Mimotope-based antigens as potential vaccine candidates in experimental murine cysticercosis.

Human cysticercosis is a public health problem caused by Taenia solium metacestodes; thus, eradication of T. solium transmission by vaccination is an urgent requirement. The Cc48 mimotope from T. solium cysticerci was tested expressed in phage particles (mCc48) and chemically synthesized (sCc48) as a vaccine candidate in experimental murine cysticercosis. For this, BALB/c mice were immunized with mCc48 (G1; n = 40), sCc48 (G2; n = 40) and phosphate-buffered saline (PBS) (G3; n = 40, positive control) and challenged with Taenia crassiceps metacestodes. Another PBS group without parasite challenge was used as a negative control (G4; n = 40). Mice were sacrificed 15, 30, 45 and 60 days post-infection for cysticerci and serum collection. Immunization efficacy was determined by cysticerci counting. Serum samples were tested by ELISA to verify antibody (IgM, IgG, IgA and IgE) and cytokine (IFNγ and IL-4) levels. The sCc48 achieved the highest rates of protection and efficacy (90 and 98%, respectively). The group immunized with mCc48 presented the highest reactivity for IgM, IgG and IgE. All groups presented IL-4, but IFNγ was quite variable among groups. The protection induced by sCc48 synthetic peptide supports further studies of this mimotope as a potential vaccine candidate against cysticercosis.

Use of polyclonal IgY antibodies to detect serum immune complexes in patients with active hookworm infection.

Definitive diagnosis of hookworm infection is usually based on the microscopic detection of eggs in a stool sample; however, several cases display a low or irregular egg output. Serodiagnosis can be a useful tool to identify these cases, but conventional tests do not differentiate past from active infections. The aim of this study was to obtain and apply egg yolk polyclonal immunoglobulin (IgY) antibodies to detect immune complexes (ICs) in serum samples from patients infected with hookworm. Hens were immunized with Ancylostoma ceylanicum saline extract, their eggs were collected and then IgY antibodies were extracted and purified. Antibody purity was tested by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis and specificity was assessed by immunoblotting and immunofluorescence. IgY production was evaluated by kinetics enzyme-linked immunosorbent assay (ELISA). Sandwich ELISA tested the ability of IgY to detect ICs in serum samples, from which diagnostic parameters were calculated. Antibody responses increased steadily from day 7 to 42. In the immunoblotting assay, IgY recognized two protein complexes. The immunofluorescence assay showed no staining in control samples. The sandwich ELISA presented a very high diagnostic value, with a sensitivity of 90% and a specificity of 86.7%. Our pioneer strategy highlights the potential use of egg yolk IgY as a diagnostic test to detect active hookworm infection.

Neurocysticercosis serodiagnosis: mimotope-based synthetic peptide as potential biomarker.

Herein, we evaluate a mimotope-based synthetic peptidenamed NC41 to diagnose neurocysticercosis (NC), a neglected parasitic disease and a major cause of epilepsy worldwide. NC41 synthetic peptide was evaluated to diagnose NC, and total saline extract from Taenia solium metacestodes (SE) was used as control. Serum samples from patients with NC (n = 40), other parasitic diseases (n = 43), and healthy individuals (n = 40) were tested. Diagnostic parameters such as sensitivity (Se), specificity (Sp), likelihood ratio (LR), and area under curve (AUC) were calculated using receiver operating characteristic (ROC) curves. The sequence from T. solium phosphoenolpyruvate carboxykinase (PEPCK) was used for epitope prediction, resulting in one high-scoring patch centered at residue L247. NC41 synthetic peptide reached high diagnostic performance (Se 97.5% and Sp 97.5%, LR+ 39 and AUC 0.997). Data from diagnostic parameters and in silico analyses proved the usefulness of NC41 synthetic peptide as a diagnostic marker for human NC.

Metaperiodate deglycosylation of Strongyloides venezuelensis larvae: Immunochemical characterization and antigen production for human strongyloidiasis diagnosis.

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.

Bacteriophage-fused peptides for serodiagnosis of human strongyloidiasis.

BACKGROUND: Strongyloidiasis, 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 FINDINGS: We 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/SIGNIFICANCE: These 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.

Reconstrução de couro cabeludo após ampla ressecção de angiossarcoma / Scalp reconstruction after wide resection of an angiosarcoma

Introdução: O couro cabeludo é uma área de difícil reconstrução devido à sua pouca elasticidade e por sobrepor-se a uma estrutura rígida e convexa. Existem diferentes técnicas cirúrgicas para reparação dos defeitos, que podem ser produto de diversas etiologias, como: traumas, deformidades e consequência de doenças, principalmente oncológicas, como é o caso do paciente apresentada neste trabalho. O angiossarcoma cutâneo é um tumor vascular maligno raro e extremamente agressivo, que afeta principalmente idosos. É caracterizado clinicamente pelo aparecimento de placas eritêmato-violáceas e de rápida evolução. O tratamento depende da extensão da doença. A maioria dos casos são tratados com ampla ressecção cirúrgica e reconstrução. O objetivo é relatar um caso de reconstrução de grande defeito do couro cabeludo depois de uma dissecção oncológica, realizado no Hospital Central do Exército (HCE) - RJ. Métodos: O caso foi tratado com enxerto autólogo e uso de matriz dérmica, em 2 tempos cirúrgicos, até a total cobertura da área lesionada. Resultados: Obteve-se resultado satisfatório após as cirurgias de enxertia de pele na área ressecada. Conclusão: O enxerto autólogo, juntamente a matriz dérmica mostrou-se uma opção viável na reconstrução do couro cabeludo.

Introduction: It is difficult to reconstruct the scalp due to its poor elasticity and presence of layers over a rigid convex structure. Different surgical techniques are used to repair defects that may develop due to several etiologies, such as trauma, deformities, and disease sequelae, especially cancer, as noted in the present case. Cutaneous angiosarcoma, a rare and extremely aggressive malignant vascular tumor that mainly develops in elderly individuals, is clinically characterized by the onset of rapidly evolving erythematous purple plaques. The treatment depends on disease extent. Most patients are treated with wide surgical resection and reconstruction. The objective is to report a case of reconstruction of a major scalp defect after an oncologic dissection performed at the Hospital Central do Exército in Rio de Janeiro. Methods: An autologous graft and dermal matrices were applied during two surgical periods till the damaged area was fully covered. Results: Satisfactory results were obtained after performing skin grafting surgery in the resected area. Conclusion: The autologous graft and dermal matrix proved to be a viable option for scalp reconstruction.

Development of specific scFv antibodies to detect neurocysticercosis antigens and potential applications in immunodiagnosis.

We have shown previously that detection of circulating antibodies against mimotopes selected by phage display were useful in neurocysticercosis diagnosis. However, circulating antigens may also be useful in patients' clinical follow-up. Therefore, we aimed to select novel combinatorial antibodies, single-chain variable fragment (scFv), which can be used for specific antigens with pre-defined affinity and specificity without prior immunization. A phage scFv antibody library was selected against Taenia solium mimotopes displayed on phages coupled in beads and total saline extract of T. solium metacestodes (S) immobilized on microtiter plate wells. After two rounds of selection, 96 phage clones were evolved and validated against each target by enzyme linked immunosorbent assay (ELISA), and dot-blot, and three specific antibodies (B6, G10 and A4) were further characterized by sequencing and indirect immunofluorescence (IFI) assays. IFI revealed tegument staining for the B6, while the others showed a non-uniform staining in the whole parasite. The selected scFvs were used to capture their antigen targets that were elucidated through mass spectrometry, and used for antibody detection in NC patients' sera by ELISA, which achieved sensitivities greater than 97% and specificities above 95%. We have successfully developed scFv antibodies against important mimotopes used in NC diagnosis, and can be further explored to detect circulating antigens for clinical follow-up of patients with NC. Our strategy also highlighted the possibility of using this combinatorial approach to select, capture and characterize specific antigens to better understand this intriguing parasite infection and disease evolution.

Taenia saginata metacestode antigenic fractions obtained by ion-exchange chromatography: potential source of immunodominant markers applicable in the immunodiagnosis of human neurocysticercosis.

The aim of this study was to fractionate and partially characterize fractions obtained from the total saline extract (SE) of Taenia saginata metacestodes after ion-exchange procedure in carboxymethyl sepharose (CM) and diethylaminoethyl sepharose (DEAE) resins, as a source of antigenic markers applicable in the immunodiagnosis of neurocysticercosis (NCC). For IgG detection by enzyme-linked immunosorbent assay (ELISA) and immunoblotting, 140 serum samples were analyzed: 45 from patients with NCC (G1), 50 from patients with other parasitic infections (G2), and 45 from healthy individuals. Sensitivity (Se), specificity (Sp), area under curve (AUC), and likelihood ratios (LR) were calculated. CM S2 and DEAE S2 fractions provided high diagnostic values (Se 88.8% and 93.4%; Sp 93.7% and 92.6%; AUC 0.965 and 0.987; LR+ 14.07 and 12.67; LR- 0.11 and 0.07, respectively). In conclusion, CM S2 and DEAE S2 fractions are important sources of specific peptides, with high efficiency to diagnose NCC.

Usefulness of concanavalin-A non-binding fraction of Strongyloides venezuelensis larvae to detect IgG and IgA in human strongyloidiasis.

Glycosylated components from Strongyloides have an important role in parasite establishment and host recognition of these substances. Considering the sugar-binding capacity of lectins, such as concanavalin-A (Con-A), IgG and IgA detection in serum samples from strongyloidiasis patients was tested using different antigenic preparations. The total saline extract (SE) of Strongyloides venezuelensis filariform larvae was fractionated in Con-A column to obtain Con-A unbound (Con-A UF) and Con-A bound (Con-A BF) fractions. Sensitivity (Se), specificity (Sp), area under the ROC curve (AUC), likelihood ratio (LR), and correlation coefficients were calculated. Con-A UF showed the highest diagnostic parameters for IgG detection (Se 95.0%, Sp 92.5%, AUC 0.99, LR 12.7) and high correlation (r = 0.700) with SE. Con-A fractions did not clearly demonstrate any usefulness for IgA detection. In conclusion, the results obtained demonstrate that Con-A UF is an important source of specific peptides efficient to detect IgG in strongyloidiasis immunodiagnosis.