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


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


CONCLUSIONS
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.
Blood Systems Research Institute, San Francisco, California; Yale School of Public Health and Yale School of Medicine, New Haven, Connecticut; Tufts University Cummings School of Veterinary Medicine, North Grafton, Massachusetts; Nantucket Cottage Hospital, Nantucket, Massachusetts.
Evan M
EM
Bloch
Tzong-Hae
TH
Lee
Peter J
PJ
Krause
Sam R
SR
Telford
Lani
L
Montalvo
Daniel
D
Chafets
Sahar
S
Usmani-Brown
Timothy J
TJ
Lepore
Michael P
MP
Busch
D000818:Animals; D041001:Babesia microti / Q000235:genetics / Q000302:isolation & purification*; D001404:Babesiosis / Q000175:diagnosis*; D006801:Humans; D051379:Mice; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity