Inactivation of Prions: Studies in France & Germany

Inactivation of Prions: Studies in France & Germany Sterilizzazione a gas plasma di perossido di idrogeno: studi di inattivazione dei prioni

Prions: A Longstanding Animal & Human Health Issue ANIMALS! 1732: Natural Scrapie / Sheep Europa! 1985: Bovine Spongiform Encephalopathy (180,000 cases more than 900,000 contaminated bovine in food) UK! 1990: Cat SE (Switzerland)! Transmissible Mink Encephalopathy (TME)! Chronic Wasting Disease (CWD) HUMANS! 1951: Kuru New Guinea! 1920: Creutzfeld-Jakob Disease (CJD) Europa! Gerstmann-Sträussler-Scheinker Syndrome (GSS)! Fatal Familial Insomnia (FFI)! 1995: Variant of CJD (vcjd)

Prion: Unconventional Transmissible Agent UNLIKE CONVENTIONAL DISEASES! No virus or microorganism! No immune reaction! No inflammation! Degeneration of the central nervous system (CNS) with lesions identified in the CNS only (spongiosis) TRANSMISSIBLE (e.g., food, blood transfusion, surgery), BUT NOT CONTAGIOUS ALWAYS FATAL! Suspected agent is a protein (prion protein)! Long asymptomatic period (several years before signs of disease appear) EXTREME RESISTANCE TO CONVENTIONAL STERILIZATION/ DISINFECTION PROCESSES With dry heat, infectivity is still detectable after:! 180 C during 24 hours! 320 C during 1 hour! 600 C during 15 minutes

Inactivation of prion protein with hydrogen peroxide gas plasma

Inactivation of prion protein with hydrogen peroxide gas plasma

PRINCIPLES Sterilization System Technology General overview! Terminal sterilization process! Low temperature gas plasma! Sterilizing agent: hydrogen peroxide VALIDATED PROCESS! Compliant with EN ISO 14937! Reaches a 10-6 Sterility Assurance Level (SAL)! CE marked CHARACTERISTICS! Low temperature; adapted for heat- and moisture-sensitive devices! Short cycles times! No toxic residuals MARKET PENETRATION! As of August 2012, more than 20,000 STERRAD System units are installed worldwide

Phases of the Study supported by Advanced Sterilization Products Irvine, California USA 2002 2005 IN VIVO TESTS - STERRAD 100S Systems / REFERENCE METHODS*! Steam (134 C, 18 ) and steam + NaOH (1N, 1h at RT)! STERRAD 100S System long cycle (1 cycle and 2 consecutive cycles)! Steam or STERRAD 100S System + alkaline (A and B, at 55 C and 70 C) or enzymatic (37 C) detergents 2005 2007 IN VIVO TESTS - STERRAD NX vs. STERRAD 100S*! STERRAD NX System Advanced cycle (1 cycle and 2 consecutive cycles)! STERRAD 100S or NX System+ alkaline detergents 2007 IN VITRO TESTS - VARIOUS STRAINS & SUPPORTS! (STAINLESS STEEL and HEAT-SENSITIVE MATERIALS)*! STERRAD 100S, STERRAD NX and STERRAD 100NX System vs. steam! STERRAD 100NX System STANDARD and FLEX cycles! STERRAD NX System ADVANCED cycle 2008 IN VITRO TESTS - VARIOUS STRAINS & SUPPORTS (SHAPE)! STERRAD 100S and STERRAD NX System vs. steam

In vivo vs. in vitro Test Methods In vivo tests:! Tests performed on living animals infected with a contaminated wire introduced in brain.! Number of days of survival of animals depends on efficiency of sterilization and/or disinfection process applied on contaminated wires before they are placed in brain.! Survival varies between 80 days (no processing) and 18 months (end of experiments). In vitro tests:! Bio-chemical analysis performed on contaminated stainless steel (wires and plates) or plastic plates covered with contaminated brain homogenates.! Tests indicate quantity of PrPres remaining on the plate after processing the plate.

Laboratories Conducting the Studies IN VIVO TESTS Directed by Klaus Roth, SMP GmbH (Tübingen, Germany) in collaboration with:! University of Tübingen! Federal Reference Center for Virus Diseases of Animals IN VITRO TESTS 1. Directed by Pascal Clayette, Ph.D., SPI-BIO, Neurovirology Laboratory (Fontenay aux-roses, France)! SPI BIO is a spinoff of CEA, one of the reference research centers for prion diseases in France.! Pascal Clayette was a close collaborator of the late Dr. Dominique Dormont, a scientist involved in prions research and expert for Afssaps and EMEA. 2. Co-Directed by Pascal Clayette and Armand Perret-Liaudet, Ph.D., Lyon University Hospital! Armand Perret-Liaudet is one of the reference prion experts in France, for AFSSaPS and EMEA.

Originality and Strengths of the Study Comparative Methodology! In vivo: 41 experimental conditions (sterilization, detergence, disinfection, combinations, different levels of concentration, temperature, etc.)! In vitro: 20 experimental conditions with 6 different strains and 3 types of materials and 2 shapes of support (quadru- or sexplicats) Coherence and Consolidation of the Results! 13 test groups with the STERRAD 100S Sterilizer! 4 test groups with the STERRAD NX Sterilizer! 2 Western Blot protocols used for the key groups (3F4 and coupled SAF83)

Bioassay: Methodology Preparation of the wires! Stainless steel wires (1.4301; Flechsig et al., 2001*). Ø : 0,3 mm / L : 5 mm! Incubation in 10% brain homogenate in phosphate buffer for 16 h! Dried at room temperature for 1 h! Treated with the different processes (except for positive controls) Implantation! Introduction in an injection needle! Use of a stereotaxic instrument! Insertion in the brain of an anesthetized hamster Monitoring! Clinical! Biochemical (Western Blots) *Transmission of scrapie by steel-surface-bound prions. Flechsig E, Hegyi I, Enari M, Schwarz P, Collinge J, Weissmann C. Mol Med. 2001 Oct;7(10):679-84.

Bioassay: Correlation Between Infectious Dose and Incubation Period! An incubation delay of 12 days = a reduction of the infection level 1 log! After 200 days, only residual infectivity detected. * Müller H, Stitz L, Wille H, Prusiner SB, Reisner D. Influence of water, fat, and glycerol on the mechanism of thermal prion inactivation. J Biol Chem 2007; 282:35855 35867

«263K/hamster model»: Brain invasion by prions after wire implantation Complete brain invasion by prions after the implantation of one 263K-contaminated steel wire Lemmer, 2008 (PET blot detection of cerebral PrPsc)

Validation of the Methodology Transmission Rate: Number of sick animals/total number of animals in group (usually 10 animals/ group). Incubation Period: Number of days between contamination and death of the animal. Incubation Delay: Additional survival period obtained by processing of wire (=incubation time- # of days of survival for hamster contaminated with a non-processed wire). Reduction Factor: Estimated reduction of initial contamination load expressed on a logarithmic scale. Twelve additional days of survival correspond approximately to 1 log reduction of infection load. Experimental method does not allow statement above 5-6 log. Negative controls (wires exposed to 10% brain homogenate) Positive controls (wires exposed to 10% 263K-infected brain homogenate) Transmission Rate (%) Incubation Period (days) Incubation Delay (days) Reduction Factor 0% 606 ± 118 - - 100% 83 ± 3 - - 10% 263K-infected brain homogenate 100% 78 ± 2 - - Wires implanted for only 5 minutes 100% 101 ± 5 18 1,5! Implantation: no effect on animals life expectancy! 5 insertion: sufficient to infect the animals

Results 1 : Steam (prion cycle) Negative controls (wires exposed to 10% brain homogenate) Positive controls (wires exposed to 10% 263K-infected brain homogenate) Transmission Rate (%) Incubation Period (days) Incubation Delay (days) Reduction Factor 0% 606 ± 118 - - 100% 83 ± 3 - - 10% 263K-infected brain homogenate 100% 78 ± 2 - - Wires implanted for only 5 minutes 100% 101 ± 5 18 1,5 Steam 134 C 18 min* 50% 428 ± 103 345 5-6! Steam 134 C / 19 minutes : 50 % transmission rate! Confirmed by other investigators who found transmission rates for steam 134 C 18 minutes between 40 and 60 % * Fichet G, Antloga K, Comoy E, Deslys JP, McDonnell G. Prion inactivation using a new gaseous hydrogen peroxide sterilisation process. J Hosp Infect 2007; 67:278 286. Fichet G, Comoy E, Duval C, et al. Novel methods for disinfection of prion-contaminated medical devices. Lancet 2004; 364:521 526. Baxter HC, Campbell GA, Whittaker AG, et al. Elimination of transmissible spongiform encephalopathy infectivity and decontamination of surgical instruments by using radiofrequency gas-plasma treatment. J Gen Virol 2005; 86:2393 2399. Jackson GS, McKintosh E, Flechsig E, et al. An enzyme-detergentmethod for effective prion decontamination of surgical steel. J Gen Virol 2005; 86: 869 878

Results 2: Influence of H 2 O 2 concentration and phase TRANSMISSION RATE (%) INCUBATION PERIOD (DAYS) INCUBATION DELAY (DAYS) REDUCTION FACTOR Liquid H 2 O 2 * (59%, RT, 10 min) STERRAD 100S Long cycle ** (gaseous H 2 O 2, 59%, 50 C, 2 x 10 min) STERRAD NX 1 Advanced cycle *** (gaseous H 2 O 2, 90%, 53 C, 2 x 7 min) 50% 443 ± 140 360 5-6 100% 99 ± 6 16 1.3 0% 570 ± 18 487 5-6 Steam 134 C 18 min 50% 428 ± 103 345 5-6 * Tests were also performed during 20 min. Results are comparable. ** Tests were first performed with STERRAD 100S GMP System and then with STERRAD 100S System. Results are comparable. *** Tests were also performed with 2 consecutive Advanced cycles: results are comparable.! At 59 % : liquid H 2 O 2 > gaseous H 2 O 2! STERRAD NX System (gaseous H 2 O 2, 90%) > STERRAD 100S System (gaseous H 2 O 2, 59%)

Results 3: Interest of cleaning and interaction between cleaning and sterilization (STERRAD 100S System) TRANSMISSION RATE (%) INCUBATION PERIOD (DAYS) INCUBATION DELAY (DAYS) REDUCTION FACTOR Enzymatic detergent (2%, 37 C, 10 min) 100% 95 ± 0 13 1.1 Enz. Det. + steam (134 C 18 min) 100% 131 ± 17 48 4.0 Enz. Det + STERRAD 100S System Long cycle 100% 111 ± 12 29 2.4 1% alkaline detergent A (55 C, 10 min) * 11% 446 ± 153 363 5-6 1% alkaline detergent A (55 C, 10 min) * plus STERRAD 100S System 1 long cycle 0% 540 ± 14 457 5-6 Steam 134 C 18 min 50% 428 ± 103 345 5-6 * Tests were also performed with the same alkaline detergent (Detergent A) followed by two consecutive STERRAD 100S System long cycles. Results are comparable to those obtained with Detergent A + 1 STERRAD 100S System long cycle. Another alkaline detergent (detergent B) was also evaluated, alone and in combination with 1 or 2 consecutive STERRAD 100S System long cycles. Results are similar to those obtained with the Alkaline detergent A.! Confirmation of interest of the cleaning phase. But alkaline > enzymatic?! Possible antagonist effect between enzymatic detergent and steam! Interest of the combination STERRAD 100S System + detergents

Results 4: STERRAD NX System In vivo With and Without Pre-treatment TRANSMISSION RATE (%) INCUBATION PERIOD (DAYS) INCUBATION DELAY (DAYS) REDUCTION FACTOR STERRAD NX System 1 Advanced cycle 1% alkaline detergent A (55 C, 10 min) plus STERRAD NX System 1 Advanced cycle* 0% 570 ± 18 487 5-6 0% 559 ± 22 476 5-6 Steam 134 C 18 min*** 50% 428 ± 103 345 5-6 * Tests were also performed with alkaline detergent B. Results are comparable to those obtained with Detergent A.! In experimental conditions STERRAD NX alone obtained complete inactivation of Prion on all hamsters instead of 50% for steam! Additional effect of detergents is not visible (since STERRAD NX System is fully effective by itself)! Alkaline detergents + STERRAD NX System: no antagonism

In vitro: Methodology Preparation of the supports! Plates in Stainless Steel, Polypropylene or Polyethylene Dimension: 9 x 9 mm Contamination: 20 µl of inoculums! Wires (Flechsig et al., 2001*) / Stainless Steel Number per experimental condition: 40 Dimension : Ø: 0,3 mm / L: 5 mm Contamination: 100 µl of inoculums! Drying: room temperature, 16 hours 20 µl of brain homog. Sheet Desorbed solution Contamination Drying Treatment Desorption Treatment! With one of the tested processes! No treatment for positive and negative controls Immunoblotting Analysis PC1 PC2 PCWB NC PCWB! Desorption (Lemmer et al., 2004**)! Detection of residual PrPres on sheets(for controls: controls of desorption)! Determination PrPres titers: limit-dilution in desorption solutions and calculation with the Spermann-Kärber formula Desorbed sheet Non-desorbed sheet 1 2 3 4 5 1 2 3 4 5 Dil. (log) *Transmission of scrapie by steel-surface-bound prions.flechsig E, Hegyi I, Enari M, Schwarz P, Collinge J, Weissmann C. Mol Med. 2001 Oct;7(10):679-84.** Lemmer K, Mielke M, Pauli G, Beekes M. Decontamination of surgical instruments from prion proteins: in vitro studies on the detachment, destabilization and degradation of PrPSc bound to steel surfaces. J Gen Virol 2004; 85:3805 3816

Results 4: In Vitro Tests Influence of Strains and Materials and Shape of Supports In vitro Strains, Material and Shape of Supports Prion strains Material Shape of Support 263K Stainless steel Plates Wires Steam 134 C/18 min. Totally efficient NP STERRAD 100S System Partially efficient Part. efficient STERRAD NX System Totally efficient Tot. efficient STERRAD 100NX System Totally Efficient Polypropylene Plates NP NP Tot. efficient NP Polyethylene Plates NP NP Tot. efficient NP 6PB1 Stainless steel Plates Tot. Efficient Tot. Efficient Tot. efficient NP vcjd Stainless steel Plates Tot. Efficient Tot. Efficient Tot. efficient NP Fukuoka Stainless steel Plates NP Tot. Efficient Tot. efficient NP C506M3 Stainless steel Plates NP Tot. Efficient Tot. efficient NP 22L Stainless steel Plates NP! Material and shape of support did not influence results Part. efficient Tot. efficient NP=Test Not Performed NP NP! STERRAD NX system obtains elimination of residual signal for all strains! 263 K and 22 L strains appear more resistant to STERRAD Systems and the hydrogen peroxide used in the technology and confirms that in vivo tests were performed with the most resistant strain

System Efficiency against Prions Gaz Plasma System! STERRAD 100S without cleaning: 1.3 log (1.8 log after 2 consecutive cycles)! Alkaline detergent + STERRAD 100S System: 5-6 log, transmission rate = 0% Gaz Plasma System(alone or combined with alkaline detergent)! No infectivity detected (bioassay): 5-6 log! Efficiency (in vitro) against 263K strain & human strains! Efficiency (in vitro) for different materials (Stainless steel, Polypropylene, Polyethylene) Gaz Plasma System! Identical efficiency (in vitro) against the 263K strain as compared to STERRAD NX System

Conclusions 1/2 Studies published in Infection Control & Hospital Epidemiology conducted by two independent laboratories demonstrate that STERRAD NX System hydrogen peroxide gas plasma technology was able to inactivate prions. The studies also found the STERRAD NX System was at least as effective, and in some conditions more effective, than conventional high-temperature steam methods. For example, the STERRAD NX System proved more effective than steam sterilization at 134 C/18 min. (prion cycle). The results of this study and other published tests prove that prion inactivation can be accomplished without the use of corrosive solutions and high temperatures

Instruction N DGS/RI3/2011/449 01/12/2011 Recommended procedures for non autoclavable medical devices

France : Standard Prion Protocol in vitro Surface + - - - - - - Suspension + + + + - - - in vivo Surface - Partial Performances Non effective Non effective > Partial < Reference Reference Partial > Partial < Reference Reference Performances Partial Elimination Total elimination Non effective Partial inactivation Total inactivation In vitro! Recommended for Screening and, in addition to in vivo, to explain mode of action and, for solutions, to differentiate between elimination and inactivation. In vivo! 2 strains : Hamsters / 263K scrapie strain + Mouse/infected bovine or human strain (mouseadapted 6PB1 BSE strain)! Used to efficacy of process by quantifying reduction of infectious load and comparing it with a minimum of 2 reference treatments : Total inactivation : NaOH Sodium hydroxide 1N / 1 hr at room T C Total inactivation : NaOCl Sodium hypochlorite 20 000 ppm /1 hr / Room T C Altough not reaching total inactivation steam 134 C / 18 min is accepted as a benchmark for «physical process» Partial activity : dilution or shorter exposure time of above processes (NaOH 0.1M 15 min. or 1 hr, NaOCl 2000 ppm or 5000 ppm 15 minutes)

Products approved for Total Inactivation Afssaps / April 2010

Conclusions 2/2 Prion risk, even low need to be considered and the standard procedures used daily to reprocess medical devices must include an inactivation phase if the device was in contact with high infectious risk tissues. The results of this study and other published tests prove that prion inactivation can be accomplished without the use of corrosive solutions and high temperatures

Grazie Mille