NEW ENTOMOLOGICAL SAMPLING METHOD FOR ARBOVIRUS SURVEILLANCE: FIELD TEST IN ITALY AND DJIBOUTY

Background: Several strategies are available in mosquito-borne disease surveillance, according to target species and surveillance goals. In this study, we propose an innovative method to simplify pathogen detection from mosquitoes. To this aim we modified a BG sentinel trap to include a sugar delivery system equipped with a nucleic acid preserving substrate (FTA-card). During sugar feeding, pathogens in mosquito saliva are released on a honey-soaked FTA-card, remaining detectable for several days at environmental conditions. To define the applicability of the system we tested the modified trap in Italy and Djibouti exploring the performance in relation to i) a standard surveillance method (analysis of mosquitoes trapped with CDC-CO2) ii) different target species iii) sampling scheme and iv) long term storage. Methods: All mosquitoes collected in Italy (Jul-Aug 2019 and 2021) and Djibouti (Jan Feb 2020) were morphologically identified to define the species. Each card was tested through RNA extraction and PCR for arboviruses detection. In samples collected in Italy we also processed mosquitoes in pools of up to 100 specimens as described for FTA-cards. Collected data were analysed through statistical software R with Mann-Whitney U test and GLM models. Results: Data collected in Italy in 2019 show that the CDC-CO2 trap catches 1.6 times more mosquitoes than the modified BG-sentinel under the same condition (GLM, p < 0.001; 95%CI=1.2-2.2). In relation to mosquito species, the proposed method collects less Cx. pipiens (median BG=96, CDC=189; Mann-Whitney U test, p<0.001), but shows best performance in trapping Ae. albopictus (median BG=7.5, CDC=1; Mann-Whitney U test, p<0.0001) and no difference for Oc. caspius (median BG=8.5, CDC=10.5; Mann-Whitney U test, p=0,8). Both trap types detect West Nile (WNV) and Usutu (USUV) viruses. Positive events from FTA-cards (N=2/72) are later and less than events reported with standard method (positive pool/tested mosquitoes, N=15/16646). In 2021 we modified the sampling scheme increasing traps and working days up to 4-day sampling. In this collection, the proposed system (N=2/50) highlight USUV circulation first of the standard method (N=2/919). FTA cards collected in Djibouti show the circulation of WNV (N=2/71) and DENV (N=1/71) in different dates and traps, according to the presence of vector species in the corresponding trap. Discussion: The detection of pathogens from FTA-cards in field show that the proposed system could be used for arbovirus surveillance. Under the same condition, our results suggest a low sensitivity of the modified trap compared to standard method, at least for USUV and WNV surveillance. The performance could be enhanced increasing the number of traps and working days and resulting particularly advantageous in long-term storage of arboviruses and in minimize the samples to detect arboviruses circulation in mosquitoes.