PWS_GEIS_Anopheles_experimental_hut_and_vector_incrimination.docx

PERFORMANCE WORK STATEMENT

Project: Characterization of Anopheles darlingi behavior as it relates to malaria vector competency using experimental huts and malaria vector incrimination in transmission hot-spots in the Peruvian Amazon Introduction The U.S. Naval Medical Research Unit No. 6 (NAMRU-6) Entomology department in Peru conducts behavioral and molecular research on malaria vectors as an essential component of malaria epidemiological research. These studies are in line with NAMRU-6 mission to research, understand and develop protective strategies against infectious diseases affecting the uniformed service members and relevant populations in the region. Therefore, it is essential to conduct our research studies on a timely, cost-efficient and high-quality manner to transfer critical data gathered to assess malaria risk to our command and relevant host-nation and regional partners to prevent and control malaria outbreaks. Achieving mission results thru solid teamwork and improved, efficient processes is essential. The requirements of this research project make it necessary to involve an outside partner who will be required to conduct the project logistics as described below.

Background Since 2,000, the Entomology department of the U.S. Naval medical Research Unit No. 6 Peru has conducted active malaria vector surveillance in remote areas of the Peruvian Amazon. Here we propose to conduct malaria vector behavioral characterization and incrimination studies in Loreto, Northern Peruvian Amazon. Overall, this study focuses on obtaining relevant information regarding Anopheles species involved in the transmission of malaria in hot-spots found in low endemic areas of the Peruvian Amazon Basin, and we also expect that results from the experimental hut study will serve to provide a model system for Anopheles surveillance to be used to assess malaria impact on military personnel and relevant civilian populations. The results of this study will help develop malaria distribution maps and contribute to medical risk assessment through identification of risk factors such as human biting rate, infected mosquito frequency and density in military-relevant settings in Peru. This information can also significantly improve current vector surveillance and control efforts by the Peruvian Ministry of Defense and Ministry of Health as it will provide baseline information critical for designing effective malaria prevention and control interventions.

The research project has two aims:

Aim 1: Characterization of Anopheles darlingi behavior in experimental huts in a rural community near Iquitos, Loreto

a) Characterization temporal patterns of Anopheles darlingi adult densities, age composition, physiological status, Plasmodium spp. infection rates and blood meal source in field populations using standardized experimental huts, and correlate these parameters with environmental variables, all recorded over a one-year period.

b) Compare An. darlingi capture efficiency between protected human landing collections (HLC) and odorant-based traps for perimeter and indoor collection using standardized experimental huts.

Aim 2: Malaria Vector characterization in transmission hot-spots in the northern Peruvian Amazon

a) Perform adult Anopheles spp. surveillance in malaria transmission areas that have highly mobile human populations (i.e. logging camps) and where vector incrimination studies are lacking. Indoor and outdoor mosquito collections will be performed using traps and human landing catch to estimate human biting rates and identify where malaria transmission is most likely to occur.

b) Identify collected adult Anopheles mosquitoes to species where possible using taxonomic keys. Conduct molecular characterization of morphologically similar species and those comprising species complexes using DNA barcoding approaches.

c) Perform incrimination testing of malaria vectors in the northern Peruvian Amazon through Plasmodium sporozoite PCR analysis on head-thorax portions of collected Anopheles. Identify blood meals in engorged Anopheles through PCR analysis and determine human blood meal index to determine potential highly effective malaria vectors.

d) Determine immature habitats, relative distribution and abundance of potential malaria vector species.

Methodology:

a) Characterization of Anopheles darlingi behavior in experimental huts in a rural community near Iquitos, Loreto The study will be conducted in a malaria endemic community, Zungaracocha (Loreto), using three existing experimental huts that replicate local housing characteristics. The huts will be employed for characterization of An. darlingi populations to include temporal adult density patterns, indoor resting behavior, age composition, physiological status, blood meal identification and Plasmodium spp. infection rates. To characterize An. darlingi temporal patterns, nocturnal (12 h) interception trap collections will be conducted for three consecutive days every other week. Evaluation of An. darlingi collection efficacy using odorant-baited traps and protected HLC placed inside the huts will be conducted during four months of peak An. darlingi densities (March to June). Trap devices will include miniature CDC light traps with UV light baited with CO2 and octenol, and Suna trap baited with CO2 and M-bita blend (ammonia, lactic acid, tetradecanoic, and 3-methyl-1-butanol). Traps and protected HLC will be rotated among huts using a Latin Square study design during a 6-day sampling period per month. Environmental parameters will be continuously recorded inside huts (temperature and humidity) and at a central location outdoors (temperature, humidity, precipitation and wind) to allow analysis of correlations with species-specific behavioral patterns and population dynamics. All wild-caught anophelines will be identified to species using site-specific morphological keys prior to further processing. Head and thorax of wild-caught An. darlingi will be tested for the presence of Plasmodium spp. using PCR methods and infection rates correlated with environmental variables. Source of host blood meals in wild-caught An. darlingi will be identified by PCR. Parity status will be determined following standard WHO techniques for estimation of field population age structure. Data of Anopheles species recorded will be entered into WRBU’s VectorMap and shared with the Peruvian Ministry of Health.

b) Malaria Vector characterization in transmission hot-spots in the northern Peruvian Amazon Study sites will include logging camps at high risk of malaria transmission in Loreto, northern Peruvian Amazon, where NAMRU-6 Parasitology Department will conduct human-based malaria surveillance activities. Adult mosquito collections will be conducted using different trapping methods. Traps will be set up in forested areas surrounding the logging camps (outdoors) and within logging camps in areas where people congregate. Two collections will be conducted at each site. HLC will be used as a control. Adult anopheline mosquitoes will be identified morphologically in a field lab, and species confirmed using taxonomic keys and PCR. Malaria infection rates in collected adult anopheline mosquitoes will be determined by PCR detection of Plasmodium spp. sporozoites at the NAMRU-6 laboratories. Blood hosts in blood-fed anophelines collected in traps will be determined by PCR. Data obtained will be entered into WRBU’s VectorMap and shared with Peruvian MoH Malaria control program. Voucher specimens will be shipped to WRBU.

Our research studies are carried out under austere field conditions in malaria-endemic communities in the periphery of Iquitos as well as deep riverine portions of the Peruvian Amazon, at our insectary in Iquitos, and at our base in Lima, Peru. Conducting our research in these areas requires flexible support that can expand and retract on short notice based on malaria epidemiology, specifically malaria vector densities and malaria incidence and prevalence. We need a solid administrative and scientific structure for conducting extensive research on tropical infectious diseases, including vector-borne diseases such as malaria in the Peruvian Amazon. We require high quality staff including entomologists, biologists, field technicians and mosquito catchers trained on mosquito vector surveillance as well as a professional driver to execute the project in austere locations in Loreto, Peru. We require collection of high quality, solid data that can be analyzed and used to generate publications in peer-reviewed journals. We also require an organization that can provide oversight for the project in remote, austere locations in the northern Peruvian Amazon and with proven hiring and purchasing capabilities in the locations proposed in this project and familiar with DoD regulations and requirements. The unique and austere conditions of our work require the NAMRU-6 Entomology department to partner with an organization that can provide personnel, materials, and logistics support on short notice in the Peruvian Amazon.

General requirements The field component of the project will be executed in rural communities in the periphery of Iquitos, Loreto and in remote, riverine communities in the northern Peruvian Amazon. The laboratory component of this project will be executed in the NAMRU-6 laboratories in Iquitos and Lima. The period of performance is from 29 September 2015 to 28 September 2016. All personnel required to access NAMRU-6 computers and other electronic devices and electronic databases, and to operate NAMRU-6 vehicles must have full US embassy security clearance. All contractor employees must fulfill NAMRU-6 Occupational Health requirements.

Performance requirements Based on project aims, we require the following elements:

· A project coordinator/supervisor. This professional shall be in charge of all the implementation activities of the protocol. Tasks include: Plan, organize, and execute field and laboratory experiments including Anopheles field collections and taxonomical identification to determine species diversity and population densities, Anopheles behavioral characterization (indoor resting behavior, entry, dispersal) in experimental huts, Anopheles ovary dissection to determine physiological status and age composition, Plasmodium detection in Anopheles by ELISA and PCR, blood meal identification in Anopheles by PCR, Anopheles molecular characterization by DNA barcoding. Train, lead and supervise field technicians and mosquito catchers on all field activities. Closely supervise proper handling of specimens and perform appropriate shipment to the Lima NAMRU-6 laboratory for processing. Prepare list of materials, supplies and equipment needed to execute experiments. Prepare purchase orders of materials, supplies and equipment. Prepare and update inventory of materials, supplies and equipment. Closely supervise the data entry process. Prepare and manage databases and conduct data analysis. Prepare project progress reports in English and Spanish. Assist in preparation of manuscripts for publication. A US embassy security clearance is required. The Coordinator shall have a university bachelor degree in the life sciences, and have experience participating in research studies for a minimum of 10 years.

· Entomology field technicians: Assist project coordinator in the execution of all field experiments including Anopheles field collections and taxonomical identification, Anopheles behavioral characterization and, Anopheles ovary dissection. Assist project coordinator in preparation of list of materials/supplies, purchase orders and inventory. Responsible for field experiment data collection and entry. Organize activities of mosquito catchers. Field technicians shall have a university bachelor degree in the life sciences or technical degree in the life sciences, and have experience participating in research studies for a minimum of 1 year. Will undergo full security screening to be able to access NAMRU-6 facilities and laboratories. US embassy security clearance is required. No English language skills are required. Fluency in Spanish is required.

· Mosquito catchers: Collect Anopheles mosquitoes by human landing catch and using different mosquito trapping methods. Assist in Anopheles behavioral experiments under supervision of Entomology field technicians and project coordinator, record behavioral data and report to Entomology field technicians. Assist in preparation of materials, supplies and equipment needed for all field experiments. A US embassy partial security clearance is required. No English language skills are required.

· A driver: Transport personnel and materials/equipment to field sites in Loreto according to the schedule established by the project coordinator. Assist in preparation and loading of materials/equipment to be transported to the field. Driver shall have the required professional driver license for transportation of people and US embassy security clearance to be able to drive NAMRU-6 vehicles.

· Field lab lease: Make necessary arrangements with the study site community to lease a field lab/space for processing field samples and to safely store project materials/equipment.

· Travel: Travel of project coordinator, field technicians and mosquito catchers to field sites in Loreto. Travel of project coordinator to attend international conference. Travel/visits within Peru and overseas to include travel tickets, lodging arrangements and costs, per diem costs and other related travel requirements.

· Supplies: Purchase and shipment of field and laboratory equipment including insect traps, microscopes, insect dissection tools, personal protective equipment, anti-malarics and any other item needed for this research project. Purchase and shipment of office supplies including notebooks, binders, printer toner, CDs, paper, markers, pencils, pens, hole puncher, stapler, scissors for events, trainings and office support. Purchase and shipment of laboratory supplies. Shipping of entomological samples, documentations and other items.

Deliverables:

General:

· Collected data must be entered into approved NAMRU-6 datasheets, and managed and stored in standard electronic forms in NAMRU-6 computers.

· Mosquito specimens collected for taxonomic identification and molecular analysis must be stored in NAMRU-6 Entomology freezers. A freezer inventory shall be prepared and regularly updated. Voucher specimens must be deposited in NAMRU-6 Entomology collection, and San Marcos University National History Museum as required by the Peruvian Ministry of Agriculture.

· Progress reports shall be submitted by the end of every quarter to NAMRU-6 Entomology Department.

· All information required for manuscript preparation must be delivered upon completion of data collection. A manuscript draft shall be delivered by the end of the study.

Specific deliverables based on project quarterly milestones:

· Anopheles darlingi behavioral and physiological pattern characterization in experimental huts and record environmental parameters.

· Identify mosquitoes (4,000 approximately) caught in traps in Zungarococha to determine species composition and seasonal densities.

· Analyze samples for Plasmodium spp. infection (200 pools), blood meal source (40 mosquitoes) and parity rates (40 mosquitoes).

· Identify adult anopheline mosquitoes (500 approximately) and rear a subset of anopheline larvae (200 approximately) to adulthood for species identification to determine mosquito species composition and seasonal densities.

· Conduct molecular species identification of Anopheles species complexes.

· Enter Anopheles surveillance data into VectorMap (WRBU’s VectorMap.com).

· Analyze data, prepare presentation for professional conference.

· Prepare manuscript for publication in peer-reviewed journal.

· Purchase field supplies and equipment prior to execution of field and laboratory work.

Special requirements

· Equipment for field and laboratory studies will be provided to contractor to execute project and achieve research project goals.

· Contractor employees shall work from 1600 to 0000 or 0000 to 0600 as required for field experiments.

· Contractor employees with full security screening will have access to NAMRU-6 Entomology facilities and allowed to use computers, cell phones and other electronics required to perform the work

· Contractor employees must fulfill NAMRU-6 occupational health program requirements including vaccinations and use of anti-malarics.