Supportive Data

The following assay verification data supports the use of this assay for clinical testing.

Accuracy/Diagnostic Sensitivity and Specificity:

Species Inclusivity:

Thirty-seven strains of free-living amoeba were tested. All 20 Acanthamoeba species and all strains of Naegleria fowleri and Balamuthia mandrillaris were detected, while other Naegleria and Balamuthia species were not detected with this assay.

Results from this free-living ameba polymerase chain reaction (PCR) assay were compared to culture results on 6 cerebrospinal fluid (CSF) specimens. All were negative by both culture and PCR (100% concordance).

Twenty-eight formalin-fixed paraffin-embedded (FFPE) tissue blocks (up to 34 years old) were tested with the free-living ameba PCR assay and results were compared to histopathologic diagnosis. Nineteen of the tissues were positive by histopathology diagnosis. Of these 19, 15 were positive by PCR (83% sensitivity). Slides were not available for review of the histopathology-positive, PCR-negative cases, and thus the presence of organisms within the blocks tested by PCR could not be confirmed. Specificity was 100%.

Supplemental Data:

Spiking studies were performed using free-living amoeba negative CSF, fresh tissue, and FFPE tissue spiked with Acanthamoeba, N fowleri, and B mandrillaris genomic DNA. The sensitivity of the PCR assay was 97% to 100% and the specificity with spiked specimens was 100%. The samples were extracted and tested in a blinded fashion.

Analytical Sensitivity/Limit of Detection:

The limit of detection (LOD) established using genomic DNA spiked into CSF is:

A polyphaga ATCC 30173 - 2.5 target copies/mcL

B mandrillaris, ATCC PRA 290 - 3.05 target copies/mcL

N fowleri, ATCC 30896 - 1.16 target copies/mcL

The LOD established using genomic DNA spiked into fresh tissue matrix is:

A polyphaga, ATCC 30173 - 6.5 target copies/mcL

B mandrillaris, ATCC PRA 290 - 1.84 target copies/mcL

N fowleri, ATCC 30896 - 4.01 target copies/mcL

The LOD established using genomic DNA spiked into formalin-fixed paraffin-embedded tissue matrix is:

A polyphaga, ATCC 30173 - 5.7 target copies/mcL

B mandrillaris, ATCC PRA 290 - 21.94 target copies/mcL

N fowleri, ATCC 30896 - 7.82 target copies/mcL

Analytical Specificity:

No PCR signal was obtained from the extracts of 33 bacterial, viral, parasitic, and fungal isolates from similar organisms or from organisms commonly found in the specimens tested.

Precision:

Interassay precision was 100% and intra-assay precision was 100%.

Reference Range:

The reference range is "negative" for this assay. This assay is intended for detection of DNA from Acanthamoeba species, N fowleri, and B mandrillaris in CSF, and fresh and FFPE tissue (skin, lung, and brain) to aid in the diagnosis of primary amebic meningoencephalitis (PAM) and granulomatous amebic encephalitis (GAE) in conjunction with clinical findings.

Reportable Range:

This is a qualitative assay, and the results are reported as negative or positive for targeted free-living amoeba (Acanthamoeba species, N fowleri, and B mandrillaris) detected.