Detection of high Leishmania infantum loads in Phlebotomus perniciosus captured in the leishmaniasis focus of southwestern Madrid region (Spain) by real time PCR

- A real time PCR was applied in female sand flies captured in a human leishmaniasis focus.
- Limit of detection achieved was 0.001 parasite/reaction, corresponding to 0.08 fg of parasite DNA.
- High parasite loads (>1000 parasites/reaction) in sand flies from the focus were observed.
- Unfed females showed significant higher parasite loads than blood-fed females.

Since 2010 a human leishmaniasis outbreak has been notified in southwestern Madrid region that still remains active. Entomological surveys have been carried out in the affected area in order to obtain information about species diversity, distribution, and density of sand flies. Moreover, molecular identification of blood meal preferences of sand flies and molecular detection of Leishmania infantum has been performed. In this work, we optimized a real time PCR assay in order to determine parasite loads in unfed and blood-fed Phlebotomus perniciosus female sand flies caught in the focus area. Results showed elevated parasite loads in nearly 70% of the studied positive sand flies. Furthermore, significantly higher parasite loads were observed in females without blood in their guts. In conclusion, high L. infantum loads found in P. perniciosus sand flies from the Madrid focus support the exceptional characteristics of this outbreak.

In this work a qPCR assay was applied in order to estimate the number of parasites in P. perniciosus females collected in the human leishmaniasis focus that is affecting southwestern Madrid region.197