Excretion-secretion products and proteases from live Sporothrix schenckii yeast phase: immunological detection and cleavage of human IgG

Antigenic preparations from Sporothrix schenckii usually involve materials from mixed cultures of yeast and mycelia presenting cross-reactions with other deep mycoses. We have standardized pure yeast phase with high viability of the cells suitable to obtain specific excretion-secretion products without somatic contaminations. These excretion-secretion products were highly immunogenic and did not produce noticeable cross-reactions in either double immunodiffusion or Western blot. The antigenic preparation consists mainly of proteins with molecular weights between 40 and 70 kDa, some of them with proteolytic activity in mild acidic conditions. We also observed cathepsin-like activity at two days of culture and chymotrypsin-like activity at four days of culture consistent with the change in concentration of different secreted proteins. The proteases were able to cleave different subclasses of human IgG suggesting a sequential production of antigens and molecules that could interact and interfere with the immune response of the host.

Frequency of human toxocariasis in a rural population from Cajamarca, Peru determined by DOT-ELISA test

The aim of this study was to estimate the frequency of human toxocariasis in Cauday district, Cajamarca, Peru, using a dot-ELISA test. From June to October 2005, a total of 256 adult subjects were studied. Blood samples were collected for serology by a dot-ELISA test and for hematological examination. Parasitological examination was also carried out in stool samples to check cross-reactions in the dot-ELISA. The frequency observed was 44.92%, with a significant higher proportion of positivity in male subjects. From subjects with positive serology, 45.6% had respiratory symptoms, 40.44% abdominal pain, 32.35% hepatic symptoms, 14.7% cutaneous signs, 13.23% ocular manifestations, 43.38% eosinophilia, and all of these were statistically associated to serology. Among the population evaluated, 90.23% (231/256) were parasitized. From subjects with positive serology, 92.17% had at least one intestinal parasite and the most frequent were: Blastocystis hominis (68.38%), Giardia lamblia (28.68%), Hymenolepis nana (20.0%), Ascaris lumbricoides (15.65%), Entamoeba histolytica/E. dispar (13.24%), Cyclospora cayetanensis (4.41%), Cryptosporidium sp. (1.47%), Enterobius vermicularis (0.87%), Strongyloides stercoralis (0.87%), Taenia sp. (0.87%), and Trichuris trichiura (0.87%). The rate of false positives in the dot-ELISA test was improved by serum absorption each with A. suum antigens, with a decrease of cross-reactions. In conclusion, human toxocariasis is highly frequent in this population and some risk factors like dog/cat ownership, presence of pets within house, and previous history of geophagia were observed in the present study.

Serology using rROP2 antigen in the diagnostic of toxoplasmosis in pregnant women

Toxoplasma gondii causes severe fetal disease during acute infection in pregnant women, thus demanding early diagnosis for effective treatment and fetus preservation. Fetal tests are inefficient and risky, and diagnosis is based on maternal IgM serology, which had weak screening ability due to increased sensitivity, with alternative IgG avidity tests. Here, we performed ELISA and avidity assays using a recombinant T. gondii antigen, rROP2, in samples from 160 pregnant women screened from a large public hospital who were referred due to positive IgM assays. IgG serology and avidity assays were compared using whole T. gondii extract or rROP2. ELISA IgG detection with rROP2 showed good agreement with assays performed with T. gondii extract, but rROP2 IgG avidity assays were unrelated to whole extract antigen IgG avidity, regardless of the chaotrope used. These data show that avidity maturation is specific to individual antigen prevalence and immune response during infection. ELISA rROP2 IgG assays may be an alternative serological test for the diagnosis of toxoplasmosis during pregnancy, although our data do not support their use in avidity assays.

Circulation of antibodies against yellow fever virus in a simian population in the area of Porto Primavera Hydroelectric Plant, São Paulo, Brazil

Yellow fever (YF) is an acute viral infectious disease transmitted by mosquitoes which occurs in two distinct epidemiological cycles: sylvatic and urban. In the sylvatic cycle, the virus is maintained by monkey's infection and transovarian transmission in vectors. Surveillance of non-human primates is required for the detection of viral circulation during epizootics, and for the identification of unaffected or transition areas. An ELISA (enzyme-linked immunosorbent assay) was standardized for estimation of the prevalence of IgG antibodies against yellow fever virus in monkey sera (Alouatta caraya) from the reservoir area of Porto Primavera Hydroelectric Plant, in the state of São Paulo, Brazil. A total of 570 monkey sera samples were tested and none was reactive to antibodies against yellow fever virus. The results corroborate the epidemiology of yellow fever in the area. Even though it is considered a transition area, there were no reports to date of epizootics or yellow fever outbreaks in humans. Also, entomological investigations did not detect the presence of vectors of this arbovirus infection. ELISA proved to be fast, sensitive, an adequate assay, and an instrument for active search in the epidemiological surveillance of yellow fever allowing the implementation of prevention actions, even before the occurrence of epizootics.

Seroprevalence of human toxocariasis in Andean communities from the Northeast of Lima, Peru

The aim of this study was to assess the seroprevalence of human toxocariasis in three Andean communities from the Northeast of Lima, Peru. A total of 303 subjects including children and adults were studied and blood samples were collected to detect anti-Toxocara antibodies by ELISA-IgG test and by hematological examination; stool samples were collected also for parasitological examination. The overall seroprevalence of toxocariasis observed in the total population was 20.46%, with a significant high proportion in children from one to 10 years old (p = 0.034). Among the subjects with positive serology, 32.26% of them had respiratory disturbances, 22.58% hepatomegaly, 17.74% ocular signs or symptoms, 14.51% abdominal pain, 9.68% neurological involvement, and 4.84% cutaneous signs, but none of these clinical features were associated to a positive serology by multivariate analysis. Furthermore, 79.03% of seropositive subjects also harbored at least one intestinal parasite, which was associated to a positive serology (p < 0.05). The presence of pets within the houses, a previous history of pica or geophagia and the use of public places were also present in this population, but only the latter was associated to the serology (p < 0.05). In conclusion, clinical, serological, and epidemiological evidences for larval Toxocara infection were found in the studied population.

Human toxocariasis: a seroepidemiological survey in the Amazonian city of Yurimaguas, Peru

The aim of this study was to determine the seroprevalence of the infection by Toxocara in the general population of the Amazonian city of Yurimaguas, Peru. From March to August 2008, a total of 300 subjects were sampled and tested by means of a Toxocara ELISA-IgG test. A clinical and epidemiological questionnaire was used to assess the symptomatology and risk factors associated with human toxocariasis. The overall rate of seropositivity was 35.66%, with a significant high proportion in children (p < 0.001). The clinical evaluation revealed that 95.33% of the seropositive group had some type of symptomatology: headache (66.36%), respiratory compromise (63.55%), abdominal pain (54.21%), cutaneous signs (40.19%) and ocular manifestations (36.45%), and almost all of them were statistically significant (p < 0.001). Furthermore, 56.07% of the seropositive subjects presented at least one intestinal pathogen parasite with predominance of helminthes, but without significant association (p = 0.334). The analysis of risk factors showed only that the use of public places and geophagia exhibited a significant association with the seropositivity (p < 0.001). Clinical, serological and epidemiological findings associated to infection with Toxocara were observed in the present study and future studies should be done to assess this serious health problem.

Hipersonolência Diurna. Narcolepsia, Qual a Etiologia?

Introdução: A narcolepsia é uma doença do sono REM com desregulação do ciclo de sono-vigília, consequente sonolência diurna e eventual associação a alucinações hipnagógicas, paralisia do sono e cataplexia. A sua prevalência é de 0,05 a 0,02% no adulto mas desconhecida na idade pediátrica. Caso clínico: Criança de seis anos, previamente saudável com sonolência excessiva até 18 horas/dia e discinésia oromandibular, desequilíbrio na marcha e movimentos coreiformes dos membros superiores. Duas semanas antes realizara vacinação para a gripe pandémica. Registou-se ainda hiperfagia diurna e nocturna durante cinco dias com resolução espontânea, episódios de cataplexia perante riso e alterações emocionais e tremor da cabeça e dos membros superiores com melhoria clínica progressiva após oito dias. Realizou RMN-CE e EEG sem alterações. O exame líquido céfalo-raquidiano e PCR para painel de vírus herpes, Mycoplasma pneumoniae e enterovírus negativas. Nesta fase realizou polissonografia com teste de latências múltiplas do sono (TLMS) sem alterações. Exame cultural do exsudado faríngeo, TASO e anticorpo AntiDnase B negativos. Da exaustiva investigação que realizou apresentava serologias ELISA e WB compatíveis com infecção por Borrelia burdorferi, pelo que cumpriu ceftriaxone 14 dias. Serologias para influenza A mostraram IgM 39 UA/mL com IgG 194 UA/mL com segunda amostra com IgM 43 UA/mL e IgG 162 UA/mL (VR IgM<20;IgG<20). O estudo da autoimunidade revelou ANA 1/320, anticorpos anticardiolipina e antinucleares extraíveis negativos. Restantes autoanticorpos e doseamento de complemento normal. Anticorpos Anti-NMDA e VKCG negativos. Doseamento de hipocretina muito diminuído com HLA DR2 e DQB1*0602 presentes. A polissonografia com TLMS, sete meses após a primeira, confirmou sonolência excessiva com quatro inícios do sono REM sugestivos de narcolepsia. Faz terapêutica com metilfenidato, a sonolência diurna diminuiu e cumpre o seu horário escolar sem limitações. Comentários: O diagnóstico de narcolepsia foi sugerido pela clínica e confirmado pelo teste de latências múltiplas. O valor de hipocretina diminuído pode sugerir uma etiologia autoimune. Uma infecção como a borreliose ou a vacinação prévia para H1N1, responsabilizada por outros casos de narcolepsia podem ter sido desencadeantes de uma alteração imunitária responsável pela doença, nesta criança com a susceptibilidade HLA DR2 e DBQ1*0602.

Epidemiological investigation of an acute case of Chagas disease in an area of active transmission in Peruvian Amazon region

The study objective was to investigate an acute case of Chagas disease in the San Pedro de Shishita community, Pebas District, in the Peruvian Amazon basin, a non-endemic area. Both parents of the index case (acute case) were thoroughly interviewed, a seroepidemiological survey was carried out in the community, parasitological exams were carried out only in relatives of the index case, and triatomine bugs were searched for inside houses, peridomiciliary, and in wild environments. Seroprevalence for IgG anti-T. cruzi antibodies was 1/104 (0.96%), using an ELISA test and an indirect immunofluorescence assay. Panstrongylus geniculatus and Rhodnius pictipes adults were found. The index case is autochthonous from San Pedro de Shishita, but the source of transmission is unknown.

Preliminary report of HIV and Toxoplasma gondii occurrence in pregnant women from Mozambique

Toxoplasmosis, a protozoan disease, causes severe disease in fetuses during pregnancy and deadly encephalitis in HIV patients. There are several studies on its seroprevalence around the world, but studies focusing on African countries are limited in number and mostly anecdotal. We studied two groups of samples from Mozambique by ELISA, using serum samples from 150 pregnant women and six Cerebrospinal fluid (CSF) samples from AIDS patients with encephalitis. HIV status was confirmed, and CD4 blood counts were obtained from HIV-positive pregnant women. IgG seroprevalence of the group as a whole was 18.7% (28/150), with a higher prevalence in HIV-positive individuals compared to those who were HIV-negative (31.3%, [18/58] vs. 10.9%, [10/92]) patients. These data may be biased due to cumulative effects of exposition affecting disease prevalence. If corrected, this data may indicate an interaction of HIV and T. gondii. Prevalence of both diseases increases with age, but this is more clearly seen for toxoplasmosis (p < 0.005) than HIV infection, possibly explained by higher transmission of HIV after childhood. In HIV patients suffering from encephalitis, CSF serology showed that 33% of specific IgG CSF had a high avidity, which was in accordance with the data from the group of pregnant women. Lower prevalence rates of both infections in older groups could be explained by more deaths in the infected groups, resulting in an artificially lower prevalence. Using CD4 counts as a marker of time of HIV infection, and correcting for age, patients with contact with T. gondii had fewer CD4 cells, suggesting prolonged HIV disease or other causes. Toxoplasma IgG prevalence is higher in HIV+ groups, which could be ascribed to HIV- and T. gondii-associated risk factors, such as exposure to higher and more diverse social contacts. The low incidence of Toxoplasma IgG in younger age groups shows that transmission could be related to better access to cyst-containing meat in adulthood, as environmental transmission due to oocysts is usually blamed for higher incidence in children. Taken together, these data support the urgent need of research in toxoplasmosis in Africa, especially in the presence of HIV epidemics.

Discrepancies and consequences of indirect hemagglutination, indirect immunofluorescence and Elisa tests for the diagnosis of Chagas disease

Using the indirect hemagglutination (IH), indirect immunofluorescence (IIF) and enzyme linked immunosorbent assay (ELISA) tests for the diagnosis of Chagas disease, 4000 serum samples were examined. This study was conducted with different purposes: clinical interest, research support and parasitological monitoring of those patients with Chagas disease who were treated with heart transplantations. The tests occurred without patient selection and in accordance with the medical requests. The results showed discrepancies and brought about several questions, considering the different results that all three methods showed when considered together. What was found brought about concerns and we suggest the adoption of different measures, aiming to avoid these mismatches in the context of this disease.

Antibody levels to hantavirus in inhabitants of western Santa Catarina State, Brazil

Hantavirus cardiopulmonary syndrome (HCPS) is an infectious disease caused by hantaviruses of the family Bunyaviridae, and is transmitted by aerosols of excreta of infected rodents. The aim of the present study was to determine antibody levels to hantavirus in the population that lives at frontier of Brazil and Argentina. Participated of the study 405 individuals living in the municipalities of Bandeirante, Santa Helena, Princesa and Tunapolis, state of Santa Catarina, Brazil. IgG antibodies to hantavirus were analyzed in sera by an ELISA that uses a recombinant N protein of Araraquara hantavirus as antigen. The results were also confirmed by immunofluorescent test. Eight individuals showed antibodies to hantavirus (1.97% positivity), with serum titers ranging from 100 to 800. Six seropositives were males, older than 30 years and farmers. Our results reinforce previous data on hantavirus circulation and human infections in the southern border of Brazil with Argentina.

Case-control study of an outbreak of acute toxoplasmosis in an industrial plant in the state of São Paulo, Brazil

Foodborne diseases represent operational risks in industrial restaurants. We described an outbreak of nine clustered cases of acute illness resembling acute toxoplasmosis in an industrial plant with 2300 employees. These patients and another 36 similar asymptomatic employees were diagnosed with anti-T. gondii IgG titer and avidity by ELISA. We excluded 14 patients based on high IgG avidity and chronic toxoplasmosis: 13 from controls and one from acute disease other than T. gondii infection. We also identified another three asymptomatic employees with T.gondii acute infection and also anti-T. gondii IgM positive as remaining acute cases. Case control study was conducted by interview in 11 acute infections and 20 negative controls. The ingestion of green vegetables, but not meat or water, was observed to be associated with the incidence of acute disease. These data reinforce the importance of sanitation control in industrial restaurants and also demonstrate the need for improvement in quality control regarding vegetables at risk for T. gondii oocyst contamination. We emphasized the accurate diagnosis of indexed cases and the detection of asymptomatic infections to determine the extent of the toxoplasmosis outbreak.

THE SCHISTOSOMULA TEGUMENT ANTIGEN AS A POTENTIAL CANDIDATE FOR THE EARLY SEROLOGICAL DIAGNOSIS OF SCHISTOSOMIASIS MANSONI

If Schistosoma mansoni infection could be detected in its early stages, especially before the egg deposition in the host tissues, the development of severe pathologic lesions could be efficiently prevented. We therefore developed an indirect enzyme-linked immunosorbent assay based on the detection of specific IgG against schistosomula antigens (ELISA-SmTeg). The assay was applied in sera samples from non-infected and infected mice collected seven and 15 days post-infection. The results were compared to the number of adult worms obtained by perfusion of the murine hepatic system 50 days post-infection. The sensitivity and specificity of the ELISA-SmTeg were 100% (p = 0.0032 and 0.0048 respectively for seven and 15 days of infection) with a cutoff value of 0.15 (p = 0.0002). Our findings show a novel low-cost serological assay using antigens which are easy to obtain, which was able to detect all the infected mice as early as seven days post-infection.

IgG AVIDITY WESTERN BLOT USING Toxoplasma gondii rGRA-7 CLONED FROM NUCLEOTIDES 39-711 FOR SERODIAGNOSIS OF ACUTE TOXOPLASMOSIS

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.

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.