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.

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.

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.

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.

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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.

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.