Development of a real‐time polymerase chain reaction assay for sensitive detection and quantitation of Babesia microti infection. Issue 10 (30th January 2013)

• Record Type: Journal Article
• Authors: Bloch, Evan M.; Lee, Tzong‐Hae; Krause, Peter J.; Telford, Sam R.; Montalvo, Lani; Chafets, Daniel; Usmani‐Brown, Sahar; Lepore, Timothy J.; Busch, Michael P.
• Is Part Of: Transfusion. Volume 53:Issue 10(2013)
• Page Start: 2299
• Page End: 2306
• Publication Date: 2013-01-30
• Subjects: Hematology -- Periodicals; Blood -- Transfusion -- Periodicals; Blood Group Antigens -- Periodicals; Blood Preservation -- Periodicals; Blood Transfusion -- Periodicals; 615
• DOI: 10.1111/trf.12098
• Languages: English
• ISSNs: 0041-1132

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="trf12098-sec-0001" sec-type="section"> <title>Background</title> <p> <italic>Babesia microti</italic>, 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.</p> </sec> <sec id="trf12098-sec-0002" sec-type="section"> <title>Study Design and Methods</title> <p>Frozen <italic>Babesia</italic>‐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 <italic>Babesia</italic>‐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.</p> </sec> <sec id="trf12098-sec-0003" sec-type="section"> <title>Results</title> <p>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</p> </sec> <sec id="trf12098-sec-0004" sec-type="section"> <title>Conclusion</title> <p>We have developed a highly sensitive and specific, quantitative real‐time PCR‐based assay for detection of <italic>B. microti</italic> that could have a useful role in blood screening. It can also be employed broadly to understand <italic>Babesia</italic> epidemiology, disease pathogenesis, and host immunology.</p> </sec> </abstract>