Parasite detection and quantification in avian blood is dependent on storage medium and duration

Studies of parasites in wild animal populations often rely on molecular methods to both detect and quantify infections. However, method accuracy is likely to be influenced by the sampling approach taken prior to nucleic acid extraction. Avian Haemosporidia are studied primarily through the screening of host blood, and a range of storage mediums are available for the short- to long-term preservation of samples. Previous research has suggested that storage medium choice may impact the accuracy of PCR-based parasite detection, however, this relationship has never been explicitly tested and may be exacerbated by the duration of sample storage. These considerations could also be especially critical for sensitive molecular methods used to quantify infection (qPCR). To test the effect of storage medium and duration on Plasmodium detection and quantification, we split blood samples collected from wild birds across three medium types (filter paper, Queen's lysis buffer, and 96% ethanol) and carried out DNA extractions at five time points (1, 6, 12, 24, and 36 months post-sampling). First, we found variation in DNA yield obtained from blood samples dependent on their storage medium which had subsequent negative impacts on both detection and estimates of Plasmodium copy number. Second, we found that detection accuracy (incidence of true positives) was highest for filter-paper-stored samples (97%), while accuracy for ethanol and Queen's lysis buffer-stored samples was influenced by either storage duration or extraction yield, respectively. Lastly, longer storage durations were associated with decreased copy number estimates across all storage mediums; equating to a 58% reduction between the first- and third-year post-sampling for lysis-stored samples. These results raise questions regarding the utility of standardizing samples by dilution, while also illustrating the critical importance of considering storage approaches in studies of Haemosporidia comparing samples subjected to different storage regimes and/or stored for varying lengths of time.