Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

Expression of TLR2 and TLR4 in lesions of patients with tegumentary american leishmaniasis

OBJECTIVES: The aim of this study was to describe the pattern of expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in skin biopsies of patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. METHODS: This prospective study evaluated 12 patients with ATL caused by Leishmania braziliensis confirmed by polymerase chain reaction. Immunohistochemistry was performed to determine the expression of TLR2 and TLR4. The number of NK cells, dendritic cells and macrophages in the tissue were calculated. The cytokine expression was determined using the anti-TNF-&#945;, anti-IFN-&#915;, anti-IL-1 and anti-IL-6. Double immunostaining reactions were used to determine the cell expressing TLR2 and TLR4. RESULTS: The numbers of cells expressing TLR2 and TLR4 were 145.48 �� 82.46 cell/mm�� and 3.26 �� 4.11 cell/mm�� respectively (p < 0.05). There was no correlation of TLR2 and TLR4 with the amount of cytokines and the number of NK cells, dendritic cells or macrophages. The double immunostaining revealed that TLR2 was expressed by macrophages. CONCLUSION: In human cutaneous leishmaniasis caused by Leishmania braziliensis, TLR2 is the most common TLR expressed during active disease, mainly by macrophages although without correlation with the amount of cytokines and number of cells.

AMERICAN CUTANEOUS LEISHMANIASIS WITH UNUSUAL CLINICAL PRESENTATION AND RESPONSE TO TREATMENT

The clinical manifestations and prognosis of cutaneous leishmaniasis (CL) can be influenced by the immune response of the patient and the species of the parasite. A case of atypical clinical presentation of CL, with development of non-characteristic lesions, poor response to therapy, and a long time to resolution is reported. Confirmatory laboratory tests included parasite detection, indirect immunofluorescence, Montenegro skin test, polymerase chain reaction, and parasite identification by multilocus enzyme electrophoresis. The parasite was identified as Leishmaniabraziliensis. The lesion was unresponsive to three complete courses of N-methylglucamine antimoniate intramuscular, and to treatment with pentamidine. The patient did not tolerate amphotericin B. The lesion finally receded after treatment with intravenous N-methylglucamine antimoniate. It is essential to ensure the accuracy of diagnosis and the appropriate treatment, which can include the use a second choice drug or a different route of administration.

Conceitos de uma popula����o local a respeito da leishmaniose mucocut��nea em uma ��rea end��mica

Uma investiga����o a respeito do que a popula����o local pensa sobre v��rios aspectos da leishmaniose mucocut��nea foi realizada em Tr��s Bra��os, Bahia, uma ��rea de alta preval��ncia de infec����o por Leishmania braziliensis braziliensis. Os resultados obtidos indicam que essa popula����o tem um entendimento razoavelmente bom sobre alguns aspectos epidemiol��gicos e tratamento dessa doen��a, mas demonstram seu pouco conhecimento concernente �� etiologia e preven����o. Esses dados poder��o ser valiosos, no futuro, em um planejamento do controle de transmiss��o da doen��a nesta comunidade.

American cutaneous leishmaniasis: presentation and problems of patient management

We report our experience with the diagnosis and treatment of 60 patients with American cutaneous leishmaniasis. They were infected in Panama (55), Brazil (4) or Colombia (I). Among 35 patients with a 3 week exposure in Panama, the mean maximum incubation period was 33 days (range 4-81 days). Diagnosis was delayed an average of 93 days after onset of skin lesions, due to the patient's delay in seeking medical attention (31 days), medical personnel's delay in considering the diagnosis (45 days), and the laboratory's delay in confirming the diagnosis (17 days). Forty-four patients (73%) developed ulcers typical of cutaneous leishmaniasis. Sixteen additional patients (27%) had atypical macular, papular, squamous, verrucous or acneiform skin lesions that were diagnosed only because leishmanial cultures were obtained. Of the 59 patients treated with pentavalent antimonial drugs, only 34 (58%) were cured after the first course of treatment. Lesions which were at least 2 cm in diameter, ulcerated, or caused by Leishmania braziliensis were less likely to be cured after a single course of treatment than were lesions smaller than 2 cm, nonulcerated or caused by Leishmania mexicana or Leishmania donovani.

Leishmaniose visceral canina: avalia����o da metodologia sorol��gica utilizada em inqu��ritos epidemiol��gicos

Foi realizado um estudo comparativo da rea����o de imunofluoresc��ncia indireta em eluatos de sangue de c��es infectados experimentalmente com diferentes tripanosomat��deos. Utilizaram-se como antigenopromastigotas de L. mexicana, L. braziliensis e L. chagasi. Os resultados mostraram que a sensibilidade do m��todo foi de 87,5% para o diagn��stico do calazar canino, independentemente do antigeno empregado; e que ocorre rea����o cruzada com Leishmaniose tegumentar em 75% dos casos e com doen��a de Chagas em 83,3%. Levantamento epidemiol��gico em ��rea de leishmaniose confirma que a rea����o de imunofluoresc��ncia em eluatos de sangue canino fornece rea����es cruzadas em c��es infectados com Leishmania braziliensis e L. chagasi. N��o se verificou rea����o cruzada pela RFC. Sugere-se a utiliza����o da rea����o de imunofluoresc��ncia nas campanhas de sa��de p��blica, mas �� de se chamar a aten����o para o fato de que as taxas de positividade n��o devem ser utilizadas como indicadores da preval��ncia do calazar canino.

Inqu��rito canino em foco recente de leishmaniose tegumentar no Munic��pio de Sabar��, regi��o metropolitana de Belo Horizonte

Em 1992, foi obsewada les��o cut��nea por Leishmania em um c��o do munic��pio de Sabar��, Minas Gerais, onde j�� haviam sido registrados casos humanos de leishmaniose tegumentar. O parasita foi caracterizado como pertencente ao subg��nero Viannia, ao qual pertence a Leishmania braziliensis, principal esp��cie encontrada tia regi��o sudeste do Brasil. Com o objetivo de determinar o papel do c��o no ciclo de transmiss��o da doen��a, foi realizado um inqu��rito canino na ��rea. Foram examinados 631 c��es, sendo a soropreval��ncia para leishmaniose igual a 3,2%. Foi observada a proximidade ou presen��a no mesmo domic��lio de c��es e pessoas doentes. Este aspecto fala a favor da transmiss��o domiciliar ou peridomiciliar, com o c��o infectado podendo atuar como fator de risco nesta ��rea periurbana. Entretanto, a baixa soropreval��ncia encontrada deve-se provavelmente ao pequeno papel deste animal na transmiss��o da doen��a neste foco recente da doen��a.

Surto de leishmaniose tegumentar americana em Canoa, Santo Amaro, Bahia, Brasil

Em 1993, um surto leishmaniose tegumentar americana (LTA) foi detectado no povoado rural de Canoa, munic��pio de Santo Amaro, Bahia. Um estudo observacional prospectivo delineou-se, com objetivo de determinar as taxas de freq����ncia e caracterizar clinicamente a doen��a. Foram acompanhados 555 indiv��duos, registrando-se 29 casos de LTA, 11 casos sugestivos de LTA pregressa e 529 sadios. Desses 529 sadios, 65 apresentaram rea����o de Montenegro positiva sem qualquer evid��ncia presente ou passada de doen��a. A preval��ncia de LTA no per��odo de estudo foi de 5,2% (29/555). A leishmania envolvida foi caracterizada como Leishmania braziliensis e o vetor, Lutzomyia intermedia. Foram detectados c��es e equ��deos infectados por leishmania O acometimento de crian��as menores de 10 anos, o acometimento igual entre os sexos e um componente de agrega����o familiar sugerem um padr��o de transmiss��o peri ou intradomiciliar.

An intermittent schedule is better than continuous regimen of antimonial therapy for cutaneous leishmaniasis in the municipality of Rio de Janeiro, Brazil

This study reviews a series of cutaneous leishmaniasis cases diagnosed and treated in outpatient units in the municipality of Rio de Janeiro, where the intermittent schedule of antimonial therapy was replaced by the continuous regimen. Both schedules were based on daily intramuscular injections of pentavalent antimonial. Forty-nine subjects received the intermittent regimen, consisting of three ten-day series alternated with ten-day rest intervals whereas seventy-one patients received the continuous regimen during 20 consecutive days. The study groups had similar composition regarding age, sex and clinical condition. The cure rate was significantly higher in the group receiving the intermittent schedule than in the group receiving continuous therapy (89.8% vs 63.3%). Moreover, loss to follow-up was significantly more frequent in the group receiving continuous therapy (19.7% vs 4.1% in the intermittent therapy). Under field conditions, the intermittent regimen provided higher effectiveness and adherence than the continuous schedule.

Efeito de uma formula����o hidrof��lica de paromomicina t��pica na leishmaniose cut��nea em pacientes com contra-indica����es de tratamento com antimonial pentavalente

Descrevem-se o efeito terap��utico e os eventos adversos associados com o uso t��pico de paromomicina 10% em gel na leishmaniose cut��nea. Quinze pacientes com leishmaniose cut��nea cumpriram os crit��rios de inclus��o descritos a seguir: contra-indica����o para o uso de antimoniato de meglumina, intradermorrea����o de Montenegro positiva e at�� quatro les��es ulceradas. A f��rmula foi prescrita duas vezes ao dia por 20 dias. Quatorze pacientes estiveram dispon��veis para a avalia����o do desfecho terap��utico e a propor����o de cura foi de 21,4% (3/14), 50% melhoraram at�� a epiteliza����o completa e a propor����o de falha foi de 28,6%. Nove pacientes que n��o apresentaram cura inicialmente foram re-tratados. Oito receberam uma nova s��rie de paromomicina t��pica e um foi tratado com antimoniato de meglumina. Dois pacientes n��o receberam novo tratamento e tiveram melhora lenta e cont��nua. Cinco de oito pacientes retratados com paromomicina t��pica alcan��aram a cura cl��nica, e tr��s apresentaram falha, incluindo um paciente que tinha apresentado melhora com o primeiro tratamento. Os eventos adversos foram leves e locais em 53,3% dos pacientes e nunca levaram �� suspens��o do tratamento.

Immunophenotyping of circulating T cells in a mucosal leishmaniasis patient coinfected with HIV

HIV coinfection modifies the clinical course of leishmaniasis by promoting a Th2 pattern of cytokine production. However, little information is available regarding the lymphocytic response in untreated coinfected patients. This work presents the immunophenotyping of Leishmania-stimulated T cells from a treatment-na��ve HIV+ patient with ML. Leishmania braziliensis antigens induced CD69 expression on CD3+CD4+ and CD3+CD8+ cells. It also increased IL-4 intracellular staining on CD3+CD4+GATA3- population and decreased the percentage of CD3+CD4+IL-17+ cells. This suggests that modulations in the IL-4R/STAT6 pathway and the Th17 population may serve as parasitic evasion mechanisms in HIV/ML. Further studies are required to confirm these results.

Cutaneous leishmaniasis in northeastern Brazil: a critical appraisal of studies conducted in State of Pernambuco

American cutaneous leishmaniasis (ACL) is a complex disease with clinical and epidemiological features that may vary from region to region. In fact, at least seven different Leishmania species, including Leishmania (Viannia) braziliensis, Leishmania (Viannia) guyanensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) naiffi, Leishmania (Viannia) shawi, Leishmania (Viannia) lindenbergi, and Leishmania (Leishmania) amazonensis, have been implicated in the etiology of ACL in Brazil, and numerous phlebotomine sandfly species of the genus Lutzomyia have been regarded as putative or proven vectors. Because ACL is a focal disease, understanding the disease dynamics at the local level is essential for the implementation of more effective control measures. The present paper is a narrative review about the ACL epidemiology in Pernambuco, northeastern Brazil. Furthermore, the need for more effective diagnosis, treatment, control and prevention strategies for the affected populations is highlighted. This paper will provide researchers with a critical appraisal of ACL in Pernambuco. Hopefully, it will also be helpful for public health authorities to improve current control strategies against ACL at the state and country levels.

CD8+ T cells in situ in different clinical forms of human cutaneous leishmaniasis

Introduction Leishmania braziliensis infection induces a large spectrum of lesions that clinically manifest as nodules or papules that progress to ulcers. Although it is already known that T helper cells predominate in the lesions, cytotoxic T cells have also been reported to be present, and their role in leishmaniasis immunopathogenesis is not well known. This study investigated the amounts of CD8+ and granzyme B+ cells in different clinical forms of human cutaneous leishmaniasis (CL). Methods Forty tissue fragments from early (E-CL) and late CL (L-CL) lesions and from disseminated leishmaniasis (DL) - papules and ulcers - were characterized. The inflamed area per fragment was calculated, and the CD8 and granzyme B expression levels in the infiltrates were quantified by counting positive cells in 15 fields. The localization of CD8 and granzyme B was graded subjectively. Results Inflammation was higher in L-CL and DL ulcers. CD8 expression was increased in late ulcerated lesions compared to recent lesions. The increase in CD8+ cells also correlated with the duration of the lesion. Papules had a higher frequency of granzyme B+ cells than E-CL lesions, although the frequency was similar to those for late and DL ulcers. CD8+ cells were mostly found in the papillary dermis. Conclusions CD8+ T and granzyme B+ cells are present in the inflammatory infiltrates of CL and DL and may participate in the immunopathogenesis of Leishmania infection.

Poor response to azithromycin in cutaneous leishmaniasis leading to a premature interruption of a multicentric phase III clinical trial in Brazil

Introduction Parenteral antimony-based compounds are still the standard of care for cutaneous leishmaniasis (CL) treatment in many countries, despite their high toxicity. Previous studies showed that oral azithromycin could be an option for CL treatment. The aim of this study was to evaluate efficacy and safety of oral azithromycin (AZ) for CL treatment compared with injectable meglumine antimoniate (MA). Methods This was a randomized, open-label, 2-arm, non-inferiority clinical trial. Treatment-na&#239;ve patients with localized CL were treated with MA (15mg/kg/day up to 1,215mg) or AZ (500mg/day) during 20 consecutive days. The primary efficacy end point was a CL cure 90 days after treatment completion. The analysis was performed with intention-to-treat (ITT) and per protocol (PP) analyses. After an anticipated interim analysis, the study was interrupted due to the high failure rate in the azithromycin group. Results Twenty-four volunteers were included in each group. The MA group had a higher cure rate than the AZ group with the ITT and PP analyses, which were 54.2% versus 20.8% [relative risk (RR) 1.97; 95% confidence intervals (95%CI) 1.13-3.42] and 72.2% versus 23.8% (RR 3.03; 95%CI 1.34-6.87), respectively. No unexpected adverse events were observed. Conclusions Azithromycin is ineffective for CL treatment and does not seem to have a role in the therapeutic arsenal for CL.

The principal reservoir host of cutaneous leishmaniasis in the urban areas of Manaus, Central Amazon of Brazil

Twenty one opossums (Didelphis marsupialis) from disturbed primary forest in the vicinity of Manaus, and seven from an isolated island of secondary vegetation within the city of Manaus, were examined for the presence of Leishmania. Of the opossums from the primary forests, 13 (61.9%) were found to be positive for Leishmania braziliensis guyanenesis. One additional opossum was found to be positive for Le. mexicana amazonensis. A simple and economical trapping technique for opossums is presented here, and a control method in special cases is suggested.