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

JONATHAN B. PARR 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

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JESUS EMMANUEL SEVILLEJA 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

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SAMIE AMIDOU 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

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CIRLE ALCANTARA 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

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SUZANNE E. STROUP 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

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ANITA KOHLI 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

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RON FAYER 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

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ALDO A. M. LIMA 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

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ERIC R. HOUPT 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

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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

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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.

Author Notes

  • 1

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    Garcia LS, Shimizu RY, 1997. Evaluation of nine immunoassay kits (enzyme immunoassay and direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvum in human fecal specimens. J Clin Microbiol 35 :1526–1529.

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    Sturbaum GD, Reed C, Hoover PJ, Jost BH, Marshall MM, Sterling CR, 2001. Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts. Appl Environ Microbiol 67 :2665–2668.

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    Fontaine M, Guillot E, 2003. An immunomagnetic separation–real-time PCR method for quantification of Cryptosporidium parvum in water samples. J Microbiol Methods 54 :29–36.

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    Higgins JA, Fayer R, Trout JM, Xiao L, Lal AA, Kerby S, Jenkins MC, 2001. Real time PCR for the detection of Cryptosporidium parvum.J Microbiol Methods 47 :323–337.

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    Cai X, Woods KM, Upton SJ, Zhu G, 2005. Application of quantitative real-time reverse transcription-PCR in assessing drug efficacy against the intracellular pathogen Cryptosporidium parvum in vitro. Antimicrob Agents Chemother 49 :4437–4442.

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    Fayer R, Trout JM, Graczyk TD, Lewis EJ, 2000. Prevalence of Cryptosporidium, Giardia and Eimeria infections in post-weaned and adult cattle on three Maryland farms. Vet Parasitol 93 :103–112.

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    Stroup SE, Roy S, McHele J, Maro V, Ntabaguzi S, Siddique A, Kang G, Guerrant RL, Kirkpatrick BD, Fayer R, Herbein J, Ward H, Haque R, Houpt ER, 2006. Real-time PCR detection and speciation of Cryptosporidium infection using scorpion probes. J Med Microbiol 55 :1217–1222.

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  • 10

    Xiao L, Escalante L, Yang C, Sulaiman I, Escalante AA, Montali RJ, Fayer R, Lal AA, 1999. Phylogenetic analysis of Cryptosporidium parasites based on the small-subunit rRNA gene locus. Appl Environ Microbiol 65 :1578–1583.

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  • 11

    Bushen OY, Kohli A, Pinkerton RC, Dupnik K, Newman RD, Sears CL, Fayer R, Lima AA, Guerrant RL, 2007. Heavy cryptosporidial infections in children in northeast Brazil: comparison of Cryptosporidium hominis and Cryptosporidium parvum.Trans R Soc Trop Med Hyg 101 :378–384.

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