The impact of pooling samples on surveillance sensitivity for the megalocytivirus Infectious spleen and kidney necrosis virus

Abstract

Movements of large volumes and species varieties make the ornamental fish industry a high‐risk pathway for the transfer of aquatic pathogens to new geographical regions and naïve hosts, potentially resulting in emergency disease events. Infectious spleen and kidney necrosis virus (genus Megalocytivirus) is considered exotic to Australia despite documented incursions since 2003. There are current import controls requiring freedom from infection for entry to Australia. The objective was to evaluate the effect of tissue pooling strategies for qPCR testing using a SYBR® assay for freedom from ISKNV at 2% expected prevalence with 95% confidence. Tissue homogenates from apparently healthy imported ornamental fish were tested as individuals and in pools of 5 and 10. Analytical sensitivity of the qPCR assay was reduced by two orders of magnitude when the nucleic acid extraction process was accounted for by spiking the plasmid in fish tissues and compared with molecular grade water. Diagnostic sensitivity of the assay was substantially reduced when testing tissues in pools compared with individual testing. For Population 1 (66% positive for ISKNV with moderate viral loads), surveillance sensitivity was only achieved using individual testing. For Population 2 (100% positive ISKNV with high viral loads), surveillance sensitivity was achieved using 260 fish in pools of 10 for a total of 26 tests or 200 fish in pools of 5 for 40 tests. Surveillance sensitivity could be maximized even when there was a reduction in pooled diagnostic sensitivity compared with diagnostic sensitivity for individual fish by increasing the sample size. Pooled sensitivity was influenced by the prevalence and variable virus load among fish with subclinical infections. Pooled testing is highly effective when the prevalence is >10% which should be informed by prior knowledge or pooling can be used for a screening test to rapidly identify populations with high prevalence.

To pool or not to pool? Guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals

“…Although pooling can be cost‐effective for surveillance studies, it is not always useful or recommended, depending on prevalence and analyte concentration (e.g. Johnson et al, ), value of the individuals sampled, and the costs of sampling and testing. In Figure , we present a theoretical schematic of how analyte load (using Ct values from qPCR tests), prevalence of infected animals in the population (as a proxy for disease state), and pool size can impact the usefulness of pooling techniques.…”

“…In this case, apparently healthy individuals or subclinically infected individuals would be appropriate for pooling studies. The number of sampled individuals per pool can affect PSe results for surveillance purposes in subclinical populations (Hall et al, ; Johnson et al, ), as a function of analyte dilution. The cost of sampling and testing will also influence the choice of pool size, which should be determined a priori as part of the sampling strategy (Hall, Soje, Kilburn, Maguire, & Murray, ).…”

“…In the simulation example we discuss later, we show how tissue weight, analyte concentration and pool size can affect these decisions for WSSV. High sensitivity and the recommendation for individual sampling is important for rare species, where each individual is valuable, and in situations where a false negative (from lower or diluted sensitivity of pools) is detrimental to decision‐makers, often for import/export purposes, particularly for low prevalence of the disease (Johnson et al, ). However, as mentioned before, the cost‐effectiveness of pooling in surveillance studies in apparently healthy (low prevalence) populations has increased the interest in using pooling for these studies for the purpose of demonstration of disease freedom.…”

“…However, these assumptions depend on the prevalence of the infectious agent in the source population and the concentration of target analyte in each specimen included in the pool (Johnson et al, ; Zainathan, Carson, Crane, & Nowak, ). In low prevalence scenarios, pools of tissues or swabs from non‐infected animals can dilute an already low concentration of an infectious agent that may be close to the detection limit or cut‐off value used to classify a test result as positive or negative (Corsin et al, ; East, ; Jansen et al, ; Johnson et al, ; Knüsel et al, ; Oidtmann et al, ). Another important factor that is often driven by prevalence and affected by analyte concentration is the number of positives per pool.…”

“…Based on the 12 papers identified in the literature search (three in the systematic review and nine by other means), no clear patterns were evident with various pooling methods (see Appendix 1), other than a negative effect of pooling on DSe (Bergmann, Riechardt, Fichtner, Lee, & Kempter, 2010;Hall, Duguid, Wallace, & Murray, 2015;Hyatt et al, 2007;Johnson et al, 2019;Lane et al, 2017;McClure, Hammell, Stryhn, Dohoo, & Hawkins, 2005;Monaghan et al, 2015). Animals, tissues, tissue homogenates, fluids or swabs were pooled, with different numbers of specimens per pool; the most used pool size was 5.…”