PWS_Sand_fly_project_final.docx

PERFORMANCE WORK STATEMENT

Project: Co-occurrence of leishmaniasis and bartonellosis in the Peru-Ecuador frontier: characterization of sand fly vectors and potential reservoirs in rural villages and military outposts Introduction The U.S. Naval Medical Research Unit No. 6 (NAMRU-6) Entomology department in Peru conducts surveillance research on leishmaniasis and bartonellosis sand fly vectors as an essential component of research on the epidemiology of vector-borne diseases. 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 leishmaniasis and bartonellosis risk of infection to our command and relevant host-nation and regional partners to prevent and control 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 1990, the Entomology department of the U.S. Naval Medical Research Unit No. 6 Peru has conducted active leishmaniasis and bartonellosis vector surveillance in geographically and ecologically distinct endemic regions of Peru. Both diseases co-exist in the northern and central Peruvian Andes, with the highest prevalence recorded in areas close to the Ecuador border. Here we propose to characterize the vectors of these diseases so that effective control strategies can be implemented in the absence of vaccines and limited access to medical treatment. Vector surveillance data in combination with epidemiological data provided by local Ministry of Health (MoH) will provide valuable information for prevention and control of leishmaniasis and bartonellosis affecting relevant military and civilian populations in the Peru-Ecuador border.

The research project has the following aims:

1. Determine the distribution and density of sand fly species in leishmaniasis and bartonellosis transmission areas in the Peru-Ecuador border, and contribute these data to WRBU’s VectorMap.

2. Identify sand flies by morphological identification and complement taxonomic identification with molecular and chemical characterization of morphologically similar sand fly species.

3. Characterize sand fly host-seeking and feeding behavior, and determine host preference and potential reservoirs by PCR analysis of blood meal sources.

4. Estimate Bartonella spp. and Leishmania natural infection rates in sand flies to identify potential regional vectors of these diseases.

5. Correlate sand fly vector data with leishmaniasis and bartonellosis incidence data.

Methodology:

Study sites Study sites will be located in areas of high risk for leishmaniasis and bartonellosis in the Peru-Ecuador border: the villages of San Pedro and San Jose de Lourdes in San Ignacio province, Cajamarca; the village of Lalaquiz in Huancabamba province, Piura.

Sand fly collection methods Three sand fly collections will be carried out at each study site during the months of February-March, April-May and July-August to account for seasonal effects on sand fly species composition and abundance. Six houses will be selected in each study site based on the occurrence of human leishmaniasis and bartonellosis cases reported by the local Ministry of Health (MINSA-DGE, DIRESA). Sand flies will be collected using the CDC light traps containing colored LED lights in intra- and peri-domiciliary areas. CDC UV traps, Mosquito Magnet traps, and Shannon traps will also be used in peridomiciliary (50 m away from the house perimeter) and extradomiciliary areas (over 50 m away from dwelling). Miniature CDC light traps will be used as a standard collection control. Additionally, direct collections will be performed in sand fly resting places. Sand fly collections will be performed for five consecutive days per site in collaboration with local MoH personnel. A Latin square design will be employed with traps being placed in peridomiciliary areas of selected houses and at least 50 meters apart to eliminate interference. The traps will be rotated among the different collection points within the intra-, peri- and extradomiciliary areas of selected houses so that each trap is tested at each point following a Latin Square design to avoid trap location bias. All traps will operate from 1800 to 0600, and insects will be collected daily in individual collection cups at the end of each collection period (0600) for all traps except the Shannon trap for which hourly collections will be conducted. Labeled (location, collection method, date) collection cups will be placed in humidified coolers and transported to the field lab where sand flies will be counted and sorted by location, collection method, sex, and gonotrophic status. Sand flies will be stored in glass vials with silica gel and taken to NAMRU-6 Lima where specimens will be identified to species.

Sand fly morphological identification Sand fly identification will be done morphologically using taxonomic keys. To preserve DNA integrity for subsequent PCRs, sand fly species identification will be carried out following a modified procedure that uses lactophenol only on specific parts of the sand fly body, thus preserving the remainder of the specimen for PCR analysis. Identified sand flies will be pooled according to species, date, site and method of collection, and preserved in plastic vials with 70% ethanol.

Sand Fly DNA Isolation for molecular species identification and pathogen detection DNA from sand fly specimens will be isolated using the QIAGEN DNeasy Tissue Kit following manufacturer’s instructions. Briefly, pooled sand flies (1-10 specimens) will be placed in a microfuge tube with ATL buffer and Proteinase K and minced until no recognizable parts remain. Samples will be incubated at 42°C overnight, buffer AL will be added and samples will be mixed with the aid of a vortex. Samples will be heated at 70°C for 10 min, and 100% EtOH will then be added. The contents of each tube will be transferred to a QIAGEN spin column placed in a collection tube, centrifuged, washed with Buffer AW1 and AW2, and centrifuged again and collected with elution buffer (100 μl).

Sand fly molecular identification Morphologically similar potential vector species will be confirmed by PCR using primers targeting Lutzomyia 18S rRNA gene sequence or cytochrome oxidase subunit I gene. PCR products will be extracted with a QIAquick Gel Extraction kit (Qiagen), or purified using ExoSapIT (Affymertix Inc.), and sequenced in both directions using a BigDye terminator cycle-sequencing kit V3.1 (Applied Biosystems™) and a 3130xl Avant-Genetic Analyzer (Applied Biosystems™). Sequences will be edited in Sequencher or BioEdit, and compared with entries in GenBank DNA using the nucleotide-nucleotide basic alignment search tool (BLAST).

PCR for Leishmania and Bartonella detection Sand flies will be PCR screened for natural Leishmania infection using primers targeting the Cyt B gene, and for Bartonella infection using primers targeting the gltA Citrate Synthase gene and NADH Dehydrogenase Gamma sub unit gene. PCR products will be visualized by UV illumination of an ethidium-bromide stained 2.5% agarose gel following electrophoresis. Positive samples are expected to generate PCR products of specific sizes not found in negative samples.

Blood meal analysis Engorged wild-caught female sand flies will be screened by PCR to determine feeding behavior. Abdomen of sand flies will be macerated and DNA extracted. Identification of bloodmeal sources will be performed using mitochondrial cytochrome oxidase subunit I (COI) and cytocrome b (Cyt B) genes. We will use specific species oligonucleotide probes for Homo sapiens, Equus caballus, Equus asinus, Capra hircus, Ovis aries, Bos taurus, Mus musculus, Rattus rattus, Cavia porcellus, Gallus gallus, Felis domesticus, Canis familiars and Sus domesticus. PCR amplicons will be sequenced, and sequences obtained will be analyzed using Sequencher software. Unknown sequences will be putatively identified by comparison in the GenBank DNA sequence database using BLAST only if they have> 90% identity (% nucleotide identity) for the targeted genes.

Our research studies are carried out under austere field conditions in leishmaniasis-endemic communities in Northern Peru, and also at our base in Lima, Peru where specimens will be identified and processed for molecular analysis. Conducting our research in the proposed study sites requires flexible support that can expand and retract on short notice based on vector densities, and leishmaniasis and bartonellosis 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 leishmaniasis and bartonellosis in Peru endemic regions. We require high quality staff including entomologists, biologists, field and laboratory technicians and mosquito catchers trained on vector surveillance. 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 northern Peru with proven hiring and purchasing capabilities in the locations proposed in this project. 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 northern Peru.

General requirements The field component of the project will be executed in rural communities in northern Peru near the Peru-Ecuador border. The laboratory component of this project will be executed in the NAMRU-6 laboratories in 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 must have full US embassy security screening. All contractor employees must fulfill NAMRU-6 Occupational Health requirements.

Performance requirements

Based on project aims, we require the following elements:

· Project coordinator/supervisor. This professional shall be in charge of all the implementation activities of the protocol. He/she will be leading and supervising field and laboratory technicians and mosquito catchers that will be collecting specimens at field sites. He/she shall provide training to the study team related to the study research activities and verify that all study personnel perform activities accordingly. He/she shall present the research protocol to the local authorities to obtain approval and coordinate sand fly collection activities in study sites. Plan, organize, and execute field and laboratory experiments including field collections and taxonomical identification to determine species diversity and population densities, Leishmania and Bartonellosis detection in sand flies by PCR, blood meal identification in sand flies by PCR, sand fly species molecular characterization by DNA barcoding. 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, and provide weekly updates on project progress to Principal Investigator. Assist in preparation of manuscripts for publication. A US embassy security screening is required. The Coordinator shall have a university bachelor degree in the life sciences and experience participating in sand fly research studies for a minimum of 8 years. Shall have level II language proficiency in English and level IV in Spanish.

· Entomology field and laboratory technicians: Assist project coordinator in the execution of all field and laboratory experiments including sand fly field collections and taxonomical identification, Leishmania and Bartonellosis detection in sand flies by PCR, blood meal identification in sand flies by PCR, sand fly species molecular characterization by DNA barcoding. Assist project coordinator in preparation of list of materials/supplies, purchase orders and inventory. Responsible for data collection and entry, and preparation of tables, graphs and other items required for project reports. Organize activities of mosquito catchers. Field technicians shall have a university bachelor degree in the life sciences and have experience participating in field and laboratory entomology research studies for a minimum of 3 years. Will undergo full security screening to be able to access NAMRU-6 facilities and laboratories. US embassy security clearance is required. Shall have level II language proficiency in English and level IV in Spanish.

· Mosquito catchers: Collect sand flies using different trapping methods. Assist in preparation of materials, supplies and equipment needed for all field experiments under supervision of project coordinator. No English language skills are required.

· Travel: Travel of project coordinator, field technicians and mosquito catchers to field sites in northern Peru. Travel of project coordinator to collaborator’s institution and to attend an 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 materials and supplies including insect trap accessories (lures, light sources, collection containers), insect dissection tools, microfuge tubes, personal protective equipment 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 and similar needed for trainings and office support. Shipping of entomological samples, documentations and other items.

Deliverables:

· Preparation and submission of research protocol in Spanish to local authorities.

· Preparation of purchase orders for supplies and equipment for field and laboratory work.

· Coordination of field collections with local authorities and training of local ministry of health personnel participating in sand fly collections.

· Collection of sand flies in study sites (3 collections per site) and sand fly morphological species identification (250-500 specimens per collection per site).

· Preparation and shipment of entomological samples, materials and equipment to and from field sites.

· Conduct molecular studies to determine natural infection of sand flies with Leishmania and Bartonella (400-600 pools).

· Conduct molecular studies to determine blood meal sources in sand flies from all collections (100-200 specimens).

· Conduct molecular studies for taxonomic discrimination between morphologically similar Lutzomyia species (30 specimens).

· Consolidate and analyze data on sand fly species identification, distribution, and leishmniasis and bartonellosis natural infection.

· Upload sand fly collection and species identification data to WRBU VectorMap.

· 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.

Special requirements

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

· Reagents for molecular studies such as DNA extraction kits, PCR reagents and other laboratory reagents shall be provided to contractor.

· 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.