No abstract

IgG avidity assays have been developed for several parasitic diseases although there are no researches focused in strongyloidiasis diagnosis. Definitive diagnosis of strongyloidiasis is based on the presence of Strongyloides larvae in stool, but majority of cases involve low and irregular larval output. While limitations of serological assays for strongyloidiasis are well known, characteristics of persons who are misdiagnosed based on negative coproparasitological tests have been little explored. The aim of the present study was to evaluate the use of IgG avidity to detect patients with active strongyloidiasis and to characterize sources of disagreement between serology and coproparasitology. A total of 80 serum samples was analyzed, 40 from patients with Strongyloides larvae in stool (G1) and 40 from individuals with negative coproparasitology, but positive serology (G2). Serum samples were analyzed in an indirect IgG avidity ELISA using urea 6M in serial double dilutions from 1:80 to 1:2560. Avidity index (AI) was calculated to each serum dilution and analyzed as screening AI (serum dilution of 1:160) or mean AI of different serum dilutions that had a positive result. Statistical analyzes were performed by Mann-Whitney's (U) and Fisher's exact tests. At screening dilution, median of AI was 68% in G1 and 88% in G2 (P<0.0001), whereas median of mean AI in G1 was 72% and in G2 94% (P<0.0001), but there was no significant differences between both AI in each patient group. A cut off value established at AI of 75% demonstrated a significant difference between groups, with G1 sera showing AI<75% and G2 sera with AI>75% (P<0.0001). In conclusion, IgG avidity assays may distinguish active infection with Strongyloides stercoralis from suspect or serologically false positive cases.

BackgroundStrongyloidiasis, a human intestinal infection caused by the nematode Strongyloides stercoralis, is frequently underdiagnosed and although its high prevalence is still a neglected parasitic disease because conventional diagnostic tests based on parasitological examination (presence of Strongyloides larvae in stool) are not sufficiently sensitive due to the low parasitic load and to the irregular larval output. There is an urgent need to improve diagnostic assays, especially for immunocompromised patients with high parasitic load as consequence of self-infection cycle, which can disseminate throughout the body, resulting in a potentially fatal hyperinfection syndrome often accompanied by sepsis or meningitis.Methods/Principal FindingsWe have performed Phage Display technology to select peptides that mimic S. stercoralis antigens, capable of detecting a humoral response in patients with strongyloidiasis. The peptides reactivity was investigated by Phage-ELISA through different panels of serum samples. We have successfully selected five peptides with significant immunoreactivity to circulating IgG from patients' sera with strongyloidiasis. The phage displayed peptides C9 and C10 presented the highest diagnostic potential (AUC>0.87) with excellent sensitivity (>85%) and good specificity (>77.5%), suggesting that some S. stercoralis antigens trigger systemic immune response.Conclusions/SignificanceThese novel antigens are interesting serum biomarkers for routine strongyloidiasis screenings due to the easy production and simple assay using Phage-ELISA. Such markers may also present a promising application for therapeutic monitoring.

Strongyloidiasis is one of the major intestinal infections in humans, and a neglected tropical disease whose diagnosis still poses a challenge. We hypothesized that diagnostic tests based on short peptides containing major epitopes may represent a promising strategy to improve strongyloidiasis detection due to reduced cross-reactivity and higher sensitivity. Our aim was to evaluate two synthetic peptides selected by phage display (C10 and D3) as potential tools for serodiagnosis of strongyloidiasis, and to predict their putative antigen target. To investigate their diagnostic potential, we have tested different panels of serum samples (n=120) by enzyme linked immunosorbent assay (ELISA) to detect specific IgG, and their diagnostic parameters were calculated. Similarities with proteins from Strongyloides stercoralis were searched and conformational epitopes were predicted and aligned to known protein structures. Both C10 and D3 achieved sensitivity of 95%, and specificities were 89.2% and 92.5%, respectively. D3 presented the highest diagnostic efficiency (93.3%). Epitope prediction for both C10 and D3 led to the alignment with the cytochrome c oxidase subunit 1 structure. In brief, we propose two synthetic peptides as new biomarkers for serodiagnosis of strongyloidiasis, which can be promptly used for ELISA and in future field sensor platforms.

Strongyloides stercoralis causes chronic asymptomatic infections in immunocompetent human hosts and systemic invasion in immunocompromised patients, developing into a fatal hyperinfection syndrome. IgA and IgG detection in saliva and serum paired samples were tested using total saline extract from Strongyloides venezuelensis (SE(*)) and its detergent phase (D) extracted with Triton X-114. Saliva and serum paired samples were obtained from: 25 patients with confirmed strongyloidiasis; 25 patients with other parasitoses and 20 from apparently healthy individuals. Sensitivity, specificity, diagnostic efficiency, positive and negative predictive values and likelihood ratio were calculated at the optimum point of reaction. Using D phase sensitivity and specificity to detect IgA in saliva were 76.0% and 88.9% and in serum 80.0% and 86.7%, respectively. To detect IgG, D phase showed sensitivity and specificity of 88.0% and 88.9% in saliva and 88.0% and 84.4% in serum, respectively. D phase proved to be specific and efficient and could be utilized as an alternative antigen for IgA and IgG detection in saliva and serum samples for strongyloidiasis diagnosis.

(1) Background: The validation of biological antigens is the study’s utmost goal in biomedical applications. We evaluated three different probes with single and multiple epitopes through electrochemical detection of specific IgG in serum for human strongyloidiasis diagnosis. (2) Methods: Screen-printed gold electrodes were used and probes consisting of two single-epitope synthetic peptides (D3 and C10) with different sequences, and a multi-epitope antigen [detergent phase (DP)—hydrophobic membrane proteins]. Human serum samples from three populations were used: Strongyloides stercoralis positive, positive for other parasitic infections and negative controls. To test the immobilization of probes onto a screen-printed gold electrode and the serum IgG detection, electrochemical analyses were carried out through differential pulse voltammetry (DPV) and the electrode surface analyses were recorded using atomic force microscopy. (3) Results: The electrochemical response in screen-printed gold electrodes of peptides D3 and C10 when using positive serum was significantly higher than that when using the DP. Our sensor improved sensitivity to detect strongyloidiasis. (4) Conclusions: Probes’ sequences are critical factors for differential electrochemical responses, and the D3 peptide presented the best electrochemical performance for strongyloidiasis detection, and may efficiently substitute whole antigen extracts from parasites for strongyloidiasis diagnosis in electrochemical immunosensors.

Strongyloidiasis is an important helminthiasis affecting million people worldwide. The aim of this study was to use sodium metaperiodate (MP) treatment to immunochemically characterize Strongyloides venezuelensis filariform larvae and use MP-treated heterologous antigen to detect IgG and subclasses in serum. Samples from individuals with definitive diagnosis of strongyloidiasis (n = 50), other parasitic diseases (n = 60) and negative endemic (n = 50) were tested. TG-ROC and two-way ANOVA were applied. MP-treatment resulted on differential localization of carbohydrates at larval structure and no carbohydrate content in saline extract (SE). Electrophoretic profiles were similar before and after treatment. ELISA sensitivity and specificity were: 90%; 88.2% for SE and 92.0%; 94.6% for MP, respectively. When using MP treated antigen we observed reduction in IgG1 and IgG3 detection in strongyloidiasis group and decrease of cross reactions in control groups. Our data demonstrate the role of carbohydrate residues in cross reactions and on the recognition of anti-Strongyloides IgG and its subclasses.

INTRODUÇÃO 1.1 -Aspectos biológicos de Strongyloides stercoralis 1.2 -Epidemiologia da estrongiloidíase humana 1.3 -Aspectos clínicos da estrongiloidíase humana 1.4 -Resposta imune do hospedeiro 1.5 -Diagnóstico da estrongiloidíase humana 1.6 -Tecnologia de exposição de biomoléculas em fagos (Phage display) e seleção (Biopanning) de peptídeos recombinantes 2 -OBJETIVOS 2.1 -Objetivos gerais 2.2 -Objetivos específicos 3 -MATERIAL E MÉTODOS 3.1 -Aspectos éticos 3.2 -Local de realização 3.3 -Normas de biossegurança 3.4 -Amostras de soro humano 3.5 -Preparação do extrato alcalino total de S. venezuelensis 3.6 -Purificação da Imunoglobulina G por microesferas magnéticas 3.7 -Dot blot para confirmação da purificação das Imunoglobulinas G 3.8 -Seleção dos Peptídeos por Phage display 3.8.1 -Biopanning 3.8.2 -Titulações 3.9 -Extração de DNA dos clones de Fagos 3.10 -Sequenciamento de DNA dos peptídeos expressos na superfície dos Fagos 3.11 -Análise de Bioinformática 3.12 -Amplificação dos clones de fagos que apresentam similaridade com proteínas de S. stercoralis 3.13 -Pré-validação por Phage-ELISA dos clones selecionados 3.14 -Amplificação dos clones de fagos considerados mais relevantes no ensaio de pré-validação 3.15 -Phage-ELISA para estudo da imunorreatividade e validação dos clones de fagos selecionados 3.16 -Phage-ELISA de competição 3.17 -Síntese química dos peptídeos expressos na superfície dos fagos selecionados que apresentaram melhores parâmetros 3.18 -ELISA para verificação da imunogenicidade dos peptídeos sintéticos 3.19 -Análise estatística dos resultados obtidos no Phage-ELISA e no ELISA de peptídeo 4 -RESULTADOS 4.1 -Produção do extrato salino total de Strongyloides venezuelensis 4.2 -Purificação de Imunoglobulina G por microesferas magnéticas e Dot blot para confirmação da purificação 4.3 -Biopanning e Titulações 4.4 -Extração de DNA 4.5 -Sequenciamento de DNA dos clones de Fagos e Análise de Bioinformática 4.6 -Pré-validação por Phage-ELISA 4.7 -Phage-ELISA para estudo da imunorreatividade e validação dos clones de fagos selecionados 4.8 -Phage-ELISA de competição 4.9 -Síntese química dos peptídeos expressos na superfície de fagos selecionados 4.10 -ELISA para verificação da imunorreatividade dos peptídeos sintéticos 5 -DISCUSSÃO 6 -CONCLUSÕES 7 -REFERÊNCIAS BIBLIOGRÁFICAS XII RESUMOEstrongiloidiase humana é uma importante parasitose intestinal em todo mundo, 50% das pessoas infectadas são assintomáticas, entretanto pode haver hiperinfecção e disseminação em pacientes imunocomprometidos, levando à morte. A detecção precoce da doença previne o desenvolvimento das síndromes de hiperinfecção e disseminação, assim, o uso de uma ferramenta diagnóstica eficiente é de grande importância para identificar e controlar esta parasitose. Devido à falta de eficiência nos testes parasitológicos e sorológicos disponíveis atualmente para detectar estrongiloidiase humana é necessário aprimorar os testes imunodiagnósticos utilizando antígenos recombinantes e sintéticos, uma vez que há limitações para obter e utilizar antí...