Canine visceral leishmaniasis in Barra de Guaratiba, Rio de Janeiro, Brazil: assessment of risk factors

Barra de Guaratiba is a coastal area of the city of Rio de Janeiro where American visceral leishmaniasis (AVL) is endemic. Although control measures including killing of dogs and use of insecticides have been applied at this locality, the canine seroprevalence remains at 25% and during 1995 and 1997 eight autochthonous human cases were notified. In order to evaluate factors related to the increase of the risk for Leishmania (Leishmania) chagasi infection in dogs we have screened 365 dogs by anti-Leishmania immunofluorescent antibody test (IFAT) and captured sandflies in the domestic and peridomestic environment. Some variables related to the infection were assessed by uni- and multivariate analysis. The distance of the residence from the forest border, its altitude and the presence of the opossum Didelphis marsupialis in the backyard, were found predictor factors for L. (L.) chagasi infection in dogs in Barra de Guaratiba. The presence of Lutzomyia longipalpis in the peridomestic environment indicates the possibility of appearence of new human cases. Our data also suggest the presence of a sylvatic enzootic cycle at this locality.

On the possibility of autochthonous Chagas disease in Roraima, Amazon region, Brazil, 2000-2001

Chagas disease has been almost entirely eradicated from the arid zones in Central and Northeastern Brazil where rare or no autochthonous cases have been reported. However, in the last 10 years the disease has increasingly been registered in the Amazon Region. Aiming to investigate the possibility of the occurrence of autochthonous cycle of Chagas disease in Roraima, triatomine collections, vectorial susceptibility studies (this one to be shown elsewhere), parasitological and serological analyses were conducted in three agricultural settlement areas (Rorainópolis, Passarão Project and Ilha Community). Blood-donor candidates were also investigated. This is the first epidemiological survey on Chagas disease conducted in agricultural settlements in Roraima. Triatomine species found were Triatoma maculata, Rhodnius pictipes, Rhodnius robustus and Panstrongylus geniculatus. Trypanosoma cruzi detection analyses included xenodiagnosis, indirect immunofluorescence, indirect hemaglutination, ELISA and kinetoplast PCR amplification. Natural triatomine infection was not found in intestinal contents. Twenty-five adult settlers (1.4% out of 1821, all > 15 year-old, 20 migrants) presented anti-T. cruzi antibodies. Two migrant settlers (from Minas Gerais and Maranhão) tested positive for more than two serological tests, besides either being positive for xenodiagnosis or PCR. Results show that Chagas disease is not endemic in the areas studied. However, all elements of the transmission cycle are present, demanding for an adequate and continuous vigilance.

Echovirus 4 associated to hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a contagious enteroviral infection occurring primarily in children and characterized by vesicular palmoplantar eruptions and erosive stomatitis. Echovirus 4 (EV-4) has been commonly associated with aseptic meningitis. The association of HFMD with EV-4 has not been reported previously. Two samples of a 14-month child who presented mild fever, sores in the mouth, rash with blisters on the palm of hands and soles of feet were sent to Enteric Viruses Laboratory of Adolfo Lutz Institute. Clinical samples were inoculated in three different cell lines, and those which presented cytopathic effect (CPE), were submitted to Indirect Immunofluorescence Assay (IFA) and "one step" RT-PCR. Agarose gel electrophoresis from RT-PCR product, showed a product with 437 bp, which is characteristic of Enterovirus group. Echovirus 4 was identified by IFA. Although HFMD is a viral infection associated mainly with Enterovirus 71 (HEV-71) and Coxsackievirus A16 (CV-A16), our results demonstrate a diversity of serotype related to HFMD and stress the importance of epidemiological surveillance to this disease and its complications.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Seroprevalence of Toxoplasma gondii infection among indian people living in Iauareté, São Gabriel da Cachoeira, Amazonas, Brazil

The goal of this survey was to estimate the seroprevalence of Toxoplasma gondii infection in Iauareté, a multiethnic Indian community in the upper Rio Negro basin. We carried out a cross-sectional survey (n = 260), in order to obtain serum samples and demographic data. The sample was randomly selected, by family conglomerate analysis. Serodiagnosis was performed by an enzyme-linked immunosorbent assay and indirect immunofluorescence. Prevalence of reactivity was 73.5% (191/260), being higher in the older-age groups, reaching 95.7% (44/46) in the group aged 50 years or more. The majority of seropositive subjects had titers equal to or less than 1:64. Seroprevalence was greater in Indians belonging to the Hupda ethnic group (p = 0.03). According to the present survey, Indian people living in Iauareté have a high prevalence of antibodies to T. gondii. Demographic concentration and urbanization within low sanitation and poor hygiene backgrounds, as well as unfiltered water consumption, may be related to the high frequency of T. gondii seroprevalence observed in the studied area.

Epidemiological and ecological aspects related to malaria in the area of influence of the lake at Porto Primavera dam, in western São Paulo State, Brazil

A study was carried out in the area of influence of the Porto Primavera Hydroelectric Power Station, in western São Paulo State, to investigate ecological and epidemiological aspects of malaria in the area and monitor the profile of the anopheline populations following the environmental changes brought about by the construction of the lake. Mosquitoes captured were analyzed by standardized indicator species analysis (ISA) before and during different flooding phases (253 m and 257 m elevations). The local human population was studied by means of parasitological (thin/thick blood smears), molecular (PCR) and serological tests. Serological tests consisted of Enzyme Linked Immunosorbent Assay (ELISA) with synthetic peptides of the circumsporozoite protein (CSP) from classic Plasmodium vivax, P. vivax variants (VK247 and "vivax-like"), P. malariae and P. falciparum and Indirect Immunofluorescence Assay (IFA) with asexual forms of P. vivax, P. malariae and P. falciparum. The results of the entomological survey indicated that, although the Anopheles darlingi population increased after the flooding, the population density remained very low. No malaria, parasite infection or DNA was detected in the inhabitants of the study area. However, there was a low frequency of antibodies against asexual forms and a significant prevalence of antibodies against P. vivax, P. vivax variants, P. falciparum and P. malariae; the presence of these antibodies may result from recent or less recent contact with human or simian Plasmodium (a parallel study in the same area revealed the existence of a sylvatic cycle). Nevertheless, these results suggest that, as in other places where malaria is present and potential vectors circulate, the local epidemiological conditions observed could potentially support the transmission of malaria in Porto Primavera Lake if infected individuals are introduced in sufficient numbers. Further studies are required to elucidate the phenomena described in this paper.

Adenovirus respiratory infection: significant increase in diagnosis using PCR comparing with antigen detection and culture methods

Adenovirus (AdV) respiratory infections are usually described as being associated with high mortality rates. Laboratory diagnosis is essential for the establishment of the appropriate therapy, and for guiding the implementation of preventive measures in order to prevent the spread of the infection. Aiming to analyze the sensitivity and specificity of the laboratorial diagnosis methods available, we compared antigen detection by indirect immunofluorescence assay (IF), and a specific nested polymerase chain reaction (PCR), to detect AdV in respiratory samples collected from patients admitted to hospital with acute respiratory disease. Positive samples were inoculated into a cell culture to confirm the results. We analyzed 381 samples from the nasopharyngeal aspirates collected during the year 2008; of these, 2.6% tested were positive for adenovirus through IF and 10% through PCR; positive isolation was obtained in 40% and 26% of these cases, respectively. Most infected patients were children under six months of age, and despite of the fact that a significant number of patients required intensive care, the mortality rate was low (5%). In conclusion, molecular methods were found to be useful for rapid diagnosis of adenovirus infections with higher sensitivity than antigen detection; their introduction permitted a significant increase in diagnoses of adenovirus infections.

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.

First isolation of dengue 4 in the state of São Paulo, Brazil, 2011

We report the first isolation of Dengue virus 4 (DENV-4) in the state of São Paulo, from two patients - one living in São José do Rio Preto and the other one in Paulo de Faria, both cities located in the Northwest region of the state. The virus isolations were accomplished in the clone C6/36 Aedes albopictus cell line, followed by indirect immunofluorescence assays, performed with type-specific monoclonal antibodies that showed positive reactions for DENV-4. The results were confirmed by Nested RT-PCR and Real-Time RT-PCR assays. The introduction of DENV-4 in a country that already has to deal with the transmission of three other serotypes increases the possibility of the occurrence of more severe cases of the disease. The importance of early detection of dengue cases, before the virus spreads and major outbreaks occur, should be emphasized.

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.

Human mucocutaneous leishmaniasis in Três Braços, Bahia - Brazil: an area of Leishmania braziliensis braziliensis transmission. II. Cutaneous disease. Presentation and evolution

The clinical records of 182 patients with cutaneous leishmaniasis probably due to Leishmania braziliensis braziliensis are analysed. 68% had a single lesion which was usually an ulceron the lower anterior tibial third. Many had short histories of one to two months and all age groups were represented 13% had closed lesions of a verrucose or plaque like nature. Evolution of these skin lesions after treatment was related to the regularity of antimony therapy. Although healing usually occurred in three months, the time to scarring after commencing treatment was variable and related to the size ofthe lesion (p < 0.01). Usually if sufficient antimony treatment was given the lesion closed. Seven of the ten patients with initially negative leishmanin skin tests converted to positive after treatment. A significant decline of indirect fluorescent antibody titres occurred in patients followed, during and after therapy.

Usefulness of serology for the evaluation of Trypanosoma cruzi transmission in endemic areas of Chagas' disease

Thirteen communities from 7 Argentinian provinces were selected for the evaluation of serology as an indicator of transmission of Chagas disease. Of the communities appraised, 6 did not have a history of previous treatment with insecticides and 7 had received sporadic or continuous insecticide treatment. The inhabitants of 20% of the houses of each locality were studied by serology. The samples were obtained byfinger pricking and 50 fil of blood were mixed with 150μl of 50% glycerine solution in tissue culture media to be assayed by Indirect Hemagglutination and Indirect Immunofluorescence tests. In untreated areas, the prevalence of infection in infants 0-4 years old was 17.5%, reaching to over 22% for the 5-9 year old group, and to 33.3% in 10-14 year old individuals. The prevalence in treated and surveyed areas was 2.6% in 0-4 year old children, 5.4% in 5-9 year old and 6,2% in 10-14 year old youngsters. The differences between both areas were statistically significant (p < 0.005). This study favors serology as a valid indicator for the evaluation of transmission of Chagas disease in rural areas.

Dot enzyme-linked immunosorbent assay (dot-ELISA) for schistosomiasis diagnosis using dacron as solid-phase

Dacron and nitrocellulose were evaluated as matrices for the dot enzyme linked immunosorbent assay (dot-ELISA) for schistosomiasis and compared to indirect immunofluorescence (IMF). Titration of sera from 18 schistosomiasis patients against soluble worm antigen preparation (SWAP) was carried out and sera from healthy individuals from non-endemic areas were used as controls. The IMF was less sensitive than the dot-ELISAs, although the difference was not statistically significant (p > 0.05). The dot-ELISA based on nitrocellulose was as sensitive as that using dacron. Stability did not differ between nitrocellulose and dacron. Specificity was lower when dacron was used than when nitrocellulose was used, although the difference was not statistically significant (p > 0.05). In conclusion, this work showed that nitrocellulose and dacron performed similarly in dot-ELISA, suggesting that they may be used alternatively in population surveillance in endemic areas.

Viral etiology of acute respiratory infections among children in Porto Alegre, RS, Brazil

Although acute respiratory infections (ARIs) are a major cause of child morbidity and mortality in Southern Brazil, little information is available on their seasonality and viral etiology. This study was conducted on children under 5 years of age with ARI to assess viral etiology in the State of Rio Grande do Sul, from 1990 to 1992. A total of 862 nasopharyngeal secretion (NPS) samples were tested using indirect immunofluorescence. The results showed that 316 (36.6%) NPS samples were positive: 26.2% for RSV, 6% for adenovirus, 1.7% for influenzaviruses, 1.5% for parainfluenzaviruses, and 1.2% for mixed infection. The mean viral prevalence rates in out-patient services, emergency wards, and in-patient hospital wards were 26.7%, 53% and 42.3%, respectively. Respiratory syncytial virus (RSV) and adenovirus accounted for 91.4 % of the viral diagnoses. RSV was more frequent in children under one year of age at the three levels of health care and was prevalent in infants under six months. Adenovirus was the most prevalent pathogen in hospitalized children, in 1992. Influenza A virus showed an increased prevalence with age among out-patient children. This study shows the annual occurence of viral respiratory infections in the coldest months, with a significant annual variation in the frequency of RSV infection.

Trypanosoma cruzi in marsupial didelphids (Philander frenata and Didelhis marsupialis): differences in the humoral immune response in natural and experimental infections

Philander frenata and Didelphis marsupialis harbor parasitism by Trypanosoma cruzi without developing any apparent disease and on the contrary to D. marsupialis, P. frenata maintains parasitism by T. cruzi II subpopulations. Here we compared the humoral immune response of the two didelphids naturally and experimentally infected with T. cruzi II group, employing SDS-PAGE/Western blot techniques and by an Indirect immunofluorescence assay. We also studied the histopathological pattern of naturally and experimentally infected P. frenata with T. cruzi. P. frenata sera recognized more antigens than D. marsupialis, and the recognition pattern did not show any change over the course of the follow up of both didelphid species. Polypeptides of 66 and 90kDa were the most prominent antigens recognized by both species in the soluble and enriched membrane fractions. P. frenata recognized intensely also a 45kDa antigen. Our findings indicate that: 1) there were no quantitative or qualitative differences in the patent or subpatent phases in the recognition pattern of P. frenata; 2) the significant differences in the recognition pattern of parasitic antigens by P. frenata and D. marsupialis sera suggest that they probably "learned" to live in harmony with T. cruzi by different strategies; 3) although P. frenata do not display apparent disease, tissular lesions tended to be more severe than has been described in D. marsupialis; and 4) Both didelphids probably acquired infection by T. cruzi after their evolutionary divergence.