RESUMO
We report a proof-of-concept study using a dipstick assay to detect Taenia solium antigen in urine samples of 30 patients with subarachnoid neurocysticercosis and 10 healthy control subjects. Strips were read in blind by two readers. The assay detected antigen in 29 of 30 cases and was negative in all 10 control samples. Although this study was performed in samples from individuals with subarachnoid neurocysticercosis who likely had high circulating antigen levels, it provides the proof of concept for a functional urine antigen point-of-care assay that detects viable cysts. Such an assay could serve to support a clinical diagnosis of suspect neurocysticercosis or to identify patients at risk of developing severe disease in areas where medical resources are limited, providing evidence to refer these individuals for imaging and specialized care as needed.
Assuntos
Cisticercose , Neurocisticercose , Taenia solium , Humanos , Animais , Neurocisticercose/diagnóstico , Sistemas Automatizados de Assistência Junto ao Leito , Ensaio de Imunoadsorção Enzimática/métodos , Antígenos de HelmintosRESUMO
Strongyloides stercoralis is a soil-transmitted nematode that can cause life-threatening conditions in immunocompromised persons. In the United States, strongyloidiasis should be considered mainly in immigrants, refugees, or travelers. The confirmatory laboratory diagnosis is usually performed by detecting larvae from the stool, duodenal material, and sputum. In persons who are immunocompromised with severe strongyloidiasis, adult worms and eggs can be detected from duodenal material. For serological diagnosis, most assays use crude antigens to detect anti-S. stercoralis IgG. Recently, recombinant proteins such as rSs-NIE-1 and rSs-IR have been used to detect IgG antibodies. We used rSs-NIE-1 and rSs-IR recombinant antigens to develop a biplex Western blot assay to detect the IgG4 antibody in individuals with strongyloidiasis. The sensitivities of rSs-NIE-1 and rSs-IR were 97.4% and 90.8%, respectively, whereas the specificities were 97.6% and 98%, respectively. In conclusion, the biplex rSs-NIE-1 and rSs-IR immunoblot performs well in detecting IgG4 antibody in S. stercoralis-infected persons.
Assuntos
Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Immunoblotting/métodos , Strongyloides stercoralis/imunologia , Estrongiloidíase/diagnóstico , Animais , Antígenos de Helmintos/genética , Fezes/parasitologia , Humanos , Immunoblotting/normas , Imunoglobulina G/sangue , Larva/imunologia , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Strongyloides stercoralis/química , Estrongiloidíase/imunologiaRESUMO
Angiostrongylus cantonensis is the main causative agent of eosinophilic meningoencephalitis (EoM) in humans. Molecular diagnostic methods are essential since the identification of larvae in cerebrospinal fluid (CSF) is extremely rare. To date, the detection of a 31 kDa antigen by Western blotting has been the primary immunodiagnostic method for EoM caused by A. cantonensis. However, cross-reactivity with other parasites has been observed. Therefore, we conducted a comparative analysis using sera from individuals with angiostrongyliasis. We also characterized proteins isolated from different cellular sources of A. cantonensis, Toxocara canis, Schistosoma mansoni, and Strongyloides stercoralis with mass spectrometry. A total of 115 cross-reactive proteins were identified. Three of these proteins, heat shock protein, an intermediate filament protein, and galectin 1, represent potential markers for cross-reactivity. In addition, synthetic peptides were generated from previously identified diagnostic targets and tested against sera from individuals infected with several other parasites. As a result, two other markers of cross-reactivity were identified: peptide #4 derived from the 14-3-3 protein and peptide #12 derived from the Lec-5 protein. In contrast, 34 proteins were exclusively present in the Angiostrongylus extracts and represent promising diagnostic molecules for specific identification of A. cantonensis infection. In particular, cytochrome oxidase subunit I is of great interest as a possible immunodiagnostic target for angiostrongyliasis.
Assuntos
Angiostrongylus cantonensis/imunologia , Antígenos de Helmintos/imunologia , Proteínas de Helminto/imunologia , Meningoencefalite/diagnóstico , Meningoencefalite/parasitologia , Infecções por Strongylida/diagnóstico , Sequência de Aminoácidos , Angiostrongylus cantonensis/química , Animais , Antígenos de Helmintos/sangue , Antígenos de Helmintos/química , Antígenos de Helmintos/isolamento & purificação , Western Blotting , Sequência Conservada , Reações Cruzadas , Eletroforese , Eletroforese em Gel Bidimensional , Proteínas de Helminto/química , Proteínas de Helminto/isolamento & purificação , Humanos , Imunoensaio , Testes Imunológicos , Espectrometria de Massas , Meningoencefalite/imunologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologiaRESUMO
Individuals infected with Strongyloides stercoralis have been reported to produce different immunoglobulins isotypes, yet few studies have evaluated their use in strongyloidiasis diagnosis. The aim of this work was to evaluate the immunoreactivity of different classes and subclasses of anti-S. stercoralis circulating antibodies in alcoholic patients by ELISA and to perform immunoblotting in samples with discordant results between parasitological and immunological methods. 345 male patients with a clinical diagnosis of alcoholism hospitalized at a reference center for alcoholics in Salvador, Bahia, Brazil, were included in this study. The fecal samples were examined by three different parasitological methods (spontaneous sedimentation, Baermann-Moraes and Agar Plate Culture methods). The ELISA was performed for the detection of IgG, IgG1, IgG4, IgE and IgA1 anti-S. stercoralis. Immunoblotting, for the detection of specific IgA1, was used to elucidate discordant results between parasitological and immunological methods. S. stercoralis infection frequency in alcoholic patients by parasitological methods was 21.4% (74/345). Although IgE-ELISA demonstrated a high sensitivity and specificity in non-alcoholic patients, about 30% (22/74) of alcoholics with larvae in feces were negative. IgG1-ELISA detected the lowest frequency of antibodies in alcoholic patients with larvae in feces, only 57% (42/74). IgG4-ELISA was the best assay for S. stercoralis infection immunodiagnosis. Immunoreactivity in the immunoblotting for IgA1 at 90, 75, 26 and/or 17â¯kDa bands was observed in 92% (33/36) of alcoholics with larvae excretion and negative ELISA for one or more antibody isotypes. In conclusion, IgG4-ELISA showed the highest sensitivity and specificity, thus demonstrating its superiority for strongyloidiasis immunodiagnosis in alcoholic and non-alcoholic individuals. Both, IgE and IgG1-ELISA presented high sensitivities and specificities for S. stercoralis infection diagnosis in non-alcoholics, however there was low reactivity in alcoholic individuals. This can be associated with an increased susceptibility to severe strongyloidiasis in these patients. IgA1-immunoblotting can be used to confirm S. stercoralis infection when there are discordant results between parasitological methods and ELISA.
Assuntos
Consumo de Bebidas Alcoólicas/imunologia , Alcoolismo/imunologia , Anticorpos Anti-Helmínticos/imunologia , Strongyloides stercoralis/imunologia , Estrongiloidíase/imunologia , Adulto , Idoso , Alcoolismo/parasitologia , Animais , Brasil , Ensaio de Imunoadsorção Enzimática/métodos , Fezes/parasitologia , Humanos , Imunoglobulina G/imunologia , Testes Imunológicos/métodos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Sensibilidade e Especificidade , Estrongiloidíase/diagnóstico , Estrongiloidíase/parasitologia , Adulto JovemRESUMO
Neurocysticercosis accounts for approximately 30% of all epilepsy cases in most developing countries. The immunodiagnosis of cysticercosis is complex and strongly influenced by the course of infection, the disease burden, the cyst location, and the immune response of the host. The main approach to immunodiagnosis should thus be to evaluate whether the serological results are consistent with the diagnosis suggested by imaging. Antibody detection is performed using lentil lectin-purified parasite antigens in an enzyme-linked immunoelectrotransfer blot format, while antigen detection uses a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA). Promising new assay configurations have been developed for the detection of both antibody and antigen, including assays based on synthetic or recombinant antigens that may reduce costs and improve assay reproducibility and multiplex bead-based assays that may provide simultaneous quantitative results for several target antigens or antibodies.
Assuntos
Cysticercus/imunologia , Imunoensaio , Testes Imunológicos , Neurocisticercose/diagnóstico , Taenia solium/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Anticorpos Anti-Helmínticos/líquido cefalorraquidiano , Anticorpos Anti-Helmínticos/imunologia , Antígenos de Helmintos/sangue , Antígenos de Helmintos/imunologia , Cysticercus/isolamento & purificação , Humanos , Neurocisticercose/parasitologia , Neurocisticercose/patologia , Reprodutibilidade dos Testes , Taenia solium/isolamento & purificaçãoRESUMO
Angiostrongylus cantonensis is a parasitic nematode and the main causative agent of human cerebral eosinophilic meningoencephalitis (EoM). A definitive diagnosis of EoM usually requires serologic or molecular analysis of the patient's clinical sample. Currently, a 31â¯kDa antigen is used in immunological tests for this purpose, however as a crude antigen preparation it may present cross-reactivity with other helminthic infections, especially echinococcosis. Heterologous expression studies using prokaryotic systems failed on producing antigenic proteins. The aim of this study was to express and purify three recombinant glycoproteins representing A. cantonensis antigens: ES-7, Lec-5, and 14-3-3, in Chinese hamster ovary (CHO) cells and ES-7 in human embryonic kidney (HEK) cells to develop a source of specific antigens to be used in the diagnosis of angiostrongyliasis. The potential diagnostic value of these three proteins was subsequently characterized in one- and two-dimensional electrophoresis and Western blot to dot blot analyses, with Angiostrongylus-positive sera, normal human sera (NHS), and a pool of Echinococcus-positive sera (included as a specificity control) used for detection. In addition, recognition of these three proteins following treatment with N-glycosidase F was examined. The ES-7 proteins that were expressed in HEK and CHO cells, and the Lec-5 protein that was expressed in CHO cells, were specifically recognized by A. cantonensis-positive sera in the 2D electrophoresis analysis. This recognition was shown to be dependent on the presence of glycidic portions, making mammalian cells a very promising source of heterologous expression antigenic proteins from Angiostrongylus.
Assuntos
Angiostrongylus cantonensis/genética , Antígenos de Helmintos/biossíntese , Proteínas de Helminto/biossíntese , Proteínas Recombinantes/biossíntese , Animais , Antígenos de Helmintos/genética , Western Blotting , Células CHO , Clonagem Molecular , Cricetulus , Expressão Gênica , Células HEK293 , Proteínas de Helminto/genética , Humanos , Immunoblotting , Proteínas Recombinantes/genética , Testes Sorológicos/métodos , Infecções por Strongylida/diagnósticoRESUMO
The primary causative agent of eosinophilic meningoencephalitis (EoM) in endemic regions is the nematode Angiostrongylus cantonensis. The occurrence of EoM was previously restricted to countries in Southeast Asia and the Pacific Islands; however, more recently, it has been reported from other regions, including Brazil. The commonly used diagnosis is detection of specific antibody reactivity to the 31 kDa antigen, which is derived from female worm somatic extracts. Here we report the occurrence of cross-reactivity to this antigen in sera from other parasitic infections, especially those that may cause EoM, such as gnathostomiasis, toxocariasis, hydatidosis and strongyloidiasis. We also demonstrated that the cross-reactivity, in part, is dependent of the concentration of antigen used in Western blot assays. We discuss the importance of these findings on the interpretation of this test.
Assuntos
Angiostrongylus cantonensis/imunologia , Antígenos de Helmintos/imunologia , Meningoencefalite/diagnóstico , Meningoencefalite/parasitologia , Infecções por Strongylida/diagnóstico , Angiostrongylus cantonensis/metabolismo , Animais , Reações Cruzadas , Humanos , Meningoencefalite/sangue , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologiaRESUMO
One of the most well-characterized tests for diagnosing neurocysticercosis (NCC) is the enzyme-linked immunoelectrotransfer blot (EITB) assay developed at the CDC, which uses lentil lectin-bound glycoproteins (LLGP) extracted from Taenia solium cysticerci. Although the test is very reliable, the purification process for the LLGP antigens has been difficult to transfer to other laboratories because of the need for expensive equipment and technical expertise. To develop a simpler assay, we previously purified and cloned the diagnostic glycoproteins in the LLGP fraction. In this study, we evaluated three representative recombinant or synthetic antigens from the LLGP fraction, individually and in different combinations, using an immunoblot assay (recombinant EITB). Using a panel of 249 confirmed NCC-positive and 401 negative blood serum samples, the sensitivity of the recombinant EITB assay was determined to be 99% and the specificity was 99% for diagnosing NCC. We also tested a panel of 239 confirmed NCC-positive serum samples in Lima, Peru, and found similar results. Overall, our data show that the performance characteristics of the recombinant EITB assay are comparable to those of the LLGP-EITB assay. This new recombinant- and synthetic antigen-based assay is sustainable and can be easily transferred to other laboratories in the United States and throughout the world.