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Production of refolded Toxoplasma gondii recombinant SAG1-related sequence 3 (SRS3) and its use for serodiagnosis of human toxoplasmosis

Mirzadeh A, Saadatnia G, Golkar M, Babaie J, Noordin R

Protein Expr Purif, 2017 05;133:66-74.
PMID: 28263855 DOI: 10.1016/j.pep.2017.03.001

SAG1-related sequence 3 (SRS3) is one of the major Toxoplasma gondii tachyzoite surface antigens and has been shown to be potentially useful for the detection of toxoplasmosis. This protein is highly conformational due to the presence of six disulfide bonds. To achieve solubility and antigenicity, SRS3 depends on proper disulfide bond formation. The aim of this study was to over-express the SRS3 protein with correct folding for use in serodiagnosis of the disease. To achieve this, a truncated SRS3 fusion protein (rtSRS3) was produced, containing six histidyl residues at both terminals and purified by immobilized metal affinity chromatography. The refolding process was performed through three methods, namely dialysis in the presence of chemical additives along with reduced/oxidized glutathione and drop-wise dilution methods with reduced/oxidized glutathione or reduced DTT/oxidized glutathione. Ellman's assay and ELISA showed that the protein folding obtained by the dialysis method was the most favorable, probably due to the correct folding. Subsequently, serum samples from individuals with chronic infection (n = 76), probable acute infection (n = 14), and healthy controls (n = 81) were used to determine the usefulness of the refolded rtSRS3 for Toxoplasma serodiagnosis. The results of the developed IgG-ELISA showed a diagnostic specificity of 91% and a sensitivity of 82.89% and 100% for chronic and acute serum samples, respectively. In conclusion, correctly folded rtSRS3 has the potential to be used as a soluble antigen for the detection of human toxoplasmosis.
Fulltext Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples

Berizi M, Babaie J, Fard-Esfahani P, Enshaeieh M, Noordin R, Saadatnia G, et al.

Iran J Parasitol, 2022 1 28;16(4):621-630.
PMID: 35082891 DOI: 10.18502/ijpa.v16i4.7875

Background: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples.
Methods: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction).
Results: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant.
Conclusion: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.
Fulltext Identification and real-time expression analysis of selected Toxoplasma gondii in-vivo induced antigens recognized by IgG and IgM in sera of acute toxoplasmosis patients

Amerizadeh A, Khoo BY, Teh AY, Golkar M, Abdul Karim IZ, Osman S, et al.

BMC Infect Dis, 2013;13:287.
PMID: 23800344 DOI: 10.1186/1471-2334-13-287

Toxoplasma gondii is an obligate intracellular zoonotic parasite of the phylum Apicomplexa which infects a wide range of warm-blooded animals, including humans. In this study in-vivo induced antigens of this parasite was investigated using in-vivo induced antigen technology (IVIAT) and pooled sera from patients with serological evidence of acute infection.
IgG avidity Western blot using Toxoplasma gondii rGRA-7 cloned from nucleotides 39-711 for serodiagnosis of acute toxoplasmosis

Deshpande PS, Kotresha D, Noordin R, Yunus MH, Saadatnia G, Golkar M, et al.

Rev Inst Med Trop Sao Paulo, 2013 4 9;55(2):79-83.
PMID: 23563759

Toxoplasmosis is an important cause of congenital infection. The present study was performed to evaluate the usefulness of recombinant (r) GRA-7 cloned from nucleotides (n) 39-711 in discriminating between acute and chronic toxoplasmosis. First, commercial IgM, IgG and IgG avidity ELISAs were used to determine the serological profile of the sera. Serum samples were from 20 symptomatic patients with acute infection (low IgG avidity, IgM positive), 10 with chronic infection (high IgG avidity, IgM negative) and 10 with indeterminate IgG avidity (IgM positive) which were tested for IgG avidity status with an in-house developed IgG avidity Western blot using the rGRA-7 recombinant antigen. All 20 sera from cases of probable acute infection showed bands which either faded out completely or reduced significantly in intensity after treatment with 8 M urea, whereas the band intensities of the 10 serum samples from chronic cases remained the same. Of the 10 sera with indeterminate IgG avidity status, after treatment with 8 M urea the band intensities with six sera remained the same, two sera had completely faded bands and another two sera had significantly reduced band intensities. Discrimination between acute and chronic toxoplasmosis was successfully performed by the in-house IgG avidity Western blot.
Fulltext Multi-Laboratory Evaluation of a Lateral Flow Rapid Test for Detection of Amebic Liver Abscess

Noordin R, Yunus MH, Saidin S, Mohamed Z, Fuentes Corripio I, Rubio JM, et al.

Am J Trop Med Hyg, 2020 12;103(6):2233-2238.
PMID: 32996457 DOI: 10.4269/ajtmh.20-0348

Independent evaluations of XEh Rapid®, an IgG4-based rapid dipstick test, were performed to assess its diagnostic performance to detect amebic liver abscess (ALA) using 405 samples at seven laboratories in four countries. The test showed high diagnostic specificity (97-100%) when tested with samples from healthy individuals (n = 100) and patients with other diseases (n = 151). The diagnostic sensitivity was tested with a total of 154 samples, and the results were variable. It was high in three laboratories (89-94%), and moderate (72%) and low (38%) in two other laboratories. Challenges and issues faced in the evaluation process are discussed. Nevertheless, XEh Rapid is promising to be developed into a point-of-care test in particular for resource-limited settings, and thus merits further confirmation of its diagnostic sensitivity.