RESUMO
Human ascariasis is a neglected tropical disease of great relevance to public health and is considered the most frequent helminthiasis in poor regions. Accurately diagnosing this parasite has been challenging due to limitations of current diagnostic methods. Immunoglobulin Y (IgY) technology is a very effective alternative for the production of highly specific and profitable antibodies. This study aimed to produce and apply anti-Ascaris suum IgY antibodies in the immunodiagnosis of human ascariasis. Five immunizations comprising total saline extract from A. suum adult life forms were given at 14-day intervals to Gallus gallus domesticus hens of the Isa Brown line. Eggs and blood samples were collected weekly and fortnightly, respectively, to monitor the production of antibodies. The specificity of antibodies was confirmed by dot-blot, kinetic enzyme-linked immunosorbent assay (ELISA), avidity ELISA, immunoblotting and indirect immunofluorescence antibody tests. The application for disease diagnosis was performed through the detection of immune complexes in human serum samples by sandwich ELISA. Peaks of IgY anti-A. suum production occurred at weeks 6 and 8. IgY showed high avidity levels after the second dose of immunization, ranging from 64% to 93%, with a mean avidity index of 78.30%. Purified IgY recognized 12 bands of proteins from A. suum saline extract. Eggs, the uterine portion and cuticles of A. suum female adult are reactive in immunofluorescence. The detection of immune complexes showed diagnostic values of 80% sensitivity and 90% specificity. In conclusion, specific IgY have been shown to be a potential immunodiagnostic tool with promising future applications in human ascariasis.
Assuntos
Anticorpos Anti-Helmínticos/biossíntese , Ascaríase/diagnóstico , Ensaio de Imunoadsorção Enzimática , Imunoglobulinas/biossíntese , Animais , Complexo Antígeno-Anticorpo , Antígenos de Helmintos/administração & dosagem , Antígenos de Helmintos/imunologia , Ascaris suum , Galinhas , Feminino , Humanos , Imunização , Testes Imunológicos/métodos , Sensibilidade e EspecificidadeRESUMO
Human strongyloidiasis is caused by helminth Strongyloides stercoralis. It has a worldwide distribution, often neglected and cause of severe morbidity. The parasitological diagnosis is hindered by the low and irregular amount of larvae in feces. The goal of the present study was to detect IgG and IgG immune complex using conventional serum samples and saliva as alternative samples. We collected samples from 60 individuals, namely: group I composed of 30 healthy individuals; and group II composed of 30 individuals eliminating S. stercoralis larvae in feces. We calculated the area under the curve, general index of diagnostic accuracy, Kappa index and determined the correlations between different diagnostic tests. The detection of IgG levels was performed by an immunoenzymatic assay with alkaline extract of S. venezuelensis larvae as antigen. Positivity of anti-S. stercoralis IgG in serum samples from group I was 3·3%, and from group II 93·3%. The detection of immune complex indicated that group I exhibited 3·3% and group II 56·7%. In the saliva samples, IgG detection was 26·7% for group I and 43·3% for group II. Immune complex was detected in 20% of group I, and 30% of group II. IgG immune complex in conventional serum samples and saliva as alternative samples can be considered biomarkers for the diagnosis of active strongyloidiasis.
Assuntos
Complexo Antígeno-Anticorpo/análise , Antígenos de Helmintos/imunologia , Imunoglobulina G/análise , Testes Imunológicos/métodos , Saliva/química , Estrongiloidíase/diagnóstico , Animais , Anticorpos Anti-Helmínticos/sangue , Complexo Antígeno-Anticorpo/sangue , Biomarcadores/análise , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Humanos , Imunoglobulina G/sangue , Larva , Strongyloides stercoralis/imunologia , Estrongiloidíase/imunologiaRESUMO
There is an increasing interest in improving neurocysticercosis (NCC) diagnosis through the search of new and alternative antigenic sources, as those obtained from heterologous antigens. The aim of this study was to obtain potential biomarkers for NCC diagnosis after gel filtration chromatography [gel filtration fraction (GFF)] from the total saline extract (SE) from Taenia saginata metacestodes, followed by protein identification and application in immunodiagnostic. SE and GFF proteic profiles were characterized in gel electrophoresis, and diagnostic performance was verified by testing 160 serum samples through enzyme-linked immunosorbent assay and immunoblotting. Sensitivity (Se), specificity (Sp) and other diagnostic parameters were calculated. Polypeptides of interest in the diagnosis of human NCC present at GFF were analysed by mass spectrometry (MS) and B-cell epitopes were predicted. GFF had the best diagnostic parameters: Se 93·3%; Sp 93%; AUC 0·990; LR+ = 13·42 and LR- = 0·07, and proved to be useful reacting with serum samples in immunoblotting. Proteic profile ranged from 64 to 68 kDa and enolase and calcium binding protein calreticulin precursor were identified after MS. The enolase and calcium-binding protein calreticulin precursor showed 18 and 10 predicted B-cell epitopes, respectively. In conclusion we identified important markers in the GFF with high efficiency to diagnose NCC.
Assuntos
Cromatografia em Gel/métodos , Proteínas de Helminto/metabolismo , Neurocisticercose/sangue , Neurocisticercose/diagnóstico , Taenia saginata/metabolismo , Animais , Biomarcadores/sangue , Fracionamento Químico , Ensaio de Imunoadsorção Enzimática , Epitopos de Linfócito B , Proteínas de Helminto/sangue , Proteínas de Helminto/genética , Humanos , Programas de Rastreamento , Modelos Moleculares , Neurocisticercose/parasitologia , Conformação Proteica , Taenia saginata/isolamento & purificaçãoRESUMO
This study aimed to compare three qualitative parasitological methods for the diagnosis of Syphacia muris infection in 30 Wistar rats (Rattus norvegicus) infected naturally. Methods of spontaneous sedimentation (Hoffman, Pons and Janer, or HPJ) and spontaneous flotation (Willis) for faecal samples and a method of taping (Graham) were performed and compared. The Graham and Willis methods were more sensitive than the HPJ method (P< 0.05). The Graham method was able to detect S. muris eggs in 100% of the samples. Eggs were detected in 83% and 60% of the samples using the Willis and HPJ methods, respectively. Method choice is important for screening for parasites of rats kept under laboratory conditions, as accurate diagnosis helps prevent future environmental contamination and infection. We concluded that the Graham method was the most efficient of those tested in this study for detection of S. muris infection in rats. This method is also rapid, inexpensive and practical, and should be implemented as a necessary measure for infection control.
Assuntos
Oxiuríase/veterinária , Oxyuroidea/isolamento & purificação , Parasitologia/métodos , Doenças dos Roedores/diagnóstico , Animais , Feminino , Masculino , Oxiuríase/diagnóstico , Oxiuríase/parasitologia , Oxyuroidea/fisiologia , Ratos , Ratos Wistar , Doenças dos Roedores/parasitologiaRESUMO
In the present study, antigens from parthenogenetic females and eggs of Strongyloides venezuelensis, or anti-parthenogenetic-female and anti-egg antigens were used to detect specific IgG and immune complex responses, respectively. Serum samples from experimentally infected immunocompetent and immunosuppressed rats were analysed on days 5, 8, 13 and 21 post-infection (dpi). An enzyme-linked immunosorbent assay (ELISA) was performed using alkaline parasite extract for specific IgG detection, and anti-parthenogenetic-female or anti-egg antigens for immune complex detection. The data were analysed using analysis of variance (ANOVA), followed by a Bonferroni test. When parthenogenetic female or egg extracts were used as antigens, specific IgGs were not detected in either immunocompetent or immunosuppressed rats. When anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used, immune complexes were detected for the duration of the infection in immunosuppressed animals and were only detected between 5 and 13 dpi in immunocompetent animals. The duration of infection was not significantly different between the immunocompetent and immunosuppressed groups when anti-parthenogenetic-female or anti-S. venezuelensis-eggs were used. Parthenogenetic female extracts yielded significant differences between antibody and immune complex responses in immunocompetent rats from 5 to 13 dpi, but only on day 5 dpi in immunosuppressed rats. Exposure to S. venezuelensis egg extract yielded significant differences in both antibody and immune complex detection between immunocompetent and immunosuppressed rats for the duration of the infection. In conclusion, ELISA using alternative antigens may be a successful strategy for identifying immune complexes in serum samples and diagnosing active strongyloidiasis, particularly under conditions of immunosuppression.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Complexo Antígeno-Anticorpo/sangue , Imunoglobulina G/sangue , Terapia de Imunossupressão , Strongyloides/imunologia , Estrongiloidíase/diagnóstico , Zigoto/imunologia , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Masculino , Ratos , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologiaRESUMO
Little is known about the actin cytoskeleton architecture in female Strongyloides venezuelensis and thus to investigate the distribution and concentration of actin, female worms were labelled with phalloidin-rhodamine and visualized under confocal microscopy. Our results demonstrate that filamentous actin accumulates in the vulva and the concentration of F-actin at this site suggests its important role, especially during oviposition, in the life cycle of S. venezuelensis.
Assuntos
Actinas/análise , Strongyloides/química , Animais , Feminino , Microscopia Confocal/métodos , Oviposição , Coloração e Rotulagem/métodos , Strongyloides/fisiologia , Vulva/químicaRESUMO
The aim of this study was to fractionate and partially characterize the antigenic extract of filariform larvae of Strongyloides venezuelensis in ion-exchange resin diethylaminoethyl sepharose (DEAE), to obtain antigenic fractions potentially applicable in immunoassays. Somatic antigen (SA) and its fractions DEAE S1 and DEAE S2 - which interacted with the resin - were evaluated by 1-dimensional electrophoresis to obtain protein profiles. SA and its fractions were tested in serum samples for IgG detection by ELISA. Serum samples (n = 155) were analysed: 50 from strongyloidiasis patients (G1), 55 from patients with other parasitic infections (G2) and 50 from healthy volunteers. Sensitivity (Se), specificity (Sp), area under curve (AUC) and likelihood ratios (LR) were calculated. The DEAE S2 fraction provided a high diagnostic value for IgG detection (Se 92·0%, Sp 91·4%, AUC 0·981, LR+ 10·75, LR - 0·09). In conclusion, the DEAE S2 fraction would probably be a source of immunodominant polypeptides for IgG detection in human strongyloidiasis serodiagnosis.
Assuntos
Antígenos de Helmintos , Cromatografia por Troca Iônica , Strongyloides/química , Estrongiloidíase/diagnóstico , Animais , Ensaio de Imunoadsorção Enzimática/normas , Humanos , Imunoglobulina G/sangue , Larva/química , Sensibilidade e Especificidade , Testes Sorológicos , Soro/parasitologiaRESUMO
The aim of this study was to use larval, parasitic female and egg antigens from Strongyloides venezuelensis to detect parasite-specific IgG and immune complexes in human serum samples by enzyme-linked immunosorbent assay (ELISA). In total, 95 serum samples were analysed, consisting of 30 patients harbouring S. stercoralis larvae, 30 healthy subjects and 35 patients with other parasites. Sensitivity, specificity and diagnostic efficiency were calculated. A significant statistical difference was found in the detection of immune complexes and antibodies in patients harbouring S. stercoralis larvae from larval and eggs antigens, with higher positivity using larval antigen. The larval antigen showed the highest values for sensitivity, specificity and diagnostic efficiency in ELISA from detection of immune complexes. For the first time we used IgG anti-larvae, IgG anti-parasitic females or IgG anti-eggs for immune complex detection. We concluded that the association of antibody and immune complex detection could be used in the diagnosis of human strongyloidiasis.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Antígenos de Helmintos , Imunoglobulina G/sangue , Strongyloides/imunologia , Estrongiloidíase/diagnóstico , Animais , Especificidade de Anticorpos , Antígenos de Helmintos/imunologia , Feminino , Humanos , Testes Imunológicos , Larva/imunologia , Óvulo/imunologia , Ratos , Ratos Wistar , Sensibilidade e Especificidade , Strongyloides/classificação , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologiaRESUMO
The objective of this review was to outline an epidemiological profile of Strongyloides stercoralis by parasitological and serological diagnosis in inhabitants, and to associate this profile with different immunosupression situations, in Brazil, over 20 years (1990-2009). The occurrence of S. stercoralis using parasitological methods was 5·5%, being 4·8% in rural and 5·0% in urban areas, characterizing the country as hyperendemic. There was a diversity of techniques used as a diagnostic tool and only 39·1% of the studies presented results based on at least 1 specific method. The occurrence increased with age, being 12·1%, for those over 60 that suggests an epidemiological condition of concern for the elderly population. Of the seroepidemiological studies in the general population the mean positivity in serum samples was 21·7% and 29·2%, using an immunofluorescence antibody test and enzyme-linked immunosorbent assay (ELISA), respectively. The occurrence of strongyloidiasis in immunosuppressed individuals was 11·8% by parasitological methods and 19·5% using immunological methods. Considering that Brazil is a tropical country and that the character of chronicity and autoinfection of the parasite that can result in severe forms of hyperinfection or dissemination makes strongyloidiasis an important medically and socially neglected problem.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Strongyloides stercoralis/fisiologia , Estrongiloidíase , Adolescente , Adulto , Fatores Etários , Idoso , Animais , Anticorpos Anti-Helmínticos/imunologia , Brasil/epidemiologia , Criança , Pré-Escolar , Bases de Dados Bibliográficas , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Hospedeiro Imunocomprometido , Imunoglobulina G/sangue , Masculino , Pessoa de Meia-Idade , Prevalência , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Testes Sorológicos , Estrongiloidíase/diagnóstico , Estrongiloidíase/epidemiologia , Estrongiloidíase/imunologia , Estrongiloidíase/parasitologiaRESUMO
Neurocysticercosis (NC), caused by Taenia solium metacestode, infects the central nervous system and is a devastating parasitic infection. Diagnosis is based on symptoms, imaging, serology and epidemiology. Current markers present variable sensitivity and specificity, frequent cross-reactions and are not able to discriminate NC clinical forms. The aim of this study was to select mimotopes of T. solium metacestode antigens that may be used in NC immunodiagnosis, specifically to discriminate between active and inactive forms. A random peptide phage display library was screened against IgY from chickens immunized with total saline extract from T. solium metacestodes and validated against 110 serum samples, classified into active NC (18), inactive NC (22), cross-reactive parasitic diseases (40) and healthy controls (30). We have successfully selected seven peptides with significant immunoreactivity to IgG of NC patients, with sensitivity ranging from 95.5% to 100% to detect the inactive form and specificity varied from 85.7% to 94.3%. One phage-displayed peptide (Cc48) can be directly used as biomarker to distinguish inactive from active forms with an accuracy of 95.7%, and this novel mimotope may also be used as an auxiliary tool to neuroimaging tests and treatment follow-up.