Original Article

Detection Cell-Free DNA (cfDNA) Using Nested-PCR as a Diagnosis Tool for Human Fascioliasis Infection


Background: We aimed to detect Fasciola specific deoxyribonucleic acid (DNA) by nested-PCR assay on human stool and urine samples and compare the results with the respective ELISA diagnostic assay.

Methods: Overall, 206 clinically suspected cases of fascioliasis were enrolled in the study. Blood samples were collected from all the patients, and serum samples were isolated. ELISA assay, using Fasciola somatic antigen (SA), was carried out to detect anti Fasciola antibodies for the collected sera. DNA was randomly extracted from 25 stool and 10 urine samples of seropositive individuals and was evaluated by conventional PCR and nested PCR methods. The nested-PCR results were confirmed by sequencing the 430 bp region of ribosomal ITSI gene. Stool and urine samples from patients with different parasitic diseases and 25 stool samples from healthy individuals served as controls. Urine samples were collected from 10 healthy controls as well.

Results: Fascioliasis was detected by ELISA in 24.8% of the individuals. Of these, 25 seropositive patients were randomly assigned to the study. Fasciola DNA was identified in the stool samples of 96% of seropositive patients by nested PCR but ova of Fasciola was detected by parasitology methods in only 20% of seropositive cases. Fasciola DNA was identified in 90% of the urine samples by nested PCR. No cross-reactions were observed with other parasites.        

Conclusion: Detection of cfDNA in stool and urine samples has high accuracy and thus can be used for the diagnosis of Fasciola infection in human.


1. Harrington D, Lamberton PHL, McGregor A (2017). Human liver flukes. Lancet Gastroenterol Hepatol, 2(9):680-89.
2. Rokni M (2008). The present status of human helminthic diseases in Iran. Ann Trop Med Parasitol, 102(4):283-95.
3. Bozorgomid A, Nazari N, Eshrat Beigom K, et al (2018). Epidemiology of fasciolosis in Kermanshah Province, western Iran. Iran J Public Health, 47(7): 967-72.
4. Fredericks D (1996). Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates. Clin Microbiol Rev, 9(1):18-33.
5. Wilson IG (1997). Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 63(10):3741-51.
6. Joshi M,Deshpande J (2010). Polymerase chain reaction: methods, principles and application. Int J Biomed Res, 2(1):81-97.
7. Kajugu P-E, Hanna R, Edgar H, et al (2015). Fasciola hepatica: Specificity of a coproantigen ELISA test for diagnosis of fasciolosis in faecal samples from cattle and sheep concurrently infected with gastrointestinal nematodes, coccidians and/or rumen flukes (paramphistomes), under field conditions. Vet Parasitol, 212(3-4):181-87.
8. Arifin MI, Höglund J, Novobilský A (2016). Comparison of molecular and conventional methods for the diagnosis of Fasciola hepatica infection in the field. Vet Parasitol, 232:8-11.
9. Yu J, Gu G, Ju S (2014). Recent advances in clinical applications of circulating cell-free DNA integrity. Lab Med, 45(1):6-12.
10. Wagner J (2012). Free DNA–new potential analyte in clinical laboratory diagnostics? Biochemia medica: Biochemia medica, 22(1):24-38.
11. Aryaeipour M, Eshrat Beigom K, Heidari Z, et al (2015). Serological study of human fasciolosis in patients referring to the school of public health, Tehran University of Medical Sciences, Tehran, Iran during 2008–2014. Iran J Parasitol 10(4):517-22.
12. Saberinasab M, Mohebali M, Molawi G, et al (2014). Seroprevalence of human fascioliasis using indirect ELISA in Isfahan district, central Iran in 2013. Iran J Parasitol, 9(4):461-65.
13. Rokni M, Massoud J, O’Neill SM, et al (2002). Diagnosis of human fasciolosis in the Gilan province of Northern Iran: application of cathepsin L-ELISA. Diagn Microbiol Infect Dis, 44(2):175-79.
14. Rokni M, Bozorgomid A, Heydarian P, et al (2018). Molecular evidence of human Fasciolosis due to Fasciola gigantica in Iran: a case report. Iran J Public Health, 47(5):750-54.
15. Ashrafi K (2015). The status of human and animal fascioliasis in Iran: A narrative review article. Iran J Parasitol, 10(3):306-28.
16. Torgerson PR, Devleesschauwer B, Praet N, et al (2015). World Health Organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases, 2010: a data synthesis. PLoS Med, 12(12):1001920.
17. Sedighe M, Dabirzadeh M, Rokni Mb, et al (2019). Identification and Phylogenetic Classification of Fasciola species Isolated from Sheep and Cattle by PCR-RFLP in Zabol, in Sistan and Baluchistan Province, Southeast Iran. Iran J Public Health, 48(5):934-42.
18. Aryaeipour M, Bozorgomid A, Kazemi B, et al (2017). Molecular and morphometrical characterization of Fasciola species isolated from domestic ruminants in Ardabil Province, Northwestern Iran. Iran J Public Health, 46(3):318-25.
19. Moazeni M, Ahmadi A (2016). Controversial aspects of the life cycle of Fasciola hepatica. Exp Parasitol, 169:81-89.
20. Forghanparast K (2001). Comparison of the formalin-ether and Kato-Katz in the parasitological diagnosis of human fascioliasis. J Med Fac, 9:1-6.
21. Rokni M, Lesan S, Massoud J, et al (2006). Comparative evaluation of Fast enzyme linked immunosorbent assay (Fast-ELISA) and standard-ELISA for the diagnosis of human hydatidosis. Iran J Public Health, 35(2):29-32.
22. Adela Valero M, Victoria Periago M, Pérez‐Crespo I, et al (2012). Assessing the validity of an ELISA test for the serological diagnosis of human fascioliasis in different epidemiological situations. Trop Med Int Health, 17(5):630-36.
23. Charlier J, De Meulemeester L, Claerebout E et al (2008). Qualitative and quantitative evaluation of coprological and serological techniques for the diagnosis of fasciolosis in cattle. Vet Parasitol, 153(1-2):44-51.
24. Gormally E, Caboux E, Vineis P, et al (2007). Circulating free DNA in plasma or serum as biomarker of carcinogenesis: practical aspects and biological significance. Mutat Res, 635(2-3):105-17.
25. Chiu RW, Chan LY, Lam NY et al (2003). Quantitative analysis of circulating mitochondrial DNA in plasma. Clin Chem, 49(5):719-26.
26. Jiang W-W, Masayesva B, Zahurak M, et al (2005). Increased mitochondrial DNA content in saliva associated with head and neck cancer. Clin Cancer Res, 11(7):2486-91.
27. Su Y-H, Wang M, Brenner DE, et al (2004). Human urine contains small, 150 to 250 nucleotide-sized, soluble DNA derived from the circulation and may be useful in the detection of colorectal cancer. J Mol Diagn, 6(2):101-07.
28. Botezatu I, Serdyuk Og, Potapova G, et al (2000). Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clin Chem, 46(8):1078-84.
29. Diehl F, Schmidt K, Durkee KH, et al (2008). Analysis of mutations in DNA isolated from plasma and stool of colorectal cancer patients. Gastroenterology, 135(2):489-98.
30. Van der Drift M, Prinsen C, Hol B, et al (2008). Can free DNA be detected in sputum of lung cancer patients? Lung Cancer, 61(3):385-90.
31. Wang Y, Springer S, Zhang M, et al (2015). Detection of tumor-derived DNA in cerebrospinal fluid of patients with primary tumors of the brain and spinal cord. Proc Natl Acad Sci, 112(31):9704-09.
32. Pajek J, Kveder R, Guček A, et al (2010). Cell‐free DNA in the Peritoneal Effluent of Peritoneal Dialysis Solutions. Ther Apher Dial, 14(1):20-26.
33. Leon SA, Revach M, Ehrlich GE, et al (1981). DNA in synovial fluid and the circulation of patients with arthritis. Arthritis Rheum, 24(9):1142-50.
34. Wu D, Chi H, Shao M, et al (2014). Prenatal diagnosis of Down syndrome using cell-free fetal DNA in amniotic fluid by quantitative fluorescent polymersase chain reaction. Chin Med J, 127(10):1897-901.
35. Suzuki N, Kamataki A, Yamaki J, et al (2008). Characterization of circulating DNA in healthy human plasma. Clinica Chimica Acta, 387(1-2):55-8.
36. Tsumita T,Iwanaga M (1963). Fate of Injected Deoxyribonucleic Acid in Mice. Nature, 198(4885):1088-89.
37. Weerakoon KG,McManus DP (2016). Cell-free DNA as a diagnostic tool for human parasitic infections. Trends Parasitol, 32(5):378-91.
38. Wichmann D, Panning M, Quack T, et al (2009). Diagnosing schistosomiasis by detection of cell-free parasite DNA in human plasma. PLoS Negl Trop Dis, 3(4):1-9.
39. Ximenes C, Brandão E, Oliveira P, et al (2014). Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems. Mem Inst Oswaldo Cruz, 109(8):978-83.
40. Akter S, Nakao R, Imasato Y, et al (2019). Potential of cell-free DNA as a screening marker for parasite infections in dog. Genomics, 111(4):906-912.
41. Repetto S, Soto CA, Cazorla SI, et al (2013). An improved DNA isolation technique for PCR detection of Strongyloides stercoralis in stool samples. Acta Tropica, 126(2):110-14.
42. Al-Soud WA,Rådström P (2000). Effects of amplification facilitators on diagnostic PCR in the presence of blood, feces, and meat. J Clin Microbiol, 38(12):4463-70.
43. Hierl T, Reischl U, Lang P, et al (2004). Preliminary evaluation of one conventional nested and two real-time PCR assays for the detection of Toxoplasma gondii in immunocompromised patients. J Med Microbiol, 53(7):629-32.
44. Hafez H, Hauck R, Lüschow D, et al (2005). Comparison of the specificity and sensitivity of PCR, nested PCR, and real-time PCR for the diagnosis of histomoniasis. Avian Dis, 49(3):366-70.
IssueVol 49 No 6 (2020) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijph.v49i6.3367
Fascioliasis Serodiagnosis Nested-PCR Human Cell-free DNA

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
ARYAEIPOUR M, KAZEMI B, BOZORGOMID A, MOHEBALI M, AZIZI H, ROKNI M. Detection Cell-Free DNA (cfDNA) Using Nested-PCR as a Diagnosis Tool for Human Fascioliasis Infection. Iran J Public Health. 2020;49(6):1148-1156.