project titled "Integrated phenotypic and genotypic Aedes aegypti insecticide resistance surveillance in military relevant regions in eastern and central Honduras”.

PERFORMANCE WORK STATEMENT

1. Introduction:

The Performance Work Statement (PWS) defines the requirements for conducting the

Study entitled “Integrated phenotypic and genotypic Aedes aegypti insecticide resistance surveillance in military relevant regions in eastern and central Honduras”.

The study is funded by Global Emerging Infections Surveillance and Response

Branch (GEIS) of the Armed Forces Health Surveillance Division (AFHSB) intramural funding. The country where the activities will be carried out is Honduras.

2. Background:

Current FHP prevention strategies against mosquito-borne diseases rely on the effectiveness of permethrin-treated uniforms as the first line of defense. Dengue fever, the most widespread Aedes-borne disease worldwide, represents a serious health threat to U.S. service members deployed to endemic areas. Joint Task Force Bravo (JTF-B), which conducts joint operations across Central America, is deployed in several dengue-endemic regions of Honduras, particularly across Comayagua, Cortés, and Olancho departments. In 2024, these departments reported 31.1% of the national dengue burden. This operational context coincides with widespread insecticide resistance in the primary dengue vector, Aedes aegypti, particularly to pyrethroids, thereby compromising the effectiveness of standard vector control tools used to protect deployed personnel. Pyrethroid resistance in Ae. aegypti is mediated by multiple mechanisms, among which mutations in the voltage-gated sodium channel gene are the most consistently validated markers. Specifically, three single nucleotide polymorphisms (SNPs) associated with knockdown resistance (kdr) have been widely reported as reliable indicators in field populations. Additional mutations have been identified more recently across the Americas; however, their functional contribution to resistance phenotypes remains unresolved, highlighting a critical gap in the current evidence base.

The primary aim of this work is to determine whether this combined entomological phenotypic–molecular approach can function as a scalable, field-adapted surveillance system. Its utility is twofold. Operationally, it will generate actionable data on resistance patterns that directly inform the effectiveness of pyrethroid-based personal protection and vector control tools used by JTF-B. Methodologically, it will assess whether kdr genotyping can serve as a proxy diagnostic for resistance in settings where insectary capacity is limited, reducing dependence on labor-intensive bioassays. If validated, this framework could be expanded across Honduras and other JTF-B deployment areas, strengthening evidence-based decision-making for vector control interventions.

Establishing Aedes aegypti insecticide resistance surveillance capacity for NAMRU SOUTH in Honduras requires the services of research institutions that can collect and identify specimens as well as conduct laboratory experiments for phenotypic and genotypic insecticide resistance assessment on these specimens. In this regard, this project will allow the execution of Aedes aegypti insecticide resistance surveillance in Honduras to generate actionable data that can be used for the benefit of the warfighter.

3. General Requirements:

Study sites will prioritize the departments of Comayagua, Cortes and Olancho. Other regions might be considered upon coordination with NAMRU SOUTH.

The contractor will provide general classes of services including (but not limited to) project management, collection and processing of entomological samples, and shipment of collected samples. In addition, the contractor shall provide Aedes-borne disease incidence data available from local health authorities to correlate with vector densities and phenotypic and genotypic status.

Monitoring activities will be conducted by NAMRU SOUTH via monthly conference calls and one monitoring visit to selected field sites during the contract period.  Monthly, short reports are expected during the conduction of the study.

4. Performance Requirements:

The study sites will be in Honduras and will prioritize the following regions:

• Comayagua, Cortes and Olancho
• Other sites might be requested by NAMRU SOUTH.

The contractor will provide general classes of services including (but not limited to) project management, coordination with local health authorities, provision of biological specimens, provision of epidemiological data, laboratory testing, shipment of samples, data analysis and reporting.

Periodic monitoring activities will be conducted by NAMRU SOUTH through in-person meetings and conference calls.

Where necessary, the contractor will secure all protocol provisions required by NAMRU SOUTH.

The following elements are required:

1. FIELD ACTIVITIES

1. Coordinate entomological collection activities with NAMRU SOUTH Entomology and local partners including Ministry of Health (MoH).
2. Conduct collections of Aedes aegypti mosquitoes in at least 10 blocks within and/or around military bases on each site, specific collection sites will be selected by convenience. At each site, both ovitraps (n=40) and adult aspiration on households (n=50) will be carried out.
3. Provide transportation and storage facilities for egg collections papers and adult mosquitoes collection from field to the laboratory.
4. Storage wild caught Ae. aegypti on DNA/RNA Shield until molecular analysis.
5. Provide field data and assist with preparation of field collection database (NAMRU SOUTH template).

1. LABORATORY ACTIVITIES

1. Hatch egg papers from each site together and rear until adulthood to obtain F0 and F1 progeny under standard insectary conditions.
2. Assess insecticide susceptibility in Ae. aegypti using CDC bottle bioassays including discriminating doses of permethrin and deltamethrin, along with increased doses (5X and 10X) for each insecticide.  Test at least 120 female individuals from each location and bioassay. Register knocked down data at diagnostic time. Subsequently, susceptible and resistant individuals will be transferred to separate holding containers with 10% sugar solution to evaluate mortality after 24 hours. After recording the mortality rate at 24 hours, both susceptible and resistant individuals will be separated and stored at -20°C until molecular analyses.
3. Perform individual DNA extraction in both adult mosquitoes from bioassays (n=240) and wild-caught mosquitoes from field (n=150). Additionally, pools for DNA extraction will be carried out in mosquitoes collected by aspiration (n=250).
4. Perform PCR and Sanger sequencing for kdr alleles detection (410, 989 and 1016) up to 240 Ae. aegypti from bioassays and up to 150 Ae. aegypti from adult aspirations.
5. Provide access to UNAH laboratory and use of equipment for dengue and other pathogen detection and bloodmeal analysis in ticks and hematophagous diptera.
6. Contact the Ministry of Health to request additional tick specimens for identification and/or pathogen screening. 

1. DATA SHARING

1. The contractor will provide collection of data information on field site location, habitat description, collection method, vector species identified, and numbers recorded per collection period per site as well as relevant epidemiological data as requested by NAMRU SOUTH Entomology.
2. Provide input for report and scientific manuscript preparation.

1. EQUIPMENT, SUPPLIES AND MATERIALS

1. The contractor will cover all the costs of equipment, supplies and materials necessary for the execution of their activities. These include (but not limited to) supplies for collection, transport, storage and identification of entomological samples, morphological identification, DNA isolation, molecular identification and pathogen screening.
2. NAMRU SOUTH Entomology will provide field equipment and/or accessories when performing joint Aedes collections.  Will also provide reagents, consumables and labor for Aedes DNA extraction and Nanopore sequencing experiments.

1. Shipments

1. Upon request and payment by NAMRU SOUTH or USDA-CMAVE, contractors shall conduct shipments of samples from field sites to USDA-CMAVE or NAMRU SOUTH, respectively.

The delivery addresses are:

1. USDA-ARS-CMAVE, Mosquito & Fly Research Unit, 1600-1700 SW 23rd Drive, Gainesville, FL, 32608, Attn.: Alden Estep, phone number +1 352-374-5806.
2. NAMRU SOUTH, Entomology Department, Av. Venezuela cuadra 36 S/N, Bellavista, Callao, Peru, Attn.: Gissella Vasquez, phone number +51 614-4143

1. The contractor shall include the following clearly labeled into sample shipments:

1. Entomological samples collected as part of the study.
2. Nucleic acid material obtained from collected samples covered under this contract.

1. Required documentation for specimen exportation. USDA-CMAVE will provide appropriate import permits.

5. DELIVERABLES:

The materials and services specifically listed and requested must be delivered in a timely manner and must be of good quality.

1. Project database that includes specimen identification, collecting technique, location, date, and other pertinent information of mosquitoes collected and following NAMRU SOUTH database format.
2. Approximately 1,500 Aedes aegypti immatures will be reared to obtain colonies for insecticide resistance assays.
3. Approximately 390 morphologically identified Aedes aegypti specimens collected across sites for ADN/RNA extraction, arbovirus screening and kdr genotyping. Additionally, 250 pools of mosquitoes will be processed. Mosquito specimens will be stored and shipped to NAMRU SOUTH or partner laboratory for further molecular analysis, as needed.
4. Pyrethroid susceptibility/resistance data for 240 adult Ae. aegypti mosquitoes from each collection site. Mortality rates and time-response curves (logistic regression) for permethrin and deltamethrin, and kdr genotyping will be included.

6. SPECIAL REQUIREMENTS: N/A

7. TASK ORDERS: N/A

8. ANNUAL SCHEDULE:

Quarter 1 and 2:

• Organizational meetings, identify VBD transmission sites focusing on dengue or other Aedes-borne infections through meetings with key contacts and MoH personnel.
• Coordinate entomological collection activities with local authorities.
• Purchase supplies and materials for field work and laboratory activities.
• Participate in monthly teleconferences with NAMRU SOUTH.
• Conduct entomological sample collection activities in endemic areas in Honduras. Pooling of entomological specimens collected for molecular testing. Perform testing and analysis in historical samples if required. Database preparation and submission to NAMRU SOUTH.
• Rear Ae. aegypti mosquito colonies to obtain F1 egg batches.
• Perform Aedes bioassay exposure and DNA extraction, PCRs kdr sequencing. Database preparation and submission to NAMRU SOUTH.
• Send epidemiological data and laboratory results to NAMRU SOUTH.
• Perform one shipment of samples to NAMRU SOUTH Lima or USDA CMAVE if required.
• Provide access to laboratory space and use of equipment for sand fly molecular testing and/or bloodmeal analysis.

Quarter 3 and 4:

• Continue entomological sample collection in Honduras.
• Continue DNA extractions and molecular testing of entomological specimens.
• Rear Ae. aegypti mosquito colonies to obtain F1 egg batches.
• Continue logistics support (lab space/equipment) for sand fly testing and bloodmeal analysis.
• Contact MoH to request Aedes aegypti for pathogen screening or insecticide bioassays.
• Participate in monthly teleconference with NAMRU SOUTH.
• Perform one shipment of samples to NAMRU SOUTH or USDA CMAVE if required.
• Provide access to laboratory space and use of equipment for sand fly molecular testing and/or bloodmeal analysis.
• Database quality control and data analysis.
• Deliver databases for field collecting data, vector species identification, DNA samples, and insecticide resistance testing to NAMRU SOUTH.
• Assist NAMRU SOUTH with data analysis, revision of study results and reporting, and manuscript preparation.

Period of performance:

The period of performance is 12 months. Include option Year 1 and Year 2.