Development Of A Rapid Test For Toxocariasis Using Recombinant Toxocara Cati Tes-120 Protein
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Date
2020-01
Authors
Tan Farrizam, Siti Naqiuyah
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Toxocariasis is a neglected zoonotic disease with worldwide distribution and
has been shown to be especially prevalent among children from socio-economically
disadvantaged populations. The causative agents are Toxocara canis and Toxocara
cati in which the definitive hosts are dogs and cats, respectively. Although it is
acknowledged that human toxocariasis is caused by both T. canis and T. cati, however,
the importance of T. cati as a zoonotic agent of the disease has been under-recognized
since T. cati TES is rarely used for Toxocara serodiagnosis and T. canis TES antigens
are generally thought to be able to detect infections by both species. Thus, it is highly
likely that some cases of T. cati infection may be missed by tests using only T. canis
antigen. Therefore, it is an urgent need for the development of diagnostic tools that use
both T. cati and T. canis recombinant antigens to enable detection of infections caused
by both species. Previously, a T. cati recombinant protein TES-120 was successfully
produced and cloned into a pGEX-4T-1 expression vector which showed good
diagnostic potential in western blot. However, the yield was low and unsuitable for
rapid test development. In this study, the T. cati DNA insert was cloned into a pET32
expression vector and two short peptides tagged variants [five residues each of aspartic
acid (D) and lysine(K)] were designed to increase the chances of obtaining a good
yield of the soluble recombinant protein. All three variants namely TES-120
cati/pET32a, TES-120 5D cati/pET32a, and TES-120 5K cati/pET32a were expressed
and purified. After the protein identities were confirmed by anti-histidine western blot
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and MALDI TOF/TOF, the immunoreactivities of all three variants against
serum samples were tested by western blot and the best variant was found to be TES-
120 cati/pET32a. Lateral flow dipstick rapid test was then developed and tested against
a panel of serum samples, with anti-human IgG4 gold conjugate as the detector.
Diagnostic seroreactivity of the newly developed recombinant TES-120 cati lateral
flow dipstick was then compared to previously developed prototype rapid test which
comprised three dipsticks separately dotted with T. canis recombinant antigens i.e.
TES-26, TES-30, and TES-120. In this study, the diagnostic sensitivity of rTES-120
cati, rTES-26, rTES-30 and rTES-120 was found to be 64.0% (14/22), 82.0% (18/22),
77.0% (17/22) and 50.0% (11/22), respectively. When the results of all four dipsticks
were combined, the rapid test successfully detected 95.0% (21/22) of the positive
serum samples. In comparison, a sensitivity of 86% (19/22) was obtained when results
of the T. cati dipstick was excluded. All four recombinant dipsticks produced 100%
specificity when tested against healthy and other helminthic infection serum samples.
Thus, as a conclusion, the inclusion of T. cati TES-120 in the lateral flow rapid test for
toxocariasis increased its diagnostic sensitivity and enabling the detection of Toxocara
infection from both species T. cati and T. canis.
Description
Keywords
Toxocariasis , Protein