# PMCA to demonstrate the efficacy of prion inactivation methods on reusable medical devices: a relevant alternative to animal bioassays

A. Igel, Mohammed Moudjou, P. Destrez, P. Clayette, Vincent Béringue

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A. Igel, Mohammed Moudjou, P. Destrez, P. Clayette, Vincent Béringue. PMCA to demonstrate the efficacy of prion inactivation methods on reusable medical devices: a relevant alternative to animal bioassays. Journal of Hospital Infection, 2024, 154, pp.60-63. 10.1016/j.jhin.2024.07.020. hal-04831377

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Short Report

# PMCA to demonstrate the efficacy of prion inactivation methods on reusable medical devices: a relevant alternative to animal bioassays

A. Igelª, M. Moudjouª, P. Destrezb,*,†, P. Clayettec,*,†, V. Béringuea,*,†

## ARTICLE INFO

**Article history:**

Received 15 May 2024

Accepted 21 July 2024

Available online 30 September 2024

**Keywords:**

Prions

263K strain

Human CJD strains

In-vitro/in-vivo comparison

Efficacy

Alternative methodology

Low temperature hydrogen

peroxide

STERRAD NX™

<figure><img src="image_4.png" /></figure>

## SUMMARY

Validation of prion inactivation processes for medical devices relies on in-vivo experimental protocols. However, bioassays are costly, long (1–2 years) and ethically disposable. Additionally, results obtained with one prion strain – for example, 263K (hamster-adapted strain originating from sheep scrapie) – cannot be easily extrapolated to relevant human prion strains, further questioning the utility of bioassays. Over the past two decades, cell-free prion amplification assays have emerged as potential alternatives to bioassays. Rather than measuring prion infectivity, they quantify prion seeding activity (i.e. the capacity to convert the normal prion protein into the disease-associated isoform). The results obtained from an optimized cell-free assay termed ‘miniaturized-bead protein misfolding cyclic amplification’ (mb-PMCA) with four processes using three different prion strains – 263K and two human prions derived from variant and sporadic Creutzfeldt–Jakob disease – were compared with published bioassays using the same three strains and processes, when available. Tests performed on reference processes (steam, sodium hydroxide, sodium hypochlorite) and low temperature H₂O₂ sterilization (STERRAD NX™ Advanced cycle) showed perfect alignment between mb-PMCA and available bioassays. STERRAD NX™ Advanced cycle was efficacious against all three prion strains. These data confirm that PMCA, particularly mb-PMCA, is a relevant alternative to animal bioassays for the assessment of prion inactivation processes, and highlight the interest of some low temperature H₂O₂ sterilization cycles.

© 2024 The Authors. Published by Elsevier Ltd

on behalf of The Healthcare Infection Society. This is an open access article
under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

<footnote>ªMolecular Virology Immunology Unit, Université Paris-Saclay, INRAE, UVSQ, Jouy-en-Josas, France</footnote>

<footnote>ᵇAdvanced Sterilization Products, Irvine, CA, USA</footnote>

<footnote>ᶜJALS, Versailles, France</footnote>

<footnote>* Corresponding authors. Addresses: Molecular Virology Immunology Unit, Université Paris-Saclay, INRAE, UVSQ, 78350 Jouy-en-Josas, France. Advanced Sterilization Products, Irvine, CA, USA. JALS, 78000 Versailles, France.</footnote>

<footnote>E-mail addresses: philippe destrez@asp.com (P. Destrez), pascal.clayette@outlook.fr (P. Clayette), vincent.beringue@inrae.fr (V. Béringue).</footnote>

<footnote>† Senior authors.</footnote>
## Introduction

The attention currently paid to human health risks associated with infectious prions, responsible for Creutzfeldt–Jakob disease (CJD), is less than after the outbreak of zoonotic variant CJD (vCJD) due to the bovine spongiform encephalopathy (BSE) epidemic in the early 1990s. However, several studies have suggested a significant number of asymptomatic vCJD cases in countries affected by BSE. Additionally, there has been a consistent increase in sporadic CJD (sCJD) mortality worldwide, and the zoonotic potential of animal prions, particularly those causing chronic wasting disease in cervids, remains uncertain. As no prophylactic therapies or preclinical routine diagnostics exist for prion diseases, the risk of inadvertent prion contamination relies solely on preventive strategies.

Preventive strategies include adequate processing of surgical devices which may have been contaminated. Unfortunately, prions resist most routine decontamination processes available to healthcare facilities. Hence, guidelines have been implemented to recommend processes with proven efficacy. However, existing recommendations are based on lengthy, costly and ethically disputable in-vivo evaluations. These infectivity bioassays involve intracranial implantation of prion-ad sorbed steel wires into laboratory animals. In-vitro prescreening is essential to minimize the risk of in-vivo failure, but conventional in-vitro tools such as western blotting that detect the disease-associated form of the prion protein (PrP<sup>Sc</sup>) are not as sensitive as bioassays. In France, the national health agency ANSM [L'Agence nationale de sécurité du médicament et des produits de santé] has defined a two-step protocol involving both in-vitro and in-vivo assessments, along with minimum reduction factor (RF) criteria that must be satisfied to be listed as a total inactivation process. The 'Standard Prion Protocol 2011' (PSP2011) is based on the internationally recognized 263K strain and hamster bioassays. PSP2011 allowed the identification of 16 prion inactivating processes that achieve total inactivation, including liquid disinfection or cleaning chemistries; low-temperature gaseous sterilization (LTS) processes, such as STERRAD NX™ Advanced cycle [Advanced Sterilization Products (ASP), Irvine, CA, USA] and STERRAD 100NX™ Flex cycle; and a combination of liquid and LTS methods].

Beyond interlaboratory variations, the effectiveness of prion inactivation procedures may depend on the host and strain [1], and the corresponding PrP<sup>Sc</sup> conformations [2]. For example, some detergent formulations effective against 263K prions, notably those listed by PSP2011, are not fully effective against vCJD prions [3] or sCJD prions [4]. Conversely, saturated steam at +121 °C and +134 °C (for 18 min) was found to be more effective against vCJD than against 263K. Consequently, the PSP was revised in 2018 to include additional in-vitro and in-vivo challenges against human prions [5]. For various reasons (contextual, economic), PSP2018 did not achieve the same success as PSP2011. In parallel, considerable progress has been made in the optimization of cell-free amplification assays of prions adsorbed on steel wires [1]. Unlike the first generation of in-vitro tools, these new methods achieve a high level of sensitivity that equals and even exceeds that of the bioassays. One of these in-vitro methods, termed 'protein misfolding cyclic amplification' (PMCA), was used by Belondrade et al. for the screening of chemical processes that

had been evaluated according to PSP2011 against vCJD prions [6]. The study also included the reference inactivation of PSP2011 and saturated steam at 134 °C for 18 min. It demonstrated perfect alignment between in-vivo and in-vitro outcomes [3]. However, a link was missing between 263K and human strain data.

This study is a pragmatic attempt to establish correlations between published bioassays with 263K, vCJD and sCJD, and available or new PMCA data obtained with an optimized, miniaturized-bead PMCA assay (mb-PMCA) to help define the place of optimized PMCA in future research. It involves a representative panel of medical device processing procedures that have been tested previously on prions. These include: (i) benchmark processes recommended by international communities and adopted by regulatory agencies (i.e. sodium hydroxide and sodium hypochlorite at partially effective and totally effective concentrations); (ii) the most common sterilization technology (i.e. saturated steam at both 121 °C and 134 °C for 18 min); and (iii) the LTS technology widely used in hospitals [i.e. hydrogen peroxide (STERRAD NX™ Advanced cycle)]. Unlike other LTS technologies, such as ethylene oxide and steam formaldehyde, which act as prion fixatives, H₂O₂ LTS (STERRAD NX™ Advanced cycle) met the total inactivation criteria [7] according to PSP2011.

## Results and discussion

The maximal median seeding dose (SD<sub>50</sub>) per wire was determined for 263K prions, and humanized (i.e. passaged in human PrP mice) vCJD and sCJD (VV2 subtype, henceforth referred to as 'VV2-sCJD') prion strains in the mb-PMCA assay (see Methods in Supplementary information). After two or three rounds of amplification, the mean SD<sub>50</sub> values were established at ~10<sup>8</sup> per wire for all three strains (Table I and Figure S1, see online supplementary material). The interassay coefficients of variation (%CV) were 5%, 11% and 3%, respectively (Table I), demonstrating low variability and confirming the reliability and robustness of the methodology.

Previously published bioassay titration with wire-bound 263K prions provided an infectious titre [50% infectious dose (ID<sub>50</sub>)] value per wire upon implantation in Golden Syrian hamsters of 10<sup>7.0 ± 1.1</sup> [7,8]. Consequently, SD<sub>50</sub> and ID<sub>50</sub> values per wire for the 263K strain are of similar magnitude.

It has been reported previously that the infectious titre of wire-bound vCJD prions derived from a World Health Organization (WHO) reference brain material was 10<sup>3.5 ± 0.5</sup> ID<sub>50</sub> per wire in bovine PrP mice [3]. In a follow-up study, PMCA was performed on the same reference material, and provided an SD<sub>50</sub> value in the range of ours (10<sup>7.3</sup> SD<sub>50</sub>/wire [6]). Bioassay of humanized vCJD prions in bovine PrP mice provided an ID<sub>50</sub> value of 10<sup>5.8</sup> per wire (article submitted for publication). Thus, the PMCA assays offer a dynamic range 1000–10,000 times higher than that of bovinized bioassays for estimating the effectiveness of decontamination processes against vCJD. While human PrP mice could theoretically be used for vCJD bioassay, their extended time to response renders them unsuitable for such studies [1].

Regarding VV2-sCJD, bioassay in transgenic mice expressing human PrP (Val<sub>129</sub> [1]) resulted in the estimation of ID<sub>50</sub> of 10<sup>4.1</sup> per wire (article submitted for publication). Therefore, the mb-PMCA assay was again far more sensitive than the bioassay.
Table I

Interexperience variability of miniaturized-bead protein misfolding cyclic amplification for the three strains tested [263K, variant Creutzfeldt–Jakob disease (vCJD) and VV2 subtype of sporadic CJD (VV2-sCJD)]

<table><thead><tr><th rowspan="2"></th><th colspan="2">263K</th><th colspan="4">vCJD</th><th colspan="4">VV2-sCJD</th></tr><tr><th>A01</th><th>A02</th><th>A01</th><th>A02</th><th>A03</th><th>A04</th><th>A01</th><th>A02</th><th>A03</th><th>A04</th></tr></thead><tbody><tr><td>SD<sub>50</sub> per wire</td><td>7.64</td><td>8.53</td><td>7.87</td><td>8.31</td><td>6.20</td><td>7.87</td><td>8.20</td><td>7.87</td><td>7.53</td><td>7.87</td></tr><tr><td>95% CI</td><td>0.94</td><td>2.12</td><td>1.13</td><td>0.69</td><td>1.39</td><td>1.39</td><td>1.96</td><td>1.39</td><td>1.39</td><td>1.79</td></tr><tr><td>N# rounds</td><td>2</td><td>2/3</td><td>3</td><td>2</td><td>2</td><td>2</td><td>3</td><td>3</td><td>2</td><td>2</td></tr><tr><td>Mean ± SD</td><td>8.1 ± 0.4</td><td></td><td>7.6 ± 0.8</td><td></td><td></td><td></td><td>7.9 ± 0.2</td><td></td><td></td><td></td></tr><tr><td>%CV</td><td>5%</td><td></td><td>11%</td><td></td><td></td><td></td><td>3%</td><td></td><td></td><td></td></tr></tbody></table>

A0N, Assay #N; SD<sub>50</sub>, maximal median seeding dose; CI, confidence interval; CV, coefficient of variation.

In summary, mb-PMCA assays appear to be highly relevant methods for evaluation of the capacity of processes to eliminate and/or inactivate prions, particularly human prions, given their dynamic range.

Next, the prionicide activity of various processes was examined using mb-PMCA. The prionicide activity of saturated steam remained incomplete but important for both 263K and vCJD prions (Table II and Figure S1, see online supplementary material; RF ~6–7 Log<sub>10</sub>), and complete for VV2-sCJD prions at +134 °C (Table II and Figure S1, see online supplementary material; RF ≥8.20). Bioassay of 263K wires treated with steam at +134 °C showed partial inactivation (~50%) with RF values >5–6 [7,9], consistent with the observations using mb-PMCA. With the WHO reference material, vCJD wires treated with steam at +134 °C exhibited no residual seeding activity nor infectivity [3,6]. The reason for the difference between human vCJD and humanized vCJD with respect to steam inactivation is not known. It may be due to the slightly higher SD<sub>50</sub> value of the humanized brain or the matrix used (human vs mouse brain).

The prionicide activity of sodium hydroxide at the 1N concentration was partial but significant for 263K and VV2-sCJD prions (RF 6–7 Log<sub>10</sub>), and complete for vCJD prions (RF ≥8.3) (Table II). At the 0.1N concentration, sodium hydroxide partially inactivated all three strains (Table II). Sodium hydroxide at the 1N concentration has been reported to

inactivate 263K infectivity, as estimated by hamster bioassay [10]. However, the complete effectiveness of the 1N NaOH treatment was not observed systematically in every in-vivo study, occasionally resulting in one or two positive animals (P. Clayette, unpublished data). For VV2-sCJD, bioassay in human PrP mice confirmed partial inactivation, with three of 14 mice implanted with wires developing the disease (article submitted for publication). For vCJD, complete inactivation at the 1N concentration was also observed with the WHO reference sample by both PMCA and bioassay [3,6]. The present bioassay also showed complete inactivation with humanized vCJD (article submitted for publication).

Bleach at 2% (20,000 ppm) proved to be completely effective against all prion strains tested in the operational conditions (Table II), as observed previously in-vitro and in-vivo ([3,6,10] and article submitted for publication), suggesting no differences between the 263K strain and human strains for bleach. Moreover, these results are consistent with the greater effectiveness of bleach compared with sodium hydroxide.

Regarding the STERRAD NX™ system, the Advanced cycle inactivated all steel wire-bound prions completely, regardless of the strain type, with an RF of ~8 (Table II and Figure S1, see online supplementary material). This correlates with previous data using a less sensitive methodology [7].

Table II

Efficacy of standard ANSM (L'Agence nationale de sécurité du médicament et des produits de santé) treatments and of STERRAD NX™ Advanced cycle against hamster and human prions

<table><thead><tr><th rowspan="2"></th><th rowspan="2"></th><th colspan="2">263K</th><th colspan="2">vCJD</th><th colspan="2">VV2-sCJD</th></tr><tr><th>A01</th><th>A02</th><th>A01</th><th>A02</th><th>A01</th><th>A02<sup>a</sup></th><th>A03</th></tr></thead><tbody><tr><td>SD<sub>50</sub> per wire</td><td></td><td>7.64</td><td>8.53</td><td>7.87</td><td>8.31</td><td>8.20</td><td>6.87</td><td>7.70</td></tr><tr><td rowspan="2">Steam</td><td>+121 °C</td><td>-</td><td>6.28</td><td>6.55</td><td>-</td><td>-</td><td>-</td><td>-</td></tr><tr><td>+134 °C</td><td>-</td><td>6.94</td><td>6.28</td><td>-</td><td>≥8.20</td><td>-</td><td>-</td></tr><tr><td>Sodium hydroxide (NaOH)</td><td>0.1 N</td><td>6.52</td><td>7.41</td><td>-</td><td>6.99</td><td>-</td><td>5.55</td><td>-</td></tr><tr><td>Sodium hypochlorite (NaOCl)</td><td>0.2%</td><td>6.33</td><td>7.22</td><td>-</td><td>≥8.31</td><td>-</td><td>6.07</td><td>-</td></tr><tr><td>STERRAD NX™</td><td>2%</td><td>-</td><td>≥7.64</td><td>≥8.53</td><td>-</td><td>≥8.31</td><td>-</td><td>≥6.87</td><td>-</td></tr><tr><td></td><td>Advanced cycle</td><td>-</td><td>≥8.53</td><td>≥7.87</td><td>-</td><td>-</td><td>-</td><td>≥7.70</td></tr></tbody></table>

A0N, Assay #N; SD<sub>50</sub>, maximal median seeding dose.

For 'Comparator' treatments: steam +134 °C/18 min/3 bars, sodium hydroxide 1 N/1 h/RT (room temperature) and sodium hypochlorite 2% (20,000 ppm)/1 h/RT; for 'Partial efficacy' treatments: steam +121 °C/20 min/3 bars, sodium hydroxide 0.1 N/15 min/RT and sodium hypochlorite 0.2% (2000 ppm)/15 min/RT); and for the STERRAD NX™ Advanced cycle using miniaturized-bead protein misfolding cyclic amplification against hamster 263K strains, humanized variant Creutzfeldt–Jakob disease (vCJD) and VV2 subtype of sporadic CJD (VV2-sCJD).

Results are expressed as reduction factors.

<footnote><sup>a</sup> Only two protein amplification rounds.</footnote>
To conclude, correlation between the mb-PMCA results and data available from the literature further strengthens the relevance of optimized cell-free prion amplification assays for assessing the effectiveness of physical or chemical inactivating processes against prions. Optimized PMCA approaches could overcome the potential drawback of low infectious titres in animal models, particularly with human prions. The high levels of prion seeding activity bound to wires, measured for all tested strains, strongly indicates the exquisite sensitivity achievable with mb-PMCA compared with former in-vitro and in-vivo evaluations. This could potentially reduce or even replace animal bioassays, offering practical advantages in terms of overall cost, ease of execution and time to completion. The results also show that the 263K strain, used by many laboratories, may remain relevant as a screening approach for saturated steam and some LTS technologies. Indeed, for chemical processes, the strain was not predictive or was less predictive of the efficacy on human prions [3,6], further justifying subsequent studies against human prions. Furthermore, the present results highlight the efficacy of routine low temperature H₂O₂ sterilization cycles (STERRAD NX™ Advanced cycle) on all tested prion strains.

**Author contributions**

Angélique Igel: methodology, investigation, experimentation, formal analysis. Mohammed Moudjou: methodology, investigation, experimentation, formal analysis. Philippe Destrez: investigation, conceptualization, writing – review and editing. Pascal Clayette: investigation, validation, conceptualization, writing – original draft, review and editing. Vincent Béringue: project administration, supervision, validation, investigation, experimentation, writing – review and editing.

**Conflict of interest statement**

Philippe Destrez is employed by ASP, and Pascal Clayette works as a scientific consultant for ASP. Angélique Igel, Mohammed Moudjou and Vincent Béringue declare no competing financial interests or personal relationships that could appear to influence the work reported in this paper.

**Funding sources**

This work was supported by ASP (Grant No. 13001513) and by an intramuros grant from INRAE (Grant No. 7813001515).

**Ethical approval**

Animal experiments were approved by INRAE Ethics Committee (Comethea; Permit No. APAFIS#29603–2021020914525215 v215/056). All experiments were

performed in biosafety level 3 facilities, adhering strictly to safety protocols.

**Data availability**

Data will be made available on request.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.jhin.2024.07.020.

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