Concurrent detection of Leishmania infantum in dogs and Leishmania DNA in peridomestic sand fly pools from a coastal focus in Costa Rica.

Canine visceral leishmaniasis caused by Leishmania infantum has recently been confirmed as autochthonous in Costa Rica, yet molecular evidence from peridomestic settings remains limited. We investigated dogs clinically suspected of leishmaniasis and screened phlebotomine sand flies collected in the same canine focus in Tamarindo (Guanacaste Province) between March and October 2025. Ten dogs were tested using a rapid serological assay and real-time PCR (qPCR) targeting kinetoplast minicircle DNA (kDNA); qPCR-positive samples were further assessed by PCR targeting the internal transcribed spacer 1 region (ITS-1 PCR) and sequencing for species confirmation. In parallel, female sand flies were morphologically identified, pooled by species/site/feeding status (2-11 per pool), and screened by kDNA qPCR; selected positive pools were evaluated by digital droplet PCR (ddPCR) to corroborate low-level detection. Blood-meal sources were investigated in qPCR-positive pools using host DNA markers. Four dogs were seropositive and qPCR-positive, and ITS-1 sequencing confirmed L. infantum in two cases, whereas other qPCR-positive dog samples did not yield ITS-1 amplicons, consistent with low parasite DNA loads. Forty-five female sand flies were collected and grouped into 13 pools; three pools (all composed of Lutzomyia serrana) were qPCR-positive near the detection limit, including one non-engorged pool, corroborated by ddPCR. Blood-meal analysis detected canine DNA in one positive pool and avian DNA in others. These findings document concurrent detection of canine L. infantum infection and Leishmania DNA in peridomestic sand fly pools from the same locality. Although the entomological findings should be interpreted cautiously due to low parasite DNA loads and the lack of species-level confirmation in sand flies, the results provide preliminary evidence consistent with local Leishmania circulation and support the need for integrated One Health surveillance of dogs and vectors in Costa Rica.