SBIR-STTR Award

Malaria caused by Plasmodium vivax threatens over 2 billion people globally and sickens tens of millions annually. Radical cure for P. vivax malaria includes therapy aimed both at the acute attack (blood schizontocidal) and against future attacks (hypnozoitocidal). The only hypnozoitocide available are 8- aminoquinolines such as primaquine or tafenoquine. However, clinicians often do not prescribe 8- aminoquinolines due to the high prevalence (8%) of individuals with various levels of inherited Glucose-6- phosphate dehydrogenase (G6PDH) deficiencies, because these drugs can cause life-threatening acute hemolytic anemia in patients with moderate to severe G6PDH deficits. There is an urgent need to quantify both Hgb and G6PDH for patients stricken with malaria or prior to the administration of 8-aminoquinilones. All current quantitative methods for G6PDH determination are laboratory based spectrophotometric methods, requiring diluents, reagents, pipettes and trained personnel. There are currently no commercially available point-of-care (POC) tests that can quantify both Hgb and G6PDH directly from a finger-stick sample. Developing a robust, quantitative assay for field use in low resource areas is a high priority for overall malaria control and elimination. In this proposal, In Vitro Diagnostic Systems (IVDS) will establish the feasibility of developing a POC test, the PreQuine Test, in which Hgb and G6PDH levels can be quantitated simultaneously from a 30 L blood stick sample. In pilot studies, we have successfully assessed Hgb and G6PDH levels using independent test strips. To develop a dual Hgb and G6PDH test strip, we propose develop a lysing protocol to maximize liberation of Hgb and G6PDH from blood samples (Aim 1), optimize Hgb and G6PDH assay conditions (Aim 2) and then combine the Hgb and G6PDH assays into a single test strip (Aim 3). The PreQuine Test results will be compared to results obtained with a calibrated hand-held meter; demonstration of concordance of the results from the PreQuine Test strip and calibrated meter to reference samples will indicate success. In Phase II, we will establish real-time and long-term stability of the PreQuine Test, implement a meter temperature compensation program, conduct pre-clinical testing as well as field testing, and use NCLIS guidelines for performance testing. The ability to identify G6PDH deficient individuals using a rapid POC test and also to monitor patients for G6PDH levels and Hgb levels during therapy would have great impact on malaria treatment strategies.

Public Health Relevance Statement:
Project Narrative Malaria caused by Plasmodium vivax threatens over 2 billion people globally. Although radical cure for P. vivax malaria include 8-aminoquinolines, clinicians often do not prescribe 8-aminoquinolines due to the high prevalence (8%) of individuals with various levels of inherited Glucose-6-phosphate dehydrogenase (G6PDH) deficiencies because these drugs can cause life-threatening acute hemolytic anemia in patients with moderate to severe G6PD deficits. In this Phase I application, propose the development of a point of care (POC) device which can provide a cost-effective, user-friendly, and rapid platform to simultaneously quantitate Hgb and G6PDH levels from finger stick blood samples, which will enable clinicians to monitor patients for G6PDH levels and Hgb levels during therapy.

Project Terms:
Affect; Aminoquinolines; Hemolytic Anemia; Biologic Assays; Bioassay; Assay; Biological Assay; Blood Reticuloendothelial System; Blood; Chemistry; Clams; Color; Confidence Intervals; Desferroxamine; Desferrioxamine B; Desferrioxamine; Desferioximine; Deferrioxamine B; Deferoximine; Deferoxamine B; Deferoxamine; Detergents; Digestion; drug/agent; Pharmaceutic Preparations; Medication; Drugs; Pharmaceutical Preparations; Fingers; Future; Genetic Screening; Glucose-6-Phosphate Dehydrogenase; EC 1.1.1.49; Glucosephosphate Dehydrogenase; glucose 6 phosphate dehydrogenase deficiency; GPD Deficiency; G6PD deficiency; Glucosephosphate Dehydrogenase Deficiency; Goals; Gold; Hand; hemoglobin level; Hemoglobin concentration result; Hemoglobin; Hemolysis; Housing; Immobilization; orthopedic freezing; In Vitro; Iron Chelating Agents; Iron Chelates; Laboratories; Malaria; Plasmodium Infections; Paludism; Vivax Malaria; Plasmodium vivax Malaria; Methods; Mutation; genome mutation; Genetic defect; Genetic Change; Genetic Alteration; Optics; optical; Patient Monitoring; Patients; Pilot Projects; pilot study; Plasmodium vivax; P. vivax; P vivax; Primaquine; Reagent; research and development; R&D; R & D; Development and Research; Resources; Research Resources; Saponins; Signal Transduction; biological signal transduction; Signaling; Signal Transduction Systems; Intracellular Communication and Signaling; Cell Signaling; Cell Communication and Signaling; Sodium Nitrite; sodium salt Nitrous acid; medical specialties; Specialty; Temperature; Testing; Time; Treatment Schedule; Treatment Regimen; Treatment Protocols; World Health Organization; glass fibers; fiberglass; NaN3; Sodium Azide; Generations; Diagnostic tests; Sodium Choleate; Sodium Cholate; Titrations; Guidelines; base; Blood Sample; Blood specimen; Area; Acute; Solid; Phase; Biological; Ensure; Compensation; Financial compensation; Training; Individual; Rural; point of care testing; Bedside Testings; Liquid substance; liquid; fluid; instrument; Diagnostic; Whole Blood; Life; programs; Inherited; Hereditary; Protocols documentation; Protocol; Reaction; System; Test Result; meter; field study; field test; field learning; field based data; Membrane; membrane structure; success; cryogenics; Devices; Human Resources; personnel; Manpower; Sampling; performance tests; G6PD gene; G6PD1; G6PD; High Prevalence; clinical test; Clinical Testing; Clinical Evaluation; research clinical testing; Monitor; developmental; Development; point of care; preclinical; pre-clinical; sub micron; submicron; cost effective; cost-effective; Prevalence; innovation; innovative; innovate; user-friendly; prototype; treatment strategy; screening; diagnostic assay; curative treatments; curative therapy; curative therapeutic; curative intervention

Malaria caused by Plasmodium vivax threatens over 2 billion people globally and sickens tens of millions annually. Radical cure for P. vivax malaria includes therapy aimed both at the acute attack (blood schizontocidal) and against future attacks (hypnozoitocidal). The only hypnozoitocides available are 8-aminoquinolines such as primaquine or tafenoquine. However, clinicians often do not prescribe 8-aminoquinolines due to the high prevalence (8%) of individuals with various levels of inherited Glucose-6-phosphate dehydrogenase (G6PD) deficiencies and 8-aminoquinolines can cause life-threatening acute hemolytic anemia in patients with moderate to severe G6PD deficiencies. There are no commercially available point-of-care (POC) tests that can quantify both Hgb and G6PD from a finger-stick sample. In Phase I, we successfully met the performance as outlined by the World Health Organization and the Program for Appropriate Technology in Health (PATH). Results of whole blood and plasma testing revealed excellent concordance between our PreQuine System and current reference methods. In Phase II, we propose to manufacture a simple-to-use inexpensive device, the PreQuine System, that can be used in the field or clinical setting, which will have a number of valuable features. First, the PreQuine System will be comprised of test strips and a hand-held meter with an on-board temperature correction algorithm. Second, the PreQuine System will be rugged, low cost, and minimally invasive for broad use in clinical settings, public health labs and targeted outreach programs. To complete development of the PreQuine System, we will: (1) Finalize Assay Development by Assessing Bioactive Components. Three commercially available diaphorases will be tested to ensure (1) a high degree of activity at the optimal pH (2) long-term stability; and (3) no inhibition in the presence of maleimide; (2) Transition from Hand Assembly to Semi-Automated Assembly of Test Strips. This will be achieved by optimizing process capabilities of coating, slitting and laminating the strips into 5-up card stock.; (3) Incorporate a Temperature Correction Factor (TFC). The PreQuine system must be enable function in a working temperature of 18°C to 40°C. The TFC will be incorporated into the software and correct for temperature differences. Finally, (4) Validation of the PreQuine System. Validation for G6PD and Hgb levels will demonstrate concordance between the manufactured test strips and reference methods. Once validated and commercialized, the PreQuine System will provide point of care diagnostic tool to identify G6PD deficient individuals who can or cannot tolerate 8-aminoquinoline treatment, which will transform malaria treatment strategies and aid in the eradication of P.vivax and P. ovale malaria.

Public Health Relevance Statement:
Project Narrative Malaria caused by Plasmodium vivax and P. Oval threatens over 2 billion people globally. Although radical cure for P. vivax malaria includes administering 8-aminoquinolines, clinicians often do not prescribe these drugs due to the high prevalence (8%) of individuals with various levels of inherited Glucose-6-phosphate dehydrogenase (G6PD) deficiencies, which can cause life-threatening acute hemolytic anemia in patients with moderate to severe G6PD deficits. In this Phase II application, we propose to complete the development effort and validate the performance of this point-of-care device for the diagnosis of G6PD using CLSI guidelines.

Project Terms:
Affect; Algorithms; Aminoquinolines; Anemia; Hemolytic Anemia; Biological Assay; Biologic Assays; Bioassay; Assay; Blood; Blood Reticuloendothelial System; Color; Diagnosis; Negatrons; Negative Beta Particle; Electrons; Enzyme Gene; Enzymes; Fingers; Future; Glucose-6-Phosphate Dehydrogenase; EC 1.1.1.49; Glucosephosphate Dehydrogenase; glucose 6 phosphate dehydrogenase deficiency; GPD Deficiency; G6PD deficiency; Glucosephosphate Dehydrogenase Deficiency; Hand; Health; Hematology Testing; Hematological Tests; Hematologic Tests; Blood Tests; hemoglobin level; Hemoglobin concentration result; Hemoglobin; In Vitro; Life Experience; Photoradiation; Light; diaphorase; NADH Diaphorase; NAD Diaphorase; Lipoyl Dehydrogenase; Lipoic Acid Dehydrogenase; Lipoamide Dehydrogenase; Dihydrolipoyl Dehydrogenase; dihydrolipoamide dehydrogenase; Plasmodium Infections; Paludism; Malaria; Plasmodium vivax Malaria; Vivax Malaria; Maleimides; Methods; Patients; Reticuloendothelial System, Serum, Plasma; Plasma Serum; Blood Plasma; Plasma; P. vivax; P vivax; Plasmodium vivax; Primaquine; Program Appropriateness; Public Health; Reagent; Sensitivity and Specificity; Software; Computer software; Technology; Temperature; Testing; Treatment Schedule; Treatment Regimen; Treatment Protocols; World Health Organization; Mediating; Guidelines; base; Acute; Clinical; Phase; Ensure; Evaluation; Individual; Measurement; enzyme activity; Bedside Testings; point of care testing; Plasmodium ovale; P. ovale; P ovale; tool; Diagnostic; Whole Blood; Life; Inherited; Hereditary; System; Test Result; meter; Membrane; membrane structure; Performance; success; cryogenics; Agreement; Devices; Sampling; outreach program; assay development; Drops; Mediator of activation protein; Mediator of Activation; Mediator; High Prevalence; Validation; Process; Development; developmental; point of care; cost; design; designing; cost effective; blind; user-friendly; prototype; commercialization; minimally invasive; treatment strategy; process optimization; point-of-care diagnostics; rate of change; preservation