Evaluation of modified Ziehl–Neelsen, direct fluorescent-antibody and PCR assay for detection of Cryptosporidium spp. in children faecal specimens
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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.
KeywordsCryptosporidium 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.
- 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
- Cole DJ (1997) Detection of Cryptosporidium parvum using the Kinyoun acid-fast stain. Proc Annu Conv AAEP 43:409–410Google Scholar
- Fayer R, Xiao L (2008) Cryptosporidium and cryptosporidiosis, 2nd edn. CRC, Boca RatonGoogle Scholar
- 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
- McDonald V (1996) Cryptosporidiosis. In: Cox FEG (ed) Welcome trust illustrated history of tropical disease. The Welcome Trust, London, pp 257–2565Google Scholar
- 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
- 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