Designing a Recombinant Multi-Epitope Antigen of Echinococ-cus granulosus to Diagnose Human Cystic Echinococcosis

  • Aliyar MIRZAPOUR Department of Medical Parasitology and Mycology, International Branch of Shahid Beheshti University of Medical Sciences, Tehran, Iran .Department of Parasitology and Mycology, School of Medicine, Mashhad Branch, Islamic Azad University, Mashhad, Iran
  • Seyyed Javad SEYYED TABAEI 3. Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Teh-ran, Iran
  • Mojgan BANDEHPOUR Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran .Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  • Ali HAGHIGHI Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Teh-ran, Iran
  • Bahram KAZEMI 3. Department of Medical Parasitology and Mycology, School of Medicine, Shahid Beheshti University of Medical Sciences, Teh-ran, Iran 4. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Keywords: Multi-epitope, Echinococcus granulosus, Hydatidosis, ELISA

Abstract

Background: Cystic echinococcosis can cause severe disease and probable death in humans. Epitopes of its antigens play a key role in the sensitivity and specificity of immunodiagnostic tests. Methods: Epitope prediction software programs predict the most antigenic linear B-cell epitopes of AgB (8 kD), Ag5, and Ag95. Six such epitopes were predicted and connected by “Gly-Ser” linker and synthesized. The purity of the concentrated recombinant multi-epitope protein was assessed by 15% SDS-PAGE. Overall, 186 serum samples were collected from the Loghman Hakim Hospital and different laboratories, Tehran, Iran, from July 2016 to February 2017. Patients infected with hepatic hydatid cysts, patients infected by other parasites and viruses, and healthy individuals were used to detect the anti-CE IgG using recombinant multi-epitope protein. Results: Forty-one samples out of 43 cases of hydatidosis were diagnosed correctly as positive, and two were negative. In addition, six negative cases of healthy individual group were diagnosed as positive and negative with rMEP-ELISA and the commercial kit, respectively. Therefore, these six samples were considered as false positive using our method. In addition, a diagnostic sensitivity of 95.3% (95% CI, 84.19% to 99.43%) and a specificity of 95.0% (95% CI, 89.43% to 98.14%) were obtained using optimum cutoff value (0.20). The sensitivity and specificity of the commercial kit was 100%. Conclusion: Our findings showed high diagnostic accuracy of the ELISA test using the developed recombinant protein, which encourages the use of this recombinant multi-epitope protein for rapid serological diagnosis of hydatidosis.

References

1. McManus DP, Zhang W, Li J, Bartley PB. Echino-coccosis. Lancet. 2003; 362(9392):1295-304.
2. Moro P, Schantz PM. Echinococcosis: a review. Int J Infect Dis. 2009; 13(2):125-33.
3. Sabzevari S, Badirzadeh A, Shahkaram R, Seyyedin M. Traumatic rupture of liver hydatid cysts into the peritoneal cavity of an 11-year-old boy: a case report from Iran. Rev Soc Bras Med Trop. 2017; 50(6):864-7.
4. Torgerson PR, de Silva NR, Fèvre EM et al. The global burden of foodborne parasitic diseases: an update. Trends Parasitol. 2014; 30(1):20-6.
5. Singh BB, Dhand NK, Ghatak S, Gill JP. Econom-ic losses due to cystic echinococcosis in India: need for urgent action to control the disease. Prev Vet Med. 2014; 113(1):1-12.
6. Valizadeh M, Haghpanah B, Badirzadeh A et al. Immunization of sheep against Echinococcus granulosus with protoscolex tegumental surface antigens. Vet World. 2017; 10(8):854-8.
7. Craig P, Hegglin D, Lightowlers M et al. Chapter Two-Echinococcosis: Control and Prevention. Adv Parasitol. 2017; 96:55-158.
8. Mandal S, Mandal MD. Human cystic echinococ-cosis: epidemiologic, zoonotic, clinical, diagnostic and therapeutic aspects. Asian Pac J Trop Med. 2012; 5(4):253-60.
9. Budke CM, Casulli A, Kern P, Vuitton DA. Cystic and alveolar echinococcosis: Successes and contin-uing challenges. PLoS Negl Trop Dis. 2017; 11(4):e0005477.
10. Marzouki A, Naam A, Abdulrazak S et al. Muscu-loskeletal Echinococcus infection as a rare first presen-tation of hydatid disease: case report. Patient Safety in Surgery. 2017; 11(1):21.
11. Gharbi HA, Hassine W, Brauner M, Dupuch K. Ultrasound examination of the hydatic liver. Radiol-ogy.1981; 139(2):459-63.
12. Balik AA, Çelebi F, Başoğlu M et al. Intra-abdominal extrahepatic echinococcosis. Surg Today. 2001; 31(10):881-4.
13. Grimm F, Maly FE, Lü J, Llano R. Analysis of specific immunoglobulin G subclass antibodies for serological diagnosis of echinococcosis by a stand-ard enzyme-linked immunosorbent assay. Clin Di-agn Lab Immunol. 1998; 5(5):613-6.
14. Virginio V, Hernandez A, Rott M et al. A set of recombinant antigens from Echinococcus granulosus with potential for use in the immunodiagnosis of human cystic hydatid disease. J Clin Exp Immu-nol.2003; 132(2):309-15.
15. Sangaran A, Sundar SB, Latha BR. Antigen based detection of cystic echinococcosis in buffaloes us-ing ELISA and Dot-EIA. J Parasit Dis. 2017; 41(1):128-30.
16. Siles-Lucas M, Casulli A, Conraths F, Müller N. Laboratory diagnosis of Echinococcus spp. in human patients and infected animals. Adv Parasitol. 2017; 96:159-257.
17. Pan W, Chen DS, Lu YJ et al. Bioinformatic pre-diction of the epitopes of Echinococcus granulosus anti-gen 5. Biomed Rep. 2017; 6(2):181-7.
18. Savardashtaki A, Sarkari B, Arianfar F, Mostafavi-Pour Z. Immunodiagnostic Value of Echinococcus Granulosus Recombinant B8/1 Subunit of Antigen B. Iran J Immunol. 2017; 14(2):111-22.
19. Hezarjaribi HZ, Fakhar M, Esboei BR et al. Sero-logical evidence of human cystic echinococcosis and associated risk factors among general popula-tion in Mazandaran Province, northern Iran. Ann Med Surg (Lond).2017; 18:1-5.
20. Woollard DJ, Gauci CG, Heath DD, Lightowlers MW. Protection against hydatid disease induced with the EG95 vaccine is associated with confor-mational epitopes. Vaccine. 2000; 19(4):498-507.
21. Ponomarenko JV, Van Regenmortel MH. B cell epitope prediction. Struct Bioinform. 2009:849-79.
22. Blythe MJ, Flower DR. Benchmarking B cell epitope prediction: underperformance of existing methods. Protein Sci. 2005; 14(1):246-8.
23. Pestka S, Rubinstein M. Protein purification process and product. Google Patents; 1981.
24. Eckert J, Deplazes P. Biological, epidemiological, and clinical aspects of echinococcosis, a zoonosis of increasing concern. Clin Microbiol Rev. 2004; 17(1):107-35.
25. Ortona E, Riganò R, Margutti P et al. Native and recombinant antigens in the Immunodiagnosis of human cystic echinococcosis. Parasite Immunol. 2000; 22(11):553-9.
26. Leggatt G, Yang W, McManus D. Serological eval-uation of the 12 kDa subunit of antigen B in Echino-coccus granulosus cyst fluid by immunoblot analysis. Trans R Soc Trop Med Hyg. 1992; 86(2):189-92.
27. Li J, Zhang W-B, Wilson M et al. A novel recom-binant antigen for Immunodiagnosis of human cystic echinococcosis. J Infect Dis. 2003; 188(12):1951-60.
28. Zhang W, Wen H, Li J et al. Immunology and Immunodiagnosis of cystic echinococcosis: an up-date. Clin Dev Immunol. 2011; 2012.
29. Zhang W, Li J, McManus DP. Concepts in immu-nology and diagnosis of hydatid disease. Clin Mi-crobiol Rev. 2003; 16(1):18-36.
30. Kordafshari S, Hosseini SH, Jalousian F et al. Eval-uation of dot immunogold filtration assay (DIGFA) by recombinant protein EPC1 for anti-Echinococcus granulosus IgG antibody. Iran J Parasitol. 2015; 10(1):30-8.
31. Fleitas AL, Randall LM, Möller MN, Denicola A. Purification of a recombinant glutathione transfer-ase from the causative agent of hydatidosis, Echino-coccus granulosus. Biochem Mol Biol Educ. 2016; 44(1):28-37.
32. Lampe K, Gottstein B, Becker T et al. Immuniza-tion of rhesus macaques with Echinococcus multilocu-laris recombinant 14-3-3 antigen leads to specific an-tibody response. J Parasitol Res. 2017; 116 (1):435-9.
33. Lorenzo C, Ferreira HB, Monteiro KM et al. Com-parative analysis of the diagnostic performance of six major Echinococcus granulosus antigens assessed in a double-blind, randomized multicenter study. J Clin Microbiol. 2005; 43(6):2764-70.
34. Rott MB, Fernández V, Farias S et al. Comparative analysis of two different subunits of antigen B from Echinococcus granulosus: gene sequences, expression in Escherichia coli and serological evaluation. Acta Trop. 2000; 75(3):331-40.
35. Carmena D, Benito A, Eraso E. [Recent advances in the immunodiagnosis of human cystic echino-coccosis]. Enferm Infecc Microbiol Clin. 2007; 25(4):263-9.
Published
2020-03-09
How to Cite
1.
MIRZAPOUR A, SEYYED TABAEI SJ, BANDEHPOUR M, HAGHIGHI A, KAZEMI B. Designing a Recombinant Multi-Epitope Antigen of Echinococ-cus granulosus to Diagnose Human Cystic Echinococcosis. Iran J Parasitol. 15(1):1-10.
Section
Original Article(s)