Leishmania RNA virus-1 is similarly detected among metastatic and non-metastatic phenotypes in a prospective cohort of American Tegumentary Leishmaniasis.

American Tegumentary Leishmaniasis (ATL) is an endemic and neglected disease of South America. Here, mucosal leishmaniasis (ML) disproportionately affects up to 20% of subjects with current or previous localised cutaneous leishmaniasis (LCL). Preclinical and clinical reports have implicated the Leishmania RNA virus-1 (LRV1) as a possible determinant of progression to ML and other severe manifestations such as extensive cutaneous and mucosal disease and treatment failure and relapse. However, these associations were not consistently found in other observational studies and are exclusively based on cross-sectional designs. In the present study, 56 subjects with confirmed ATL were assessed and followed out for 24-months post-treatment. Lesion biopsy specimens were processed for molecular detection and quantification of Leishmania parasites, species identification, and LRV1 detection. Among individuals presenting LRV1 positive lesions, 40% harboured metastatic phenotypes; comparatively 58.1% of patients with LRV1 negative lesions harboured metastatic phenotypes (p = 0.299). We found treatment failure (p = 0.575) and frequency of severe metastatic phenotypes (p = 0.667) to be similarly independent of the LRV1. Parasite loads did not differ according to the LRV1 status (p = 0.330), nor did Leishmanin skin induration size (p = 0.907) or histopathologic patterns (p = 0.780). This study did not find clinical, parasitological, or immunological evidence supporting the hypothesis that LRV1 is a significant determinant of the pathobiology of ATL.

High-resolution melting analysis identifies reservoir hosts of zoonotic Leishmania parasites in Tunisia.

BACKGROUND: Leishmaniasis is endemic in Tunisia and presents with different clinical forms, caused by the species Leishmania infantum, Leishmania major, and Leishmania tropica. The life cycle of Leishmania is complex and involves several phlebotomine sand fly vectors and mammalian reservoir hosts. The aim of this work is the development and evaluation of a high-resolution melting PCR (PCR-HRM) tool to detect and identify Leishmania parasites in wild and domestic hosts, constituting confirmed (dogs and Meriones rodents) or potential (hedgehogs) reservoirs in Tunisia. METHODS: Using in vitro-cultured Leishmania isolates, PCR-HRM reactions were developed targeting the 7SL RNA and HSP70 genes. Animals were captured or sampled in El Kef Governorate, North West Tunisia. DNA was extracted from the liver, spleen, kidney, and heart from hedgehogs (Atelerix algirus) (n = 3) and rodents (Meriones shawi) (n = 7) and from whole blood of dogs (n = 12) that did not present any symptoms of canine leishmaniasis. In total, 52 DNA samples were processed by PCR-HRM using both pairs of primers. RESULTS: The results showed melting curves enabling discrimination of the three Leishmania species present in Tunisia, and were further confirmed by Sanger sequencing. Application of PCR-HRM assays on reservoir host samples showed that overall among the examined samples, 45 were positive, while seven were negative, with no Leishmania infection. Meriones shawi were found infected with L. major, while dogs were infected with L. infantum. However, co-infections with L. major/L. infantum species were detected in four Meriones specimens and in all tested hedgehogs. In addition, multiple infections with the three Leishmania species were found in one hedgehog specimen. Sequence analyses of PCR-HRM products corroborated the Leishmania species found in analyzed samples. CONCLUSIONS: The results of PCR-HRM assays applied to field specimens further support the possibility of hedgehogs as reservoir hosts of Leishmania. In addition, we showed their usefulness in the diagnosis of canine leishmaniasis, specifically in asymptomatic dogs, which will ensure a better evaluation of infection extent, thus improving elaboration of control programs. This PCR-HRM method is a robust and reliable tool for molecular detection and identification of Leishmania and can be easily implemented in epidemiological surveys in endemic regions.

Pathogenic role of mitogen activated protein kinases in protozoan parasites.

Protozoan parasites with complex life cycles have high mortality rates affecting billions of human lives. Available anti-parasitic drugs are inadequate due to variable efficacy, toxicity, poor patient compliance and drug-resistance. Hence, there is an urgent need for the development of safer and better chemotherapeutics. Mitogen Activated Protein Kinases (MAPKs) have drawn much attention as potential drug targets. This review summarizes unique structural and functional features of MAP kinases and their possible role in pathogenesis of obligate intracellular protozoan parasites namely, Leishmania, Trypanosoma, Plasmodium and Toxoplasma. It also provides an overview of available knowledge concerning the target proteins of parasite MAPKs and the need to understand and unravel unknown interaction network(s) of MAPK(s).

Culicoides Latreille (Diptera: Ceratopogonidae) as potential vectors for Leishmania martiniquensis and Trypanosoma sp. in northern Thailand.

Biting midges of genus Culicoides (Diptera: Ceratopogonidae) are the vectors of several pathogenic arboviruses and parasites of humans and animals. Several reports have suggested that biting midges might be a potential vector of Leishmania parasites. In this study, we screened for Leishmania and Trypanosoma DNA in biting midges collected from near the home of a leishmaniasis patient in Lamphun province, northern Thailand by using UV-CDC light traps. The identification of biting midge species was based on morphological characters and confirmed using the Cytochrome C oxidase subunit I (COI) gene. The detection of Leishmania and Trypanosoma DNA was performed by amplifying the internal transcribed spacer 1 (ITS1) and small subunit ribosomal RNA (SSU rRNA) genes, respectively. All the amplified PCR amplicons were cloned and sequenced. The collected 223 biting midges belonged to seven species (Culicoides mahasarakhamense, C. guttifer, C. innoxius, C. sumatrae, C. huffi, C. oxystoma, and C. palpifer). The dominant species found in this study was C. mahasarakhamense (47.53%). Leishmania martiniquensis DNA was detected in three samples of 106 specimens of C. mahasarakhamense tested indicating a field infection rate of 2.83%, which is comparable to reported rates in local phlebotomines. Moreover, we also detected Trypanosoma sp. DNA in one sample of C. huffi. To our knowledge, this is the first molecular detection of L. martiniquensis in C. mahasarakhamense as well as the first detection of avian Trypanosoma in C. huffi. Blood meal analysis of engorged specimens of C. mahasarakhamense, C. guttifer, and C. huffi revealed that all specimens had fed on avian, however, further studies of the host ranges of Culicoides are needed to gain a better insight of potential vectors of emerging leishmaniasis. Clarification of the vectors of these parasites is also important to provide tools to establish effective disease prevention and control programs in Thailand.

Methodological Appraisal of Literature Concerning the Analysis of Genetic Variants or Protein Levels of Complement Components on Susceptibility to Infection by Trypanosomatids: A Systematic Review.

Background: Trypanosomatids are protozoa responsible for a wide range of diseases, with emphasis on Chagas Disease (CD) and Leishmaniasis, which are in the list of most relevant Neglected Tropical Diseases (NTD) according to World Health Organization (WHO). During the infectious process, immune system is immediately activated, and parasites can invade nucleated cells through a broad diversity of receptors. The complement system - through classical, alternative and lectin pathways - plays a role in the first line of defense against these pathogens, acting in opsonization, phagocytosis and lysis of parasites. Genetic modifications in complement genes, such as Single Nucleotide Polymorphisms (SNPs), can influence host susceptibility to these parasites and modulate protein expression. Methods: In March and April 2021, a literature search was conducted at the PubMed and Google Scholar databases and the reference lists obtained were verified. After applying the inclusion and exclusion criteria, the selected studies were evaluated and scored according to eleven established criteria regarding their thematic approach and design, aiming at the good quality of publications. Results: Twelve papers were included in this systematic review: seven investigating CD and five focusing on Leishmaniasis. Most articles presented gene and protein approaches, careful determination of experimental groups, and adequate choice of experimental techniques, although several of them were not up-to-date. Ten studies explored the association of polymorphisms and haplotypes with disease progression, with emphasis on lectin complement pathway genes. Decreased and increased patient serum protein levels were associated with susceptibility to CD and Visceral Leishmaniasis, respectively. Conclusion: This systematic review shows the influence of genetic alterations in complement genes on the progression of several infectious diseases, with a focus on conditions caused by trypanosomatids, and contributes suggestions and evidence to improve experimental design in future research proposals.

In Silico Characterization of Calcineurin from Pathogenic Obligate Intracellular Trypanosomatids: Potential New Biological Roles.

Calcineurin (CaN) is present in all eukaryotic cells, including intracellular trypanosomatid parasites such as Trypanosoma cruzi (Tc) and Leishmania spp. (Lspp). In this study, we performed an in silico analysis of the CaN subunits, comparing them with the human (Hs) and looking their structure, post-translational mechanisms, subcellular distribution, interactors, and secretion potential. The differences in the structure of the domains suggest the existence of regulatory mechanisms and differential activity between these protozoa. Regulatory subunits are partially conserved, showing differences in their Ca2+-binding domains and myristoylation potential compared with human CaN. The subcellular distribution reveals that the catalytic subunits TcCaNA1, TcCaNA2, LsppCaNA1, LsppCaNA1_var, and LsppCaNA2 associate preferentially with the plasma membrane compared with the cytoplasmic location of HsCaNAα. For regulatory subunits, HsCaNB-1 and LsppCaNB associate preferentially with the nucleus and cytoplasm, and TcCaNB with chloroplast and cytoplasm. Calpain cleavage sites on CaNA suggest differential processing. CaNA and CaNB of these trypanosomatids have the potential to be secreted and could play a role in remote communication. Therefore, this background can be used to develop new drugs for protozoan pathogens that cause neglected disease.

Leishmania Parasite: the Impact of New Serum-Free Medium as an Alternative for Fetal Bovine Serum

Background: Flagellated protozoan of the genus Leishmania is the causative agent of vector-borne parasitic diseases of leishmaniasis. Since the production of recombinant pharmaceutical proteins requires the cultivation of host cells in a serum-free medium, the elimination of FBS can improve the possibility of large-scale culture of Leishmania parasite. In the current study, we aimed at evaluating a new serum-free medium in Leishmania parasite culture for future live Leishmania vaccine purposes. Methods: Recombinant L. tarentolae secreting PpSP15-EGFP and wild type L. major were cultured in serum-free (complete serum-free medium [CSFM]) and serum-supplemented medium. The growth rate, protein expression, and infectivity of cultured parasites in both conditions was then evaluated and compared. Results: Diff-Quick staining and epi-fluores¬cence microscopy examination displayed the typical morphology of L. major and L. tarentolae-PpSP15-EGFP promastigote grown in CSFM medium. The amount of EGFP expression was similar in CSMF medium compared to M199 supplemented with 5% FBS in flow cytometry analysis of L. tarentolae-PpSP15-EGFP parasite. Also, a similar profile of PpSP15-EGFP proteins was recognized in Western blot analysis of L. tarentolae-PpSP15-EGFP cultured in CSMF and the serum-supplemented medium. Footpad swelling and parasite load measurements showed the ability of CSFM medium to support the L. major infectivity in BALB/C mice. Conclusion: This study demonstrated that CSFM can be a promising substitute for FBS supplemented medium in parasite culture for live vaccination purposes.

Chemical control and insecticide resistance status of sand fly vectors worldwide.

BACKGROUND: Phlebotomine sand flies are prominent vectors of Leishmania parasites that cause leishmaniasis, which comes second to malaria in terms of parasitic causative fatalities globally. In the absence of human vaccines, sand fly chemical-based vector control is a key component of leishmaniasis control efforts. METHODS AND FINDINGS: We performed a literature review on the current interventions, primarily, insecticide-based used for sand fly control, as well as the global insecticide resistance (IR) status of the main sand fly vector species. Indoor insecticidal interventions, such as residual spraying and treated bed nets are the most widely deployed, while several alternative control strategies are also used in certain settings and/or are under evaluation. IR has been sporadically detected in sand flies in India and other regions, using non-standardized diagnostic bioassays. Molecular studies are limited to monitoring of known pyrethroid resistance mutations (kdr), which are present at high frequencies in certain regions. CONCLUSIONS: As the leishmaniasis burden remains a major problem at a global scale, evidence-based rational use of insecticidal interventions is required to meet public health demands. Standardized bioassays and molecular markers are a prerequisite for this task, albeit are lagging behind. Experiences from other disease vectors underscore the need for the implementation of appropriate IR management (IRM) programs, in the framework of integrated vector management (IVM). The implementation of alternative strategies seems context- and case-specific, with key eco-epidemiological parameters yet to be investigated. New biotechnology-based control approaches might also come into play in the near future to further reinforce sand fly/leishmaniasis control efforts.

Metabolomic Reprogramming of C57BL/6-Macrophages during Early Infection with L. amazonensis.

Leishmania survival inside macrophages depends on factors that lead to the immune response evasion during the infection. In this context, the metabolic scenario of the host cell-parasite relationship can be crucial to understanding how this parasite can survive inside host cells due to the host's metabolic pathways reprogramming. In this work, we aimed to analyze metabolic networks of bone marrow-derived macrophages from C57BL/6 mice infected with Leishmania amazonensis wild type (La-WT) or arginase knocked out (La-arg-), using the untargeted Capillary Electrophoresis-Mass Spectrometry (CE-MS) approach to assess metabolomic profile. Macrophages showed specific changes in metabolite abundance upon Leishmania infection, as well as in the absence of parasite-arginase. The absence of L. amazonensis-arginase promoted the regulation of both host and parasite urea cycle, glycine and serine metabolism, ammonia recycling, metabolism of arginine, proline, aspartate, glutamate, spermidine, spermine, methylhistidine, and glutathione metabolism. The increased L-arginine, L-citrulline, L-glutamine, oxidized glutathione, S-adenosylmethionine, N-acetylspermidine, trypanothione disulfide, and trypanothione levels were observed in La-WT-infected C57BL/6-macrophage compared to uninfected. The absence of parasite arginase increased L-arginine, argininic acid, and citrulline levels and reduced ornithine, putrescine, S-adenosylmethionine, glutamic acid, proline, N-glutamyl-alanine, glutamyl-arginine, trypanothione disulfide, and trypanothione when compared to La-WT infected macrophage. Moreover, the absence of parasite arginase leads to an increase in NO production levels and a higher infectivity rate at 4 h of infection. The data presented here show a host-dependent regulation of metabolomic profiles of C57BL/6 macrophages compared to the previously observed BALB/c macrophages infected with L. amazonensis, an important fact due to the dual and contrasting macrophage phenotypes of those mice. In addition, the Leishmania-arginase showed interference with the urea cycle, glycine, and glutathione metabolism during host-pathogen interactions.

Innate immune response: ally or enemy in cutaneous leishmaniasis?

Cutaneous leishmaniasis (CL) is an infectious and neglected disease caused by parasites of the genus Leishmania, which produces a wide spectrum of cutaneous manifestations. CL research has shown that the innate immune activity of cells such as neutrophils, natural killers, macrophages, dendritic cells and the complement system are capable of controlling this infection. However, Leishmania can also modulate the immune activity of these cells to promote its own survival and proliferation at the intracellular level. This review discusses the role of the innate immune response in the control and spread of this infection.

Frequency and factors associated of potential zoonotic pathogens (Borrelia spp., Rickettsia spp., Leishmania spp., and Anaplasma phagocytophilum) in equids in the state of Bahia, Brazil.

BACKGROUND: Currently, various zoonotic diseases are classified as emerging or reemerging. Because equids have a direct relationship with various vectors, they are possibly more frequently exposed to zoonotic agents than are humans. The undeniable importance of diseases such as human granulocytic anaplasmosis, spotted fever, and leishmaniasis for both public and animal health, as well as the possibility of equids acting as sources, reservoirs, or even sentinels for these pathogens, justifies the detection of their frequency and factors associated with infection in equids from northeastern Brazil. METHODS: Blood samples were collected from 569 equids (528 horses, 33 donkeys, and 8 mules), 516 from a rural area and 53 from an urban area. Pathogen detection was carried out as follows: Borrelia spp. and Rickettsia spp., serological analysis; Leishmania spp., serological analysis and polymerase chain reaction (PCR); Anaplasma phagocytophilum, PCR. Determination of associated factors was carried out through generalized linear models. RESULTS: The frequencies of positivity for the pathogens observed in equids were as follows: Borrelia spp., 13.9% (79/569); Leishmania spp., 3.5% (20/569); Rickettsia spp. 33.4% (190/569). Regarding factors associated with infection, male sex was associated with protection against Borrelia spp.; donkeys and mules were associated with protection against Rickettsia spp., while a younger age was a risk factor. The infection of A. phagocytophilum was not detected in the sampled population. Co-infection was detected in 5.1% (29/569) of the animals. CONCLUSIONS: Most of the studied pathogenic agents are present in the prospected area, indicating a possible risk for both human and animal health. This demonstrates that equids can be considered important sentinels in the assessment of pathogens with zoonotic potential in the region.

Avaliação da tendência temporal e da prevalência de leishmaniose visceral canina e humana dentre os anos de 2010 a 2019 em municípios de Minas Gerais ­ Brasil / Evaluation of the temporal trend and prevalence of canine and human visceral leishmaniosis between 2010 and 2019 in municipalities of Minas Gerais - Brazil / Evaluación de la tendencia temporal y de la prevalencia de leishmaniosis visceral canina y humana entre los años 2010 a 2019 en los municipios de Minas Gerais ­ Brasil

Leishmanioses são causadas por protozoários do gênero Leishmania, parasitos que infectam grande número de mamíferos, incluindo o homem. A Leishmaniose Visceral (LV) é a forma mais severa da doença e invariavelmente leva ao óbito, se não diagnosticada e tratada precocemente. O objetivo deste estudo foi realizar uma análise de natureza documental, descritiva e analítica, de abordagem quantitativa das informações contidas no banco de dados Fundação Ezequiel Dias/Gerenciador de Ambientes Laboratoriais (FUNED/GAL), da Secretaria Estadual de Saúde de Minas Gerais. Os dados epidemiológicos que compõe este estudo são registros de casos positivos e negativos da cidade de Patos de Minas-MG e 18 municípios das mesorregiões do Triângulo Mineiro e Alto Paranaíba, Noroeste de Minas e Norte de Minas. No período compreendido entre janeiro de 2010 e junho de 2019 foram notificados um total de 1170 indivíduos suspeitos de Leishmaniose Visceral Canina e/ou Humana. 304 (25,98%) indivíduos receberam o resultado positivo, enquanto 866 foram negativos, e em alguns casos, inconclusivos. Os dados obtidos no estudo revelaram a tendência temporal crescente e alta prevalência da doença, mostrando que a doença está em expansão na região estudada onde o cão é o principal reservatório doméstico da doença, permanecendo como principal elo de ligação entre o protozoário e o hospedeiro humano.(AU)

Leishmaniasis are caused by protozoa of the genus Leishmania, parasites that infect a large number of mammals, including humans. Visceral Leishmaniasis (VL) is the most severe form of the disease and invariably leads to death if not diagnosed and treated early. The objective of this study was to carry out a documentary, descriptive and analytical analysis, with a quantitative approach to the information contained in the Fundação Ezequiel Dias/Manager of Laboratory Environments (FUNED/GAL) database from the Minas Gerais State Health Secretariat. The epidemiological data that make up this study are records of positive and negative cases in the city of Patos de Minas - MG and of 18 municipalities in the mesoregions of the Triângulo Mineiro and Alto Paranaíba, Northwest of Minas and North of Minas. In the period between January 2010 and June 2019, a total of 1170 individuals suspected of Canine and/or Human Visceral Leishmaniasis were notified. A total of 304 (25.98%) individuals received a positive result, while 866 were considered negative, and in some cases, inconclusive. The data obtained in the study revealed the growing temporal trend and high prevalence of the disease, showing that the disease is expanding in the studied region where the dog is presented as the main domestic reservoir of the disease, remaining as the main link between the protozoan and the human host.(AU)

Leishmaniosis son causadas por protozoos del género Leishmania, parásitos que infectan a un gran número de mamíferos, incluyendo el hombre. La Leishmaniosis Visceral (LV) es la forma más grave de la enfermedad e invariablemente conduce a la muerte, si no se la diagnostica y la trata a tiempo. El objetivo de ese estudio fue realizar un análisis de naturaleza documental, descriptivo y analítico, de enfoque cuantitativo de las informaciones contenidas en la base de datos Fundação Ezequiel Dias/Gerente de Ambientes de Laboratorio (FUNED /GAL), de la Secretaría de Salud del Estado de Minas Gerais. Los datos epidemiológicos que conforman este estudio son registros de casos positivos y negativos en la ciudad de Patos de Minas-MG y 18 municipios de las mesorregiones del Triângulo Mineiro y Alto Paranaíba, Noroeste de Minas y Norte de Minas. En el período comprendido entre enero de 2010 y junio de 2019, se notificó a un total de 1170 personas sospechosas de Leishmaniosis Visceral Canina y/o Humana. 304 (25,98%) individuos recibieron un resultado positivo, mientras que 866 fueron negativos y, en algunos casos, no concluyentes. Los datos obtenidos en el estudio revelaron la tendencia creciente temporal y la alta prevalencia de la enfermedad, mostrando que la enfermedad se está expandiendo en la región estudiada donde el perro es el principal reservorio doméstico de la enfermedad, permaneciendo como principal conexión entre el protozoo y el hospedero humano.(AU)

Alkyl-Resorcinol Derivatives as Inhibitors of GDP-Mannose Pyrophosphorylase with Antileishmanial Activities.

Leishmaniasis is a vector-borne disease caused by the protozoan parasite Leishmania found in tropical and sub-tropical areas, affecting 12 million people around the world. Only few treatments are available against this disease and all of them present issues of toxicity and/or resistance. In this context, the development of new antileishmanial drugs specifically directed against a therapeutic target appears to be a promising strategy. The GDP-Mannose Pyrophosphorylase (GDP-MP) has been previously shown to be an attractive therapeutic target in Leishmania. In this study, a chemical library of 5000 compounds was screened on both L. infantum (LiGDP-MP) and human (hGDP-MP) GDP-MPs. From this screening, oncostemonol D was found to be active on both GDP-MPs at the micromolar level. Ten alkyl-resorcinol derivatives, of which oncostemonols E and J (2 and 3) were described for the first time from nature, were then evaluated on both enzymes as well as on L. infantum axenic and intramacrophage amastigotes. From this evaluation, compounds 1 and 3 inhibited both GDP-MPs at the micromolar level, and compound 9 displayed a three-times lower IC50 on LiGDP-MP, at 11 µM, than on hGDP-MP. As they displayed mild activities on the parasite, these compounds need to be further pharmacomodulated in order to improve their affinity and specificity to the target as well as their antileishmanial activity.

Diagnostic usefulness of immunohistochemical evaluation of CD1a antigen and polyclonal anti-leishmania antibodies in cutaneous leishmaniasis.

BACKGROUND: Different immunohistochemical markers to detect amastigotes in cutaneous leishmaniasis have been proposed with variable diagnostic usefulness. OBJECTIVES: To evaluate the diagnostic usefulness of immunohistochemical amastigotes identification by specific polyclonal anti-Leishmania antibodies and CD1a expression (clone EP3622) in a series of PCR confirmed cutaneous leishmaniasis. MATERIALS AND METHODS: Thirty-three skin samples corresponding to PCR confirmed cutaneous leishmaniasis were included in the study. All samples were stained with Hematoxylin-eosin and Giemsa. Moreover, immunohistochemical studies with anti-CD1a and anti-Leishmania antibodies were performed. The patients clinical features and the observed histopathological features were also recorded. RESULTS: From the selected 33 biopsies, Leishmania spp. amastigotes were detected in 48.4% of cases with conventional Hematoxylin-eosin stain and in 57.5% of cases by Giemsa staining. In 31/33 cases, anti-CD1a allowed us to identify parasitic structures, and in 33/33 cases amastigotes were detected with anti-Leishmania antibodies. Concordance between both techniques, anti-CD1a and anti-Leishmania, was 94% [CI 95%: (79,8%-99,3%)] ; p value <0.05. The sensitivity of anti-CD1a in comparison with the PCR was 94%, with a positive predictive value of 100%. Two cases of low parasitic index were negative for CD1a immunostaining. In cases with high parasitic index, anti-CD1a stained amastigotes in superficial and deep dermis. Only a few cases were originally diagnosed with the available histological techniques, needing PCR for Leishmania spp. CONCLUSIONS: Anti-CD1a antibody seems to be a useful technique to identify amastigotes when PCR and anti-Leishmania antibodies are not available. The sensitivity to detect amastigotes is increased when the CD1a immunostaining is added to the classical Haematoxylin - eosin and Giemsa staining.

Retrospective evaluation of vector-borne pathogens in cats living in Germany (2012-2020).

BACKGROUND: Blood-feeding arthropods can transmit parasitic, bacterial, or viral pathogens to domestic animals and wildlife. Vector-borne infections are gaining significance because of increasing travel and import of pets from abroad as well as the changing climate in Europe. The main objective of this study was to assess the percentage of cats with positive test results for selected vector-borne pathogens in Germany and explore any possible association of such results with time spent abroad. METHODS: This retrospective study included test results from cats included in the "Feline Travel Profile" established by the LABOKLIN laboratory at the request of veterinarians in Germany between April 2012 and March 2020. This diagnostic panel includes the direct detection of Hepatozoon spp. and Dirofilaria spp. via PCR as well as indirect detection assays (IFAT) for Ehrlichia spp. and Leishmania spp. The panel was expanded to include an IFAT for Rickettsia spp. from July 2015 onwards. RESULTS: A total of 624 cats were tested using the "Feline Travel Profile." Serum for indirect detection assays was available for all 624 cats; EDTA samples for direct detection methods were available from 618 cats. Positive test results were as follows: Ehrlichia spp. IFAT 73 out of 624 (12%), Leishmania spp. IFAT 22 out of 624 (4%), Hepatozoon spp. PCR 53 out of 618 (9%), Dirofilaria spp. PCR 1 out of 618 cats (0.2%), and Rickettsia spp. IFAT 52 out of 467 cats (11%) tested from July 2015 onwards. Three cats had positive test results for more than one pathogen before 2015. After testing for Rickettsia spp. was included in 2015, 19 cats had positive test results for more than one pathogen (Rickettsia spp. were involved in 14 out of these 19 cats). CONCLUSIONS: At least one pathogen could be detected in 175 out of 624 cats (28%) via indirect and/or direct detection methods. Four percent had positive test results for more than one pathogen. These data emphasize the importance of considering the above-mentioned vector-borne infections as potential differential diagnoses in clinically symptomatic cats.

Diagnosis and identification of Leishmania species in patients with cutaneous leishmaniasis in the state of Roraima, Brazil's Amazon Region.

BACKGROUND: Cutaneous leishmaniasis (CL) is an endemic disease in Brazil that is highly prevalent in the northern region of the country. Although there is a continuous and growing number of cases registered in the state of Roraima, there is limited information regarding the species of Leishmania that affect the human population. In this study, we aimed to characterize which Leishmania species cause human disease in those presenting with cutaneous leishmaniasis in endemic areas of the State of Roraima. METHODS: We conducted a prospective surveillance study between 2016 to 2018 in health centers located in the State of Roraima, Brazil. Participants with clinical suspicion of CL were enrolled and provided lesion samples for parasitological detection by microscopy. A subset of the samples was tested by polymerase chain reaction and sequencing of the internal transcribed spacer 1 (ITS-1 PCR) for molecular species identification. RESULTS: A total of 262 participants were enrolled in this study. Of those, 129 (49.27%) were positive by parasitological examination. Most positive subjects (81.58%) were male, and most cases presented a single lesion (80.26%). ITS-1 PCR and sequencing on a subset of 76 samples allowed us to detect nine different species of Leishmania: L. (V.) braziliensis, L (V.) panamensis, L. (V.) guyanensis, L. (V.) naiffi, L. (V.) shawi, L.(V.) utingensis, L. (V.) lindenbergi, L. (L.) amazonensis and L. (L.) mexicana. CONCLUSIONS: Our study provides the first assessment of circulating species of Leishmania in the State of Roraima, Brazil, and shows the high diversity in this region. This study opens the path for further research on the transmission of leishmaniasis in the northernmost Brazilian State including vector and reservoir surveillance as well as for intensification of investigation and control activities against CL in the region.

Role of host genetics and cytokines in Leishmania infection.

Cytokines and chemokines are important regulators of innate and specific responses in leishmaniasis, a disease that currently affects 12 million people. We overviewed the current information about influences of genetically engineered mouse models of cytokine and chemokine on leishmaniasis. We found that genetic background of the host, parasite species and sub-strain, as well as experimental design often modify effects of genetically engineered cytokine genes. Next we analyzed genes and QTLs (quantitative trait loci) that control response to Leishmania species in mouse in order to establish relationship between genetic control of cytokine expression and organ pathology. These studies revealed a network-like complexity of the combined effects of the multiple functionally diverse QTLs and their individual specificity. Genetic control of organ pathology and systemic immune response overlap only partially. Some QTLs control both organ pathology and systemic immune response, but the effects of genes and loci with the strongest impact on disease are cytokine-independent, whereas several loci modify cytokines levels in serum without influencing organ pathology. Understanding this genetic control might be important in development of vaccines designed to stimulate certain cytokine spectrum.

The different faces of the NLRP3 inflammasome in cutaneous Leishmaniasis: A review.

Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease caused by Protozoa of the genus Leishmania. Clinical manifestations of this disease are the result of a complex interplay of diverse factors, including the genetic background and the immune status of the host. Understanding the impact of these factors on the CL pathology may provide new targets to manage the infection and improve clinical outcome. The NLRP3 inflammasome, an innate immune complex of several cell types, seems to be involved in the CL physiopathology. Current studies of its role show contradictory effects of this complex on the evolution of Leishmania infection in mice and humans. In this review, we discuss the data regarding different roles of the NLRP3 inflammasome in murine and human CL.

Regulation of macrophage subsets and cytokine production in leishmaniasis.

Macrophages are host cells for parasites of the genus Leishmania where they multiply inside parasitophorous vacuoles. Paradoxically, macrophages are also the cells responsible for killing or controlling parasite growth, if appropriately activated. In this review, we will cover the patterns of macrophage activation and the mechanisms used by the parasite to circumvent being killed. We will highlight the impacts of the vector bite on macrophage activation. Finally, we will discuss the ontogeny of macrophages that are infected by Leishmania spp.