Abstract
BackgroundSuboptimal agreement between molecular assays for the detection of Acanthamoeba spp. in clinical specimens has been demonstrated, and poor assay sensitivity directly imperils the vision of those affected by amoebic keratitis (AK) through delayed diagnosis. We sought to develop and validate a single Taqman real time PCR assay targeting the Acanthamoeba 18S rRNA gene that could be used to enhance sensitivity and specificity when paired with reference assays.MethodsBiobanked DNA from surplus delinked AK clinical specimens and 10 ATCC strains of Acanthamoeba was extracted. Sequence alignment of 66 18S rRNA regions from 12 species of Acanthamoeba known to cause keratitis informed design of a new TaqMan primer set. Performance of the new assay was compared to the 2 assays used currently in our laboratory.ResultsAmong 24 Acanthamoeba-positive and 83 negative specimens by the CDC reference standard, performance characteristics of the newly designed primer set were as follows: sensitivity 100%, specificity 94%, PPV 82.8%, and NPV 100%. Compared to culture, sensitivity of the new primer set was 100%, and specificity 96%. No cross-reactivity of the primer set to non-acanthamoebae, including Balamuthia and Naegleria, was found.ConclusionsWe have validated a real time PCR assay for the diagnosis of AK, and in doing so, have overcome important barriers to rapid and sensitive detection of acanthamoebae, including limited sensitivity and specificity of commonly used assays.
Highlights
Suboptimal agreement between molecular assays for the detection of Acanthamoeba spp. in clinical specimens has been demonstrated, and poor assay sensitivity directly imperils the vision of those affected by amoe‐ bic keratitis (AK) through delayed diagnosis
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 polymerase chain reaction (PCR) [6, 7], others are used on an end-point platform [8, 9]
Between January 2012 and May 2015, 97 surplus specimens submitted for Acanthamoeba diagnostics were biobanked as follows: corneal scrapings (n = 87), contact lenses in saline (n = 4) or contact lens solution (n = 6)
Summary
Suboptimal agreement between molecular assays for the detection of Acanthamoeba spp. in clinical specimens has been demonstrated, and poor assay sensitivity directly imperils the vision of those affected by amoe‐ bic keratitis (AK) through delayed diagnosis. Other more detectable corneal pathogens, such as herpes simplex virus, cause a similar type of keratitis, and empiric treatment for these more common organisms leads to Traditional diagnosis of AK has relied on historic parasitologic techniques such as culture, but while highly specific, this method is insensitive, inefficient, labor intensive, and requires technical expertise for interpretation Molecular diagnostic techniques, such as polymerase chain reaction (PCR) performed directly on clinical specimens such as corneal scrapings and contact lens casings, are increasingly preferred over culture as Karsenti et al BMC Res Notes (2017) 10:355 they provide faster turnaround time (hours vs days) and eliminate the need for skilled microscopists. 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]
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