K. Larry Hammell

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.

Heart and skeletal muscle inflammation (HSMI) disease diagnosed on a British Columbia salmon farm through a longitudinal farm study

Heart and skeletal muscle inflammation (HSMI) is an emerging disease of marine-farmed Atlantic Salmon (Salmo salar), first recognized in 1999 in Norway, and later also reported in Scotland and Chile. We undertook a longitudinal study involving health evaluation over an entire marine production cycle on one salmon farm in British Columbia (Canada). In previous production cycles at this farm site and others in the vicinity, cardiac lesions not linked to a specific infectious agent or disease were identified. Histologic assessments of both live and moribund fish samples collected at the farm during the longitudinal study documented at the population level the development, peak, and recovery phases of HSMI. The fish underwent histopathological evaluation of all tissues, Twort’s Gram staining, immunohistochemistry, and molecular quantification in heart tissue of 44 agents known or suspected to cause disease in salmon. Our analysis showed evidence of HSMI histopathological lesions over an 11-month timespan, with the prevalence of lesions peaking at 80–100% in sampled fish, despite mild clinical signs with no associated elevation in mortalities reported at the farm level. Diffuse mononuclear inflammation and myodegeneration, consistent with HSMI, was the predominant histologic observation in affected heart and skeletal muscle. Infective agent monitoring identified three agents at high prevalence in salmon heart tissue, including Piscine orthoreovirus (PRV), and parasites Paranucleospora theridion and Kudoa thyrsites. However, PRV alone was statistically correlated with the occurrence and severity of histopathological lesions in the heart. Immunohistochemical staining further localized PRV throughout HSMI development, with the virus found mainly within red blood cells in early cases, moving into the cardiomyocytes within or, more often, on the periphery of the inflammatory reaction during the peak disease, and reducing to low or undetectable levels later in the production cycle. This study represents the first longitudinal assessment of HSMI in a salmon farm in British Columbia, providing new insights on the pathogenesis of the disease.