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.

Leishmania species infection of bats: A systematic review.

A wide variety of mammals, including domestic and wild species, have been considered potential hosts and reservoirs for Leishmania. Bats have longevity, dispersal capacity, and adaptability to synotropic environments, characteristics that may favor their role in maintaining the life cycle of parasites. Therefore, the objective of this study was to carry out a worldwide systematic review of the occurrence of Leishmania species in bats, as well as to identify associations between eating habits and the type of sample collected with the occurrence of the infection. Data were obtained from a bibliographic search for studies that used molecular methods to identify parasites, employing the keywords "bats" AND "Leishmania" and their synonyms. We found 68 original studies, of which 20 were included in this review. Most studies were conducted in Brazil (60 %) and only 10 % were conducted in Old World countries. In all, 48 bat species were recorded that hosted seven Leishmania species, resulting in 62 different host-parasite interactions, and the Leishmania infantum interaction with bat species presented higher frequency. There was no significant difference between Leishmania species richness, infection percentage, and type of sample analyzed, but in general, it is observed that the use of different biological samples seems to expand the possibility of parasite detection. The patterns observed here indicate that bats can become infected with a wide variety of Leishmania species and likely play an important role in maintaining the parasite's life cycle. Thus, we suggest that studies aimed at understanding the transmission cycle of leishmaniasis include the investigation of bats as potential hosts or reservoirs of Leishmania.

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.

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.

Molecular survey of Leishmania spp. in skin samples of capybaras (Hydrochoerus hydrochaeris) from different areas of Brazil / Investigação molecular de Leishmania spp. em amostras de pele de capivaras (Hydrochoerus hydrochaeris) de diferentes regiões do Brasil

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the genus Leishmania, with some species of rodents being incriminated as reservoirs. The capybara is the largest extant rodent species in the world and is widely distributed in South America. The occurrence of infection by Leishmania spp. was investigated in capybaras captured in Brazil during 2015­2019 from established populations in five highly anthropic areas of the state of São Paulo and two natural areas of the states of Mato Grosso and Mato Grosso do Sul. A total of 186 individuals were captured and subjected to abdominal skin biopsy. All skin samples were Leishmania kDNA-negative, suggesting that capybaras have no role in the transmission cycles of Leishmania species in the studied areas despite the well-known role of other rodents in the life cycle of Leishmania spp.(AU)

As leishmanioses compreendem um espectro de doenças causadas por protozoários do gênero Leishmania e algumas espécies de roedores são incriminadas como reservatórios de Leishmania spp. As capivaras compreendem a maior espécie de roedores existentes e são amplamente distribuídas na América do Sul. Para investigar a ocorrência de infecção por Leishmania spp. em capivaras, durante os anos de 2015-2019 capivaras foram capturadas em cinco áreas antrópicas do estado de São Paulo e em duas áreas naturais dos estados do Mato Grosso e do Mato Grosso do Sul, todos esses ambientes com populações de capivaras estabelecidas. Um total de 186 indivíduos foram capturados e submetidos à biópsia de pele abdominal. Todas as amostras de pele foram negativas para o alvo kDNA, assim, os dados sugerem que nas áreas estudadas as capivaras não têm papel no ciclo de transmissão de espécies de Leishmania spp., apesar do papel bem conhecido de outros roedores no ciclo de vida de Leishmania spp.(AU)

Leishmania diversity in bats from an endemic area for visceral and cutaneous leishmaniasis in Southeastern Brazil.

This study aimed to determine the occurrence of Leishmania infection in bats in urban and wild areas in an endemic municipality for visceral and cutaneous leishmaniasis in Minas Gerais, Brazil. Between April 2014 to April 2015, 247 bats were captured and classified into 26 species belonging to Phyllostomidae (90.7%), Vespertilionidae (8.1%) and Molossidae (1.2%) families. Blood samples from 247 bats were collected and submitted to nested-PCR, targeting the variable V7-V8 region of the SSU rRNA gene, followed by sequencing of the PCR product. The overall infection rate of Leishmania spp. in bats was 4.4%. Of the eleven bats infected, ten were frugivorous bats: Artibeus planirostris (8/11), Artibeus lituratus (1/11) and Artibeus cinereus (1/11) and one a nectarivorous bat (Glossophaga soricina). None of the individuals exhibited macroscopic alterations in the skin, spleen or liver. Phylogenetic analysis separated Leishmania species in clades corresponding to the subgenera Viannia, Leishmania, and Mundinia, and supported that the isolates characterized in the present study clustered closely with Leishmania (Viannia) sp., Leishmania (Leishmania) infantum and Leishmania (Leishmania) amazonensis. Here we report for the first time the bat Artibeus cinereus as a host of Leishmania (Leishmania) amazonensis. In the study we found that the mean abundance of bats did not differ in wild habitats and urban areas and that bat-parasite interactions were similarly distributed in the two environments. On the other hand, further studies should be conducted in more recent times to verify whether there have been changes in these parameters.

Cross-sectional study on Brucella spp., Leptospira spp. and Salmonella spp. in bats from Montes Claros, Minas Gerais, Brazil.

The understanding on the role of bats in the ecology of zoonotic diseases, especially its relevance as a carrier of pathogens, is important for the determination of preventive measures considering the One Health context. The present study aimed to investigate the presence of Brucella spp., Leptospira spp. and Salmonella spp. in blood (n = 163), liver (n = 35) and spleen (n = 62) samples from bats captured in Montes Claros, Minas Gerais, Brazil. Only Salmonella spp. was found in a blood sample of an insectivorous female bat of the species Lasiurus blossevilli, evidencing the capacity of this animal species to host this pathogen. In conclusion, our results in bats from Montes Claros indicate that they do not act as hosts for Brucella spp. and Leptospira spp., although being potential carriers of Salmonella spp. in a low prevalence.

Correlations between tissue parasite load and common clinical signs in dogs naturally infected by Leishmania infantum.

qPCR is being used for the quantification of parasite load in different tissues of dogs infected by Leishmania infantum with or without clinical manifestations. It may be employed in the diagnosis, monitoring of the infection during treatment, and clinical studies for validation of vaccines. Aimed at enhancing the molecular diagnosis and the subsequent monitoring of the infection, this study evaluated the parasite load in several tissues from dogs infected by Leishmania infantum, showing different clinical status. Thus, the qPCR was performed on skin, conjunctival swab, popliteal lymph node, and bone marrow puncture samples taken from 65 dogs naturally infected by L. infantum. Dogs were divided into three groups per clinical score: group 1 (n = 12), included animals with zero points and no clinical manifestations of the disease; group 2 (n = 35), included animals with a score ranging from 1 to 5 points and moderate clinical manifestations; and group 3 (n = 18), included dogs with a score ranging from 6 to 11 points and intensive clinical manifestations. Another analysis was performed classifying the animals into two groups, considering the presence of, or lack of clinical signs of the disease. Analyses of these results showed that the skin was the tissue with a higher parasite load, followed by popliteal lymph node and bone marrow punctures, and conjunctival swab samples having the lowest loads. Furthermore, the skin was also the tissue with the highest parasite load when evaluating the groups individually. Animals in group 3, with intensive clinical manifestations, showed a higher parasite load in different tissues when compared to animals from groups 1 and 2. Finally, animals with clinical manifestations of the disease showed a higher parasite load when compared to dogs with no manifestations. The importance of the dog as a reservoir of L. infantum in nature is reinforced by the demonstration of skin having the highest amount of parasites/µL in this study's analysis, as well as the fact that skin is the main point of access to the parasite vector. Also, a strong and positive correlation between the intensity of clinical manifestations and the increase of parasite load in the skin was observed. In conclusion, skin was the tissue that was demonstrated to be the best option for the molecular diagnosis of L. infantum infection in dogs with varying clinical statuses used in this study.

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.

Serological study of feline leishmaniasis and molecular detection of Leishmania infantum and Leishmania braziliensis in cats (Felis catus).

Blood samples and swabs from ocular conjunctiva and mouth were obtained from 64 cats. Of 64 serum samples, 19 were positive for Leishmania antibodies by ELISA (29.80%). Eight cats were positive by PCR (12.5%) in swab samples from mouth and/or ocular mucosa. Poor kappa agreement between serological and molecular results (k = 0.16) was obtained. From five positive PCR samples one was L. braziliensis and four were L. infantum. Phylogenetic analysis performed with the five isolates of Leishmania, showed that samples of L. infantum isolated from the cats were phylogenetically close to those isolated from domestic dogs in Brazil, while the L. braziliensis is very similar to the one described in humans in Venezuela. The study demonstrated that, despite high seropositivity for Leishmania in cats living in the study region, poor agreement between serological and molecular results indicate that positive serology is not indicative of Leishmania infection in cats. Parasite DNA can be detected in ocular conjunctiva and oral swabs from cats, indicating that such samples could be used for diagnosis. Results of phylogenetic analyzes show that L. infantum circulating in Brazil is capable of infecting different hosts, demonstrating the parasite's ability to overcome the interspecies barrier.

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.

Comparative PCR-based diagnosis for the detection of Leishmania infantum in naturally infected dogs.

The applicability of molecular biology/PCR for canine visceral leishmaniasis diagnosis presents challenges, mainly due to the diversity of targets described. The objectives of this study were to compare the sensitivities and reliability of five targets (kDNA/120, kDNA/145, ITS1, hsp70/234 and hsp70/1300) in four different tissue samples (bone marrow, popliteal lymph node, skin and conjunctival swab). Sixty-five dogs (32 males and 33 females) naturally infected with Leishmania infantum and ten dogs without infection were examined. Dogs were characterized by serological and parasitological methods. The parasitological test was considered the gold standard for analysis. All tests presented high specificity 100% (95% CI 0.72-1), and variable sensitivity. The targets kDNA/145, ITS1, kDNA/120, hsp70/234 and hsp70/1300 detected 100% (65/65), 93.4% (61/65), 92.3% (60/65), 84.61% (55/65) and 72.3% (77/65) of positive animals respectively. The performance of PCR methods was analyzed in two different scenarios. The highest sensitivity value identified in all scenarios studied was kDNA/145. Our results suggest that popliteal lymph node and conjunctival swab samples, besides being less invasive collections, represent a good substratum for PCR-based diagnosis, and the target kDNA/145 is the best choice for detecting L. infantum DNA in naturally infected dogs.

Evaluation of the euthanasia of seropositive dogs for canine visceral leishmaniasis as the only method of controling the disease in the enzootic area in the Midwestern Minas Gerais / Avaliação da eutanásia de cães soro reagentes para leishmaniose visceral canina como único método de controle da doença em área enzoótica no Centro Oeste de Minas Gerais

Human Visceral Leishmaniasis (VL) is expanding, and it is distributed in the five geographic regions of Brazil, with the Northeast being the region that presents the most significant number of registered cases. The main urban reservoir of the etiological agent of VL is the domestic dog, and it is known that canine cases precede human cases. One of the control actions of VL, recommended by the Ministry of Health, is performing the euthanasia of seropositive dogs. In 2013, in the municipality of Iguatama/MG, the first canine serological survey was carried out in the city, with a prevalence of 8.3% of seropositive dogs for Leishmania infantum. Of the seropositive animals for Canine Visceral Leishmaniasis (CVL) in this survey, 84% were euthanized, and 16% died within two years after diagnosis. No other control measures for CVL were taken in the municipality. The objectives of the present study were to determine the current prevalence of seropositive dogs for CVL in the municipality of Iguatama and to observe the impact of euthanasia of seropositive dogs to VL as the only control measure performed in this municipality, which is considered an enzootic area of the disease. For this, a new canine epidemiological survey was carried out in the municipality of Iguatama, following the guidelines of the Manual of Surveillance and Control of Visceral Leishmaniasis. Blood samples were collected from 270 dogs and tested by DPP® immunochromatographic test and by Enzyme-Linked Immunosorbent Assay (ELISA). The animals that presented inconclusive results had new blood samples collected and analyzed. The only animal that showed a positive result in DPP® and undetermined in the ELISA, in the first evaluation, became seropositive four months later, so this study reinforces the recommendation of the Ministry of Health to reassess dogs that present inconclusive results for CVL. From a total of 270 samples, 21 (7.8%) were reagents in both tests. Thus, the prevalence of seropositive dogs for CVL, in 2017, in the urban area of the municipality of Iguatama was 7.8%. The Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) molecular technique confirmed infection by L. infantum in the ten dogs sampled with CVL-positive serology. The comparison between the current prevalence (after euthanasia) with that found in 2013 (8.3%), there was no significant difference (p=0.764). The prevalence of CVL in the urban area of the municipality of Iguatama is still high, making it possible to presume that the euthanasia of seropositive dogs for CVL, and the absence of monitoring or other interventions measures, did not contribute to decrease the transmission of the parasite and was not sufficient for the control of CVL in the municipality of Iguatama, an area considered as non-endemic to CVL until 2013.(AU)

A Leishmaniose visceral humana (LV) está em franca expansão e distribuída nas cinco regiões geográficas do Brasil, o Nordeste sendo a região em que há o maior número de casos registrados. O principal reservatório urbano do agente etiológico da LV é o cão doméstico e sabe-se que casos caninos antecedem o aparecimento de casos humanos. Uma das ações de controle da LV, preconizada pelo Ministério da Saúde, é a eutanásia dos cães soro reagentes. Em 2013, no município de Iguatama/MG, foi realizado o primeiro inquérito sorológico canino na cidade, sendo constatada uma prevalência de 8,3% de cães soro reagentes para Leishmania infantum. Dos animais com sorologia positiva para Leishmaniose visceral canina (LVC), neste inquérito, 84% foram eutanasiados e 16% foram a óbito em, no máximo, dois anos após o diagnóstico. Nenhuma outra medida de controle para LVC foi realizada no município. Os objetivos deste estudo foram determinar a prevalência atual de cães soro reagentes para LVC no município de Iguatama e observar o impacto das eutanásias de cães soro reagentes para LV como única medida de controle realizada neste município, considerado área enzoótica para a doença. Para isso, foi realizado um novo inquérito epidemiológico canino no município de Iguatama, seguindo as normas do Manual de Vigilância e Controle da Leishmaniose Visceral. Foram colhidas amostras sanguíneas de 270 cães que foram examinadas pelo teste imunocromatográfico DPP® e pelo ensaio imunoenzimático (ELISA). Os animais que apresentaram resultados inconclusivos tiveram novas amostras de sangue coletadas e analisadas. O único animal que apresentou resultado positivo no DPP® e indeterminado no ELISA, na primeira avaliação, soro converteu quatro meses depois, o que reforça a recomendação do Ministério da Saúde de reavaliar os cães que apresentam resultado inconclusivo para LVC. Do total das 270 amostras, 21 foram reagentes nos dois testes. Portanto, a prevalência de cães soro reagentes para LVC, em 2017, na área urbana do município de Iguatama foi igual a 7,8%. A infecção pela espécie Leishmania infantum foi confirmada através da técnica molecular de PCR-RFLP em 10 cães amostrados com sorologia positiva para LVC. A comparação entre a prevalência atual (depois das eutanásias) com a encontrada em 2013 (8,3%), não mostrou diferença significativa (p=0,764). Uma vez que a soro prevalência de LVC na área urbana do município de Iguatama ainda é alta, pode-se supor que a eutanásia dos cães soro reagentes para LVC, somada à ausência de monitoramento e de outras medidas de intervenção, não contribuiu para a diminuição da transmissão do parasito, e não foi suficiente para o controle de LVC no município de Iguatama, uma área considerada, até 2013, indene para LVC.(AU)

Serological study of feline leishmaniasis and molecular detection of Leishmania infantum and Leishmania braziliensis in cats (Felis catus) / Pesquisa sorológica da leishmaniose felina e detecção molecular de Leishmania infantum e Leishmania braziliensis em gatos (Felis catus)

Abstract Blood samples and swabs from ocular conjunctiva and mouth were obtained from 64 cats. Of 64 serum samples, 19 were positive for Leishmania antibodies by ELISA (29.80%). Eight cats were positive by PCR (12.5%) in swab samples from mouth and/or ocular mucosa. Poor kappa agreement between serological and molecular results (k = 0.16) was obtained. From five positive PCR samples one was L. braziliensis and four were L. infantum. Phylogenetic analysis performed with the five isolates of Leishmania, showed that samples of L. infantum isolated from the cats were phylogenetically close to those isolated from domestic dogs in Brazil, while the L. braziliensis is very similar to the one described in humans in Venezuela. The study demonstrated that, despite high seropositivity for Leishmania in cats living in the study region, poor agreement between serological and molecular results indicate that positive serology is not indicative of Leishmania infection in cats. Parasite DNA can be detected in ocular conjunctiva and oral swabs from cats, indicating that such samples could be used for diagnosis. Results of phylogenetic analyzes show that L. infantum circulating in Brazil is capable of infecting different hosts, demonstrating the parasite's ability to overcome the interspecies barrier.

Resumo Amostras de sangue e swabs da conjuntiva ocular e oral foram obtidas de 64 gatos. Das 64 amostras de soro, 19 foram positivas para anticorpos contra Leishmania por ELISA (29,80%). Oito gatos foram positivos por PCR (12,5%) em amostras de swab da boca e / ou mucosa ocular. Demonstrou-se baixa concordância kappa entre os resultados sorológicos e moleculares (k = 0,16). Das cinco amostras positivas para PCR, uma era L. braziliensis e quatro eram L infantum. A análise filogenética realizada com os cinco isolados de Leishmania, mostrou que amostras de L. infantum, isoladas dos gatos, eram filogeneticamente próximas às isoladas de cães domésticos do Brasil enquanto L. braziliensis era muito semelhante ao descrito em humanos na Venezuela. O estudo demonstrou que, apesar da alta soropositividade para Leishmania, em gatos que vivem na região do estudo, pouca concordância entre os resultados sorológicos e moleculares indica que a sorologia positiva não é indicativa de infecção por Leishmania em gatos. O DNA do parasita pode ser detectado na conjuntiva ocular e nas zaragatoas orais de gatos, indicando que essas amostras podem ser usadas para o diagnóstico. . Resultados de análises filogenéticas mostram que L. infantum, circulando no Brasil, é capaz de infectar diferentes hospedeiros, demonstrando a capacidade do parasita de superar a barreira interespécies.

Performance of different serological tests in the diagnosis of natural infection by Leishmania infantum in dogs.

Visceral leishmaniasis (VL) is a zoonosis caused by the parasite Leishmania infantum and the dog is its main reservoir in rural and urban areas. The diagnosis of infection is mainly based on the presence of anti-Leishmania IgG antibodies in the serum of infected dogs. In this study, the sensitivity and specificity of qualitative rapid tests (RTs) dual path platform (DPP) Bio-Manguinhos, rapid enzyme-linked immunosorbent assay (ELISA) IDEXX, Kalazar Detect and ALERE, as well as quantitative ELISA Bio-Manguinhos and in-house indirect immunofluorescence assay (IFA) tests were analyzed in sera from infected and uninfected dogs. Serial dilutions of the in-house IFA were compared with RTs and ELISA Bio-Manguinhos. The results showed that none of the tests reached 100% sensitivity and specificity. There was no statistical difference between the analyzed RTs. The most sensitive test was the DPP Bio-Manguinhos (97.9%), while the rapid ELISA IDEXX showed higher specificity (100%). In the treatment setting of infected and/or diseased animals, quantitative tests for monitoring the evolution of antibody titers are required, which indicates the maintenance of in-house IFA in animal handling. Furthermore, we demonstrate that the RTs present higher sensitivity in serum samples with superior antibody titers obtained in the in-house IFA. However, the RTs exhibited false negatives in samples with low titers of antibodies. Among the RTs, only the DPP Bio-Manguinhos presented better performance in this situation. Therefore, the use of RTs for the diagnosis of VL in dogs with low titers of antibodies, such as asymptomatic, should be carefully evaluated.

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake.

BACKGROUND: In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES: The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS: The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS: Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS: There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

Intraspecies Polymorphisms in the Lipophosphoglycan of L. braziliensis Differentially Modulate Macrophage Activation via TLR4.

Lipophosphoglycan (LPG) is the major Leishmania surface glycoconjugate having importance during the host-parasite interface. Leishmania (Viannia) braziliensis displays a spectrum of clinical forms including: typical cutaneous leishmaniasis (TL), mucocutaneous (ML), and atypical lesions (AL). Those variations in the immunopathology may be a result of intraspecies polymorphisms in the parasite's virulence factors. In this context, we evaluated the role of LPG of strains originated from patients with different clinical manifestations and the sandfly vector. Six isolates of L. braziliensis were used: M2903, RR051 and RR418 (TL), RR410 (AL), M15991 (ML), and M8401 (vector). LPGs were extracted and purified by hydrophobic interaction. Peritoneal macrophages from C57BL/6 and respective knock-outs (TLR2-/- and TLR-4-/-) were primed with IFN-γ and exposed to different LPGs for nitric oxide (NO) and cytokine production (IL-1ß, IL-6, IL-12, and TNF-α). LPGs differentially activated the production of NO and cytokines via TLR4. In order to ascertain if such functional variations were related to intraspecies polymorphisms in the LPG, the purified glycoconjugates were subjected to western blot with specific LPG antibodies (CA7AE and LT22). Based on antibody reactivity preliminary variations in the repeat units were detected. To confirm these findings, LPGs were depolymerized for purification of repeat units. After thin layer chromatography, intraspecies polymorphisms were confirmed especially in the type and/size of sugars branching-off the repeat units motif. In conclusion, different isolates of L. braziliensis from different clinical forms and hosts possess polymorphisms in their LPGs that functionally affected macrophage responses.

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.

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment. OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil. METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake. FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport. MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.