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Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography.

ABSTRACT: Strongyloidiasis is a neglected tropical disease that can cause fatal complications due to hyperinfection and disseminated strongyloidiasis in immunocompromised patients. We used two Strongyloides stercoralis recombinant antigenic proteins, L3NieAg.01 (NIE) and IgG-immunoreactive antigen (SsIR), to develop the recombinant antigen-based immunochromatography test (ICT) kit. We constructed and compared kits using either the NIE (NIE ICT kit) or the SsIR (SsIR ICT kit) antigens and a kit using a mixture of both (NIE-SsIR ICT kit) for detection of anti-Strongyloides IgG antibody in human serum samples. Serum samples from normal healthy individuals (Group I, n = 40), proven strongyloidiasis patients (Group II, n = 100), and those with other parasitic infections (Group III, n = 154) were evaluated. Sensitivity and specificity were 81.0% and 84.0% for the NIE ICT kit, 89.0% and 83.5% for the SsIR ICT kit, and 95.0% and 90.2% for the NIE-SsIR ICT kit, respectively. The NIE-SsIR ICT kit provided the best diagnostic results; it can supplement stool examination for clinical diagnosis and can be used to screen for asymptomatic S. stercoralis infection in people at risk in endemic areas. The NIE-SsIR ICT kit can also be used in large-scale sero-epidemiological investigations in endemic areas without the need for additional facilities or ancillary supplies.

SUBMITTER: Boonroumkaew P

PROVIDER: S-EPMC10723528 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography.

Boonroumkaew Patcharaporn P Sadaow Lakkhana L Janwan Penchom P Rodpai Rutchanee R Sanpool Oranuch O Buadee Punyisa P Suprom Chanida C Thanchomnang Tongjit T Intapan Pewpan M PM Maleewong Wanchai W

Parasite (Paris, France) 20231213

Strongyloidiasis is a neglected tropical disease that can cause fatal complications due to hyperinfection and disseminated strongyloidiasis in immunocompromised patients. We used two Strongyloides stercoralis recombinant antigenic proteins, L3NieAg.01 (NIE) and IgG-immunoreactive antigen (SsIR), to develop the recombinant antigen-based immunochromatography test (ICT) kit. We constructed and compared kits using either the NIE (NIE ICT kit) or the SsIR (SsIR ICT kit) antigens and a kit using a mix ...[more]

PMID: 38099622

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Project description: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.

Project description:BackgroundThe sensitivity of parasitological and molecular methods is unsatisfactory for the diagnosis of strongyloidiasis, and serological techniques are remaining as the most effective diagnostic approach. The present study aimed to design and produce a chimeric recombinant antigen from Strongyloides stercoralis immunoreactive antigen (SsIR) and Ss1a antigens, using immune-informatics approaches, and evaluated its diagnostic performance in an ELISA system for the diagnosis of human strongyloidiasis.Methodology/principal findingsThe coding sequences for SsIR and Ss1a were selected from GenBank and were gene-optimized. Using bioinformatics analysis, the regions with the highest antigenicity that did not overlap with other parasite antigens were selected. The chimeric recombinant antigen SsIR- Ss1a, was constructed. The solubility and physicochemical properties of the designed construct were analyzed and its tertiary structures were built and evaluated. The construct was expressed into the pET-23a (+) expression vector and the optimized DNA sequences of SsIR-Ss1a (873 bp) were cloned into competent E. coli DH5α cells. Diagnostic performances of the produced recombinant antigen, along with a commercial kit were evaluated in an indirect ELISA system, using a panel of sera from strongyloidiasis patients and controls. The physicochemical and bioinformatics evaluations revealed that the designed chimeric construct is soluble, has a molecular with of 35 KDa, and is antigenic. Western blotting confirmed the immunoreactivity of the produced chimeric recombinant antigen with the sera of strongyloidiasis patients. The sensitivity and specificity of the indirect ELISA system, using the produced SsIR-Ss1a chimeric antigen, were found to be 93.94% (95% CI, 0.803 to 0.989) and 97.22% (95% CI, 0.921 to 0.992) respectively.Conclusions/significanceThe preliminary findings of this study suggest that the produced SsIR-Ss1a chimeric antigen shows promise in the diagnosis of human strongyloidiasis. However, these results are based on a limited panel of samples, and further research with a larger sample size is necessary to confirm its accuracy. The construct has potential as an antigen in the ELISA system for the serological diagnosis of this neglected parasitic infection, but additional validation is required.

Project description:Strongyloides stercoralis infection is a neglected tropical disease with a global distribution. Serodiagnosis is a sensitive method, but improving its performance and simplifying into a point-of-care test (POCT) are needed. This study aimed to improve the diagnostic performance of serological tests using partially purified Strongyloides ratti antigen in an enzyme-linked immunosorbent assay (ELISA) and an immunochromatographic test (ICT). Crude S. ratti antigen was purified by an IgG affinity column to partition the antigen into flow-through, washing fraction (WF), and elution fractions. Optimized ELISA and ICT using crude and antigen fractions were used to analyze sera from three groups of subjects. Group 1 comprised subjects with proven strongyloidiasis, Group 2 were subjects with other parasitic infections, and Group 3 were negative parasitic infections. The diagnostic performance and Kappa agreement of the serological tests were analyzed and compared, using larvae detection as the reference test (fecal examination). The results showed that the WF was the most efficient antigen in terms of sensitivity and specificity, as determined by the ELISA and ICT. Kappa's agreement between fecal examination and WF-ELISA was moderate (Kappa = 0.52), and WF-ICT was almost perfect (Kappa = 0.94). The WF antigen reduced cross-reactivity to other parasitic infections, that is, Opisthorchis viverrini, Taenia spp., and hookworms, compared to crude S. ratti antigen when assessed by ELISA and ICT. We concluded that the WF of purified S. ratti improved the ELISA and ICT diagnostic performance, and the latter assay format could be used as a POCT for screening and controlling strongyloidiasis.IMPORTANCEThis study aimed to improve the serological diagnosis of strongyloidiasis, a disease caused by infection with the intestinal nematode Strongyloides stercoralis, by evaluating the impact of Strongyloides ratti antigen purification using an IgG affinity column for detecting parasite-specific IgG in serum via enzyme-linked immunosorbent assay (ELISA) and immunochromatographic test (ICT) formats. Compared to crude S. ratti antigen, the washing fraction (WF) of the purified antigen demonstrated significantly improved sensitivity and specificity in both ELISA and ICT, achieving strong diagnostic concordance with the gold-standard fecal examination. Furthermore, the WF antigen fraction exhibited reduced cross-reactivity with coinfections caused by the liver fluke (Opisthorchis viverrini), tapeworms (Taenia spp.), and hookworms. These findings underscore antigen purification as a promising strategy to enhance the accuracy of strongyloidiasis serodiagnosis.

Project description:The rapid detection and identification of infectious disease pathogens is a critical need for healthcare in both developed and developing countries. As we gain more insight into the genomic basis of pathogen infectivity and drug resistance, point-of-care nucleic acid testing will likely become an important tool for global health. In this paper, we present an inexpensive, handheld, battery-powered instrument designed to enable pathogen genotyping in the developing world. Our Microfluidic Biomolecular Amplification Reader (µBAR) represents the convergence of molecular biology, microfluidics, optics, and electronics technology. The µBAR is capable of carrying out isothermal nucleic acid amplification assays with real-time fluorescence readout at a fraction of the cost of conventional benchtop thermocyclers. Additionally, the µBAR features cell phone data connectivity and GPS sample geotagging which can enable epidemiological surveying and remote healthcare delivery. The µBAR controls assay temperature through an integrated resistive heater and monitors real-time fluorescence signals from 60 individual reaction chambers using LEDs and phototransistors. Assays are carried out on PDMS disposable microfluidic cartridges which require no external power for sample loading. We characterize the fluorescence detection limits, heater uniformity, and battery life of the instrument. As a proof-of-principle, we demonstrate the detection of the HIV-1 integrase gene with the µBAR using the Loop-Mediated Isothermal Amplification (LAMP) assay. Although we focus on the detection of purified DNA here, LAMP has previously been demonstrated with a range of clinical samples, and our eventual goal is to develop a microfluidic device which includes on-chip sample preparation from raw samples. The µBAR is based entirely around open source hardware and software, and in the accompanying online supplement we present a full set of schematics, bill of materials, PCB layouts, CAD drawings, and source code for the µBAR instrument with the goal of spurring further innovation toward low-cost genetic diagnostics.

Project description:BackgroundRapid point-of-care tests provide plausible diagnostic strategy for hepatitis B surface antigen (HBsAg) in low resource areas. However, their utility depends upon their overall performance. Our objective was to meta-analyze the diagnostic accuracy of rapid point-of-care tests for HBsAg.MethodsLiterature search was done with the help of a metasearch engine Mettā, a query interface for retrieving articles from five leading medical databases. Studies that employed rapid point-of-care tests for detection of HBsAg and compared the results with reference test were included. Two reviewers performed quality assessment of the studies and extracted data for estimating test accuracy. Twenty-seven studies were meta-analyzed and stratified by multiple parameters.ResultsTwenty-seven studies had evaluated 49 test brands and generated 76 data points. Sensitivity of individual tests varied widely and were heterogeneous (range 43.5%-99.8%); while specificity estimates were more robust and close to 100% (range 90%-100%). Overall pooled sensitivity, specificity, positive likelihood ratio (LR), negative LR and diagnostic odds ratio for all tests were 97.1% (95% CI, 96.1%-97.9%), 99.9% (CI, 99.8%-100%), 118.4 (CI, 84.7-165.5), 0.032 (CI, 0.023-0.045) and 4094.7 (CI, 2504.1-6600.8) respectively. This suggested high pooled accuracy for all studies. We found substantial heterogeneity between studies. Three factors (study location, reference standard and study score) appeared most strongly associated with test estimates and observed heterogeneity. The Determine test showed consistency in performance in studies done across developed and developing countries and the Determine and the BinaxNOW tests had significantly higher estimates than pooled estimates of remaining tests. Tests revealed analytical sensitivity of 4 IU/ml against manufacturer's claim of 0.5 IU/ml; reduced sensitivity with HBsAg mutants and poor performance in seroconversion panels.ConclusionsTests with better analytical sensitivity need to be developed and their feasibility and outcomes in various clinical settings need to be addressed.

Dataset Information

Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography.

Publications

Improved diagnostic sensitivity of human strongyloidiasis using point-of-care mixed recombinant antigen-based immunochromatography.

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