Patrícia Flávia Quaresma

Molecular diagnosis of canine visceral leishmaniasis: Identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR.

The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.

Natural infection of Lutzomyia neivai and Lutzomyia sallesi (Diptera: Psychodidae) by Leishmania infantum chagasi in Brazil.

ABSTRACT Natural infections with Leishmania were found in females of the phlebotomine sand flies Lutzomyia neivai (Pinto) (=Nyssomyia neivai) and Lutzomyia sallesi (Galvão & Coutinho) (=Evandromyia sallesi) (Diptera: Psychodidae) from Lassance, in the Brazilian state of Minas Gerais. Promastigotes were found in the pyloric region of the former species and in the abdominal midgut of the latter species. Insects found to be infected by microscopic examination were macerated in saline solution and inoculated into hamsters. Subsequent analysis by polymerase chain reaction-restriction fragment length polymorphism revealed both isolates to belong to the species Leishmania infantum chagasi Cunha & Chagas.

Wild, synanthropic and domestic hosts of Leishmania in an endemic area of cutaneous leishmaniasis in Minas Gerais State, Brazil

Domestic, synanthropic and wild hosts of Leishmania spp. parasites were studied in an area endemic for American tegumentary leishmaniasis (ATL), specifically in northern Minas Gerais State, Brazil. Domestic dogs and small forest mammals are reservoir hosts for L. (Leishmania) infantum. However, the role that these animals play in the transmission cycle of the Leishmania spp. that cause cutaneous leishmaniasis is not well known. This study evaluated 72 rodents, 25 marsupials and 98 domestic dogs found in two villages of the Xakriabá Indigenous Territory, an area of intense ATL transmission. A total of 23 dogs (23.47%) were shown to be positive according to at least one test; 8 dogs (8.16%) tested positive in a single serological test and 15 dogs (15.31%) tested positive by IFAT and ELISA. Eleven dogs were euthanised to allow for molecular diagnosis, of which nine (81.8%) tested positive by PCR for Leishmania in at least one tissue. Seven animals were infected only with L. (L.) infantum, whilst two displayed a mixed infection of L. (L.) infantum and L. (V.) braziliensis. Isoenzymatic characterisation identified L. (L.) infantum parasites isolated from the bone marrow of two dogs. Of the 97 small mammals captured, 24 tested positive for Leishmania by PCR. The results showed that L. (V.) braziliensis, L. (L.) infantum and L. (V.) guyanensis are circulating among wild and synanthropic mammals present in the Xakriabá Reserve, highlighting the epidemiological diversity of ATL in this region.

Relationship of Leishmania-specific IgG levels and IgG avidity with parasite density and clinical signs in canine leishmaniasis.

The clinical status and tissue parasite burden of the skin and spleen of 40 dogs naturally infected with Leishmania chagasi (syn. Leishmania infantum), together with 5 uninfected control dogs, were assessed. On the basis of the clinical evaluation, infected dogs were classified as asymptomatic (AD) or symptomatic (SD). Infected animals were also grouped according to their parasite load as exhibiting low (LP), medium (MP) and high (HP) parasitism. The results indicated a high parasite load in the skin samples of SD animals in relation to the AD group. The serum immunoglobin isotype profiles of the studied animals revealed increased levels of IgG(1) in the AD and LP dogs, whereas high levels of IgG(2) were correlated with SD and HP dogs. The avidity index (AI) of IgG(total) in the SD group was high in comparison of that of the AD group. Moreover, animals with a larger parasite burden either in the spleen or skin showed higher AI values than animals with lower parasitism. Based on these findings, it is suggested that CVL commences with an asymptomatic clinical form with low parasitism, high production of IgG(1) and low affinity of IgG(total) molecules, and evolves into a symptomatic clinical form with higher parasitism intensity, higher IgG(2) levels, and high affinity of IgG(total).

Natural Leishmania sp. reservoirs and phlebotomine sandfly food source identification in Ibitipoca State Park, Minas Gerais, Brazil.

Leishmania spp are distributed throughout the world and different species are associated with varying degrees of disease severity. However, leishmaniasis is thought to be confined to areas of the world where its insect vectors, sandflies, are present. Phlebotomine sandflies obtain blood meals from a variety of wild and domestic animals and sometimes from humans. These vectors transmit Leishmania spp, the aetiological agent of leishmaniasis. Identification of sandfly blood meals has generally been performed using serological methods, although a few studies have used molecular procedures in artificially fed insects. In this study, cytochrome b gene (cytB) polymerase chain reaction (PCR) was performed in DNA samples isolated from 38 engorged Psychodopygus lloydi and the expected 359 bp fragment was identified from all of the samples. The amplified product was digested using restriction enzymes and analysed for restriction fragment length polymorphisms (RFLPs). We identified food sources for 23 females; 34.8% yielded a primate-specific banding profile and 26.1% and 39.1% showed banding patterns specific to birds or mixed restriction profiles (rodent/marsupial, human/bird, rodent/marsupial/human), respectively. The food sources of 15 flies could not be identified. Two female P. lloydi were determined to be infected by Leishmania using internal transcribed spacer 1 and heat shock protein 70 kDa PCR-RFLP. The two female sandflies, both of which fed on rodents/marsupials, were further characterised as infected with Leishmania (Viannia) braziliensis. These results constitute an important step towards applying methodologies based on cytB amplification as a tool for identifying the food sources of female sandflies.

Gene expression profile of cytokines and chemokines in skin lesions from Brazilian Indians with localized cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a chronic inflammatory disease caused by dermotropic Leishmania species belonging to the Viannia subgenera, with Leishmania (V.) braziliensis considered the main agent in Brazil. After infection, a local inflammatory process is initiated, inducing the expression of several cytokine/chemokine genes. We evaluated the immunity to CL of patients living in the indigenous community Xakriabá, Minas Gerais state, Brazil, by performing detailed analyses of the mRNA expression of different cytokines and chemokines in CL lesions, considering the time evolution (recent or late). We also studied the profile of the inflammatory infiltrate by histopathological analysis. The histopathological features of recent CL lesions showed an intense inflammatory reaction, characterized by the presence of both mononuclear and polymorphonuclear cells, whereas late CL lesions exhibited a predominance of mononuclear leukocytes. The gene expression of cytokines/chemokines in skin biopsies from the CL group showed higher transcript levels of modulatory (IL10 and TGFB1), anti-inflammatory (IL4), and pro-inflammatory (TNF, IFNG, IL12B, CCL2, CCL3, CCL5, CXCL10) biomarkers in recent lesions than in late lesions. Our findings suggest that differential gene expression of cytokines and chemokines found in skin lesions from CL patients is associated with time evolution of lesions.

Ecological aspects of the Phlebotominae fauna (Diptera: Psychodidae) in the Xakriabá Indigenous Reserve, Brazil

BackgroundSand fly collections were performed to study ecological aspects of the Phlebotominae fauna of the Xakriabá Indigenous Reserve, an area with endemic cutaneous leishmaniasis, located in the state of Minas Gerais, Brazil.MethodsThe collections were performed in peridomicile areas and along trails previously selected for the study of wild and synanthropic Leishmania hosts. Differences in the distribution patterns of the sand fly species as well as in species richness and abundance between the different ecotopes were investigated during both rainy and dry seasons over the course of the study period.ResultsA total of 8,046 sand flies belonging to 11 genera and 28 species were collected. Lutzomyia longipalpis and Nyssomyia intermedia were the most abundant species in peridomicile areas, whereas Martinsmyia minasensis and Lutzomyia cavernicola were the most abundant species among the different trail ecotopes.ConclusionThe different composition of the sand fly fauna observed in the peridomicile areas and in the trails during the study, reinforces the importance of sampled different areas in a phlebotomine fauna survey. The presence of Lutzomyia longipalpis and Ny. Intermedia most abundant in peridomicile can be important to Leishmania infantum and Leishmania braziliensis transmission in the Imbaúbas native village.

Molecular Detection of Leishmania in Phlebotomine Sand Flies (Diptera: Psychodidae) from a Cutaneous Leishmaniasis Focus at Xakriabá Indigenous Reserve, Brazil

Autochthonous cases of American cutaneous leishmaniasis (ACL) have been reported since 2001 in the Xakriabá Indigenous Reserve located in the municipality of São João das Missões in northern Minas Gerais state, Brazil. In order to study the presence of Leishmania DNA in phlebotomine sand flies, six entomological collections were carried out from July 2008 through July 2009, using 40 light traps placed in peridomicile areas of 20 randomly selected houses. From October 2011 through August 2012, another six collections were carried out with 20 light traps distributed among four trails (five traps per trail) selected for a previous study of wild and synanthropic hosts of Leishmania. A total of 4,760 phlebotomine specimens were collected belonging to ten genera and twenty-three species. Single female specimens or pools with up to ten specimens of the same locality, species and date, for Leishmania detection by molecular methods. Species identification of parasites was performed with ITS1 PCR-RFLP using HaeIII enzyme and genetic sequencing for SSU rRNA target. The presence of Leishmania DNA was detected in eleven samples from peridomicile areas: Lu. longipalpis (two), Nyssomyia intermedia (four), Lu. renei (two), Lu. ischnacantha, Micropygomyia goiana and Evandromyia lenti (one pool of each specie). The presence of Leishmania DNA was detected in twelve samples from among the trails: Martinsmyia minasensis (six), Ny. intermedia (three), Mi. peresi (two) and Ev. lenti (one). The presence of Leishmania infantum DNA in Lu. longipalpis and Leishmania braziliensis DNA in Ny. intermediasupport the epidemiological importance of these species of sand flies in the cycle of visceral and cutaneous leishmaniasis, respectively. The results also found other species associated with Leishmania DNA, such as Mt. minasensis and Ev. lenti, which may participate in a wild and/or synanthropic cycle of Leishmania transmission in the studied area.

Hourly Activity and Natural Infection of Sandflies (Diptera: Psychodidae) Captured from the Aphotic Zone of a Cave, Minas Gerais State, Brazil

Sandflies are holometabolous insects that are of great epidemiological importance in the neotropical region as vectors of leishmaniases. Caves are ecotopes that significantly differ from external environments and, among the insects that live or visit their internal area and adjacent environment, sandflies are commonly found. Based on this context, the objective of this work was to examine the period of activity of sandflies in the cave environment in the aphotic zone. Thus, four sandfly captures were conducted, one in each season of the year, in a cave where studies on the bioecological aspects of sandfly fauna have been conducted since 2008. In this same study, we have also noticed the presence of flagellates in some captured females. Catches were carried out for 24 hours using a Shannon trap, light bait, and cave walls were actively searched. We collected a total of 638 sandflies, representing 11 species. The most abundant species and with more intense period of activity were, in descending order: Lu. cavernicola (62%), Ev. spelunca (16%) and Ev. sallesi (14%). A total of 69 females were dissected to check for natural infection, and in five specimens we found living flagellated forms: two Ev. spelunca, two Ev. sallesi and one Sc. sordellii. This study shows that the activity of some species caught in the aphotic zone of the cave, especially Lu. cavernicola, differs from what has already been reported in previous sandfly captures, which are almost always conducted at night and during twilight. The existence of sandflies that were naturally infected with flagellates and the lack of awareness regarding the behaviour of sandflies in cave environments are strong indicators of the need for further study on this group of insects in this ecotope, as a safety measure to protect the visitors of such environment.

Phlebotomine fauna in the urban area of Timóteo, State of Minas Gerais, Brazil

This work is characterized by an entomological research and an investigation on whether seasonal behaviours can be associated to the phlebotomine fauna found in the urban area of Timóteo-MG - an endemic focus of tegumentary leishmaniasis (TL). The analysis of the seasonal behaviour of sand flies has taken into account the following climatic variables: rainfall, relative humidity and temperature. Automatic light traps were installed in households between 2009 and 2010. The sand fly species with the highest number captured was Lutzomyia whitmani (66.5%), a TL vector species, whose abundance has provided strong evidences that this species is the main vector of TL in the municipality of Timóteo, with its cycle of transmission developing in its urban area. Amongst the results observed in the analyses of seasonal behaviour, only temperature conveyed particular association between seasonal occurrence of sand flies and climate variables. The findings of this study may assist the local epidemiological surveillance agency in defining strategies and directing efforts for controlling these insects.