LIMITATION OF PRIMERS USED IN PCR FOR THE CHARACTERIZATION OF LEISHMANIA INFANTUM.

Conventional PCR provides Leishmania species characterization with even a small amount of biological material. Species-specific primers have been a widely used alternative; however, nonspecific amplifications are a reality, interfering with PCR efficiency. In endemic areas with multiple etiological agents for leishmaniasis, there is a requirement for higher specificity of primers. This study evaluates 3 pairs of primers described for the identification and characterization of Leishmania infantum. Primers RV1/RV2, LEISH1/LEISH2, and FLC2/RLC2 were used with the DNA of L. infantum, Leishmania amazonensis, and Leishmania braziliensis. An initial temperature curve was performed (52-62 C) to determine the optimal annealing temperature, followed by a dilution curve of Leishmania DNA (500 pg/µl, 50 pg/µl, 5 pg/µl, 500 fg/µl, 50 fg/µl, 5 fg/µl, and 0.5 fg/µl) to be used for analytical sensitivity. RV1/RV2 PCR amplified L. infantum and L. amazonensis at all analyzed temperatures; LEISH1/LEISH2 PCR amplified all 3 species of Leishmania, although at some temperatures L. infantum was specifically amplified, and, finally, FLC2/RLC2 PCR amplified only L. infantum at all temperatures analyzed. In terms of sensitivity, RV1/RV2 PCR detected 1 fg of L. infantum DNA and 100 pg of L. amazonensis DNA; LEISH1/LEISH2 PCR detected 1 fg of L. infantum DNA, 100 fg of L. amazonensis DNA, and 10 fg of L. braziliensis DNA; and FLC2/RLC2 PCR detected 10 fg of L. infantum DNA. Thus, PCR with FLC2/RLC2 primers is best suited for the molecular characterization of L. infantum, especially in areas where there is an incidence of more than 1 Leishmania species, such as South America.

Canine visceral leishmaniasis diagnosis by UV spectroscopy of blood serum and machine learning algorithms.

Visceral leishmaniasis (VL) is a zoonotic disease caused by the protozoan Leishmania infantum, and dogs are considered the main urban hosts for future disease transmission. The first and most effective control against the spread of disease relies on identifying infected animals, followed by their treatment or sacrifice, to reduce the protozoan reservoirs. Despite the availability of various diagnostic tests for VL in dogs the development of a quick and accurate diagnosis is essential from a public health and ethical point of view. Here we analyze the use of UV-Vis spectroscopy as an alternative diagnostic method for VL diagnosis by using the antigen-antibody interaction in canine blood serum and machine learning algorithms. The main UV spectra in the 220 to 280 nm range exhibit nine electronic absorption bands, but no significative difference could be identified between the positive and negative group spectra. Finally, UV pre-proceed spectra by SNV (standard normal variate) were submitted to principal component analysis followed by Linear SVM algorithm, the prediction model was tested in a leave-one-out cross-validation and external validation test reaching an overall accuracy of 75%.

Geographic distribution of human leishmaniasis and phlebotomine sand flies in the State of Mato Grosso do Sul, Brazil.

BACKGROUND: In the State of Mato Grosso do Sul, Brazil, sand flies and cases of visceral (VL) and cutaneous (CL) leishmaniases have been reported in almost all municipalities. The aim of this study was to analyze the geographic distribution of VL and CL in relation the sand fly species found in the municipalities of Mato Grosso do Sul. METHODS: We analyzed VL and CL cases from 2001 to 2018 using data from the Notifiable Diseases Information System (SINAN). Data collected since 2003 on the presence of sand fly vectors (proven or suspected) were provided by the State Health Secretariat. RESULTS: A total of 3566 and 3030 cases of VL and CL, respectively, were reported from 2001 to 2018. The municipalities with the most reported cases of VL were Campo Grande (2495), Três Lagoas (442), Corumbá (140) and Aquidauana (136); and those for CL were Campo Grande (635) and Bodoquena (197). The following sand fly species with vector potential were found in 59 municipalities (74.7%): Lutzomyia longipalpis, Lutzomyia cruzi, Nyssomyia whitmani, Migonemyia migonei, Nyssomyia neivai, Pintomyia pessoai, Bichromomyia flaviscutellata and Pintomyia fischeri. Sand flies were present in six municipalities where no cases of VL were reported and in two municipalities where no cases of CL were reported. CONCLUSIONS: Our results indicate that the geographical distribution of VL and CL in Mato Grosso do Sul expanded during the study period, and highlight the presence of sand fly vectors in municipalities where these diseases are currently considered to be non-endemic.

Data on the differentiation among Leishmania (Viannia) spp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis in Brazilian clinical samples using real-time PCR.

This article contains the data regarding Leishmania species identification in human and canine clinical samples from a Brazilian region endemic for Leishmania (Viannia) spp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis, using a previously developed approach involving two qPCR assays (qPCR-ML and qPCR-ama). The data are related to the article "Real-time PCR to differentiate among Leishmania (Viannia) subgenus, Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis: application on Brazilian clinical samples" [1], and include also details of clinical evaluation/diagnosis of human patients and primer sequences used in the qPCR assays. The Leishmania species has been determined in 27 canine samples and 11 human samples, exploiting HRM analysis of qPCR-ML and Cq values of qPCR-ML and qPCR-ama, as reported previously [2]. The qPCR data were in agreement with the species characterization obtained with other methods such as conventional species-specific PCR, ITS1 PCR-RFLP or DNA sequencing. Despite the limited number of clinical samples, these data are encouraging for a potential application in regions where L. (Viannia) spp., L. (L.) infantum and L. (L.) amazonensis are co-endemic.

Characterization of Leishmania species from Central-West Region of Brazil.

Leishmaniasis represents a complex of diseases with a broad clinical spectrum and epidemiological diversity. Involving a variety of agents, reservoirs, and vectors, cutaneous leishmaniasis (CL) presents different transmission patterns, which makes its control especially difficult. Epidemiological characterization with identification of existing Leishmania species in a particular region is essential for understanding this parasitosis epidemiology. Because similar results may be observed in places with overlapping transmission of visceral and cutaneous leishmaniasis, contributing to knowledge about the epidemiological cycle of cutaneous leishmaniasis is of global interest. This study proposes to identify the different species of Leishmania present in human CL cases in the state of Mato Grosso do Sul, Brazil. Slides of imprint tissue were selected from patients diagnosed with CL from 2009 to 2013 at the Laboratório de Parasitologia Clínica, Universidade Federal de Mato Grosso do Sul. The slides were scraped and extracted genetic material was amplified with PCR addressing the Leishmania spp. kDNA region. The reaction product was subjected to RFLP for the specific identification of Leishmania. Forty-three samples (82.7%) amplified with PCR were considered positive. Of these, it was possible to determine the species in 40 samples (76.9%) through analysis of electrophoresis profiles. Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis, and Leishmania (Leishmania) infantum were identified as the etiologic agents of CL in Mato Grosso do Sul. The results of this study permit a more detailed understanding of CL epidemiology and the geographical distribution of its agents.

First record of Leishmania braziliensis presence detected in bats, Mato Grosso do Sul, southwest Brazil.

Leishmaniasis, a zoonotic disease caused by parasites of the genus Leishmania, has expanded beyond its natural range and is becoming increasingly urban. Using PCR and PCR-RFLP, we detected Leishmania (Viannia) braziliensis in two bats (Chiroptera) in Mato Grosso do Sul, Brazil, an endemic area. This is the first record of L. (V.) braziliensis in bats. It is also the first record of any Leishmania sp. in bats in the state. The animals testing positive were found in both a rural site and an urban site. These results indicate the need for further research into the viability of Leishmania in bats and could potentially have implications for public health in Mato Grosso do Sul, given the large populations of urban bats, their mobility, and their ability to roost at close proximity to humans within residences and other buildings.