Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cássia dos Coqueiros, SP) determined by ELISA and immunoblotting using Taenia crassiceps antigens

Considering the impact of cysticercosis on public health, especially the neurologic form of the disease, neurocysticercosis (NC), we studied the frequency of positivity of anti-Taenia solium cysticercus antibodies in serum samples from 1,863 inhabitants of Cássia dos Coqueiros, SP, a municipal district located 80 km from Ribeirão Preto, an area considered endemic for cysticercosis. The 1,863 samples were tested by enzyme linked immunosorbent assay (ELISA) using an antigenic extract from Taenia crassiceps vesicular fluid (Tcra). The reactive and inconclusive ELISA samples were tested by immunoblotting. Of the 459 samples submitted to immunoblotting, 40 were strongly immunoreactive to the immunodominant 18 and 14 kD peptides. Considering the use of immunoblotting as confirmatory due to its high specificity, the anti-cysticercus serum prevalence in this population was 2.1%.

Heterologous antigen extract in ELISA for the detection of human IgE anti-Strongyloides stercoralis

Strongyloides ratti larval extract was used for the standardization of ELISA to detect genus-specific IgE in human strongyloidiasis. Forty serum samples from monoinfected patients shedding S. stercoralis larvae (Group I), 40 from patients with other intestinal parasites (Group II), and 40 from copronegative healthy subjects (Group III) were analyzed. Genus-specific IgE levels (ELISA Index: EI) were significantly higher in the group I (EI = 1.43) than groups II (EI = 0.70) and III (EI = 0.71), showing positivity rates of 55%, 2.5% and 0%, respectively. Similarly, sera from copropositive patients had significantly higher levels of total IgE (866 IU/mL) as compared to those from group II (302 IU/mL) and III (143 IU/mL). A significant positive correlation was found between levels of Strongyloides specific-IgE and total IgE in sera from patients with strongyloidiasis. In conclusion, S. ratti heterologous extract showed to be a useful tool for detecting genus-specific IgE by ELISA, contributing for a better characterization of the immune response profile in human strongyloidiasis.

Immunoprecipitation techniques and Elisa in the detection of anti-Fonsecaea pedrosoi antibodies in chromoblastomycosis

Chromoblastomycosis (CBM) is a chronic subcutaneous infection caused by several dematiaceous fungi. The most commonly etiological agent found in Brazil is Fonsecaea pedrosoi, which appears as thick walled, brownish colored cells with transverse and longitudinal division in the lesions, called "muriform cells". This disease is found worldwide but countries like Madagascar and Brazil have highest incidence. Diagnosis is made by clinical, direct and histopathologic examination and culture of specimens. Serological tests have been used to identify specific antibodies against Fonsecaea pedrosoi antigens, as well as immunotechniques have been used for CBM serological identification and diagnosis. In the present study double immunodiffusion (DID), counterimmunoelectrophoresis (CIE) and immunoenzymatic test (ELISA) have been used to evaluate humoral immune response in patients with CBM caused by F. pedrosoi. Metabolic antigen was used for immunoprecipitation tests (DID and CIE) while somatic antigen for ELISA. Our results demonstrated 53% sensitivity and 96% specificity for DID, while CIE presented 68% sensitivity and 90.5% specificity. ELISA demonstrated 78% sensibility and 83% specificity. Serological tests can be a useful tool to study different aspects of CBM, such as helping differential diagnosis, when culture of the pathogenic agent is impossible.

Evaluation of an in-house specific immunoglobulin G (IgG) avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection

This article describes the standardization and evaluation of an in-house specific IgG avidity ELISA for distinguishing recent primary from long-term human cytomegalovirus (HCMV) infection. The test was standardized with the commercial kit ETI-CYTOK G Plus (Sorin Biomedica, Italy) using 8 M urea in phosphate-buffered saline to dissociate low-avidity antibodies after the antigen-antibody interaction. The performance of the in-house assay was compared to that of the commercial automated VIDAS CMV IgG avidity test (bioMérieux, France). Forty-nine sera, 24 from patients with a recent primary HCMV infection and 25 from patients with a long-term HCMV infection and a sustained persistence of specific IgM antibodies, were tested. Similar results were obtained with the two avidity methods. All 24 sera from patients with recently acquired infection had avidity indices compatible with acute HCMV infection by the VIDAS method, whereas with the in-house method, one serum sample had an equivocal result. In the 25 sera from patients with long-term infection, identical results were obtained with the two methods, with only one serum sample having an incompatible value. These findings suggest that our in-house avidity test could be a potentially useful tool for the immunodiagnosis of HCMV infection.

A simple and cheaper in house varicella zoster virus antibody indirect ELISA

We have developed a cheaper an simple in house indirect ELISA that uses the live attenuated VZV vaccine as a coating antigen. The alternative ELISA had an agreement of 94% when compared with a commercial VZV ELISA kit. Moreover, our ELISA proved to be more reliable than the kit when assessing true negative samples. By adding a standard serum, we were able to produce results in international units per millilitre. Also, the addition of an extra step with 8M urea allowed the assessment of VZV IgG avidity without excessive costs. The cost per sample to test VZV IgG was 2.7 times cheaper with our ELISA, allowing the testing of many samples without the burden of production of VZV antigen in the laboratory.

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 .

Serodiagnosis of neurocysticercosis in patients with epileptic seizure using ELISA and immunoblot assay

Sera from 88 patients from Santa Catarina and São Paulo states of Brazil, with epileptic seizures who underwent cerebral computed tomography (CT) were analyzed for the detection of antibodies to T. solium cysticercus by ELISA and Immunoblot (IB) with the following antigens: Taenia solium cysticercus total saline (Tso), Taenia crassiceps cysticercus vesicular fluid (Tcra-vf) and T. crassiceps cysticercus glycoproteins (Tcra-gp). ELISA carried out with Tso, Tcra-vf and Tcra-gp antigens showed 95%, 90% and 80% sensitivities, respectively, and 68%, 85% and 93% specificities, respectively. In the epileptic patients group, ELISA positivity was 30%, 51% and 35% with Tso, Tcra-vf and Tcra-gp antigens respectively. Considering the IB as the confirmatory test, the positivity was 16% (14/88) in the epileptic patients total group and 22% (12/54) in the epileptic patients with positive CT and signals of cysticercosis. We found a significant statistical correlation among ELISA or IB results and the phase of the disease when any antigens were used (p < 0.05). We emphasize the need to introduce in the laboratory routine the search for neurocysticercosis (NC) in patients presenting with epileptic seizures because of the high risk of acquiring NC in our region and its potential cause of epilepsy.

Dot-Elisa for evaluation of hydatid cyst wall, protoscoleces and hydatid cyst fluid antigens in the serodiagnosis of cystic echinococcosis

The aim of the present study is to evaluate cyst wall and protoscolex as an alternate source of antigen in serodiagnosis of cystic echinococcosis (CE). A total of 90 blood samples, 30 each of confirmed CE cases, disease controls and healthy controls were collected. Dot-ELISA using cyst wall, protoscolex and cyst fluid were used to demonstrate anti-hydatid antibodies. The sensitivity of Dot-ELISA using cyst wall, protoscolex and cyst fluid was 96.66%, 86.66% and 93.33% respectively and the specificity of the assay was 70% for Dot-ELISA using cyst fluid, protoscolex and cyst wall antigens. Results of the present study show that cyst wall and protoscolex can also be an useful source of antigen in detection of hydatid antibodies in the serodiagnosis of CE.

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.

High diagnostic efficiency of IgM-ELISA with the use of multiple antigen peptides (MAP1) from T. gondii ESA (SAG-1, GRA-1 and GRA-7), in acute toxoplasmosis

The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

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.

Evaluation of the ELISA-F29 test as an early marker of therapeutic efficacy in adults with chronic Chagas disease

This work compared the time at which negative seroconversion was detected by conventional serology (CS) and by the ELISA-F29 test on a cohort of chronic chagasic patients treated with nifurtimox or benznidazole. A retrospective study was performed using preserved serum from 66 asymptomatic chagasic adults under clinical supervision, and bi-annual serological examinations over a mean follow-up of 23 years. Twenty nine patients received trypanocide treatment and 37 remained untreated. The ELISA-F29 test used a recombinant antigen which was obtained by expressing the Trypanosoma cruzi flagellar calcium-binding protein gene in Escherichia coli. Among the untreated patients, 36 maintained CS titers. One patient showed a doubtful serology in some check-ups. ELISA-F29 showed constant reactivity in 35 out of 37 patients and was negative for the patient with fluctuating CS. The treated patients were divided into three groups according to the CS titers: in 13 they became negative; in 12 they decreased and in four they remained unchanged. ELISA-F29 was negative for the first two groups. The time at which negativization was detected was significantly lower for the ELISA-F29 test than for CS, 14.5 ± 5.7 and 22 ± 4.9 years respectively. Negative seroconversion was observed in treated patients only. The results obtained confirm that the ELISA-F29 test is useful as an early indicator of negative seroconversion in treated chronic patients.