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Sensitive microscopic quantification of surface-bound prion infectivity for the assessment of surgical instrument
T J Secker1, R C Hervé1, C W Keevil1
1Environmental Healthcare Unit, School of Biological Sciences, University of Southampton, Southampton, UK.
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.
Insights
A new cell-based method quantifies prion infectivity without proteinase K, offering greater sensitivity for detecting contamination on surgical instruments. This approach validates decontamination efficacy, crucial for reprocessing reusable surgical tools.
Area of Science:
- Prion biology
- Infectious disease research
- Biomedical engineering
Background:
- Pathogenic prions (PrPSc) are amyloid proteins that contaminate surgical surfaces, posing challenges for instrument reprocessing.
- Current in vitro detection methods may underestimate prion infectivity due to proteinase K digestion, potentially leading to false negatives.
Purpose of the Study:
- To develop a sensitive in-situ method for quantifying prion amyloid accumulation without proteinase K treatment.
- To dynamically measure prion infection in cells exposed to infected tissues and contaminated stainless-steel surfaces.
Main Methods:
- N2a cells were infected with the 22L prion strain, either in solution or dried on stainless-steel wires.
- Prion amyloid aggregate accumulation was directly measured over passages using sensitive fluorescence microscopy.
Main Results:
- A 10-log dynamic range was established for testing residual prion infectivity.
- The method validated variable decontamination efficacy of commercial cleaning chemistries against prions.
Conclusions:
- The cell-based infectivity assay avoids proteinase K digestion, enhancing sensitivity for prion detection.
- This low-cost, ethically sound method is adaptable for different prion strains and superior for testing cleaning efficacy compared to measuring total residual proteins.
