Rapid and Highly Sensitive Detection of Variant Creutzfeldt - Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies

Abstract

The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10−8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.

Rapid amplification of prions from variant Creutzfeldt–Jakob disease cerebrospinal fluid

“…Based on the molecular susceptibility of PrP C by the abnormal and misfolded isoform PrP Sc , the in vitro conversion systems such as Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking Induced Conversion (RT-QuIC) have provided important advances in the development of diagnostic opportunities for several protein misfolding disorders including human prion diseases [7][8][9][10][11][12][13][14][15].…”

Correlation between Bioassay and Protein Misfolding Cyclic Amplification for Variant Creutzfeldt-Jakob Disease Decontamination Studies

“…Stainless steel wires (catalog no. FE245102; Goodfellow, England) (diameter, 0.15 mm) were prepared as previously described (32). Briefly, batches of wires were cut into 3-mm pieces and contaminated in 96-well plates (one wire per well) with serial dilutions of vCJD-IBH (10 Ϫ1 to 10 Ϫ8 ) in order to evaluate bioassay sensitivity (Table 1).…”

“…Before processing, 96-well plates containing contaminated wires were dried at room temperature. Wires were then exposed to the different formulations or procedures for prion disinfection listed in Table 2 and Table S1 in the supplemental material as previously described (32). Briefly, test wires were incubated in 200 l of the different treatment solutions and handled according to the manufacturer's instructions.…”

“…Surf-PMCA. Surf-PMCA was performed as previously described (32). Briefly, wires contaminated with 10 Ϫ7 dilution of vCJD-IBH or the negative PMCA control, hu-NBH, were serially amplified using normal brain homogenate (NBH) from humanized transgenic mice (M129 allele, tg650 line) as the substrate for PMCA.…”

“…In a previous study, by using PMCA associated with contaminated steel wires (Surf-PMCA), we were able to show that the sensitivity of the Surf-PMCA method allows discrimination of decontamination treatments with respect to their effectiveness on vCJD prions by monitoring residual seeding activity (RSA) on wires (32). However, the results obtained with prion-specific commercial solution showed variable effectiveness to alter the seeding activity of the vCJD prion.…”