First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil.

BACKGROUND: Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES: In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS: A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS: We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS: To the best of our knowledge, this is the first detection of LRV2 in the New World.

Chemokines in Leishmaniasis: Map of cell movements highlights the landscape of infection and pathogenesis.

Pathogen interactions with the host immune response components are critical for establishing protective immunity and pathological responses against Leishmania parasites. A predominant proinflammatory profile associated with enhanced phagocytosis trigger a cell-mediated immune response that is relevant to infection control. On the other hand, an anti-inflammatory phenotype, correlated with a predominant modulated/regulatory response, favors intracellular proliferation of Leishmania parasites and disease progression. In this context, chemokines play an important role in determining cellular composition at inflammatory sites. Leishmania infection induces the expression of various chemokines and chemokine receptors in the mammalian host, which can subvert the host immune responses. Indeed, the balance and dynamic changes in cytokines and chemokines may control or predict the disease outcome. In this review, we address our current knowledge regarding the chemokines and chemokines receptors' role in the immunopathogenesis of Tegumentary and Visceral Leishmaniasis.

Distinct immunomodulatory properties of extracellular vesicles released by different strains of Acanthamoeba.

Free living amoeba of the genus Acanthamoeba are opportunist protozoan involved in corneal, systemic, and encephalic infections in humans. Most of the mechanisms underlying intraspecies variations and pathogenicity are still unknown. Recently, the release of extracellular vesicles (EVs) by Acanthamoeba was reported. However, comparative characterization of EVs from distinct strains is not available. The aim of this study was to evaluate EVs produced by Acanthamoeba from different genotypes, comparing their proteases profile and immunomodulatory properties. EVs from four environmental or clinical strains (genotypes T1, T2, T4, and T11) were obtained by ultracentrifugation, quantitated by nanoparticle tracking analysis and analyzed by scanning and transmission electron microscopy. Proteases profile was determined by zymography and functional properties of EVs (measure of nitrite and cytokine production) were determined after peritoneal macrophage stimulation. Despite their genotype, all strains released EVs and no differences in size and/or concentration were detected. EVs exhibited a predominant activity of serine proteases (pH 7.4 and 3.5), with higher intensity in T4 and T1 strains. EVs from the environmental, nonpathogenic T11 strain exhibited a more proinflammatory profile, inducing higher levels of Nitrite, tumor necrosis factor alpha and interleukin-6 via TLR4/TLR2 than those strains with pathogenic traits (T4, T1, and T2). Preincubation with EVs treated with protease inhibitors or heating drastically decreased nitrite concentration production in macrophages. Those data suggest that immunomodulatory effects of EVs may reflect their pathogenic potential depending on the Acanthamoeba strains and are dependent on protease integrity.

Distinct genetic profiles of Leishmania (Viannia) braziliensis associate with clinical variations in cutaneous-leishmaniasis patients from an endemic area in Brazil.

American tegumentary leishmaniasis (ATL) samples obtained from the lesions of patients with typical (n = 25, 29%), atypical (n = 60, 69%) or both (n = 2%) clinical manifestations were analysed by multilocus enzyme electrophoresis, hsp70 restriction-fragment length polymorphism (PCR-RFLP), hsp70 sequencing and phylogenetics methods. The hsp70 PCR-RFLP analysis revealed two different profiles whose the most samples differed from those expected for Leishmania braziliensis and the other Leishmania species tested: of 39 samples evaluated, two (5%) had a restriction profile corresponding to L. braziliensis, and 37 (95%) had a restriction profile corresponding to a variant pattern. A 1300-bp hsp70 gene fragment was sequenced to aid in parasite identification and a phylogenetic analysis was performed including 26 consensus sequences from the ATL patient's samples and comparing to other Leishmania and trypanosomatids species. The dendrogram allowed to observe a potential population structure of L. braziliensis complex in the studied region, emphasizing that the majority of clinical samples presented a variant genetic profile. Of interest, the L. braziliensis diversity was associated with different clinical manifestations whose parasites with hsp70 variant profile were associated with atypical lesions. The results may be helpful to improve the diagnosis, treatment and control measures of the ATL in endemic areas.

Genetic variant strains of Leishmania (Viannia) braziliensis exhibit distinct biological behaviors.

A variety of clinical forms of American cutaneous leishmaniasis (ACL) caused by Leishmania braziliensis, as well as differing immune responses of patients, have been reported for an ACL focus in the state of Minas Gerais, Brazil. In addition, two genetic profiles of L. braziliensis have been described, of which one variant profile (hsp70-variant) has been associated with atypical lesions. We investigated the biological behavior of genetic variant strains of L. braziliensis isolated from patients with different clinical manifestations of ACL. Experimental infections were performed with golden hamsters for five L. braziliensis strains in standardized doses of 1 × 106 parasites per inocula. The characteristics of skin lesions, histopathological features, and parasite burden were independently analyzed at 30 and 60 days post-infection. The data revealed distinct patterns in the onset time of visible skin lesions as well as in lesion size and parasite burden among the strains. The extent and density of the inflammatory infiltrate differed among strains, although cellular composition of granulomas appeared similar. Multivariate analysis indicated the occurrence of two clusters: one comprising native strains (cluster 1) and one comprising the reference strain (cluster 2). Within cluster 1, the genetic variants of L. braziliensis did not group with the non-variant strain suggesting that the distinct patterns of biological behavior of these strains could be associated with the known genetic diversity previously described for them.

Seasonality of sand flies (Diptera: Psychodidae) and Leishmania DNA detection in vector species in an area with endemic visceral leishmaniasis.

BACKGROUND: Leishmaniases are a serious health problem in southeast Brazil, including the city of Belo Horizonte (BH), Minas Gerais state (MG), where there are high rates of incidence and mortality due to visceral leishmaniases. BH is divided into nine sanitary districts (SD) of which one, the Venda Nova SD, was selected for this study because it has high rates of positivity for canine leishmaniasis and high incidence of human leishmaniasis. OBJECTIVES: This study aimed to survey the sand fly fauna in Venda Nova SD from August 2011 to July 2013 and perform a descriptive analysis of the vector population. METHODS: The sampling was carried out using automatic HP light traps at all covered areas of the Venda Nova SD, in a total of eighteen light traps. Sampled specimens were identified following Galati (2003), and females were submitted to molecular techniques for the detection and identification of Leishmania DNA. A simple environmental description was done for it area and Kernel estimation was used to infer vector density for each study site. FINDINGS: A total of 2,427 sand fly specimens belonging to eight species and five genera were collected of which 95.3% were Lutzomyia longipalpis. The seasonal variation curve was delineated by this species. Lu. longipalpis was the most abundant at all collection points and in all months of the study, and exhibited a natural infection rate of 1.01% for Leishmania infantum and 1.77% for Leishmania braziliensis. MAIN CONCLUSIONS: The results show the presence and adaptation of Lu. longipalpis to the anthropic environment of BH and reinforces its role as the main vector of L. infantum in the region.

Clinical and Laboratory Response of Domiciled Dogs with Visceral Leishmaniasis Treated with Miltefosine and Allopurinol.

Canine visceral leishmaniasis (CVL) remains a significant disease worldwide. In Brazil, its treatment is performed using miltefosine, which has demonstrated promising outcomes in dogs. This study represents the first attempt to treat and monitor dogs with CVL in natural conditions over the course of one year. The dogs were divided into two groups: G1 received miltefosine and allopurinol for 28 days, while G2 received miltefosine for 28 days, followed by allopurinol for one year. The follow-up involved clinical, hematological, and biochemical evaluations, as well as the detection of Leishmania DNA in skin and bone marrow samples. By the end of the follow-up, dogs in G2 exhibited improved staging compared to their initial conditions, whereas those in G1 showed worsened staging. Leishmania DNA in skin and bone marrow decreased between 6 and 12 months after treatment. Our observations indicate that the treatment using miltefosine reduces the detection of the parasite in the skin and bone marrow for up to one year following its administration. The continuous use of allopurinol contributes to control of the disease in dogs. These findings provide valuable insights into the response of dogs treated in natural conditions, offering essential information for veterinarians and public health authorities.

Phenotypical Differences between Leishmania (Leishmania) amazonensis PH8 and LV79 Strains May Impact Survival in Mammal Host and in Phlebotomine Sand Flies.

We previously showed that L. (Leishmania) amazonensis promastigotes and amastigotes of the PH8 strain generated larger lesions in mice than LV79, and that lesion-derived amastigotes from the two strains differ in their proteomes. We recently reported that PH8 promastigotes are more phagocytized by macrophages. Promastigotes' membrane-enriched proteomes showed several differences, and samples of each strain clustered based on proteomes. In this paper, we show phenotypic differences between PH8 and LV79 promastigotes that may explain the higher virulence of PH8. We compared in vitro macrophage infections by day 4 (early) and day 6 (late stationary phase) cultures, resistance to complement, and LPG characteristics. PH8 promastigotes showed a higher infectivity and were more resistant to murine complement. LPG was different between the strains, which may influence the interaction with macrophages and survival to complement. We compared the infection of the permissive vector Lutzomyia longipalpis. PH8 was more abundant in the vector's gut 72 h after feeding, which is a moment where blood digestion is finished and the parasites are exposed to the gut environment. Our results indicate that PH8 promastigotes are more infective, more resistant to complement, and infect the permissive vector more efficiently. These data suggest that PH8 is probably better adapted to the sand fly and more prone to survive in the vertebrate host.

Clinical evolution of equine leishmaniasis with self-limiting cutaneous disease caused by Leishmania infantum in northeastern Brazil: A case report.

Leishmania infantum infections have long been described in humans and dogs worldwide, but characterization of equine cases remains scarce. We describe the clinical evolution of a natural L. infantum infection to contribute to the diagnostic knowledge and epidemiology of equine leishmaniasis (EL). An auction-acquired four-year-old Mangalarga Marchador mare from Pernambuco state, presented a few subcutaneous nodules on the head and neck upon arrival at the purchaser's stud at Bahia state, in November of 2019. They progressed to multiple ulcerated and non-ulcerated nodules and spread to both right limbs in seven weeks. Hematology revealed anemia, lymphocytosis, monocytosis, and elevated plasma fibrinogen. Histopathology of the biopsied nodules identified a granulomatous dermatitis with macrophages containing Leishmania amastigotes. PCR detected Leishmania in skin lesions, but not in blood or spleen aspirate samples; ITS1 PCR-RFLP and DNA sequencing confirmed L. infantum species. A topical antiseptic and insect-repellent therapy and a monthly follow-up were established. All lesions improved progressively, without specific anti-Leishmania treatment, and 14 months later there was a consistent resolution. This first description of EL by L. infantum in an endemic area is relevant to emphasize the need for epidemiological studies, and to enhance clinicians' awareness for differential diagnosis.

Phlebotominae Fauna (Diptera: Psychodidae) and Molecular Detection of Leishmania (Kinetoplastida: Trypanosomatidae) in Urban Caves of Belo Horizonte, Minas Gerais, Brazil.

Sand flies are often collected in urban areas, which has several implications for the risk of transmission of Leishmania Ross, 1903, to humans and other mammals. Given this scenario, we describe the sand fly fauna of caves and their surroundings in Mangabeiras Municipal Park (MMP) and Paredão Serra do Curral Park (PSCP), both located in the urban area of Belo Horizonte, Minas Gerais, Brazil, an endemic focus of visceral and cutaneous leishmaniasis. Collections were conducted monthly from November 2011 to October 2012, using CDC light traps exposed for two consecutive nights in four caves and their surroundings. Nonsystematized collections using Shannon traps and active searches were also performed around the caves. The presence of Leishmania DNA in collected female sand flies was evaluated by ITS1-PCR. A total of 857 sand flies representing fourteen species were collected in MMP, of which Evandromyia edwardsi (Mangabeira, 1941) was the most abundant. Leishmania amazonensis was detected in Brumptomyia nitzulescui (Costa Lima, 1932) and Ev. edwardsi, with the latter also having Leishmania braziliensis, Leishmania infantum, and Leishmania sp. A total of 228 sand flies representing four species were collected in PSCP, of which Sciopemyia microps (Mangabeira, 1942) was the most abundant. No females from PSCP were positive for Leishmania-DNA. Studies aimed at describing sand fly faunas of cave environments and detecting Leishmania are essential to understanding the relationship between these insects and this ecotope and assessing and monitoring areas that may pose risks to the health of visitors and employees.

Sand fly bioecological aspects and risk mapping of leishmaniasis by geographical information systems approach in a mineral exploration area of Brazil.

Epidemiological studies of leishmaniasis in areas of great human influence and environmental change serve as important tools for the implementation of effective control plans. Mining is currently a major economic activity in Brazil with the municipality of Pains, in the state of Minas Gerais, being one of the main lime producing municipalities in the country. This study aimed to map areas of potential transmission risks within the municipality of Pains using an epidemiological approach in association with the ecological study of sand flies. Twelve samplings carried out between May 2015 and April 2016 collected a total of 12,728 sandflies, comprising 2,854 females (22.42%) and 9,874 males (77.58%), of 20 species belonging to ten genera. The most abundant species was Lutzomyia longipalpis (80%). Leishmania DNA was detected in seven pools of female sand flies with an infection rate of 0.37%. Geoprocessing and the use of maps revealed that vector sand flies are distributed throughout the urban area, as are cases of canine and human leishmaniasis. However, the greatest abundances of sand flies were at sampling points at the border of the urban area. Higher densities of sand flies and the presence of Leishmania DNA may be correlated with extensive degradation by limestone mining. Integrated and multidisciplinary research approaches are necessary to better understand how the impacts of environmental change influence these insect vectors of leishmaniasis.

Potential antigenic targets used in immunological tests for diagnosis of tegumentary leishmaniasis: A systematic review.

Immunological tests may represent valuable tools for the diagnosis of human tegumentary leishmaniasis (TL) due to their simple execution, less invasive nature and potential use as a point-of-care test. Indeed, several antigenic targets have been used with the aim of improving the restricted scenario for TL-diagnosis. We performed a worldwide systematic review to identify antigenic targets that have been evaluated for the main clinical forms of TL, such as cutaneous (CL) and mucosal (ML) leishmaniasis. Included were original studies evaluating the sensitivity and specificity of immunological tests for human-TL, CL and/or ML diagnosis using purified or recombinant proteins, synthetic peptides or polyclonal or monoclonal antibodies to detect Leishmania-specific antibodies or antigens. The review methodology followed PRISMA guidelines and all selected studies were evaluated in accordance with QUADAS-2. Thirty-eight original studies from four databases fulfilled the selection criteria. A total of 79 antigens were evaluated for the detection of antibodies as a diagnostic for TL, CL and/or ML by ELISA. Furthermore, three antibodies were evaluated for the detection of antigen by immunochromatographic test (ICT) and immunohistochemistry (IHC) for CL-diagnosis. Several antigenic targets showed 100% of sensitivity and specificity, suggesting potential use for TL-diagnosis in its different clinical manifestations. However, a high number of proof-of-concept studies reinforce the need for further analysis aimed at verifying true diagnostic accuracy in clinical practice.

Anti-mitochondrial Tryparedoxin Peroxidase Monoclonal Antibody-Based Immunohistochemistry for Diagnosis of Cutaneous Leishmaniasis.

Cutaneous leishmaniasis (CL) remains a globally spreading public health problem. Among Latin America countries, Brazil has the greatest number of recorded CL cases with several Leishmania species being associated with human cases. Laboratory diagnosis is one of the major challenges to disease control due to the low accuracy of parasitological techniques, the restricted use of molecular techniques, and the importance of differential diagnosis with regard to several dermatological and systemic diseases. In response, we have developed and validated an immunohistochemistry (IHC) technique for CL diagnosis using anti-mTXNPx monoclonal antibody (mAb). Recombinant Leishmania-mTXNPx was produced and used as an immunogen for mAb production through the somatic hybridization technique. The viability of mAb labeling of Leishmania amastigotes was tested by IHC performed with skin biopsies from hamsters experimentally infected with Leishmania amazonensis, Leishmania braziliensis, and Leishmania guyanensis. The enzymes horseradish peroxidase (IHC-HRP) and alkaline phosphatase (IHC-AP), both biotin-free polymer detection systems, were used in the standardization step. The IHC was further validated with skin biopsies from 49 CL patients diagnosed by clinical examination and quantitative real-time polymerase chain reaction and from 37 patients presenting other dermatological infectious diseases. Other parasitological techniques, such as direct examination and culture, were also performed for confirmed CL patients. Histopathology and IHC were performed for all included patients. Overall, the highest sensitivity was observed for IHC-AP (85.7%), followed by IHC-HRP (79.6%), direct examination (77.6%), histopathological examination (HE; 65.3%), and in vitro culture (49%). Only IHC and HE presented specificity over 90% and were able to detect CL patients regardless of parasite burden (odds ratio > 1.94; 95%CI: 0.34-11.23). A significant increase in positivity rates was observed when IHC-AP was combined with direct examination (95.9%) and HE (93.9%). The IHC techniques evaluated in here detected the main Leishmania species causing CL in Brazil and can support diagnostic strategies for controlling this neglected disease, especially if used in combination with other approaches for an integrative laboratorial diagnosis.

Feline Leishmaniasis Caused by Leishmania infantum: Parasite Sequencing, Seropositivity, and Clinical Characterization in an Endemic Area From Brazil.

Zoonotic leishmaniasis caused by Leishmania infantum is a disease of One Health concern since human and animal cases and environmental damage are interconnected. L. infantum has a complex epidemiological cycle with multiple hosts, including mammals-humans, domestic, and wild animals-and arthropod vectors. Knowledge on mammal infections in endemic areas is crucial for developing control strategies. This work aimed to detect and characterize L. infantum infection in domestic cats from areas where human and canine leishmaniasis cases occur. No cases of feline leishmaniasis (FeL) had been previously reported in those areas. Five municipalities from Bahia state were chosen, comprising 2,480.8 km2 with 1,103,866 inhabitants. Ninety domiciliated and/or sheltered cats underwent clinical examination and serology by a rapid reference test recommended by the Brazilian government. Cytology, PCR, and parasite DNA sequencing were performed in bone marrow samples. Rapid tests detected antibodies in 5.6% (5/90) of the cats. Leishmania infantum infection was confirmed in 7.8% (7/90) of the cats by PCR, sequencing, and parasite isolation. Three out of the five municipalities (60%) had infected cats, and PCR positivity varied from 6.9 to 29%. One cat was categorized as harboring active L. infantum infection with amastigote forms in bone marrow smears. No clinical signs were detected at the first clinical exam, but 1 month later the cat developed severe FeL. The cat isolate was grown in culture, typed and its DNA sequence was homologous to the L. infantum reference strain (PP75). In conclusion, cats are potential hosts and may acquire L. infantum in endemic areas where canine and human cases occur. For cats, the need for surveillance, differential diagnosis and clinical care is highly recommended since a fast clinical progression of FeL developed in a subclinical animal. An accurate standardized immunodiagnostic assay for FeL is warranted.

Potential Vectors of Leishmania Parasites in a Recent Focus of Visceral Leishmaniasis in Neighborhoods of Porto Alegre, State of Rio Grande do Sul, Brazil.

In view of recent cases of human and canine visceral leishmaniasis reported in Porto Alegre, Rio Grande do Sul, Brazil, we investigated the sand fly fauna inhabiting the neighborhoods of Morro Santana and Jardim Carvalho, Brazil, continuing a series of entomological surveys aimed to identify potential vectors of Leishmania (Ross, 1903) parasites. Sand flies were collected monthly from October 2016 to October 2017 using CDC light traps in the intradomiciliary and peridomiciliary environments of seven residences. Sand fly abundance was correlated to climatic variables. Females were pooled by species, location, and date for Leishmania DNA molecular screening using ITS1 and kDNA polymerase chain reaction. In total, 501 sand flies from five species were collected in which Lutzomyia gaminarai (Cordero, Vogelsang & Cossio, 1928) (Diptera: Psychodidae) (78%) was the most abundant species in the intradomiciliary sites while Migonemyia migonei (Franca, 1920) (Diptera: Psychodidae) (43.3%) was the most abundant in the peridomiciliary sites. A higher number of sand flies were collected during the warmest months, from December to March (Mann-Whitney statistical test - P < 0.001). Leishmania infantum DNA was detected in Lu. gaminarai (2), Pintomyia fischeri (Pinto, 1926) (1) and Mg. migonei (1). Leishmania braziliensis DNA was detected in Lu. gaminarai (1) and Pi. fischeri (1). Our results add support to the possible vector role of Pi. fischeri in the epidemiological cycle of Le. infantum in Brazil. Furthermore, the first documented detection of Leishmania DNA in Lu. gaminarai may be indicative of multiple vectors being involved in the Leishmania cycle within Porto Alegre.

Biodiversity of Hematophagous Diptera (Culicidae and Psychodidae) in a Small Hydroelectric Power Station and Surrounding Area in the State of Minas Gerais, Brazil.

Hydroelectric power stations may affect the population dynamics of mosquitoes and sand flies, a group with impact on public health due to the possibility to transmit pathogens to humans. This work characterized and compared the fauna of mosquitoes and sand flies in a hydroelectric power station and peridomicile areas in the State of Minas Gerais, Brazil. Insect collections were performed in August 2015 at dry season and February 2016 in rainy season. Ten HP light traps were set at each of two sites for three consecutive days in each of two seasons (dry and rainy). Furthermore, collections with Shannon traps were made in each sampling area (hydropower plant and peridomicile area) from 4:00 p.m. being shut down at 8:00 p.m. for two consecutive days in each of two seasons (dry and rainy). In total, 1,222 insects from 13 genera and 27 species were collected. The most plentiful species were respectively Culex declarator (Dyar and Knab, 1906) and Pintomyia pessoai (Coutinho & Barretto, 1940). A high number of insects (78.5%) were collected during the rainy period (P < 0.05). About equitability, statistical significance was observed in the peridomicile area (dry season J = 0.75 and rainy season J = 0.82). The highest values of species diversity were observed in the hydropower plant (H = 2.68) and peridomicile area (H = 2.38) both in the rainy season with statistical significance comparing with dry season. Our results demonstrate that the occurrence of vector species in this region increases the potential risk of transmission of pathogens, especially arbovirus and Leishmania Ross, 1903.

Level of Knowledge and Risk Factors for Visceral Leishmaniasis in a Mining Area of Minas Gerais State, Brazil.

Aiming to optimize and adjust leishmaniasis prevention and control measures for the resident population of Pains, state of Minas Gerais, Brazil, a structured questionnaire containing conceptual questions and questions about household characteristics was used to evaluate knowledge level and exposure risk. A total of 396 individuals were interviewed revealing unscientific and fragmented knowledge about the subject for most of the studied population. The female population was found to have 1.68 times more chance of knowing about the disease than the male population, while highly educated individuals were found to have 2.92 times more chances of knowing about leishmaniasis compared to basic educated individuals. All of the respondents reported the presence of, at least, one risk factor, while ages ≥40 years were considered a protective factor compared to younger ages, indicating that older individuals are more likely to recognize risks and protect themselves against leishmaniasis. These results will contribute to the production of didactic materials for the population with respect to their previous knowledge and will provide a basis for control and prophylactic measures.

Diversity of phlebotomine sand flies and molecular detection of trypanosomatids in Brumadinho, Minas Gerais, Brazil.

This study aimed to describe the sand fly fauna and detect trypanosomatids in these insects from Casa Branca, state of Minas Gerais, Brazil, an endemic area of both visceral (VL) and tegumentary leishmaniasis (TL). Sand flies were collected bimonthly from May 2013 to July 2014, using automatic light traps exposed for three consecutive nights in peridomiciliary areas of nine houses with previous reports of VL and TL. ITS1-PCR and DNA sequencing were performed for trypanosomatids identification. A total of 16,771 sand flies were collected belonging to 23 species. The most abundant species was Nyssomyia whitmani (Antunes & Coutinho, 1939) (70.9%), followed by Lutzomyia longipalpis (Lutz & Neiva, 1912) (15.2%) and Migonemyia migonei (França, 1920) (9.1%). Leishmania amazonensis DNA was detected in Ny. whitmani (four pools) and Le. braziliensis DNA was detected in Psychodopygus lloydi (one pool). In seven pools of Ny. whitmani and in one pool of Lu. longipalpis positive for Leishmania DNA, the parasite species was not determined due to the low quality of the sequences. Moreover, DNA of Herpetomonas spp. was detected in Ny. whitmani (two pools) and Cortelezzii complex (one pool). DNA of Crithidia spp. was detected in Ny. whitmani and Ps. lloydi (both one pool). Our results suggest that Ny. whitmani may be involved in the transmission of Le. amazonensis in the study area. The molecular detection of Le. amazonensis suggests the presence of this species in a sylvatic cycle between vertebrate and invertebrate hosts in the region of Casa Branca. Our data also reveal the occurrence of other non-Leishmania trypanosomatids in sand flies in Casa Branca District.

Ecology and Molecular Detection of Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatida) in Wild-Caught Sand Flies (Psychodidae: Phlebotominae) Collected in Porto Alegre, Rio Grande do Sul: A New Focus of Visceral Leishmaniasis in Brazil.

Phlebotomine sand flies are hematophagous insect vectors of the protozoan parasites of the genus Leishmania Ross, 1903 (Kinetoplastida: Trypanosomatida) that infect mammals, including humans, causing leishmaniasis. In Porto Alegre, Brazil, three autochthonous cases of human visceral leishmaniasis were reported in 2016 through 2017. We analyzed for the presence of Leishmania DNA in sand flies collected at the neighborhood of Agronomia, Porto Alegre, Brazil. Phlebotomine sand flies were collected at three sites from October 2014 to September 2015. Female sand flies were pooled in numbers from 1 to 20 depending upon species, locality, and date; all were screened for Leishmania infection by the amplification of the ITS1 region. In total 518 phlebotomine sand flies were collected: Psathyromyia lanei (Barretto and Coutinho, 1941) (Diptera: Psychodidae) (30.5%), Brumptomyia sp. (França and Parrrot, 1921) (Diptera: Psychodidae) (25.7%), Migonemyia migonei (Franca, 1920) (Diptera: Psychodidae) (21.4%), Pintomyia fischeri (Pinto, 1926) (Diptera: Psychodidae) (21.4%), and Nyssomyia neivai (Pinto, 1926) (Diptera: Psychodidae) (1.0%). Most sand flies were collected during the hot and rainy season from October 2014 to April 2015. Of the 113 pools analyzed, five pools of Pi. fischeri were PCR-positive with the amplicons possessing sequences similar (>95%) to that of Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatida). These results represent the first molecular detection of Le. infantum in Pi. fischeri. It is possible that Pi. fischeri is involved in the transmission cycle of Le. infantum in the studied area; however, further studies are needed to establish the true role of Pi. fischeri in the visceral leismaniasis cycle.

Molecular variants of Leishmania (Viannia) braziliensis trigger distinct patterns of cytokines and chemokines expression in golden hamster.

Cutaneous leishmaniasis (CL) mainly caused by Leishmania braziliensis is a chronic inflammatory disease widely spread in Brazil. Genetic variant strains of this parasite have been associated with atypical clinical manifestations of CL in an endemic area in Brazil. Furthermore, these strains have presented distinct biological behaviors in golden hamster, suggesting differential activation of the immune response. In the present study we proposed to evaluate the localized immune response in golden hamsters infected with known molecular variant strains of L. braziliensis, in distinct time points post-infection (PI). Detailed analyses of the mRNA expression of cytokines and chemokines in hamster-skin lesions were performed. Heat map matrix and hierarchical cluster analysis were carried out to segregate the strains due to mRNA expression. Distinct patterns of immune response were found in both time points, more evident in the recent-phase disease (30 days-PI). At this time point, the genetic variant strains expressed high levels of tnfα, il12 and tgfß whilst the non-variant strain expressed ifnγ, il6, il4, il10, il13 and ccl17. The hierarchical clustering highlights this distinct pattern in which all genetic variant strain was grouped in the cluster I and the non-variant strain grouped into the cluster II. At late-phase disease (60 days-PI) all isolates expressed high levels of il4 and il10. The non-variant strain shown a significant reduced expression of ifnγ, il6, ccl17, and ccl22 whilst distinct patterns were observed for the genetic variant strains. For the first time, a large panel of cytokines and chemokines mRNA-expression was analyzed in experimental trials using golden hamsters as animal model and genetic variant strains of L. braziliensis. Our findings suggest that genetic variant strains of L. braziliensis are able to trigger differential gene expression of cytokines and chemokines in the skin lesion from infected hamsters. The parasite intrinsic ability to activate distinct pathways in the host-parasite interaction may be associated to the large spectrum of clinical manifestation observed in CL-patients.