A new real-time PCR assay for improved detection of the parasite Babesia microti

Abstract

Babesiosis is an emerging zoonosis with important public health implications, as the incidence of the disease has risen dramatically over the past decade. Because the current gold standard for detection of Babesia is microscopic examination of blood smears, accurate identification requires trained personnel. Species in the genus cannot be distinguished microscopically, and Babesia can also be confused with the early trophozoite stage (ring forms) of Plasmodium parasites. To allow more accurate diagnosis in a format that is accessible to a wider variety of laboratories, we developed a real-time PCR assay targeting the 18S rRNA gene of Babesia microti, the dominant babesiosis pathogen in the United States. The real-time PCR is performed on DNA extracted from whole-blood specimens and detects Babesia microti with a limit of detection of ∼100 gene copies in 5 μl of blood. The real-time PCR assay was shown to be 100% specific when tested against a panel of 24 organisms consisting of Babesia microti, other Babesia species, Plasmodium species, tick-borne and other pathogenic bacteria, and other blood-borne parasites. The results using clinical specimens show that the assay can detect infections of lower parasitemia than can be detected by microscopic examination. This method is therefore a rapid, sensitive, and accurate method for detection of Babesia microti in patient specimens.

Fig 1

Patient specimens identified as positive for Babesia sp. in New York State from 1999 to 2010.

Fig 2

Diagnostic region targeted by this assay in B. microti and other members of the genus. Bases which differ from the sequence of B. microti (boxed) are highlighted. GenBank accession numbers, in order: AY027815, AY048113, GU647159, U16370, M93660, X59604, Z15104, AF175300, U16369, GQ411405, AJ871610, Z15105, DQ200887, AB049999, AF244912, AY260176, DQ312442, AY726009, DQ111765, AY533146, L19079, DQ111764, AY596279, EU376017, EF551335, AY260180, HQ150006, AF419313.

Fig 3

C_T values of patient specimens tested from 2009 to 2011. Samples lying to the left of the dotted line were reported as positive. Those to the right were retested. In each case, one replicate gave a high positive C_T result, whereas the duplicate reaction was negative. Upon retesting both replicates were negative.