Field study of the improved rapid sand fly exposure test in areas endemic for canine leishmaniasis.

Author summary Canine leishmaniasis (CanL) is a widespread severe chronic disease in dogs, caused by the protozoan parasite Leishmania infantum. Leishmania parasites are transmitted by the bite of phlebotomine sand flies, of which the most important one in southwestern Europe is Phlebotomus perniciosus. A sand fly bite is accompanied by the inoculation of saliva, which elicits a specific antibody response in the host. Past studies have used these antibodies as markers of exposure to sand flies. Recently, a rapid sand fly exposure test was prepared using one salivary protein of P. perniciosus, the SP03B protein. This rapid test possessed a high sensitivity and specificity when screening experimentally exposed versus non-bitten dogs. However, since naturally exposed dogs possess a higher variability of antibodies against sand fly saliva, we have optimized the previously proposed rapid test and evaluated its detectability on field samples. In this study we present the optimized rSP03B sero-strip that possesses a higher specificity and an ability to detect seropositive dogs from a naturally exposed dog population in a similar fashion as standard serological methods. Furthermore, we have optimized it with whole canine blood which renders it suitable for use in field conditions. Background Canine leishmaniasis (CanL) is a severe chronic disease caused by Leishmania infantum and transmitted by sand flies of which the main vector in the Western part of the Mediterranean basin is Phlebotomus perniciosus. Previously, an immunochromatographic test (ICT) was proposed to allow rapid evaluation of dog exposure to P. perniciosus. In the present study, we optimized the prototype and evaluated the detection accuracy of the ICT in field conditions. Possible cross-reactions with other hematophagous arthropods were also assessed. Methodology/Principal findings The ICT was optimized by expressing the rSP03B protein in a HEK293 cell line, which delivered an increased specificity (94.92%). The ICT showed an excellent reproducibility and inter-person reliability, and was optimized for use with whole canine blood which rendered an excellent degree of agreement with the use of serum. Field detectability of the ICT was assessed by screening 186 dogs from different CanL endemic areas with both the SGH-ELISA and the ICT, and 154 longitudinally sampled dogs only with the ICT. The ICT results corresponded to the SGH-ELISA for most areas, depending on the statistical measure used. Furthermore, the ICT was able to show a clear seasonal fluctuation in the proportion of bitten dogs. Finally, we excluded cross-reactions between non-vector species and confirmed favorable cross-reactions with other L. infantum vectors belonging to the subgenus Larroussius.