Serodiagnosis Of Strongyloidiasis: Identification Of Cdna Clones, Production Of Recombinant Antigens And Immunoassay Development
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Date
2016-03
Authors
Arifin, Norsyahida
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Strongyloidiasis is a human parasitic disease caused by the nematode
Strongyloides stercoralis. Infection by this parasite can cause long-term infection in
humans or can disseminate to other organs, especially in individuals with
immunosuppression, which commonly results in fatal outcomes. The majority of
patients are asymptomatic or present with non-specific gastrointestinal complaints,
and there is no gold standard method to rule out the infection. Definitive diagnosis is
usually made by a combination of clinical signs and symptoms, microscopic
identification, and serology test. To date, the available commercial tests are based on
native parasite antigen extract, but such tests have problems of cross-reactivity with
other helminthic infections. A recombinant antigen-based test is a good alternative
for improved diagnostic specificity and standardized test quality, thus, the present
study was conducted to achieve this goal. The initial stage of the study involved
testing serum samples with the in-house IgG-, IgG4- and IgE-ELISAs in addition to
a commercial IgG-ELISA kit. The results showed that the IgG-ELISA assay
demonstrated the highest sensitivity using either in-house or commercial assays
(84.6%, n=26), whereas the IgG4-ELISA displayed the highest specificity of 92.7%
(n=55). The serum samples were categorized into different groups based on the IgG
and IgG4 results for use in the subsequent immunoscreening experiments. Primary
immunoscreening of the S. stercoralis phage cDNA library resulted in the selection of 20 cDNA clones from each IgG- and IgG4-phage immunoblot. These clones were
subjected to secondary and tertiary immunoscreenings using individual pre-adsorbed
serum samples. Finally, two cDNA clones namely Ss3a and SsIa were selected as
having high potential diagnostic value. The Ss3a DNA insert was highly similar to a
S. stercoralis hypothetical protein, while the SsIa DNA insert was identical to an
immunoglobulin-binding protein of S. ratti. The Ss3a gene was recombinantly
expressed as a GST-fusion protein in the pET42a vector and validated by MALDI
TOF/TOF analysis. The diagnostic value of rSs3a protein was determined by western
blot analysis using individual serum samples. Subsequently, a one-hour lateral flow
dipstick test was produced with diagnostic sensitivity and specificity of 100%,
respectively. Meanwhile, the SsIa gene was fused to a dual protein tag in the pET28b
vector and expressed as a His-fusion protein and verified by western blot analysis
and MALDI-TOF TOF. The rSs1a was then developed into a rapid (15 min) lateral
flow dipstick test, which gave a sensitivity rate of 90% (n=30) and a specificity rate
of 98% (n=46). This study has successfully discovered two infection markers and
produced two new recombinant proteins of potential diagnostic value. Proof-ofconcept
of their usefulness in a lateral flow dipstick test for serodiagnosis of
strongyloidiasis was also demonstrated, and in combination may serve as a good
rapid test for of human strongyloidiasis.
Description
Keywords
Strongyloides stercoralis