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Multiplex Real-Time PCR Assay for Simultaneous Detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri

Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia,¹ Atlanta Research and Education Foundation in conjunction with the Atlanta VA Medical Center, Decatur, Georgia²

Received 26 April 2006/ Returned for modification 7 July 2006/ Accepted 25 July 2006

Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris occur throughout the world and pose many diagnostic challenges. To date, at least 440 cases of severe central nervous system infections caused by these amebas have been documented worldwide. Rapid and specific identification of these free-living amebas in clinical samples is of crucial importance for efficient case management. We have developed a triplex real-time TaqMan PCR assay that can simultaneously identify Acanthamoeba spp., B. mandrillaris, and N. fowleri in the same PCR vessel. The assay was validated with 22 well-characterized amebic strains harvested from cultures and nine clinical specimens that were previously characterized by in vitro culture and/or immunofluorescence assay. The triplex assay demonstrated high specificity and a rapid test completion time of less than 5 h from the reception of the specimen in the laboratory. This assay was able to detect one single ameba per sample analyzed, as determined with cerebrospinal fluid spiked with diluted cultured amebas. This assay could become useful for fast laboratory diagnostic assessment of amebic infections (caused by free-living amebas) in laboratories with adequate infrastructure to perform real-time PCR testing.

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