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Parasitology

Detection of Balamuthia Mitochondrial 16S rRNA Gene DNA in Clinical Specimens by PCR

Shigeo Yagi, Gregory C. Booton, Govinda S. Visvesvara, Frederick L. Schuster
Shigeo Yagi
1California Department of Health Services, Viral and Rickettsial Disease Laboratory, 850 Marina Bay Parkway, Richmond, California 94804
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  • For correspondence: syagi@dhs.ca.gov
Gregory C. Booton
2Department of Evolution, Ecology and Organismal Biology, The Ohio State University, Columbus, Ohio 43210
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Govinda S. Visvesvara
3Centers for Disease Control and Prevention, Division of Parasitic Diseases, Atlanta, Georgia 30341
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Frederick L. Schuster
1California Department of Health Services, Viral and Rickettsial Disease Laboratory, 850 Marina Bay Parkway, Richmond, California 94804
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DOI: 10.1128/JCM.43.7.3192-3197.2005
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  • FIG. 1.
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    FIG. 1.

    Indirect immunofluorescence-stained section of necrotic brain tissue from patient 4 (Table 1). The dark circles seen in the section are the lumens of blood vessels; the bright-staining profiles surrounding the vessels and scattered throughout the tissue are Balamuthia amebas. In all the types of amebic encephalitides, the amebas are typically found in large numbers in the perivascular regions of the brain.

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    FIG. 2.

    PCR gel showing bands at ∼1,075 bp for Balamuthia mitochondrial DNA. Lane 1, marker; lane 2, DNA isolated from brain tissue of Balamuthia patient 4; lane 3, negative control (water); lane 4, positive control (using DNA extracted from cultured Balamuthia). The arrow in lane 1 (marker) indicates ∼1,075 bp.

  • FIG. 3.
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    FIG. 3.

    Agarose gel showing banding at ∼1,075 bp for Balamuthia DNA in CSF samples. Lane 1, CSF from patient 2 (Table 1); lane 2, CSF from patient 3; lane 3, positive control, using DNA extracted from cultured Balamuthia; lane 4, same as lane 3 but at 1/10 concentration; lane 5, negative control (water); lane 6, marker. The arrow in lane 6 (marker) indicates ∼1,075 bp.

  • FIG. 4.
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    FIG. 4.

    Agarose gel showing CSF samples spiked with Balamuthia DNA. Lanes 1 and 12, markers; lane 2, CSF with DNA extracted from 100 amebas; lane 3, CSF with DNA from 50 amebas; lane 4, CSF with DNA from 12 amebas; lane 5, CSF with DNA from 3 amebas; lane 6, CSF with DNA from 0.75 ameba; lane 7, CSF with DNA from 0.2 ameba; lane 8, DNA from 0.05 ameba; lane 9, Hanks' basal salt solution; lane 10, negative control (water); lane 11, positive control (DNA from Balamuthia harvested from culture). Arrow in lane 1 (marker) indicates ∼1,075 bp.

Tables

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  • TABLE 1.

    Clinical samples from Balamuthia cases: IIF and culture results and materials for PCR

    Case no.Sex/age (yr)LocationaTiterb for:Availabilityd of:Resultse for:
    BalamuthiaAcanthamoebaUnfixed tissueCSFCulture attemptsH&E slidesSlides for IIFcSpecimens for PCR
    1Male/64San Diego Co., CA12816/32Yes (brain)NABrain, (−)(+)Brain, (+); kidney, (−); lung, (−)Brain, (+); kidney, (−); lung, (−)
    2fMale/2.5Bexar Co., TX25632NAYesCSF, (−)(+)Brain, (+)CSF (+)
    3fMale/7Orange Co., CA2562NAYesCSF, (−)(+)Brain, (+)CSF (+)
    4Male/7Santa Clara Co., CA25632Yes (brain)NABrain, (−)(+)Brain, (+); kidney, (−); lung, (−)Brain, (+); kidney, (−); lung, (+)
    5Female/3Sonoma Co., CA128/25632Yes (brain)YesBrain, (+); CSF, (−)(+)Brain, (+)Brain, (+); CSF, (+)
    • ↵ a County and state from which specimen originated.

    • ↵ b Antibody levels expressed as reciprocal titers. Threshold for positive titers, 1:128.

    • ↵ c Unstained slides with tissue sections as noted.

    • ↵ d NA, not available; yes, available.

    • ↵ e (−), unsuccessful isolation and/or detection of amebas or ameba DNA; (+), successful isolation and/or detection of amebas.

    • ↵ f Not included in the CEP.

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Detection of Balamuthia Mitochondrial 16S rRNA Gene DNA in Clinical Specimens by PCR
Shigeo Yagi, Gregory C. Booton, Govinda S. Visvesvara, Frederick L. Schuster
Journal of Clinical Microbiology Jul 2005, 43 (7) 3192-3197; DOI: 10.1128/JCM.43.7.3192-3197.2005

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Detection of Balamuthia Mitochondrial 16S rRNA Gene DNA in Clinical Specimens by PCR
Shigeo Yagi, Gregory C. Booton, Govinda S. Visvesvara, Frederick L. Schuster
Journal of Clinical Microbiology Jul 2005, 43 (7) 3192-3197; DOI: 10.1128/JCM.43.7.3192-3197.2005
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KEYWORDS

DNA, Mitochondrial
encephalitis
Lobosea
polymerase chain reaction
RNA, Ribosomal, 16S

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