Re-evaluation of an Acanthamoeba Molecular Diagnostic Algorithm following an Atypical Case of Amoebic Keratitis.

Lau R1, Cunanan M1, Jackson J2, Ali IK2, Chong-Kit A1, Gasgas J1, Tian J1, Ralevski F1, Boggild AK3.

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Abstract

BACKGROUND:

Amoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential to limiting ocular morbidity. We undertook a quality improvement initiative around the molecular detection of Acanthamoebae in our laboratory due to an unusual case of discordance.

METHODS:

Nine ATCC strains of Acanthamoeba and 40 delinked, biobanked surplus corneal scrapings were retrieved and analyzed for the presence of Acanthamoebae by 4 separate real time PCR assays. The assay used by the Free Living and Intestinal Amebas laboratory of the CDC was considered the reference standard, and performance characteristics of each individual assay, and pairs of assays were calculated. Outcome measures were sensitivity, specificity, and positive predictive value (PPV) and negative (NPV) predictive value.

RESULTS:

Of 49 included specimens, 14 (28.6%) were positive by the gold standard assay, and 35 (71.4%) were negative. Sensitivity of the individual test assays ranged from 64.3% to 92.9% compared to the gold standard, while specificity ranged from 88.6% to 91.4%. PPV and NPV ranged from 69.2% to 78.6%, and 86.1% to 96.9%, respectively. Combinations of assay pairs led to improved performance with sensitivities ranging from 92.9% to 100%, and specificity from 97.1% to 100%. ATCC and clinical strains of Acanthamoeba that failed detection by certain individual assays included A. castellani, A. culbertsoni, and A. lenticulata. In 3 clinical specimens, false negativity of the gold standard assay could not be excluded.

CONCLUSIONS:

Molecular diagnostic approaches, especially combinations of both highly sensitive and specific assays, offer a reasonably performing, operator independent, and rapid strategy for the detection of Acanthamoebae from clinical specimens, and are likely more practical than either culture or direct microscopic detection.