TO OR NOT TO POOL?: GUIDELINES FOR POOLING DECISIONS IN AQUATIC ANIMAL HEALTH SURVEILLANCE

For surveillance purposes (presence or absence of pathogens), testing of pooled specimens from aquatic animals is preferable to testing individuals when the prevalence of infected samples is low and concentration of the target analyte is high. However, use of pooling raises important questions: for which combinations of prevalence and load can pooling be justified; should pooling recommendations differ for systemic vs. localized infections; are there published data to support recommendations for pooling of 5 specimens?  

A systematic review of published studies in aquatic animals was undertaken using standard search terms in PubMed and CAB Abstracts. OIE-listed and non-listed diseases were included. Abstracts were screened and relevant information was extracted and summarized from the full papers. An expert group was convened to review the collated evidence and develop guidelines for designing pooling studies.

Fourteen relevant papers were identified in the search, many involving qPCR. Most authors concluded that pooling reduced sensitivity compared with testing of individuals. Supporting data for pooling was limited. Only two papers specifically mentioned the concept of pooled sensitivity and pooled specificity. These two outbreak studies provided useful comparative data (virus isolation and qPCR for SAV and ISAV), but no papers reported Ct values.

Pooling is logistically necessary for testing early life stages but is optional in other situations. Scientific evidence about pooling that can withstand legal scrutiny is needed, especially for OIE-listed diseases The preferred design is field studies with parallel testing of both individual specimens and these same specimens in randomly-created pools that mimic real-life surveillance scenarios supported with the respective cost and sensitivity data. Individual and pooled Ct values should be reported even if the final decision is presence/absence. Analyte loads are likely to be much lower several months after outbreaks and much more reflective of surveillance scenarios. Use of experimentally-infected animals and spiking experiments were considered inferior sources of data to evaluate effects of pooling. Studies are also needed to describe the distribution of Ct values in naturally-infected but healthy aquatic animals, as this information can provide a sound basis for simulation studies of potential effects of pooling and inform laboratory experiments.