up to $150,000 for up to 6 months
PROPOSALS THAT EXCEED THE BUDGET OR PROJECT DURATION LISTED ABOVE MAY NOT BE FUNDED.
Background
This topic proposal will evaluate the methodological limitations and benefits of direct highly multiplexed digital quantification (HMDQ) of molecules present in samples used in the diagnosis of infectious diseases. Although many multiplexed, quantitative assays have been described for infectious diseases, they suffer from several limitations: 1) Mixed infections are often not detected because only specific agents that are suspected to be present, based on clinical or epidemiological considerations, are tested. 2) Many tests also require multiple controls for quantitation making them difficult to do in a conventional multiplex format. 3) Most platforms are suitable for antigen detection or for nucleic acid detection and use different approaches that are difficult to do simultaneously and do not provide absolute quantitation. 4) Finally, multiplexed assays are often limited in the number that can be performed so that different targets from the same agent or different genotypes cannot be assessed during the initial screen. For example, it is estimated that as many as nine bacterial, viral, and parasitic agents may be present in ticks during feeding and because many of these are pathogenic for humans, domestic animals, or companion animals, it is not uncommon to find co-infections in hosts. The tick vector (Ixodes scapularis) of Lyme disease alone is estimated to cause more than 300,000 cases annually but this vector can also transmit Anaplasma phagocytophilum, Babesia sp., Bartonella, Ehrlichia muris subsp euclairensis and several viruses. The mosquito vector of Zika virus can also transmit several important human viral diseases including dengue and chikungunya virus. Many of the vector-borne diseases present a wide range of symptoms that are often confused with other diseases and co-infections are particularly difficult to recognize. Mixed vector-borne infections present significant diagnostic difficulties for physicians, even when a primary disease agent is recognized, especially as some co-infecting agents require very different therapeutic agents and/or the agents are resistant to those treatments.
Project Goals
The goals of the proposed research are to rapidly, simultaneously, and cost-effectively detect and accurately quantify multiple antigen (protein, carbohydrate) and nucleic acid (DNA, RNA) target molecules used in the primary diagnosis of vector-borne infectious diseases caused by viruses, bacteria, and parasites. The technology should ultimately incorporate innovations which enable large numbers of clinical samples and pools of vectors to be analyzed. The platform must incorporate an open architecture enabling the user to augment or change the specific target molecules as diagnostic and epidemiological interests for emerging vector-borne infectious diseases change (e. g., new agents or genetic types or alternative diagnostic targets are identified). Given the increasing number of studies attempting to relate specific host molecular changes (miRNA, cytokine and other effector molecule gene expression) to specific infectious diseases, the platform must be compatible with performing assays for these biomarkers. The platform and methodology employed must also be compatible with FDA approvals for clinical diagnostic assays for both infectious agents and host biomarkers.
Specific Project Goals:
• Develop assays suitable for use with pools of different vectors and obtain quantitative data from assays.
• Develop assays suitable for use with clinical samples obtained from different vector-borne diseases and obtain quantitative data from assays.
• Expand the range of assays available and move toward commercialization of a subset of those assays.
Phase I Activities and Expected Deliverables:
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• To demonstrate the accurate and simultaneous detection and quantitation of at least 10 (3 viral, 4 bacterial, 3 parasitic) vector-borne disease agents found in pools of ticks and mosquitos (assays and associated data). Commercial reagents or in-house generated reagents may be utilized but the targets must include both antigens and nucleic acids and both types of pools.
• To demonstrate the accurate and simultaneous detection and quantitation of at least 10 (3 viral, 4 bacterial, 3 parasitic) disease agents found in clinical samples originating from 5 different vector-borne diseases transmitted by both ticks and mosquitos (assays and associated data). Commercial reagents or in-house generated reagents may be utilized but the targets must include both antigens and nucleic acids and at least two types of clinical samples (e.g., biopsy tissue, blood, urine). Commercial reagents or in-house generated reagents may be utilized.
Impact
One significant impact of this technology would be to avoid the bias introduced by PCR amplification of nucleic acids from various diagnostic samples where sparse amounts of target DNA limit the laboratorians’ ability to detect it. Bias is introduced by the amplification method, the presence of molecules that interfere with amplification or which provide incorrect products. The ability to directly detect and count DNA, RNA, and protein molecules could greatly increase the speed in which samples are analyzed, the accuracy of the results obtained, and provide the ability to compare relative counts of different types of molecules in the same clinical sample and at different time points during the infection to monitor the patient’s response. A highly multiplexed assay system has the capacity to improve QC in standardizations by increasing the numbers of controls, and can detect multiple mixed co-infections and contaminants simultaneously. This approach can significantly improve the quality of outbreak investigations and is a greatly superior methodology for complex diagnostic samples.
Commercialization Potential
Numerous companies have developed multiplexed platforms for detection of biomolecules. These include microarrays of various types (for both nucleic acid and proteins), flow cytometry, qPCR and ddPC approaches, as well as second (Next Generation) sequencing platforms. Microscopy and direct optical mapping methods can also be highly multiplexed. Some of these approaches have been commercialized in the cancer diagnostic field. These advances are thus heavily covered by commercial and university patents. Companies successful in achieving the goals outlined above should be able to develop strong commercial markets given the number of cases of arbovirus (West Nile, dengue, Zika), rickettsial, parasitic (e. g., malaria, Chagas), and other bacterial etiological agents of interest (e.g., Lyme disease, plague, tularemia).
