American cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis resistant to meglumine antimoniate, but with good response to pentamidine: a case report

This is a case report of a Brazilian soldier with cutaneous leishmaniasis. The lesion relapsed following two systemic treatments with meglumine antimoniate. The patient was treated with amphotericin B, which was interrupted due to poor tolerance. Following isolation of Leishmania sp., six intralesional infiltrations of meglumine antimoniate resulted in no response. Leishmania sp promastigotes were again isolated. The patient was submitted to intramuscular 4mg/kg pentamidine. Parasites from the first and second biopsies were identified as Leishmania (Viannia) braziliensis; those isolated from the first biopsy were more sensitive to meglumine antimoniate in vitro than those isolated from the second biopsy. No relapse was observed.

Phlebotomine sandfly fauna and natural Leishmania infection rates in a rural area of Cerrado (tropical savannah) in Nova Mutum, State of Mato Grosso in Brazil

Introduction American cutaneous leishmaniasis (ACL) has been reported in every municipality of the State of Mato Grosso, Brazil, but the transmission epidemiology remains poorly understood. Our study was developed in a rural area of the Nova Mutum municipality where four autochthonous cases of ACL were reported in 2009. Our aims were to describe the local phlebotomine sandfly fauna and to investigate the infection rates and infecting Leishmania species in the captured sandflies. Methods Entomological captures were performed bimonthly at 10 fixed sites close to the edge of a forested area between June 2011 and April 2012. Results A total of 3,743 phlebotomine sandflies belonging to 31 distinct species were captured. Approximately 75% of the specimens were females. The most abundant species (45.4%) was Lutzomyia antunesi, which was consistently captured at every site. Species that are epidemiologically important for ACL, such as L. flaviscutellata, L. whitmani and L. umbratilis, were also captured. L. antunesi and L. ubiquitalis were naturally infected by Leishmania braziliensis or Le. guyanensis, with minimum infection rates of 0.88% and 6.67%, respectively. Surprisingly, L. antunesi was infected by Le. infantum (synonym chagasi). Conclusions The natural infection of L. antunesi and L. ubiquitalis by Leishmania sp. suggests that these species might play a role in the zoonotic cycle of ACL in Nova Mutum. The presence of Le. infantum in L. antunesi suggests that there may be a risk of an outbreak of visceral leishmaniasis (VL) in Nova Mutum.

Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Neospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs

Introduction: The aim of this study was to evaluate the serological cross-reactivity between Leishmania sp. and other canine pathogens. Methods: Positive serum samples for Ehrlichia canis, Babesia canis, Toxoplasma gondii, Neospora caninum and Trypanosoma cruzi were tested using three serological methods enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and Kalazar Detect™, for canine visceral leishmaniasis. Results: Of the 57 dog samples tested, 24 (42.1%) tested positive using one of the three serological methods: 10/57 (17.5%) for ELISA, 11/57 (19.3%) for IFAT and 3/57 (5.3%) for Kalazar Detect™. Conclusions: Our results demonstrated that the presence of other infectious agents may lead to cross-reactivity on leishmaniasis serological tests.

Molecular detection of Trypanosoma sp. and Blastocrithidia sp. (Trypanosomatidae) in phlebotomine sand flies (Psychodidae) in the Federal District of Brazil

Abstract: INTRODUCTION : This study describes the occurrence of trypanosomatids in phlebotomines in Brasília, Brazil. METHODS : Two hundred and ten females of 13 sand fly species were analyzed by polymerase chain reaction (PCR) using different molecular markers (D7 24Sα rRNA, kDNA, and ITS1) and sequencing. RESULTS : PCR revealed trypanosomatid-positive samples from Nyssomyia whitmani and Evandromyia evandroi, which were negative by kDNA and ITS1 Leishmania-specific PCRs. DNA sequence analysis of D7 24Sα rRNA amplicons indicated the occurrence of Blastocrithidia sp. and Trypanosoma sp. in Nyssomyia whitmani and Evandromyia evandroi, respectively. CONCLUSIONS : Two trypanosomatid species other than Leishmania sp. were found to circulate in sand flies in Central Brazil.

Intradermorreação de Montenegro em cães (Mammalia: Canidae) experimentalmente inoculados por Leishmaniaguyanensis e Leishmania braziliensis (Kinetoplastida: Trypanosomatidae), principais agentes causadores de Leishmaniose Tegumentar na Amazônia

O teste de intradermorreação de Montenegro é utilizado para detectar infecção por Leishmania em humanos. A técnica se baseia numa reação de hipersensibilidade tardia. Os antígenos de Montenegro utilizados no experimento são soluções de antígenos homólogos brutos de Leishmania (Viannia) braziliensis e L.(V)guyanensis. Este experimento demonstrou que os animais inoculados com as espécies de Leishmania inoculadas desenvolveram enduração no local do teste mais acentuada que os animais controle. Os resultados sugerem que o teste cutâneo pode vir a ser indicado como método auxiliar de diagnóstico em cães infectados por Leishmania sp.

Leishmaniasis dissemineted by Leishmania braziliensis in a mare (Equus cabalus) immunotherapy and chemotherapy assays

Cutaneous disseminated lesions caused by Leishmania sp. were found in a pregnant mare (Equus cabalus) from a rural city in the State of rio de Janeiro, Brazil. Before delivering, treatment was undertaken by immunotherapy followed by chemotherapy. Histopatology and serology were performed during treatment, as well as the biochemical characterization of the parasite (L. braziliensis) that was isolated from one of the lesions.

Cotton Rats (Sigmodon hispidus) and Black Rats (Rattus rattus) as Possible Reservoirs of Leishmania spp. in Lara State, Venezuela

A total of 519 wild animals belonging to eleven species were collected during a two year study in a cutaneous leishmaniasis endemic area in Venezuela (La Matica, Lara State). The animals were captured in home-made Tomahawk-like traps baited with maize, bananas or other available local fruits, and parasites were isolated from 27 specimens. Two different species were found naturally infected with flagellates, i.e., cotton rats (Sigmodon hispidus) and black rats (Rattus rattus). Characterization of the parasites using PCR, kDNA restriction pattern and hybridization with species-specific probes revealed the presence of Leishmania (L.) mexicana in three of the black rats and Leishmania (V.) braziliensis in two others. The latter species was also identified in the single positive specimen of S. hispidus. The results suggested both species of animals as possible reservoirs of Leishmania sp.

Serologic evidence of Leishmania infection in free-ranging wild and domestic canids around a Brazilian National Park

Transmission of disease between wildlife, domestic animals, and humans is of great concern to conservation issues and public health. Here we report on the prevalence of anti-Leishmania sp. antibodies in 21 wild canids (7 Chrysocyon brachyurus, 12 Cerdocyon thous, and 2 Lycalopex vetulus) and 74 free domestic dogs (Canis familiaris) sampled around the Serra do Cipó National Park. In dogs, the apparent prevalence was 8.1% and in wild canids it was 19% (2 crab-eating foxes, C. thous, and 2 maned wolves, C. brachyurus). Management of the domestic dog population with evaluation of incidence changes in humans and wildlife, and enlightenment on the role of wild reservoirs are essential issues for future action and research.

Leishmania infection in humans, dogs and sandflies in a visceral leishmaniasis endemic area in Maranhão, Brazil

Leishmania infection in humans, dogs and sandflies was examined in the endemic visceral leishmaniasis (VL) municipality of Raposa, state of Maranhão, Brazil. In this study, we examined Leishmania chagasi infection in the blood serum of both humans and Canis familiaris and the natural Leishmania sp. infection rate in the sandfly vector, Lutzomyia longipalpis. Enzyme-linked immunosorbent assay, indirect immunofluorescence reaction and polymerase chain reaction were performed to detect Leishmania infections in humans, dogs and sandflies, respectively. Overall, 186 out of 986 studied human beings were infected with L. chagasi parasites, representing an infection prevalence of 18.9%. An even higher infection rate was detected in dogs, where 66 (47.8%) out of 138 were infected. Among all Lu. longipalpis captured (n = 1,881), only 26.7% were females. The Leishmania infection frequency for the vector Lu. longipalpis was 1.56%. Remarkably, all infected sandflies were found in the peridomiciliary area. Furthermore, a high incidence of asymptomatic forms of VL in the human and canine populations was observed. The results of this study suggest autochthonous transmission of L. chagasi in this endemic area for visceral leishmaniasis because infection by Leishmania sp. was identified in all important elements of the transmission chain.

Diagnóstico molecular e frequência de anticorpos anti-Leishmania infantum chagasi em cães do município de Belém, Pará

A leishmaniose visceral é uma enfermidade cujo agente etiológico no Brasil é o protozoário Leishmania infantum chagasi. Os cães são considerados reservatórios urbanos da doença, sendo indicadores da ocorrência de casos humanos. O presente trabalho teve como objetivo diagnosticar a infecção por L. infantum chagasi em cães domiciliados e errantes do município de Belém, estado do Pará, através da reação em cadeia da polimerase (PCR) e da reação de imunofluorescência indireta (RIFI), empregando dois antígenos distintos. Amostras de sangue venoso de cães adultos, sem distinção de sexo ou raça, de diferentes bairros e épocas do ano da cidade de Belém-PA, foram colhidas em tubos sem e com anticoagulante para obtenção do soro e do DNA, respectivamente. Esses animais foram divididos em dois grupos: cães errantes capturados pelo Centro de Controle de Zoonoses (Grupo A) e cães domiciliados (Grupo B). Os soros foram analisados através do teste de RIFI para pesquisa de IgG utilizando-se dois antígenos distintos: 1) antígeno do kit Bio-Manguinhos/FIOCRUZ (Ag-PRO) contendo formas promastigotas de Leishmania sp. (complexo major-like); 2) Antígeno do Instituto Evandro Chagas (Ag-AMA) constituído por formas amastigotas de L. infantum chagasi. A avaliação dos dois antígenos foi realizada com as amostras reagentes a partir da titulação 1:80. Já a PCR foi realizada a partir do DNA extraído do sangue total dos animais e amplificado utilizando-se os iniciadores RV1e RV2. Das 335 amostras analisadas, 10,4% (35/335) foram reagentes na RIFI (Ag-PRO) e 0,9% (3/335) reagiram com o Ag-AMA. A distribuição das amostras positivas se deu da seguinte forma: Grupo A 14,8% (25/169) com Ag-PRO e 1,2% (2/169) com Ag-AMA; Grupo B 6% (10/166) com Ag-PRO e 0,6% (1/166) com Ag-AMA; sendo que todas as amostras positivas pelo teste de RIFI com o Ag-AMA também reagiram com o Ag-PRO e em nenhuma das amostras foi detectado o DNA de L. infantum chagasi. Os achados do presente estudo indicam que Belém ainda pode ser considerada área não endêmica para leishmaniose visceral canina e que a natureza do antígeno influencia no resultado da RIFI para a pesquisa de anticorpos anti-L. infantum chagasi em cães, sendo que a RIFI que utiliza formas promastigotas de Leishmania major-like como antígeno deve ser utilizada com cautela como método diagnóstico confirmatório em estudos epidemiológicos em áreas não endêmicas para LVC.

Anti-Leishmania titers and positive skin tests in patients cured of kala-azar

Visceral leishmaniasis (VL), also known as kala-azar, is an important public health problem. If not treated, virtually all clinically symptomatic patients die within months. The diagnosis is based on the Montenegro skin test (MST) and anti-Leishmania titers. Nevertheless, the time required for cured individuals living in a leishmaniasis-endemic area to present a positive skin test and negative anti-Leishmania serology is known. To determine the cellular and humoral immune response profile in relation to different times post-VL cure, a cross-sectional study was conducted on subjects from a kala-azar endemic area in Paço do Lumiar, MA, Brazil, on the basis of 1995-2005 notifications reported by the National Health Foundation/Regional Coordination of Maranhão. We visited cured individuals with a history of VL within the last 10 years. Seventy-four subjects (30 females) ranging in age from 1 to 44 years were included, all of them symptom free at the time of the study. A cellular immune response was observed in 73 (98.6%) subjects, whereas no significant antibody titers were detected by indirect immunofluorescence (IIF) in the sera of 69 (93.2%) cases. Ten years post-cure, 39 (52%) subjects had a positive MST and negative IIF reaction, while in one subject the skin and anti-Leishmania serology tests were negative. Two other subjects were positive in both tests 1 year after cure. These data suggest that a cellular immune response may still be present in subjects cured of VL regardless of post-cure time, and that the parasite persists in the host after clinical cure of the disease. This would explain the persistence of significant Leishmania sp antibody titers in some subjects after treatment.

Vigilância de leishmaniose visceral americana em cães de área não endêmica, São Paulo

Realizou-se inquérito sorológico em cães domiciliados e errantes do Município de São José do Rio Preto, SP, para identificar animais infectados e detectar a possibilidade de transmissão da leishmaniose visceral americana. De novembro de 1998 a junho de 2000, 2.104 amostras de soros foram testadas por meio da reação de imunofluorescência indireta, empregando-se como antígeno formas promastigotas de Leishmania (L.) chagasi. Observaram-se 2.092 amostras não reagentes e 12 reagentes. Dos cães com sorologia positiva foi possível realizar raspados de lesão em três animais. O material fixado em lâminas foi corado por Giemsa e, em apenas um, foram encontradas formas amastigotas características de Leishmania sp. Este resultado indica a necessidade de manutenção da vigilância sorológica canina e entomológica no município de São José do Rio Preto, a fim de detectar, precocemente, qualquer alteração na epidemiologia local.

Azithromycin in the treatment of mucosal leishmaniasis

This report describes three elderly patients with mucosal form of American tegumentary leishmaniasis associated with chronic cardiopathy. Due to the known toxicity of classical drugs with activity against Leishmania sp., the patients received three oral courses of azithromycin therapy in single 500 mg daily dose during ten days, every other month. All lesions healed after the third series. One of the patients relapsed and a new series of azithromycin was prescribed. Azithromycin may be an alternative drug for the treatment of leishmaniasis in special situations due to its optimal mucosal and intraphagocyte concentration, single daily posology, high tolerance and oral administration. The mechanism of this drug on Leishmania sp. is unknown at present.

Low efficacy of azithromycin to treat cutaneous leishmaniasis in Manaus, AM, Brazil

An open trial to evaluate the azithromycin efficacy in cutaneous leishmaniasis patients was carried out in Manaus (AM), where Leishmania (Viannia) guyanensis is the main etiologic agent. Forty-one patients with skin lesions of less than 12 weeks duration, without specific treatment for the last three months and a positive imprint to Leishmania sp. were included. From these, 31 (75.6%) were male with median age of 30.2. All of them received a daily-single oral dose of 500 mg of azithromycin for ten days. At 25th day, 16 (39%) presented therapeutic failure and received intramuscular pentavalent antimonial, four were considered lost, 21, that had improved or were inaltered received another ten-day series of azithromycin and were monthly followed, but nine (21.9%) of them presented a poor clinical response and switched to intramuscular pentavalent antimonial on day 55. Of the 12 remaining cases evaluated on day 55, despite of clinical improvement, three asked for antimony therapy and 9 (21.9%) continued the follow-up but, only three were cured on 55th, 85th and 115th days, and six did not come back for final evaluation. The intention-treatment overall response rate was 22% and whole cure was seen in three (7.3%) of cases. Thus, azithromycin showed a low efficacy to treat cutaneous leishmaniasis in Manaus.

IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.