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Detection and Differentiation of Babesia microti and Other Pathogenic Babesia Species by a Single-Amplicon, Dual-Probe Real-Time PCR Assay

Session: Poster Abstract Session: Diagnostic Microbiology: Viruses/Fungal/AFB/Parasitic
Saturday, October 11, 2014
Room: The Pennsylvania Convention Center: IDExpo Hall BC
Background: Babesiosis is an emerging zoonotic disease caused by infections with the apicomplexa, Babesia. Babesiosis is most frequently acquired through bites with infected ticks but may also be transmitted through blood transfusion or vertical infection. In the U.S., most human infections are caused by Babesia microti, but infections with Babesia duncani (previously WA-1) and the unnamed taxon MO-1 are increasingly recognized. Microscopic examination of blood films, the reference method for diagnosis of babesiosis, is labor intense, only enables genus-level identification, and may pose difficulties distinguishing Babesia spp. from Plasmodium falciparum. Most available real-time PCRs only detect Babesia microti. Thus, we developed a multiplex, real-time PCR for detection and differentiation of Babesia microti (channel 1) and other human pathogenic Babesia spp. (channel 2).

Methods: A 190bp segment of the 18S rRNA gene (18S) was amplified with a common set of PCR primers and detected by two different probes: (1) Babesia microti, (2) other Babesia spp. of human relevance.  Test performance was established with 39 whole blood specimens from patients with known B. microti infections diagnosed by microscopy (n=8) and PCR (n=31). Whole blood was also spiked with B. duncani organism (ATCC PRA-302) and plasmids covering the 18S target of MO-1, B. divergens, and EU-1.

Results: Analytical sensitivity for both channels was between 285-435 copies/mL (8-13 copies/reaction).  All positive whole blood samples produced the expected results. Positive patient specimens had a mean of 2.6E+08 copies/mL (range 2.5E+03 to 1.2E+10). No cross-reactivity was observed with 56 other human pathogens, including the 5 relevant Plasmodium spp., Trypanosoma cruzi, and Leishmania infantum

Conclusion: The incidence of babesiosis has increased in recent years and previously unrecognized species have been shown to cause human infections. We describe the first multiplex, real-time PCR assay for sensitive detection of all Babesia species of known human relevance using an innovative single-amplicon, dual-probe design. Given the highly conserved 18S target, this assay will enable us to identify and monitor circulation of unusual Babesia species by subsequent 18S sequencing.

Brianne Couturier, Ph.D.1, Kimberly Kalp1, Christine Ginocchio, PhD2 and Robert Schlaberg, MD, MPH3, (1)Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT, (2)North Shore Health System Core Laboratory, Lake Success, NY, (3)Pathology, University of Utah, Salt Lake City, UT


B. Couturier, Gen-Probe: Collaborator, Kits supplied for study

K. Kalp, None

C. Ginocchio, None

R. Schlaberg, Epoch Biosciences: Collaborator, Research reagents

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