Location: Mosquito and Fly Research
Project Number: 6036-32000-052-102-I
Project Type: Interagency Reimbursable Agreement
Start Date: Aug 1, 2022
End Date: Sep 30, 2022
Objective:
The overall objective of this project is to collaborate with vector control agencies in Panama to determine the effects of the recently identified invasion of new resistance alleles on our capabilities to assess and control insecticide resistance.
Subobjectives:
1) Conduct quarterly mosquito surveillance trapping in Panama with Gorgas Institute partners focusing on the collection of Aedes aegypti as the primary vector species.
2) Gather collection data from local (MOH & Gorgas Institute) ongoing, surveillance for input into a WRBU gap analysis for Panama to guide improved future surveillance.
3) Conduct simultaneous pathogen and kdr detection in Aedes aegypti using the sample workflow currently in place.
4) Update and validate existing NAMRU6 and USDA resistance assessment protocols to include surveillance for the invasive Indopacific resistance mutations.
Approach:
Study site: Surveillance sites in Panama will be a mix of sites already in use by GMI and local vector control operations. Use of previously surveilled sites will allow assessment of changes over time with respect to species diversity and kdr alleles.
Study period: Mosquito collections will be performed from April-September 2022.
Study size: Attempts will be made to collect at least 30 Aedes aegypti from each surveillance site during each collection period. Additional mosquitoes and other host seeking hematophagous diptera collected in traps will be cataloged, identified, and reported.
Sample and data collection: Arthropod surveillance collections will be made using a variety of trapping methods depending on local conditions. Adults, whether emerged or trapped, will be frozen on dry ice using the CryoCube system or placed into nucleic acid preservative to prevent degradation of RNA pathogens. Samples will remain frozen/preserved. Legs will be removed from individual Aedes aegypti and banked for kdr testing when samples are pooled for pathogen testing.
Laboratory analysis: Toxicologic characterization of field collections will be conducted at GMI. Samples for molecular and pathogen analysis will be processed using the standard sample workflow for arthropod surveillance specimens in the laboratory at GMI in Panama, or in the US at USDA-CMAVE. USDA-CMAVE has APHIS approved permits and laboratories for sample importation. Pooled samples for pathogen testing will be purified using the Zymo Quick RNA/DNA kit or equivalent. The RNA specimens will be stored at -80C until use. DNA samples will be stored at -20C at USDA-CMAVE for long-term storage and use in future studies. Purified RNA will be used for RT-PCR based detection of DENV, ZIKV, CHIK, and MAYV using SOPs and primers already in use. Samples that give positive initial RT-PCR results will be confirmed with a second assay using a second amplicon. Assay will include positive and negative controls with purified virus provided by BEIResources. Samples with 2 arbovirus positives will be sequenced to provide more information on the pathogen. This sequencing will be conducted on the Oxford Nanopore Technologies GridION platform using an adaptive sequencing protocol with the Ae aegypti host genome as the negative database and custom adaptors to enhance the unamplified signal of the pathogen. Leg samples for genetic resistance testing will homogenized in 96 well plates following the procedure described in Estep et al (2018) and currently in place in NAMRU6 and USDA CMAVE. This kdr assay will be modified using the primers of Saavedra-Rodriguez (2008) to assess the presence of Indopacific mutations. Samples positive for Indopacific mutations will be amplified by PCR and sent for Sanger sequencing to confirm the presence.
