1. Air Force I Institute for Operational Health Col James Swaby 12 February 2004 AFPMB – Diagnostics Committee Briefing AF RAPID: Honduras, CONUS, Iraq, Thailand

2. 2 Ruggedized Advanced Pathogen Identification System (RAPID) Polymerase Chain Reaction- PCR Fluorometric, thermocycler Easy-to-use software allows the RAPID to automatically collect and interpret data, and then report results RAPID is 1 st tactical, field sustainable, biological detection device in DOD Idaho Technology, Inc.

3. 3 Diseases have produced more morbitity & mortality than battle & non-battle injuries combined in major U.S. wars Vectorborne diseases continue to impact military forces Prevention necessary to maintain the health & safety of combat forces * Injuries in the Military; A Hidden Epidemic, AFEB, Nov 96 Vectorborne Disease and the DoD

4. 4 Dengue and Dengue Hemorrhagic Fever Dengue virus and its mosquito vectors are distributed worldwide in tropical environments Dengue is an increasing problem globally and is now the most important vectorborne viral disease

5. 5 Consequences 20 million cases annually in over 100 countries 24,000 deaths 2.5 billion people at risk No vaccine is available- supportive care only Classical dengue fever (DF) & dengue hemorrhagic fever (DHF)

6. 6 Dengue & Dengue Hemorrhagic Fever Dengue is primarily an urban disease of the tropics DF & DHF are caused by one of four closely related, but antigenically distinct, virus serotypes of the genus Flavivirus, (DEN-1, DEN-2, DEN-3, and DEN-4) Viruses that cause it are maintained in a cycle that involves humans and Aedes aegypti, a domestic, day-biting mosquito that prefers to feed on humans

7. 7 Dengue & Dengue Hemorrhagic Fever Infection with any one of these serotypes does not provide cross-protective immunity Persons living in a dengue-endemic area can have four dengue infections during their lifetimes & increased risk of DHF Infection with a dengue virus serotype can produce a spectrum of clinical illness, ranging from a nonspecific viral syndrome to severe and fatal hemorrhagic disease Important risk factors for DHF include the strain and serotype of the virus involved, as well as the age, immune status, and genetic predisposition of the patient.

8. 8 Vectors Aedes aegypti is the primary vector of dengue; Aedes albopictus is a secondary vector Aedes mosquitoes are day-flying, artificial container breeding species Anthrophilic species

9. 9 Military Importance Historically, dengue fever is second only to malaria as a source of morbidity & mortality among deployed U.S. military forces U.S. military forces remain at significant risk for infection when working in dengue endemic areas Public law 105-85 US military advisers in fight against Abu Sayyaf guerrillas- Philippines

10. 10 Operational Difficulties of Mosquito Surveillance Sorting and testing individual mosquitoes can be an impractical and time consuming task Medical personnel on deployment may not have entomology background

11. 11 Detection of Dengue Fever Virus & Aedes Mosquito Vectors Using the RAPID Dengue Virus Accurate results in 2 hours, or less RAPID allows detection of both virus and vector without sorting Development of genetic probes for dengue virus and Aedes vectors allows for detection of their RNA & DNA, respectively with PCR technique using RAPID PCR RNA

12. 12 The Honduras Problem DF and DHF are major public health problems in Central America, including Honduras

13. 13 Dengue/Aedes Assays General technique Reverse transcription-PCR Real-time fluorescence RAPID Assays were evaluated under both lab & field conditions

14. 14 Ae. aegypti Assay An Ae. aegypti specific fluorogenic probe hydrolysis (TAqMan) PCR assay was developed for real-time sampling on the RAPID

15. 15 Dengue Assays Dengue virus Universal & dengue serotypes 1-4 (DEN 1-4) were developed for screening and serotype identification of infected mosquito & human sera using the RAPID

16. 16 Lab & Field Evaluations Five basic experiments: I. Evaluation of Aedes aegypti assay specificity II. Evaluation of Aedes aeqypti assay sensitivity in mixed mosquito pools III. Evaluation of dengue virus assay specificity (serotypes I-IV, Universal) occurring in Aedes aegypti IV. Evaluation of dengue virus assay sensitivity in mixed mosquito pools V. Evaluation of dengue virus assays on human sera

17. 17 Aedes aegypti Assays Specificity testing Extract genomic DNA from 1-2 mosquitoes of different species. Performed real time PCR on the RAPID to determine cross reactivity. Sensitivity testing Extract genomic DNA from pools with up to 30 mosquitoes and one A. aegypti in each pool. Performed real time PCR on the RAPID to test assay sensitivity.

18. 18 Dengue Virus Assays RNA extracted from mosquitoes and tested for Dengue 1, 2, 3, and 4 subtypes. USAMRIID inoculated Aedes aegypti with dengue (1-4), yellow fever, St. Louis encephalitis, and West Nile viruses (Flaviviridae) Specificity Testing Individual infected Aedes aegypti tested for cross-reactivity to YF, SLE, WN Sensitivity Testing Mixed mosquito pools of 10, 25, and 50 with one dengue infected mosquito inserted in each pool. Human sera were tested in the lab & under field conditions in Honduras

19. 19 Honduras Field Collections Breeding sources were abundant Few personal protective measures were evident--bed nets were rare

20. 20 Honduras Field Collections Adult and immature mosquitoes were collected from homes in Honduras and from other breeding sources Mosquitoes were returned to the lab alive for ID and testing

21. 21 Honduras Field Lab Mosquitoes were identified, sorted & and blind pools were prepared for analysis

22. 22 Aedes aegypti Assay Lab based testing of Ae. aegypti, Cx. pipiens, Cx. quinqefasciatus, An. stephensi and Oc. taeniorhynchus individual and mixed pools (n-10) showed 100% concordance in sensitivity and specificity Limit of detection of Ae. aegypti egg pools was 5 individual eggs Field testing in Honduras of panels of individual and mixed pools (n=30) adult Ae. aegypti and Culex spp. Larvae, pupae and adults showed 100% concordance in sensitivity and 97% for specificity with one false positive Blind panels (n=16) demonstrated 90% concordance in sensitivity, and 88% specificity

23. 23 RAPID Output- Aedes Assay

24. 24 Dengue Assay Validation testing was accomplished with a blind panel of 27 dengue virus-infected and 21 non-dengue Flavivirus-infected mosquitoes (YF, WN, SLE), and 8 dengue viremic and 31 non-dengue febrile patient sera samples Flavivirus infected mosquitoes showed the DEN universal assay in vitro sensitivity was 100% and specificity was 96% Each DEN serotype assay in vitro sensitivity was 100%; Den 2 & 4 were 100% specific; DEN 1 & 3 were 98% specific.

25. 25 Dengue Assay No cross reactivity occurred with other Flavivirus spp. We were not able to detect Dengue virus in field- collected Ae. aegypti

26. 26 RAPID Output- Dengue Assay

27. 27 Benefits  Detection of dengue virus and its vectors using the RAPID is a valuable tool for protecting force health and preventing mission crippling disease  Accurate and fast disease and vector assessments for area of operations  Compliance with U.S. public law 105- 85

28. 28 USAFA & F.E. Warren AFB assessment Jul 03 Hantavirus and Plague surveillance Follow-up plague survey at Colorado Springs – Aug 03

29. 29 USAFA & F.E. Warren AFB assessed Jul 03 Samples analyzed for Yersinia pestis and Hantavirus Samples negative for Y. pestis using RAPID Confirmation testing performed by CDC All samples negative Hantavirus results pending

30. 30 Operational Mission to Tallil AB, Iraq Jul 03 Two person VBDST Collect specimens, evaluated surveillance/control Developed Leishmaniasis and sandfly probes One member returned to conduct field evaluations Oct 03 1 st FY 04 team on-site

31. 31 Thailand Dengue Field Trials Aug 03 RAPID PCR probe field trials Dengue universal & serotype 1,2,3,4 probes Aedes aegypti probe Identified serotype 4 in a wild Aedes aegypti

32. 32 CANARY Bioagent-Identification Sensor Bioagent B Cell Pathogen binds to B-cell Approach Tests Against Tularemia Prototype Sensor Detects important pathogens causing: smallpox, tularemia, VEE, plague, brucellosis, cholera, foot-and-mouth Very low false positive rate (0.4% over 1288 trials) Low-cost: requires only 1 droplet/test 0 Time (sec) 10 100 1000 10000 100 200 Signal (Photons/sec) 0 600 60 # bacterial particles Light emitted Best speed & sensitivity for pathogen-ID test MIT Lincoln Laboratory

33. 33 Thailand Field Trials Aug 04 $200K to lyophilize B-cell for CANARY field trials Comparative field evaluation of: CANARY: rugged commercial version RAPID Lyophilized B-Cells: Dengue, Salmonella, Shigella PCR probes: Dengue, Salmonella, Shigella Also conduct JE PCR probe trials

34. 34 The assay architect: Jim McAvin Field crews: Lt Col John Putnam, Lt Col Jim Jones, Major Doug Burkett, Major David Bowles, Major Miguel Quintana, Major Jamie Blow, Capt Keith Blount, Don Lowe Lab Analyses Dr. Kent Lohman Liz Chong Cadet Melissa Morlock Cadet Christopher Hart Mosquito/virus sources Dr. Jimmy Olson, Texas A&M University Linda J. McCuiston, Rutgers University Capt (S) Mary Ann Haberman,AFIOH Det 3, KADENA AB U.S. Army Medical Research Institute of Infectious Diseases Acknowledgements

35. 35 Questions?