The Impact of Different Copro-preservation Conditions on Molecular Detection of Cryptosporidium Species
AbstractBackground: We aimed to evaluate different copro-preservation conditions along the duration of one month for a better outcome of molecular diagnosis of Cryptosporidium species.Methods: Ten samples out of 380 fresh stool samples collected from patients with diarrhea proved positive after direct examination, concentration, staining and confirmed by immunochromatographic test. The study was conducted at the Diagnostic and Research Unit of Parasitic diseases, Faculty of Medicine, Ain Shams University at the time interval from July 2014 to December 2015. Each stool sample was preserved in five different conditions; freezing at -20 ºC, 70% ethyl alcohol, 10% formalin, 2.5% potassium dichromate (K dichromate) at 4 ºC and 2.5% K dichromate at room temperature (RT). Then DNA extraction and nested PCR, with Cryptosporidium oocyst wall protein (COWP) gene were done from each sample at zero time (fresh specimens) as a standard for comparison with the preservation conditions at 10, 20 and 30 d.Results: Sensitivity of studied preservative conditions along the whole study duration showed best outcome from freezing at -20 °C (80%) then K dichromate (4 °C) (73.3%) followed by K dichromate (RT) (66.7%), then alcohol (33.3%), while formalin was the worst (0%) with a highly significant comparative outcome between the different conditions. Along the three extraction intervals, K dichromate (RT), unlike all the rest of conditions lacks the consistent preservative action.Conclusion: Our study highlights freezing at -20 ºC to be the most suitable condition for preservation followed by K dichromate at 4 °C, K dichromate at RT, then 70% ethyl alcohol. Formalin (10%) is better to be avoided.
Checkley W, White AC Jr, Jaganath D et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium. Lancet Infect Dis. 2015; 15(1): 85–94.
Fayer R, Morgan U, Upton SJ. Epidemiology of Cryptosporidium: transmission, detection and identification. Int J Parasitol. 2000; 30 (12-13): 1305-22.
Chen XM, Keithly JS, Paya CV et al. Cryptosporidiosis. N Engl J Med. 2002; 346(22): 1723–31.
Kaushik K, Khurana S, Wanchub A et al. Evaluation of staining techniques, antigen detection and nested PCR for the diagnosis of cryptosporidiosis in HIV seropositive and seronegative patients. Acta Trop. 2008; 107(1):1–7.
Uppal B, Singh O, Chadha S et al. A comparison of Nested PCR assay with conventional techniques for diagnosis of intestinal cryptosporidiosis in AIDS cases from Northern India. J Parasitol Res. 2014; 2014:706105.
Savioli L, Smith H, Thompson A. Giardia and Cryptosporidium join the 'Neglected Diseases Initiative'. Trends Parasitol. 2006; 22(5): 203-8.
Chalmers RM, Davies AP. Minireview: Clinical cryptosporidiosis. Exp Parasitol. 2010; 124(1): 138-146.
Weber R, Bryan RT, Bishop HS et al. Threshold of detection of Cryptosporidium oocysts in human stool specimens: evidence for low sensitivity of current diagnostic methods. J Clin Microbiol. 1991; 29(7): 1323-7.
Mansfield LS, Gajadhar AA. Cyclospora cayetanensis, a food-and waterborne coccidian parasite. Vet Parasitol. 2004; 126 (1-2): 73-90.
Chacon-Cruz E. Intestinal protozoal disease. E- Medicine Pediatrics 2014; http://emedicine.medscape.com/ article/999282-clinical.
Kurniawan A, Dwintasari SW, Soetomenggolo HA, Wanandi SI. Detection of Cryptosporidium sp infection by PCR and modified acid fast staining from potassium dichromate preserved stool. Med J Indones. 2009; 18 (3): 147- 151.
AL-Braiken FA, Amin A, Beeching NJ et al. Detection of Cryptosporidium among diarrhoeic and assymptomatic chidren in Jeddah, Saudi Arabia. Ann Tropic Med Parasitol. 2003; 97(5):505-10.
Dillingham RA, Lima AA, Guerrant RL. Cryptosporidiosis: epidemiology and impact. Microbes Infect. 2002; 4(10): 1059-66.
Caccio SM, Thompson RC, McLauchlin J et al. Unravelling Cryptosporidium and Giardia epidemiology. Trends Parasitol. 2005; 21(9):430-7.
El-Badry AA, AL-Ali KH, Mahrous AS. Molecular identification and prevelance of Giardia lamblia and Cryptosporidium in duodenal aspirate in Al-Madinah. J Med Biomedical Sci. 2010;1 (2): 47-52.
Surl CG, Jung BD, Park BK, Kim HC. Resistance of Cryptosporidi¬um parvum oocysts following commercial bleach treatment. Ko¬rean J Vet Res. 2011;51: 101-105.
Oikarinen S, Tauriainen S, Viskari H et al. PCR in¬hibition in stool samples in relation to age of infants. J Clin Vi¬rol. 2009; 44(3): 211-4.
Hawash Y. DNA Extraction from Protozoan Oocysts/Cysts in Feces for Diagnostic PCR. Korean J Parasitol. 2014;52(3): 263-71.
Schrader C, Schielke A, Ellerbroek L et al. PCR inhibitors- occurrence, properties and removal. J Appl Microbiol. 2012; 113(5): 1014-26.
Holland JL, Louie L, Simor AE et al. PCR detection of Escherichia coli O157:H7 directly from stools: evaluation of commercial extraction methods for purifying fecal DNA. J Clin Microbiol. 2000; 38(11): 4108-13.
Kuk S, Yazar S, Cetinkaya U. Stool sample storage conditions for the preservation of Giardia intestinalis DNA. Mem Inst Oswaldo Cruz 2012; 107(8): 965-8.
Wilke H, Robertson LJ. Preservation of Giardia cysts in stool samples for subsequent PCR analysis. J Microbiol Methods 2009;78(3): 292-6.
Dirim Erdoğan D, Dağci H, Turgay N et al. The molecular diagnosis of cryptosporidiosis in fresh and formalin preserved fecal samples. Turkiye Parazitol Derg. 2009; 33(2):120-4.
Pietrzak-Johnston SM, Bishop H, Wahlquist S et al. Evaluation of commercially available preservatives for laboratory detection of helminths and protozoa in human fecal specimens. J Clin Microbiol. 2000; 38(5): 1959-64.
CDC “Centers for Disease Control and Prevention” 2013: Diagnostic procedures/stool specimens/ molecular diagnosis. http://www.cdc.gov/dpdx/ diagnostic Proce-dures/stool/moleculardx.html.
U. M. Morgan, L. Pallant, B. W. Dwyer et al. Comparison of PCR and microscopy for detection of Cryptosporidium parvum in human fecal specimens: clinical trial. J Clin Microbiol. 1998; 36(4): 995–998.
Zhao GH, Ren WX, Gao M et al. Genotyping Cryptosporidium andersoni in Cattle in Shaanxi Province, Northwestern China. PloS One. 2013; 8(4), e60112.
Rahman MT, Uddin MS, Sultana R, et al.Polymerase Chain Reaction (PCR): A Short Review. AKMMC J. 2013; 4(1): 30-36.
Helmy YA, Krücken J, Nöckler K et al. Molecular epidemiology of Cryptosporidium in livestock animals and humans in the Ismailia province of Egypt. Vet Parasitol. 2013; 193(1-3): 15-24.
Kato S, Lindergard G, Mohammed HO. Utility of the Cryptosporidium oocyst wall protein (COWP) gene in a nested PCR approach for detection infection in cattle. Vet Parasitol. 2003; 111 (2-3):153-9.
Gobet P, Toze S. Sensitive genotyping of Cryptosporidium parvum by PCR-RFLP analysis of the 70-kilodalton heat shock protein (HSP70) gene. FEMS Microbiol Lett. 2001;200(1): 37- 41.
Feng Y, Dearen T, Cama V et al. 90-Kilodalton heat shock protein, HSP 90 as a target for genotyping Cryptosporidium spp. known to infect humans. Eukaryot cell. 2009; 8(4):478-82.
Sulaiman IM, Lal AA, Xiao L. Molecular phylogeny and evolutionary relationships of Cryptosporidium parasites at the actin locus. J Parasitol. 2002; 88(2): 388-94.
Spano F, Putignani L, McLauchlin J et al. PCR-RFLP analysis of the Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairi and C. parvum, and between C. parvum isolates of human and animal origin. FEMS Microbiology Lett. 1997;150(2): 209-217.
S. Pedraza-Díaz, C. Amar, G. L. Nichols, J. McLauchlin. Nested Polymerase Chain Reaction for Amplification of the Cryptosporidium Oocyst Wall Protein Gene. Emerg Infect Diseases. 2001; 7(1): 49-56.
Xiao L, Ryan UM. Molecular epidemiology. In: Cryptosporidium and Cryptosporidiosis, Fayer, R.; Xiao, L. (eds.), 2nd ed. 2008; CRC Press and IWA Publishing, Boca Raton, Florida, P.:119-151.
WHO. Techniques of collection, preparation, and examination of samples, fecal specimens. In (Basic laboratory methods in medical parasitology, Parasitology -laboratory manuals). WHO 1991 Geneva, Typest in India, Printed in England. P:10-23.
Cheesbrough MDirect examination of faeces and concentration techniques. In: District Laboratory Practice in Tropical Countries. Part 1, Cheesbrough, M (ed.), 2nd ed. 2009; Cambridge University Press, New York, P. 196-198.
Henriksen SA, Pohlenz JF. Staining of Cryptosporidia by a modiﬁed Ziehl- Neelsen technique. Acta Vet Scand. 1981; 22(3-4): 594-6.
QIAamp® DNA Stool Handbook: For DNA purification from stool samples, 2nd ed. 2010.
Chevallet M, Luche S, Rabilloud T. Silver staining of proteins on polyacrylamide gels. Nat Protocol. 2006; 1(4): 1852-8.
Johnson DW, Pieniazek NJ, Griffin DW et al. Development of a PCR protocol for sensitive detection of Cryptosporidium in water samples. Appl Environ Microbiol. 1995; 61(11): 3849-55.
Xiao L, Morgan UM, Fayer R et al. Cryptosporidium systematics and implications for public health. Parasitol Today. 2000; 16(7): 287-92.
Jongwutiwes S, Tiangtip R, Yentakarm S et al. Simple method for long-term copro-preservation of Cryptosporidium oocysts for morphometric and molecular analysis. Trop Med Int Health. 2002;7(3): 257–64.
Ramos F, Zurabian R, Morán P et al. The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica. Trans R Soc Trop Med Hyg. 1999;93(3):335-6.
Gonçalves EM, Araújo RS, Orban M et al. Protocol for DNA extraction of Cryptosporidium spp. oocysts in fecal samples. Rev Inst Med Trop Sao Paulo. 2008;50(3):165-7.
Piggott MP. Effect of sample age and season of collection on the reliability of microsatellite genotyping of faecal DNA. Wildl Res. 2004; 31: 485-493.
Molina N, Polverino D, Minvielle M et al. PCR amplifi¬cation of triosephosphate isomerase gene of Giardia lamblia in formalin-fixed feces. Rev Latinoam Microbiol. 2007;(1-2):6-11.
Minvielle MC, Molina NB, Polverino D et al. First genotyping of Giardia lamblia from human and animal feces in Argentina, South America. Mem Inst Oswaldo Cruz. 2008; 103(1):98-103.