Open Collections

UBC Faculty Research and Publications

Development and validation of a real-time PCR assay for the detection of clinical acanthamoebae Karsenti, Nessika; Lau, Rachel; Purssell, Andrew; Chong-Kit, Ann; Cunanan, Marlou; Gasgas, Jason; Tian, Jinfang; Wang, Amanda; Ralevski, Filip; Boggild, Andrea K

Abstract

Background: 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 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. Methods: Biobanked 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. Results: Among 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. Conclusions: We 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.

Item Metadata

Title
Development and validation of a real-time PCR assay for the detection of clinical acanthamoebae
Creator
Karsenti, Nessika; Lau, Rachel; Purssell, Andrew; Chong-Kit, Ann; Cunanan, Marlou; Gasgas, Jason; Tian, Jinfang; Wang, Amanda; Ralevski, Filip; Boggild, Andrea K
Publisher
BioMed Central
Date Issued
2017-07-28
Description
Background: 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 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. Methods: Biobanked 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. Results: Among 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. Conclusions: We 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.
Subject
Acanthamoebae; Amoebic keratitis; Corneal ulcer; Molecular diagnosis; Ophthalmology
Genre
Article
Type
Text
Language
eng
Date Available
2018-05-16
Provider
Vancouver : University of British Columbia Library
Rights
Attribution 4.0 International (CC BY 4.0)
DOI
10.14288/1.0366893
URI
http://hdl.handle.net/2429/65949
Affiliation
Medicine, Faculty of; Non UBC
Citation
BMC Research Notes. 2017 Jul 28;10(1):355
Publisher DOI
10.1186/s13104-017-2666-x
Peer Review Status
Reviewed
Scholarly Level
Faculty
Copyright Holder
The Author(s)
Rights URI
http://creativecommons.org/licenses/by/4.0/
Aggregated Source Repository
DSpace

Item Media

Item Citations and Data

Rights

Attribution 4.0 International (CC BY 4.0)