Development of a rapid serological assay for the diagnosis of strongyloidiasis using a novel diffraction-based biosensor technology.

BACKGROUND: Strongyloidiasis is a persistent human parasitic infection caused by the intestinal nematode, Strongyloides stercoralis. The parasite has a world-wide distribution, particularly in tropical and subtropical regions with poor sanitary conditions. Since individuals with strongyloidiasis are typically asymptomatic, the infection can persist for decades without detection. Problems arise when individuals with unrecognized S. stercoralis infection are immunosuppressed, which can lead to hyper-infection syndrome and disseminated disease with an associated high mortality if untreated. Therefore a rapid, sensitive and easy to use method of diagnosing Strongyloides infection may improve the clinical management of this disease. METHODOLOGY/PRINCIPAL FINDINGS: An immunological assay for diagnosing strongyloidiasis was developed on a novel diffraction-based optical bionsensor technology. The test employs a 31-kDa recombinant antigen called NIE derived from Strongyloides stercoralis L3-stage larvae. Assay performance was tested using retrospectively collected sera from patients with parasitologically confirmed strongyloidiasis and control sera from healthy individuals or those with other parasitoses including schistosomiasis, trichinosis, echinococcosis or amebiasis who were seronegative using the NIE ELISA assay. If we consider the control group as the true negative group, the assay readily differentiated S. stercoralis-infected patients from controls detecting 96.3% of the positive cases, and with no cross reactivity observed in the control group These results were in excellent agreement (κ = 0.98) with results obtained by an NIE-based enzyme-linked immunosorbent assay (ELISA). A further 44 sera from patients with suspected S. stercoralis infection were analyzed and showed 91% agreement with the NIE ELISA. CONCLUSIONS/SIGNIFICANCE: In summary, this test provides high sensitivity detection of serum IgG against the NIE Strongyloides antigen. The assay is easy to perform and provides results in less than 30 minutes, making this platform amenable to rapid near-patient screening with minimal technical expertise

Analysis of ELISA results using FhCL1 antigen and anti-total-IgG as secondary antibody.

<p>A) Histogram of control and <i>Fasciola-</i>positive serum samples. B) Histogram of normalized total IgG for negative control and <i>Fasciola</i> positive samples. Dash line represents cut-off for negative samples.</p

Analysis of ELISA data using FhCL1 antigen and secondary antibodies specific for IgG1, IgG2 and IgG4+IgG1 isotypes.

<p>Histograms of ELISA data obtained for Fasciola-positive and control patient sera using A) anti-IgG1, B) anti-IgG2, and C) IgG4+IgG1 secondary antibodies. Black bars represent sera from control patients while white bars represent sera from positive patients.</p

Scattergraphs for ELISA data using various secondary antibodies.

<p>Data obtained using secondary antibodies specific for anti-IgG4 (A), anti-IgG2 (B) and anti-IgG1 (C) compared to data obtained using anti-total IgG. Blue circles represent sera from control patients while green circles represent Fasciola-positive patients.</p

Comparison of ELISA data obtained using secondary antibodies specific for various isotypes.

<p>Absorbance between positive infected sera and negative control sera using anti-total IgG and the different serotypes.</p

Box plots of ELISA data using sera from non-infected control patients, <i>F. hepatica</i>-infected patients, and patients with various parasitic diseases.

<p>A: ELISA using anti-total IgG as secondary antibody and B: ELISA using anti-IgG4 as secondary antibody. The dashed line represents the cut-off for negative samples. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002414#s3" target="_blank">Results</a> are obtained from three independent experiments conducted in duplicate.</p

Sensitivity and specificity analysis for ELISA using anti-IgG4 as secondary antibody.

<p>A) Histogram showing negative sample where two fall out of the cut-off limit. B) Histogram showing Fasciola-positive and control negative sera. Discrimination between negatives and positives exists; however, a small space exists between them that cannot be noticed from the graph. Dashed line represents the cut-off for negatives sample.</p

New metabolic signature for Chagas disease reveals sex steroid perturbation in humans and mice

The causative agent of Chagas disease (CD), Trypanosoma cruzi, claims thousands of lives each year. Current diagnostic tools are insufficient to ensure parasitological detection in chronically infected patients has been achieved. A host-derived metabolic signature able to distinguish CD patients from uninfected individuals and assess antiparasitic treatment efficiency is introduced. Serum samples were collected from chronic CD patients, prior to and three years after treatment, and subjected to untargeted metabolomics analysis against demographically matched CD-negative controls. Five metabolites were confirmed by high-resolution tandem mass spectrometry. Several database matches for sex steroids were significantly altered in CD patients. A murine experiment corroborated sex steroid perturbation in T. cruzi-infected mice, particularly in male animals. Proteomics analysis also found increased steroidogenesis in the testes of infected mice. Metabolic alterations identified in this study shed light on the pathogenesis and provide the basis for developing novel assays for the diagnosis and screening of CD patients

Somatotroph pituitary adenoma with acromegaly and autosomal dominant polycystic kidney disease: SSTR5 polymorphism and PKD1 mutation

A 39-year-old woman with autosomal dominant polycystic kidney disease (ADPKD) presented with acromegaly and a pituitary macroadenoma. There was a family history of this renal disorder. She had undergone surgery for pituitary adenoma 6 years prior. Physical examination disclosed bitemporal hemianopsia and elevation of both basal growth hormone (GH) 106 ng/mL (normal 0-5) and insulin-like growth factor (IGF-1) 811 ng/mL (normal 48-255) blood levels. A magnetic resonance imaging scan disclosed a 3.0 cm sellar and suprasellar mass with both optic chiasm compression and left cavernous sinus invasion. Pathologic, cytogenetic, molecular and in silico analysis was undertaken. Histologic, immunohistochemical and ultrastructural studies of the lesion disclosed a sparsely granulated somatotroph adenoma. Standard chromosome analysis on the blood sample showed no abnormality. Sequence analysis of the coding regions of PKD1 and PKD2 employing DNA from both peripheral leukocytes and the tumor revealed the most common PKD1 mutation, 5014_5015delAG. Analysis of the entire SSTR5 gene disclosed the variant c.142C &gt; A (p.L48M, rs4988483) in the heterozygous state in both blood and tumor, while no pathogenic mutations were noted in the MEN1, AIP, p27Kip1 and SSTR2 genes. To our knowledge, this is the fourth reported case of a GH-producing pituitary adenoma associated with ADPKD, but the first subjected to extensive morphological, ultrastructural, cytogenetic and molecular studies. The physical proximity of the PKD1 and SSTR5 genes on chromosome 16 suggests a causal relationship between ADPKD and somatotroph adenoma.FAPESP (Sao Paulo State Research Foundation)Sao Paulo State Research Foundation (FAPESP)CNPq (National Research Foundation)CNPq (National Research Foundation)NIDDK [DK058816]NIDDKMayo PKD Translational CenterMayo PKD Translational Center [DK090728