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Development of a real-time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection

Authors

  • Evan M. Bloch,

    Corresponding author
    1. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    2. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    3. Nantucket Cottage Hospital, Nantucket, Massachusetts
    • Blood Systems Research Institute, San Francisco, California
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  • Tzong-Hae Lee,

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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  • Peter J. Krause,

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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  • Sam R. Telford III,

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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  • Lani Montalvo,

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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  • Daniel Chafets,

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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  • Sahar Usmani-Brown,

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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  • Timothy J. Lepore,

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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  • Michael P. Busch

    1. Blood Systems Research Institute, San Francisco, California
    2. Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut
    3. Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts
    4. Nantucket Cottage Hospital, Nantucket, Massachusetts
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Address reprint requests to: Evan M. Bloch, MD, MS, Blood Systems Research Institute, 270 Masonic Avenue, San Francisco, CA 94118; e-mail: ebloch@bloodsystems.org.

Abstract

Background

Babesia microti, the most frequently implicated pathogen in transfusion-transmitted babesiosis, is widely endemic in the Northeast and upper Midwestern United States. High seroprevalence in endemic areas limits antibody-based donor screening. A high-performance molecular test is needed to identify donors in the preseroconversion window phase as well as to discriminate past serologic exposure with parasite clearance from continued parasitemia.

Study Design and Methods

Frozen Babesia-spiked whole blood was microcentrifuged, and the supernatant transferred and microcentrifuged again to concentrate the parasite. The DNA was extracted and amplified using real-time polymerase chain reaction (PCR) using Babesia-specific primers. The assay was employed in three series of experiments: 1) a validation and optimization spiking experiment, 2) a blinded serial dilution probit analysis to determine the limit of detection, and 3) evaluation of two blinded panels of clinical samples from possible babesiosis cases.

Results

At a decreasing inoculum of 445, 44.5, and 4.45 copies/mL, the assay had positive rates of 100, 97.5, and 81%, respectively. The blinded probit analysis demonstrated a detection rate of 95 and 50% at 12.92 and 1.52 parasites/2 mL of whole blood, respectively. Evaluation of clinical samples showed 13 of 21 samples to be positive, with a range of 85 to 4.8 million parasites/mL. There were no positives detected among 48 healthy donors

Conclusion

We have developed a highly sensitive and specific, quantitative real-time PCR-based assay for detection of B. microti that could have a useful role in blood screening. It can also be employed broadly to understand Babesia epidemiology, disease pathogenesis, and host immunology.

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