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Col James Swaby

12 February 2004AFPMB – Diagnostics Committee Briefing

AF RAPID: Honduras, CONUS, Iraq, Thailand

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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.

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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

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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

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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

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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

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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

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The Honduras Problem

DF and DHF are major public health problems in Central America, including Honduras

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Dengue/Aedes Assays

General technique Reverse transcription-PCR Real-time fluorescence RAPID

Assays were evaluated under

both lab & field conditions

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Ae. aegypti Assay

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

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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

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Lab & Field Evaluations

Five basic experiments:I. Evaluation of Aedes aegypti assay specificityII. Evaluation of Aedes aeqypti assay sensitivity in mixed mosquito

poolsIII. Evaluation of dengue virus assay specificity (serotypes I-IV,

Universal) occurring in Aedes aegyptiIV. Evaluation of dengue virus assay sensitivity in mixed mosquito

poolsV. Evaluation of dengue virus assays on human sera

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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.

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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

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Honduras Field Collections

Breeding sources were abundant

Few personal protective measures were evident--bed nets were rare

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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

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Honduras Field Lab

Mosquitoes were identified, sorted & and blind pools were prepared for analysis

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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

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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.

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Dengue Assay

No cross reactivity occurred with other Flavivirus spp.

We were not able to detect Dengue virus in field-collected Ae. aegypti

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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

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USAFA & F.E . Warren AFB assessment Jul 03

• Hantavirus and Plague surveillance• Follow-up plague survey at Colorado

Springs – Aug 03

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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

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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

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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

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CANARY Bioagent-Identification Sensor

Bioagent BCell

Pathogen bindsto 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

0Time (sec)

10

100

1000

10000

100 200

Sig

nal

(P

ho

ton

s /se

c)

0

600

60

# bacterial particles

Light emitted

Best speed & sensitivity for pathogen-ID test

MIT Lincoln Laboratory

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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

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Questions?