A duplex real-time PCR assay for the detection and differentiation of
Leishmania infantum and Leishmania tarentolae in vectors and potential
Leishmanioses are vector-borne diseases caused by Leishmania spp., which
are transmitted by phlebotomine sand flies (Diptera, Psychodidae). The
recent reports in humans of Leishmania tarentolae, which is primarily
found in cold-blooded animals, and Leishmania infantum in Sergentomyia
minuta spurred us to develop an internal transcribed spacer 1-based
duplex quantitative real-time PCR (dqPCR) assay for the detection and
differentiation between these Leishmania spp. The specificity of dqPCR
was assessed by processing DNA samples from Phlebotomus spp. (n=188) and
Se. minuta (n=171) and from tissues (i.e., heart, liver, muscle, lungs,
spleen, kidney, eggs) of Podarcis siculus (n=4) and Tarentola
mauritanica (n=3). In the absence of naturally infected and/or
co-infected lizards, DNA from cultured L. infantum and L. tarentolae
were spiked into tissues of lizards and used as controls. The analytical
sensitivity of the dqPCR, assessed using 10-fold serial dilutions of DNA
from both Leishmania spp. and spiked DNA samples from lizards was 2.3 x
10-7 ng/2 µl for L. infantum and 2.1 x 10-7 ng/2 µl for L. tarentolae.
With the spiked DNA samples, the dqPCR detected up to 2.6 x 10-6 ng/2 µl
of L. infantum and up to 2.1 x 10-7 ng/2 µl of L. tarentolae. Of 359
phlebotomine sand flies tested, five (3.6%) and two (1.4%) Ph.
perniciosus scored positive for L. infantum and L. tarentolae,
respectively. Similarly, of 171 Se. minuta, 56 (32.7%) and six (3.5%)
scored positive for L. tarentolae and L. infantum, respectively.
Co-infection with both Leishmania spp. was detected in two Se. minuta
(1.2%). Out of seven reptiles tested, four P. siculus were positive for
L. tarentolae. The newly dqPCR herein described may represent an
improvement in the diagnosis of L. infantum and L. tarentolae and may
assist in identifying the role of lizards as reservoirs and Se. minuta
as vector, for these Leishmania spp.