Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters.

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.

LEISHMANICIDAL ACTIVITY in vivo OF A MILTEFOSINE DERIVATIVE IN Mesocricetus auratus.

Visceral leishmaniasis (VL) is a chronic and systemic disease; if untreated, it can cause death in a large number of cases. The therapy is based on the use of antimonials, which have been used for over 50 years. However, cases of resistance have been reported in some countries. In this context, miltefosine (MIL) was introduced to treat antimonial unresponsive cases. Nonetheless, in recent years MIL unresponsive and relapse cases of VL have increasingly been reported. In the current study, the therapeutic potential of compound 5-(4-(3-methanesulfonatepropyl)-1H-1,2,3-triazol-1-yl)dodecyl methanesulfonate (C11), an MIL derivative, was assessed in an experimental VL hamster model. For this purpose, golden hamsters (Mesocricetus auratus) were infected with Leishmania (L.) infantum chagasi and treated daily for 10 days with C11 and MIL administered orally; in addition, Glucantime (GLU), peritoneal route, were administered at 15, 10, 50 mg/kg body weight/day, respectively. Twenty four hours after the end of treatment the animals were euthanatized; and the specimens were collected to evaluate the relative mRNA expression of cytokines IFN-γ, TNF-α, IL-17, TGF-ß, IL-4 and IL-10 in fragments of the spleen and liver; moreover, the parasitism in these organs was evaluated as well as the main histopathological alterations. The C11-treated animals showed greater expression of IL-17 and TNF-α cytokines and reduced expression of IL-10 in the spleen in comparison to the infected untreated group (UTG) (p <0.05). The C11 and GLU groups showed a significant reduction in the IgG levels in comparison to the UTG group (p <0.05). Moreover, the C11-treated animals had fewer parasites in the spleen than the UTG animals (reduction of 95.9%), as well as a greater preservation of white pulp architecture in the spleen than the UTG, GLU and MIL groups (p <0.05). For the liver, the animals from the C11 and MIL groups showed a significant increase in TNF-α relative expression in comparison to the UTG animals, which would explain the increase in the number of granulomas and the reduction in the parasitic load (p <0.05). Combined, these findings indicate that C11 is an interesting compound that should be considered for the development of new drugs against VL, mainly due to its leishmanicidal effect and immunostimulating action.

Evidence of Leishmania (Leishmania) infantum infection in dogs from Juiz de Fora, Minas Gerais State, Brazil, based on immunochromatographic dual-path platform (DPP®) and PCR assays.

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

EVIDENCE OF Leishmania (Leishmania) infantum INFECTION IN DOGS FROM JUIZ DE FORA, MINAS GERAIS STATE, BRAZIL, BASED ON IMMUNOCHROMATOGRAPHIC DUAL-PATH PLATFORM (DPP®) AND PCR ASSAYS / Evidência de infecção por Leishmania (Leishmania) infantum em cães de Juiz de Fora, Minas Gerais, Brasil, detectada pelo teste imunocromatográfico rápido DPP e PCR

In Brazil, domestic dogs are branded as the primary reservoir for zoonotic visceral leishmaniasis, due to the clear positive correlation observed between human and canine infection rates. This study aimed to carry out a serological survey of canine visceral leishmaniasis (CVL) in dogs housed at a public kennel in the municipality of Juiz de Fora, Minas Gerais State, Brazil, using the immunochromatographic TR DPP® CVL rapid test. Additionally, conventional and/or real time PCR assay was used to detect and confirm L. infantum infection in the DPP positive dogs only. Of the 400 dogs studied, most did not present clinical signs for CVL (p < 0.05), and fifteen (3.8%) were seropositive in the DPP test. There was no statistically significant difference between the DPP seropositive dogs and the clinical signs of the disease (p > 0.05). Both conventional and real time PCR tests confirmed L. infantum infection in nine (75.0%) of the twelve DPP seropositive dogs that remained alive during the follow-up period. This study is the first seroepidemiologic survey of CVL held in the city of Juiz de Fora, and the results reinforce the idea that this disease is currently in a process of expansion and urbanization in Brazil. Furthermore, this study highlights the use of the DPP test as an alternative for diagnosing CVL in large and mid-sized cities, due to its ease of implementation.

No Brasil, cães domésticos são considerados como os principais reservatórios da leishmaniose visceral zoonótica, devido à clara correlação positiva existente entre as curvas de infecção humana e canina. Este estudo objetivou a realização de um inquérito sorológico da leishmaniose visceral canina (LVC) em cães abrigados em um canil público de Juiz de Fora, Minas Gerais, Brasil, através do teste rápido imunocromatográfico TR DPP®. Adicionalmente, a PCR convencional e/ou em tempo real foi usada para detectar/confirmar a infecção por L. infantum apenas nos animais DPP positivos. Dos 400 cães estudados, a maioria não apresentou sinais clínicos para a LVC (p < 0,05) e quinze (3,8%) foram sororreativos ao DPP. Não houve diferença estatisticamente significativa entre os cães com DPP positivo e os sinais clínicos para a doença (p > 0,05). PCR convencional e em tempo real confirmaram a infecção por L. infantum em nove (75,0%) dos doze animais DPP positivos que permaneceram vivos durante o estudo. Este é o primeiro estudo soroepidemiológico sobre LVC realizado no município de Juiz de Fora, e os resultados reforçam a idéia de que esta doença está em processo de expansão e urbanização no Brasil. Além disto, este estudo destaca o uso do DPP como uma alternativa para o diagnóstico da LVC em cidades de médio e grande porte, devido à facilidade de execução.

Effectiveness of liposomal buparvaquone in an experimental hamster model of Leishmania (L.) infantum chagasi.

The objective of this study was to develop a novel liposomal formulation, containing phosphatidylserine (PS), of buparvaquone (BPQ) and to evaluate its in vivo effectiveness in Leishmania (L.) infantum chagasi-infected hamsters. The activity of BPQ was evaluated against both the promastigote forms of different Leishmania species and the intracellular amastigotes of L. (L.) infantum chagasi. Buparvaquone was entrapped in PS-liposomes (BPQ-PS-LP), and the drug was quantified by ultra-high-performance liquid chromatography. The treatment was quantified by detecting the RNA of the living amastigotes in the spleen and the liver by real-time PCR. In vitro assays with L. (L.) infantum chagasi intracellular amastigotes were performed in peritoneal macrophages for the evaluation of the 50% inhibitory concentration (IC(50)). BPQ-PS-LP at 0.33 mg/kg/day for eight consecutive days reduced the number of amastigotes by 89.4% (P<0.05) in the spleen and by 67.2% (P>0.05) in the liver, compared to 84.3% (P<0.05) and 99.7% (P<0.05), respectively, following Glucantime® treatment at 50 mg/kg/day. Free BPQ at 20 mg/kg/day failed to treat the hamsters when compared to the untreated group. BPQ was significantly (P<0.05) selective against L. (L.) infantum chagasi intracellular amastigotes, with an IC(50) value of 1.5 µM; no in vitro mammalian cytotoxicity could be detected. Other cutaneous species were also susceptible to BPQ, with IC(50) values in the range 1-4 µM. BPQ-PS-LP caused a significant reduction in the parasite burden at a 60-fold lower dose than did the free BPQ. These results show the potential of PS-liposome formulations for the successful targeted delivery of BPQ in visceral leishmaniasis.

In vitro and experimental therapeutic studies of the calcium channel blocker bepridil: detection of viable Leishmania (L.) chagasi by real-time PCR.

The need for novel and efficacious drugs against neglected parasitic diseases, such as Leishmaniasis and American Trypanosomiasis, is certainly apparent. In this work, we evaluated the in vitro potential of the calcium channel blocker bepridil against Leishmania spp. and Trypanosoma cruzi parasites and exploited an experimental assay using a hamster model with Leishmania (L.) chagasi, with a real-time PCR method for therapeutic evaluation. Bepridil was in vitro effective against promastigotes and intracellular amastigotes of L. (L.) chagasi, with 50% inhibitory concentration (IC(50)) values of 3.81 and 21.55 µM, respectively. Leishmania (L.) amazonensis, L. (L.) major and L. (V.) braziliensis promastigotes and T. cruzi trypomastigotes were also susceptible to bepridil, with in vitro selectivity toward parasites and IC(50) values in the range of 3 to 7 µM. The mammalian cytotoxicity using LLC-MK2 cells resulted in an IC(50) value of 62.67 µM. However, bepridil showed lack of activity at 12 mg/kg in the experimental hamster model infected with L. (L.) chagasi parasites. However, the real-time PCR was a promising tool for the accurate and fast quantification of RNA of living parasites in the liver and spleen of infected hamsters after treatment, eliminating time-consuming light microscopy evaluations.

Detection of Leishmania (Leishmania) infantum RNA in fleas and ticks collected from naturally infected dogs.

The occurrence of the insect vector (sand flies) with low rates of Leishmania infection, as well as autochthonous transmission in the absence of the natural vector in dogs, have been reported. These unexpected data suggest a hypothesis of other arthropods as a possible way of Leishmania transmission. The prevalence of Leishmania (Leishmania) infantum in fleas and ticks collected from dogs with canine visceral leishmaniasis (CVL), as well as parasite viability, were evaluated herein. The presence of L. (L.) infantum was assayed by PCR and ELISA in ectoparasites and biological samples from 73 dogs living in a Brazilian endemic area. As the occurrence of Leishmania DNA in ticks and fleas is expected given their blood-feeding habits, we next investigated whether parasites can remain viable inside ticks. PCR and ELISA confirmed that 83% of the dogs had CVL. Fleas and ticks (nymphs, male and female adults) were collected in 55% and 63% of the 73 dogs, respectively. Out of the 60 dogs with CVL, 80% harbored ectoparasites infected with L. (L.) infantum. The infection rates of the ectoparasites were 23% and 50% for fleas and ticks, respectively. The RNA analysis of the extract from ticks left in laboratory conditions during 7 to 10 days after removal from CVL dogs showed that parasites were alive. In addition, live parasites were also detected inside adult ticks recently molted in laboratory conditions. These findings indicate a higher infection rate of L. (L.) infantum in ticks and fleas, but they do not conclusively demonstrate whether these ticks can act as vectors of CVL, despite the fact that their rates were higher than those previously described in Lutzomyia longipalpis. The presence of viable L. (L.) infantum in ticks suggests the possible importance of dog ectoparasites in CVL dissemination.

Diagnosis of cerebral toxoplasmosis in AIDS patients in Brazil: importance of molecular and immunological methods using peripheral blood samples.

Cerebral toxoplasmosis is the most common cerebral focal lesion in AIDS and still accounts for high morbidity and mortality in Brazil. Its occurrence is more frequent in patients with low CD4(+) T-cell counts. It is directly related to the prevalence of anti-Toxoplasma gondii antibodies in the population. Therefore, it is important to evaluate sensitive, less invasive, and rapid diagnostic tests. We evaluated the value of PCR using peripheral blood samples on the diagnosis of cerebral toxoplasmosis and whether its association with immunological assays can contribute to a timely diagnosis. We prospectively analyzed blood samples from 192 AIDS patients divided into two groups. The first group was composed of samples from 64 patients with cerebral toxoplasmosis diagnosed by clinical and radiological features. The second group was composed of samples from 128 patients with other opportunistic diseases. Blood collection from patients with cerebral toxoplasmosis was done before or on the third day of anti-toxoplasma therapy. PCR for T. gondii, indirect immunofluorescence, enzyme-linked immunosorbent assay, and an avidity test for toxoplasmosis were performed on all samples. The PCR sensitivity and specificity for diagnosis of cerebral toxoplasmosis in blood were 80% and 98%, respectively. Patients with cerebral toxoplasmosis (89%) presented higher titers of anti-T. gondii IgG antibodies than patients with other diseases (57%) (P<0.001). These findings suggest the clinical value of the use of both PCR and high titers of anti-T. gondii IgG antibodies for the diagnosis of cerebral toxoplasmosis. This strategy may prevent more invasive approaches.