Description:
OBJECTIVE: To develop a system that has high-throughput capability for extracting nucleic acids from arthropods, to include ticks, mosquitoes, and sand flies. The system must then be able to deposit purified nucleic acids into multiple type of receiver (i.e., tubes or plates) for future diagnostic testing systems. DESCRIPTION: The rapid identification of relevant arthropod transmitted pathogens and the determination of potential human disease risk, especially in hostile environments, is of great importance to the U.S. military. In some cases, determining if a given pathogen is present in a given area is important for emphasizing the level of personal protective equipment (PPE) or personal protective measures (PPM) necessary for a given area. To determine if an arthropod-borne pathogen is present in a given area, hundreds (and even thousands) of pools of arthropods would need to be screened in order to conduct risk assessments for that given area. The screening of large numbers of arthropods is necessary to determine the infection rate, and thus the risk from acquiring an infection from the bite of an arthropod. Currently, vector surveillance is conducted by manually pooling and processing thousands arthropods for subsequent testing. To alleviate this bottleneck, a system that has high-throughput capability for extracting nucleic acids from arthropods (to include ticks, mosquitoes, and sand flies), followed by automated sample preparation would help alleviate this issue. The system must then be able to deposit purified nucleic acids into multiple type of receiver (i.e., microcentrifuge/PCR tubes, real-time PCR capillaries, or 96-well plates) for current and future diagnostic testing systems. The development of a processing/purification system would enable Preventive Medicine Units to screen more arthropods for more pathogens in less time and with less logistical needs, thus resulting in better vector surveillance, diagnostic testing, and risk assessments. Requirement: To extract nucleic acids from arthropods in a high-throughput manner for diagnostic applications using arthropods collected during military deployments. The unit/system would need to be fieldable, with minimal logistical requirements, and should be designed for far forward applications in a deployed setting. Desired Capability/Concept of Final Product: The primary vision for the final product would be a unit/system that would sit on a regular laboratory bench (or smaller in size) where the investigator would load pools of arthropods into it using the developer"s designated tubes or plates. The arthropods would consist of soft-bodied arthropods (e.g., mosquitoes, sand flies, and swollen blood-filled ticks) or hard-bodied arthropods (e.g., adult hard ticks). The unit would then process the arthropods and would produce purified nucleic acids that would then be deposited into microcentrifuge tubes, real-time PCR capillary tubes, or 96-well plates for diagnostic testing. The diagnostic testing is not a part of this topic. The unit/system should be rapid, cost effective, and easy to use, with reagents that are stable at elevated temperatures for extended periods of time (e.g. 40oC for 2 years). Technical Risk: There is a degree of technical risk associated with this project. Currently, there is no single unit/system that fulfills the requirements of this proposal. The candidate contractor is expected to use innovative and in-house or associated expertise to develop a prototype that meets the needs of the Department of Defense. Access to Government Facilities and Supplies: The candidate contractor should coordinate with the Contracting Officer"s Representative (COR) to determine if any support is available for supplying arthropods or other testing materials. PHASE I: The selected contractor will determine the feasibility of the concept by developing a prototype unit/system where pools of arthropods are loaded into it using the developer"s designated tubes or plates. The unit would then process the arthropods and would produce purified nucleic acids that would then be deposited into microcentrifuge tubes, real-time PCR capillary tubes, or 96-well plates for diagnostic testing. The unit/system should be rapid, cost effective, and easy to use, with reagents that are stable at elevated temperatures for extended periods of time (e.g. 40oC for 2 years). For Phase I, the prototype should be able to process up to 48 pools of arthropods in less than 4 hours and should be able to deliver purified nucleic acids into microcentrifuge/PCR tubes (Threshold). The contractor will conduct initial laboratory evaluations of the prototype device with both soft-bodied and hard-bodied arthropods and will supply a written report to the COR. By conclusion of Phase I, the contractor will provide a prototype unit/system to the COR for evaluation. The degree to which the prototype unit/system meets the desired capability as outlined above will be evaluated at a government laboratory. Data from this independent evaluation will be used in the determination of a Phase II awardee, if applicable. PHASE II: The prototype unit/system should be able to process 960 pools of arthropods in less than 8 hours and should be able to deliver purified nucleic acids into microcentrifuge/PCR tubes, PCR capillaries, or 96-well plates (Objective) and should be fieldable. The prototype should consist of a single unit without the need for manual transfer of sample/material between the stages (Objective). The selected contractor will conduct comprehensive laboratory evaluations of the unit/system performance characteristics (to include, but is not limited to the number of arthropods in a single pool, reliability, ease of use, and range of usable work conditions, e.g., conducting the processing where the sun is shining on the work area and where the work is conducted at 35oC in a dusty environment). The selected contractor will also conduct stability testing of the unit/system/components/ associated reagents as part of Phase II. The stability testing should be conducted under both real-time and accelerated conditions (e.g. attempt to force the unit/system/ components/associated reagents to fail under a broad range of temperature and humidity conditions). PHASE III: During this phase, the performance of the unit/system should be evaluated in a variety of field studies that will conclusively demonstrate that the unit/system meets the requirements of this topic. By the conclusion of Phase III, the selected contractor will have completed the development of the unit/system and will successfully commercialized the product. The contractor should provide a report that summarizes the performance of the unit/system to the Armed Forces Pest Management Board (AFPMB) and will request a National Stock Number (NSN) be assigned. The contractor should coordinate in advance with the COR for any support required from the Walter Reed Army Institute of Research (WRAIR) or from the US Army Medical Research Institute of Infectious Diseases (USAMRIID). Military Application: Once an NSN as been assigned to the unit/system the AFPMB will work with the appropriate organizations to have the unit/system incorporated into the appropriate"sets, kits, and outfits"that are used by deployed Preventive Medicine Units. Commercial Applications: This unit/system will be made available for non-military purposes, such as for use by commercial pest controllers or non-governmental organizations (NGO"s) in areas of the world where arthropod-borne diseases are an issue. We envision that the contractor that develops the unit/system will be able to market it to a variety of commercial, governmental, and non-governmental vector control organizations and testing facilities, and that this market will be adequate to sustain the continued production of the unit/system. By the end of Phase III, the selected contractor will be able to make this unit/system available to potential end-user customers throughout the world. REFERENCES: 1. O'Guinn M, Lee JS, Kondig JP, Fernandez R, and Carbajal F. 2004. Field detection of eastern equine encephalitis virus in the Amazon Basin region of Peru using reverse transcription-polymerase chain reaction adapted for field identification of arthropod-borne pathogens. Am J Trop Med Hyg 70: 164-171. 2. Pages F, Faulde M, and Orlandi-Prarole P. 2010. The past and present threat of vector-borne diseases in deployed troops. Clin Microbiol Infect 16: 209-224. 3. Gu W, Lampman R, Novak RJ, 2004. Assessment of arbovirus vector infection rates using variable size pooling. Med Vet Entomol 18: 200-4. 4. Philip Samuel P, Tyagi BK, 2006. Diagnostic methods for detection & isolation of dengue viruses from vector mosquitoes. Indian J Med Res 123: 615-28. 5. Lundstrom JO, 1999. Mosquito-borne viruses in Western Europe: a review. J Vector Ecol 24: 1-39.
