Development of Ss-NIE-1 recombinant antigen based assays for immunodiagnosis of strongyloidiasis.

Strongyloides stercoralis is a widely distributed parasite that infects 30 to 100 million people worldwide. In the United States strongyloidiasis is recognized as an important infection in immigrants and refugees. Public health and commercial reference laboratories need a simple and reliable method for diagnosis of strongyloidiasis to identify and treat cases and to prevent transmission. The recognized laboratory test of choice for diagnosis of strongyloidiasis is detection of disease specific antibodies, most commonly using a crude parasite extract for detection of IgG antibodies. Recently, a luciferase tagged recombinant protein of S. stercoralis, Ss-NIE-1, has been used in a luciferase immunoprecipitation system (LIPS) to detect IgG and IgG4 specific antibodies. To promote wider adoption of immunoassays for strongyloidiasis, we used the Ss-NIE-1 recombinant antigen without the luciferase tag and developed ELISA and fluorescent bead (Luminex) assays to detect S. stercoralis specific IgG4. We evaluated the assays using well-characterized sera from persons with or without presumed strongyloidiasis. The sensitivity and specificity of Ss-NIE-1 IgG4 ELISA were 95% and 93%, respectively. For the IgG4 Luminex assay, the sensitivity and specificity were 93% and 95%, respectively. Specific IgG4 antibody decreased after treatment in a manner that was similar to the decrease of specific IgG measured in the crude IgG ELISA. The sensitivities of the Ss-NIE-1 IgG4 ELISA and Luminex assays were comparable to the crude IgG ELISA but with improved specificities. However, the Ss-NIE-1 based assays are not dependent on native parasite materials and can be performed using widely available laboratory equipment. In conclusion, these newly developed Ss-NIE-1 based immunoassays can be readily adopted by public health and commercial reference laboratories for routine screening and clinical diagnosis of S. stercoralis infection in refugees and immigrants in the United States

Cross-reactivity of SS-NIE-1 ELISA and Luminex.

<p>NT = Not tested</p><p>Cross-reactivity of SS-NIE-1 ELISA and Luminex.</p

Performance of SS-NIE-1 ELISA and Luminex.

<p>Note: * CI = confidence interval</p><p>Performance of SS-NIE-1 ELISA and Luminex.</p

Baylisascaris procyonis Roundworm Seroprevalence among Wildlife Rehabilitators, United States and Canada, 2012–2015

Baylisascaris procyonis roundworms can cause potentially fatal neural larva migrans in many species, including humans. However, the clinical spectrum of baylisascariasis is not completely understood. We tested 347 asymptomatic adult wildlife rehabilitators for B. procyonis antibodies; 24 were positive, suggesting that subclinical baylisascariasis is occurring among this population

Development of a Luminex Bead Based Assay for Diagnosis of Toxocariasis Using Recombinant Antigens Tc-CTL-1 and Tc-TES-26.

The clinical spectrum of human disease caused by the roundworms Toxocara canis and Toxocara cati ranges from visceral and ocular larva migrans to covert toxocariasis. The parasite is not typically recovered in affected tissues, so detection of parasite-specific antibodies is usually necessary for establishing a diagnosis. The most reliable immunodiagnostic methods use the Toxocara excretory-secretory antigens (TES-Ag) in ELISA formats to detect Toxocara-specific antibodies. To eliminate the need for native parasite materials, we identified and purified immunodiagnostic antigens using 2D gel electrophoresis followed by electrospray ionization mass spectrometry. Three predominant immunoreactive proteins were found in the TES; all three had been previously described in the literature: Tc-CTL-1, Tc-TES-26, and Tc-MUC-3. We generated Escherichia coli expressed recombinant proteins for evaluation in Luminex based immunoassays. We were unable to produce a functional assay with the Tc-MUC-3 recombinant protein. Tc-CTL-1 and Tc-TES-26 were successfully coupled and tested using defined serum batteries. The use of both proteins together generated better results than if the proteins were used individually. The sensitivity and specificity of the assay for detecting visceral larval migrans using Tc-CTL-1 plus Tc-TES-26 was 99% and 94%, respectively; the sensitivity for detecting ocular larval migrans was 64%. The combined performance of the new assay was superior to the currently available EIA and could potentially be employed to replace current assays that rely on native TES-Ag

Specificity of the Toxocariasis Luminex assay.

<p>Specificity of the Toxocariasis Luminex assay.</p

Performance of Tc-CTL-1 and Tc-TES-26 Luminex based assays.

<p>Note</p><p>* = 95% Confidence Interval</p><p>Performance of Tc-CTL-1 and Tc-TES-26 Luminex based assays.</p

Identified proteins from mass spectrometry analysis.

<p>Note</p><p>+ = Molecular Weight</p><p>* = Sequence Coverage</p><p>Identified proteins from mass spectrometry analysis.</p

2-D gel electrophoresis, silver staining and western blotting of <i>Toxocara canis</i> Excretory Secretory Antigens (TES-Ag).

<p>The TES-Ag sample was separated and analyzed using 2D gel electrophoresis and western blotting. Three of the 2DE gels were transferred to nitrocellulose membranes and probed with a strong EIA positive <i>Toxocara</i> human sera pool (A), a negative human serum sample (B), and <i>Baylisascaris procyonis</i> positive serum (C).A reference gel was stained using silver stain (D). The circled spots in D represent proteins that were excised and subjected to mass spectrometry analysis.</p