Evolution of IgG antibody response against Toxoplasma gondii tissue cyst in acute and chronic human infections

The recognition profile of the tissue cysts antigens by IgG antibodies was studied during acute and chronic human toxoplasmic infection. Thus the IgG response against Toxoplasma gondii was investigated by immunoblotting in two patients accidentally infected with the RH strain as well as in group of naturally infected patients at acute and chronic phase. There was an overall coincidence of molecular mass among antigens of tachyzoites and tissue cysts recognized by these sera, however, they appear not to be the same molecules. The response against tissue cysts starts early during acute infection, and the reactivity of antibodies is strong against a wide range of antigens. Six bands (between 82 and 151 kDa) were exclusively recognized by chronic phase sera but only the 132 kDa band was positive in more than 50% of the sera analysed. A mixture of these antigens could be used to discriminate between the two infection phases. The most important antigens recognized by the acute and the chronic phase sera were 4 clusters in the ranges 20-24 kDa, 34-39 kDa, 58-80 kDa and 105-130 kDa as well as two additional antigens of 18 and 29 kDa. Both accidentally infected patients and some of the naturally infected patients showed a weak specific response against tissue cyst antigens.

Giardia duodenalis: INTER-STRAIN VARIABILITY OF PROTEINS, ANTIGENS, PROTEASES, ISOENZYMES AND NUCLEIC ACIDS

Giardia duodenalis isolates from asymptomatic or symptomatic patients and from animals present similarities and differences in the protein composition, antigenic profile, pattern of proteases and isoenzymes, as well as in nucleic acids analysis. In the present overview, these differences and similarities are reviewed with emphasis in the host-parasite interplay and possible mechanisms of virulence of the protozoon.

Detection of a Giardia lamblia coproantigen by using a commercially available immunoenzymatic assay, in Belo Horizonte, Brazil

It is known that fecal examination to detect Giardia lamblia cysts or trophozoites produces a high percentage of false-negative results. A commercially available immunoenzymatic assay (ProSpecT Giardia Microplate Assay, Alexon, Inc., BIOBRÁS) to detect G. lamblia specific coproantigen was evaluated for the first time in Brazil. A total of 90 specimens were tested. Each specimen was first tested as unpreserved stool, and then it was preserved in 10% Formalin to be tested 2 months later. The assay was able to identify all the 30 positive patients (sensitivity = 100.0%) by visual or spectrophotometric examination in the unpreserved specimens and was negative in 57 of the 60 patients without G. lamblia (specificity = 95.0%). The assay identified 27 of the 30 positive patients (sensitivity = 90.0%) and was negative in 59 of the 60 negatives (specificity = 98.3%) in the preserved stools according to both readings. A marked difference was observed in the optical densities in both groups, preserved and unpreserved stools, when the G. lamblia-positive specimens were compared to the negative or positive for other intestinal parasites than G. lamblia. The assay seems a good alternative for giardiasis diagnosis, especially when the fecal examination was repeatedly negative and the patient presents giardiasislike symptoms.

Cyclospora cayetanensis in sputum and stool samples

We report the observation of acid-fast Cyclospora cayetanensis oocysts in a sputum sample. The patient, a 60 year-old, HIV negative man, was successfully treated for pulmonary tuberculosis during 1997. On February 1998, he was admitted to our center due to loss of weight, cough with purulent expectoration, dysphonia and a radiological picture of pulmonary fibrosis. Bacilloscopic study of sputum (negative for acid-fast bacilli) stained with Ziehl-Neelsen technique showed large (8-10 µm) spherical, acid-fast Cyclospora cayetanensis oocysts. No other pathogens were isolated on cultures from this sample or from laryngeal biopsy. Serial parasitologic studies showed C. cayetanensis and also eggs of Trichuris trichiura, Ascaris lumbricoides and Hymenolepis nana and of Entamoeba coli cysts. The patient lives in the outskirts of Buenos Aires in a brick-made house with potable water and works as builder of sewers. He travelled in several occasions to the rural area of province of Tucumán which has poor sanitary conditions. C. cayetanensis is an emergent agent of diarrhea and as far as we know this is the first time the parasite is observed in respiratory samples.

Arbitrarily primed PCR fingerprinting of RNA and DNA in Entamoeba histolytica

Differences were detected in the gene expression of strains of E. histolytica using RNA (RAP-PCR) and DNA fingerprinting (RAPD). Analysis of the electrophoretic profiles of the gels revealed some polymorphic markers that could be used in the individual characterization of the strains. The 260 bands generated by using five different primers for RAP-PCR, as well as RAPD, were employed in the construction of dendograms. The dendogram obtained based on the RAPD products permitted the distinction of symptomatic and asymptomatic isolates, as well the correlation between the polymorphism exhibited and the virulence of the strains. The dendogram obtained for the RAP-PCR products did not show a correlation with the virulence of the strains but revealed a high degree of intraspecific transcriptional variability that could be related to other biological features, whether or not these are involved in the pathogenesis of amebiasis.

Characterization and optimization of bovine Echinococcus granulosus cyst fluid to be used in immunodiagnosis of hydatid disease by ELISA

The aim of this work was to assess the influence in the diagnostic value for human hydatid disease of the composition of bovine hydatid cyst fluid (BHCF) obtained from fertile (FC) and non-fertile cysts (NFC). Eight batches from FC and 5 from NFC were prepared and analysed with respect to chemical composition: total protein, host-derived protein, carbohydrate and lipid contents. No differences were observed in the first two parameters but carbohydrate and lipid contents were shown to be higher in batches from FC than in those from NFC. Bands of 38 and 116 kD in SDS-PAGE profiles were observed to be present in BHCF from FC only. Two pools were prepared from BHCF batches obtained from FC (PFC) and NFC (PNFC), respectively. Antigen recognition patterns were analysed by immunoblot. Physicochemical conditions for adsorption of antigens to the polystyrene surface (ELISA plates) were optimized. The diagnostic value of both types of BHCF as well as the diagnostic relevance of oxidation of their carbohydrate moieties with periodate were assessed by ELISA using 42 serum samples from hydatid patients, 41 from patients with other disorders, and 15 from healthy donors. Reactivity of all sera against native antigen were tested with and without free phosphorylcholine. The best diagnostic efficiency was observed using BHCF from periodate-treated PFC using glycine buffer with strong ionic strength to coat ELISA plates.

Outbreaks of human-herpes virus 6 (HHV-6) infection in day-care centers in Belém, Pará, Brazil

A total of 730 children aged less than 7 years, attending 8 day-care centers (DCCs) in Belém, Brazil were followed-up from January to December 1997 to investigate the occurrence of human-herpes virus 6 (HHV-6) infection in these institutional settings. Between October and December 1997 there have been outbreaks of a febrile- and -exanthematous disease, affecting at least 15-20% of children in each of the DCCs. Both serum- and- plasma samples were obtained from 401 (55%) of the 730 participating children for the detection of HHV-6 antibodies by enzyme-linked immunosorbent assay (ELISA), and viral DNA amplification through the nested-PCR. Recent HHV-6 infection was diagnosed in 63.8% (256/401) of them, as defined by the presence of both IgM and IgG-specific antibodies (IgM+/IgG+); of these, 114 (44.5%) were symptomatic and 142 (55.5%) had no symptoms (p = 0.03). A subgroup of 123 (30.7%) children were found to be IgM-/IgG+, whereas the remaining 22 (5.5%) children had neither IgM nor IgG HHV-6- antibodies (IgM-/IgG-). Of the 118 children reacting strongly IgM-positive ( > or = 30 PANBIO units), 26 (22.0%) were found to harbour the HHV-6 DNA, as demonstrated by nested-PCR. Taken the ELISA-IgM- and- nested PCR-positive results together, HHV-6 infection was shown to have occurred in 5 of the 8 DCCs under follow-up. Serological evidence of recent infections by Epstein-Barr virus (EBV) and parvovirus B19 were identified in 2.0% (8/401) and 1.5% (6/401) of the children, respectively. Our data provide strong evidence that HHV-6 is a common cause of outbreaks of febrile/exanthematous diseases among children attending DCCs in the Belém area.

Study of a cutaneous leishmaniasis outbreak in General Vedia, Province of Chaco, 1996

Between March and July 1996, a focalized epidemic outbreak of cutaneous leishmaniasis in General Vedia, province of Chaco, associated to the gallery forest of the Oro river was verified. The incidence rate in the area, which was 0-2/000 cases in preceding years, reached 8/000 cases in 1996. The risk of symptomatic infection was similar between sexes, but was different when analizing the different age groups by sex, suggesting a greater relative importance of the peridomestic transmission for the females and of the transmission in the subtropical forest for the males. Specimens of Lutzomyia intermedia, a species already incriminated as a vector of Leishmaniasis in other provinces of northern Argentina, were captured and identified in the focus locality in May 1996. The possible causes of the outbreak related to the climatic variables and the vector abundance are analyzed and the results in the framework of possible preventive and control activities are discussed.

Analysis of proteins from membrane and soluble fractions of Giardia duodenalis trophozoites of two Brazilian axenic strains

In the present study, we have analyzed by sodium docecyl sulphate - polyacrilamide gel electrophoresis (SDS-PAGE), immunoblotting and Concanavalin A blotting (Con A blotting) proteins of membrane fractions and soluble fractions obtained from Giardia duodenalis trophozoites of two axenic strains isolated in Brazil from a symptomatic (BTU-11) and an asymptomatic patient (BTU-10), as compared to the reference strain Portland 1. Both Brazilian strains showed a complex and homogeneous electrophoretic pattern of proteins, but some differences could be observed. Several glycoproteins were detected, particularly the proteins of 81, 72, 59 kDa and the protein of 62 kDa in the membrane proteins and cytosol, respectively. Many antigenic components were revealed by anti-Giardia rabbit IgG antibodies in the immunoblotting analysis. Among these components, the membrane protein of 32 kDa and the cytosol protein of 30 kDa could be related to giardin, as previously demonstrated.

Penicillin at the late stage of leptospirosis: a randomized controlled trial

There is evidence that an early start of penicillin reduces the case-fatality rate of leptospirosis and that chemoprophylaxis is efficacious in persons exposed to the sources of leptospira. The existent data, however, are inconsistent regarding the benefit of introducing penicillin at a late stage of leptospirosis. The present study was developed to assess whether the introduction of penicillin after more than four days of symptoms reduces the in-hospital case-fatality rate of leptospirosis. A total of 253 patients aged 15 to 76 years with advanced leptospirosis, i.e., more than four days of symptoms, admitted to an infectious disease hospital located in Salvador, Brazil, were selected for the study. The patients were randomized to one of two treatment groups: with intravenous penicillin, 6 million units day (one million unit every four hours) for seven days (n = 125) and without (n = 128) penicillin. The main outcome was death during hospitalization. The case-fatality rate was approximately twice as high in the group treated with penicillin (12%; 15/125) than in the comparison group (6.3%; 8/128). This difference pointed in the opposite direction of the study hypothesis, but was not statistically significant (p = 0.112). Length of hospital stay was similar between the treatment groups. According to the results of the present randomized clinical trial initiation of penicillin in patients with severe forms of leptospirosis after at least four days of symptomatic leptospirosis is not beneficial. Therefore, more attention should be directed to prevention and earlier initiation of the treatment of leptospirosis.

First report of a human case of polycystic echinococcosis due to Echinococcus vogeli from neotropical area of Peru, South America

We report a human case of polycystic hidatidosis due to Echinococcus vogeli from Contamana (Department of Loreto) village located in the central jungle of Peru. The patient is a 44 year-old lady, teacher, who carried a painless liver mass since a year ago. She was submitted to abdominal surgery and the liver mass was removed and showed multiple cysts containing colorless liquid as is showed in the polycystic hidatidosis. The morphology and measure of the hooks obtained from the liquid contained in the cysts are from Echinococcus vogeli. It is the first report of this parasitism in Perú.

Study of enteroparasites infection frequency and chemotherapeutic agents used in pediatric patients in a community living in Porto Alegre, RS, Brazil

Parasitic infections caused by intestinal protozoan and helminths affect more than two billion people worldwide and chemotherapy is the most commonly used therapeutic procedure. Considering the problems created by parasitic infections and the incorrect use of drugs, the aim of this work was to detect the frequency of enteroparasites infection and to estimate the use of chemotherapeutic agents in children living in the periphery of the city of Porto Alegre, RS, Brazil. Ninety-six preschool age children, who had parasitological exams and who used antiparasitic drugs, were analyzed. The efficacy of treatment was evaluated by stool examination repeated six months after treatment. The same diagnostic test was used to evaluate parasitological cure, which was defined as absence of eggs and cysts in the stool. From these children, 79 (82.3%) were contaminated by some species of parasite, the most prevalent were Ascaris lumbricoides, Trichuris trichiura and Giardia lamblia. The most commonly used drugs were mebendazole (86% of prescriptions) and metronidazole (30.3%). The cure rate in the 79 children, examined 6 months after treatment, was 65.3% for A. lumbricoides and 66.1% for T. trichiura. This study suggests that a continuous education program regarding the prevention and treatment of parasitic infections is an essential tool for their eradication.

Childhood astrovirus-associated diarrhea in the ambulatory setting in a Public Hospital in Cordoba city, Argentina

Human astroviruses have been increasingly identified as important agents of diarrheal disease in children. However, the disease burden of astrovirus infection is still incompletely assessed. This paper reports results on the epidemiological and clinical characteristics of astrovirus-associated diarrhea, as well as the impact of astrovirus infection on the ambulatory setting at a Public Hospital in Córdoba city, Argentina. From February 2001 through January 2002, 97 randomly selected outpatient visits for diarrhea among children < 36 months old were enrolled. A single specimen of stool from each child was collected and tested for astrovirus antigen by enzyme immunoassay. Astroviruses were detected in 12.37% of the diarrheal episodes. All the positive cases occurred in children 4 to 18 months, but the highest rate was in children aged 4 to 6 months (23.80%). The clinical symptoms of astrovirus associated-diarrhea were fever 41.66%, vomiting 25.00% and dehydration 8.33%; overall 16.66% required hospitalization. Astrovirus was identified through the year and no seasonally pattern was detected (cool semester 15.21% versus warm semester 9.80% p > 0.05). According to our estimation about one out of seventy-four children in this cohort would be assisted annually for an astroviral-diarrheal episode in the Public Hospital and one out of eight diarrheal cases could be attributed to astrovirus infection. Astrovirus is a common symptomatic infection in pediatric outpatient visits in the public hospital in the study area, contributing 12.37% of the overall morbidity from diarrhea.

Dot-ELISA for the diagnosis of neurocysticercosis

The aim of the present study was to standardize and evaluate dot-Enzyme linked immunosorbent assay (Dot-ELISA), a simple and rapid test for the detection of cysticercus antibodies in the serum for the diagnosis of neurocysticercosis (NCC). The antigen used in the study was a complete homogenate of Cysticercus cellulosae cysts obtained from infected pigs and dotted on to nitrocellulose membrane. Test sera were collected from the patients of NCC, and control sera from patients with other diseases and healthy students and blood donors of the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Hospital, Pondicherry, during a study period from 2001 to 2003. Dot-ELISA detected antibodies in 14 of 25 (56%) in clinically suspected cases of NCC, 13 of 23 (56.5%) in CT/MRI proven cases of NCC and 2 of 25 (8%) each in non-cysticercal CNS infection controls and healthy controls. The test showed a sensitivity of 56.25%, specificity of 92%, positive predictive value of 87.09%, and negative predictive value of 70.76%. Results of the present study shows that the Dot-ELISA as a simple test can be used in the field or poorly equipped laboratories for diagnosis of NCC .

Toxoplasma gondii in fresh pork sausage and seroprevalence in butchers from factories in Londrina, Paraná State, Brazil

The aims of this study were to verify the presence of Toxoplasma gondii cysts in fresh pork sausage and the presence of antibodies against T. gondii in serum of workers from factories with Municipal Inspection Service, in Londrina, PR, Brazil. 149 samples of sausage were collected from eight factories and blood samples from 47 workers. We also took information about the practices that were adopted in the factories and the workers' habits that could influence the prevalence of toxoplasmosis. After bioassay in mice, 13 (8.7%) sausage samples were positive, in one of them T. gondii was isolated and in the other 12 the mice seroconverted. Of 47 workers, 36 (76.6%) worked in sausage production and 11 (23.4%) were involved in other functions; 59.5% (28/47), 55.5% (20/36) and 72.7% (8/11), respectively, had T. gondii antibodies. There were no significant differences in the variables of industries' practices and workers' habits related to T. gondii infection. We concluded that fresh pork sausage could be important in the transmission of toxoplasmosis.