Detection and quantification of Cryptosporidium in HCT-8 cells and human fecal specimens using real-time polymerase chain reaction.
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.
Parr
Jonathan B
JB
University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Sevilleja
Jesus Emmanuel
JE
Samie
Amidou
A
Amidou
Samie
S
Alcantara
Cirle
C
Stroup
Suzanne E
SE
Kohli
Anita
A
Fayer
Ron
R
Lima
Aldo A M
AA
Houpt
Eric R
ER
Guerrant
Richard L
RL
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
D017931:DNA Primers; D016054:DNA, Protozoan; D012965:Sodium Chloride