title: Detection and quantification of Cryptosporidium in HCT-8 cells and human fecal specimens using real-time polymerase chain reaction.
abstract: Cryptosporidium is a significant cause of diarrheal illness worldwide, especially among children and immunocompromised patients. Currently used diagnostic techniques are time-consuming, require skilled technicians, and are not useful for quantification of oocysts in fecal and environmental samples. In this study, we examined the use of a real-time polymerase chain reaction (PCR) assay for detecting and quantifying Cryptosporidium parvum in three distinct and progressively more complex matrices: phosphate-buffered saline (PBS), HCT-8 cells (human ileocecal carcinoma), and human fecal specimens. A reliable standard curve was generated using the PBS samples spiked with pure oocysts, and oocyst starting quantities were calculated for the infected HCT-8 cell and spiked fecal samples. The assay detected Cryptosporidium in samples infected/spiked with > or =10(3) oocysts/sample and detected both C. hominis and C. parvum in clinical specimens. This assay is useful in a variety of samples in the research laboratory and will likely prove to be a useful tool in the clinical laboratory.
authors.affiliation: University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Parr
authors.forename: Jesus Emmanuel
authors.initials: JE
authors.lastname: Sevilleja
authors.forename: Amidou
authors.initials: A
authors.lastname: Samie
authors.forename: Samie
authors.initials: S
authors.lastname: Amidou
authors.forename: Cirle
authors.initials: C
authors.lastname: Alcantara
authors.forename: Suzanne E
authors.initials: SE
authors.lastname: Stroup
authors.forename: Anita
authors.initials: A
authors.lastname: Kohli
authors.forename: Ron
authors.initials: R
authors.lastname: Fayer
authors.forename: Aldo A M
authors.initials: AA
authors.lastname: Lima
authors.forename: Eric R
authors.initials: ER
authors.lastname: Houpt
authors.forename: Richard L
authors.initials: RL
authors.lastname: Guerrant
mesh_terms: D000818:Animals; D002417:Cattle; D045744:Cell Line, Tumor; D003457:Cryptosporidiosis / Q000175:diagnosis* / Q000469:parasitology; D003458:Cryptosporidium / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000302:isolation & purification; D005243:Feces / Q000469:parasitology*; D005260:Female; D020459:Genes, rRNA / Q000235:genetics; D006801:Humans; D007223:Infant; D033661:Oocysts; D016133:Polymerase Chain Reaction / Q000379:methods*; D012965:Sodium Chloride
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D012965:Sodium Chloride