Abstract
A variable clinical picture of cyclosporiasis including gastrointestinal tract (GIT) symptomatic or asymptomatic beside extraintestinal consequences suggests a possibility of heterogenicity of Cyclospora cayetanensis. The present work aimed to explore the possibility of genetic variation of C. cayetanensis using high-resolution melting (HRM) curve of polymerase chain reaction (PCR) amplified 18S rRNA genes. DNAs extracted from the stool samples of 70 cyclosporiasis patients were amplified and scanned by PCR/HRM curve. The results showed that there are four different genotypic profiles of C. cayetanensis with presence of mixed ones. Although Tm of all profiles was within the same range, they were discerned by plotting of the temperature-shifted florescence difference between normalized melting curves (dF/dT). Genotypic profile I was found alone in 40 % of patients and mixed with genotypic profile II and/or III in 25.7 % of patients, followed by genotypic profile II in 14.3 % then genotypic profile III and IV (10 % each). A significant relation was found between genotypic profiles and GIT symptomatic status as profile I and profile II were mostly detected in patients with acute GIT symptoms without or with chronic illness, respectively, while profile IV cases only were GIT asymptomatic. Statistical significance relations between genotypic profiles and age, gender, residence and oocyst shape index were determined. In conclusion, PCR/HRM proved a wide variation on C. cayetanensis genes that could be reflected on its pathogenic effects and explaining the variability of the clinical manifestations presented by cyclosporiasis patients.
Similar content being viewed by others
References
Abdel-Wahab AM, El-Sharkawy SG, Rayan H, Hussein EM (2008) Detection of Cyclospora cayetanensis infections among diarrheal children attending Suez Canal University Hospital. PUJ 1(1):37–46
Adam RD, Ortega YR, Gilman RH, Sterling CR (2000) Intervening transcribed spacer region 1 variability in Cyclospora cayetanensis. J Clin Microbiol 38:2339–2343
Akosy U, Marangi M, Papini R, Ozkoc S, Bayram Delibas S, Giangaspero A (2014) Detection of Toxoplasma gondii and Cyclospora cayetanensis in Mytilus galloprovincialis from Izmir Province coast (Turkey) by real time PCR/high resolution melting analysis (HRM). Food Microbiol 44:128–135
Alakpa GE, Fagbenro-Beyioku AF (2002) Cyclospora cayetanensis and intestinal parasitic profile in stool samples in Lagos. Nigeria Acta Protozoologica 41(3):221–227
Berlin GW, Peter JB, Gagne C, Conteas CN, Ash LR (1998) Autofluorescence and the detection of Cyclospora oocysts. Emerg Infect Dis 4:127–128
Bern C, Hernandez B, Lopez MB, Arrowood MJ, Alvarez M, Merida AM, Hightower AW, Venczel L, Herwaldt BL, Klein RE (1996) Epidemiologic studies of Cyclospora cayetanensis in Guatemala. Emerg Infect Dis 5:766–74
Bienz M, Siles-Lucas M, Muller N (2001) Use of novel DNA melting profile assay for the identification of PCR-amplified genomic sequences encoding for variant-specific surface proteins from the cloned GS/M-83-H7 line of Giardia lamblia. Parasitol Res 87:1011–1015
Chacin-Bonilla L (2010) Epidemiology of Cyclospora cayetanensis: a review focusing in endemic areas. Acta Trop 115:181–93
Chacin-Bonilla L, Barrios F, Sanchez Y (2007) Epidemiology of Cyclospora cayetanensis infection in San Carlos Island, Venezuela: strong association between socio-economic status and infection. Trans R Soc Trop Med Hyg 101:1018–24
Crist A, Morningstar C, Chambers R, Fitzgerald T, Stoops D, Deffley M (2004) Outbreak of cyclosporiasis associated with snow peas. Morb Mortal Wkly Rep 53(37):876–8
Eberhard ML, Ortega YR, Hanes DE, Nace EK, Do RQ, Robl MG, Won KY, Gavidia C, Sass NL, Mansfield K, Gozalo A, Griffiths J, Gilman R, Sterling CR, Arrowood MJ (2000) Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals. J Parasitol 86:577–582
Garcia LS (2007) Diagnostic Medical Parasitology, 5th ed. ASM Press, Washington DC, p 60-105
Garcia-Lopez HL, Rodreguiz-Tovar LE, Medina-De la Garza CE (1996) Identification of Cyclospora in poultry. Emeg Infect Dis 2:356–357
Giangaspero A, Marangi M, Arace E (2015) Cyclospora cayetanensis travels in tap water on Italian trains. J Water Health 3(1):210–216
Herrmann MG, Durtschi JD, Wittwer CT, Voelkerding KV (2007) Expanded instrument comparison of amplicon DNA melting analysis for mutation scanning and genotyping. Clin Chem 8:1544–1548
Herwaldt BL (2000) Cyclospora cayetanensis: a review focusing on the outbreaks of cyclosporiasis in the 1990s. Clin Infect Dis 31:1040–57
Hussein E, El-Moamly A, Dawoud H, Fahmy H, El-Shal H, Sabek N (2007) Real-time PCR and flow cytometry in detection of Cyclospora oocysts in fecal samples of gastrointestinal symptomatic and asymptomatic pediatrics patients. J Egypt Soc Parasitol 37(1):151–170
Hussein EM, Al-Mohammed HI, Hussein AM (2009) Genetic diversity of Dientamoeba fragilis isolates of irritable bowel syndrome patients by high-resolution melting-curve (HRM) analysis. Parasitol Res 105(4):1053–60
Jeffery N, Gasser R, Steer P, Noor-Mohammadi A (2007) Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting curve analysis of the v1hA gene single-copy region. Microbiol 153:2679–2688
Lainson R (2005) The genus Cyclospora: (Apicomplexa), with a description of Cyclospora schneideri n.sp. in the snake from Amazonian Brazil. Mem Inst Oswaldo Cruz 100:103–15
Lalonde LF, Gajadhar AA (2011) Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis. J Parasitol 97(4):725–735
Lalonde LF, Reyes J, Gajadhar AA (2013) Application of qPCR assay with melting curve analysis for detection and differentiation of protozoan oocysts in human fecal samples from Dominican Republic. Am J Trop Med Hyg 89(5):892–898
Legua P, Seas C (2013) Cystoisospora and Cyclospora. Curr Opin Infect Dis 26:479–483
Mangold K, Manson R, Koay E, Stephens K, Regner M, Thomson R, Peterson L, Kaul K (2005) Real-time PCR for detection and identification of Plasmodium spp. J Clin Microbiol 43(5):2435–2440
Mansfield LS, Gajadhar AA (2004) Cyclospora cayetanensis to food and waterborne coccidian parasite. Vet Parasitol 126:73–90
Massoud NM, Said DE, El-Salamouny AR (2012) Prevalence of Cyclospora cayetanensis among symptomatic and asymptomatic immune-competent children less than five years of age in Alexandria, Egypt. Alexandria Journal of Medicine 48:251–259
Murray P, Baron E, Jorgensen H, Landry M, Pfaller M (2007) Manual of clinical microbiology, 9th ed. ASM Press, Washington DC
Nicolas L, Milon G, Prina E (2002) Rapid differentiation of old world Leishmania species by LightCycler polymerase chain reaction and melting curve analysis. J Microbiol Methods 51:295–299
Olivier C, Van de Pas SP, Lepp W, Yoder K, Relman DA (2001) Sequence variability in the first internal transcribed spacer region within and among Cyclospora species is consistent with polyparasitism. Int J Parasitol 31:14751487
Ortega YR, Sanchez R (2010) Update on Cyclospora cayetanensis, a food-borne and waterborne parasite. Clin Microbiol Rev 23:218–234
Ortega YR, Sterling CR, Gilman RH, Cama VA, Diaz F (1993) Cyclospora species—a new protozoan pathogen of humans. N Engl J Med 328:1308–1312
Pangasa A, Jex AR, Campbell BE, Bott NJ, Whipp M, Hogg G, Stevens MA, Gasser RB (2009) High resolution melting curve (HRM) analysis for the diagnosis of cryptosporoidiosis in humans. Mollecular and Cellular Probes 23:10–15
Reed G, Kent J, Wittwer C (2007) High-resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics 8:597–608
Relman DA, Schmidt TM, Gajadhar A, Sogin M, Cross J, Yoder K, Sethabutr O, Echeverria P (1996) Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species. J Infect Dis 173:440–445
Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245(2):154–160
Robinson B, Saint C, Monis P (2006) Rapid, sensitive and discriminating identification of Naegleria spp. by real-time PCR and melting-curve analysis. App Environ Microbiol 72:5857–5863
Sheilds JM, Olson BH (2003) PCR-restriction fragment length polymorphism method for detection of Cyclospora cayetanensis in environmental waters without microscopic confirmation. Appl Environ Microbiol 69(8):4662–4669
Sherchand JB, Cross JH, Jimba M, Sherchand S, Shrestha MP (1999) Study of Cyclospora cayetanensis in health care facilities, sewage water and green leafy vegetables in Nepal. Southeast Asian J Trop Med Public Health 30:58–63
Sulaiman IM, Ortega Y, Simpson S, Kerdahi K (2014) Genetic characterization of human-pathogenic Cyclospora cayetanensis parasites from three endemic regions at the 18S ribosomal RNA locus. Infect Genet Evol 22:229–234
Tan TC, Suresh KG, Smith HV (2008) Phenotypic and genotypic characterization of Blastocystis hominis isolates implicates subtype 3 as a subtype with pathogenic potential. Parasitol Res 104(1):85–93
Varma M, Hester JD, Schaefer FW III, Ware MW, Lindquist HD (2003) Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay. J Microbiol Methods 53:27–36
Wang KX, Li CP, Wang J, Tian Y (2002) Cyclospora cayetanensis in Anhui, China. World J Gastroenterol 8:1144–1148
Windsor J, Macfarlane L, Hughes-Thapa G, Jones S, Whiteside T (2003) Detection of Dientamoeba fragilis by culture. Br J Biomed Sci 60:79–83
Wittwer C, Reed G, Gundry C, Vandersteen J, Pryor J (2003) High resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49:853–860
Zepra R, Uchima N, Huicho L (1995) Cyclospora cayetanensis associated with watery diarrhoea in Peruvian patients. Am J Trop Med Hyg 98:325–329
Zhang P, Liu Y, Alsarakibi M, Li Y, Li G (2012) Application of HRM assays with Eva Green dye for genotyping Giardia duodenalis zoonotic assemblages. Parasitol Res 111:2157–2163
Zhou Y, Biao LV, Wang Q, Jian F, Zhang L, Ning C, Fu K, Wang Y, Qi M, Yao H, Zhao J, Zhang X, Sun Y, Shi K, Arrowood MJ, Xiao L (2011) Prevalence and molecular characterization of Cyclospora cayetanensis, Henen, China. Emerg Infect Dis 17:1887–1890
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Hussein, E.M., El-Moamly, A.A., Mahmoud, M.A. et al. Wide genetic variations at 18S ribosomal RNA locus of Cyclospora cayetanensis isolated from Egyptian patients using high resolution melting curve. Parasitol Res 115, 2797–2806 (2016). https://doi.org/10.1007/s00436-016-5029-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-016-5029-y