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A ir F orce I nstitute for O perational H ealth

A ir F orce I nstitute for O perational H ealth. 12 February 2004 AFPMB – Diagnostics Committee Briefing AF RAPID: Honduras, CONUS, Iraq, Thailand. Col James Swaby. Ruggedized Advanced Pathogen Identification System (RAPID). Polymerase Chain Reaction- PCR Fluorometric, thermocycler

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A ir F orce I nstitute for O perational H ealth

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

  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 1st tactical, field sustainable, biological detection device in DOD Idaho Technology, Inc.

  3. 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

  4. 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

  5. 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

  6. 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

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

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

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

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

  11. 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

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

  13. 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.

  14. 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

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

  16. 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

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

  18. 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

  19. 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.

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

  21. 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

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

  23. 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 resultspending

  24. 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 • 1st FY 04 team on-site

  25. 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

  26. # bacterial particles 60 600 0 CANARY Bioagent-Identification Sensor Approach Prototype Sensor Pathogen binds to B-cell Bioagent B Cell Light emitted Best speed & sensitivity for pathogen-ID test • 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 Tests Against Tularemia 10000 1000 Signal (Photons/sec) 100 10 100 200 0 MIT Lincoln Laboratory Time (sec)

  27. 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

  28. Questions?

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