Journal of Parasitic Diseases

, Volume 40, Issue 3, pp 958–963 | Cite as

Evaluation of modified Ziehl–Neelsen, direct fluorescent-antibody and PCR assay for detection of Cryptosporidium spp. in children faecal specimens

  • S. Aghamolaie
  • A. Rostami
  • Sh. Fallahi
  • F. Tahvildar Biderouni
  • A. Haghighi
  • N. SalehiEmail author
Original Article


To determine the sensitivity and specificity of routine screening methods for cryptosporidiosis, three methods including conventional modified Ziehl–Neelsen (MZN), direct fluorescent-antibody (DFA) and Nested-PCR assay compared together. To this end, their ability to identify the low concentrations of Cryptosporidium spp. oocysts in children fecal samples was evaluated. The sample population of this study was children under 12 years old who had diarrhea and referred to pediatric hospitals in Tehran, Iran. 2,510 stool specimens from patients with diarrhea were screened for Cryptosporidium oocysts by concentration method and MZN. To determine sensitivity and specificity, Nested-PCR and DFA were performed on 30 positive and 114 negative samples which previously had been proved by MZN. By using the microscopic method, DFA assay and PCR analysis, a total of 30 (1.2 %), 28 (1.1 %) and 32 (1.27 %) positive samples were detected respectively. According to the results, the sensitivity, specificity, and positive and negative predictive values of the Nested-PCR assay were 100 %, compared to 94, 100, 100, and 98 %, respectively, for MZN and 87.5, 100, 100, and 96 %, respectively, for DFA. Results of the present study showed that the Nested-PCR assay was more sensitive than the other two methods and laboratories can use the Nested-PCR method for precise diagnosis of Cryptosporidium spp. However, regarding the costs of Nested-PCR and its unavailability in all laboratories and hospitals, MZN staining on smears has also enough accuracy for Cryptosporidium diagnosis.


Cryptosporidium Modified Zeihl–Neelsen PCR Direct fluorescent-antibody 



This research was part of master’s (MSc) thesis and financially supported by Vice Chancellors for Education of Shahid Beheshti University of Medical Sciences and approved by Ethical Committee of Shahid Beheshti University of Medical Sciences, Tehran, Iran. The authors wish to thank the Shahid Fahmideh, Mofid pediatric hospitals and Mahak Medical Center staffs in Tehran city, for their great assistance.


  1. Barugahare R, Dennis MM, Becker JA, Sˇlapeta J (2011) Detection of Cryptosporidium molnari oocysts from fish by fluorescent-antibody staining assays for Cryptosporidium spp. affecting humans. App Environ Microbiol 77:1878–1880CrossRefGoogle Scholar
  2. Barwick RS, Levy DA, Craun GF, Beach MJ, Calderon RL (2000) Surveillance for waterborne-disease outbreaks-United States, 1997–1998. MMWR Morb Mortal Wkly Rep 49:1–21Google Scholar
  3. Bialeka R, Bindera N, Dietzb K, Joachimc A, Knobloch J, Zelcka UE (2002) Comparison of fluorescence, antigen and PCR assays to detect Cryptosporidium parvum in fecal specimens. Diagn Microbiol Infect Dis 43:283–288CrossRefGoogle Scholar
  4. Cole DJ (1997) Detection of Cryptosporidium parvum using the Kinyoun acid-fast stain. Proc Annu Conv AAEP 43:409–410Google Scholar
  5. Fayer R, Ungar BL (1986) Cryptosporidium spp. and cryptosporidiosis. Microbiol Rev 50:458–483PubMedPubMedCentralGoogle Scholar
  6. Fayer R, Xiao L (2008) Cryptosporidium and cryptosporidiosis, 2nd edn. CRC, Boca RatonGoogle Scholar
  7. Fayer R, Morgan U, Upton SJ (2000) Epidemiology of Cryptosporidium: transmission, detection and identification. Int J Parasitol 30:1305–1322CrossRefPubMedGoogle Scholar
  8. Garcia LS, Shimizu RY (1997) Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens. J Clin Microbiol 35:1526–1529PubMedPubMedCentralGoogle Scholar
  9. Garcia LS, Brewer TC, Bruckner DA (1987) Fluorescence detection of Cryptosporidium oocysts in human fecal specimens by using monoclonal antibodies. J Clin Microbiol 25:119–121PubMedPubMedCentralGoogle Scholar
  10. Garcia LS, Shum AC, Bruckner DA (1992) Evaluation of a new monoclonal antibody combination reagent for direct fluorescence detection of Giardia cysts and Cryptosporidium oocysts in human fecal specimens. J Clin Microbiol 30:3255–3257PubMedPubMedCentralGoogle Scholar
  11. Kaushik K, Khurana S, Wanchu A, Malla N (2008) Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients. Parasitol Res 107:1–7Google Scholar
  12. Lumb R, Erlich J, Davidson GP (1985) Cryptosporidia detection. Med J Aust 142:329–330PubMedGoogle Scholar
  13. McDonald V (1996) Cryptosporidiosis. In: Cox FEG (ed) Welcome trust illustrated history of tropical disease. The Welcome Trust, London, pp 257–2565Google Scholar
  14. Morgan UM, Constantine CC, Forbes DA, Thompson RCA (1997) Differentiation between human and animal isolates of Cryptosporidium parvum using rDNA sequencing and direct PCR analysis. J Parasitol 83:825–830CrossRefPubMedGoogle Scholar
  15. Morgan UM, Pallant L, Dwyer BW, Forbes DA, Rich G, Thompson RCA (1998) Comparision of PCR and microscopy for detection of Cryptosporidium parvum in human fecal Specimens. J Clin Microbiol 36:995–998PubMedPubMedCentralGoogle Scholar
  16. Paul S, Chandra D, Tewari AK, Banerjee PS, Ray DD, Boral R (2009) Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines. Vet Parasitol 164:291–295CrossRefPubMedGoogle Scholar
  17. Scheffler EH, VanEtta LL (1994) Evaluation of rapid commercial enzyme immunoassay for detection of Giardia lamblia in formalin-preserved stool specimens. J Clin Microbiol 32:1807–1808PubMedPubMedCentralGoogle Scholar
  18. Sterling CR, Arrowood MJ (1986) Detection of Cryptosporidium spp. infection using a direct immunofluorescent assay. Ped infect dis 5:139–142CrossRefGoogle Scholar
  19. Vohra P, Singla P, Sharma M, Yadav A, Chaudhary U (2012) Comparison of direct Immunofluorescence, iodine-saline wet mount and modified acid fast staining methods for detection of Cryptosporidium and Giardia spp. in human fecal specimens. JEMDS 1:285–289CrossRefGoogle Scholar
  20. Xiao L, Bern C, Limor J, Sulaiman I, Roberts J, Checkley W et al (2001) Identification five types of Cryptosporidium parasites in children in Lima, Peru. J Infect Dis 183:492–497CrossRefPubMedGoogle Scholar
  21. Zaidah AR, Chan YY, SitiAsma H, Shukri A, Nurhaslindawati A, Salleh M, Zeehaida M, Lalitha P, Mustafa M, Ravichandran M (2008) Detection of Cryptosporidium parvum in HIV-infected patients in Malaysia using a molecular approach. South Asian J Trop Med Pub Health 39:140–151Google Scholar
  22. Ziegler PE, Santucci F, Lindergard G, Nydam DV, Wade SE, Schaaf SL, Chang YF, Mohammed HO (2007) Evaluation of polymerase chain reaction diagnosis of Cryptosporidium spp. in dairy cattle and wildlife. Vet Ther 8:148–159PubMedGoogle Scholar
  23. Zimmerman SK, Needham CA (1995) Comparison of conventional stool concentration and preserved-smear methods with Merifluor Cryptosporidium/Giardia direct immunofluorescence assay and ProSpecT Giardia EZ microplate assay for detection of Giardia lamblia. J Clin Microbiol 33:1942–1943PubMedPubMedCentralGoogle Scholar

Copyright information

© Indian Society for Parasitology 2014

Authors and Affiliations

  • S. Aghamolaie
    • 1
  • A. Rostami
    • 1
  • Sh. Fallahi
    • 2
  • F. Tahvildar Biderouni
    • 1
  • A. Haghighi
    • 1
  • N. Salehi
    • 3
    Email author
  1. 1.Department of Parasitology and MycologyShahid Beheshti University of Medical SciencesTehranIran
  2. 2.Department of Parasitology and MycologyLorestan University of Medical SciencesKhorramabadIran
  3. 3.Department of Medical MicrobiologyObihiro Medical UniversityHokkaidoJapan

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