1. DEPARTMENT OF HEALTH RESEARCH INSTITUTE FOR TROPICAL MEDICINE Ebola Virus Disease Diagnosis Catalino S. Demetria, DVM

2. 2 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Diagnosis Assays Antigen Detection ELISA (Direct ELISA, Sandwich ELISA) IgM ELISA and IgG ELISA (Indirect ELISA) Polymerase Chain Reaction Virus Isolation

3. 3 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Antigen ELISA (Sandwich, Direct ELISA) 3 Principle of the test

4. 4 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Indirect ELISA (antibody detection) 4

5. 5 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Laboratory Diagnosis of EVD 5 Example of a positive microtiter plate

6. 6 RESEARCH INSTITUTE FOR TROPICAL MEDICINE PCR 6

7. 7 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Laboratory Diagnosis and Surveillance of Ebola Reston Virus Real time-Polymerase Chain Reaction

8. 8 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Diagnostic Sample Required 2ml of Whole Blood (Violet top, with EDTA) 2 ml of Serum (Yellow top with serum separator or red top) Samples are inactivated prior to testing Chemical inactivation for PCR Heat inactivation for Serology (56˚C for 30 minutes)

9. 9 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Diagnosis tests used according to stage of the Disease Acute phase 1.Antigen ELISA 2.PCR 3.IgM ELISA Timing of sample collection is crucial Convalescent Phase 1.IgG ELISA

10. 10 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Advantages and Limitations of the Assays AssayAdvantagesLimitations Antigen ELISACan detect all Ebola species Has allowance for genetic changes Can be set up in the field Can not be used to speciate the virus since it is a cocktail of different antibodies, may not detect small viral copies PCRSensitive, can detect small viral copies, fast turn around time for real time PCR Can not accommodate changes in sequence (mutation) and difficult to set up in the field IgM ELISAUseful in the acute phase, can be used as a complimentary test for antigen ELISA and PCR IgM does not persist long the immune system

11. 11 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Advantages and Limitations of the Assays AssayAdvantagesLimitations IgG ELISACan be used to monitor the progress (convalescence of the patient), IgG persist longer in the immune system, has allowance for viral mutations Not a point of care test Virus IsolationHigh sensitivity, “gold standard” especially in novel outbreak of pathogens Needs specialized facility (BSL4) Needs specialized training Needs strict biosafety and biosecurity measures

12. 12 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Interpretation of Results AssayResults Antigen ELISAPositive Viral antigen detected Negative No viral antigen present PCRViral RNA detected No Viral RNA present IgM ELISAIgM detected (Acute phase) No IgM detected IgG ELISAIgG detected (Exposure to disease, convalescent stage No IgG detected

13. 13 RESEARCH INSTITUTE FOR TROPICAL MEDICINE Cut-off and Turn around time Running days: Monday to Saturday Cut off: 10 am Turn around time: 48 hours upon receipt of the sample by RITM

14. 14 RESEARCH INSTITUTE FOR TROPICAL MEDICINE THANK YOU! 14