Design and Optimization of an IgG Avidity ELISA Based on Toxoplasma gondii Lysate Antigen for Differentiating Acute and Chronic Infections in Pregnant Women
-
Mostafa Tork
,
Shahabeddin Sarvi
,
Hossein Asgarian-Omran
,
Mitra Sadeghi
,
Bahare Basirpour
,
Eisa Nazar
,
Seyed Abdollah Hosseini
,
Ahmad Daryani
,
Sargis A. Aghayan
Abstract
Background: Toxoplasmosis is a significant global health concern. Definitive diagnosis of toxoplasmosis is a serious challenge due to false positive serological results. This study focuses on optimization and standardization of an in-house IgG avidity ELISA for Toxoplasma gondii using freshly prepared Toxoplasma lysate antigen (TLA).
Methods: Samples were collected during routine pregnancy screening. Following tachyzoite purification, TLA concentration was determined by the Bradford assay at 20 μg/mL. The indirect ELISA assay was then performed with a urea treatment step (6 M). Using 75 serum samples classified as acute or chronic toxoplasmosis, the IgG avidity test was performed by ELISA in two rows of microplates with 6 M urea and PBS, and then the avidity index was calculated.
Results: After calculating the cutoff values, the sensitivity and specificity of the in-house Toxoplasma IgG avidity kit were 92% (95% CI: 74–99%) and 100% (95% CI: 86–100%), respectively, with an area under the ROC curve (AUC) of 0.977 (95% CI: 0.951–1.000).
Conclusion: The results of the IgG avidity test designed in this study along with standardizing the method and interpreting the results showed the promising performance of this assay to differentiate the acute from the chronic stages of toxoplasmosis.
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2. Stelzer S, Basso W, Silván JB, et al. Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact. Food Waterborne Parasitol. 2019;15:e00037.
3. Hill D, Dubey JP. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect. 2002;8(10): 634-40.
4. Dubey JP. Toxoplasmosis of animals and humans: CRC press; 2016.
5. Abdoli A, Barati M, Dalimi A, Pirestani M, Hoseini Shokouh SJ. Toxoplasmosis among patients with immunocompromising conditions: a snapshot. J Arch Mil Med. 2016;4(4): e41832.
6. Teimouri A, Goudarzi F, Goudarzi K, et al. Toxoplasma gondii infection in immunocompromised patients in Iran (2013–2022): a systematic review and meta-analysis. Iran J Parasitol. 2022;17(4):443- 457.
7. Dalimi A, Abdoli A. Latent toxoplasmosis and human. Iran J Parasitol. 2012;7(1): 1–17.
8. Montoya J, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965-76.
9. Saadatnia G, Golkar M. A review on human toxoplasmosis. Scand J Infect Dis. 2012;44(11):805-14.
10. Horváth KN, Szénási Z, Danka J, Kucsera I. Value of the IgG avidity in the diagnosis of recent toxoplasmosis: A comparative study of four commercially available anti-Toxoplasma gondii IgG avidity assays. Acta Parasitol. 2005;50(3):255-60.
11. Julliac B, Théophile H, Begorre M, Richez B, Haramburu F. Side effects of spiramycin masquerading as local anesthetic toxicity during labor epidural analgesia. Int J Obstet Anesth. 2010;19(3):331-2.
12. Hedman K, Lappalainen M, Seppäiä I, Mäkelä O. Recent primary Toxoplasma infection indicated by a low avidity of specific IgG. J Infect Dis. 1989;159(4):736-40.
13. Teimouri A, Mohtasebi S, Kazemirad E, Keshavarz H. Role of Toxoplasma gondii IgG avidity testing in discriminating between acute and chronic toxoplasmosis in pregnancy. J Clin Microbiol. 2020;58(9): e00505-20.
14. Flori P, Bellete B, Crampe C, et al. A technique for dating toxoplasmosis in pregnancy and comparison with the Vidas anti-Toxoplasma IgG avidity test. Clin Microbiol Infect. 2008;14(3):242-9.
15. Hedman K, Seppälä I. Recent rubella virus infection indicated by a low avidity of specific IgG. J Clin Immunol. 1988;8:214-21.
16. Paul M. Immunoglobulin G avidity in diagnosis of toxoplasmic lymphadenopathy and ocular toxoplasmosis. Clin Diagn Lab Immunol. 1999;6(4):514-8.
17. Montazeri M, Ebrahimzadeh MA, Ahmadpour E, et al. Evaluation of propranolol effect on experimental acute and chronic toxoplasmosis using quantitative PCR. Antimicrob Agents Chemother. 2016;60(12):7128-33.
18. Dempster RP. Toxoplasma gondii: purification of zoites from peritoneal exudates by eight methods. Exp Parasitol. 1984;57(2):195-207.
19. Ferra BT, Holec-Gąsior L, Gatkowska J, et al. The first study on the usefulness of recombinant tetravalent chimeric proteins containing fragments of SAG2, GRA1, ROP1 and AMA1 antigens in the detection of specific anti-Toxoplasma gondii antibodies in mouse and human sera. PLoS One. 2019;14(6):e0217866.
20. Rahbari AH, Keshavarz H, Shojaee S, Mohebali M, Rezaeian M. IgG avidity ELISA test for diagnosis of acute toxoplasmosis in humans. Korean J Parasitol. 2012;50(2):99-102.
21. Teimouri A, Abbaszadeh Afshar MJ, Mohtasebi S, et al. 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. J Clin Microbiol. 2021;59(8):e0041621.
22. Pietkiewicz H, Hiszczyńska-Sawicka E, Kur J, et al. Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis. Parasitol Res. 2007;100:333-7.
23. Holec-Gąsior L, Drapała D, Lautenbach D, Kur J. Toxoplasma gondii: usefulness of ROP1 recombinant antigen in an immunoglobulin G avidity assay for diagnosis of acute toxoplasmosis in humans. Pol J Microbiol. 2010;59:307-10.
24. Del Bono V, Canessa A, Bruzzi P, Fiorelli MA, Terragna A. Significance of specific immunoglobulin M in the chronological diagnosis of 38 cases of toxoplasmic lymphadenopathy. J Clin Microbiol. 1989;27(9):2133-5.
25. Bobic B, Šibalić D, Djurkovic-Djakovic O. High levels of IgM antibodies specific for Toxoplasma gondii in pregnancy 12 years after primary Toxoplasma infection: case report. Gynecol Obstet Invest. 1991;31(3):182-4.
26. Gras L, Gilbert R, Wallon M, et al. Duration of the IgM response in women acquiring Toxoplasma gondii during pregnancy: implications for clinical practice and cross-sectional incidence studies. Epidemiol Infect. 2004;132(3):541-8.
27. Shah K, Maghsoudlou P. Enzyme-linked immunosorbent assay (ELISA): the basics. Br J Hosp Med (Lond). 2016;77(7):C98-101.
28. Wu K, Chen X-G, Li H, et al. Diagnosis of human toxoplasmosis by using the recombinant truncated surface antigen 1 of Toxoplasma gondii. Diagn Microbiol Infect Dis. 2009;64(3):261-6.
29. Taylor DW, Evans CB, Aley SB, Barta JR, Danforth HD. Identification of an apically‐located antigen that is conserved in sporozoan parasites. J Protozool. 1990;37(6):540-5.
30. Hassi A, Müller W, Aspöck H. An identical epitope in Pneumocystis carinii and Toxoplasma gondii causing serological cross reactions. Parasitol Res. 1991;77:351-2.
31. Buffolano W, Beghetto E, Del Pezzo M, et al. Use of recombinant antigens for early postnatal diagnosis of congenital toxoplasmosis. J Clin Microbiol. 2005;43(12):5916-24.
32. Beghetto E, Spadoni A, Bruno L, et al. Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products. J Clin Microbiol. 2006;44(6):2133-40.
33. Saeij JP, Boyle JP, Boothroyd JC. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol. 2005;21(10):476-81.
34. Frenkel J, Dubey J, Hoff R. Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni. J Protozool. 1976;23(3):421-4.
35. Dubey JP. The history of Toxoplasma gondii the first 100 years. J Eukaryot Microbiol. 2008;55(6):467-75.
36. Blake C, Gould BJ. Use of enzymes in immunoassay techniques A review. Analyst. 1984;109(5):533-47.
37. Proll G, Ehni M. Immunoassays. Handbook of Spectroscopy: Second, Enlarged Edition. 2014;1313-34.
38. Lilyanna S, Ng EMW, Moriguchi S, et al. Variability in microplate surface properties and its impact on ELISA. J Appl Lab Med. 2018;2(5):687-99.
39. Costa JG, Peretti LE, García VS, et al. P35 and P22 Toxoplasma gondii antigens abbreviate regions to diagnose acquired toxoplasmosis during pregnancy: toward single-sample assays. Clin Chem Lab Med. 2017;55(4):595-604.
2. Stelzer S, Basso W, Silván JB, et al. Toxoplasma gondii infection and toxoplasmosis in farm animals: Risk factors and economic impact. Food Waterborne Parasitol. 2019;15:e00037.
3. Hill D, Dubey JP. Toxoplasma gondii: transmission, diagnosis and prevention. Clin Microbiol Infect. 2002;8(10): 634-40.
4. Dubey JP. Toxoplasmosis of animals and humans: CRC press; 2016.
5. Abdoli A, Barati M, Dalimi A, Pirestani M, Hoseini Shokouh SJ. Toxoplasmosis among patients with immunocompromising conditions: a snapshot. J Arch Mil Med. 2016;4(4): e41832.
6. Teimouri A, Goudarzi F, Goudarzi K, et al. Toxoplasma gondii infection in immunocompromised patients in Iran (2013–2022): a systematic review and meta-analysis. Iran J Parasitol. 2022;17(4):443- 457.
7. Dalimi A, Abdoli A. Latent toxoplasmosis and human. Iran J Parasitol. 2012;7(1): 1–17.
8. Montoya J, Liesenfeld O. Toxoplasmosis. Lancet. 2004;363(9425):1965-76.
9. Saadatnia G, Golkar M. A review on human toxoplasmosis. Scand J Infect Dis. 2012;44(11):805-14.
10. Horváth KN, Szénási Z, Danka J, Kucsera I. Value of the IgG avidity in the diagnosis of recent toxoplasmosis: A comparative study of four commercially available anti-Toxoplasma gondii IgG avidity assays. Acta Parasitol. 2005;50(3):255-60.
11. Julliac B, Théophile H, Begorre M, Richez B, Haramburu F. Side effects of spiramycin masquerading as local anesthetic toxicity during labor epidural analgesia. Int J Obstet Anesth. 2010;19(3):331-2.
12. Hedman K, Lappalainen M, Seppäiä I, Mäkelä O. Recent primary Toxoplasma infection indicated by a low avidity of specific IgG. J Infect Dis. 1989;159(4):736-40.
13. Teimouri A, Mohtasebi S, Kazemirad E, Keshavarz H. Role of Toxoplasma gondii IgG avidity testing in discriminating between acute and chronic toxoplasmosis in pregnancy. J Clin Microbiol. 2020;58(9): e00505-20.
14. Flori P, Bellete B, Crampe C, et al. A technique for dating toxoplasmosis in pregnancy and comparison with the Vidas anti-Toxoplasma IgG avidity test. Clin Microbiol Infect. 2008;14(3):242-9.
15. Hedman K, Seppälä I. Recent rubella virus infection indicated by a low avidity of specific IgG. J Clin Immunol. 1988;8:214-21.
16. Paul M. Immunoglobulin G avidity in diagnosis of toxoplasmic lymphadenopathy and ocular toxoplasmosis. Clin Diagn Lab Immunol. 1999;6(4):514-8.
17. Montazeri M, Ebrahimzadeh MA, Ahmadpour E, et al. Evaluation of propranolol effect on experimental acute and chronic toxoplasmosis using quantitative PCR. Antimicrob Agents Chemother. 2016;60(12):7128-33.
18. Dempster RP. Toxoplasma gondii: purification of zoites from peritoneal exudates by eight methods. Exp Parasitol. 1984;57(2):195-207.
19. Ferra BT, Holec-Gąsior L, Gatkowska J, et al. The first study on the usefulness of recombinant tetravalent chimeric proteins containing fragments of SAG2, GRA1, ROP1 and AMA1 antigens in the detection of specific anti-Toxoplasma gondii antibodies in mouse and human sera. PLoS One. 2019;14(6):e0217866.
20. Rahbari AH, Keshavarz H, Shojaee S, Mohebali M, Rezaeian M. IgG avidity ELISA test for diagnosis of acute toxoplasmosis in humans. Korean J Parasitol. 2012;50(2):99-102.
21. Teimouri A, Abbaszadeh Afshar MJ, Mohtasebi S, et al. 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. J Clin Microbiol. 2021;59(8):e0041621.
22. Pietkiewicz H, Hiszczyńska-Sawicka E, Kur J, et al. Usefulness of Toxoplasma gondii recombinant antigens (GRA1, GRA7 and SAG1) in an immunoglobulin G avidity test for the serodiagnosis of toxoplasmosis. Parasitol Res. 2007;100:333-7.
23. Holec-Gąsior L, Drapała D, Lautenbach D, Kur J. Toxoplasma gondii: usefulness of ROP1 recombinant antigen in an immunoglobulin G avidity assay for diagnosis of acute toxoplasmosis in humans. Pol J Microbiol. 2010;59:307-10.
24. Del Bono V, Canessa A, Bruzzi P, Fiorelli MA, Terragna A. Significance of specific immunoglobulin M in the chronological diagnosis of 38 cases of toxoplasmic lymphadenopathy. J Clin Microbiol. 1989;27(9):2133-5.
25. Bobic B, Šibalić D, Djurkovic-Djakovic O. High levels of IgM antibodies specific for Toxoplasma gondii in pregnancy 12 years after primary Toxoplasma infection: case report. Gynecol Obstet Invest. 1991;31(3):182-4.
26. Gras L, Gilbert R, Wallon M, et al. Duration of the IgM response in women acquiring Toxoplasma gondii during pregnancy: implications for clinical practice and cross-sectional incidence studies. Epidemiol Infect. 2004;132(3):541-8.
27. Shah K, Maghsoudlou P. Enzyme-linked immunosorbent assay (ELISA): the basics. Br J Hosp Med (Lond). 2016;77(7):C98-101.
28. Wu K, Chen X-G, Li H, et al. Diagnosis of human toxoplasmosis by using the recombinant truncated surface antigen 1 of Toxoplasma gondii. Diagn Microbiol Infect Dis. 2009;64(3):261-6.
29. Taylor DW, Evans CB, Aley SB, Barta JR, Danforth HD. Identification of an apically‐located antigen that is conserved in sporozoan parasites. J Protozool. 1990;37(6):540-5.
30. Hassi A, Müller W, Aspöck H. An identical epitope in Pneumocystis carinii and Toxoplasma gondii causing serological cross reactions. Parasitol Res. 1991;77:351-2.
31. Buffolano W, Beghetto E, Del Pezzo M, et al. Use of recombinant antigens for early postnatal diagnosis of congenital toxoplasmosis. J Clin Microbiol. 2005;43(12):5916-24.
32. Beghetto E, Spadoni A, Bruno L, et al. Chimeric antigens of Toxoplasma gondii: toward standardization of toxoplasmosis serodiagnosis using recombinant products. J Clin Microbiol. 2006;44(6):2133-40.
33. Saeij JP, Boyle JP, Boothroyd JC. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol. 2005;21(10):476-81.
34. Frenkel J, Dubey J, Hoff R. Loss of stages after continuous passage of Toxoplasma gondii and Besnoitia jellisoni. J Protozool. 1976;23(3):421-4.
35. Dubey JP. The history of Toxoplasma gondii the first 100 years. J Eukaryot Microbiol. 2008;55(6):467-75.
36. Blake C, Gould BJ. Use of enzymes in immunoassay techniques A review. Analyst. 1984;109(5):533-47.
37. Proll G, Ehni M. Immunoassays. Handbook of Spectroscopy: Second, Enlarged Edition. 2014;1313-34.
38. Lilyanna S, Ng EMW, Moriguchi S, et al. Variability in microplate surface properties and its impact on ELISA. J Appl Lab Med. 2018;2(5):687-99.
39. Costa JG, Peretti LE, García VS, et al. P35 and P22 Toxoplasma gondii antigens abbreviate regions to diagnose acquired toxoplasmosis during pregnancy: toward single-sample assays. Clin Chem Lab Med. 2017;55(4):595-604.
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| Issue | Vol 21 No 3 (2026) | |
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How to Cite
1.
Tork M, Sarvi S, Asgarian-Omran H, Sadeghi M, Basirpour B, Nazar E, Hosseini SA, Daryani A, A. Aghayan S. Design and Optimization of an IgG Avidity ELISA Based on Toxoplasma gondii Lysate Antigen for Differentiating Acute and Chronic Infections in Pregnant Women. Iran J Parasitol. 2026;21(3):326-334.
