Visceral leishmaniasis represents an important
public health issue in different parts of the world, requiring that
measures be put in place to control the spread of the
disease worldwide. The canine
leishmaniasis diagnosis is not easy based on clinical signs, since
dogs may not develop the
infection with recognizable signs. Thus, the laboratorial
diagnosis is essential to ascertain the
incidence and
prevalence of canine
leishmaniasis especially in areas with major control efforts. Although, the
diagnosis can be performed by the use of different approaches, the molecular
methods such as
PCR have become an indispensable tool for
leishmaniases diagnosis. A TaqMan assay for
real-time PCR (Linj31-qPCR) was developed to determine the
parasite occurrence in clinical cases of
leishmaniasis. The assay targets an L. (L.) infantum hypothetical
protein region. The
specificity of the assay was verified by using
Leishmania World Health Organization reference
strains including
parasites belonging to subgenus L. (
Leishmania), subgenus L. (Viannia), other
Leishmania species and
Trypanosoma cruzi. The
sensitivity was verified by using isolates of L. (L.) amazonensis and L. (L.) infantum. The usefulness of the assay for
diagnosis was ascertained by testing 277 samples from
dogs in regions endemic for visceral and/or
cutaneous leishmaniasis and from regions in which
leishmaniasis was not endemic in São Paulo
State,
Brazil.
Diagnosis of canine
visceral leishmaniasis (CVL) was determined on these
animals by conventional
PCR and three
serological tests. The
dog samples were divided into four groups. I,
dogs with CVL (n = 101); II,
dogs with other
diseases and without CVL (n = 97); III,
dogs with American
cutaneous leishmaniasis (n = 7), and, IV,
dogs without CVL (n = 72) from areas where
leishmaniasis was not endemic as
control group. Results indicated that Linj31-qPCR was able to identify
parasites belonging to subgenus L. (
Leishmania) with no cross-amplification with other
parasite subgenera. The Linj31-qPCR detected
Leishmania parasites DNA in 98% of samples from Group I. In conclusion this
methodology can be used as routine diagnostic tools to detect
parasites from subgenus
Leishmania.