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 enzyme-linked immunosorbent assay (ELISA) for measuring Toxoplasma-specific IgG, IgM, and IgG avidity has been developed and assessed based on use of various Toxoplasma 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% sensitivity and 96.6% specificity compared to those of 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 those using TLAs.

INTRODUCTION

Toxoplasma gondii, the causative agent of toxoplasmosis, is a globally distributed parasite that can infect humans and warm-blooded animals. Humans become infected through ingesting contaminated food/water with oocysts excreted in cat feces or raw/undercooked meats containing tissue cysts. Although the primary infection usually causes benign mononucleosis-like symptoms in immunocompetent individuals, it can be complicated and life-threatening in immunocompromised patients such as organ transplant recipients, cancer patients, and those infected with human immunodeficiency virus (HIV) (1–3). Primary infection in pregnant women may be challenging and cause serious problems due to vertical transmission of rapidly replicating tachyzoites via the placenta which may lead to irreversible defects in the fetus, including abortion, stillbirth, or neonatal abnormalities. These highlight the importance of accurate discrimination between the primary and chronic infections, especially in pregnancy (1–3).

Toxoplasmosis can be diagnosed through isolation of the parasite from blood and body fluids as well as use of molecular methods and serological assays, which are currently reported as the major routine diagnostics of toxoplasmosis (4). Several serological assays, including commercial kits and automated platforms, are available for the diagnosis of T. gondii infections. Toxoplasma lysate antigens (TLAs) from the parasite tachyzoites are the most predominant components used in commercial serological assays. However, these antigens are not pure and may be associated with nonparasitic impurities such as culture media and host cell-derived components (5). Production methods of such antigens are time and labor consuming and may vary between various laboratories with potential biohazard challenges (6). Furthermore, standardization of such assays with TLAs is challenging and affected by insufficient specificity and inability to accurately discriminate between acute and chronic infections (7, 8).

A recently developed approach to improve diagnosis of T. gondii infections and discriminate between the acute and chronic infections is use of recombinant antigens instead of TLAs. Primary studies on plausibility of supplanting native antigens were carried out by Tenter and Johnson in 1991, who used two recombinant fusion proteins to improve serologic tests for diagnosis of T. gondii in human sera (9). Since 1991, complementary studies have verified effective uses of recombinant proteins to detect T. gondii-specific antibodies (10–15). Use of recombinant antigens facilitates standardization of the method. Precise antigen composition of the assay has been described, and more than one antigen can be used. More importantly, use of recombinant antigens that are specific for the acute or chronic stages of the infection could serve as a tool to discriminate between these stages (14). Of these recombinant proteins, surface antigens (SAGs), matrix antigens (MAGs), microneme proteins (MICs), rhoptry proteins (ROPs), and dense granule antigens (GRAs) are the most commonly used proteins in literature (13, 14).

Diagnosis of recently acquired T. gondii infections is based primarily on the detection of specific IgM antibodies, seroconversion, or significant increases in specific IgG antibody titers. Since seroconversion, as the most reliable serological marker, and increases in IgG titers are seldom demonstrable, detection of T. gondii-specific IgM antibodies is the most frequently used serological approach for the diagnosis of acute infections (4, 7, 16). However, IgM may be detected in individuals for a long time following the primary infections (16–18). Moreover, it has been reported that natural IgM may interact with Toxoplasma antigens in the absence of the infection (19–21). These reports on T. gondii IgM emphasize the importance of setting up a reliable laboratory tool to efficiently diagnose primary T. gondii infections (21).

Recently, Toxo IgG avidity assay has been described as a reliable and standard diagnostic tool for improved estimation of the infection acquisition time and identification of primary T. gondii infections in patients (21, 22). Furthermore, IgG avidity tests can provide confirmatory evidences of acute infections and discriminate between the reactivations and primary infections (23, 24). Avidity is described as the aggregate potency of polyclonal IgG antibody binding to antigens. Stronger bonds indicate longer durations of the infection. Low Toxo IgG avidity indices show possible recent infections, which do not exclude older infections. In contrast, high Toxo IgG avidity indices exclude recent infections of less than 4 months (23). Promising results of the authors’ previous studies (25–27) on use of recombinant proteins for serodiagnosis of T. gondii infections in humans as well as expensive commercial assays for the assessment of Toxoplasma-specific IgG, IgM, and IgG avidity encouraged carrying out this study. Therefore, the aim of this study was to develop an economical in-house enzyme-linked immunosorbent assay (ELISA) for the assessment of Toxoplasma-specific IgG, IgM, and IgG avidity based on various T. gondii antigens, including SAG1, GRA7, a combination of SAG1 and GRA7 (SAG1+GRA7), and TLAs for the discrimination of acute from chronic T. gondii infections.

MATERIALS AND METHODS

Ethical statement.

The study was carried out based on the ethical standards by institutional and/or national research committees and Helsinki Declaration, 1964. The animal procedures were carried out based on the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (U.S. NIH) and approved by the Ethical Committee of Tehran University of Medical Sciences, Tehran, Iran.

Study design and clinical sample collections.

The study was designed using 138 human sera from various clinical laboratories, from May 2017 to March 2018. In the current study, a set of well-characterized human sera were used, which were verified by the authors in previous studies (25, 28–30). Human sera were assessed for anti-Toxoplasma IgG and IgM using ELISA commercial kits (Trinity Biotech, New York, USA) according to the manual instructions. Results were interpreted as follows using the manufacturer’s recommendations: sera with <0.9 immune status ratio (ISR) were reported as negative, 0.9 to 1.1 as equivocal, and >1.1 as positive (28). All IgG-positive samples, regardless of the IgM results, were assessed for Toxo IgG avidity using VIDAS Toxo IgG avidity kit (bioMérieux, Marcy l’Etoile, France) based on the manual instructions. Interpretation of IgG avidity results was carried out as follows based on the manufacturer’s recommendations: values of <20% indicated low avidity, values of 20 to 30% indicated borderline, and values of >30% indicated high avidity of specific IgG (23). Sera were classified into three major groups as follows based on the results from commercial assays and clinical criteria (31). (i) Acute infection sera (AIS), including 29 sera with IgG⁺, IgM⁺, and low avidity profiles, represented acute infections. Lymphadenopathy, as an acute toxoplasmosis sign, was reported in patients of this group. No immunocompromised patients were included in this group, and therefore none of the patients were treated with pyrimethamine-sulfadiazine. (ii) Chronic infection sera (CIS), including 69 sera with IgG⁺, IgM⁻, and high avidity profiles, represented chronic infections. Follow-up of the samples revealed no increase in specific IgG levels or presence of IgM in this group. In general, 44 sera belonged to participants with a positive IgG history for >1 year and no IgM detected. (iii) Negative sera (NS), including 40 sera with no anti-T. gondii IgG and IgM, belonged to healthy individuals with no reported illnesses. To ensure blinded analyses, each sample was batched with a unique identification code and then used in in-house ELISA for the detection of human Toxoplasma-specific IgG and IgM. Furthermore, IgG-positive samples were assessed further using in-house IgG avidity.

Expression and purification of recombinant SAG1 and GRA7.

Recombinant expression plasmids were successfully constructed and purified in laboratory as described previously (25). Briefly, gene products were purified and cloned into pET28a vectors at specified restriction sites. The recombinant plasmids containing SAG1 or GRA7 genes were verified using colony PCR, restriction enzymes digestion, and sequencing. Escherichia coli BL21(DE3) pLysS (Promega, USA) was used to express recombinant antigens as described previously. Recombinant SAG1 and GRA7 proteins were purified using a purification system (Qiagen, Germany) and Ni-NTA resin based on the manufacturer’s instructions. Recombinant proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12% polyacrylamide gel and stained with Coomassie brilliant blue dye. Concentrations of the purified proteins were assessed using the Bradford method and bovine serum albumin (BSA) as standard. Immunoreactivity of the recombinant proteins was assessed using Western blotting. The protocol is described fully in a previous study by the authors (25).

Preparation of TLAs.

TLAs were prepared by sonicating the purified T. gondii tachyzoites RH strain collected from infected BALB/c mice peritoneal fluids (25). Supernatants were filtered through Whatman number 1 paper filters, divided into small aliquots, and stored at −20°C until use. Protein quantity of the samples was assessed using Bradford assay.

Assessment of in-house IgM and IgG ELISA.

To assess levels of specific IgM and IgG antibodies in the sera using recombinant and TLA antigens, immunoenzymatic assay was used based on a protocol previously described with minor modifications (26, 27). Briefly, serological Maxisorb 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated with recombinant proteins or TLAs in coating buffer (50 mM NaHCO₃ [pH 9.6]) at final concentrations of 5 and 2.5 μg/ml, respectively. These optimal concentrations were selected during preliminary assays (data not shown). After an overnight incubation at 4°C, plates were washed three times with washing buffer (0.1% Triton X-100 in phosphate-buffered saline [PBS]) and blocked by adding 200 μl of blocking solution (5% nonfat dry milk and 0.05% Tween 20 in PBS) per well. Then, wells were washed three times and incubated at 37°C for 1 h with human sera diluted 1:100 in blocking solution. Plates were washed three times with washing buffer and incubated with 100 μl of anti-human IgG/IgM conjugated with horseradish peroxidase (HRP) (Dako, Denmark) that was diluted 1:5,000 in blocking solution. After 30 min of incubation at 37°C, plates were washed and incubated for 30 min at room temperature with ortho-phenylenediamine (OPD) (Sigma-Aldrich, USA) as the substrate. After 45 min of incubation at 37°C in dark, reactions were stopped by adding 0.1 M sulfuric acid solution and the optical density (OD) was measured using automated ELISA reader (Biotek, USA) at 492 nm. Each serum sample was assessed twice. For each sample, results were reported as the mean OD of duplicate wells. Twenty negative sera from the NS group were assessed using in-house IgM and IgG ELISA to calculate cutoff values. Cutoff values were calculated as the mean OD plus 2 standard deviation (SD) values of Toxoplasma-negative sample reactivity. Values greater than the cutoff were reported as positive.

Assessment of in-house IgG avidity.

Maxisorb 96-well plates (Nunc, Roskilde, Denmark) were coated with recombinant proteins or TLAs in coating buffer (50 mM NaHCO₃ [pH 9.6]) at final concentrations of 5 and 2.5 μg/ml, respectively. The optimal concentrations were selected during the preliminary assessments (data not shown). After an overnight incubation at 4°C, plates were washed three times with washing buffer (0.1% Triton X-100 in PBS) and blocked by adding 200 μl of blocking solution (5% nonfat dry milk and 0.05% Tween 20 in PBS) per well. Then, wells were washed three times and incubated at 37°C for 1 h with human sera diluted 1:100 in blocking solution. Samples were assessed in two rows, including row A and row B. Row A was incubated with 200 μl of dissociation buffer (6 M urea and 0.05% Tween 20 in PBS) per well, and row B was incubated with 200 μl of regular washing solution (0.05% Tween 20 in PBS) per well. After 30 min of incubation at 37°C with vigorous shaking, plates were washed once with washing buffer and ELISA was carried out using procedures described previously. The OD of each well was measured using automated ELISA reader at 492 nm. Each serum sample was assessed twice. Results were represented as the avidity indices (AI) calculated using the following formula (22).

$$\text{AI\hspace{0.17em}}\left(\%\right)=\left(\frac{\text{OD\hspace{0.17em}of\hspace{0.17em}washed\hspace{0.17em}urea\hspace{0.17em}well}}{\text{OD\hspace{0.17em}of\hspace{0.17em}washed\hspace{0.17em}regular\hspace{0.17em}buffer\hspace{0.17em}well}}\right)\times \text{\hspace{0.17em}1}00$$

Cutoff values were calculated for each dilution as the mean OD plus 2 standard deviation (SD) values of the Toxoplasma-negative samples reactivity, with or without urea treatments. The AIs of <0.5 were considered low avidity, those of 0.5 to 0.6 were considered borderline/intermediate avidity, and those of >0.6 were considered high avidity. This classification was established based on the results achieved previously (32, 33). One low-avidity and one high-avidity sample were used as controls.

Statistical analysis.

Significance of associations between various assays was analyzed using McNemar chi-square test. To assess agreements between various assays, interrater agreement (kappa) was calculated and kappa values (κ) were considered as follows based on the Altman assessment (34): poor agreement (κ < 0.20), fair agreement (κ = 0.21 to 0.40), moderate agreement (κ = 0.41 to 0.60), good agreement (κ = 0.61 to 0.80), and very good agreement (κ = 0.81 to 1.00). Expected performances of in-house ELISA at various cutoff points were assessed using receiver operating characteristic (ROC) curves. Moreover, relative sensitivity and specificity of TLAs, SAG1, GRA7, and SAG1+GRA7 were calculated using 95% confidence intervals (35).

RESULTS

In-house IgM and IgG ELISA.

A total of 138 sera in three groups of AIS, CIS, and NS were assessed by in-house IgM and IgG ELISA using TLAs, SAG1, GRA7, and SAG1+GRA7 as coated antigens for diagnosis of toxoplasmosis. Results from the comparison of various antigens of in-house ELISA with those of commercial ELISA for the detection of Toxo IgM and IgG are presented in Tables 1 and 2. Very good agreements were found between in-house IgM and IgG ELISA using TLAs, SAG1, GRA7, and SAG1+GRA7 with commercial ELISA (κ > 0.80). The highest agreements were reported between in-house ELISA using SAG1+GRA7 and commercial ELISA with scores of 0.92 (95% confidence interval [CI] 0.86 to 1.00) and 0.98 (95% CI 0.94 to 1.00) for Toxo IgM and IgG, respectively. McNemar chi-square test showed no significant differences between the positive and negative results through comparing various antigens of in-house ELISA with those of commercial ELISA in detection of Toxo IgM and IgG (P > 0.05). Estimated sensitivities and specificities with the most appropriate cutoff values for various antigens were achieved from ROC analysis (Table 3). Calculated cutoff values were similar to those achieved from ROC analysis. The 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 (Table 3). Furthermore, AUC was calculated to be 0.99, 0.99, 0.98, and 0.99 for IgG-TLAs, IgG-SAG1, IgG-GRA7, and IgG-SAG1+GRA7, respectively (Table 3). Results of the study showed that GRA7 included 100% sensitivity for the detection of Toxo IgM, while SAG1 included 89.7% sensitivity (Table 3). 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 antigen, followed by TLAs (97.9%). For the detection of Toxo IgG, sensitivity achieved with GRA7 antigen was the lowest (93.8%) (Table 3). Furthermore, sensitivities and specificities with the most appropriate cutoff values for various antigens were estimated based on interactive dot diagrams (Fig. 1 and 2).

TABLE 1.

Results of Toxoplasma gondii IgM detection using in-house ELISA of TLAs, SAG1, GRA7, and SAG1+GRA7, compared to results of commercial ELISAs^a

Antigen	In-house ELISA results	Commercial ELISA results		Kappa agreement (95% CI)
		No. of positive samples	No. of negative samples
TLAs	No. of positive samples	28	6	0.86 (0.75–0.96)
	No. of negative samples	1	103
	Total	29	109
SAG1	No. of positive samples	26	4	0.86 (0.74–0.96)
	No. of negative samples	3	105
	Total	29	109
GRA7	No. of positive samples	29	4	0.92 (0.86–1.00)
	No. of negative samples	0	105
	Total	29	109
SAG1+GRA7	No. of positive samples	28	3	0.92 (0.86–1.00)
	No. of negative samples	1	106
	Total	29	109

^aTLAs, Toxoplasma lysate antigens; CI, confidence interval.

TABLE 2.

Results of Toxoplasma gondii IgG detection using in-house ELISA of TLAs, SAG1, GRA7, and SAG1+GRA7, compared to results of commercial ELISAs^a

Antigen	In-house ELISA	Commercial ELISA		Kappa agreement (95% CI)
		No. of positive samples	No. of negative samples
TLAs	No. of positive samples	96	0	0.97 (0.92–1.00)
	No. of negative samples	2	40
	Total	98	40
SAG1	No. of positive samples	95	1	0.93 (0.86–1.00)
	No. of negative samples	3	39
	Total	98	40
GRA7	No. of positive samples	92	2	0.86 (0.77–0.96)
	No. of negative samples	6	38
	Total	98	40
SAG1+GRA7	No. of positive samples	98	1	0.98 (0.94–1.00)
	No. of negative samples	0	39
	Total	98	40

^aTLAs, Toxoplasma lysate antigens; CI, confidence interval.

TABLE 3.

In-house IgM and IgG ELISA using TLAs, SAG1, GRA7, and SAG1+GRA7 for the detection of Toxoplasma gondii IgM and IgG^a

In-house ELISA type	Antigen	Calculated cutoff	ROC cutoff	AUC (95% CI)	Sensitivity (%)	Specificity (%)
IgM	TLAs	0.64	0.63	0.98 (0.94–0.99)	96.6	94.5
	SAG1	0.42	0.45	0.97 (0.93–0.99)	89.7	96.3
	GRA7	0.39	0.45	0.99 (0.97–1.00)	100.0	96.3
	SAG1+GRA7	0.41	0.41	0.99 (0.96–1.00)	96.6	97.2
IgG	TLAs	0.51	0.69	0.99 (0.96–1.00)	97.9	100.0
	SAG1	0.27	0.27	0.99 (0.96–1.00)	96.9	97.5
	GRA7	0.26	0.25	0.98 (0.94–0.99)	93.88	95.0
	SAG1+GRA7	0.30	0.28	0.99 (0.97–1.00)	100.0	97.5

^aTLAs, Toxoplasma lysate antigens; ROC, receiver operating characteristic; AUC, area under curve.

FIG 1.

Immunoreactivities of TLAs, SAG1, GRA7, and SAG1+GRA7 in IgM-ELISA using AIS, CIS, and NS. Horizontal lines represent cutoff values.

FIG 2.

Immunoreactivities of TLAs, SAG1, GRA7, and SAG1+GRA7 in IgG ELISA using AIS, CIS, and NS. Horizontal lines represent cutoff values.

In-house IgG avidity.

Details of IgG avidity ELISAs using various antigens to discriminate acute and chronic phases of T. gondii infections are shown in Table 4. The ability of various antigens to discriminate between the serum samples with low and high AI was assessed via two methods. The first method was based on the assumption that borderline avidity ranged from 0.5 to 0.6, while the second method was based on ROC analysis. In general, 29 sera from AIS group with low avidity reacted with TLAs and SAG1, GRA7, and SAG1+GRA7 antigens at low avidity in 25, 24, 26, and 27 cases, respectively, using in-house IgG avidity. Of 69 sera from patients with high avidity (CIS group), 66, 65, 65, and 67 sera reacted at high avidity with in-house ELISA IgG avidity using TLAs, SAG1, GRA7, and SAG1+GRA7, respectively. As shown in Table 4, the range of values for sera classified in the borderline avidity group varies depending on the antigens. Using the second method, ROC analysis was carried out after calculating cutoff values. It was assumed that borderline avidity fell between cutoff values of ±10%. Sample distribution analysis calculated AUC values of 0.95 (0.89 to 0.98), 0.97 (0.91 to 0.99), 0.96 (0.91 to 0.98), and 0.98 (0.93 to 0.99) for TLAs, SAG1, GRA7, and SAG1+GRA7, respectively. Combined recombinant proteins, SAG1+GRA7, showed the greatest potential for assessing avidity of IgG antibodies. This combination was characterized by 97.1% sensitivity and 96.6% specificity, compared to those of VIDAS Toxo IgG avidity (Table 4). Moreover, estimated sensitivities and specificities of IgG avidity with the most appropriate cutoff values for various antigens were carried out based on interactive dot diagrams (Fig. 3).

TABLE 4.

In-house IgG avidity ELISA using TLAs, SAG1, GRA7, and SAG1+GRA7 to discriminate between acute and chronic phases of Toxoplasma gondii infections^a

Antigen	Calculated cutoff (low, high)	ROC cutoff	Undetermined zone (borderline AI)	Avidity index (AI)		AUC (95% CI)	Sensitivity (%)	Specificity (%)	Agreement (95% CI)
				AIS (n = 29) (low, border, high)	CIS (n = 69) (low, border, high)
TLAs	<0.5, >0.6	0.52	0.46–0.57	25, 2, 2	3, 0, 66	0.95 (0.89–0.98)	95.7	93.1	0.87 (0.76–0.98)
SAG1	<0.5, >0.6	0.55	0.49–0.60	24, 1, 4	2, 2, 65	0.97 (0.91–0.99)	97.1	86.2	0.85 (0.73–0.97)
GRA7	<0.5, >0.6	0.47	0.43–0.52	26, 1, 2	0, 4, 65	0.96 (0.91–0.98)	97.1	93.1	0.93 (0.84–1.00)
SAG1+GRA7	<0.5, >0.6	0.57	0.51–0.62	27, 1, 1	2, 0, 67	0.98 (0.93–0.99)	97.1	96.6	0.95 (0.88–1.00)

^aAIS, 29 acute infection sera with IgG⁺, IgM⁺, and low avidity profiles; CIS, 69 chronic infection sera with IgG⁺, IgM⁻, and high avidity profiles.

FIG 3.

Immunoreactivities of TLAs, SAG1, GRA7, and SAG1+GRA7 in in-house IgG avidity assay using AIS (0) and CIS (1). Horizontal lines represent cutoff values.

DISCUSSION

Diagnosis of toxoplasmosis, which is often asymptomatic, is based most commonly on serological methods to detect T. gondii-specific IgG and IgM antibodies. Available immunoassays are based mainly on TLAs, which are highly sensitive and specific diagnostic tools (4, 36). However, it is well known that whole somatic antigen-based assays may vary significantly between the laboratories or batches and hence are difficult to standardize. Previous studies have shown that recombinant proteins may be attractive alternatives for TLAs used in commercial serological assays to detect anti-T. gondii IgG and IgM antibodies (8). Use of recombinant proteins in such assays yields improved reproducibility, easier standardization, and the ability to consistently choose similar compositions of the protein preparation (37). The SAG1 is one of the most immunogenic and stage-specific T. gondii antigens, which is present in tachyzoites but not in bradyzoites. This antigen has been used extensively as a serological marker for the diagnosis of anti-T. gondii antibodies in acute and chronic infections (8, 38). Moreover, GRA7, a potent antigen expressed in all infectious stages of T. gondii, can trigger significant humoral and cellular immune responses against toxoplasmosis, producing strong antibody responses in the acute phase of the infection (8). These facts highlight significance of the selection of SAG1 and GRA7 as antigen candidates in the present study. The highest sensitivity (100%) for the detection of Toxo IgG was recorded using SAG1+GRA7, followed by TLAs (97.9%) and SAG1 (96.9%), compared to those using commercial ELISAs. However, sensitivity achieved using GRA7 antigen was the lowest (93.8%). In contrast, GRA7 included the highest and SAG1 included the lowest sensitivities (100 and 89.7%, respectively) for the detection of Toxo IgM. It has been verified that SAG1 antigens are able to detect IgG reactivity in chronic stages of the infection. However, no reactivity was sometimes reported in chronic stages of the infection (5, 8, 38). Controversial findings in the diagnostic ability of SAG1 in human sera may be affected by various factors, including selected gene fragments, cloning stratagems, and preparations of this multipart molecules in various studies as well as sensitivity and specificity of the selected diagnostic methods by the specific studies (38).

In the current study, the highest agreements were reported between the in-house ELISA using SAG1+GRA7 and the commercial ELISA with scores of 0.92 (95% CI 0.86 to 1.00) and 0.98 (95% CI 0.94 to 1.00) for Toxo IgM and IgG, respectively. Recombinant proteins are used frequently as ELISA antigens to detect Toxo IgG and IgM in the majority of studies, mostly coated on ELISA plates alone. However, these proteins can be coated in several combinations, such as 2 to 3 proteins (8, 38). It has been shown that combination of two recombinant antigens of H4/GST and H11/GST in ELISA can result in a higher sensitivity (81.3%) than either H4/GST or H11/GST can (54 and 61%, respectively) (9, 39). In studies, combinations of recombinant proteins such as GRA7, GRA8, ROP1, and SAG1 have been reported as antigen preparations for the detection of IgG and IgM against T. gondii in human sera (5). Results have verified the potential use of two or three combined recombinant antigens to boost the sensitivity rates compared to those of crude TLA preparations. Additionally, numerous combinations of the recombinant proteins have been recommended for the detection of anti-Toxoplasma IgG antibodies, including GRA7, GRA8, and SAG1; GRA7, GRA8, SAG2, and H4; SAG1, GRA1, and GRA7; and SAG1 and GRA5 mixed with MAG1, GRA2, or ROP1 (8, 38). Results from previous studies have verified that combination of recombinant T. gondii antigens results in enhanced relative sensitivities of the assays. A recommended combination of the recombinant antigens, including at least one of GRA7, GRA8, SAG2, and H4 proteins, can be used in differentiation of early and past infections (40). More recently, Reynoso-Palomar et al. (41) investigated uses of single and combined SAG1, GRA1, and GRA7 in IgG ELISA for serodiagnosing T. gondii infections in jaguars in 10 Mexican zoos. Results showed that a combination of SAG1 and GRA7 included the highest sensitivity (97.5%) and specificity (91.6%) as well as an almost perfect agreement (96.2%) and a kappa value of 0.89 for the detection of Toxo IgG. They reported high serodiagnostic performances of IgG ELISA using a combination of recombinant GRA7 and SAG1 antigens for the detection of T. gondii infection in jaguars and possibly other domestic animals (41). In the authors’ previous study, results showed that in-house dot ELISA based on a combination of SAG1 and GRA7 was a promising diagnostic tool with a similar sensitivity to that of TLAs, which could be used for the serodiagnosis of toxoplasmosis in fields as well as less-equipped laboratories (25).

Antibody avidity assessment is described as a significant additional serological marker in immunodiagnosis of infectious diseases as low- and high-avidity antibodies increase principally in recent and long-term infections (23). Use of IgG avidity assays to verify or exclude acute T. gondii infections in IgM-positive results strengthens the serodiagnosis assay and can avoid unnecessary invasive procedures or treatments in pregnant women and immunocompromised patients (23). In this study, an in-house specific IgG avidity ELISA for discriminating recent from long-term T. gondii infections was standardized and assessed. Performance of the in-house assay was compared with that of a commercial VIDAS Toxo IgG avidity test. A very good agreement was seen between the in-house IgG avidity assay using TLAs, SAG1, GRA7, and SAG1+GRA7 and the commercial VIDAS Toxo IgG (κ > 0.80) for the discrimination of samples from acute and chronic phases of T. gondii infections. The combined recombinant proteins, SAG1+GRA7, showed a great potential for assessing avidity of IgG antibodies with 97.1% sensitivity and 96.6% specificity, followed by GRA7 (97.1% sensitivity and 93.1% specificity), compared to VIDAS Toxo IgG avidity. The efficacy of single antigen-based avidity assays has been described previously in several of studies. In a study by Marcolino et al. (42), p16, p32, and p38 were investigated using human sera in IgG immunoblotting avidity assays. The p38 band was reported as a good antigenic marker for the detection of low-avidity IgG in acute T. gondii infections. In another study, Elyasi et al. (43) assessed the practicality of avidity ELISA based on a recombinant GRA6 antigen for discriminating between acute and chronic toxoplasmosis. They reported a satisfactory clinical performance of GRA6 avidity assay compared to that of commercially available Euroimmune avidity ELISA for the exclusion of early infections of less than 4 months. Efficiencies of two combinations of recombinant antigens (ROP1+SAG2 and ROP1+GRA6) in IgG avidity assay were investigated by Drapała et al. (44). Results revealed that these two mixtures of antigens have high sensitivities (100 and 95.4%, respectively) for the detection of acute T. gondii infections, suggesting that novel IgG avidity assays using combinations of recombinant antigens could be useful for the discrimination of T. gondii infection phases. As GRA7 is found in parasitophorous vacuoles and cytoplasm of the T. gondii tachyzoite-infected host cells, it is concluded why the IgG antibody response against GRA7 is significantly higher in early stages of toxoplasmosis and thus can be an adequate candidate for the detection of acute T. gondii infections (45). In a complementary study by Pfrepper et al. (13), it was shown that GRA7 and/or GRA8 were entirely present in human sera at the initial stages of IgG responses, in contrast to MAG1 and SAG1. In time course studies, IgG antibody against GRA7 and GRA8 matured significantly earlier than that against SAG1 and MAG1. These recombinant antigens were used in a commercial immunoblot avidity kit (recomLine, MIKROGEN, Germany). Findings from the current study are supported by other independent findings, revealing that the GRA7 protein reacts further with acute sera than with chronic sera (5, 46). Disparities in ranges for low- and high-avidity antibodies are anticipated and possibly associated with several parameters, including patient heterogeneity, immune status at the time of blood collection, time of blood sampling relative to the onset of infection, assessment techniques, antigen preparation, types and concentrations of hydrogen bond-disrupting agents, and methods of calculating antibody avidity (47).

After several years of studying recombinant antigens, no protein has been identified and practically used as a selective marker for discriminating between the acute and chronic phases of T. gondii infections. However, the potential diagnostic values of recombinant proteins in detection of anti-T. gondii IgM or IgG antibodies have been verified in several studies (8, 35, 48). Since the ideal selection marker of acute and chronic phases of T. gondii infections has not already been investigated, it is essential to assess the maturation dynamics of IgG antibodies. To date, only a few studies have recommended recombinant proteins to assess avidity of IgG antibodies (10, 15, 37, 49, 50). This is possibly due to the difficulties in clarification of results from such experiments, based on several assumptions that must be made at the initial stages of the assay. In IgG ELISA avidity assay, it is critical that low, borderline, or high IgG avidity are determined accurately. Although commercial assays can describe how the results are interpreted, studies have suggested that the borderline avidity for recombinant proteins should be 0.3 to 0.4 (15, 32, 49). Conversely, according to the results by Costa et al. (51), it seems that assessment of borderline avidity is associated with the specificity of antigens used in the experiments. A limitation of the study included lack of specificity assessment using samples with antibodies against other closely related or potentially cross-reactive pathogens. However, in the authors’ previous study, no cross-reactivity was seen against human sera corresponding to parasitic infections other than toxoplasmosis using SAG1 and GRA7 proteins (25).

Conclusion.

In general, the current study has demonstrated that a combination of SAG1 and GRA7 recombinant antigens can successfully be used in serodiagnosis of T. gondii infections. Results from IgM, IgG, and IgG avidity ELISAs using a combination of SAG1 and GRA7 recombinant antigens were similar to results from the commercial assays using TLAs. The preliminary results have promised better discriminations between acute and chronic infections using a combination of SAG1 and GRA7 recombinant antigens compared to those using TLAs. Values of SAG1 and GRA7 recombinant antigens for diagnostic purposes increase since large volumes of highly purified materials are produced. Thus, this assay can be further standardized and is relatively economical to produce in large scales. The present results suggest that although an in-house IgG avidity assay with the highlighted antigens has shown the greatest potential for the discrimination of recent from long-term T. gondii infections, complementary studies are necessary to show exact increase times of conversions from low to high avidities after infections. Moreover, further studies on the presence of other antigens may help improve accuracy of the ELISA and IgG avidity assays for the diagnosis of T. gondii infections. Therefore, further assessments with extended pools of sera and other research methodologies are necessary before use of a combination of SAG1 and GRA7 recombinant antigens (rather than TLAs) in clinical diagnosis of T. gondii infections. Development of a rapid lateral-flow format of this assay is promising for use in settings with less equipment and/or trained personnel.

Data availability.

Data generated in this study are included in the published article. Data analyzed during the current study are publicly available via Figshare Repository, https://figshare.com/s/ef25602b09c7ac4266f1.