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Title: DETECTION AND QUANTIFICATION OF CRYPTOSPORIDIUM IN HCT-8 CELLS AND HUMAN FECAL SPECIMENS USING REAL-TIME PCR

Author
item PARR, JONATHAN - UNIVERSITY OF VIRGINIA
item SEVILLEJA, JESUS EMMANUEL - NIH, MANILA, PHILLIPINES
item AMADOU, SAMIE - U. OF VERDA,SOUTH AFRICA
item ALCANTARA, CIRLE - NIH, MANILA, PHILLIPINES
item STROUP, SUZANNE - UNIVERSITY OF VIRGINIA
item Fayer, Ronald
item HOUPT, ERIC - UNIVERSITY OF VIRGINIA
item GUERRANT, RICHARD - UNIVERSITY OF VIRGINIA

Submitted to: American Journal of Tropical Medicine and Hygiene
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/22/2007
Publication Date: 5/1/2007
Citation: Parr, J.B., Sevilleja, J., Amadou, S., Alcantara, C., Stroup, S.E., Fayer, R., Houpt, E.R., Guerrant, R.L. 2007. Detection and quantification of Cryptosporidium in HCT-8 cells and human fecal specimens using Real-Time PCR. American Journal of Tropical Medicine and Hygiene. 76:938-942.

Interpretive Summary: Cryptosporidium is a significant cause of diarrheal illness worldwide, especially among children and immunocompromised patients. Current diagnostic techniques are time-consuming, require skilled technicians, and are not useful for quantification of oocysts in fecal and environmental samples. This study examined the utility of a real-time polymerase chain reaction assay for detecting and quantifying Cryptosporidium parvum oocysts in three distinct and complex matrices: phosphate buffered saline (PBS), HCT-8 cells, and human fecal specimens. Reliable baseline data were generated using PBS spiked with pure oocysts, and oocyst starting quantities were calculated for the infected HCT-8 cell and spiked fecal samples. The assay detected oocysts in infected HCT-8 cells and feces spiked with 100 or more oocysts, a range well below typical oocyst numbers found in natural infections. Analysis of clinical diarrhea specimens confirmed the usefullness of this assay for detecting Cryptosporidium with a sensitivity of 89% and specificity of 100% versus microscopy. This assay can be useful for detection of oocysts in a variety of matrices in the research laboratory and possibly in the clinical laboratory.

Technical Abstract: Currently used diagnostic techniques for Cryptosporidium are time-consuming, require skilled technicians, and cannot quantitate oocysts in fecal and environmental samples. In this study, we examined the utility of a real-time PCR assay using SYBR Green for detecting and quantifying Cryptosporidium parvum oocysts in three progressively more complex matrices: PBS, HCT-8 cells, and human feces. A reliable standard curve was generated using PBS spiked with oocysts, and oocyst starting quantities were calculated for the infected HCT-8 cells and spiked feces. Average differences between known and calculated starting quantities were 1.4±0.3 logs oocysts/sample for HCT-8 cells (1x105 cells/sample) and 2.2±0.5 logs oocysts/sample for the spiked feces (200mg stool and 200µL oocysts in PBS/sample). Despite losses, the assay detected oocysts in infecetd HCT-8 cells and feces spiked with '102 oocysts/sample, a range well below typical shedding in natural infections. Analysis of clinical diarrhea specimens confirmed the utility of this assay for detecting multiple Cryptosporidium species, including C. hominis and C. parvum, and demonstrated a sensitivity of 89% and specificity of 100% versus fluorescence microscopy. 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.