Reevaluation of an Acanthamoeba Molecular Diagnostic Algorithm following an Atypical Case of Amoebic Keratitis

Abstract

dAmoebic keratitis (AK) is a potentially blinding infection, the prompt diagnosis of which is essential for limiting ocular morbidity. We undertook a quality improvement initiative with respect to the molecular detection of acanthamoebae in our laboratory because of an unusual case of discordance. Nine ATCC strains of Acanthamoeba and 40 delinked, biobanked, surplus corneal scraping specimens were analyzed for the presence of acanthamoebae with four 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 the performance characteristics of each individual assay and pairs of assays were calculated. Outcome measures were sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Of 49 included specimens, 14 (28.6%) were positive by the gold standard assay, and 35 (71.4%) were negative. The sensitivities of the individual assays ranged from 64.3% to 92.9%, compared to the gold standard, while the specificities ranged from 88.6% to 91.4%. The PPVs and NPVs ranged from 69.2% to 78.6% and from 86.1% to 96.9%, respectively. Combinations of assay pairs led to improved performance, with sensitivities ranging from 92.9% to 100% and specificities ranging from 97.1% to 100%. ATCC and clinical strains of Acanthamoeba that failed to be detected by certain individual assays included Acanthamoeba castellanii, Acanthamoeba culbertsoni, and Acanthamoeba lenticulata. For three clinical specimens, false negativity of the gold standard assay could not be excluded. Molecular diagnostic approaches, especially combinations of highly sensitive and specific assays, offer a reasonably performing, operator-independent, rapid strategy for the detection of acanthamoebae in clinical specimens and are likely to be more practical than either culture or direct microscopic detection.A moebic keratitis (AK) is a potentially blinding eye infection caused by the parasite Acanthamoeba, which is a ubiquitous, free-living organism found in soil and other environmental sources (1). This infection usually occurs in the context of contact lens use, and outbreaks of AK have been linked to contact lens solutions that are inefficient in killing acanthamoebae adhering to the contact lenses during washing with amebacontaminated water, including the most recent outbreak in the United States, which affected 138 people (2) and led to the recall of several contact lens solutions and products by both the FDA and Health Canada (3, 4). Delays in diagnosis have been associated frequently with poor visual outcomes and more severe clinical progression in AK (5, 6). Traditional diagnostic procedures include direct microscopic examination of corneal scrapings or contact lens fluids stained with Giemsa stain, periodic acid-Schiff stain, hematoxylin and eosin, or acridine orange and culture of specimens on nonnutrient agar overlaid with Escherichia coli or Klebsiella pneumoniae; both methods are limited by poor sensitivity, operator dependence,...

Development and validation of a real-time PCR assay for the detection of clinical acanthamoebae

“…AK is a potentially blinding ocular infection caused by environmentally ubiquitous acanthamoebae, whose varied adaptations to specific niches and opportunistic proclivities, as well as genetic heterogeneity challenge the development of both highly sensitive and specific molecular assays for their clinical detection. We have previously demonstrated that the Riviere assay, which has an excellent LOD, failed to detect specific clinically relevant strains of Acanthamoeba [ 5 ]. Due to the typically minuscule volume of specimen attainable from the cornea of patients with AK, any molecular assay implemented clinically for acanthamoebae detection must demonstrate, in addition to broad species sensitivity, a very low LOD.…”

“…Thus, neither assay could stand alone for the diagnosis of AK in a clinical laboratory. The Riviere primer set [ 6 ] was designed by aligning sequences of 6 different 18S rRNA genomes, thus, while sensitive from a LOD perspective, the assay appears to be unable to detect the broad range of Acanthamoeba species and genotypes known to cause AK, as previously demonstrated [ 5 , 10 , 13 ].…”

“…In addition 10 ATCC strains of Acanthamoeba , as listed in Table 1 , were also used for the validation. ATCC stains were cultured according to the recommended specifications, in peptone-yeast-glucose (PYG) medium [ 5 ].…”

“…However, no single molecular assay has proven sufficiently sensitive for the range of known clinical and environmental acanthamoebae that can contaminate fresh water and contact lenses [ 4 – 7 ]. For clinical specimens, including corneal scrapings, contact lenses, and contact lens casings, individual PCR-based detection assays have reported sensitivities ranging from 64.3 to 93% compared to reference-level or composite diagnostics [ 4 , 5 ], and specificities of 88.6–100% [ 4 , 5 ]. Additionally, efficient clinical composite use of several assays in order to mitigate the risk of false negativity and positivity has been hindered by the validation of such assays on different diagnostic platforms: while some assays employ real time PCR (qPCR) [ 6 , 7 ], others are used on an end-point platform [ 8 , 9 ].…”

“…In our clinical reference laboratory, we routinely employ two molecular assays (the “Qvarnstrom” and “Riviere” assays; 6,7] as well as culture for the diagnosis of AK, however, this approach is time consuming, particularly when we need to arbitrate discordance with use of a third end-point PCR-based assay [ 5 , 8 ]. The Riviere assay [ 6 ] was designed as a molecular diagnostic test specific to both trophozoites and cysts of Acanthamoeba spp.…”