Helena Keiko Toma

Investigation of Chagas disease in four periurban areas in northeastern Brazil: epidemiologic survey in man, vectors, non-human hosts and reservoirs

Chagas disease was investigated in four periurban areas of Ceará state, northeastern Brazil, through serological, parasitological and molecular methods in humans, reservoirs and vectors. A cross-sectional survey revealed a seroprevalence rate of 1.2% (13/1076 residents, six also proving positive through PCR). Trypanosoma cruzi infection was not detected in children under 10 years old. Triatoma pseudomaculata prevailed in the peridomiciles: 63 specimens, 69% (34/49) infected with trypanosomatids. Rhodnius nasutus was captured in Copernicia prunifera palm trees (n=280; 25.0% infected with trypanosomatids) and inside dwellings (n=8, all uninfected). Trypanosoma cruzi seropositive reservoirs, represented by Didelphis albiventris (n=27), Rattus rattus (n=24), Thrichomys laurentius (n=2), Mus musculus (n=1) and Monodelphis domestica (n=1), were identified. Among domestic dogs (n=96) seroprevalence reached 21.9%. Miniexon multiplex PCR assays characterized TcI in triatomines. Both TcI and TcII were detected in wild mammal hosts. We conclude that Trypanosoma cruzi circulates within a domestic zoonotic cycle, requiring continuous surveillance. Insecticide application to domiciles does not appear to prevent continuous reintroduction of wild triatomine specimens, presenting a challenge to authorities involved in Chagas disease control.

Characterization of Leishmania sp. strains isolated from autochthonous cases of human cutaneous leishmaniasis in Santa Catarina State, southern Brazil

Four Leishmania sp. samples were isolated from autochthonous human cases of American cutaneous leishmaniasis (ACL) in Santa Catarina State, southern Brazil. These strains were characterized using indirect immunofluorescence with a panel of Leishmania-specific monoclonal antibodies (MAbs), and by PCR amplification and hybridization assay of the mini-exon gene with group specific probes. The results obtained with the MAbs were in agreement with the genetic marker. Two isolates (MHOM/BR/89/JSC89-H1 and MHOM/BR/89/JSC89-H2) were identified as L. (Leishmania) amazonensis and two (MHOM/BR/96/LSC96-H3 and MHOM/BR/97/LSC97-H4) as L. (Viannia) braziliensis. The southernmost autochthonous cases of ACL in Brazil are due to two different Leishmania sp. species, confirming the spreading of ACL on the American continent.

Detection and molecular characterization of piroplasms species from naturally infected dogs in southeast Brazil

Rangelia vitalii is a protozoon described from dogs in the south and southeast regions of Brazil. It is phylogenetically related to Babesia spp. that infects dogs, but data on this enigmatic parasite is still limited. The aim of this work was to detect piroplasm species in dogs in the state of Rio de Janeiro, Brazil, by 18S rRNA gene-based PCR assay, restriction fragment length polymorphism (RFLP) and sequence analyses. Of 103 dogs examined, seven (6.8%) were positive for Babesia spp. by PCR. The amplified products were digested by restriction enzymes to differentiate the Babesia species, and one sample was identified as Babesia vogeli. The pattern observed for the other six amplification products did not match with pattern described for large Babesia infecting dogs. Sequencing analysis confirmed these six samples as R. vitalii, with high homologies (99-100%) with a sequence from south Brazil. This study confirms the presence of Babesia vogeli and Rangelia vitalii circulate in domestic dogs in Teresópolis, Rio de Janeiro, Brazil.

Isolation of Trypanosoma caninum in domestic dogs in Rio de Janeiro, Brazil.

SUMMARY The domestic dogs involvement with different members of the Trypanosomatidae family has been the focus of several studies due to this animals close proximity to man. Recently this animal has been infected by a new Trypanosoma species (T. caninum), described in Rio de Janeiro and 19 similar isolates were later obtained. The objective of this study was to identify these isolates. All samples were isolated from intact skin cultures and analysed morphologically, by biochemical isoenzyme electrophoresis assays and by several molecular PCR assays. Additionally, anti-Leishmania sp. antibodies were assessed using the indirect Immunofluorescence Antibody Test (IFAT) in all animals. The methodologies employed to identify the isolates, including partial nucleotide sequences of 18S rRNA gene, indicated patterns identical to T. caninum and patterns different from the other species, including T. cruzi and T. rangeli samples. A phylogenetic tree constructed with the partial 18S ribosomal sequence shows that T. caninum is clustered with T. pestanai. Ten (52.6%) animals presented anti-Leishmania sp. antibodies with titres varying from 1:40 to 1:320. Thus, the hypothesis that this protozoan has disseminated among the dogs in Rio de Janeiro must be considered. The importance of a correct diagnosis in those animals and the possible consequences in the areas where visceral leishmaniasis is found are discussed here.

Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: evidence for oxidatively DNA damage generation.

Although titanium dioxide (TiO(2)) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO(2) is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO(2)-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO(2) associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO(2) plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO(2) protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO(2) plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO(2) plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

Correlation between populations of Rhodnius and presence of palm trees as risk factors for the emergence of Chagas disease in Amazon region, Brazil

Among the states that comprise the legally defined Amazon region of Brazil, Pará has presented the highest occurrences of acute cases of Chagas disease over the last two decades. These cases have been attributed to consumption of fruits from native palm trees. In surveys in rural and wild areas of the municipality of Oriximiná, Pará, triatomine fauna, their main ecotopes and the infection rate due to Trypanosoma cruzi were identified using active and passive search methods: manual capture and Noireau traps, respectively. A total of 582 ecotopes were surveyed using 1496 Noireau traps. Out of 442 specimens collected, 289 were identified as Rhodnius robustus and 153 as Rhodnius pictipes. The infection rate caused by T. cruzi was 17.4%. The food sources of the triatomines were found to be birds, hemolymph, horses, and rodents. The association between R. robustus and inajá palm trees (Attalea marita), which are abundant in rural areas, was confirmed. On the other hand, R. pictipes is found in several palm tree species, such as inajá (A. marita), mucajá (Acrocomia aculeata), murumuru (Astrocaryum murumuru) and patauá (Oenocarpus bataua), and in bromeliads in wild areas. These occurrences of triatomine species in regions with or without T. cruzi infection, in the vicinity of the main settlement of the municipality, suggest that there is a need for entomological and epidemiological surveillance in this region.

Frequency and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba hartmanni in the context of water scarcity in northeastern Brazil

This study aimed to estimate the frequency, associated factors, and molecular characterisation of Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, andEntamoeba hartmanni infections. We performed a survey (n = 213 subjects) to obtain parasitological, sanitation, and sociodemographic data. Faecal samples were processed through flotation and centrifugation methods.E. histolytica, E. dispar, E. moshkovskii, and E. hartmanni were identified by nested-polymerase chain reaction (PCR). The overall prevalence of infection was 22/213 (10.3%). The infection rate among subjects who drink rainwater collected from roofs in tanks was higher than the rate in subjects who drink desalinated water pumped from wells; similarly, the infection rate among subjects who practice open defecation was significantly higher than that of subjects with latrines. Out of the 22 samples positive for morphologically indistinguishableEntamoeba species, the differentiation by PCR was successful for 21. The species distribution was as follows: 57.1% to E. dispar, 23.8% to E. histolytica, 14.3% toE. histolytica and E. dispar, and 4.8% E. dispar and E. hartmanni. These data suggest a high prevalence of asymptomatic infection by the group of morphologically indistinguishable Entamoeba histolytica/dispar/moshkovskiicomplex and E. hartmanni species. In this context of water scarcity, the sanitary and socioenvironmental characteristics of the region appear to favour transmission.

Trypanosoma cruzi : parasitaemia produced in mice does not seem to be related to in vitro parasite-cell interaction

The interaction of Trypanosoma cruzi strains producing subpatent or high parasitaemia in mice with mouse macrophages, Vero and L929 cells was evaluated using tissue culture trypomastigotes. Macrophages were the cells most readily infected while Vero cells presented the highest parasite intracellular multiplication rates. Subpatent strains were equal or more infective than the high parasitaemia. Due to the small number of strains, no correlation could be established between the zymodemes and parasitaemia or parasite-cell interaction in vitro. However parasitaemia in mice does not seem to be related to in vitro parasite-cell interaction.

Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plasmodium spp.)

Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods. Considering the negative impact of avian malaria on penguins, the aim of this study was to detect the presence of Plasmodium spp. in Magellanic penguins using Polymerase Chain Reaction (PCR) and by verifying clinical, hematological, and biochemical alterations in blood samples as well as to verify the likely prognosis in response to infection. Blood samples were obtained from 75 penguins to determine packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), uric acid, total protein, albumin, globulin and aspartate aminotransferase (AST) activity levels. Whole blood samples were used for PCR assays. Plasmodium spp. was detected in 32.0% of the specimens using PCR and in 29.3% using microscopic analyses. Anorexia, diarrhea and neurological disorders were more frequent in penguins with malaria and a significant weight difference between infected and non-infected penguins was detected. PCV and MCV rates showed no significant difference. RBC and WBC counts were lower in animals with avian malaria and leukopenia was present in some penguins. Basophil and lymphocyte counts were lower in infected penguins along with high monocyte counts. There was no significant difference in AST activities between infected and non-infected animals. There was a significant increase in uric acid values, however a decrease in albumin values was observed in infected penguins. Based on this study, we concluded that Plasmodium spp. occurs in Magellanic penguins of rehabilitation centers in Southeastern Brazil, compromising the weight of infected animals with clinical alterations appearing in severe cases of this disease. It was also noted that, although the hematological abnormalities presented by these animals may not have been conclusive, leukopenia, monocytosis and the decrease of basophils and lymphocytes revealed an unfavorable prognosis, and Plasmodium spp. infections may progress with elevated uric acid concentration and low albumin levels.

Aflagellar epimastigote forms are found in axenic culture of Trypanosoma caninum.

Representatives of the genus Trypanosoma have been traditionally found in epimastigote, espheromastigote and trypomastigote flagellated forms in axenic cultures. Trypanosoma caninum is a trypanosomatid that has recently been reported infecting dogs in endemic areas of canine leishmaniasis in Brazil. It presents specific biological characteristics and it is found exclusively on healthy skin. Here, we describe the evolutive forms of this parasite showing not only the forms commonly found in culture, but also epimastigote forms with no free flagellum. The study was conducted using scanning and transmission electron microscopy and, we demonstrate that typical flagellated epimastigotes originate from forms without flagellum, although the latter may remain without differentiation in the culture. Two hypotheses are considered and discussed in this paper: (i) the aflagellated epimastigotes are a typical developmental forms of T. caninum and (ii) the emergence of these aflagellated forms could be resultant from a disturbed process during cell division caused by interfering specific proteins, which leads to inability to form and regulate the flagellum length. In any case, considering that T. caninum is a parasite that is still little studied, the information brought by our study adds data which may be useful to clarify aspects on the cell cycle of this intriguing parasite that has been found in different regions of Brazil.