Hostname: page-component-78c5997874-g7gxr Total loading time: 0 Render date: 2024-11-15T13:20:59.919Z Has data issue: false hasContentIssue false
  • English
  • Français

Molecular detection of Cyclospora cayetanensis in human stool specimens using UNEX-based DNA extraction and real-time PCR

Published online by Cambridge University Press:  08 November 2017

Yvonne Qvarnstrom Open the ORCID record for Yvonne Qvarnstrom [Opens in a new window] ,
Theresa Benedict ,
Paula L. Marcet ,
Ryan E. Wiegand ,
Barbara L. Herwaldt  and
Alexandre J. da Silva
Yvonne Qvarnstrom*
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Theresa Benedict
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Paula L. Marcet
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Ryan E. Wiegand
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Barbara L. Herwaldt
Affiliation:
Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA 30329, USA
Alexandre J. da Silva
Affiliation:
Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, 8301 Muirkirk Rd, Laurel, MD 20708, USA
*
Author for correspondence: Yvonne Qvarnstrom, E-mail: bvp2@cdc.gov
Get access Rights & Permissions [Opens in a new window]

Abstract

Cyclospora cayetanensis is a coccidian parasite associated with diarrheal illness. In the USA, foodborne outbreaks of cyclosporiasis have been documented almost every year since the mid-1990s. The typical approach used to identify this parasite in human stools is an examination of acid-fast-stained smears under bright-field microscopy. UV fluorescence microscopy of wet mounts is more sensitive and specific than acid-fast staining but requires a fluorescence microscope with a special filter not commonly available in diagnostic laboratories. In this study, we evaluated a new DNA extraction method based on the Universal Nucleic Acid Extraction (UNEX) buffer and compared the performances of four published real-time polymerase chain reaction (PCR) assays for the specific detection of C. cayetanensis in stool. The UNEX-based method had an improved capability to recover DNA from oocysts compared with the FastDNA stool extraction method. The best-performing real-time PCR assay was a C. cayetanensis-specific TaqMan PCR that targets the 18S ribosomal RNA gene. This new testing algorithm should be useful for detection of C. cayetanensis in human stool samples.

Keywords

coccidiaCyclospora cayetanensismolecular diagnosticsstool DNA extraction
Type
Research Article
Information
Parasitology , Volume 145 , Issue 7 , June 2018 , pp. 865 - 870
Check for updates
Copyright
Copyright © Cambridge University Press 2017 

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Berlin, OG, Peter, JB, Gagne, C, Conteas, CN and Ash, LR (1998) Autofluorescence and the detection of Cyclospora oocysts. Emerging Infectious Diseases 4, 127–128.CrossRefGoogle ScholarPubMed
Centers for Disease Control and Prevention (2004) Outbreak of cyclosporiasis associated with snow peas–Pennsylvania, 2004. MMWR Morbidity and Mortality Weekly Report 53, 876–878.Google Scholar
Centers for Disease Control and Prevention (2013) Outbreaks of cyclosporiasis–United States, June–August 2013. MMWR Morbidity and Mortality Weekly Report 62, 862.Google Scholar
Clopper, CJ and Pearson, ES (1934) The use of confidence or fiducial limits illustrated in the case of the binomial. Biometrika 26, 404–413.CrossRefGoogle Scholar
da Silva, AJ, Bornay-Llinares, FJ, Moura, IN, Slemenda, SB, Tuttle, JL and Pieniazek, NJ (1999) Fast and reliable extraction of protozoan parasite DNA from fecal specimens. Molecular Diagnosis 4, 57–64.Google Scholar
Duffy, T, Cura, CI, Ramirez, JC, Abate, T, Cayo, NM, Parrado, R, Bello, ZD, Velazquez, E, Munoz-Calderon, A, Juiz, NA, Basile, J, Garcia, L, Riarte, A, Nasser, JR, Ocampo, SB, Yadon, ZE, Torrico, F, de Noya, BA, Ribeiro, I and Schijman, AG (2013) Analytical performance of a multiplex real-time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples. PLOS Neglected Tropical Diseases 7, e2000. doi:10.1371/journal.pntd.0002000.Google Scholar
Eberhard, ML, da Silva, AJ, Lilley, BG and Pieniazek, NJ (1999) Morphologic and molecular characterization of new Cyclospora species from Ethiopian monkeys: C. cercopitheci sp.n., C. colobi sp.n., and C. papionis sp.n. Emerging Infectious Diseases 5, 651–658.Google Scholar
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 and Arrowood, MJ (2000) Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals. Journal of Parasitology 86, 577–582.Google Scholar
Eberhard, ML, Njenga, MN, DaSilva, AJ, Owino, D, Nace, EK, Won, KY and Mwenda, JM (2001) A survey for Cyclospora spp. in Kenyan primates, with some notes on its biology. Journal of Parasitology 87, 1394–1397. doi:10.1645/0022-3395(2001)087[1394:ASFCSI]2.0.CO;2.Google Scholar
Erickson, MC and Ortega, YR (2006) Inactivation of protozoan parasites in food, water, and environmental systems. Journal of Food Protection 69, 2786–2808.CrossRefGoogle ScholarPubMed
Griner, PF, Mayewski, RJ, Mushlin, AI and Greenland, P (1981) Principles of test selection and use. Annals of Internal Medicine 94, 559–563.Google Scholar
Hall, RL, Jones, JL and Herwaldt, BL (2011) Surveillance for laboratory-confirmed sporadic cases of cyclosporiasis–United States, 1997-2008. MMWR Surveillance Summary 60, 1–11.Google Scholar
Hall, RL, Jones, JL, Hurd, S, Smith, G, Mahon, BE and Herwaldt, BL (2012) Population-based active surveillance for Cyclospora infection–United States, Foodborne Diseases Active Surveillance Network (FoodNet), 1997-2009. Clinical Infectious Diseases 54(suppl. 5), S411–417. doi: 10.1093/cid/cis049.CrossRefGoogle ScholarPubMed
Herwaldt, BL (2000) Cyclospora cayetanensis: a review, focusing on the outbreaks of cyclosporiasis in the 1990s. Clinical Infectious Diseases 31, 1040–1057.Google Scholar
Ho, AY, Lopez, AS, Eberhart, MG, Levenson, R, Finkel, BS, da Silva, AJ, Roberts, JM, Orlandi, PA, Johnson, CC and Herwaldt, BL (2002) Outbreak of cyclosporiasis associated with imported raspberries, Philadelphia, Pennsylvania, 2000. Emerging Infectious Diseases 8, 783–788.Google Scholar
Jin, Y, Hein, MJ, Deddens, JA and Hines, CJ (2011) Analysis of lognormally distributed exposure data with repeated measures and values below the limit of detection using SAS. Annals of Occupational Hygiene 55, 97–112. doi: 10.1093/annhyg/meq061.Google Scholar
Lalonde, LF and Gajadhar, AA (2011) Detection and differentiation of coccidian oocysts by real-time PCR and melting curve analysis. Journal of Parasitology 97, 725–730. doi: 10.1645/GE-2706.1.Google Scholar
Li, N, Ye, J, Arrowood, MJ, Ma, J, Wang, L, Xu, H, Feng, Y and Xiao, L (2015) Identification and morphologic and molecular characterization of Cyclospora macacae n. sp. from rhesus monkeys in China. Parasitology Research 114, 1811–1816. doi: 10.1007/s00436-015-4367-5.Google Scholar
Marangi, M, Koehler, AV, Zanzani, SA, Manfredi, MT, Brianti, E, Giangaspero, A and Gasser, RB (2015) Detection of Cyclospora in captive chimpanzees and macaques by a quantitative PCR-based mutation scanning approach. Parasites and Vectors 8, 274. doi: 10.1186/s13071-015-0872-8.Google Scholar
Monteiro, L, Bonnemaison, D, Vekris, A, Petry, KG, Bonnet, J, Vidal, R, Cabrita, J and Megraud, F (1997) Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. Journal of Clinical Microbiology 35, 995–998.Google Scholar
Pieniazek, NJ, Slemenda, SB, da Silva, AJ, Alfano, EM and Arrowood, MJ (1996) PCR confirmation of infection with Cyclospora cayetanensis. Emerging Infectious Diseases 2, 357–359.Google Scholar
Relman, DA, Schmidt, TM, Gajadhar, A, Sogin, M, Cross, J, Yoder, K, Sethabutr, O and Echeverria, P (1996) Molecular phylogenetic analysis of Cyclospora, the human intestinal pathogen, suggests that it is closely related to Eimeria species. Journal of Infectious Diseases 173, 440–445.Google Scholar
Shields, JM, Joo, J, Kim, R and Murphy, HR (2013) Assessment of three commercial DNA extraction kits and a laboratory-developed method for detecting Cryptosporidium and Cyclospora in raspberry wash, basil wash and pesto. Journal of Microbiological Methods 92, 51–58. doi: 10.1016/j.mimet.2012.11.001.Google Scholar
Varma, M, Hester, JD, Schaefer, FW, Ware, MW 3rd and Lindquist, HD (2003) Detection of Cyclospora cayetanensis using a quantitative real-time PCR assay. Journal of Microbiological Methods 53, 27–36.CrossRefGoogle ScholarPubMed
Verweij, JJ, Laeijendecker, D, Brienen, EA, van Lieshout, L and Polderman, AM (2003) Detection of Cyclospora cayetanensis in travellers returning from the tropics and subtropics using microscopy and real-time PCR. International Journal of Medical Microbiology 293, 199–202.Google Scholar