Sensitive microscopic quantification of surface-bound prion infectivity for the assessment of surgical instrument decontamination procedures.

ABSTRACT

BACKGROUND: Pathogenic prions (PrPSc) are amyloid-rich hydrophobic proteins which bind avidly to surgical surfaces and represent some of the most difficult targets during the reprocessing of reusable surgical instruments. In-vitro methods to amplify and detect the presence of otherwise undetectable prion contamination are available, but they do not measure associated infectivity. Most of these methods rely on the use of proteinase K, however this can lead to the loss of a substantial portion of PrPSc, potentially producing false negatives. AIM: To develop a sensitive in-situ method without proteinase treatment for the dynamic quantification of amyloid accumulation in N2a #58 cells following 22L-prion infection from infected tissues and spiked stainless-steel surfaces. METHODS: We spiked cultures of N2a #58 cells with the 22L prion strain in solution or dried on stainless-steel wires and directly measured the accumulation of prion amyloid aggregates over several passages using highly sensitive fluorescence microscopy. FINDINGS: We demonstrated a 10-log dynamic range using our method to test residual prion infectivity, that was validated to show variable decontamination efficacy against prions from commercially available cleaning chemistries. CONCLUSIONS: The new cell-based infectivity method presented here avoids partial or possibly total proteinase K digestion of PrPSc in samples for greater sensitivity, in addition to low cost, no ethical concerns, and adaptability to detect different prion strains. This method can be used to test cleaning chemistries' efficacy with greater sensitivity than measuring total residual proteins, which may not correlate with residual prion infectivity.