Nellie Gagné

Selection of a cutoff value for real-time polymerase chain reaction results to fit a diagnostic purpose: analytical and epidemiologic approaches

Diagnostic laboratories frequently select a subjective cutoff value for real-time amplification assays, above which a threshold cycle (Ct) value is deemed false. Commonly, higher Ct values are interpreted as amplification or fluorescence artifacts, or cross contaminations. Although the implementation of Ct cutoff might be reasonable, its justification and selection should be based on evidence. The current article reviewed evidence-based strategies to select Ct cutoffs grouped in analytical and epidemiologic approaches. Analytical strategies use criteria gathered during the assay development and include fluorescence threshold, reaction end-cycle, limit of detection, and artifact investigation. Variability in amplification efficacy across test runs may induce some instability in an intended Ct cutoff and requires some standardization or normalization procedures. Epidemiologic strategies use criteria based on either the probability or the cost of a false test result associated with a specified cutoff. Cutoffs, depending on the intended purpose of the test, can be selected graphically to minimize the probability of either false-positive or false-negative results by using two-graph receiver operating characteristics curves. The assays diagnostic sensitivity and specificity may vary with the tested population, thus, the estimated two-graph receiver operating characteristics curve is population dependent and should be established for the targeted population. Although the selection of a cutoff based on misclassification cost depends on infection prevalence, the selection based on predictive values does not. To optimize the test average diagnostic performance, the Ct cutoff should be selected when diagnostic odds ratio is maximal. Epidemiologic approaches were illustrated by selecting Ct cutoffs for a real-time assay for Infectious salmon anemia virus.

Transcriptional response of Atlantic salmon (Salmo salar) after primary versus secondary exposure to infectious salmon anemia virus (ISAV).

Following an infection with a specific pathogen, the acquired immune system of many teleostean fish, including salmonids, is known to retain a specific memory of the infectious agent, which protects the host against subsequent infections. For example, Atlantic salmon (Salmo salar) that have survived an infection with a low-virulence infectious salmon anemia virus (ISAV) isolate are less susceptible to subsequent ISAV infections. A greater understanding of the mechanisms and immunological components involved in this acquired protection against ISAV is fundamental for the development of efficacious vaccines and treatments against this pathogen. To better understand the immunity components involved in this observed resistance, we have used an Atlantic salmon DNA microarray to study the global gene expression responses of preexposed Atlantic salmon (fish having survived an infection with a low-virulence ISAV isolate) during the course of a secondary infection, 18 months later, with a high-virulence ISAV isolate. We present global gene expression patterns in both preexposed and naïve fish, following exposure by either cohabitation with infected fish or by direct intra-peritoneal injection of a high-virulence ISAV isolate. Our results show a clear reduction of ISAV viral loads in head-kidney of secondary infected fish compared to primary infected fish. Further, we note a lower-expression of many antiviral innate immunity genes in the secondary infected fish, such as the interferon induced GTP-binding protein Mx, CC-chemokine 19 and signal transducer and activator of transcription 1 (STAT 1), as well as MHC class I antigen presentation involved genes. Potential acquired immunity genes such as GILT, leukocyte antigen transcript CD37 and Ig mu chain C region membrane-bound form were observed to be over-expressed in secondary infected fish. The observed differential gene expression profile in secondary and primary infected fish head-kidney provides great insight into immunity components involved during primary and secondary ISAV infection.

Use of a third class in latent class modelling for the diagnostic evaluation of five infectious salmon anaemia virus detection tests

In the absence of a reference standard, a latent class model (LCM) was used in this study to assess diagnostic sensitivity (DSe) and specificity (DSp) of a recently developed reverse-transcription polymerase chain reaction (RT-PCR) for infectious salmon anaemia virus (ISAV). The study included 4 populations of Atlantic salmon, and to ensure the identifiability of the LCM, four additional detection methods were used in parallel including real-time RT-PCR (qRT-PCR), virus isolation (VI), indirect fluorescent antibody test (IFAT), and a lateral flow immunoassay (LFI). While a conventional LCM assumes DSe and DSp to be constant across the populations, Nérette et al. (2008) previously reported concerns about non-constant DSp of RT-PCR, which detects viral RNA from both active and inactive viral particles. It was suspected that some ISAV recovered fish may carry residual RNA and may be more likely to test positive compared to naïve fish. The various mixture distributions of the two sub-classes of non-infected fish would lead to a non-constant combined DSp estimate across populations. Within a Bayesian framework, the conventional two-class LCM was extended to three classes of infection stages (naïve non-infected, recovered non-infected carrying RNA, and infected). The resulting analysis confirmed the existence of three classes of fish with substantially different test performances for ISAV. For infected fish, DSe of RT-PCRs and VI approximated 90%, and antibody based assays were the least sensitive (DSe around 65%). Regardless of the test, the DSp estimates on naïve fish were all above 98% with LFI being in average the most specific. Only RT-PCR and qRT-PCR tested positive with the additional class of recovered fish (DSp around 30%). The true infectious status of this sub-class (i.e. viral RNA carriers) is debatable and requires further knowledge about ISAV infection dynamics at the fish level. Promising applications of multiple class estimates require adjustments of traditional test interpretation and further epidemiological knowledge of the infection dynamics at the population level.

Traditional descriptive analysis and novel visual representation of diagnostic repeatability and reproducibility: application to an infectious salmon anaemia virus RT-PCR assay.

As a component of diagnostic test evaluation, the estimation of repeatability and reproducibility of an assay is necessary to assess the robustness and the transferability of the method among laboratories. Respectively defined as the agreement within and between laboratories, repeatability and reproducibility of a qualitative diagnostic test are traditionally reported using observed proportion of agreement or Kappa values. Applied to a recently designed RT-PCR assay for the detection of infectious salmon anaemia virus, repeatability only within a national reference laboratory and reproducibility with two additional independent regional laboratories were investigated. Homogenization of fish kidney tissue was conducted to potentially provide more uniform submission material, and to assess the effect of homogenization on laboratory comparability. Comparison of agreement between non-homogenized and homogenized tissue samples revealed different patterns of test results and unexpected alterations of agreement due to homogenization. This observation may be explained by cross-contamination of some samples during the homogenization process. One of the laboratories was in clear disagreement with the two others and impacted the overall reproducibility of the assay. Agreement levels were visually described using a novel tree-shape representation inspired from phylogenetic studies. The resulting phylogram illustrated the proximity of test findings between repeated samples within a laboratory and between laboratories, and facilitated the interpretation of the agreement levels.

Complete sequencing of Tunisian redspotted grouper nervous necrosis virus betanodavirus capsid gene and RNA-dependent RNA polymerase gene.

Finfish nodaviruses (betanodaviruses) can cause highly destructive infections in numerous species of farmed marine fish larvae and juveniles worldwide. The betanodavirus genome consists of two single-stranded positive-sense RNA molecules (RNA1 and RNA2). The virus can be classified into four genotypes based on the partial sequences of the coat protein (CP) gene (T2 and T4 regions). Currently, genomic sequence information for RNA1 regions of RNA2 outside of T2 and T4 is less well documented. This study reports on the characterization of the full RNA2 sequence of a Tunisian betanodavirus with a length of 1433 nt, containing a 339 amino acid open-reading frame encoding the CP, and typing to the redspotted grouper nervous necrosis virus Ia genotype following phylogenetic analysis. The homology of the capsid protein to other betanodaviruses or alphanodaviruses was compared. In addition, a full length RNA1 sequence of 3104 nt encoding a 982 amino acid RNA-dependent RNA polymerase was obtained.

Overview of infectious salmon anaemia virus (ISAV) in Atlantic Canada and first report of an ISAV North American-HPR0 subtype

The infectious salmon anaemia virus (ISAV) is an important viral disease of farmed Atlantic salmon that has caused considerable financial losses for salmon farmers around the world, including Atlantic Canada. It is listed as a notifiable disease by the World Organization for Animal Health, and to this day, culling of infected cages or farms remains the current practice in many countries to mitigate the spread of the virus. In Atlantic Canada, ISAV was first detected in 1996 and continues to be detected. While some outbreaks seemed to have arisen from isolated infections of unknown source, others were local clusters resulting from horizontal spread of infection. This study provides a description of the detected ISAV isolates in Atlantic Canada between 2012 and 2016, and explores the phylogenetic relatedness between these ISAV isolates. A key finding is the detection for the first time of a North American-HPR0 ISAV subtype, which was predicted to exist for many years. Through phylogenetic analysis, a scenario emerges with at least three separate incursions of ISAV in Atlantic Canada. An initial ISAV introduction follows a genotypic separation between North America and Europe which resulted in the NA and EU genotypes known today; this separation predates the salmon aquaculture industry. The second incursion of ISAV from Europe to North America led to a sublineage in Atlantic Canada consisting of EU-HPR∆ isolates detected in Nova Scotia and New Brunswick, and the predominant form of ISAV-HPR0 (EU). Finally, we observed what could be the third and most recent incursion of ISAV in Newfoundland, in the form of an isolate highly similar to ISAV EU-HPR0 isolates found in the Faroe Islands and the one isolate from Norway.

Development of environmental DNA (eDNA) methods for detecting high-risk freshwater fishes in live trade in Canada

Preventing the arrival, establishment, and spread of aquatic invasive species is an important step in protecting our aquatic environments. The use of detection tools, like DNA barcoding technologies, high-throughput sequencing and environmental DNA (eDNA) monitoring, is becoming increasingly important in preventing the introduction of potential invasive species. The combination of eDNA with realtime PCR (qPCR) provide the opportunity to have a rapid and specific detection. In this study, we developed a DNA sequence library that has sufficient depth and species coverage such that high-risk species can be confidently discriminated from legitimately imported and native species. A total of 12 species-specific qPCR assays were developed for the detection of 13 potential invasive species (pAIS) in bulk water samples. Detection of these species was also compared with a HTS approach. We have demonstrated the high sensitivity of qPCR assays using eDNA at very low densities, suggesting we could detect a low number of individuals mixed with non-target species in a simulated live shipment. For the detection of a targeted list of species, qPCR is advantageous. The mini-barcodes developed in this project offered a good sensitivity of detection, and HTS is a discovery tool that can be desirable when unlisted or numerous species need to be identified.

Performance characteristics of polymerase chain reaction and histological methods for the detection of Haplosporidium nelsoni in the eastern oyster (Crassostrea virginica).

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.