Iranian Journal of Parasitology 2011. 6(4):1-7.

A Sensitive and Specific PCR Based Method for Identification of Cryptosporidium Sp. Using New Primers from 18S Ribosomal RNA
A Bairami Kuzehkanan, M Rezaeian, H Zeraati, M Mohebali, Z Babaei, L Kashi, M Heydarnezhadi, S Rezaie


Background: The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidi­osis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.

Methods: A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neel­sen staining method. Total DNA was extracted by QIA amp DNA stool mini kit was carried out by using designed prim­ers.

Results: Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopi­cally were posi­tive. The described primary and nested PCR method could detect all Cryptospori­dium positive samples from human and cattle. Regards to suspected negative samples in pri­mary PCR examination, the Nested PCR could ap­prove two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was nega­tive in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100 %.

Conclusion: Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.


Cryptosporidium; PCR; Identification

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Zhu G, Janet SK, Herve Ph. What is the phylogenetic position of Cryptosporidium? Int J Syst Evol Microbiol. 2000; 50: 1673–1681.

MacKenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med. 1994; 331: 161-7.

Dupont HL, Chappell CL, Sterling CR, Okhuysen PC, Rose B and JakubowskiW.The infectivity of Cryptosporidium parvum in healthy volunteers. N Engl J Med. 1995; 332: 855-859.

Savioli L, Smith H, Thompson A. Giardia and Cryptosporidium join the ‘Neglected Diseases Initiative’. Trends Parasitol. 2006; 22 (5): 203-208.

White AC Jr. Cryptosporidiosis (Cryptosporidium hominis, Cryptosporidium parvum, other species). In: Mandell GL, Bennett JE, Dolin R, editors. Principles and Practice of Infectious Diseases. 6th ed. Philadelphia: Elsevier Churchill Livingstone; 2005. P. 3215-3228.

Bushen OY, Lima AA, Guerrant RL. Cryptosporidiosis. In: Guerrant RL, Walker DH, Weller PF, editors. Tropical Infectious Diseases. Philadelphia: Elsevier Churchill Livingstone; 2006. P. 1003-1014.

KosekM, Alcantara C, Lima AA, Guerrant RL. Cryptosporidiosis: an update. Lancet Infect Dis. 2001; 1(4): 262-9.

CookGC. Opportunistic parasitic infections associated with the acquired immune deficiency syndrome (AIDS). Q J Med. 1987; 65: 967–983.

Xiao L, Escalante L, Yang C. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol. 1999; 65: 1578–1583.

Arrowood MJ. In vitro cultivation of Cryptosporidium species. ClinMicrobiol Rev. 2002; 15: 390–400.

Weber R, Bryan RT, Bishop HS, Wahlquist SP, Sullivan JJ, Juranek DD. Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J ClinMicrobiol. 1991; 29: 1323–1327.

Garcia LS, Brewer TC, Bruckner DA. Fluorescence detection of Cryptosporidium oocysts in human fecal specimens using monoclonal antibodies. J Clin Microbiol. 1987; 25: 119–121.

Kehl KSC, Cicirello H, Havens PL. Comparison of four different methods for the detection of Cryptosporidium species. J Clin Microbiol. 1995; 33: 416–418.

Xiao L, Lal AA, Jiang J. Detection and differentiation of Cryptosporidium oocysts in water by PCR-RFLP. Methods Mol Biol. 2004; 268: 163–176.

Sterling CR, Arrowood MJ. Detection of Cryptosporidium sp. infections using a direct immunofluorescent assay. Pediatr Infect Dis J. 1986; 5: 139–142.

Stibbs HH, Ongerth JE. Immunofluorescence detection of Cryptosporidium oocysts in fecal smears. J Clin Microbiol. 1986; 24: 517–521.

Graczyk TK, Cranfield MR, Fayer R. Evaluation of commercial enzyme immunoassay (EIA) and immunofluorescent antibody (FA) test kits for detection of Cryptosporidium oocysts of species other than Cryptosporidium parvum. Am J Trop Med Hyg. 1996; 54: 274–279.

Johnson, D.W, N.J. Pieniazek, D.W. Griffin, L.Misener, and J.B.Rose. Development of a PCR Protocol for Sensitive Detection of Cryptosporidium Oocysts in Water Samples. Applied and Environmental Microbiology. 1995 ; 61:3849-3855.

Haque R, Roy S, Siddique A et al. Multiplex real-time PCR assay for detection of Entamoeba histolytica, Giardia intestinalis, and Cryptosporidium spp. Am J Trop Med Hyg 2007; 76: 713–717.

Morgan UM, Pallant L, Dwyer BW, Forbes DA, Rich G, Thompson RCA. Comparison of PCR and Microscopy for Detection of Cryptosporidium parvum in Human Fecal Specimens. J Clin Microbiol. 1998; 36(4): 995–998.

Ridley DS, Hawgood BC. The value of formol- ether concentration of fecal cysts and ova. J Clin Patho. 1956; l(9): 74-76.

O'Donoghue PJ. Cryptosporidium and cryptosporidiosis in man and animals. Int J Parasitol. 1995; 25: 139–95.

FallA, Thompson RC, Hobbs RP, Morgan- Ryan UM. Morphology is not a reliable tool for delineating species within Cryptosporidium. J Parasitol. 2003; 89: 399–402.

Clark DP. New insights into human cryptosporidiosis. Clin Microbiol Rev. 1999; 12: 554–63.

Rodriguez-Hernandez J, Canutblasco A, Ledesmagarcia M, Martinsanchez A M. Cryptosporidium oocysts in water for human consumption comparison of staining methods. Eur J Epidemiol. 1994; 10: 215–218.

Meamar AR, Guyot K, Certad G, Dei-Cas E, MohrazM,MohebaliM,Mohammad K, Mehbod AA, Rezaie S, Rezaian M. Molecular Characterization of Cryptosporidium Isolates from Humans and Animals in Iran. Appl Environ Microbiol. Feb. 2007; 73(3): 1033–1035.

PirestaniM, Sadraei J,Dalimi-aslAH, Zavvar M, Vaeznia H. Molecular characterization of Cryptosporidium isolates from human and bovine using 18s rRNA gene in Shahriar county of Tehran, Iran. Parasitol Res. 2008; 103: 467–472.

Keshavarz A, Haghighi A, Athari A, Kazemi B, Abadi A, Nazemalhosseini-Mojarad E. Prevalence and molecular characterization of bovine Cryptosporidium in Qazvin province, Iran. Vet Parasitol. 2009; 160: 316–318.


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