Assessment of an In-House Enzyme-Linked Immunosorbent Assay and IgG Avidity Test Based on SAG1 and GRA7 Proteins for Discriminating between Acute and Chronic Toxoplasmosis in Humans

Abstract

To improve serodiagnostic methods for diagnosis of acute from chronic toxoplasmosis, an economical in-house ELISA for measuring Toxoplasma -specific IgG, IgM and IgG avidity has been developed and assessed based on use of various T. gondii antigens, including SAG1, GRA7 and a combination of SAG1 and GRA7 (SAG1+GRA7) as well as Toxoplasma lysate antigens (TLAs). Performances of in-house IgM, IgG and IgG avidity assays were compared to those of ELISA commercial kits and VIDAS Toxo IgG avidity. A set of 138 sera from patients with acquired T. gondii infection and seronegative people were assessed. Receiver operating characteristic (ROC) analysis revealed an area under curve (AUC) of 0.98, 0.97, 0.99 and 0.99 for IgM-TLAs, IgM-SAG1, IgM-GRA7, and IgM-SAG1+GRA7, respectively. Furthermore, AUC was calculated as 0.99, 0.99, 0.98 and 0.99 for IgG-TLAs, IgG-SAG1, IgG-GRA7 and IgG-SAG1+GRA7, respectively. The current study showed that GRA7 included 100% sensitivity for the detection of Toxo IgM, while SAG1 included 89.7% sensitivity. Furthermore, the highest specificity (97.2%) to detect Toxo IgM was achieved using SAG1+GRA7 antigen. For the detection of Toxo IgG, the highest sensitivity (100%) was recorded for SAG1+GRA7 followed by TLAs (97.9%). The SAG1+GRA7 showed the greatest potential for assessing avidity of IgG antibodies with 97.1% of sensitivity and 96.6% of specificity, compared to VIDAS Toxo IgG avidity. The preliminary results have promised better discriminations between acute and chronic infections using a combination of SAG1 and GRA7 recombinant antigens, compared to TLAs.

Utility of blood as the clinical specimen for the diagnosis of ocular toxoplasmosis using uracil DNA glycosylase-supplemented loop-mediated isothermal amplification and real-time polymerase chain reaction assays based on REP-529 sequence and B1 gene

“…A similar study by Zhang et al revealed that the detection limits of LAMP and conventional PCR assays using REP-529 target were 1 and 10 pg, respectively [ 43 ]. Various target genes such as SAG1, SAG2, SAG3, SAG4, ITS, B1, REP-529, P30, GRA4, GRA6 and GRA7 have been used to detect toxoplasmosis [ 44 – 48 ]. The results have shown that both REP-529 and B1 are highly sensitive; REP-529 is more sensitive than B1 gene.…”

“…The SAG1, SAG2, SAG3, SAG4, GRA4, GRA6 and GRA7 genes include only one copy in genome of T. gondii , which are specific as well [ 46 ]. Studies have shown that the number of copies of each gene fragment includes direct relationships with the diagnosis sensitivity [ 48 , 52 ]. Another factor that is important in selecting appropriate gene fragments is specificity [ 55 ].…”

IgG Avidity Test as a Tool for Discrimination between Recent and Distant Toxoplasma gondii Infection—Current Status of Studies

“…However, due to the various limitations of avidity tests based on these antigenic preparation, new diagnostic tools that might improve the performance of avidity tests to distinguish between recent and past phases of toxoplasmosis are tested. In the past 20 years, several studies have reported the use of single recombinant antigens [ 44 , 60 , 61 , 62 , 63 , 64 , 65 , 66 ], mixture of proteins [ 44 , 67 , 68 ] or chimeric proteins [ 69 ] in the determination of IgG avidity ( Table 1 ). All these antigens have been obtained in Escherichia coli expression systems and purified by affinity chromatography.…”

“…These results indicate that the ARCHITECT Toxo IgG Avidity assay can not only reliably detect acute phase samples much earlier than the Vidas system, but can also be performed longer for past infection samples with low-level IgG antibodies. Another study reported that the SAG1 recombinant protein mixed with the recombinant GRA7 showed potential for assessing avidity of IgG antibodies [ 68 ]. These preliminary results showed better discrimination between acute and chronic infection using the SAG1 + GRA7 combination than those using whole-cell antigens.…”

Application of Toxoplasma gondii-specific SAG1, GRA7 and BAG1 proteins in serodiagnosis of animal toxoplasmosis

“…Moreover, during T. gondii infections, the positivity for IgM antibody is proposed to be a marker of acute infection due to the occurrence of IgM antibody within days to a couple of weeks, and IgG antibodies are often interpreted as rising to protective levels after infection and remaining detectable for years, while the positivity for both IgG and IgM antibodies resulted is generally considered to indicate chronic reactivated cases (Kimbita et al, 2001;Dösķaya et al, 2014;Dhakal et al, 2015). IgM antibodies may be detected in humans or animals for a long time following primary T. gondii infections (Del Bono et al, 1989;Fricker-Hidalgo et al, 2013;Dhakal et al, 2015;Ybanez et al, 2020;Teimouri et al, 2021), and the natural IgM antibody may interact with parasite antigens in the absence of the Toxoplasma infection (Potasman et al, 1986;Sensini et al, 1996.;Liesenfeld et al, 1997;Dhakal et al, 2015;Teimouri In this work, although it is shown that Tibetan sheep, yaks, cows, and cattle were overall low both for T. gondii IgG and IgM positivity, there was no significant difference among rSAG1-, rGRA7-, and rBAG1-ELISAs. These results indicate that T. gondii may be infected at low titers in the animals collected in this study.…”