1. USAF JBAIDS/M1M Proficiency Test Program Presented By Thomas Shaak, Maj, USAF, BSC Deputy Chief, Division of Wound Biology and Translational Research Armed Forces Institute of Pathology Washington, DC

2. Purpose of AFPT Program AFPT program for the JBAIDS/M1M is a corner stone of HSMR-LBDT capability. The PT challenges ensure that in the unlikely event of Biowarfare (BW)/Toxin attack we can accurately indentify the presence of BW agents.

3. AFPT Program 55 Air Force Sites (Bagram and Moody) Biological and Toxin testing Platforms JBAIDS proficiency testing M1M proficiency testing AFIP support

4. JBAIDS/M1M JBAIDS-uses principles of real-time PCR for presumptive identification of biological warfare agents M1M-uses principles of immuno-based ECL for detection of pathogens and toxins in biological, environmental and food samples. Both systems allow for rapid ID and diagnostic confirmation of biological pathogens

5. JBAIDS PT: Program Details 3 PT events per fiscal year Real-time PCR platform (JBAIDS) Testing Format  2 (or more) Unknowns (multiple simulated matrices)  4 Organisms (B.a (2), F.t (1), Y.p (2), Orthopox(1))  4 Runs per sample (PTC, NTC, Unk1, Unk2; pos, neg, and unknowns run in duplicate) Unknown sample QC done by AFIP  8% of samples made, tested for QC Results submitted to PT Project Manager  Due 10 business days from the time of receipt of PT samples by the HSMR-LBDT sites

6. JBAIDS PT: Program Details Supplies  Unknown samples (2+) Inactivated antigen  Critical Reagent Program  Variety of matrices Provided by AFIP  Shipped via FEDEX  Notification of receipt of samples required from HSMR-LBDT

7. JBAIDS PT: Program Details Reagents  JBAIDS Assay Targets Lyophilized reagents require reconstitution  Reconstitution water provided in assay kit  Reconstitution buffer provided in assay kit Provided by AFIP (including inhibition and extraction controls)  Prior provided by individual sites  Extraction Kits for different matrices (QFLOW, Scoop, VIBE, and Swipe) Provided by individual sites

8. QFlow Sample Extraction Matrices  Whole blood (DNA)  Food (DNA) Water Tuna Ground Beef Salad  Gastric Wash (DNA)  Water (DNA)  Air into PBS (DNA/RNA)

9. Scoop & Vibe Extractions Matrices for Scoop Extraction  Soil  Stool  Sand (new) Matrices for Vibe Extraction  Whole Blood  Nasal Swab  Sputum

10. Swipe Sample Extraction Matrices  Nasal/Pus Swab  Surface Swab  Powder  Culture Samples

11. JBAIDS Results and Scoring Submitting Results  Fax to AFIP: Result Forms Conditions & Progress Logs JBAIDS report  Forward JBAIDS run files or paper reports (.ixo) to AFIP Scoring  Grades will be determined based on: JBAIDS report Result form responses  >80% constitute passing grade for PT

12. M1M PT: Program Details Approximately 47 sites (Bagram) Three PT events per fiscal year  M1M PT will be delivered with JBAIDS PT M1M PT will be scored independently of JBAIDS PT Immunoassay platform testing format  One unknown  Three target toxins Ricin Bot A SEB (Staphylococcus enterotoxin B)

13. M1M PT: Program Details Supplies  Unknowns Non-infectious, lyophilized material Provided by AFIP  Reagents and Instrument supplies Provided by individual sites

14. M1M Results and Scoring Submitting results  Fax to AFIP Result form Conditions and progress logs  Forward to AFIP M1M run files or paper reports Grading  Pass/Fail Scoring Based on number of unknowns and assays With only 3 possible answers, one incorrect result causes the percentage to fall below 80%

15. AFIP Support USAF PT Project Manager (Ellen LaMorena)  Notification of shipment of PT thru email  Technical support Via email Via phone  Tracking information for shipments will be made available upon request  Distribution of results to individual sites and MAJCOM consultants via email  Limited number of replacement PT samples will be available upon request  AFIP will provide additional testing samples for additional training

16. AFIP Support New Site Visits  Remediation or retraining  Site request required (notification of Sq/cc)  E-mail AFIP support to initiate  Notify Chain of Command of the date  AFIP provides onsite training, evaluation, report and outbrief to site command

17. Failures  (Insufficient) Less than 80% in a PT period requires: Investigation of failure Perform any appropriate action to correct problem Complete Exception response form Return form to AFPT Project Manager  (Poor) Less than 80% in two out of three consecutive PT periods requires: The discontinuation of testing until MAJCOM approves resumption Investigation of failure

18. Failure (cont.) Perform any appropriate action to correct problem Complete Exception response form Forward completed Exception report including investigation results, corrective action proposals, etc. to MAJCOM for review and signature MAJCOM will decide if appropriate action has been taken and if testing should resume Forward forms to AFIP PT Coordinator after MAJCOM approval Within three days of resumption of testing forward Exception response form to the CCLM office

19. Failures (cont.) (Unsatisfactory) Less than 80% in three out of four consecutive PT periods requires:  Complete the steps for poor performance listed previously in addition to The Director of the CCLM will review investigations and other corrective action documentation The decision to resume testing will be made by the Director of the CCLM

20. Common Errors for PT Failure to follow SOP Using wrong extraction kit (JBAIDS) False positives/negatives – In a real scenario-a false positive could create an international incident and a false negative could lead to unnecessary casualties Transcription errors – Switching answers – Skipping answers (leaving results blank)

21. Results from AFPT-July 2009 JBAIDS PT:  54 out of 54 HSMR-LBDT sites participated  93% passing (4 out of 54 failed) 4 failed sites: 4 false negative responses 2 sites were second failures All sites repeated and did not pass

22. Results from AFPT July 2009 M1M PT:  46 HLD sites currently have M1M platform  36 out of 46 HLD sites participated 100% passing (36 out of 36) 10 non-participating sites--due to lack of reagents or instrument maintenance

23. Changes to AFPT Scoring No Changes

24. Future Direction of PT More challenging Inhibitors

25. Questions???