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In Brazil, Leishmania amazonensis is the etiological agent of cutaneous and diffuse cutaneous leishmaniasis. The state of Maranhão in the Northeast of Brazil is prevalent for these clinical forms of the disease and also has high rates of HIV infection. Here, we characterized the drug susceptibility of a L . amazonensis clinical isolate from a 46-year-old man with diffuse cutaneous leishmaniasis coinfected with HIV from this endemic area. This patient underwent several therapeutic regimens with meglumine antimoniate, liposomal amphotericin B, and pentamidine, without success. In vitro susceptibility assays against promastigotes and intracellular amastigotes demonstrated that this isolate had low susceptibility to amphotericin B, when compared with the reference strain of this species that is considered susceptible to antileishmanial drugs. Additionally, we investigated whether the low in vitro susceptibility would affect the in vivo response to amphotericin B treatment. The drug was effective in reducing the lesion size and parasite burden in mice infected with the reference strain, whereas those infected with the clinical isolate and a resistant line (generated experimentally by stepwise selection) were refractory to amphotericin B treatment. To evaluate whether the isolate was intrinsically resistant to amphotericin B in animals, infected mice were treated with other drugs that had not been used in the treatment of the patient (miltefosine, paromomycin, and a combination of both). Our findings demonstrated that all drug schemes were able to reduce lesion size and parasite burden in animals infected with the clinical isolate, confirming the amphotericin B-resistance phenotype. These findings indicate that the treatment failure observed in the patient may be associated with amphotericin B resistance, and demonstrate the potential emergence of amphotericin B-resistant L . amazonensis isolates in an area of Brazil endemic for cutaneous leishmaniasis.

In Brazil, cutaneous leishmaniasis is endemic and may be caused by Leishmania amazonensis. This species is the second most prevalent species in that country, and it is responsible for localized cutaneous and diffuse cutaneous leishmaniasis. Pentavalent antimony is still the first-line drug for cutaneous leishmaniasis treatment in Brazil. In this study, we investigated the in vitro susceptibility to antimony of a panel of L. amazonensis strains and clinical isolates responsible for cutaneous and diffuse cutaneous leishmaniasis. There was a significant variation in susceptibility to antimony not only within these strains and isolates evaluated at either promastigote or intracellular amastigote stages, but also between the two parasite stages for some of these strains and isolates. Additionally, we investigated whether this in vitro susceptibility variation to antimony would affect the in vivo response to treatment, using an experimental BALB/c mouse model of cutaneous leishmaniasis infected with three strains differing in susceptibility. Despite antimony could mildly reduce the lesion size in mice infected with one of these strains, no significant reduction in the parasite burden was found in treated animals, and they were completely refractory to drug treatment. These findings indicate that antimony treatment, even at high dosages via the intraperitoneal route, was not effective against L. amazonensis infection in this animal model. Finally, this study provides a preclinical dataset of the activity of antimony against a panel of strains and isolates of a species responsible for localized cutaneous and diffuse cutaneous leishmaniasis in Brazil.

BACKGROUND: Several species of protozoa cause acute or chronic gastroenteritis in humans, worldwide. The burden of disease is particularly high among children living in developing areas of the world, where transmission is favored by lower hygienic standards and scarce availability of safe water. However, asymptomatic infection and polyparasitism are also commonly observed in poor settings. Here, we investigated the prevalence of intestinal protozoa in two small fishing villages, Porto Said (PS) and Santa Maria da Serra (SM), situated along the river Tietê in the State of São Paolo, Brazil. The villages lack basic public infrastructure and services, such as roads, public water supply, electricity and public health services. METHODS: Multiple fecal samples were collected from 88 individuals in PS and from 38 individuals in SM, who were asymptomatic at the time of sampling and had no recent history of diarrheal disease. To gain insights into potential transmission routes, 49 dog fecal samples (38 from PS and 11 from SM) and 28 river water samples were also collected. All samples were tested by microscopy and PCR was used to genotype Giardia duodenalis, Blastocystis sp., Dientamoeba fragilis and Cryptosporidium spp. RESULTS: By molecular methods, the most common human parasite was Blastocystis sp. (prevalence, 45% in PS and 71% in SM), followed by D. fragilis (13.6% in PS, and 18.4% in SM) and G. duodenalis (18.2% in PS and 7.9% in SM); Cryptosporidium spp. were not detected. Sequence analysis revealed large genetic variation among Blastocystis samples, with subtypes (STs) 1 and 3 being predominant, and with the notable absence of ST4. Among G. duodenalis samples, assemblages A and B were detected in humans, whereas assemblages A, C and D were found in dogs. Finally, all D. fragilis samples from humans were genotype 1. A single dog was found infected with Cryptosporidium canis. River water samples were negative for the investigated parasites. CONCLUSIONS: This study showed a high carriage of intestinal parasites in asymptomatic individuals from two poor Brazilian villages, and highlighted a large genetic variability of Blastocystis spp. and G. duodenalis.

SUMMARY Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation. RESUMO A toxoplasmose cerebral pode ser altamente debilitante e ocasionalmente fatal em pessoas com deficiências do sistema imune. Nesse estudo, nós avaliamos a resposta de anticorpos das subclasses da IgG para o Toxoplasma gondii em 19 amostras de líquido cefalorraquidiano (LCR) de pacientes com toxoplasmose cerebral que apresentavam uma reação IgG anti-T. gondii positiva com ELISA padronizada com uma preparação antigênica de cistos. Não foram encontradas diferenças significativas entre as taxas de positividade e as concentrações de anticorpos (média aritmética das absorbâncias das reações ELISA, MEA) para IgG1 e IgG2, mas as taxas de positividade e valores MEA para estas duas subclasses de IgG foram significativamente superiores aos da IgG3 e IgG4. A resposta marcante de anticorpos IgG2 em LCR de pacientes com toxoplasmose cerebral merece investigação adicional.

Giardia is a pathogenic protozoan that has caused giardiasis outbreaks worldwide, and this water-borne disease may occur due to faults in water supply and treatment systems. To support surveillance and control programs, the monitoring of this parasite is essential in water samples. Although Giardia cyst detection is usually based on standard light microscopy, the resulting low contrasted cysts together with a wide array of particles of comparable size to the target protozoan demands a high level of observer skill and experience, as well as a long time to process the individual samples. The visualization of this protozoan can be significantly improved by using staining procedures, e.g., Lugol’s iodine in brightfield or fluorescence-based methods such as fluorescence-isothiocyanate (FITC) and 4′,6-diamidino-2-phenylindole (DAPI). However, the significant costs make fluorescence microscopy difficult to be performed in less-developed countries. Accordingly, the present study evaluated the effectiveness of inexpensive darkfield (DF) setups to acquire higher contrasted images of unstained cyst samples as a direct and rapid method for monitoring protozoa. Two low-cost custom-made DF illumination modes, transmitted and reflected, were evaluated on a standard light microscope. Examination of purified Giardia cyst suspensions with both DF setups revealed a direct correlation between morphological appearance and uptake of DAPI. Images captured under transmitted illumination showed higher contrast and sharpness when compared to the reflected images. DF microscopy might provide a simple, direct, and inexpensive method for observing Giardia cysts, which shows basic aspects of their intracellular structure, although the applicability of the method to raw water concentrates remains to be demonstrated.

The consumption of oysters and cockles, which are usually eaten raw or lightly-cooked, can cause outbreaks of human diseases, especially if these shellfish are harvested from polluted areas. In Brazil data about the occurrence of pathogens, like hepatitis A virus, in shellfish have been reported but research on natural contamination for pathogenic protozoa is still non-existent. Cryptosporidium oocyst contamination of oysters (Crassostrea rhizophorae) and cockles (Tivela mactroides) was evaluated during two different periods in a coastal area from São Paulo, Brazil. From June to November 2005, and from July to December 2006, 180 mollusks were harvested for tissue examination. The gills and gastrointestinal tract (n = 36 pools) were carefully extracted from the animals and homogenized in a tissue homogenizer by adding surfactant Tween 80 (0.1%). Immunofluorescence assays were performed and Cryptosporidium oocysts were detected in 50.0% of gill pools of cockles and 10.0% of gill pools of oysters. In order to evaluate seawater quality in shellfish growing areas, total levels of thermotolerant coliforms, Escherichia coli and enterococci were determined. This is the first time that Cryptosporidium oocysts were found in shellfish from the coastal region of Brazil, and to the best of our knowledge it is also the first report in Latin America and the case might be of public health importance, reflecting the extension of the contamination on seafood, requiring a need for quality control standards.

A presença de espécies dos protozoários Cryptosporidium e de Giardia em água, principalmente as destinadas ao consumo humano, assumiu nos últimos 22 anos grande importância em Saúde Pública devido à veiculação hídrica de suas formas infectantes. Esses protozoários causam gastroenterites no ser humano e em diversas espécies animais. A transmissão desses protozoários ocorre pela via fecal-oral mediante a ingestão de água ou de alimentos contaminados, ou pelo contato direto (pessoa-a-pessoa ou animal-pessoa; transmissão zoonótica). A persistência de oocistos e cistos no ambiente e a grande resistência aos processos de desinfecção contribuem com a veiculação hídrica desses protozoários. Numerosos surtos de gastroenterite aconteceram nos últimos 25 anos devido à ingestão de água de consumo e involuntariamente águas recreacionais contaminadas. No Brasil é crescente a degradação de águas superficiais e subterrâneas devido à deficiência de infra-estrutura no sistema de esgotamento sanitário. Análises realizadas pela Sabesp sobre a qualidade microbiológica da água proveniente das fontes naturais da cidade de Campos do Jordão (nordeste do estado de São Paulo) revelam que 13, de um total de 22 fontes, apresentaram padrão de qualidade impróprio para o consumo humano. Considerando que a população local utiliza amplamente essa água para o consumo (ingestão e preparo de alimentos) e que a existência de situações que implicam em risco potencial de contaminação das mesmas são freqüentes no município, o objetivo deste trabalho foi investigar a ocorrrência de Cryptosporidium sp. e de Giardia sp. em amostras procedentes das principais fontes, durante 12 meses. Para tanto, utilizou-se a técnica de filtração em membranas para concentração e visualização dos protozoários aplicando anticorpos monoclonais fluorescentes. No período compreendido entre junho de 2003 e maio de 2004, foi possível detectar a presença de, pelo menos, um dos protozoários patogênicos em 25,0% (3/12) das fontes estudadas, com concentrações que variaram de 0,2 a 0,3 oocistos de Cryptosporidium sp. e 0,07 a 0,1 cistos de Giardia sp. por litro. O inquérito parasitógico realizado em duas comunidades rurais pertencentes ao vii município revelou que 49,2% (91/185) das pessoas apresentavam parasitoses intestinais. A faixa etária dos participantes variou de 0 a 72 anos. As prevalências dos protozoários patogênicos foram: Cryptosporidium sp. (8,1%), Giardia duodenalis (5,9%), Blastocystis hominis (4,9%) e Entamoeba histolytica/dispar (2,7%); e dos não patogênicos: Entamoeba coli (19,5%), Iodamoeba bütschlii (4,3%) e Endolimax nana (0,5%). E para os helmintos intestinais foram: Ascaris lumbricoides (14,9%); Trichuris. trichiura (9,7%); ancilostomideo (1,1%), Taenia sp. (1,1%), Enterobius vermicularis (1,1%) e larva de nematódeo (1,1%). As diferenças estatísticas não foram significantes para as parasitoses observadas quanto ao sexo (p=0,453) e as diferentes faixas etárias (p=0,213). As condições ambientais (relevo, disponibilidade hídrica, ocupação desordenada, áreas de pastagem) e focos de contaminação (esgoto sanitário; fezes de animais) existentes próximo às nascentes foram fatores que contribuíram com a contaminação das águas. A ausência de água tratada, de sistema de escoamento sanitário e as condições sócio-econômicas das comunidades rurais incluídas no inquérito parasitológico podem ter contribuído para a elevada prevalência das parasitoses intestinais.

Introdução: O basquetebol requer dos praticantes boa capacidade de impulsão e agilidade, e a suplementação com creatina (Cr) está associada a melhoras no desempenho muscular. Objetivo: Investigar o efeito da suplementação de Cr nos praticantes de basquetebol. Métodos: Participaram 16 homens, com média de idade de 21±2,34 anos, organizados em dois grupos randomicamente, suplementados conforme modelo duplo-cego, recebendo o grupo placebo (GP) 5 gramas de talco nutricional; e o grupo experimento (GE), 5 gramas de Cr, durante 30 dias. Realizaram-se os mesmos testes de impulsão vertical e agilidade (quatro cantos), antes e após suplementação. Resultado: Houve melhora significativa na impulsão vertical, comparando-se os períodos pré e pós-suplementação de Cr (p=0,018). Nos testes de agilidade, não foram encontradas diferenças estatísticas entre períodos pré e pós-suplementação. Conclusão: A suplementação de Cr proporcionou maior rendimento no salto vertical, melhorando o desempenho motor da força explosiva.