Kings Arms Dr

DEVELOPMENT OF A DUAL-IDDS PROBE MULTIPLEX TEST FOR DETECTING ENDEMIC FUNGAL AGENTS - DEVELOPMENT OF A DUAL-IDDS PROBE MULTIPLEX TEST FOR DETECTING ENDEMIC FUNGAL AGENTS CONFIDENTIAL PI: SHAFER, DAVID A., PHD PROJECT SUMMARY IN THIS STUDY, WE PROPOSE DEVELOPING A SIMPLE, LYOPHILIZED MOLECULAR TEST FOR DETECTING THE CAUSATIVE AGENTS OF VALLEY FEVER (COCCIDIOIDOMYCOSIS), HISTOPLASMOSIS, OR BLASTOMYCOSIS, BASED ON OUR PROPRIETARY INTERNAL DNA- DETECTION SWITCH (IDDS) PROBE TECHNOLOGY. TIMELY DIAGNOSIS OF THESE FUNGAL INFECTIONS IS KEY FOR SELECTING EFFECTIVE TREATMENTS AND PREVENTING SEVERE MORBIDITY OR DEATH. OVER 500,000 SUCH CASES ARE THOUGHT TO OCCUR IN THE US ANNUALLY, ALTHOUGH ONLY ONE FDA-APPROVED MOLECULAR TEST EXISTS FOR DIAGNOSING VALLEY FEVER, AND NO FDA- APPROVED MOLECULAR TESTS EXIST FOR DIAGNOSING HISTOPLASMOSIS OR BLASTOMYCOSIS. THE SYMPTOMS OF COCCIDIOIDOMYCOSIS, HISTOPLASMOSIS, AND BLASTOMYCOSIS ARE FREQUENTLY VAGUE AND CHEST X-RAYS DO NOT DISTINGUISH BETWEEN THESE AND OTHER LUNG DISEASES. THUS, DIFFERENTIAL DIAGNOSIS BETWEEN THESE DISEASES AND OTHER THAT PRESENT WITH SIMILAR SYMPTOMS (ACUTE RESPIRATORY DISTRESS SYNDROME, LUNG CANCER, LYMPHOMA, PERICARDITIS, SARCOIDOSIS, OR SOLITARY PULMONARY NODULES) IS KEY FOR PROACTIVE HEALTHCARE INTERVENTIONS. MOREOVER, DIAGNOSIS USING CULTURE- OR SEROLOGY-BASED METHODS MAY HAVE A TURNAROUND TIME OF WEEKS OR MONTHS, OR SHOW FALSE-POSITIVE RESULTS, AND THE ONLY RELATED FDA-APPROVED TEST (THE GENESTAT.MDX COCCIDIOIDES ASSAY) IS ONLY APPROVED FOR USE ON THE MANUFACTURER’S INSTRUMENT, WHICH DISSUADES FROM USE AT REFERENCE LABS. GENETAG TECHNOLOGY (WWW.GENETAGTECH.COM) SPECIALIZES IN DEVELOPING SENSITIVE AND RELIABLE QPCR PROBE SYSTEMS. OUR ERROR-RESISTANT IDDS PROBE SYSTEM EMPLOYS A FLUORESCENTLY LABELED TARGET SPECIFIC PROBE AND A SLIGHTLY MISMATCHED QUENCHER-LABELED ANTIPROBE. IN THE ABSENCE OF THE INTENDED TARGET, THE ANTIPROBE HYBRIDIZES TO THE PROBE, QUENCHING ITS FLUORESCENCE AND PREVENTING OFF-TARGET DETECTION. WE HAVE ALSO DEVELOPED DUAL-IDDS PROBE ASSAYS FOR INCREASED SPECIFICITY AND AUTOMATIC CONFIRMATION OF THE TEST RESULTS. THIS ADDED LAYER OF SPECIFICITY IS KEY FOR ACCURATE DIAGNOSIS, ESPECIALLY WITH LOW LEVELS OF FUNGAL INFECTION. REDUNDANT DETECTION AT MULTIPLE TARGET SITES GREATLY REDUCES THE NEED FOR REFLEX/CONFIRMATORY TESTING. OUR SPECIFIC AIMS ARE (1) TO DEVELOP A 3-TUBE DUAL-IDDS PROBE ASSAY FOR C. IMMITIS, C. POSADASII, H. CAPSULATUM, AND B. DERMATITIDIS, AND (2) TO EVALUATE KEY PERFORMANCE CHARACTERISTICS OF THE MULTIPLEX TEST FOR ENDEMIC FUNGAL AGENTS IN LYOPHILIZED FORMAT. SPECIFICALLY, IN AIM 1, WE WILL (I) DEVELOP LOCKED NUCLEIC ACID (LNA)-ENHANCED DUAL-IDDS PROBES FOR DIAGNOSTIC SEQUENCE-SPECIFIC TARGETS IN EACH PATHOGEN, (II) CONVERT THE DUAL-IDDS PROBES TO LYOPHILIZED FORMAT, AND (III) CONDUCT CROSS-REACTIVITY TESTING WITH THE LYOPHILIZED ASSAYS TO ASSESS FALSE-POSITIVE DETECTION OF OTHER PATHOGENS COMMONLY FOUND IN BRONCHOALVEOLAR FLUID. IN AIM 2, WE WILL DETERMINE THE (I) LINEARITY, (II) LIMIT OF DETECTION (LOD), (III) PRECISION, (IV) EFFECTS OF INTERFERING SUBSTANCES (IF ANY), AND (V) STABILITY OF THE DUAL-IDDS PROBE ASSAYS. SUCCESS IN THIS ENDEAVOR WILL JUSTIFY EXPANDED TESTING WITH CLINICAL SPECIMENS IN PHASE II THROUGH A BSL III- CERTIFIED CONTRACT RESEARCH ORGANIZATION.

DEVELOPMENT OF A DUAL-IDDS PROBE MULTIPLEX TEST FOR DETECTING ENDEMIC FUNGAL AGENTS - DEVELOPMENT OF A DUAL-IDDS PROBE MULTIPLEX TEST FOR DETECTING ENDEMIC FUNGAL AGENTS CONFIDENTIAL PI: SHAFER, DAVID A., PHD PROJECT SUMMARY IN THIS STUDY, WE PROPOSE DEVELOPING A SIMPLE, LYOPHILIZED MOLECULAR TEST FOR DETECTING THE CAUSATIVE AGENTS OF VALLEY FEVER (COCCIDIOIDOMYCOSIS), HISTOPLASMOSIS, OR BLASTOMYCOSIS, BASED ON OUR PROPRIETARY INTERNAL DNA- DETECTION SWITCH (IDDS) PROBE TECHNOLOGY. TIMELY DIAGNOSIS OF THESE FUNGAL INFECTIONS IS KEY FOR SELECTING EFFECTIVE TREATMENTS AND PREVENTING SEVERE MORBIDITY OR DEATH. OVER 500,000 SUCH CASES ARE THOUGHT TO OCCUR IN THE US ANNUALLY, ALTHOUGH ONLY ONE FDA-APPROVED MOLECULAR TEST EXISTS FOR DIAGNOSING VALLEY FEVER, AND NO FDA- APPROVED MOLECULAR TESTS EXIST FOR DIAGNOSING HISTOPLASMOSIS OR BLASTOMYCOSIS. THE SYMPTOMS OF COCCIDIOIDOMYCOSIS, HISTOPLASMOSIS, AND BLASTOMYCOSIS ARE FREQUENTLY VAGUE AND CHEST X-RAYS DO NOT DISTINGUISH BETWEEN THESE AND OTHER LUNG DISEASES. THUS, DIFFERENTIAL DIAGNOSIS BETWEEN THESE DISEASES AND OTHER THAT PRESENT WITH SIMILAR SYMPTOMS (ACUTE RESPIRATORY DISTRESS SYNDROME, LUNG CANCER, LYMPHOMA, PERICARDITIS, SARCOIDOSIS, OR SOLITARY PULMONARY NODULES) IS KEY FOR PROACTIVE HEALTHCARE INTERVENTIONS. MOREOVER, DIAGNOSIS USING CULTURE- OR SEROLOGY-BASED METHODS MAY HAVE A TURNAROUND TIME OF WEEKS OR MONTHS, OR SHOW FALSE-POSITIVE RESULTS, AND THE ONLY RELATED FDA-APPROVED TEST (THE GENESTAT.MDX COCCIDIOIDES ASSAY) IS ONLY APPROVED FOR USE ON THE MANUFACTURER’S INSTRUMENT, WHICH DISSUADES FROM USE AT REFERENCE LABS. GENETAG TECHNOLOGY (WWW.GENETAGTECH.COM) SPECIALIZES IN DEVELOPING SENSITIVE AND RELIABLE QPCR PROBE SYSTEMS. OUR ERROR-RESISTANT IDDS PROBE SYSTEM EMPLOYS A FLUORESCENTLY LABELED TARGET SPECIFIC PROBE AND A SLIGHTLY MISMATCHED QUENCHER-LABELED ANTIPROBE. IN THE ABSENCE OF THE INTENDED TARGET, THE ANTIPROBE HYBRIDIZES TO THE PROBE, QUENCHING ITS FLUORESCENCE AND PREVENTING OFF-TARGET DETECTION. WE HAVE ALSO DEVELOPED DUAL-IDDS PROBE ASSAYS FOR INCREASED SPECIFICITY AND AUTOMATIC CONFIRMATION OF THE TEST RESULTS. THIS ADDED LAYER OF SPECIFICITY IS KEY FOR ACCURATE DIAGNOSIS, ESPECIALLY WITH LOW LEVELS OF FUNGAL INFECTION. REDUNDANT DETECTION AT MULTIPLE TARGET SITES GREATLY REDUCES THE NEED FOR REFLEX/CONFIRMATORY TESTING. OUR SPECIFIC AIMS ARE (1) TO DEVELOP A 3-TUBE DUAL-IDDS PROBE ASSAY FOR C. IMMITIS, C. POSADASII, H. CAPSULATUM, AND B. DERMATITIDIS, AND (2) TO EVALUATE KEY PERFORMANCE CHARACTERISTICS OF THE MULTIPLEX TEST FOR ENDEMIC FUNGAL AGENTS IN LYOPHILIZED FORMAT. SPECIFICALLY, IN AIM 1, WE WILL (I) DEVELOP LOCKED NUCLEIC ACID (LNA)-ENHANCED DUAL-IDDS PROBES FOR DIAGNOSTIC SEQUENCE-SPECIFIC TARGETS IN EACH PATHOGEN, (II) CONVERT THE DUAL-IDDS PROBES TO LYOPHILIZED FORMAT, AND (III) CONDUCT CROSS-REACTIVITY TESTING WITH THE LYOPHILIZED ASSAYS TO ASSESS FALSE-POSITIVE DETECTION OF OTHER PATHOGENS COMMONLY FOUND IN BRONCHOALVEOLAR FLUID. IN AIM 2, WE WILL DETERMINE THE (I) LINEARITY, (II) LIMIT OF DETECTION (LOD), (III) PRECISION, (IV) EFFECTS OF INTERFERING SUBSTANCES (IF ANY), AND (V) STABILITY OF THE DUAL-IDDS PROBE ASSAYS. SUCCESS IN THIS ENDEAVOR WILL JUSTIFY EXPANDED TESTING WITH CLINICAL SPECIMENS IN PHASE II THROUGH A BSL III- CERTIFIED CONTRACT RESEARCH ORGANIZATION.

DEVELOPING A MULTIPLEX DIAGNOSTIC TEST FOR SNPS RELATED TO DRY EYE DISEASE - DEVELOPING A MULTIPLEX DIAGNOSTIC TEST FOR SNPS RELATED TO DRY EYE DISEASE CONFIDENTIAL PI: SHAFER, DAVID A., PHD PROJECT SUMMARY: DRY EYE DISEASE (DED) IS ONE OF THE MOST COMMON OPHTHALMIC CONDITIONS, AFFECTING 1 IN 5 ADULTS WORLDWIDE AND 5–15% OF ALL ADULTS IN THE UNITED STATES. DED IS CHARACTERIZED BY INCREASED TEAR FILM OSMOLARITY AND INFLAMMATION ON THE OCULAR SURFACE, WHICH MANIFESTS IN PATIENTS AS VARIOUS SYMPTOMS OF DISCOMFORT. A MAJOR OBJECTIVE OF NOTICE OF SPECIAL INTEREST NOT-EY-21-007 IS TO VALIDATE DIAGNOSTIC BIOMARKERS ASSOCIATED WITH THE RISK OF DEVELOPING DED IN ASYMPTOMATIC INDIVIDUALS. DED DIAGNOSIS IS CHALLENGING DUE TO THE COMPLEX ETIOLOGICAL MECHANISMS UNDERLYING THE DISEASE, A POOR CORRELATION BETWEEN CLINICAL SIGNS AND SYMPTOMS, AND THE LACK OF A GOLD STANDARD FOR DIAGNOSIS. DED DIAGNOSIS AND CLINICAL EVALUATION ARE SUBJECTIVE AND NORMALLY DEPEND ON PATIENT-REPORTED SYMPTOMS. HOWEVER, DIAGNOSIS BASED ON SYMPTOMS ALONE IS UNRELIABLE BECAUSE THE SYMPTOMS OF DED OVERLAP WITH THOSE OF OTHER OCULAR CONDITIONS. THUS, A SIGNIFICANT NEED EXISTS FOR RELIABLE METHODS TO IDENTIFY INDIVIDUALS AT RISK FOR DEVELOPING DED OR TO SUPPORT THE DIAGNOSIS OF DED. GENETIC FACTORS CONTRIBUTE TO THE SYMPTOMS AND SIGNS OF DED. SPECIFICALLY, SINGLE-NUCLEOTIDE POLYMORPHISMS (SNPS) IN THE IL1B, IL6R, MUC1, TNF-A, TNFAIP3, AND VDR GENES HAVE BEEN IMPLICATED AS RISK FACTORS ASSOCIATED WITH DED. THEREFORE, DEVELOPING A NUCLEIC ACID AMPLIFICATION TEST FOR THESE SNPS SHOULD HELP IDENTIFY AT-RISK INDIVIDUALS AND SUPPORT DED DIAGNOSIS. GENETAG TECHNOLOGY HAS INVENTED SEVERAL QPCR PROBE SYSTEMS WITH A NOVEL ERROR-PREVENTION MECHANISM, WHICH PROVIDES EXCELLENT SPECIFICITY. OUR INTERNAL DNA-DETECTION SWITCH (IDDS) PROBE SYSTEM COMPRISES A FLUOR-LABELED PROBE AND A QUENCHER-LABELED ANTIPROBE THAT IS NEARLY COMPLEMENTARY TO THE PROBE. IN THE ABSENCE OF THE INTENDED TARGET, THE PROBE AND ANTIPROBE BIND TOGETHER, WHICH QUENCHES FLUORESCENCE AND PREVENTS FALSE-POSITIVE RESULTS. IN THIS STUDY, WE WILL GENERATE A DIAGNOSTIC TEST FOR DED RISK ALLELES USING THE IDDS PROBE SYSTEM. IN SPECIFIC AIM 1, WE WILL DEVELOP A LYOPHILIZED 8-TUBE DUAL-IDDS PROBE ASSAY FOR SNPS LINKED TO DRY EYE DISEASE. THE KEY MILESTONES OF AIM 1 ARE TO (I) DEVELOP DUAL-IDDS PROBE ASSAYS FOR SNPS LINKED TO DED, (II) CONVERT THE ASSAYS TO LYOPHILIZED FORMAT, AND (III) CONFIRM THE PERFORMANCE OF EACH ASSAY FOLLOWING LYOPHILIZATION. IN SPECIFIC AIM 2, WE WILL EVALUATE KEY PERFORMANCE CHARACTERISTICS OF THE MULTIPLEX DRY EYE DISEASE TEST. THE AIM 2 MILESTONES ARE TO DETERMINE THE (I) LINEARITY, (II) LIMIT OF DETECTION (LOD), (III) PRECISION, AND (IV) STABILITY OF THE DUAL-IDDS PROBE ASSAYS WITH HUMAN GENOMIC DNA SAMPLES, HOMOZYGOUS FOR EACH ALLELE OF INTEREST. SUCCESS IN PHASE I WILL SUPPORT EXPANDED PHASE II TESTING, WHICH WILL BE CONDUCTED THROUGH A CONTRACT RESEARCH ORGANIZATION. IN PHASE II, EXPANDED ASSAY CHARACTERIZATION (ANALYTICAL REACTIVITY, CROSS-REACTIVITY, CARRYOVER, CROSS-CONTAMINATION, ASSAY CUT-OFF) WILL BE PERFORMED USING CLINICAL SPECIMENS. PROJECT SUCCESS WILL FACILITATE DEVELOPMENT OF THE FIRST MOLECULAR DIAGNOSTIC TEST FOR SNP ALLELES LINKED TO DED SUSCEPTIBILITY, WHICH WILL SUPPORT PATIENT DIAGNOSIS AND IMPROVE PATIENT CARE.

DEVELOPMENT OF A DUAL-IDDS PROBE MULTIPLEX TEST FOR DETECTING ENDEMIC FUNGAL AGENTS - DEVELOPMENT OF A DUAL-IDDS PROBE MULTIPLEX TEST FOR DETECTING ENDEMIC FUNGAL AGENTS CONFIDENTIAL PI: SHAFER, DAVID A., PHD PROJECT SUMMARY IN THIS STUDY, WE PROPOSE DEVELOPING A SIMPLE, LYOPHILIZED MOLECULAR TEST FOR DETECTING THE CAUSATIVE AGENTS OF VALLEY FEVER (COCCIDIOIDOMYCOSIS), HISTOPLASMOSIS, OR BLASTOMYCOSIS, BASED ON OUR PROPRIETARY INTERNAL DNA- DETECTION SWITCH (IDDS) PROBE TECHNOLOGY. TIMELY DIAGNOSIS OF THESE FUNGAL INFECTIONS IS KEY FOR SELECTING EFFECTIVE TREATMENTS AND PREVENTING SEVERE MORBIDITY OR DEATH. OVER 500,000 SUCH CASES ARE THOUGHT TO OCCUR IN THE US ANNUALLY, ALTHOUGH ONLY ONE FDA-APPROVED MOLECULAR TEST EXISTS FOR DIAGNOSING VALLEY FEVER, AND NO FDA- APPROVED MOLECULAR TESTS EXIST FOR DIAGNOSING HISTOPLASMOSIS OR BLASTOMYCOSIS. THE SYMPTOMS OF COCCIDIOIDOMYCOSIS, HISTOPLASMOSIS, AND BLASTOMYCOSIS ARE FREQUENTLY VAGUE AND CHEST X-RAYS DO NOT DISTINGUISH BETWEEN THESE AND OTHER LUNG DISEASES. THUS, DIFFERENTIAL DIAGNOSIS BETWEEN THESE DISEASES AND OTHER THAT PRESENT WITH SIMILAR SYMPTOMS (ACUTE RESPIRATORY DISTRESS SYNDROME, LUNG CANCER, LYMPHOMA, PERICARDITIS, SARCOIDOSIS, OR SOLITARY PULMONARY NODULES) IS KEY FOR PROACTIVE HEALTHCARE INTERVENTIONS. MOREOVER, DIAGNOSIS USING CULTURE- OR SEROLOGY-BASED METHODS MAY HAVE A TURNAROUND TIME OF WEEKS OR MONTHS, OR SHOW FALSE-POSITIVE RESULTS, AND THE ONLY RELATED FDA-APPROVED TEST (THE GENESTAT.MDX COCCIDIOIDES ASSAY) IS ONLY APPROVED FOR USE ON THE MANUFACTURER’S INSTRUMENT, WHICH DISSUADES FROM USE AT REFERENCE LABS. GENETAG TECHNOLOGY (WWW.GENETAGTECH.COM) SPECIALIZES IN DEVELOPING SENSITIVE AND RELIABLE QPCR PROBE SYSTEMS. OUR ERROR-RESISTANT IDDS PROBE SYSTEM EMPLOYS A FLUORESCENTLY LABELED TARGET SPECIFIC PROBE AND A SLIGHTLY MISMATCHED QUENCHER-LABELED ANTIPROBE. IN THE ABSENCE OF THE INTENDED TARGET, THE ANTIPROBE HYBRIDIZES TO THE PROBE, QUENCHING ITS FLUORESCENCE AND PREVENTING OFF-TARGET DETECTION. WE HAVE ALSO DEVELOPED DUAL-IDDS PROBE ASSAYS FOR INCREASED SPECIFICITY AND AUTOMATIC CONFIRMATION OF THE TEST RESULTS. THIS ADDED LAYER OF SPECIFICITY IS KEY FOR ACCURATE DIAGNOSIS, ESPECIALLY WITH LOW LEVELS OF FUNGAL INFECTION. REDUNDANT DETECTION AT MULTIPLE TARGET SITES GREATLY REDUCES THE NEED FOR REFLEX/CONFIRMATORY TESTING. OUR SPECIFIC AIMS ARE (1) TO DEVELOP A 3-TUBE DUAL-IDDS PROBE ASSAY FOR C. IMMITIS, C. POSADASII, H. CAPSULATUM, AND B. DERMATITIDIS, AND (2) TO EVALUATE KEY PERFORMANCE CHARACTERISTICS OF THE MULTIPLEX TEST FOR ENDEMIC FUNGAL AGENTS IN LYOPHILIZED FORMAT. SPECIFICALLY, IN AIM 1, WE WILL (I) DEVELOP LOCKED NUCLEIC ACID (LNA)-ENHANCED DUAL-IDDS PROBES FOR DIAGNOSTIC SEQUENCE-SPECIFIC TARGETS IN EACH PATHOGEN, (II) CONVERT THE DUAL-IDDS PROBES TO LYOPHILIZED FORMAT, AND (III) CONDUCT CROSS-REACTIVITY TESTING WITH THE LYOPHILIZED ASSAYS TO ASSESS FALSE-POSITIVE DETECTION OF OTHER PATHOGENS COMMONLY FOUND IN BRONCHOALVEOLAR FLUID. IN AIM 2, WE WILL DETERMINE THE (I) LINEARITY, (II) LIMIT OF DETECTION (LOD), (III) PRECISION, (IV) EFFECTS OF INTERFERING SUBSTANCES (IF ANY), AND (V) STABILITY OF THE DUAL-IDDS PROBE ASSAYS. SUCCESS IN THIS ENDEAVOR WILL JUSTIFY EXPANDED TESTING WITH CLINICAL SPECIMENS IN PHASE II THROUGH A BSL III- CERTIFIED CONTRACT RESEARCH ORGANIZATION.