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Am. J. Trop. Med. Hyg., 76(5), 2007, pp. 938-942
Copyright © 2007 by The American Society of Tropical Medicine and Hygiene

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DETECTION AND QUANTIFICATION OF CRYPTOSPORIDIUM IN HCT-8 CELLS AND HUMAN FECAL SPECIMENS USING REAL-TIME POLYMERASE CHAIN REACTION

JONATHAN B. PARR, JESUS EMMANUEL SEVILLEJA, SAMIE AMIDOU, CIRLE ALCANTARA, SUZANNE E. STROUP, ANITA KOHLI, RON FAYER, ALDO A. M. LIMA, ERIC R. HOUPT, AND RICHARD L. GUERRANT*
University of Virginia School of Medicine, Charlottesville, Virginia; National Institute of Health-University of the Philippines, Manila, Philippines; Center for Global Health, Division of Infectious Disease and International Health, University of Virginia, Charlottesville, Virginia; University of Venda for Science and Technology, Department of Microbiology, Thohoyandou, South Africa; Division of Infectious Disease and International Health, University of Virginia, Charlottesville, Virginia; United States Department of Agriculture, Beltsville, Maryland; Clinical Research Unit & Institute of Biomedicine, Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil

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 ≥103 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.

Received August 28, 2006. Accepted for publication February 13, 2007.

Acknowledgments: The authors thank Relana Fitzgerald for help with statistical calculations, Leah Barrett for helpful advice, and Mary Ann Winecoff for editing assistance.

Financial support: This research was supported by National Institutes of Health Mid-Atlantic Regional Center of Excellence (MARCE) for Biodefense and Emerging Infectious Diseases Research Grant U54 AI57168.

* Address correspondence to Richard L. Guerrant, Center for Global Health, Division of Infectious Diseases and International Health, University of Virginia, MR4, Lane Road, Room 3148, Charlottesville, VA 22908. E-mail: guerrant{at}virginia.edu; rlg9a{at}virginia.edu

Authors’ addresses: Jonathan Parr, Jesus Emmanuel Sevilleja, Samie Amidou, Cirle Alcantara, Suzanne E. Stroup, Anita Kohli, Eric R. Houpt, and Richard L. Guerrant, Center for Global Health, Division of Infectious Diseases and International Health, University of Virginia, MR4, Lane Road, Room 3148, Charlottesville, VA 22908, Telephone: 434-924-5242, Fax: 434-977-5323. Ron Fayer, U.S. Department of Agriculture, Beltsville, MD 20705. Aldo A. M. Lima, Clinical Research Unit and Institute of Biomedicine, Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Fortaleza, CE, Brazil.






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