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Journal of Clinical Microbiology, July 2003, p. 3175-3180, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3175-3180.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Environmental Isolation of Balamuthia mandrillaris Associated with a Case of Amebic Encephalitis

Frederick L. Schuster,1* Thelma H. Dunnebacke,1 Gregory C. Booton,2 Shigeo Yagi,1 Candice K. Kohlmeier,1 Carol Glaser,1 Duc Vugia,3 Anna Bakardjiev,4 Parvin Azimi,4 Mary Maddux-Gonzalez,5 A. Julio Martinez,6,{dagger} and Govinda S. Visvesvara7

California Department of Health Services, Viral and Rickettsial Disease Laboratory, Richmond,1 California Department of Health Services, Disease Investigations and Surveillance Branch, Berkeley,3 Children's Hospital of Oakland, Oakland,4 Sonoma County Department of Health Services, Santa Rosa, California,5 Department of Molecular Genetics, Ohio State University, Columbus, Ohio,2 University of Pittsburgh School of Medicine, Presbyterian University Hospital, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania,6 Centers for Disease Control and Prevention, Atlanta, Georgia7

Received 25 October 2002/ Returned for modification 3 January 2003/ Accepted 6 April 2003

This report describes the first isolation of the ameba Balamuthia mandrillaris from an environmental soil sample associated with a fatal case of amebic encephalitis in a northern California child. Isolation of the ameba into culture from autopsied brain tissue confirmed the presence of Balamuthia. In trying to locate a possible source of infection, soil and water samples from the child's home and play areas were examined for the presence of Balamuthia. The environmental samples (plated onto nonnutrient agar with Escherichia coli as a food source) contained, in addition to the ameba, a variety of soil organisms, including other amebas, ciliates, fungi, and nematodes, as contaminants. Presumptive Balamuthia amebas were recognized only after cultures had been kept for several weeks, after they had burrowed into the agar. These were transferred through a succession of nonnutrient agar plates to eliminate fungal and other contaminants. In subsequent transfers, axenic Naegleria amebas and, later, tissue cultures (monkey kidney cells) served as the food source. Finally, the amebas were transferred to cell-free axenic medium. In vitro, the Balamuthia isolate is a slow-growing organism with a generation time of ~30 h and produces populations of ~2 x 105 amebas per ml. It was confirmed as Balamuthia by indirect immunofluorescence staining with rabbit anti-Balamuthia serum and human anti-Balamuthia antibody-containing serum from the amebic encephalitis patient. The environmental isolate is similar in its antimicrobial sensitivities and identical in its 16S ribosomal DNA sequences to the Balamuthia isolate from the deceased patient.

* Corresponding author. Mailing address: Viral and Rickettsial Disease Laboratory, California Department of Health Services, 850 Marina Bay Parkway, Richmond, CA 94804. Phone: (510) 307-8651. Fax: (510) 307-8599. E-mail: fschuste{at}dhs.ca.gov.

{dagger} Deceased.

Journal of Clinical Microbiology, July 2003, p. 3175-3180, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3175-3180.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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