Potential immunological markers for diagnosis of human strongyloidiasis using heterologous antigens.

Strongyloides venezuelensis is a parasitic nematode of rodents that is frequently used to obtain heterologous antigens for immunological diagnosis of human strongyloidiasis. The aim of this study was to identify antigens from filariform larvae of S. venezuelensis for immunodiagnosis of human strongyloidiasis. Soluble and membrane fractions from filariform larvae of S. venezuelensis were obtained in phosphate saline (SS and SM) and in Tris-HCl buffer (TS and TM), and were analysed by Western blotting. Different antigenic components were recognized by IgG antibodies from the sera of strongyloidiasis patients. Highest recognition was observed for a 30-40 kDa mass range present in all antigenic fractions. The band encompassing this mass range was then excised and subjected to mass spectrometry for protein identification. Immunoreactive proteins identified in the soluble fractions corresponded to metabolic enzymes, whereas cytoskeletal proteins and galectins were more abundant in the membrane fractions. These results represent the first approach towards identification of S. venezuelensis antigens for use in immunodiagnostic assays for human strongyloidiasis.

Diagnosis of the strongyloid nematode Strongyloides venezuelensis in experimentally infected rats.

Strongyloides venezuelensis is an intestinal nematode of rats, frequently used as a model for studying human and animal strongyloidiasis. In the present study, we evaluated parasitological, serological and molecular methods for the diagnosis of experimental S. venezuelensis in rats, Rattus norvegicus. Blood and faecal samples were collected and analysed up to 60 days post infection (pi) with adult worm recovery occurring from 5 to 45 days pi. Using an enzyme-linked immunosorbent assay (ELISA), serum levels of IgG antibodies increased up to 28 days pi, thereafter decreasing by day 60 pi. Polymerase chain reaction (PCR) assays detected S. venezuelensis DNA in faecal samples of rats from 5 to 21 days pi. The present study therefore represents the first step towards improving the diagnosis of experimental strongyloidiasis.

Behavioural changes and muscle strength in Rattus norvegicus experimentally infected with Toxocara cati and T. canis.

Toxocara canis and Toxocara cati are nematode parasites in dogs and cats, respectively, transmitted by ingestion of embryonated eggs, transmammary and transplacental (T. canis) routes and paratenic host predation. Many parasites use mechanisms that change the behaviour of their hosts to ensure continued transmission. Several researchers have demonstrated behavioural changes in mouse models as paratenic hosts for T. canis. However, there have been no studies on behavioural changes in laboratory rats (Rattus norvegicus) experimentally infected with T. cati. This study investigated behavioural changes and muscle strength in male and female rats experimentally infected with T. cati or T. canis in acute and chronic phases of infection. Regardless of sex, rats infected with T. cati showed a greater decrease in muscle strength 42 days post infection compared to rats infected with T. canis. However, behavioural changes were only observed in female rats infected with T. canis.

Conventional PCR for molecular diagnosis of human strongyloidiasis.

Strongyloidiasis is frequently asymptomatic and diagnosis of latent infection is difficult due to limitations of current parasitological and serological methods. This study aimed to verify the use of conventional polymerase chain reaction (PCR) assay for molecular diagnosis of Strongyloides stercoralis infection. Fresh stool samples were obtained from 103 individuals: 33 S. stercoralis positive, 30 positive for other parasites and 40 negative for parasitological methods. These samples were examined by the Lutz, Rugai and agar plate culture methods and conventional PCR assay. Two sets of primers (S. stercoralis species-specific and genus-specific sets), located in the 18S ribosomal RNA gene, were used for PCR. Of the 33 samples positive for S. stercoralis by parasitological methods, 28 (84.8%) were also detected by PCR assay using species-specific primers and 26 (78.8%) using genus-specific primers. Among the stool samples negative by parasitological methods, seven (17.5%) were positive by PCR using species-specific primers and two (5.0%) using genus-specific primers. In conclusion, the conventional PCR assay described in this study using a species-specific primer pair provided a molecular method for S. stercoralis diagnosis in human stool samples.

Schistosomiasis mansoni -- clinical features.

In the present review, we will discuss the Schistosoma mansoni form, which is the most widely distributed schistosome in humans and is found both in the Old and New Worlds. The main features of the natural history of mansonic schistosomiasis are reviewed, with emphasis on the clinical forms of the disease, their diagnosis and treatment.

Repeated mass treatment of schistosomiasis mansoni: experience in hyperendemic areas of Brazil. 3. Techniques for assessment and surveillance.

Severe morbidity due to schistosomiasis mansoni is now quite rare in Brazil; thus it is proposed that surveillance plus selective and multidisciplinary intervention limited to areas at high risk be the next step. Such a policy has to be carried out periodically and on a state-wide scale, and therefore will have to rely upon cheap and time-saving sampling procedures. Clinical features cannot at present be used as indicators, with the exception of grossly enlarged livers, which are more frequent in areas at high risk. Overall prevalence rates for a county or even city may also be misleading, even if broken down into units of smaller size. Thus, egg counts in the young age groups remain the best indicators for the detection of areas at high risk (i.e., the main foci of transmission). Egg counts performed on pooled specimens adequately reflect the risk rating of a particular neighbourhood, and have a favourable cost-benefit ratio. In addition, it is suggested that field personnel be trained in the empirical recognition of potential foci; such 'screening by suspicion' has been tested and found to be reliable.

Schistosomiasis mansoni in the "Baixada Ocidental Maranhense", state of Maranhão, Brazil: cross-sectional studies performed in 1987 and 1993.

A cross-sectional study on the prevalence of schistosomiasis mansoni in three sites of the "Baixada Ocidental Maranhense" was carried out in 1993 in: Alegre (in the municipality of São Bento), Aliança (in Cururupu) and Coroatá II (in the municipality of São João Batista). Results were compared to those of another study performed at the same sites and in similar conditions, in 1987. The entire population of the three sites, with few exceptions, was submitted to fecal tests using the Kato-Katz method and immediate intradermal tests for schistosomiasis in both studies. Subjects with positive results in one of these tests were clinically evaluated by a physical examination. In 1993, the total of 827 subjects were submitted to fecal examination and 826 to intradermal test. Schistosoma mansoni eggs were found in the feces of 154 (18.6%) subjects, while 478 (57.9%) subjects presented a positive intradermal test. Stool examination was carried out in 367 subjects in Alegre with a positivity rate of 14.9%; the intradermal test, performed in 366 subjects, was positive in 47.5% of the cases. In Aliança, 277 subjects had their feces examined and were submitted to an intradermal test, with a positivity rate of 34.4% and 70.7%, respectively. Finally in Coroatá II, 183 inhabitants submitted to fecal and intradermal tests had positivity rates of 2.2% and 59.0%, respectively. When the present data were compared to those obtained in the survey performed in 1987, a significant decrease in the prevalence of infection by S. mansoni was observed in Alegre and Coroatá II, and a prevalence increase in Aliança.

[Giardiasis in nurseries supported by the São Paulo Municipal Prefecture, 1982/1983]. / Giardíase em creches mantidas pela prefeitura do Município de São Paulo, 1982/1983.

Parasitological analysis was carried out during 1982 and 1983 in 3,076 children aged under 7 years, who were enrolled in 52 day nurseries supported by Municipal Prefecture of São Paulo and 50 food handlers (cooking employees) working at 18 day nurseries. The prevalence of Giardia lamblia infection was studied according to sex, age group and seasonal variations, and attained 32.80% among children and 4.0% among adult population. The occurrence of giardiasis was most frequent in age group corresponding to 1-4 years old. The study of seasonal variations showed that, in 1982, the prevalence of G. lamblia was high in the autumn and in the spring and, in 1983, giardiasis was more prevalent in the autumn and in the summer. Statistical analysis indicated non association between sex and prevalence for Giardia infection, and there was also no significative difference on the distribution of G. lamblia prevalence in the four regions analysed in the present study (p > 0.05). The most probable mode of acquisition of Giardia infection, in the nurseries analysed in the present study was person-to-person, as factors, like environmental contamination and others, associated with the spreading of giardiasis through water, reported in other countries, are unlike from those verified in our Municipality. On the other hand, there is evidence of no significant role of food handlers in Giardia transmission, because of the rate of infection in this group was very low.

Effect of wide spectrum anti-helminthic drugs upon Schistosoma mansoni experimentally infected mice.

Mebendazole, albendazole, levamisole and thiabendazole are well known as active drugs against several nematode species, and against cestodes as well, when the first two drugs are considered. None of the drugs have proven activity, however, against trematodes. We tested the effect of these drugs on the fecal shedding of schistosome eggs and the recovering of adult schistosomes, after portal perfusion in Schistosoma mansoni experimentally infected mice. Balb/c mice infected with 80 S. mansoni cercariae were divided into three groups, each in turn subdivided into four other groups, for each tested drug. The first group was treated with each one of the studied drugs 25 days after S. mansoni infection; the second group was submitted to treatment with each one of the drugs 60 days after infection. Finally, the third group, considered as control, received no treatment. No effect upon fecal shedding of S. mansoni eggs and recovering of schistosomes after portal perfusion was observed when mice were treated with either mebendazole or albendazole. Mice treated with either levamisole or thiabendazole, on the other hand, showed a significant reduction in the recovering of adult schistosomes after portal perfusion, mainly when both drugs were given during the schistosomula evolution period, i.e., 25 days after cercariae penetration, probably due to unspecific immunomodulation.

Persistence of specific antibody response in different experimental infections of mice with Toxocara canis larvae.

Anti-Toxocara antibody production and persistence were studied in experimental infections of BALB/c mice, according to three different schedules: Group I (GI)-25 mice infected with 200 T. canis eggs in a single dose; Group II (GII) 25 mice infected with 150 T. canis eggs given in three occasions, 50 in the 1st, 50 in the 5th and 50 in the 8th days; Group III (GIII)-25 mice also infected with 150 T. canis eggs, in three 50 eggs portions given in the 1st, 14th and 28th days. A 15 mice control group (GIV) was maintained without infection. In the 30th, 50th, 60th, 75th, 105th and 180th post-infection days three mice of the GI, GII and GIII groups and two mice of the control group had been sacrificed and exsanguinated for sera obtention. In the 360th day the remainder mice of the four groups were, in the same way, killed and processed. The obtained sera were searched for the presence of anti-Toxocara antibodies by an ELISA technique, using T. canis larvae excretion-secretion antigen. In the GI and GII, but not in the GIII, anti-Toxocara antibodies had been found, at least, up to the 180th post-infection day. The GIII only showed anti-Toxocara antibodies, at significant level, in the 30th post-infection day.

Human parasitism by Phagicola sp (Trematoda, Heterophyidae) in Cananéia, São Paulo state, Brazil.

We report one case of parasitism by Phagicola sp. (Trematoda, Heterophyidae) in a 31 year-old woman who, in 1987, travelled and stayed several months in the municipality of Cananéia (SP), where she ingested, in various occasions, raw mullet (Mugil sp.). The patient referred mild intestinal pain and laboratory examinations showed eggs of Phagicola sp. in the stools and a slight increase in eosinophil blood levels (8%). After treatment with praziquantel (75 mg/kg per day for three days) all the symptoms and signs disappeared. This is, certainly, the first record of human infection by Phagicola sp. in Brazil and, perhaps, in countries other than the U.S.A. where unclear references to a few human cases were reported in the South-eastern region.

Hepatitis B and C virus markers among patients with hepatosplenic mansonic schistosomiasis.

PURPOSE: To evaluate the frequency and the consequences of the co-infection of hepatitis B and C viruses in patients with hepatosplenic schistosomiasis (HSS). METHODS: B and C serologic markers, exposure to risk factors, biochemical assays, upper gastrointestinal endoscopies, and abdominal ultrasonograms were evaluated in 101 patients with HSS from 1994 to 1997. Whenever possible, PCR was tested and histopathological studies were reviewed. RESULTS: At least one HBV virus marker was found in 15.8%, and anti-HCV was detected in 12.9% of the subjects. The seropositive subjects tended to be older than the seronegative ones. A history of blood transfusion was significantly related to the presence of anti-HCV. Three (18.75%) out of 16 subjects exposed to B virus were HBsAg positive. Eleven (84.6%) out of thirteen patients who were anti-HCV positive demonstrated viral activity. Patients with ongoing viral infection presented a higher average level of liver aminotransferases, a higher frequency of cell decompensation and a higher rate of chronic hepatitis. Portal hypertension parameters were not influenced by viral exposure. CONCLUSIONS: The rate of hepatitis B and C viruses serologic markers observed in the patients with HSS was higher than the control group. The co-infection was responsible for a higher frequency of cell decompensation.

Autochthonous acute Chagas' disease in São Paulo State, Brazil: epidemiological aspects.

Since the beginning of the seventies the natural transmission of Chagas' infection has been considered to be under control in the State of São Paulo and not even a case of American Trypanosomiasis, transmitted by triatomine bugs, has been detected by the epidemiological surveillance system. This situation justifies the report of a case of acute Chagas' disease that occurred in a forest area considered free of domiciliary triatomines along the Southern seacoast of São Paulo State. In May, 1995 the presence of trypomastigote forms of Trypanosoma cruzi had been diagnosed in a retired 57 year-old male patient, born and living in Santos (São Paulo State), complaining of fever, fatigue and malaise. The patient reported that 40 days before he had participated with 17 friends in a 7-day excursion in a forest area of the municipalities of Itanhaém and Peruíbe. During this period the group had been lodged in three houses located within the forest. Eight days after the end of the excursion the patient began to have fever, malaise and fatigue. During the next 31 days he had received medical care both as an inpatient and an outpatient, without any significant improvement. After the detection of T. cruzi trypomastigotes in his blood stream the patient began to be treated with benzonidazole in a hospital but died 8 days after the beginning of treatment. The epidemiological investigation carried out showed no signs of the presence of triatomine bugs in the three houses where the group had been lodged, or any indication of Chagas' infection in other excursionists.

Visceral larva migrans: a seroepidemiological survey in five municipalities of São Paulo state, Brazil.

An enzyme-linked immunosorbent assay (ELISA), employing antigens from Toxocara canis larvae and the absortion of suspected sera with Ascaris lumbricoides extracts was used in a seroepidemiological study performed in five municipalities of São Paulo State, Brazil (São Paulo, Campinas, Santos, Marília and Presidente Prudente) in order to determine the frequency of antibodies to Toxocara. In 2,025 blood samples collected, 806 proceeded from male subjects and 1,219 from females; 483 samples were collected from subjects under 15 years of age and the remaining 1,542 from subjects aged 15 years or over. Among the 2,025 sera investigated, 3.60% had antibodies to Toxocara at significant levels. A moderate predominance of infection with Toxocara among male subjects (3.72%) was observed, although the difference was not statistically significant when this rate was compared with that for female (3.28%). Related to age, a higher frequency of positive results was detected among subjects under 15 years (6.41%) against the older group (2.53%). A trend of more elevated rates of infection was observed in municipalities with high demographic densities (São Paulo, Campinas and Santos). Nevertheless, such findings only appeared to be statistically significant in subjects younger than 15 years.

Standardization of dot-ELISA for the serological diagnosis of toxocariasis and comparison of the assay with ELISA.

The dot-enzyme-linked immunosorbent assay (dot-ELISA) was standardized using somatic (S) and excretory-secretory (ES) antigens of Toxocara-canis for the detection of specific antibodies in 22 serum samples from children aged 1 to 15 years, with clinical signs of toxocariasis. Fourteen serum samples from apparently normal individuals and 28 sera from patients with other pathologies were used as controls. All samples were used before and after absorption with Ascaris suum extract. When the results were evaluated in comparison with ELISA, the two tests were found to have similar sensitivity, but dot-ELISA was found to be more specific in the presence of two antigens studied. Dot-ELISA proved to be effective for the diagnosis of human toxocariasis, presenting advantages in terms of yield, stability, time and ease of execution and low cost.

[Cryptosporidium parasitism in children with acute diarrhea]. / Parasitismo por Cryptosporidium sp. em crianças com diarréia aguda.

During the period from August 1987 to July 1990, 241 fecal samples collected from 1 to 48 months old children with acute diarrhea and examined in the Child's Institute of Hospital das Clínicas of Faculdade de Medicina da Universidade de São Paulo, were submitted to parasitological tests in the Enteroparasitosis Section of the Instituto Adolfo Lutz. Fourty two (17.43%) fecal samples showed Cryptosporidium sp. oocysts by carbol-fucsin stain. Parasitism by Cryptosporidium sp. was more frequent during the period comprised from March to May, in the studied three years. The authors discussed the Cryptosporidium sp. in association with other agents.

[Infection by Clonorchis sinensis in Asian immigrants in Brazil. Treatment with praziquantel]. / Infecção por Clonorchis sinensis em imigrantes asiáticos no Brasil. Tratamento com praziquantel.

Fifteen adult patients with assymptomatic infection due to Clonorchis sinensis, diagnosed by coprological examination, were studied. They all came from Asia (twelve from Taiwan, two from South Korea and one from Hong Kong) and were examined at the Adolfo Lutz Institute and the Department of Infectious Diseases, School of Medicine, University of São Paulo, in São Paulo, Brazil. Six patients were women and nine men. All studied patients were admitted to hospital and treated with praziquantel (60 mg/kg). Previous to treatment and on the 15th, 30th and 60th days after praziquantel administration, patients were submitted to quantitative stool examinations, according to Kato-Katz's technique and to hematological and biochemical serum analysis. After a 60 day follow-up nine patients (60%) were negative for C. sinensis eggs in stools. Those not cured after praziquantel administration (six patients, 40%) revealed a sharp decline in faecal elimination of C. sinensis eggs.

Evaluation of anti-Schistosoma mansoni IgG antibodies in patients with chronic schistosomiasis mansoni before and after specific treatment.

The circumoval precipitin test (COPT), enzyme-linked immunosorbent assay (ELISA) and the immunoblotting anti-adult worm antigen (AWA) and soluble egg antigen (SEA) tests were applied to 17 chronically schistosome-infected patients for the detection of anti-Schistosoma mansoni antibodies before and on four occasions after oxamniquine administration over a period of six months. Compared to a control group, schistosomiasis patients showed high levels of IgG antibodies in AWA and SEA-ELISA. A decrease in IgG levels was observed six months after treatment, although negative reactions were not obtained. Significant decreases in IgG1, IgG3 and, mainly, IgG4, but not anti-SEA IgG2 levels were observed six months after treatment, again without negativity. Analysis of anti-AWA IgG antibodies by immunoblotting before treatment showed a 31 kDa strand in 14 patients (82%) which disappeared in three cases up to six months after treatment; furthermore, anti-SEA IgG antibodies showed the same band in nine patients (53%) before treatment, which disappeared in only four cases up to six months after treatment.

Gelatin particle indirect agglutination test for serodiagnosis of schistosomiasis: comparative study with enzyme-linked immunosorbent assay.

A new serological test, the gelatin particle agglutination test (GPAT), was used for the serodiagnosis of schistosomiasis mansoni. This technique showed the sensitivity (90.6%) and specificity (97.8%) close to those of enzyme-linked immunosorbent assay. The GPAT can be easily and rapidly performed without specialized equipment, by using lyophilized antigen-coated gelatin particles. The test also seems to be useful for mass screening of Schistosoma infection in field conditions.

[Immunodiagnosis of human leptospirosis by ELISA-IgM, employing different antigenic preparations from prevalent serovars of Leptospira interrogans]. / Imunodiagnóstico da leptospirose humana através do teste ELISA-IgM, empregando-se diferentes preparações antigênicas a partir de sorotipos prevalentes de Leptospira interrogans.

A comparative study among different serovars of Leptospira interrogans was performed in order to prepare antigens to detect IgM antibodies by ELISA in early and late phase of human leptospirosis. Ten serovars were chosen among the most prevalent detected by microscopic seroagglutination (SAM) in São Paulo city. Using ELISA-IgM five of them showed better results (canicola, hebdomadis, icterohaemorrhagiae, cynopteri and brasiliensis). These ones were also studied in a pool. The non-treated antigens showed higher reactivity than the Triton X-100 (4%/50 degrees C/4h). ELISA-IgM using individually or pool of non-treated antigens proved to be reliable with high sensitivity and should be used for an earlier diagnosis of leptospirosis, as a trial test. Faster diagnostic elucidation can be useful to detect epidemic situations, so, allowing epidemiological surveillance interventions.