Simple Filter Paper as a Shipping and Storage Medium for Human Enteric Viruses

Introduction: Over 100 types of pathogenic enteric viruses have been found in sewage-contaminated drinking water environments. Sample collection from remote environmental sites can be problematic due to the need for an uninterrupted cold chain to guarantee sample suitability for molecular detection.

Purpose: The purpose of this work was to investigate the feasibility of using filter paper as an easy and inexpensive method to collect environmental samples for enteric virus testing, as an alternative to refrigerated transport and storage.

Methods: Different concentrations (10³ to 10⁶ PFU/ml) of two enteric virus surrogates (F-RNA coliphages MS2 and Qbeta) diluted in buffer or a 10% bovine manure slurry were maintained at 4°C for 37 days. Ten µl aliquots of the dilutions were also spotted on individual 6 mm diameter filter paper (Whatman No. 1) circles to give final amounts of 10¹ to 10⁴PFU, dried and stored at 37°C for 37 days. Triplicate samples of the liquid (10 µl) or disk samples taken at 0, 6, 13 and 37 days were analyzed by real time RT-PCR.

Results: The minimum amount of MS2 or Qbeta detected consistently by RT-PCR in control samples was 10² PFU.  There was no difference (P > 0.01) in detection level on any sampling day for phage MS2 between liquid samples or the filter disk samples for the buffer or manure slurry samples.  For Qbeta, the detectable amount of bacteriophage spotted on filter paper at 37°C was on average one log lower (10³ PFU) than the amount detected in samples stored in liquid form at 4°C. This difference was statistically significant at 0 (P = 0.0005) and 37 d (P = 0.006) in the buffer samples and at 0 d (P = 0.0032) in the manure slurry samples.

Significance: These results suggest that filter paper may be used as a storage and transport method for enteric viruses, providing a sampling alternative when refrigeration of the sample is not possible.