title: Performance characteristics of polymerase chain reaction and histological methods for the detection of Haplosporidium nelsoni in the eastern oyster (Crassostrea virginica).
abstract: Fitness for purpose and validation are increasingly becoming a benchmark in the development of test methods for the diagnosis of infectious diseases in aquatic animals. The design of the evaluation and the analysis of data are critical to demonstrate test method performance characteristics and fitness for purpose, as stated in the World Organization for Animal Health pathway for test validation. Three test methods for the detection of the oyster parasite Haplosporidium nelsoni were selected for the validation study described herein: histology, end-point polymerase chain reaction (PCR), and real-time PCR (qPCR). Preliminary work evaluated the analytical sensitivity and specificity of the PCR and qPCR assay in development. The following stage used test results on 100 oysters in 3 different laboratories to assess diagnostic sensitivity (DSe), diagnostic specificity (DSp), repeatability, and reproducibility. Repeatability and reproducibility were within 68-95%. The final part of the project evaluated DSe and DSp using test results on 400 oysters and results from the first 100 oysters tested. In the absence of a 100% gold standard test, latent class modeling methods were explored to characterize the tests (i.e., Bayesian analyses). For both PCR methods, DSe was >90%, and in the 60% range for histology, whereas DSp was >90% for all methods. Based on the results of this validation, a threshold cycle value of 30 for qPCR corresponds to the limit of sensitivity for histology where unreliable detection becomes more frequent, thus providing a threshold helpful in diagnostic settings where both histology and qPCR are used.
authors.affiliation: Fisheries and Oceans Canada, Gulf Fisheries Centre, Moncton, New Brunswick, Canada (Gagné, Veniot, Stephenson)AquaEpi Research, Charlottetown, Prince Edward Island, Canada (McClure) nellie.gagne@dfo-mpo.gc.ca.
authors.forename: Nellie
authors.initials: N
authors.lastname: Gagné
authors.affiliation: Fisheries and Oceans Canada, Gulf Fisheries Centre, Moncton, New Brunswick, Canada (Gagné, Veniot, Stephenson)AquaEpi Research, Charlottetown, Prince Edward Island, Canada (McClure).
authors.forename: Anne
authors.initials: A
authors.lastname: Veniot
authors.affiliation: Fisheries and Oceans Canada, Gulf Fisheries Centre, Moncton, New Brunswick, Canada (Gagné, Veniot, Stephenson)AquaEpi Research, Charlottetown, Prince Edward Island, Canada (McClure).
authors.forename: Mary
authors.initials: M
authors.lastname: Stephenson
authors.affiliation: Fisheries and Oceans Canada, Gulf Fisheries Centre, Moncton, New Brunswick, Canada (Gagné, Veniot, Stephenson)AquaEpi Research, Charlottetown, Prince Edward Island, Canada (McClure).
authors.forename: Carol
authors.initials: C
authors.lastname: McClure
mesh_terms: D000818:Animals; D001499:Bayes Theorem; D002170:Canada; D049896:Crassostrea / Q000469:parasitology*; D041602:Haplosporida / Q000235:genetics / Q000302:isolation & purification*; D011528:Protozoan Infections / Q000469:parasitology*; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
keywords: Haplosporidium nelsoni; histology; multinucleated sphere unknown disease; oysters; polymerase chain reaction; real-time polymerase chain reaction; validation