Accuracy evaluation of diagnostic methods for visceral leishmaniasis in adult patients with and without HIV infection: clinical management implications.

Visceral leishmaniasis (VL) poses a serious health threat, particularly when untreated, necessitating accurate diagnosis. While the gold-standard method involves identifying amastigotes in bone marrow aspirate (BMA), this procedure is invasive and occasionally contraindicated. Additionally, when VL is associated with HIV infection the serologies accuracies could be affected. This study aims to evaluate and compare diagnostic methods for VL in patients with and without HIV coinfection. We enrolled prospectively 127 consecutive adult VL patients, 48 (37.8%) of whom had HIV coinfection, in Brazil's Midwestern region, where VL is endemic. Parasitological examination served as the reference standard for accuracy analysis, with index tests including immunofluorescent antibody test (IFAT), immunochromatographic test with rK39 protein (rK39-ICT), and blood polymerase chain reaction (PCR). Specificity assessment involved 430 healthy blood donors from the same endemic area. Ninety-two patients had parasitologically confirmed VL. Among HIV-uninfected patients, rK39-ICT exhibited sensitivity comparable to PCR (93.6%; 95% CI: 83.6-100 vs. 97.8%; 95% CI: 93.6-99.2, respectively) and superior to IFAT (71.1%; 95% CI: 57.9-84.3). However, in HIV-infected patients, rK39-ICT sensitivity was notably lower than PCR (40.0%; 95% CI: 22.5-57.5 vs. 97.4%; 95% CI: 92.5-98.9) and similar to IFAT (67.5%; 95% CI: 52.9-82.0). Combining two serological tests in parallel identified 82.1% of parasitologically confirmed VL cases, with a negative likelihood ratio significantly lower than either test alone. No test achieved a specificity of 90%, and there were no significant differences in specificity observed among the index tests. The positivity rate of parasitological examination in the 127 VL patients was higher in HIV-infected compared to HIV-uninfected patients, 91.3% (95% CI: 83.2-99.4) versus 67.6% (95% CI: 56.9-78.3), respectively. These findings underscore the necessity of accounting for HIV infection when choosing VL diagnostic methods. Although rK39-ICT provides reliable results in HIV-uninfected patients, BMA examination remains crucial for accurate diagnosis in individuals with HIV/AIDS. In cases where bone marrow aspiration is contraindicated, employing IFAT and rK39-ICT in parallel could be considered, as the occurrence of both positive results is uncommon in healthy individuals from endemic areas.

Feline immunodeficiency virus, feline leukemia virus and Leishmania spp. prevalence in cats from shelters in Mato Grosso do Sul, Brazil.

Diseases such as those caused by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) represent health problems for cats. Feline leishmaniasis (FL) has been reported in several cities across the country. The objective was to carry out a clinical-epidemiological and laboratory study of FIV, FeLV and FL in cats from shelters in Dourados, Mato Grosso do Sul, Brazil. Blood samples and swabs from the conjunctival and nasal mucosa were obtained from 75 cats, from four animal shelters. Serology for FIV and FeLV was performed. For Leishmania, polymerase chain reaction (PCR) was performed on blood, conjunctiva and nasal mucosa. In the immunochromatographic serological test, seven cats tested positive for FIV and none for FeLV. No samples was positive in PCR for Leishmania. The study showed that despite the presence of human and canine leishmaniasis in the studied region, Leishmania spp. were absent in the cats studied. To avoid an increase in contagion in shelters, it is essential isolate cats with FIV.

First molecular typing of cryptococcemia-causing cryptococcus in central-west Brazil.

Molecular epidemiology studies on cryptococcemia are limited. This study aimed to describe the clinical features of patients with bloodstream infections by Cryptococcus sp. in a public tertiary hospital in Mato Grosso do Sul, as well as identify the fungus' molecular type and determine its antifungal susceptibility. Molecular typing was performed using URA5 restriction fragment length polymorphism PCR, and antifungal susceptibility was determined by microdilution method standardized by the Clinical and Laboratory Standards Institute. Over 14 years, 48 patients were diagnosed with cryptococcemia. The majority (72.9 %) was male with a median age of 40 years; 81.3 % of the patients had HIV/AIDS and 72.9 % died. Cryptococcus neoformans was the most commonly isolated species (97.9 %). Molecular analysis identified the genotypes C. neoformans VNI (93.7 %), C. neoformans VNII (4.2 %), and Cryptococcus gattii VGII (2.1 %). In vitro, these fungi were not resistant to fluconazole, itraconazole, voriconazole, and amphotericin B. This is the first description of the molecular types of cryptococcemia agents in central-west Brazil. Its high lethality, especially in HIV-negative patients, suggests that early diagnosis and prompt antifungal therapy are crucial for a good clinical outcome.

Asymptomatic Leishmania infection in humans: A systematic review.

BACKGROUND: Leishmaniasis is a highly prevalent neglected tropical disease. It mainly presents as two forms: cutaneous and visceral leishmaniasis, the latter being the most severe form. However, asymptomatic cases of Leishmania infection result in an increase in the underreporting and transmission of the protozoan OBJECTIVES: In this study, articles on the incidence of asymptomatic Leishmania infection were systematically reviewed. METHODS: The publications identified in the Medline/PubMed and Science Direct databases included 4568 articles. Inclusion, exclusion, and eligibility criterion analysis resulted in 83 articles being retained. These studies were mostly performed in Brazil (n = 26) and India (n = 15). RESULTS: Several detection techniques have been used for diagnosis. Among the species found were L. infantum and L. donovani, which result in visceral leishmaniasis, and L. amazonensis, L. braziliensis, and L. panamensis. The incidence rates varied between the analyzed locations, largely due to sampling and the presence or absence of endemism in the regions. The largest populations analyzed were in two studies performed in India and Nepal. One of these studies evaluated 32,529 people and the incidence rate was 8.3% (n = 2702), while the other study evaluated 21,267 people and the incidence rate was 1.76% (n = 375). Only 14.28% of the studies investigated leishmaniasis in blood donors. Preexisting diseases have also been reported. CONCLUSION: The findings of this systematic review present the incidence of cases of asymptomatic Leishmania infection worldwide, in addition to detailing the studies and offering information for researchers and health authorities to seek alternatives to reduce the number of leishmaniasis cases.

Sand Fly Fauna, Spatial Distribution of Lutzomyia longipalpis (Diptera: Psychodidae), and Climate Factors in Dourados, Brazil.

Studies of the geographic distribution of sand flies and the factors associated with their occurrence are necessary to understand the risk of leishmaniasis transmission. The objective of this study was to characterize the sand fly fauna, particularly the spatial distribution of Lutzomyia longipalpis (Lutz & Neiva), and correlate these with climate factors in the Dourados municipality, Brazil. The collection of sand flies was carried out with CDC Light Traps over two periods: at six sites for three consecutive nights each month from August 2012 to July 2013; and at four other sites for two consecutive nights each month from April 2017 to February 2018. We collected 591 sand flies in the first period and 121 in the second period for a total of 712 sand flies; 697 of the total collected were Lu. longipalpis. The minimum and maximum sand fly infestation rate (sites with vector presence) was 11.1% and 83.33% in the first period, and 0% and 50.0% in the second period. No sand flies with Leishmania were identified via PCR. Lu. longipalpis presented an aggregate disposition with excellent adjustment. Rainfall and relative humidity were the abiotic factors that influenced the vector infestation level. The aggregate distribution for this species was predicted by the environmental factors that favor the proliferation of Lu. longipalpis. The results of this study should assist in devising measures to control sand flies in Dourados, Brazil.

Leukocyte Behavior in Mesenteric Microcirculation upon Experimental By Leishmania Spp. in BALB/c Mice.

BACKGROUND: We aimed to determine the cellular recruitment (leukocyte rolling and adhesion) by which the Leishmania (Viannia) braziliensis, L. (Leishmania) amazonensis, and L. (Leishmania) major species in the mesenteric microcirculation of BALB/c mice. METHODS: Five experimental groups were considered: group 1 (L. braziliensis); group 2 (L. amazonensis); group 3 (L. major); group 4 (control group with PBS); group 5 (negative control group), analyzed 3, 6, 12, and 24 h after parasite inoculation. RESULTS: Infections by the different Leishmania species caused an increase in the number of rolling leukocytes: L. braziliensis a peak at 6 h; L. amazonensis and L. major a peak at 3 h. The Leishmania infections induced leukocyte adhesion: L. major and L. amazonensis showed an increase after 3 and 6 h, respectively. CONCLUSION: The kinetics of cellular recruitment in Leishmania infections, leading to infection susceptibility or resistance, indicates that distinct mechanisms regulate the initial response to Leishmania infection and determine its course.

Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs.

The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.

Asymptomatic Leishmania infection in HIV-positive outpatients on antiretroviral therapy in Pernambuco, Brazil.

BACKGROUND: Visceral leishmaniasis (VL) in HIV-positive individuals is a global health problem. HIV-Leishmania coinfection worsens prognosis and mortality risk, and HIV-Leishmania coinfected individuals are more susceptible to VL relapses. Early initiation of antiretroviral therapy can protect against Leishmania infection in individuals living in VL-endemic areas, and regular use of antiretrovirals might prevent VL relapses in these individuals. We conducted a cross-sectional study in Petrolina, Brazil, an VL-endemic area, to estimate the prevalence of asymptomatic Leishmania cases among HIV-positive outpatients. METHODS: We invited any HIV-positive patients, aged ≥ 18-years-old, under antiretroviral therapy, and who were asymptomatic for VL. Patients were tested for Leishmania with enzyme-linked immunosorbent assays (ELISA)-rK39, immunochromatographic test (ICT)-rK39, direct agglutination test (DAT), latex agglutination test (KAtex), and conventional polymerase chain reaction (PCR). HIV-Leishmania coinfection was diagnosed when at least one VL test was positive. RESULTS: A total of 483 patients were included. The sample was predominantly composed of single, < 48-years-old, black/pardo, heterosexual males, with fewer than 8 years of schooling. The prevalence of asymptomatic HIV-Leishmania coinfection was 9.11% (44/483). HIV mono-infected and HIV-Leishmania coinfected groups differed statistically significantly in terms of race (p = 0.045), marital status (p = 0.030), and HIV viral load (p = 0.046). Black/pardo patients, married patients, and those with an HIV viral load up to 100,000 copies/ml presented higher odds for HIV-Leishmania coinfection. CONCLUSIONS: A considerable number of asymptomatic Leishmania cases were observed among HIV-positive individuals in a VL-endemic area. Given the potential impact on transmission and health costs, as well as the impact on these coinfected individuals, studies of asymptomatic Leishmania carriers can be useful for guiding public health policies in VL-endemic areas aiming to control and eliminate the disease.

Asymptomatic Leishmania infection in blood donors from a major blood bank in Northeastern Brazil: a cross-sectional study.

This study has estimated the risk of Leishmania transmission via blood transfusion in one of the largest blood banks in Northeastern Brazil, where visceral leishmaniasis is endemic. Five hundred blood samples from donors were tested for circulating Leishmania spp. DNA by real-time PCR. Positive samples were tested by a species-specific conventional PCR targeting Leishmania infantum . Overall, 6.2% (95% CI: 4.1-8.3%) of the samples carried Leishmania DNA and in one sample the species was confirmed as L. infantum . No statistically significant differences were found in relation to gender, sex, education level, incomeas well as the place of residence between positive and negative blood donors. Our results confirm the presence of asymptomatic Leishmania carriers among blood donors in a large blood bank in Northeastern Brazil. Considering the studied population, we estimate that for every 1,000 blood donors screened, 41 to 83 will be positive for Leishmania DNA. This finding reinforces the urgent need for elaborating specific Blood bank guidelines to allow the early detection of asymptomatic Leishmania carriers among blood donors before their blood products are transfused to uninfected individuals.

Feline immunodeficiency virus, feline leukemia virus and Leishmania spp. prevalence in cats from shelters in Mato Grosso do Sul, Brazil / Vírus da imunodeficiência felina, vírus da leucemia felina e Leishmania spp. em gatos de abrigos de Mato Grosso do Sul, Brasil

Abstract Diseases such as those caused by feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) represent health problems for cats. Feline leishmaniasis (FL) has been reported in several cities across the country. The objective was to carry out a clinical-epidemiological and laboratory study of FIV, FeLV and FL in cats from shelters in Dourados, Mato Grosso do Sul, Brazil. Blood samples and swabs from the conjunctival and nasal mucosa were obtained from 75 cats, from four animal shelters. Serology for FIV and FeLV was performed. For Leishmania, polymerase chain reaction (PCR) was performed on blood, conjunctiva and nasal mucosa. In the immunochromatographic serological test, seven cats tested positive for FIV and none for FeLV. No samples was positive in PCR for Leishmania. The study showed that despite the presence of human and canine leishmaniasis in the studied region, Leishmania spp. were absent in the cats studied. To avoid an increase in contagion in shelters, it is essential isolate cats with FIV.

Resumo Doenças como as causadas pelos vírus da imunodeficiência felina (FIV) e vírus da leucemia felina (FeLV) representam problemas de saúde para os gatos. A leishmaniose felina (LF) tem sido relatada em diversas cidades do país. O objetivo deste trabalho foi realizar um estudo clínico-epidemiológico e laboratorial de FIV, FeLV e LF em gatos de abrigos em Dourados, Mato Grosso do Sul, Brasil. Amostras de sangue e swabs da mucosa conjuntival e da mucosa nasal foram obtidas de 75 gatos, dos quatro abrigos de animais. Foi feita a sorologia para FIV e FeLV. Para Leishmania foi realizada a reação em cadeia da polimerase (PCR) em sangue, conjuntiva e mucosa nasal. No teste sorológico imunocromatografico, sete gatos apresentaram resultado positivo para FIV e nenhum para FeLV. Nenhuma amostra foi positiva na PCR para Leishmania. O estudo demonstrou que apesar da presença de leishmaniose humana e canina, na região estudada, não foi encontrado Leishmania spp. nos gatos analisados. Para evitar o aumento do contágio em abrigos é fundamental isolar os gatos com FIV.

Leishmania infection in blood donors: A new challenge in leishmaniasis transmission?

Transfusion-transmitted leishmaniasis has been a concern in regions endemic for the disease. Whether immediate or delayed, the risks posed by this mode of transmission call for careful assessment. The purpose of this study was to detect Leishmania infection in blood donors living in an endemic area and to investigate progression to the disease in these individuals. Immunofluorescent antibody test, enzyme-linked immunosorbent assay, leishmaniasis rapid test, and the polymerase chain reaction were applied to 430 donors in an initial evaluation. Of those donors with at least one positive test, 50 were reevaluated four years later by the same methods, as were 25 controls who had been negative on the same tests. In the first evaluation, Leishmania infection was detected in 41.4% (95% CI: 36.7-46.1) of donors (n = 430). None of the 75 reevaluated individuals had developed the disease, but retesting revealed positivity in at least one test in 36.0% (95% CI: 25.1-46.9) of donors. Of the 50 initially testing positive, 50% remained so on retesting. Of the 25 initially negative controls, two tested positive in the subsequent evaluation. The severity of the parasitosis and the risk of transfusion transmission warrant investigation of the potential inclusion of methods for Leishmania detection into blood banks for effective screening of infected donors.

PCR sensitivity of peripheral blood of dogs co-infected with Leishmania spp. and Ehrlichia spp. in endemic area of Brazil.

INTRODUCTION: Peripheral blood of 400 dogs infected with Leishmania and Ehrlichia were analyzed using polymerase chain reaction (PCR), and clinical signs were characterized. METHODS: PCR and parasitological tests were conducted. RESULTS: PCR was positive for Leishmania in 84.75%, and parasitological tests showed that 63.25% and 31.75% were positive for Leishmania and Ehrlichia, respectively. All animals showed more than three clinical signs. PCR results were negative for Leishmania in 15.25% of the samples. CONCLUSIONS: Conventional PCR of peripheral blood can be used for diagnosing canine visceral leishmaniasis in combination with other techniques, especially in uncertain cases that need species identification.

Polymerase chain reaction using conjunctival swab samples for detecting Leishmania DNA in dogs / Reação em cadeia da polimerase usando amostras de Swab de conjuntiva para detecção de DNA de Leishmania em cães

Abstract The dog is the main domestic reservoir of Leishmania and font of infection for the vector, constituting an important host for the transmission of the parasite to humans. Non-invasive collection of swab samples for leishmaniasis diagnosis has been a promising alternative. This study analyzed the positivity of polymerase chain reaction (PCR) for the diagnosis of canine leishmaniasis in conjunctiva samples. DNA extraction was performed using SDS 20% and PCR was performed using 13A/13B primers that amplify 120-bp of Leishmania kDNA. Of the 77 dogs analyzed, 50 (64.93%) had ocular changes: 25 (32.47%) dogs had periocular lesion, 41 (53.25%) dogs had purulent eye discharge, and 17 (22.08%) dogs had both signals. PCR was positive in 35 dogs (45.45%), and there was no significant difference between dogs with and without ocular signals (p=0.4074). PCR positivity was significant higher in dogs without periocular injury (p=0.0018). Conjunctive PCR, a less invasive, fast, and painless collection technique, is indicated to complement the diagnosis, especially in dogs without periocular injury, independent of the presence of purulent eye discharge.

Resumo O cão é o principal reservatório doméstico de Leishmania e também fonte de infecção para o vetor, constituindo um importante hospedeiro para a transmissão do parasita ao homem. A coleta não invasiva de amostras em swab para diagnóstico das leishmanioses tem sido uma alternativa promissora. Este estudo analisou a positividade da reação em cadeia da polimerase (PCR) para o diagnóstico de leishmaniose canina em amostras de conjuntiva. A extração do DNA foi realizada com SDS 20%. A PCR foi realizada com primers 13A/13B que amplificam 120-pb do kDNA de Leishmania. Dos 77 cães analisados, 50 (64,93%) tiveram alterações oculares; 25 (32,47%) cães tiveram uma lesão periocular; 41 (53,25%) tiveram secreção ocular purulenta e 17 (22,08%) cães tiveram ambos os sinais. A PCR foi positiva em 35 cães (45,45%) e não houve diferença significativa em cães com e sem sinais oculares (p = 0,4074). A positividade da PCR foi significativamente maior em cães sem lesão periocular (p = 0,0018). PCR em conjuntiva, uma técnica de coleta menos invasiva, rápida e indolor, é indicada para complementar o diagnóstico, principalmente em cães sem lesão periocular, independentemente da presença de secreção ocular purulenta.

Leishmania infantum Genetic Diversity and Lutzomyia longipalpis Mitochondrial Haplotypes in Brazil.

Leishmania infantum is the etiological agent of visceral leishmaniasis (VL) in the Americas with domestic dogs being its major reservoir hosts. The main VL vector is the sandfly Lutzomyia longipalpis, while other Lutzomyia species may play a role in disease transmission. Although the genetic structure of L. infantum populations has been widely evaluated, only a few studies have addressed this subject coupled to the genetic structure of the respective sandfly vectors. In this study, we analyzed the population structure of L. infantum in three major VL endemic areas in Brazil and associated it with Lutzomyia longipalpis geographic structure.

PCR sensitivity of peripheral blood of dogs co-infected with Leishmania spp. and Ehrlichia spp. in endemic area of Brazil

Abstract INTRODUCTION Peripheral blood of 400 dogs infected with Leishmania and Ehrlichia were analyzed using polymerase chain reaction (PCR), and clinical signs were characterized. METHODS PCR and parasitological tests were conducted. RESULTS PCR was positive for Leishmania in 84.75%, and parasitological tests showed that 63.25% and 31.75% were positive for Leishmania and Ehrlichia, respectively. All animals showed more than three clinical signs. PCR results were negative for Leishmania in 15.25% of the samples. CONCLUSIONS Conventional PCR of peripheral blood can be used for diagnosing canine visceral leishmaniasis in combination with other techniques, especially in uncertain cases that need species identification.

Comparison of indirect ELISA based on recombinant protein NcSRS2 and IFAT for detection of Neospora caninum antibodies in sheep.

Neospora caninum, an Apicomplexan parasite that can causes abortion, is responsible for considerable economic and reproductive losses in livestock. The purpose of the present study was to determine whether recombinant NcSRS2 is a suitable indirect ELISA antigen for determining specific immune response to N. caninum in sheep. A total of 441 serum samples were subjected to IFAT and rNcSRS2 based-ELISA, with both tests performing similarly. The sensitivity and specificity of indirect ELISA were 98.6 and 98.3%, respectively. The kappa index shows 0.98 concordance between the two tests, which is considered excellent. Seroprevalences of 30.8 and 32.0% were detected by IFAT and indirect ELISA, respectively, showing these tests did not differ significantly on this measure (p > 0.05). Serological analysis showed that HisG tag was detected by Western Blotting recognizing rNcSRS2 protein. The potential value of rNcSRS2-based ELISA as a highly specific and sensitive tool for serological diagnosis is also supported by the strong agreement found between IFAT and ELISA. The results support the potential use of recombinant protein NcSRS2 as an antigen in indirect ELISA in sheep.

[Identification of Leishmania species isolated in human cases in Mato Grosso do Sul, by means of the polymerase chain reaction]. / Identificação de espécies de Leishmania isoladas de casos humanos em Mato Grosso do Sul por meio da reação em cadeia da polimerase.

Leishmaniases are endemic zoonoses in the State of Mato Grosso do Sul. Their etiological agents in this region of Brazil are Leishmania (Leishmania) chagasi, Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. The polymerase chain reaction (PCR) is a tool with high specificity and sensitivity for identifying Leishmania species. This study examined 39 cryopreserved isolates of Leishmania that had been collected by bone marrow aspiration and/or lesion biopsy, depending on the clinical suspicion. The isolates were subjected to DNA extraction and PCR using the following primers: RV1/RV2 for identifying Leishmania (Leishmania) chagasi, a1/a2 for Leishmania (Leishmania) amazonensis and b1/b2 for Leishmania (Viannia) braziliensis.Leishmania (Leishmania) chagasi was the only species identified in the 37 cases of visceral leishmaniasis.Leishmania (Leishmania) amazonensis was identified in two isolates from patients with a diagnosis of cutaneous leishmaniasis. The results obtained confirm that it is possible to use these three pairs of primers as a tool for characterizing Leishmania isolates.

Presence of amastigotes in the central nervous system of hamsters infected with Leishmania sp. / Presença de amastigotas em sistema nervoso central de hamster infectado com Leishmania sp.

Visceral leishmaniasis (VL) is a severe chronic disease caused by Leishmania (Leishmania) infantum chagasi. Better knowledge on the effects caused by this disease can help develop adequate clinical management and treatment. Parasitological and immunohistochemical studies were performed golden hamsters Mesocricetus auratus infected with bone marrow from individuals with VL in the State of Mato Grosso do Sul, central-west Brazil. The effects of parasitism in the spleen, liver, kidneys, lungs, heart and brain of the animals were examined. Eighteen hamsters were inoculated intraperitoneally, and six healthy animals were used as negative controls. The animals were kept in the animal house and checked for clinical signs. Specimens of each organ were examined for the presence of amastigotes. Immunohistochemical technique was performed in all brain specimens and organs negative on the direct examination of parasites. Direct examination of amastigotes was positive in the spleen and liver of all infected animals; 33.3 percent showed the parasite in the kidneys and lungs, and 16.7 percent in the heart. Parasitic forms were seen in 83.3 percent (15/18) of the brain examined. Immunohistochemistry confirmed the results of the direct examination, except in two specimens of lung tissue and in the brain specimens. Other studies are needed to further clarify the effect of the parasite in the central nervous system.

A leishmaniose visceral (LV) é uma doença crônica grave, causada pelo parasito Leishmania (Leishmania) infantum chagasi. Esclarecer as alterações provocadas pela doença é fundamental para que se adotem condutas clínicas e de tratamento adequadas. Com o objetivo de analisar a infecção experimental em hamsters da linhagem golden, Mesocricetus auratus, infectados com tecido de medula óssea de pacientes com LV no Estado de Mato Grosso do Sul, foram realizados estudos parasitológicos e de imunomarcação. Foi verificada a distribuição do parasitismo no baço, fígado, rim, pulmão, coração e encéfalo desses animais. Foram utilizados 18 hamsters experimentalmente inoculados via intra-peritoneal, e seis animais sadios como controles negativos. Os animais foram mantidos em biotério de experimentação e observados, em busca de alterações clínicas. Com fragmentos de cada órgão, procedeu-se a confecção de lâminas por aposição para pesquisa de amastigotas. Nos órgãos com resultado negativo na pesquisa direta do parasito, e em todas as amostras de encéfalo, foi realizada a técnica de imunohistoquímica. A pesquisa direta de amastigotas foi positiva no baço e fígado de todos os animais infectados; 33,3 por cento apresentaram o parasito em rim e pulmão, e 16,7 por cento no coração. Quando realizada a pesquisa em encéfalo, formas parasitárias foram observadas em 83,3 por cento (15/18) dos animais. A imunomarcação confirmou os resultados da pesquisa direta, exceto em duas amostras de tecido pulmonar e nas amostras de encéfalo. Mais estudos são necessários, para esclarecer o real papel do parasito no sistema nervoso central.

Comparison of indirect ELISA based on recombinant protein NcSRS2 and IFAT for detection of Neospora caninum antibodies in sheep / Comparação entre ELISA baseado no antígeno recombinante NcSRS2 e RIFI para detecção de anticorpos de Neospora caninum em ovinos

Neospora caninum, an Apicomplexan parasite that can causes abortion, is responsible for considerable economic and reproductive losses in livestock. The purpose of the present study was to determine whether recombinant NcSRS2 is a suitable indirect ELISA antigen for determining specific immune response to N. caninum in sheep. A total of 441 serum samples were subjected to IFAT and rNcSRS2 based-ELISA, with both tests performing similarly. The sensitivity and specificity of indirect ELISA were 98.6 and 98.3 percent, respectively. The kappa index shows 0.98 concordance between the two tests, which is considered excellent. Seroprevalences of 30.8 and 32.0 percent were detected by IFAT and indirect ELISA, respectively, showing these tests did not differ significantly on this measure (p > 0.05). Serological analysis showed that HisG tag was detected by Western Blotting recognizing rNcSRS2 protein. The potential value of rNcSRS2-based ELISA as a highly specific and sensitive tool for serological diagnosis is also supported by the strong agreement found between IFAT and ELISA. The results support the potential use of recombinant protein NcSRS2 as an antigen in indirect ELISA in sheep.

Neospora caninum é um parasito Apicomplexa que pode causar abortos e é reconhecido como agente importante responsável por perdas econômicas e reprodutivas. Este estudo avaliou a proteína recombinante NcSRS2 como antígeno para ELISA indireto na determinação de resposta imune para N. caninum em ovinos. 441 amostras de soro foram analisadas por IFAT e ELISA indireto com rNcSRS2 e ambos os testes revelaram comportamento similar. A sensibilidade e especificidade de ELISA indireto foram 98,6 e 98,3 por cento, respectivamente. O índice kappa mostrou uma concordância entre os dois testes com valor de 0,98, que é considerado excelente. Prevalências de 30,8 e 32,0 por cento detectadas por IFAT e ELISA indireto, respectivamente, mostraram que os testes não diferiram significativamente nesse aspecto (P > 0.05). A análise sorológica revelou que os anticorpos específicos da cauda de histidina reconheceu por Western Blotting a proteína recombinante NcSRS2. O valor potencial do ELISA indireto baseado no antígeno rNcSRS2 como ferramenta altamente específica e sensível para diagnóstico sorológico é também reforçado pela alta concordância dos valores obtidos com IFAT e com ELISA indireto. Esses resultados respaldam o uso potencial da proteína rNcSRS2 como antígeno em ELISA indireto em ovinos.

Identificação de espécies de Leishmania isoladas de casos humanos em Mato Grosso do Sul por meio da reação em cadeia da polimerase / Identification of Leishmania species isolated in human cases in Mato Grosso do Sul, by means of the polymerase chain reaction

As leishmanioses são zoonoses endêmicas em Mato Grosso do Sul e têm por agentes etiológicos nessa região Leishmania (Leishmania) chagasi, Leishmania (Leishmania) amazonensis e Leishmania (Viannia) braziliensis. Como método para identificação de espécies de Leishmania, a reação em cadeia da polimerase é uma ferramenta com elevada especificidade e sensibilidade. Analisaram-se 39 isolados de Leishmania criopreservados, obtidos por meio de aspirado medular e/ou biópsia de lesão, conforme a suspeita clínica. Os isolados foram submetidos à extração de DNA e à reação em cadeia da polimerase com os iniciadores: RV1/RV2 para Leishmania (Leishmania) chagasi, a1/a2 para a identificação de Leishmania (Leishmania) amazonensis e b1/b2 para Leishmania (Viannia) braziliensis. Leishmania (Leishmania) chagasi foi a única espécie identificada em 37 casos de leishmaniose visceral. Leishmania (Leishmania) amazonensis foi identificada em dois isolados de pacientes com diagnóstico de leishmaniose tegumentar. Os resultados obtidos confirmam a possibilidade do uso dos três pares de iniciadores como uma ferramenta na caracterização de isolados de Leishmania.

Leishmaniases are endemic zoonoses in the State of Mato Grosso do Sul. Their etiological agents in this region of Brazil are Leishmania (Leishmania) chagasi, Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis. The polymerase chain reaction (PCR) is a tool with high specificity and sensitivity for identifying Leishmania species. This study examined 39 cryopreserved isolates of Leishmania that had been collected by bone marrow aspiration and/or lesion biopsy, depending on the clinical suspicion. The isolates were subjected to DNA extraction and PCR using the following primers: RV1/RV2 for identifying Leishmania (Leishmania) chagasi, a1/a2 for Leishmania (Leishmania) amazonensis and b1/b2 for Leishmania (Viannia) braziliensis.Leishmania (Leishmania) chagasi was the only species identified in the 37 cases of visceral leishmaniasis.Leishmania (Leishmania) amazonensis was identified in two isolates from patients with a diagnosis of cutaneous leishmaniasis. The results obtained confirm that it is possible to use these three pairs of primers as a tool for characterizing Leishmania isolates.