1 / 28

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

zeno
Download Presentation

A ir F orce I nstitute for O perational H ealth

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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?

More Related