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Parasitology

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

Yvonne Qvarnstrom, Govinda S. Visvesvara, Rama Sriram, Alexandre J. da Silva
Yvonne Qvarnstrom
1Division 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
2Atlanta Research and Education Foundation in conjunction with the Atlanta VA Medical Center, Decatur, Georgia
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Govinda S. Visvesvara
1Division 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
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Rama Sriram
1Division 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
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Alexandre J. da Silva
1Division 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
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  • For correspondence: abs8@cdc.gov
DOI: 10.1128/JCM.00875-06
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    FIG. 1.

    Amplification curves from cultured trophozoites, serially diluted in parasite-free CSF. Negative samples were unspiked CSF. Each sample was amplified in parallel in the single-target assay with just one set of primers and probe (dashed lines) and in the triplex assay containing oligonucleotides for detection of all three variants of amebas (solid lines). Only the fluorescence signal from the relevant probe in the triplex reactions is displayed in each figure (the signals from the other two probes remained below threshold for all samples throughout all runs). All experiments were repeated three times; the average results are displayed. dRn, baseline subtracted fluorescence reading normalized to a reference dye.

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    FIG. 2.

    Amplification curves from triplex real-time PCR on brain tissue samples from patient 4 (closed symbols) and 5 (open symbols). Amplification signals from unprocessed tissue are displayed as boxes, those from proteinase-treated tissue as circles, those from DNA extracted from the tissue samples as triangles, those from DNA extracted from cultured N. fowleri (positive control) as multiplication symbols, and those from water (negative control) as a dashed line. Only the fluorescence signal from the N. fowleri-specific probe is displayed (the signals from the other two probes remained below threshold for all samples throughout the run). dRn, baseline subtracted fluorescence reading normalized to a reference dye.

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  • TABLE 1.

    Cultured amebas included in the evaluation

    SampleGenus and/or speciesSource(s) (source/gender/state/country)aGenotypeResult of real-time PCR
    Triplex assay from this studyAssay by Riviere et al. (30)
    V006 Acanthamoeba Brain tissue/F/GA/USAT1 Acanthamoeba Acanthamoeba
    V062 Acanthamoeba Corneal scraping/M/MA/USAT4 Acanthamoeba Acanthamoeba
    V369 Acanthamoeba Brain tissue/M/NE/USAT10 Acanthamoeba Negative
    V409 Acanthamoeba Horse brain/CA/USAT10 Acanthamoeba Negative
    V499 Acanthamoeba Water/NY/USAT7 Acanthamoeba Negative
    V550 Acanthamoeba Skin/CA/USAT4 Acanthamoeba Acanthamoeba
    V532 Acanthamoeba Water/NM/USAT4 Acanthamoeba Negative
    V039 B. mandrillaris Mandrill brain/CA/USA B. mandrillaris Negative
    V451 B. mandrillaris Brain tissue/F/NY/USA B. mandrillaris Negative
    V416 B. mandrillaris Brain tissue/F/Australia B. mandrillaris Negative
    V020 N. fowleri CSF/M/TX/USAI N. fowleri Negative
    V212 N. fowleri CSF/M/MexicoI N. fowleri Negative
    V511 N. fowleri CSF/M/GA/USAI N. fowleri Negative
    V515 N. fowleri CSF/M/AZ/USAIII N. fowleri Negative
    V551 N. fowleri CSF/M/Colombia N. fowleri Negative
    CAMP N. fowleri CSF/F/CA/USAII N. fowleri Negative
    7615250 N. lovaniensis Thermal water/BelgiumNegativeNegative
    V419 N. dunnebackei Water/CA/USANegativeNegative
    EG N. gruberi Soil/CA/USANegativeNegative
    400 N. jadini Swimming pool/BelgiumNegativeNegative
    PP 397 N. italica Water, mud/ItalyNegativeNegative
    AB-T-F3 N. australiensis Drainage water/AustraliaNegativeNegative
    • ↵ a Some source parameters are omitted as necessary. F, female; M, male; USA, United States of America.

  • TABLE 2.

    Results from clinical samples

    Sample no.Source (gender/animal, state/country)aResultb
    Triplex real-time PCRIFACulture
    1CSF (M, AZ)NegativeNegativeNegative
    2CSF (horse, CA)NegativeNegativeNegative
    3CSF (F, CO)NegativeNegativeNegative
    4Brain tissue (M, OK) N. fowleri Positive N. fowleri
    5Brain tissue (M, OK) N. fowleri Positive N. fowleri
    6CSF (M, Japan)NegativeNegativeNegative
    7CSF (M, ND)NegativeNegativeNA
    8CSF (M, CO)NegativeNegativeNegative
    9CSF (M, TX) N. fowleri Positive N. fowleri
    • ↵ a F, female; M, male.

    • ↵ b NA, not available.

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Multiplex Real-Time PCR Assay for Simultaneous Detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri
Yvonne Qvarnstrom, Govinda S. Visvesvara, Rama Sriram, Alexandre J. da Silva
Journal of Clinical Microbiology Oct 2006, 44 (10) 3589-3595; DOI: 10.1128/JCM.00875-06

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Multiplex Real-Time PCR Assay for Simultaneous Detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri
Yvonne Qvarnstrom, Govinda S. Visvesvara, Rama Sriram, Alexandre J. da Silva
Journal of Clinical Microbiology Oct 2006, 44 (10) 3589-3595; DOI: 10.1128/JCM.00875-06
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KEYWORDS

Acanthamoeba
Naegleria fowleri
Reverse Transcriptase Polymerase Chain Reaction

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