Ebolavirus [EBOV] and Marburgvirus [MARV] are filoviruses that cause severe hemorrhagic fevers in humans and primates and are listed among the urgently concerning pathogens prioritized in the WHO R&D Blueprint. Genomic sequences of the EBOV and MARV genera differ by >55%, and within each genera the species sequence diversity threshold is >23% thus detection by gold standard qPCR-based methods has been encumbered by significant genetic variability between filoviruses. Clinical diagnosis of EBOV and MARV is difficult as the early stages (i.e., pre- hemorrhagic) present with non-specific symptoms associated with a range of febrile illnesses that are displayed by other infectious agents endemic to affected areas e.g., malaria, yellow fever, dengue fever, Crimean-Congo hemorrhagic (CCHFV) or Lassa fevers. In order to isolate infected individuals, effective control during an EBOV or MARV outbreak can only be achieved by implementing rapid and accurate diagnostics with consistently reliable performance. Aldatu has pioneered the use of PANDAA technology, which enables probe-based qPCR for target detection in highly variable genomic regions by simultaneously adapting and amplifying diverse templates. PANDAA uniquely mitigates the presence of genetic polymorphisms to allow otherwise divergent templates to be detected by fluorescent probes. As such, PANDAA-enabled qPCR is an ideal solution for universal detection of pathogens with significant strain, lineage, and/or sub-type sequence diversity. Aldatu is uniquely positioned to deliver a rapid pan- filovirus qPCR-based assay that is rapid, sensitive molecular diagnostic for the detection and differentiation of EBOV and MARV with superior performance compared to existing diagnostics and won't be affected by genetic changes in new viral variants. PANDAA has been successfully applied to subtype-independent detection of more than fifteen drug resistance mutation (DRM) targets in HIV. Recently, we developed the first pan-lineage assay for Lassa fever virus (LASV), another WHO priority pathogen with high outbreak potential. We propose to leverage the unique capabilities of PANDAA to develop a rapid, sensitive molecular diagnostic assay for filovirus detection, and the first with pan-species coverage of EBOV and MARV, through the following specific aims: (1) development of a pan- filovirus PANDAA assay, leveraging proven techniques and proprietary PANDAA reagent design; (2) analytical validation including confirmation of species inclusivity and high specificity; (3) multiplexing of the PANDAA-Filovirus assay with existing assays for LASV and CCHFV to produce a viral hemorrhagic fever (VHF) panel, including the thermostabilization of the both PANDAA-Filovirus and PANDAA-VHF assays, to meet the requirements of diagnostics targeted to LMICs; (4) GMP manufacturing of the thermostabilized PANDAA-Filovirus and PANDAA-VHF assays and (5) assay validation using samples representing a broad variety of circulating species and geographies, with multi- site evaluations of the test kits at reference labs at CDC- and WHO-affiliated partner institutions. As the first pan- filovirus assay, we will provide a rapid, standardized testing option for all regions that can be deployed using pre- existing qPCR equipment in central labs to radically improve the diagnostic workflow and epidemic preparedness.
Public Health Relevance Statement: Ebolavirus [EBOV] and Marburgvirus [MARV] are filoviruses that cause severe hemorrhagic fevers in humans and primates. Clinical diagnosis of EBOV and MARV is difficult as the early stages (i.e., pre-hemorrhagic) present with non-specific symptoms associated with a range of febrile illnesses that are also displayed by other infectious agents endemic to affected areas e.g., malaria, yellow fever, dengue fever, influenza, Crimean-Congo hemorrhagic or Lassa fevers. In order to isolate infected individuals, effective control during an EBOV or MARV outbreak can only be achieved by implementing rapid and accurate diagnostics with consistently reliable performance. We propose to leverage the unique capabilities of PANDAA, our qPCR-enabling technology that mitigates the effects of genetic variability on test performance, to develop a rapid, sensitive molecular diagnostic assay for the detection and differentiation of EBOV and MARV with superior performance compared to existing diagnostics and won't be affected by genetic changes in new viral variants. PANDAA will provide a rapid, standardized testing option for all endemic regions and can be deployed using pre-existing qPCR equipment in central labs to radically improve the diagnostic workflow.
Project Terms: Affect; Binding Sites; Combining Site; Reactive Site; Biological Assay; Assay; Bioassay; Biologic Assays; Biological Sciences; Biologic Sciences; Bioscience; Life Sciences; Buffers; Centers for Disease Control and Prevention (U.S.); CDC; Centers for Disease Control; Centers for Disease Control and Prevention; United States Centers for Disease Control; United States Centers for Disease Control and Prevention; Congo; Disease Outbreaks; Outbreaks; Drug resistance; drug resistant; resistance to Drug; resistant to Drug; Ebola virus; EBOV; Ebola-like Viruses; ebolavirus; Epidemic; Equipment; Fever; Pyrexia; febrile; febris; Fluorescent Probes; Genome; Geography; Goals; Gold; Hemorrhage; Bleeding; blood loss; Crimean Hemorrhagic Fever; Congo Virus Infection; Congo-Crimean Hemorrhagic Fever; Viral Hemorrhagic Fevers; hemorrhagic fever; HIV; AIDS Virus; Acquired Immune Deficiency Syndrome Virus; Acquired Immunodeficiency Syndrome Virus; Human Immunodeficiency Viruses; LAV-HTLV-III; Lymphadenopathy-Associated Virus; Virus-HIV; Human; Modern Man; Influenza; Grippe; Institutes; Lassa Fever; Lassa disease; Lassa virus; Lassa fever virus; Malaria; Paludism; Plasmodium Infections; Marburgvirus; Frankfurt-Marburg Syndrome Virus; Marburg; Marburg virus; Marburg-like Viruses; Methods; Mutation; Genetic Alteration; Genetic Change; Genetic defect; genome mutation; polymorphism; Genetic Polymorphism; Primates Mammals; Primates; Production; Quality Control; diagnostic kit; test kit; Diagnostic Reagent Kits; Reagent; Development and Research; R & D; R&D; research and development; Non-Polyadenylated RNA; RNA Gene Products; Ribonucleic Acid; RNA; Specificity; Standardization; Technology; Testing; Work; Yellow Fever; Pan Genus; Chimp; Chimpanzee; Pan Species; Custom; base; evaluation/testing; improved; Site; Area; Variant; Variation; Filovirus; Filoviridae; Ensure; Evaluation; Blood Serum; Serum; Individual; Collaborations; Genetic; infectious organism; Infectious Agent; clinical diagnosis; Diagnostic; Consensus; Reaction; Techniques; System; Country; Viral; Performance; thermolability; thermostability; novel; Position; Positioning Attribute; molecular diagnostic assays; Molecular Diagnostic Testing; Sampling; performance tests; response; Genomics; Institution; Preparedness; Readiness; Detection; Reproducibility; Genomic Segment; genomic region; Validation; Molecular; Process; Development; developmental; design; designing; pathogen; innovation; innovate; innovative; Pathogen detection; prototype; resistance mutation; resistant mutation; low and middle-income countries; LMIC; molecular diagnostics; associated symptom; co-morbid symptom; co-occuring symptom; comorbid symptom; concurrent symptom; cooccuring symptom; symptom association; symptom comorbidity; priority pathogen; in silico; Dengue Fever; Dengue disease; breakbone fever; Rapid diagnostics; detection assay; detection limit; accurate diagnostics; multiplex assay; design verification; design validation
