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Identification and Preliminary Evaluation of a Novel Recombinant Protein for Serodiagnosis of Strongyloidiasis.

ABSTRACT: Strongyloides stercoralis is a human parasite that can cause a long-term infection. In immunosuppressed patients, strongyloidiasis may be fatal when there is overwhelming autoinfection resulting in the migration of large numbers of larvae through many organs. Definitive diagnosis is still a challenge, and a combination of symptoms, microscopic identification, and serology test results are often used to arrive at a clinical decision. However, intermittent larval excretion, low parasite burden, and occult infections are challenges with parasitological diagnosis of infection with S. stercoralis. Meanwhile, serologic tests using immunoglobulin G and parasite antigen extract have problems of cross-reactivity with other helminthic infections. Recombinant antigen-based serodiagnosis is a good alternative to overcome the laboratory diagnostic issues. Herein, we report on the isolation of cDNA clone encoding an antigen of potential diagnostic value identified from immunoscreening of a S. stercoralis cDNA library. The translated protein had highest similarity to Strongyloides ratti immunoglobulin-binding protein 1. The recombinant antigen produced, rSs1a, was assessed using western blot and enzyme-linked immunosorbent assay. The latter showed 96% diagnostic sensitivity and 93% specificity; thus, rSs1a has good potential for use in serodiagnosis of human strongyloidiasis.

SUBMITTER: Arifin N

PROVIDER: S-EPMC5928823 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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Identification and Preliminary Evaluation of a Novel Recombinant Protein for Serodiagnosis of Strongyloidiasis.

Arifin Norsyahida N Yunus Muhammad Hafiznur MH Nolan Thomas J TJ Lok James B JB Noordin Rahmah R

The American journal of tropical medicine and hygiene 20180208 4

<i>Strongyloides stercoralis</i> is a human parasite that can cause a long-term infection. In immunosuppressed patients, strongyloidiasis may be fatal when there is overwhelming autoinfection resulting in the migration of large numbers of larvae through many organs. Definitive diagnosis is still a challenge, and a combination of symptoms, microscopic identification, and serology test results are often used to arrive at a clinical decision. However, intermittent larval excretion, low parasite bur ...[more]

PMID: 29436335

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Project description:BackgroundBrucellosis is an important zoonotic disease caused by different Brucella species and human brucellosis is commonly prevalent in different states of India. Among various Brucella species, B. melitensis is most pathogenic to human and included as category B biothreat which can cause infection through aerosol, cut, wounds in skin and contact with infected animals. The diagnosis of human brucellosis is very important for proper treatment and management of disease as there is no vaccine available for human use. The present study was designed to clone, express and purify immunodominant recombinant omp2a (rOmp2a) porin protein of B. melitensis and to evaluate this new antigen candidate for specific serodiagnosis of human brucellosis by highly sensitive iELISA (indirect enzyme linked immunosorbent assay).MethodOmp2a gene of B. melitensis 16 M strain was cloned and expressed in pET-SUMO expression system. The recombinant protein was purified under denaturing conditions using 8 M urea. The purified recombinant protein was confirmed by western blotting by reacting with anti-HIS antibody. The sero-reactivity of the recombinant protein was also checked by reacting with antisera of experimentally infected mice with B. melitensis 16 M at different time points. Serodiagnostic potential of recombinant porin antigen was tested against 185 clinical serum samples collected from regions endemic to brucellosis in southern part of India by iELISA. The samples were grouped into five groups. Group 1 contained cultured confirmed positive serum samples of brucellosis (n = 15), group 2 contained sera samples from positive cases of brucellosis previously tested by conventional methods of RBPT (n = 28) and STAT (n = 26), group 3 contained sera samples negative by RBPT(n = 36) and STAT (n = 32), group 4 contained sera samples of other febrile illness and PUO case (n = 35) and group 5 contained confirmed negative sera samples from healthy donors (n = 23).ResultThe rOmp2a was found to be immunoreactive by iELISA and western blotting. The test showed a sensitivity of 93.75% and specificity of 95.83% when tested against 185 serum samples. For determination of statistical significance between experimental groups and control groups, Student's t test was performed on the data.ConclusionOmp2a emerges as a potential antigen candidate for serodiagnosis of human brucellosis.

Project description:Diagnosis of fascioliasis with high sensitivity and specificity antigens play a vital role in the management of the disease. Majority of commercial serological tests use F. hepatica native antigens and indicate wide diversities in test accuracy. Nowadays, recombinant antigens have been introduced as diagnostic reagents offer better test standardization. A combination of highly pure recombinant antigens associated with correct folding will leads to improve specificity and sensitivity of ELISA for diagnosis of Fascioliasis. In this article, Fasciola hepatica saposin-like protein 2 (SAP-2), ferritin protein (Ftn-1) and leucine aminopeptidase (LAP) recombinant antigens were considered as tools for the detection of F. hepatica immunoglobulin G antibodies in persons with chronic human fasciolasis. The recombinant antigens were obtained as fusion proteins, expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC). The refolding processes of denatured recombinant proteins were performed using dialysis method in the presence of chemical additives, and reduced/oxidized glutathione (in vitro). The immunoreactivity of the recombinant antigens was assessed individually and in a combination compared with excretory/secretory antigen (E/S) in an enzyme-linked immunosorbent assay (ELISA) test. The experiments were optimized using 213 serum samples from humans, including patients with chronic fascioliasis, patients with other parasitic diseases, and healthy subjects. The results indicated 95% sensitivity and 98% specificity for rtFhSAP-2, 96% sensitivity and 91% specificity for E/S, 80% and 83.3% for rtFhFtn-1, 84% and 88% for FhLAP, and also, 96% and 95% for combination of recombinant antigens, respectively. In conclusion, the results of this investigation showed that rtFhSAP-2 with the highest specificity and acceptable sensitivity has a considerable superiority compared to mentioned antigens and even in combination with these antigens in serodiagnosis of human fascioliasis.

Dataset Information

Identification and Preliminary Evaluation of a Novel Recombinant Protein for Serodiagnosis of Strongyloidiasis.

Publications

Identification and Preliminary Evaluation of a Novel Recombinant Protein for Serodiagnosis of Strongyloidiasis.

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