Redefining diarrheal disease surveillance: Long term nucleic acids stability with simple, low-cost stool preservation

Testing nucleic acid (NA) from dried stool spots (DSS) on Whatman903 filter paper provides a simple approach to detect enteric pathogens, especially in low-resource settings where cold-chain transport and lab infrastructure are limited. DSS samples are easily collected, stored, transported, and processed. This study evaluates NA stability from DSS samples stored under different times and temperatures.

DSS were prepared by spotting stool from patients infected with Shigella spp. (dsDNA), rotavirus (dsRNA) and norovirus (ssRNA) onto filter paper. Pathogen detection using NA extracted from DSS at Time Zero (T0) and after storage at room temperature (RT), 4°C, -20°C or -80°C for 3-6 months, 6-12 months, and 12-18 months was compared to NA column-extracted from stool (the gold standard) using qPCR.

Cycle threshold (CT) values for each pathogen remained stable over time and temperature. RT-stored DSS performed similarly to cold- or frozen-stored samples. Shigella DSS showed no significant difference from column-extracted NA Mean CT after 18 months of storage: 28.33 RT vs 28.64 GS, 95% CI: (-0.44, 0.84). Rotavirus DSS showed lower CTs than column-extracted samples; mean CT after 18 months: 19.40 RT vs 26.92 GS, 95% CI: (6.57, 8.66), indicating increased nucleic acid yield from DSS. Norovirus DSS showed higher CTs from DSS, with consistent CT from DSS from time zero through 18 months: 30.63 RT vs 25.79 GS, 95% CI: (-5.98, -4.06). This suggests initial NA loss upon spotting, but CTs remained stable thereafter for the duration of the study.

DSS samples remained stable for NA extraction at RT for at least 18 months for Shigella and rotavirus. Norovirus detection from DSS showed slight initial degradation but remained detectable with stable CTs over time. DSS thus provides a practical, low-cost tool to enhance stool-based pathogen detection in global diarrheal surveillance.