Pedro Luiz Silva Pinto

THE FIRST CASE OF Angiostrongylus cantonensis EOSINOPHILIC MENINGITIS DIAGNOSED IN THE CITY OF SÃO PAULO, BRAZIL

INTRODUCTION Angiostrongylus cantonensis is a natural parasite found in lung arteries of rats, which in humans may cause eosinophilic meningitis. OBJECTIVE To report the first case of eosinophilic meningitis caused by Angiostrongylus cantonensis in the city of São Paulo, Brazil. CASE REPORT A male patient, 11 years old, living in the southern area of São Paulo, was admitted to the Pediatric Emergency Department with ongoing headaches for three days, but no fever or any other complaint. The presence of snails and rodents was reported in the peridomicile. The child was awake, lucid, oriented; muscular strength preserved, isochoric, photo reagent pupils and terminal nuchal rigidity - Glasgow Coma Scale (GCS) = 15. The laboratory tests showed a mild leukocytosis with 1736 eosinophils/mm3 and the CSF analysis disclosed 160 leukocytes/mm3 with 36% of eosinophils. The bacterial culture was negative. Computed Cerebral Tomography showed no alterations. The RT-PCR assay for detecting Angiostrongylus cantonensis larvae and DNA was negative. ELISA antibodies for IgG anti-A. cantonensis was negative in serum and undetermined in CSF and samples collected five days after the onset of symptoms. Seroconversion was observed in the sample collected 135 days later. CONCLUSION the epidemiological and clinical data, the CSF alterations with eosinophilia and the seroconversion strongly suggest Angiostrongylus cantonensis eosinophilic meningitis.

Ação de raios gama sobre formas sanguicólas de Trypanosoma cruzi. Estudo experimental em camundongos

Blood samples of animals infected with T. cruzi — Y strain, were submitted to gamma radiation in dosage of 200 and 300 krad. In order to verify the efficacy of this method to eliminante the parasite the blood samples were inoculated in to mice and the following parameters were used: parasitemia, hemoculture, xenodiagnosis, subinoculaticn, reinoculation of mice with a virulent strain and anatomy pathological study. The blood samples exposed to the different radiation dosages and inoculated at two different periods were unable to induce a subsequent infection in all animals tested.Amostras de sangue de animais infectados com cepa Y de Trypanosoma cruzi foram submetidas, respectivamente, a 200 e 300 krad de radiacao gama. Para verificar a eficacia do metodo na eliminacao do parasita, o material foi inoculado em camundongos e os parâmetros utilizados na avaliacao foram: parasitemia, cultura, xenodiagnostico, subinoculacao, reinoculacao com cepa virulenta e exame anatomo-patologico das visceras. Os sangues expostos as duas diferentes intensidades de radiacao e inoculados em dois periodos apos o processo, mostraram-se inocuos quanto a capacidade de produzir infeccao nos animais

Evaluation of real-time PCR assay to detect Schistosoma mansoni infections in a low endemic setting

BackgroundSchistosomiasis constitutes a major public health problem, and 200 million people are estimated to be infected with schistosomiasis worldwide. In Brazil, schistosomiasis has been reported in 19 states, showing areas of high and medium endemicity and a wide range of areas of low endemicity (ALE). Barra Mansa in Rio de Janeiro state has an estimated prevalence of 1%. ALE represent a new challenge for the helminth control because about 75% of infected individuals are asymptomatic and infections occur with a low parasite load (<100 eggs per gram of feces), causing a decrease in sensitivity of stool parasitological techniques, which are a reference for the laboratory diagnosis of this helminth. The objective of this study was to evaluate the performance of a TaqMan quantitative polymerase chain reaction (qPCR) technique in serum and feces DNA samples using the techniques of Kato-Katz (KK), Hoffman, Pons and Janer (HH) as references, during an epidemiological survey using fecal samples and sera from randomized residents from an ALE.MethodsA cross-sectional study conducted from April to December 2011 using a probabilistic sampling that collected 572 fecal and serum samples. The laboratory diagnostic techniques used were: KK, HH and qPCR (feces and serum).ResultsWe obtained the following results using the different diagnostic techniques: KK and HH, 0.9% (n =5); qPCR-feces, 9.6% (n =55); and qPCR-serum, 1.4% (n =8). The qPCR-feces presented the highest positivity, whereas the techniques of HH and KK were the least sensitive to detect infections (0.8%). Compared to HH and KK, qPCR-feces showed a statistically significant difference in positivity (p <0.05), although with poor agreement.ConclusionThe positivity rate presented by the qPCR approach was far higher than that obtained by parasitological techniques. The lack of adequate surveillance in ALE of schistosomiasis indicates a high possibility of these areas being actually of medium and high endemicity. This study presents a control perspective, pointing to the possibility of using combined laboratory tools in the diagnosis of schistosomiasis in ALE.

Synthesis and evaluation of new oxamniquine derivatives

Oxamniquine derivatives were synthesized and evaluated as novel schistosomicide agents. Oxamniquine (1,2,3,4-tetrahydro-2-[[(1-methylethyl)amino]methyl]-7-nitro-6-quinolinemethanol) was submitted to the Mannich reaction, using formaldehyde, paraformaldehyde and acetaldehyde as reagents, and gave three unexpected products: two of them were cyclized on the alkylamine side chain and another etherified on the aminequinolinemethanol group. The three compounds were biologically evaluated using mice infected with Schistosoma mansoni and showed promising activities, but had higher toxicities. For studies on structure-activity relationships, results demonstrate that the side alkylamine group can be modified with preserved activity, but that this modification is associated with increased toxicity.

Pesquisa de ovos de helmintos e de cistos de protozoários em dinheiro

Em virtude da elevada prevalencia de parasitoses intestinais no Brasil e da possivel participacao de objetos na transmissao dessas afeccoes, foi desenvolvida pesquisa envolvendo dinheiro difundido na coletividade. Foram analisadas, quanto a presenca de ovos de helmintos e de cistos de protozoarios, 1.003 cedulas e 1.011 moedas, de valores e procedencias diversos. Do estudo resultou o encontro de cistos de Entamoeba histolytica em duas eventualidades, de ovos de Ascaris lumbricoides larvado e infertil, de ovo de Taenia sp. morfologicamente integro e de outros elementos nao patogenicos. Ficou salientada a importância do dinheiro circulante na epidemiologia das enteroparasitoses, tendo havido sugestao de novos trabalhos de natureza congenere, inclusive delineando diretrizes de ordem profilatica.Em virtude da elevada prevalencia de parasitoses intestinais no Brasil e da possivel participacao de objetos na transmissao dessas afeccoes, foi desenvolvida pesquisa envolvendo dinheiro difundido na coletividade. Foram analisadas, quanto a presenca de ovos de helmintos e de cistos de protozoarios, 1.003 cedulas e 1.011 moedas, de valores e procedencias diversos. Do estudo resultou o encontro de cistos de Entamoeba histolytica em duas eventualidades, de ovos de Ascaris lumbricoides larvado e infertil, de ovo de Taenia sp. morfologicamente integro e de outros elementos nao patogenicos. Ficou salientada a importância do dinheiro circulante na epidemiologia das enteroparasitoses, tendo havido sugestao de novos trabalhos de natureza congenere, inclusive delineando diretrizes de ordem profilatica.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Dot-ELISA for the detection of IgM and IgG antibodies to Schistosoma mansoni worm and egg antigens, associated with egg excretion by patients

Human schistosomiasis, caused by Schistosoma mansoni, is highly prevalent in Brazil and usually diagnosed by time consuming stool analysis. Serological tests are of limited use in this disease, mainly for epidemiological studies, showing no discrimination between previous contact with the parasite and active infections. In the present study, we standardized and compared a Dot-ELISA for IgM and IgG antibodies against S. mansoni antigens from eggs and worms with a routine IgG and IgM immunofluorescence assay using similar antigens, in the study of sera from 27 patients who had quantified egg stool excretion. The positivity obtained for IgG Dot-ELISA was 96.3% and 88.9% for IgM Dot-ELISA with worm antigen and 92.6% and 90.9% with egg antigen. The IFI presented similar positivities using worm antigen, 92.6% (IgG) and 96.3% (IgM), and lower results with egg antigen, 77.8%(IgG and IgM). The patients studied were divided into two groups according to their egg excretion, with greater positivity of serological tests in higher egg excreters. When comparing the quantitative egg excretion and the serological titers of the patients, we detected a correlation only with IgM Dot-ELISA, with r = 0.552 (p = 0.0127). These data show that Dot-ELISA can be used for the detection of specific antibodies against S. mansoni in sera from suspected patients or in epidemiological studies and, with further purification of egg antigen and larger samples, IgM Dot-ELISA could be a possible tool for rough estimates of parasite burden in epidemiological studies.

Estudo experimental sobre possível atividade da violeta de genciana na profilaxia da transmissão da toxoplasmose por transfusão de sangue

The Authors proposed a study for the evaluation of the prevention of toxoplasmose transmission through blood transfusion by the gentians violet as has been made for Chagass Disease. Experiments with mice showed that the gentians violet added to the blood in the concentration of 1/1000 keeped in refrigerator for 48h prevents the transmission.Devido a evidencias sugestivas da possibilidade de transmissao da toxoplasmose por transfusao de sangue, os autores se propuseram avaliar o papel preventivo da violeta de genciana, a semelhanca do que ja e estabelecido para a doenca de Chagas. O experimento em camundongos revelou a acao profilatica da violeta de genciana quando adicionado ao sangue a ser transfundido na concentracao de 1/1000 e permanencia por 48 horas na geladeira.

Detection of Schistosoma mansoni infection by TaqMan® Real-Time PCR in a hamster model

An experimental study in hamsters was performed to evaluate the capability for detecting Schistosoma mansoni DNA in serum and fecal samples during the pre and post-egg-laying periods of infection using TaqMan® Real-Time PCR system (qPCR), was compared with the circumoval precipitin test (COPT) and the Kato-Katz technique, especially among individuals with low parasitic burden. Twenty-four hamsters were infected with cercariae. Three hamsters were sacrificed per week under anesthesia, from 7 days post infection (DPI) up to 56 DPI. A serum sample and a pool of feces were collected from each hamster. The presence of S. mansoni eggs in fecal samples was evaluated by Kato-Katz method and in the hamsters gutby histopathology. Detection of S. mansoni DNA was performed using qPCR and S. mansoni antibody using COPT. The first detection of eggs in feces by Kato-Katz method and S. mansoni DNA in feces by qPCR occurred 49 DPI. Nevertheless, S. mansoni DNA was detected in serum samples from 14 up to 56 DPI. COPT was positive at 35 DPI. The results not only confirm the reliability of S. mansoni DNA detection by qPCR, but also demonstrate that serum is a trustworthy source of DNA in the pre patent infection period.

Estudo sobre a eventual ação da artemisinina na infecção experimental de camundongos pelo Toxoplasma gondii

Because of taxonomic similarities to previous data found in the literature, and with the aim of improving treatment of toxoplasmosis, we considered it of interest to assay artemisinin, an effective anti-maiarial agent, for the treatment of experimental infection in mice by Toxoplasma gondii. Different amounts of the anti-parasitic agent were administered, including after previous contact in vitro with toxoplasma and stimulated macrophage production. With the methodology used artemisinin was not effective. Suggestions for further studies are made.