Original Article

A Triplex Nested PCR for Diagnosis of Plasmodium, Babesia and Toxoplasma in Blood to Promote Safety of Blood Transfusion Samples


Background: The risk of transmission of some infectious agents has always been an important life-threatening side effect of blood transfusion. The aim of this study was to develop a triplex nested PCR (tnPCR) to assess the presence of protozoan parasites Plasmodium, Toxoplasma and Babesia in blood samples concurrently. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of developed tnPCR method compared to gold standard methods were determined for all three parasite genera.

Methods: After selecting the genus level specific primers and setting up of tnPCR, blood samples were collected partially from Isfahan Blood Transfusion Organization (IBTO). Some different samples from human and animal were tested by this method in comparison with the gold standard methods (microscopic method for Plasmodium, Babesia and ELISA for Toxoplasma) in 2021.

Results: This tnPCR works well and the sensitivity, specificity, PPV, NPV and accuracy, of this molecular method were all 100% for Plasmodium spp., 93.33%, 99.16%, 93.33%, 99.16% and 99.25% for Babesia spp. and 100%,98.5%, 85.72%, 98.5% and 100% for Toxoplasma gondii respectively compared to standard methods. In average there were 100%, 99.22%, 95.24%, 99.5% and 99.75% contingency for all three parasites (α<0.05). The designed and provided method can detect one, two, or all three potentially dangerous pathogens simultaneously in one tube and one-step, in biological specimens as well as blood.  

Conclusion: The developed tnPCR worked well. It could be recommended for facilitating test, saving time, reducing the expense and cross contamination, subsequently the promotion of blood transfusion safety.

1. O’Grady NP, Barie PS, Bartlett JG, et al. Guidelines for evaluation of new fever in critically ill adult patients: 2008 update from the american college of critical care medicine and the infectious diseases society of america. Crit Care Med. 2008;36:1330-49.
2. Storch EK, Custer BS, Jacobs MR, et al. Review of current transfusion therapy and blood banking practices. Blood Rev. 2019;38:100593.
3. World Health Organization (2021). Guidance on increasing supplies of plasma-derived medicinal products in low-and middle-income countries through fractionation of domestic plasma. WHO 2021.
4. McCullough J. Transfusion‐transmitted diseases. Transfusion-Transmitted Diseases. 2021;422-452.
5. Busch MP, Bloch EM, Kleinman S. Prevention of transfusion-transmitted infections. Blood. 2019;133(17):1854-1864.
6. Sarmah P, Dan M, Adapa D, et al. A review on common pathogenic microorganisms and their impact on human health. Electronic Journal of Biology. 2018;14(1):50-58.
7. Brouwer EE, van Hellemond JJ, van Genderen PJ, et al. A case report of transfusion-transmitted Plasmodium malariae from an asymptomatic non-immune traveller. Malaria J. 2013;12(1):1-6.
8. Lima GF, Sanchez MCA, Levi JE, et al. Asymptomatic infections in blood donors harbouring Plasmodium: An invisible risk detected by molecular and serological tools. Blood Transfus. 2018;16:17-25.
9. Mardani A. Prevention strategies of transfusion-transmitted parasitic infections (ttpis): Strengths and challenges of current approaches, and evaluation of the strategies implemented in Iran. Parasite Epidemiol Control. 2020;9:e00141.
10. Schnittger L, Rodriguez AE, Florin-Christensen M, et al. Babesia: A world emerging. Infect Genet Evol. 2012;12(8):1788-809.
11. Hill DE, Dubey JP. Toxoplasma gondii. Foodborne Parasites. Springer; 2018:119-138.
12. Sarkari B, Shafiei R, Zare M, et al. Seroprevalence and molecular diagnosis of Toxoplasma gondii infection among blood donors in southern Iran. J Infect Dev Ctries. 2014;8(4):543-7.
13. Derouin F, Pelloux H, Parasitology ESGoC. Prevention of toxoplasmosis in transplant patients. Clin Microbiol Infec. 2008;14:1089-101.
14. Siegel SE, Lunde MN, Gelderman AH, et al. Transmission of toxoplasmosis by leukocyte transfusion. Blood. 1971;37(4):388-94.
15. Tonnetti L, Townsend RL, Deisting BM, et al. The impact of Babesia microti blood donation screening. Transfusion. 2019;59:593-600.
16. Leiby D. Babesiosis and blood transfusion: Flying under the radar. Vox sang. 2006;90(3):157-65.
17. Bloch EM, Herwaldt BL, Leiby DA, et al. The third described case of transfusion‐transmitted Babesia duncani. Transfusi. 2012;52:1517-22.
18. Doni NY, Zeyrek FY, Seyrek A. Detection of Plasmodium using filter paper and nested pcr for patients with malaria in sanliurfa, in turkey. Malar J. 2016; 15(1):299.
19. Kim JY, Cho SH, Joo HN, et al. First case of human babesiosis in korea: Detection and characterization of a novel type of Babesia sp.(ko1) similar to ovine Babesia. J Clin Microbiol. 2007;45(6):2084-7.
20. Hierl T, Reischl U, Lang P, et al. Preliminary evaluation of one conventional nested and two real-time pcr assays for the detection of Toxoplasma gondii in immunocompromised patients. J Med Microbiol. 2004;53:629-632.
21. Bankole Henry O, Richard O, Eguagie Osareniro O, et al. Asymptomatic malaria among blood donors in Benin city Nigeria. Iran J Parasitol. 2014 9(3):415-422
22. Owusu-Ofori AK, Parry C, Bates I. Transfusion-transmitted malaria in countries where malaria is endemic: A review of the literature from sub-saharan africa. Clin Inft Dis. 2010;51(10):1192-8.
23. Thomas J, Ayieko P, Ogero M, et al. Blood transfusion delay and outcome in county hospitals in kenya. Am J Trop Med Hyg. 2017; 96(2):511-517.
24. Eskandarian A. Seroepidemiology of toxoplasmosis in admitted pregnant women in maternity ward of kowsar teaching and cure center in qazvin-2006. Iran J Med Microbiol. 2009;3:73-79.
25. Zakeri S, Najafabadi ST, Zare A, et al. Detection of malaria parasites by nested pcr in south-eastern, Iran: Evidence of highly mixed infections in Chahbahar district. Malar J. 2002; 1:2.
26. Lam W, Yeung AC, Tang JW, et al. Rapid multiplex nested pcr for detection of respiratory viruses. J Clin Microbiol. 2007;45(11):3631-3640.
27. Pöschl B, Waneesorn J, Thekisoe O, et al. Comparative diagnosis of malaria infections by microscopy, nested pcr, and lamp in northern thailand. The Am J Trop Med Hyg. 2010; 83(1):56-60.
28. Eskandarian AA, Jafarnezghad G-A, Akbari M. Seroprevalence of Toxoplasma-specific antibodies in patients suspected to have active toxoplasmosis: A cross-sectional survey. Adv Biomed Res. 2014; 3:236.
29. Ormazdi H, Sanikhani N, Hadighi R, et al. Investigation of antibodies (IGG and IGM) against Toxoplasma gondii in blood donors referred to Tehran Blood Transfusion Organization by ELISA. Urmia Medi J. 2010;21:212-216.
30. Gholami M, Mohammadi A, Fallah N. Seroprevalence of toxoplasmosis in blood donors of Hamadan transfusion center in 2013. Yafte. 2015; 17(2): 113-122.
31. Ferdowsi S, Farsi L, Tajallai SM, et al. Seroprevalence anti-Toxoplasma gondii antibodies and anti-epstein-barr virus (ebv) antibody among volunteer blood donors referred gonabad blood transfusion organization. Zabol Univer Med Sci. 2013;2:60-69.
32. Eskandarian AA. Development of an efficient multiplex semi-nested pcr for convenient use in urine samples for diagnosis of toxoplasmosis. Medical Express. 2014;1:39-42.
33. Eskandarian AA, Keshavarz H, Basco LK, et al. Do mutations in Plasmodium falciparum dihydropteroate synthase and dihydrofolate reductase confer resistance to sulfadoxine-pyrimethamine in Iran? Trans R Soc Trop Med Hyg. 2002;96(1):96-8.
34. Khamesipour F, Doosti A, Koohi A, et al. Determination of the presence of Babesia species in blood samples of cattle, camel and sheep in Iran by pcr. Archives of Biological Sciences. 2015;67(1):83-90.
IssueVol 17 No 2 (2022) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v17i2.9533
Plasmodium Babesia Toxoplasma Nested PCR Blood transfusion

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Eskandarian A. A Triplex Nested PCR for Diagnosis of Plasmodium, Babesia and Toxoplasma in Blood to Promote Safety of Blood Transfusion Samples. Iran J Parasitol. 2022;17(2):174-183.