Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (Viannia) braziliensis

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100% positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100% positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100% positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95% of Kala-azar and 35% of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15% of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

Predictive value of serologic tests in the diagnosis and follow-up of patients with paracoccidioidomycosis

A serologic study was undertaken in a group of 43 patients with active paracoccidioidomycosis who were treated in the same form (ketoconazole), for identical periods of time (6 months), and folio wed-up for various periods posttherapy. The tests employed were agar gel immunodiffusion (AGID) and complement fixation (FC). Also studied were 50 sera from patients with proven histoplasmosis and pulmonary aspergilloma, 30 patients with culturaly proven tuberculosis as well as 92 specimens from healthy individuals, residents in the endemic area for paracoccidioidomycosis. A single lot of yeast filtrate antigen was used throughout the study. The value of each test was measured according to GALEN and GAMBINO6. Both tests were highly sensitive, 89 and 93% respectively. Regarding their specificity, the AGID was totally specific while the CF exhibited 96.6% and 97% specificity in front of tuberculosis patients and healthy individuals respectively and 82% in comparison with patients with other mycoses. The concept of predictive value, that is, the certainty one has in accepting a positive test as diagnostic of paracoccidioidomycosis, favored the AGID procedure (100%) over the CF test. The latter could sort out with 93% certainty a patient with paracoccidioidomycosis among a group of healthy individuals and with 97.5% in the case of TB patients; when the group in question was composed by individuals with other deep mycoses, such certainty was lower (81%). The above results indicate that both the AGID and the CF tests furnish results of high confidence; one should not relay, however, in the CF alone as a means to establish the specific diagnosis of paracoccidioidomycosis.

Use of saprophytic leptospira strains in the serodiagnosis of experimental leptospirosis in guinea-pigs (Cavia sp)

The efficiency of four Leptospira biflexa strains (Buenos Aires, Patoc 1, Rufino and São Paulo) as single antigen in the serodiagnosis in guinea-pigs experimentally infected with seven Leptospira interrogans serovars (canicola, grippotyphosa, hardjo, icterohaemorrhagiae, pomona, tarassovi and wolffi) was evaluated by the microscopic agglutination test. The four saprophytic strains were not able to reveal antibody titres in sera of guinea-pigs experimentally infected with Leptospira interrogans. Serological cross-reactions were observed between strains Patoc 1 and São Paulo and between serovars wolffi and hardjo.

Monoclonal antibody to serotype 17 of Neisseria meningitidis and their prevalence in Brazilian States

Neisseria meningitidis are gram-negative diplococci responsible for cases of meningococcal disease all over the world. The epidemic potential of N. meningitidis serogroup B and C is clearly a function of their serotype antigens more than of their capsular polysaccharides. Until recently, hiperimmune sera were used to detect typing antigens on the bacteria. The advent of monoclonal antibodies (MAbs) offered the opportunity to eliminate many of the cross-reactions and have improved the accuracy and reproducibility of meningococcal serotyping. We have produced a MAb to the outer membrane protein of the already existent serotype 17 that have been detected by the use of hiperimmune rabbit sera. The prevalence of this serotype epitope is low in the Brazilian strains. By using the MAb 17 we could not decrease the percentage of nontypeable serogroup C strains. However, there were a decreasing in nontypeable strains to 13% into serogroup B strains and to 25% into the other serogroups.

Chagas' disease in the Brazilian Amazon: II. A serological survey

A serological survey, involving indirect immunofluorescence testing of blood sera samples, was carried out on the residents of one in every five dwellings in the town of Barcelos (in the northern part of the State of Amazonas, on the right bank of the Rio Negro, 490 Km from Manaus by river) and on the rural populations of the villages of Piloto and Marará (also on the right bank of the Rio Negro, 30 minutes by boat from Barcelos). A total of 710 sera samples were tested, 628 from the resident population in the town of Barcelos, 35 from Piloto and 47 from Marará. The tests were carried out using human anti-gammaglobulin type IgG (Biolab) and antigen from formolized culture of T.cruzi Y strain. The sera were serially diluted from 1:40 to 1:320 in PBS 7.2. Of the 710 samples examined 89(12.5%) were positive for anti-T.cruzi antibodies: 2 of these (2.2%) at a dilution of 1:320; 12(13.4%) at 1:160; 38 (42.6%) at 1:80; and the remainder at 1:40, giving a median serological dilution of 1:80. The following questions are discussed: the high serological prevalence for Chagas'infection found in our survey; the possibility of serological cross-reactions; the need for confirmatory tests for the positives reactions; and the strong correlation between our results and preliminary epidemiological data (such as the level of human contact with wild triatominae, know locally as "Piacava's lice". We draw attention to the isolation by xenodiagnosis of one strain of T.cruzi from a patient with positive serology for Chagas' infection.

ELISA test for the diagnosis of cysticercosis in pigs using antigens of Taenia solium and Taenia crassiceps cysticerci

In the present study ELISA was standardized for the diagnosis of swine cysticercosis based on necropsy parameters and confirmed positive and negative control sera. Serum samples from pigs with other infections were also assayed to determine possible cross-reactions. Four antigens were assayed: from Taenia crassiceps vesicular fluid (VF-Tcra) and crude larvae extract (T-Tcra), and from Taenia solium extracts of scolex (S-Ts) and of larvae (T-Ts). A checkerboard evaluation of antigen, serum and conjugate dilutions, as well as the use of Tween-20 and skim cow milk in wash and blocking solution had a marked effect on improving ELISA performance. All the antigens showed a good performance, but VF-Tcra was the best, with 96.0% and 80.0% sensitivities for cut-offs respectively at 2sd and 3sd, and corresponding specificities of 97.5% and 100.0%. Cross-reactivity was observed only with hydatidosis and ascaridiosis. In view of the high performance observed, the ELISA test should be recommended for the diagnosis of cysticercosis in suspected swine in slaughterhouses and for the screening of cysticercosis in swine production. These results will support integrated measures of cysticercosis control throughout the chain of swine production, effectively contributing to public health.

Borrelia burgdorferi antibodies in dogs from Cotia county, São Paulo State, Brazil

Dogs sera samples collected from Cotia County, São Paulo were tested using indirect immunoenzymatic test (ELISA) in order to study Lyme disease serology in dogs. ELISA method was standardized and G39/40 North American strain of Borrelia burgdorferi was used as antigen. Positive results were confirmed employing the Western blotting technique. Because of the possibility of cross-reactions, sera were also tested for different serological strains of Leptospira interrogans and L. biflexa using microscopic sera agglutination test. Twenty-three of 237 (9.7%) serum samples were positive in the ELISA; 20 of them (86.9%) were confirmed by the Western blotting, what suggests that Cotia may be a risk area for Lyme disease. Although 4 samples (1.7%) were positive for Lyme disease and leptospirosis, no correlation was found between the results (X² = 0.725; p = 0.394) what suggests absence of serological cross reactivity.

Serotyping and evaluation of the virulence in mice of Streptococcus suis strains isolated from diseased pigs

A total of 110 strains of Streptococcus suis, isolated from diseased pigs in Brazil were serotyped and analyzed for virulence. Serotyping of the strains resulted in the following classification: 42 strains of serotype 2 (38.2%), 10 strains of serotype 14 (9.1%), seven strains of serotype 9 (6.4%), three strains each of serotype 7 and 11 (2.7%), two strains each of serotype 1 and 8 (1.8%) and one strain each of serotypes ½, 3, 5, 6 and 10 (0.9%). Cross reactions among serotypes 1, 14 and 7 were observed in 21 strains (19.1%). Only 41.9% of the strains were lethal for mice using the pathogenicity test.

Sharing of antigens between Plasmodium falciparum and Anopheles albimanus

The presence of common antigens between Plasmodium falciparum and Anopheles albimanus was demonstrated. Different groups of rabbits were immunized with: crude extract from female An. albimanus (EAaF), red blood cells infected with Plasmodium falciparum (EPfs), and the SPf66 synthetic malaria vaccine. The rabbit's polyclonal antibodies were evaluated by ELISA, Multiple Antigen Blot Assay (MABA), and immunoblotting. All extracts were immunogenic in rabbits according to these three techniques, when they were evaluated against the homologous antigens. Ten molecules were identified in female mosquitoes and also in P. falciparum antigens by the autologous sera. The electrophoretic pattern by SDS-PAGE was different for the three antigens evaluated. Cross-reactions between An. albimanus and P. falciparum were found by ELISA, MABA, and immunoblotting. Anti-P. falciparum and anti-SPf66 antibodies recognized ten and five components in the EAaF crude extract, respectively. Likewise, immune sera against female An. albimanus identified four molecules in the P. falciparum extract antigen. As far as we know, this is the first work that demonstrates shared antigens between anophelines and malaria parasites. This finding could be useful for diagnosis, vaccines, and the study of physiology of the immune response to malaria.

Clinical and serological evidence of Toxocara infection in school children from Morrope District, Lambayeque, Peru

The aim of this study was to estimate the frequency of human toxocariosis in a child population from Morrope district, Lambayeque, Peru. From October to December 2005, 182 school children (96 male and 86 female) were studied. Blood samples were collected for Toxocara ELISA-IgG test and hematological examination. Additionally, stool samples were collected for coproparasitological examination to check cross reactions. We found frequency of positives in 32.4% (59/182) with a significant higher proportion of positivity in male children (p < 0.00001). 71.2% of the children with positive serology (52 male and seven female), were between five and 10 years old, 77.96% had respiratory symptoms, 61.02% had ocular manifestations, 38.98% had hepatic symptoms, 38.98% had mild or moderate eosinophilia, signs statistically associated with seropositivity. 83.5% of studied population had some intestinal parasite, such as: Blastocystis hominis (53.3%), Giardia lamblia (31.3%), Entamoeba coli (29.1%), Entamoeba histolytica/E. dispar (1.1%), Hymenolepis nana (5.49%), and Ascaris lumbricoides (3.3%), but they had not any association with serology results. The ownership of dogs or/and cats were significantly associated with seropositivity to anti-Toxocara antibodies although the presence of such pets within the house was not. In conclusion, clinical and serological evidence of Toxocara infection exists in the studied population.

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.

Eclectic feeding behavior of Lutzomyia (Nyssomyia) intermedia (Diptera, Psychodidae, Phlebotominae) in the transmission area of American cutaneous leishmaniasis, state of Paraná, Brazil

Introduction The blood meal source of sandflies provides valuable information about the vector/host interaction and allows for an understanding of American cutaneous leishmaniasis (ACL) transmission mechanisms. The aim of this study was to identify the blood meal sources of Lutzomyia (Nyssomyia) intermedia in an endemic area of leishmaniasis in Brazil's State of Paraná using a precipitin test. Methods Sandflies were collected in the rural locality of Epitácio Pessoa within the City of Adrianópolis, State of Paraná, in southern Brazil. A total of 864 female sandflies were captured, and 862 (99.8%) were identified as L. intermedia species. However, two unidentified specimens were considered to be part of the genus Lutzomyia. Results Among the females examined, 396 specimens presented reactions to a certain type of tested antiserum, and most (67.9%) reacted to the simple type. These sandflies fed mainly on the blood of birds, opossums, and rodents, but specimens that fed on the blood of humans, dogs, horses, cattle, and cats were also found. Among the cross-reactions found (32.1%), bird/rodent, bird/opossum, bird/dog, bird/human, and horse/dog cross-reactions were the most common. Conclusions These results demonstrate a tendency in the eclectic feeding behavior of L. intermedia and support its potential role as a vector for ACL in the study area.

Serodiagnosis of chronic Chagas infection by using EIE-Recombinant-Chagas-Biomanguinhos kit

A kit based on an enzyme immunoassay, EIE-Recombinant-Chagas-Biomanguinhos, developed by the Oswaldo Cruz Foundation, was evaluated for the serodiagnosis of chronic Chagas disease. Evaluation was performed with 368 serum samples collected from individuals living in an endemic area for Chagas disease: 131 patients in the chronic phase with confirmed clinical, epidemiological, and serological diagnosis (indirect immunofluorescence, indirect hemagglutination or enzyme-linked immunosorbent assay) and 237 nonchagasic seronegative individuals were considered negative control. The EIE-Recombinant-Chagas-Biomanguinhos kit showed high sensitivity, 100% (CI 95%: 96.4-100%) and high specificity, 100% (CI 95%: 98-100%). The data obtained were in full agreement with clinical and conventional serology data. In addition, no cross-reaction was observed with sera from patients with cutaneous (n=14) and visceral (n=3) leishmaniasis. However, when these sera were tested by conventional serological assays for Chagas disease, cross-reactions were detected in 14.3% and 33.3% of the patients with cutaneous and visceral leishmaniasis, respectively. No cross-reactions were observed when sera from nonchagasic seronegative patients bearing other infectious disease (syphilis, n=8; HTLV, n=8; HCV, n=7 and HBV, n=12) were tested. In addition, sera of patients with inconclusive results for Chagas disease by conventional serology showed results in agreement with clinical evaluation, when tested by the kit. These results are relevant and indicate that the refered kit provides a safe immunodiagnosis of Chagas disease and could be used in blood bank screening.