Julia Maria Costa-Cruz

Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB) using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence antibody test (IFAT) results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals). S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.

Hydrophobic fractions from Strongyloides venezuelensis for use in the human immunodiagnosis of strongyloidiasis

The objective of the present research was to evaluate detergent and aqueous phases of total saline (TS) and alkaline extracts of Strongyloides venezuelensis for human strongyloidiasis immunodiagnosis. Total extracts and detergent and aqueous antigenic fractions were separated using Triton X-114 and were examined by enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) tests to detect immunoglobulin G (IgG). Serum samples were obtained from 120 individuals: 40 strongyloidiasis patients (group I), 40 patients with other parasitic diseases (group II), and 40 apparently healthy individuals (group III). Each extract provided a different profile of antigenic components as recognized by IgG in IB. The detergent fraction of the TS extract demonstrated the highest sensitivity and specificity for ELISA and IB. The results indicated that the detergent saline fraction, purified from S. venezuelensis, furnished the most valid results for the strongyloidiasis immunodiagnosis and could be employed as an alternative antigen and as a useful source of specific polypeptides.

Application of synthetic peptides to the diagnosis of neurocysticercosis

We tested the possible diagnostic utility of five Taenia saginata oncosphere‐derived synthetic peptides in T. solium neurocysticercosis (NC). The five peptides correspond to protein sequences with high antigenic indexes that were cloned from a T. saginata oncosphere cDNA library. The test samples consisted of cerebrospinal fluid (CSF) samples randomly collected from patients referred from Mexican and Brazilian neurological institutes. Indirect enzyme‐linked immunosorbent assays (ELISA) were carried out with the peptides either unconjugated or coupled to carrier proteins, and were compared with results obtained using T. solium cyst fluid as a positive control. For active inflammatory NC, the higher sensibility (93%) and specificity (85%) was obtained with peptides HP6‐2 and Ts45W‐1, respectively, coupled to ovalbumin, in both Mexican and Brazilian patients. Examining the results of the individual peptide assays in combination, in some instances, improved the sensitivity to 100%.

ELISA and Western Blotting tests in the detection of IgG antibodies to Taenia solium metacestodes in serum samples in human neurocysticercosis

Summary A comparative study of total saline extract (SE) and cyst vesicular fluid (VF) of Taenia solium metacestodes by ELISA and Western blotting assay (WB) tests was conducted to detect IgG in sera for diagnosis of human cysticercosis. Sera were obtained and analysed by ELISA in 1 : 20 and 1 : 100 dilutions from 208 individuals: 22 confirmed neurocysticercosis (NC) (group 1), 101 suspected NC (group 2), 55 with various intestinal parasitosis (group 3) and 30 healthy individuals (group 4). The WB test was carried out on SE and VF extracts with and without reducing agent, 2‐β‐mercaptoethanol (2‐ME) in 20 sera of each group. WB using extracts without 2‐ME and ELISA at 1 : 100 dilution were compared in 20 sera from each group; sensitivity and specificity were calculated using samples from groups 1, 3 and 4. By ELISA, in the 1 : 100 sera dilution reactivity was reduced for both antigens without changes in the sensitivity of the test. By WB, antigens treated with 2‐ME demonstrated low specificity. For SE and VF antigens, the proteins of 24, 39–42, 47–52, 56, 64–68, 126–155 kDa and 18, 24, 26–28, 32–36, 47–52, 75 kDa, respectively, were considered immunodominant markers, with high indices of specificity, suggesting a profile for NC patients. However, as the sensitivity was found to be low, it might still not be a definitive test for NC when used alone. These data suggest WB as an indicative test to determine exposure to T. solium. ELISA and WB together may supply reliable results for the diagnosis of human cysticercosis, since appropriate purified antigens are not available yet.

Ocorrência de cisticercose em necropsias realizadas em Uberlândia, Minas Gerais, Brasil

3937 autopsies were performed between 1971 and 1993 in the Servico de Anatomia Patologica of the Hospital de Clinicas of the Fundacao de Assistencia, Estudo e Pesquisa de Uberlândia, Universidade Federal de Uberlândia, in Minas Gerais, Brazil. At this Service of Pathology are realized all the autopsies of the municipal district of Uberlândia. The analysis of 2862 concluded autopsy reports, of death above the age of one year, disclosed 39 cases (1.4%) of cysticercosis. The age range was 16 to 83 years and 66.6% were males; 82.1 % of the patients were from Minas Gerais State, 15.4% were from Goias State, and in one case (2.5%) the origin was not registered. From these 39 cases, 35 (89.7%) showed central nervous system involvement, isolated or in association to other clinical forms of the disease; in 9 occurred the isolated or associated cardiac form; in 4 the muscular form, isolated or associated, was found; 4 presented the isolated or associated visceral form. In only 7 (17.9%) cases, the cysticercosis was assumed to be the direct cause of the death.

Detection of IgG in cerebrospinal fluid for diagnosis of neurocysticercosis: evaluation of saline and SDS extracts from Taenia solium and Taenia crassiceps metacestodes by ELISA and immunoblot assay

We compared saline (S) and sodium dodecyl sulphate (SDS) extracts from Taenia solium (homologous species – HO) and Taenia crassiceps (heterologous species – HE) metacestodes in order to detect IgG by ELISA and immunoblot assay (IBA) in cerebrospinal fluid (CSF) for the diagnosis of human neurocysticercosis (NC). CSF samples were obtained from 93 patients. Of these, 40 had NC, five had a diagnosis of probable NC, nine had central nervous system schistosomiasis or strongyloidiasis and 39 had other neurological alterations. Samples were analysed by ELISA and the results were compared with IBA in all samples with confirmed and probable NC diagnosis, in all samples with other central nervous system parasitic infection, and in 10 of those with another neurological alterations. ELISA sensitivity was 100%, 85%, 95% and 87.5% for the S‐HO, S‐HE, SDS‐HO and SDS‐HE extracts, respectively, and ELISA specificity was 100% for S‐HO, S‐HE, SDS‐HO extracts and 97.9% for SDS‐HE antigen. Immunodominant peptides detected by IBA were, by decreasing percentage of recognition: 64–68 and 45u2003kDa for S‐HO; 108–114, 92–95, 64–68, 83 and 88u2003kDa for S‐HE; 64–68, 108–114, 77 and 86u2003kDa for SDS‐HO; and 108–114, 88 and 92–95u2003kDa for SDS‐HE. Overall the homologous antigenic extracts showed higher sensitivity than the heterologous extracts in the diagnosis of NC in CSF samples. The heterologous extracts contained most of the immunodominant peptides presented in the homologous extracts, which are recognized by IgG antibodies in CSF samples.

Parasitological and immunological diagnosis of Strongyloides stercoralis in patients with gastrointestinal cancer

This study examined the frequency of Strongyloides stercoralis infection in patients with gastrointestinal cancer through parasitological and immunological tests. A total of 77 patients were evaluated, 33 with gastrointestinal cancer and 44 controls with other types of cancers. All the patients were undergoing chemotherapy and 14 (18.2%) were receiving concomitant radiotherapy. For a parasitological diagnosis, we applied the Baermann and Lutz methods. The immunological diagnosis involved the indirect fluorescence antibody test (IFAT) and enzyme linked immunosorbent assay (ELISA) to detect IgG antibodies using Strongyloides ratti antigens. The frequency of positive S. stercoralis in gastrointestinal cancer diagnosed by parasitological methods was 3 cases (9.1%), by serology it was 8 cases (24.2%). In the control group 1 case (2.3%) of S. stercoralis was diagnosed by parasitological methods and 2 cases (4.5%) by immunological tests (p<0.05). Patients with gastrointestinal cancer had a 6.7-fold greater chance of testing positive for S. stercoralis infection. Our data highlight the importance of parasitological and immunological diagnosis for S. stercoralis in patients with gastrointestinal cancer living in endemic areas of strongyloidiasis, since they have a higher risk of becoming infected with S. stercoralis than patients with other types of cancer.

A Glance at Taenia Saginata Infection, Diagnosis, Vaccine, Biological Control and Treatment

The Taenia saginata taeniasis-cysticercosis complex is a cosmopolitan zoonosis of great medical, veterinary and economic importance where humans play an important role as the carrier of adult stage and cattle as carrier of the larval stage of the parasite. Here we reviewed aspects concerning diagnosis, vaccine development, biological control and treatment of the disease.

Specific IgG and IgA to larvae, parthenogenetic females, and eggs of Strongyloides venezuelensis in the immunodiagnosis of human strongyloidiasis.

The aim of this study was to detect levels of IgG and IgA by enzyme-linked immunosorbent assay (ELISA) using alkaline extracts of larvae, adult female worms, and eggs of Strongyloides venezuelensis as antigen. One hundred twenty serum samples divided into 3 groups were analysed: group I (40 strongyloidiasis patients), group II (40 patients with other parasitic infections), and group III (40 healthy subjects). Statistical variations were analyzed using analysis of variance. There was a significant statistical difference (P < 0.001) in the detection of antibodies in group I between larvae and female antigens and between larvae and egg antigens, with higher positivity using larvae antigen. The larvae antigen showed the highest values for sensitivity, specificity, and diagnostic efficiency in ELISA. This study is the first that examines the use of adult female worm and egg antigens to detect antibodies for human strongyloidiasis diagnosis compared with the larval extract. By comparing all 3 extracts, larval antigens demonstrated better diagnostic parameters.

Taenia solium metacestode immunodominant peptides recognized by IgG antibodies in cerebrospinal fluid and serum paired samples from patients with active and inactive neurocysticercosis

The aim of this study was to test if serological distinction between patients with active and inactive neurocysticercosis (NCC), could be accomplished by the recognition of immunodominant peptides in total saline antigenic extract of Taenia solium metacestodes by IgG antibody in cerebrospinal fluid (CSF) and serum paired samples. CSF and serum samples of 10 each, active NCC patients, inactive NCC, and individuals with other neurological disorders, were used to recognize the antigenic peptides by western blot (WB). In the active NCC the 28-32 and 39-42 kDa peptides were more frequently detected in CSF than in sera (p < 0.05). The 47-52, 64-68, and 70 kDa antigens showed high frequencies in both samples from patients with active NCC. All the CSF samples of inactive NCC and other neurological disorder (control) patients tested negative, while serum samples from these last two groups recognized mainly the 80, 86, 95, and 98 kDa bands. This finding eliminates the use of the high molecular weigh bands (>or= 80 kDa) for diagnosis of NCC. The final conclusions were that the difference between active and inactive NCC may be done with the detection of peptides only in the CSF samples and that the 47-52, 64-68, and 70 kDa bands may be included as specific markers for active NCC when detected in CSF samples by WB using total saline extract of T. solium metacestode.