Engineering & Science Advisory
Committee into the Decontamination of
Surgical Instruments Including Prion
Removal (ESAC-Pr)
New Technologies Working Group
Report on Prion Inactivating Agents
Published August 2008
1
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Title
ESAC-Pr New Technologies Working Group Report on Prion Inactivating
Agents
Author
Dr Geoff Ridgway
Publication Date
01 Aug 2008
Target Audience
NHS Trust CEs, Foundation Trust CEs , Allied Health Professionals, Scientific
Research Facilities, Private Industry, Trust Decontamination Leads, and
Theatre Managers
Circulation List
Medical Directors, Directors of PH
Description
The report gives advice on the applicability of the various anti-prion
technologies that are on, or close to market as part of the surgical instrument
decontamination cycle.
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Contents
1.
Introduction
2.
The decontamination cycle
3.
The use of models for evaluation of prion inactivation
4.
Inactivation of TSE prions: general considerations
5.
The physical destruction or denaturation of prions
6.
The chemical inactivation or denaturation of prions
7.
Protein loads on surgical and dental instruments
8.
Detection of protein
9.
Requirements of a prion inactivating agent or process
for use in the decontamination cycle
10.
Incorporation of prion inactivating technologies into the
decontamination cycle
11.
Coatings and barriers
12.
Conclusions and recommendations for future research
13.
Conflicts of interest
14.
Acknowledgements
15.
References
3
1.
Introduction
In October 2007, ESAC-Pr commissioned a New Technologies Working
Group, charged with producing advice to the ESAC-Pr Industry and Scientific
Sub-Committee’s on the applicability of the various anti-prion technologies
that are on, or close to coming on the market, as part of the Surgical
Instrument Decontamination Cycle. The advice will subsequently be made
available to all involved in the decontamination of reusable medical
equipment, reflecting the growing interest in alternative methods to standard
decontamination procedures.
The guidance will be against the background of the SEAC recommendations
and be consistent with NICE Guidance (NICE 2006) and the 2006 report from
ESAC-Pr.
In summary, these require that data on the prion inactivation activity of a
product or method should:
•
•
•
•
quantify the reduction in the titre of infectivity as a result of the
overall decontamination process, when this is modified by the use
of the new technologies.
not be restricted to the use of hamster adapted scrapie prions, as
studies show that sporadic CJD and BSE prions may be more
resistant to conventional methods of decontamination than the
hamster scrapie model may indicate in some cases.
include evaluation by other bioassays including other animal
models such as mice as these provide the most relevant and
robust system to quantify the effectiveness of a decontamination
technology to remove or deactivate TSE infectivity.
In addition to evaluation of the effectiveness of new
decontamination technologies, it is important to establish that
these technologies do not compromise the removal of other
infectious agents or add residues that would affect patient safety.
The SEAC guidance also noted, “cell-based infection assays with a
sufficiently large dynamic range could, with further development, provide a
useful tool for preliminary assessment of the effectiveness of new
decontamination technologies prior to more extensive evaluation using
infectivity bioassays. Biochemical assays to measure PrPSc concentrations
are less useful as they may not accurately reflect the effect of a
decontamination treatment on infectivity owing to lack of sensitivity. However,
biochemical assays may be useful to examine the mechanism of action of a
decontamination treatment to monitor the performance of a previously
validated decontamination technology when in use in Sterile Service
Departments (SSDs)”. Presently although cell-based infection assays exist for
scrapie strains, widely used assays for BSE or human derived material are
not yet available.
4
The working group is required to advise on a recommended way forward
to utilise the available evidence to use such technologies as part of the
decontamination process for reusable medical equipment, to give
guidance on their incorporation into the decontamination cycle and to
advise on the direction of future research needs with regard to the
practical inactivation of prions in the decontamination environment.
Terminology
The terminology for the causal agents for the group of diseases, which include
scrapie in sheep and goats as the archetype, Bovine Spongiform
Encephalopathy (BSE) and Creutzfeldt Jakob Disease (CJD), has become
confusing. The term "prion" was originally derived from "protein infection" and
relates specifically to the hypothesis that the infectious agents of these
diseases is comprised solely of protein and exclude a nucleic genome. Its use
infers that the prion hypothesis is proven. Although there is a substantial
amount of evidence in support of the prion hypothesis, the hypothesis as it is
currently formulated fails to account fully for the diversity in properties of the
causal agents, including their differences in physical and chemical stability.
Other hypotheses may yet describe more successfully the structure of the
causal agents. Accordingly, some scientists in the field still prefer to describe
the group of diseases as the Transmissible Spongiform Encephalopathies
(TSE) and the causal agents as "TSE agents", as more neutral terms.
To avoid confusion, the term “prion” has been retained in this report.
2.
The Decontamination Cycle
The decontamination cycle for reusable medical equipment is defined as the
whole process from instrument acquisition, through preliminary processing,
disinfection, sterilization and distribution to the point of use. The process is
represented diagrammatically in Figure 1 below:
5
Figure 1
3.
The Use of Models for Evaluation of Prion Inactivation
This section reviews and assesses the TSE (prion) models available to
assess methods of TSE inactivation and their relevance to surgical instrument
sterilization. A priori, there are two aspects in choosing relevant models: those
that are most resistant to the inactivation method or those that are closest to
the sources of TSE of greatest concern (e.g. vCJD).
Available TSE animal models
A TSE model comprises a source of TSE infection and an appropriate
experimental animal host in which the TSE agent can be replicated and
assayed. TSE isolates vary in phenotypic properties (as with other infectious
organisms). These properties can and have been maintained on serial
passage and after high dilution “cloning” to provide a series of experimental
“TSE strains”, analogous to virus strains. In practice, the choice of model is
influenced by the length of incubation period and the titre of infectivity that can
be obtained in an infected tissue.
Animal models of TSE disease have been developed, primarily using
experimental mice but also using hamsters and experimental sheep flocks.
6
The host gene PrP is polymorphic in conventional experimental mice and two
alleles, which control relative incubation periods, have been identified. This
has allowed the phenotypic characterisation of many TSE strains that are
maintained by serial passage in a defined PrP genotype. Many experiments
have also been conducted with hamsters infected with the 263K strain of TSE
agent since the agent replicates to very high titres (up to 1010 intracerebral (ic)
ID50 per gram) and this model has shorter incubation periods than TSE
models in conventional mice. Changes in TSE agent properties may also
occur on passage in different PrP genotypes. Recently it has been found that
bank voles infected with sporadic CJD produce a model of disease with
relatively short incubation periods (Nonno et al., 2006) although they replicate
vCJD very poorly compared with models of TSE in conventional strains of
mice.
The development of transgenic technology in the last 10 to 20 years has
allowed the construction of mouse models, which contain PrP genes from
other species, either by homologous recombination to produce a direct
replacement of the endogenous mouse gene or by insertion of additional
genes to produce multicopy transgenic animals. Transgenic mice allow the
passage and assay of primary sources of TSE without the attenuating effects
of the species barrier at primary passage due to differences between species
in PrP protein sequence. Additional copies of the PrP gene shorten incubation
periods and can sometimes lead to shortened lifespan or other side effects.
An example of a useful model is that of multicopy transgenic mice carrying the
bovine PrP gene in which BSE has been assayed with a titre of 107.7 ic ID50
per gram and a minimum incubation period of 230 days (Buschmann &
Groschup, 2005).
There are a range of sources of TSE infection available. Primary sources of
infection include variant CJD (vCJD), sporadic CJD (sCJD) and various forms
of CJD associated with mutation of the PrP gene, (gCJD), from humans, BSE
from cattle, scrapie from sheep and goats and chronic wasting disease (CWD)
from three species of North American deer. When addressing questions
relating to a specific TSE (e.g. vCJD) it would be most appropriate to use it as
a source in experiments. However, the amount of material available may be
limited and its use may be ethically constrained. Moreover, the relevance to
other TSE sources may be poorly understood if the model has been poorly
characterised in comparison with other TSE models.
Available experimental models include mouse passaged strains 301C, 301V
and 6PB1 derived from BSE; ME7, 22C, and 22A derived from sheep scrapie;
and 79A and 139A derived from the drowsy goat source. RML also derives
from an early passage of the drowsy goat source so its phenotypic properties
are likely to be similar to 79A and 139A. These models are well developed
and their differential biological properties characterised. They can be used to
establish a range of physicochemical and biochemical properties. Hamster
passaged strain 263K (also derived from the drowsy goat source) has been
used in many inactivation experiments. The relevance of 263K to other TSE
agents is not established although it appears to be less resistant to
autoclaving than 301V in VM mice or 22A in VM mice (Fernie et al., 2007).
7
Non-rodent models are also available, such as non-human primates and
sheep. Although they can replicate many aspects of human disease, their cost
and the incubation times may prohibit their use.
TSE animal models: methodology
The delivery of test solutions is normally as serial dilutions of the sample,
starting at the highest concentration tolerated by the host. Intracerebral (ic)
injection is normally the most efficient route, but the route may be
compromised by the requirement to use smaller volumes than can be used
intraperitoneally (ip) and toxic components in the inoculums may have a
greater effect ic than by other routes. Intraperitoneal injection, although less
sensitive than ic, may be a better model for general surgery (Baxter, Campbell
et al 2005).
Single dilutions of inoculums can be used to measure incubation periods and
calculate the titre from dose response curves. Such measurements can be
compromised by changes in the dose response curve due to chemical
modification of the inoculums. For example for the same dose of infectivity,
heat treatments can often extend incubation periods. Shifts in dose response
curves after heat inactivation were first demonstrated by Dickinson et al
(1969) and subsequently by others, including Taylor et al., (2002) and Fernie
et al., (2007).
Although this approach reduces the number of animals per measurement, it
must be used with caution. At very low TSE agent concentrations, a single
large group of animals can be used. Such an approach has been used to
measure successfully small amounts of TSE infectivity in blood derived
samples (Gregori et al., 2004).
Recently, solid delivery supports e.g. stainless steel wires, steel spheres and
plastic wires have been implanted in the brain successfully, to test the amount
of TSE infectivity that can bind to surfaces and the degree to which infectivity
can be inactivated when bound (Flechsig et al., 2001; Baxter et al 2005;
Fichet et al., 2007a) . The wire method appears currently to be the best
standardised in vivo model for evaluating prion inactivation ability (Fichet et
al., 2007a). Spheres have the advantages of being easier to handle and they
can be implanted at several sites.
TSE models: In vitro models
Although it is generally recognised that abnormal forms of the host protein
PrP are closely associated with the TSE infectious agent, the relationship
between them is complex. In the 263K hamster model there are about 10,000
molecules of PrP per unit of TSE infectivity (this is about 0.6 femptogrammes
or 0.02 attamoles of PrPSc) and under certain conditions much of the PrP can
be separated from infectious agent. In addition, the biochemical properties of
PrPSc may not always correlate with the inactivation properties of infectious
agent (Barron et al., 2007). Accordingly, it may be helpful to conduct
preliminary inactivation experiments using in vitro PrPSc assays, it is essential
8
to validate findings with measurements of infectivity directly (i.e. using
bioassay) (Sutton et al., 2006).
Recent developments in the field include the discovery of an in vitro
conversion of PrP and amplification of the amount of PrPSc present in a
sample. This method is called protein missfolding cyclic amplification (PMCA)
(Jones et al., 2007). The relationship between PMCA products and TSE
infectivity is poorly understood. It may ultimately provide a method for
assaying TSE infectivity but it has yet to be validated for inactivation
experiments. It seems unlikely that PMCA will be useful on surfaces, and its
validity as an assay for instruments is therefore questionable. Cell assays of
infectivity are also showing promise (Mahal et al., 2007).
Summary (The Use of Models)
To measure TSE infectivity sensitively ultimately still requires animal assays
and TSE models that are most relevant to the problem should be selected.
The assays are based on assaying serial dilutions of sample, but incubation
period measurements can sometimes be used if no confounding problems
have been identified. The measurements are usually recorded on a
logarithmic scale; hence the assays are highly sensitive but are of limited
accuracy for measuring small biochemical changes.
The limit of detection is determined by the lowest dilution assayed. The
clearance of an experiment, i.e. the reduction in titre that can be
demonstrated for a process, is determined by starting titre of the untreated
sample and limit of detection. Only quantitative reductions in TSE infectivity
can be measured. There can be no measurement or guarantee of absolute
sterility as samples with no detectable infectivity may be just below the limit of
detection, hence there is no qualitative guarantee of removal of infectivity. An
emerging problem is determination of a correct end-point – death, clinical
disease or pathological changes in the brain. There is accumulating evidence
that clinically well mice can have CNS lesions. Thus, clinical disease rather
than death is a more applicable end-point.
The choice of model is constrained by that available and existing knowledge
about them. Ideally, they should model the problem to be addressed most
closely and/or exhibit TSE model characteristics with the most extreme
properties relevant to the processes to be assessed. Practical concerns about
their use include titre in model, availability, and safety issues. Finally, it is
helpful if appropriate quantitative risk assessments to determine the amount
of inactivation required have been performed to determine whether the model
can demonstrate the desired reduction in infectivity.
In selecting model TSE systems for use in evaluating TSE inactivation
experiments, it is important to ensure that the systems to be modelled are all
included. For example should the models only reflect the properties of primary
transmissions of vCJD, or should they also include secondary vCJD, and
other CJD infections as well? In the absence of a useable TSE model
9
identical to vCJD in humans, TSE models most likely to have high resistance
to the technology under test should be used. The range of inactivation
properties of TSEs to different mechanisms of inactivation requires further
examination.
4.
Inactivation of TSE prions: General Considerations
•
•
•
•
•
•
•
•
•
Removal of prions solely with water is not effective due to
hydrophobicity of the protein. Dilution in water may increase affinity for
surfaces.
BSE related TSE strains such as vCJD may be the most thermostable.
High pH acts synergistically with heat - but is it practical to use
routinely?
Methods using detergent inactivation and protease digestion look
promising. However, some enzyme preparations may increase
resistance to steam sterilization, whilst others show expected reduction
in risk, cumulative on sterilization.
There is a substantial risk that some inactivation methods whilst
reducing the titre will leave a residuum that is then very thermostable.
Inactivation protocols should be based on the most stable TSE models
(more data needed).
Dehydration and other protein fixation treatments may compromise
inactivation protocols.
The efficacy of inactivation protocols should be quantified and
compared to required degree of TSE inactivation, established by risk
assessment.
Hydrolysis may be a logical approach (McDonnell, 2007).
For the development of decontamination, processes there are three major
requirements: efficacy (to meet the need of the product), compatibility, and
safety. In addition, there are regulatory considerations. In the EU these may
include the various directives, such as Medical Devices, Detergents,
Machinery, Biocides and Registration, Evaluation and Authorisation of
Chemicals (REACH); (emphasis on chemical methods is on environmental
safety). Additional considerations include cost/time for using the product,
universal precautions (does it fit into current practices) and science issues
(e.g. water quality, test methods, the nature of the infectious agent etc). All
need to be balanced for successful adoption and practical use.
It is also important to understand not only the differences between the state of
the agent (e.g. liquid or gas), process control (temperature, time,
concentration etc.), but also water quality (as an example in cleaning),
formulation effects (defined as a combination of ingredients, including active
and inert ingredients, into a product for its intended use). Ultimately, one
needs to know how they affect the goal (e.g., a non-foaming cleaner that
removes all types of organic and potentially inorganic soils, can tolerate water
hardness, has broad spectrum compatibility, a fast cycle time and deactivates
prions).
10
Mechanisms of TSE Inactivation
In general, methods of TSE inactivation include destructive procedures which
include combustion (incineration), oxidation (e.g. with hypochlorite) and
hydrolysis (at extreme pH, or with proteases). Techniques that are likely to be
milder probably denature TSE agents and include heat when the sample is
hydrated and exposure to denaturing chemicals (e.g. strong detergents,
chaotropes). Variables that may affect the success of treatments include
biological properties such as TSE agent strain, inoculation route, host
genotype, and tissue type. The dynamics and kinetics of the inactivation
process may be affected by time of exposure, concentration of inactivating
chemicals, temperature, and pH.
5.
The Physical Destruction or Denaturation of Prions
This section reviews physical methods for the denaturation or destruction of
prions.
The following is a list summarising the mechanisms available for the removal
of prion infectivity. It should be noted that specific technologies may involve
one or more of these mechanisms.
Physical Destruction
• Combustion (incineration)
• Oxidation (e.g. hypochlorite, hydrogen peroxide, sodium hydroxide)
• Hydrolysis (e.g. extreme pH, proteases)
• Gas plasma
• Ozone
Denaturation
• Heat (hydrated - autoclaving): Dependent on TSE strain and host PrP
genotype. Limited by formation of highly thermostable forms of TSE
agent, e.g. by dehydration
• Chemical exposure (pH)
Dependent on TSE strain
May be limited by formation of pH-stable forms of agent
• Chemical
exposure
(detergents
e.g.
SDS
and
alkaline
detergents/proteases)
Concentration, pH and temperature dependent
Probably TSE agent dependent
In addition, when reviewing the activity of the various technologies against
prions, the following variables must be considered:
•
•
11
Biological: Agent strain, Host genotype, Inoculation route, Tissue,
(Tissue state).
Dynamics and kinetics: Time , Concentration, Temperature and pH
Heat Inactivation
Heat inactivation under hydrated conditions appears to follow second order
kinetics, hence there is little effect of time after initial exposure and the
inactivation curve is biphasic with respect to time (see original data: (Kimberlin
et al., 1983), and re-analysis (Somerville, 2002). An experiment in which 10
TSE models, comprising of five mouse passaged TSE agent strains in two
PrP genotypes of mouse showed a major difference in resistance to
autoclaving between TSE strains, which was little affected by PrP genotype
(Taylor et al., 2002). These results reflect the fundamental thermostability
properties of TSE models (Somerville, unpublished). An autoclaving
experiment with BSE and related sources has shown that after autoclaving at
134°C or 137°C for 3 minutes the BSE titre was reduced by an average for
BSE of 1.6 log ID50, (2 samples, vCJD = 2.8 log ID50, (4 samples) and 301V =
3.5 log ID50. (Fernie and Somerville, unpublished data). These and other data
show that BSE and related TSE sources have higher thermostability
properties than other TSE sources and in particular, they exhibit survival of
substantial amounts of infectivity after autoclaving. Varying the conditions of
autoclaving has little effect on the degree to which TSE agents were
inactivated. However, dehydration and tissue fixation can markedly increase
resistance to heat inactivation (Fernie et al., 2007), perhaps because the size
of heat-resistant subpopulations are increased by some physical or chemical
treatments.
Effect of Hypochlorite (bleach)
Recent results and surveys of the literature demonstrated that sodium
hypochlorite treatment markedly reduces TSE infectivity but TSE infectivity
may still be detectable. Increasing the concentration and time slightly
increases the degree of inactivation. However, initial studies with hypochlorite
indicate that it might not be as efficient as first thought. There are indications
of differences in TSE strain sensitivity to hypochlorite. pH may have a major
inactivating effect (pH of hypochlorite solution ≈ pH 12.5) - see below.
pH Inactivation
There is a synergistic effect of heat and high pH. In one experiment there was
a significant reduction in TSE infectivity with time at pH 13, but not at pH 12 or
lower (Somerville et al unpublished). Repeated use of alkali autoclaving does
not appear to damage good quality steel instruments, but removes all
detectable infectivity. Lower temperatures can achieve similar levels of
inactivation. Preliminary analysis indicated differences in sensitivity to high pH
between TSE strains at 20 °C, which correlates with the differential survival of
TSE infectivity in hypochlorite (Somerville et al unpublished). High
temperatures reduce the pH of TSE inactivation. It is possible that partial
inactivation by high pH (e.g. between pH 12 and 13 for 301V) may stabilise
TSE agents, e.g. increase resistance to autoclaving. Inactivation involving pH
is considered further under chemical inactivation below.
12
Sodium Dodecyl Sulphate (SDS) Inactivation
The kinetics of SDS inactivation appears to be monophasic and TSE strain
and/or PrP genotype dependent with time. Monophasic inactivation of 301V
with increasing temperature, in 1% SDS, at pH10 has also been observed.
Denaturing inactivation in solution using detergents could be optimised.
Electro-elution
Electro-elution is a novel method for protein removal from metallic surfaces. A
low current is passed between electrodes in a bath of sodium carbonate.
Experiments against a test soil of alcohol fixed porcine blood have shown that
the process efficiently removed soil form the surface of the test piece within 5
minutes (Plinston et al 2007). To date, prion studies have been restricted to a
scrapie derived agent with Western Blot analysis. The process could be
adapted as a cold pre-wash for surgical instruments. Further research with
BSE derived strains in animal models is anticipated.
Implications for CJD Inactivation
BSE related TSE strains e.g. vCJD may be more thermostable than other
TSE sources. However, qualitative problems for other TSEs, including other
types of CJD remain the same. High pH acts synergistically with heat, but
methods using hot alkali may not be practical to use routinely. However,
methods using detergent inactivation under mild alkaline conditions, possibly
combined with protease digestion may be worth further investigation (Sutton
et al., 2006).
Ideally, inactivation protocols should be tested on the most resistant TSE
models available, if they have been identified. The thermostability properties
of a range of TSE models have been characterised to some extent and form a
basis for the selection of appropriate models. However, much less information
is available for other mechanisms of inactivation, e.g. with high pH or strong
detergent. It would be desirable to compare inactivation in a range of TSE
models. Dehydration and other protein fixation treatments may compromise
inactivation protocols. The efficacy of inactivation protocols should be
quantified and compared with the required degree of TSE inactivation, as
established by risk assessment.
6.
The Chemical Inactivation or Denaturation of Prions
This section reviews the status of chemicals with defined prion inactivating
properties that are at or near to being placed on the market. For the European
market, this means that the product will require CE marking. However, CE
marking does not assess the scientific qualities of the product (see description
of CE Marking below).
13
General considerations
The use of chemical agents in the decontamination cycle must be assessed
against a background of thorough cleaning this being the underlying principle.
For each chemical product, the assessment for prion inactivating properties
must involve testing against more than one prion strain and, as noted above,
must involve more than one animal model. For reasons stated previously,
animal studies must not be restricted to the use of hamster adapted scrapie
prions.
The process of testing must be undertaken in a washer disinfector that has
satisfied both ISO 15883-1 and HTM 2030 requirements. Such testing will
require specialist containment facilities and experience of handling washer
disinfectors, prion agents and access to animal facilities. It is recommended
that GLP-compliant animal facilities are used to support the quality control of
data emerging from the studies. Whilst the study itself is not suitable to be run
to GLP, due to the nature of the spike materials, an enhanced quality system
with study protocol, locally controlled documents and audit protocols is
essential to support the study.
Consideration of use may require incorporation of the issue of wet (moist)
versus dry for surgical instruments (ESAC-Pr Autumn Report 2006). There is
evidence that scrapie infected brain tissue dried on surfaces is 1,000-10,000
fold more resistant to inactivation than wet tissue by heat based inactivation
methods. (Fernie et al 2007).
Common laboratory detergents such as SDS and CTAB removed some, but
not all, proteins. Commercial detergents such as Hamo 54 and Decon90 were
better, but SolidMetalPro removed more than 99.9% of detectable protein.
Commercially available thermostable enzymes can effectively reduce
infectivity without causing damage to instruments.
To date, the majority of products developed for prion inactivation have only
been used as a pre-soak. Work in hand includes Dupont currently looking at
the use of Rely On in an Ophthalmic Department, Steris investigating the use
of HamoTM 100 in their own washer disinfectors using the cleaning efficacy
requirements for both EN ISO 15883-1 (Anonymous 2005) and HTM 2030
(Anonymous 1997) and Dr Weigert evaluating neodisher SeptoClean in an
SSD.
The problems surrounding the soaking of instruments are detailed below. In
order to provide validated evidence that prion decontamination products will
be effective in washer disinfectors against prions then the only way of
achieving this is to actually undertake the investigations required.
It is apparent that there needs to be greater discussion between the
disinfectant product manufacturers, washer disinfector manufacturers and the
end users, particularly the Decontamination Leads and the Sterile Services
Managers. The overwhelming conclusion of these professionals is that using
14
these products, as a manual pre-soak is not a viable option in operating
departments or in SSDs. It is not possible to validate reliably the soaking of
instruments in open containers of chemical. Further, the question of
penetration of chemical into serrations and box joints cannot be guaranteed.
Therefore, it is vital that chemicals intended for this purpose are incorporated
into the existing decontamination cycle practices i.e. as part of the washer
disinfector process.
The following reviews the range, type and effectiveness of chemical and
biological products currently available commercially for the decontamination of
prions. The information included here is based on what is available in the
public domain and the manufacturers of each of the products were consulted
on the information that was initially presented.
The following products were reviewed by the Group:
1.
2.
3.
4.
5.
6.
7.
1.
Hamo™ 100 Prion Inactivating Detergent, Steris Corporation
Prionzyme M™ Genencor
Rely+On (TM) Prion inactivator, DuPont
neodisher SeptoClean, Dr Weigert
PRIOX™, InPro biotechnology
Deconex, Borer Chemie AG (Bellimed UK)
Ozone steriliser, TSO-3
Product: Hamo™ 100 Prion Inactivating Detergent, Steris Corporation
This product is supplied by Steris Ltd, UK (www.steris.com) and is composed
of an alkaline detergent. The laboratory work was carried out at CEA, France.
Methodology:
Tested in vitro (316 stainless steel coupons or glass slides - 20µl of a 10%
brain homogenate solution in PBS) using prion agents 263K (Fichet et al
2004), sCJD, vCJD and BSE 6PB1 (Fichet et al 2007a) with results assessed
by Western Blot analysis.
Tested in vivo, using prion agents 263K scrapie in hamsters (>5.6-log
inactivation) and BSE 6PB1 in mouse (>5.5-log inactivation), using 316
stainless steel wires and polypropylene wires (data not shown) (Fichet et al
2007b)
Product tested above as a manual pre-soak: Temperature > 43°C, pH10.5,
1.6%, 15 minutes or at 0.8% for 7.5mins.
Hospital trials: (Theatre Sterile Services Unit at Basingstoke and North
Hampshire NHS Foundation Trust) have been undertaken with Hamo T-21
washer disinfectors: pre-rinse with water < 45ºC for 30 seconds, wash at 43ºC
for 7 min (0.45 Hamo 100) (Fichet, Harrison and McDonnell 2007).
15
CE marked* - Yes
Rapid Review Panel¥ – not been submitted
2. Product: Prionzyme M™, Genencor International
This
product
is
supplied
by
Genencor
International,
USA
(www.genencor.com). The laboratory work was carried out under commercial
contract at the Health Protection Agency, Porton Down, Salisbury (HPA). The
product is composed of a single biological protease from Bacillus lentus.
Methodology:
The product has been tested in vitro using Western Blot detection
(suspension only) and in vivo using BSE-301V and VM mice (suspension
only); >7-log inactivation.
Application (from product label). Pre-soak: Temperature 60°C, pH 12 for 30
minutes.
CE marked* - Yes
Rapid Review Panel¥ – Recommendation 2
3. Product: Rely+On ™ Prion inactivator DuPont
This product is supplied by DuPont (www.relyon.dupont.com) and is
composed of proteases and SDS. The laboratory work was carried out at the
MRC Prion Unit (Jackson et al., 2005).
Methodology:
Pre-soak for 10 minutes at 50°C in a pH neutral solution.
The product has been tested in vitro using vCJD with Western Blot analysis.
The in vivo work has been carried out using RML scrapie in Tg20 mice.
Hospital trials: The product has been on trial at the Ophthalmic Department at
St Mary's Hospital Paddington for 3 months to gain user feedback as to
product handling and instrument compatibility - no results to date.
CE marked* - Yes
Rapid Review Panel¥ - Recommendation 3
4. Product: neodisher SeptoClean, Dr Weigert
This product is supplied by Dr Weigert and is a liquid alkaline cleaner (>pH10,
based on potassium hydroxide with surfactants) and the work was carried out
at the Robert Koch Institute, Berlin, Germany.
Methodology:
Tests carried out in vitro and in vivo with dilutions using 263K scrapie/hamster
(Baier et al 2004). Tests carried out in vitro using the 263K scrapie/hamster
model 0.5%, at 55°C for 5min and 1% at 60°C for 10-min (Lemmer et al.,
16
2004)]. In vivo tests with stainless steel wires using the 263K scrapie/hamster
model work are on going.
Application: Recommended for use in washer disinfectors and for rigid
endoscopes. 1%, at 55°C, 10-30 min, pH12.
Currently being considered for use by the Vernon Carus Super Centre at
Chorley, Lancashire.
The Notified Body for Medical Devices in Germany has approved the
declaration of destabilisation, inactivation and decontamination of prions for
neodisher SeptoClean at room temperature and at 55 – 60°C.
CE marked* - Yes
Rapid Review Panel¥ - not been submitted
5. PRIOX™, InPro biotechnology
This product, an acidic SDS, is supplied by InPro biotechnology, the work was
carried out by the Institute for Neurodegenerative Disease, San Francisco,
California.
Methodology:
Application (4% SDS plus 1% Acetic acid; pH 4.5) at 65°C or 121°C / 134°C
autoclaving validated against sCJD.
In vivo using the wire model with Sc237 in either hamsters or transgenic mice
TG7 and Tg 23372 (Peretz et al., 2006).
CE marked* - No
Rapid Review Panel¥ - not been submitted
6. Deconex, Borer Chemie AG (Germany), Bellimed (UK)
This is an alkaline product. The application is as 0.5%, 10 min, 70°C, pH >11
and has been tested in a custom made washer disinfector at SMP Gmbh,
Tubingen, Germany.
In vivo testing using stainless steel wires & 263K scrapie / hamster model
(Rosenberg et al 2005). Estimated 5-6 log reduction in infectivity.
CE marked* - No
Rapid Review Panel¥ - not been submitted
Other references e.g. Yan et al 2004, Yoshika et al., 2007, have described
other detergent or enzyme products and approaches.
*CE marking
CE marking is a declaration by the manufacturer that the product meets all the
appropriate provisions of the relevant legislation implementing certain
17
European Directives. CE marking gives companies easier access into the
European market to sell their products without adaptation or rechecking. The
initials "CE" do not stand for any specific words but are a declaration by the
manufacturer that his product meets the requirements of the applicable
European Directive(s).
¥ Rapid Review Panel
The Rapid Review Panel (RRP) has been convened by the HPA at the
request of The Department of Health. The panel provides a prompt
assessment of new and novel equipment, materials, and other products or
protocols that may be of value to the NHS in improving hospital infection
control and reducing hospital acquired infections. The panel will not conduct
evaluations of products but will review information and evidence provided and
makes recommendations to the Department of Health. It is not within the
remit of the RRP to evaluate clinically or undertake the evaluation of products
within the NHS. It is for the manufacturer to initiate and complete such
trials/evaluations. Furthermore, it is not within the remit of the panel to
influence procurement and the “uptake” of products into the NHS once
recommendations are formulated. The RRP is an independent arms-length
review panel that does not have a role in the procurement of products in the
NHS and does not “champion” specific products once evaluated.
The following recommendations may be made:
1. Basic research and development, validation and recent in use evaluations
have shown benefits that should be available to NHS bodies to include as
appropriate in their cleaning, hygiene or infection control protocols.
2. Basic research and development has been completed and the product may
have potential value; in use evaluations/trials are now needed in an NHS
clinical setting.
3. A potentially useful new concept but insufficiently validated; more research
and development is required before it is ready for evaluation in practice.
4a. Not a significant improvement on equipment/materials/products already
available which claim to contribute to reducing health care associated
infection; no further consideration needed.
4b. Unlikely to contribute to the reduction of health care associated infection;
no further consideration needed.
5. Insufficient clarity/evidence presented to enable full review of the product.
6. An already well established product that does not merit further
consideration by the Panel.
7. The product is not sufficiently related to infection control procedures to
merit consideration by the Panel.
Those products falling into the first recommendation will be considered for fast
tracking into the future work plans of the NHS Purchasing and Supply Agency
(PASA) and the National Institute for Clinical Excellence (NICE).
18
Other technologies
Hydrogen peroxide technologies
1. Hydrogen peroxide/gas plasma technology:
The production of hydroxyl and peroxide radicals following application of radio
frequency generated gas plasma to an injection of hydrogen peroxide in a
high vacuum produces an environment hostile to micro-organisms. This is the
principle of the SterradTM, marketed by Advanced Sterilization Product, a
Division of Johnson and Johnson. The SterradTM NX Advanced cycle machine
has been evaluated in vivo against the 263K scrapie hamster model, using
both brain homogenate and steel wires, and in vitro against the 6PB1 BSE
derived strain and a strain of vCJD. For both 2 and 4 injections of hydrogen
peroxide significant inactivation of infectivity was obtained in both in vivo and
in vitro experiments. The SterradTM, 100NX was tested only in vitro but similar
results to the SterradTM, NX model were obtained and the SterradTM, 100NX
employs the same cycle technology as the SterradTM, NX with a larger
chamber (Legros, Ridgway, and personal communication).
2. Vapour phase hydrogen peroxide under vacuum (instrument
decontamination)
Fichet et al (2007) describe the use of vapour phase hydrogen peroxide under
vacuum in a dedicated chamber, for the decontamination of reusable medical
equipment. Activity was evaluated in vitro and in vivo (steel wires) with the
263K hamster scrapie model and with the 6PB1 BSE strain and the TGB1
variant mouse model. Vapour phase hydrogen peroxide was completely
effective with both a three phase and six phase injection. In contrast, liquid
phase hydrogen peroxide was not effective.
Ozone
This product is supplied by TSO-3, Quebec, Canada (www.tso3.com). The
laboratory work is being carried at the HPA under contract to the Department
of Health. The 125L Ozone Sterilizer is a low-temperature, high capacity,
general purpose sterilizer designed for use in all medical facilities. This
reliable, easy-to-use system provides safe, efficacious sterilization of highdemand heat and moisture-sensitive instruments. The 125L is being assessed
for it’s capability to reduce the infectivity of prions.
Previous in vitro work has been carried out at CEA, France using RML strain.
The in vivo work is being carried out using surgical steel wires with BSE-301V
and VM mice and results will be published in due course (Hesp et al., 2007).
Gas plasma technologies
Radio frequency gas plasma for instrument cleaning
Two different gas-plasma technologies are relevant to decontamination: Low
pressure plasmas (Baxter et al., 2005; 2006) (< 3 Torr), and atmospheric
pressure plasmas (Yu et al., 2006). Only the former has been validated
against a TSE agent, the 263K scrapie agent in vivo.
19
Gas plasmas are used successfully to clean complex industrial surfaces and
initial work has shown that they can remove all detectable organic material
from stainless steel surfaces. Under suitable conditions, atmospheric plasmas
can penetrate lumens, but the efficacy of this for the removal of biological
material has yet to be accessed.
The current status of RF Gas-Plasma Methodology indicates that:
• Contamination can be removed to less than 1pg/mm2.
• Reprocessed surgical instruments can be efficiently cleaned by RF gas
plasma.
• The efficacy of protein removal is 1000 - 10,000 fold better than that
achieved by current SSD procedures and enzymatic procedures
(Baxter, Campbell, Richardson et al., 2006).
• There is no evidence of damage to the instruments.
• The process for removal of TSE infectivity can be validated. In vivo
assays of TSE infected brain tissue adsorbed on stainless steel and
treated with RF gas-plasma show elimination of TSE infectivity.
• In the Sterile Services context, gas-plasma decontamination would be
a finishing process – to reduce contamination to sub nanogram
amounts, and would likely be placed between washing and steam
sterilization and might only be applied to high-risk instruments.
• By measuring the amount of CO2 and other gases coming off the
instrument, it may be possible to determine when the end of the
process has been achieved, although how practical this will be in
practice remains to be determined.
• Plasma technologies have the additional advantage that they can
penetrate narrow lumens and can destroy organic material. However,
the extent to which penetration of lumens is effective is the subject of
further discussion for both atmospheric and low pressure gas plasma
systems.
• There are three research groups currently in this field, two of which
already have links with commercial companies.
7.
Protein Loads on Surgical and Dental Instruments
Between 2 and 60 mg of tissue are deposited on surgical instruments after
use. Some instruments have over 1 mg attached, and all tonsillectomy
instruments examined had >0.5 mg attached. Most processed surgical
instruments had 0.2 - 0.5mg of extractable protein attached to them (Baxter,
Baxter et al., 2006, Murdoch et al., 2006; Lipscomb, et al., 2006).
In a recent assessment of dental instruments, protein residues left after
cleaning (manual, manual combined with ultrasonic and also washer
disinfectors) were highly variable from instrument to instrument, regardless of
type (range 0 - 3.8 mg of protein per instrument). This variability was reduced
somewhat by use of an automated washer disinfector, but by no means
eliminated (Bennett et al., 2007).
20
8.
Detection of protein
One fundamental problem is that some methods only work in solution. The
relevance of a solution method to measuring surface contamination is doubtful
in that it assumes that everything can be removed for analysis. Tautologically,
were this the case, then the problem of surface contamination would not exist.
However, the following points have emerged from the Group’s deliberations:•
•
•
•
•
•
•
9.
Ninhydrin is of little value.
Readily available dyes such as orthophthalaldehyde (OPA) can detect
50ng of protein, in solution.
High sensitivity ELISAs can detect 5-10 pmoles/mm2. This is a solution
technique, and although it has been used on surfaces, it is relatively
insensitive in this situation.
Episcopic differential interference contrast (EDIC) microscopy, a
surface technique, can detect single “infectious units” containing 1 pg
protein/mm2 and can “see” in 3D. This technique has been used on
instruments.
Fluorescein based technologies can detect 100 attomoles/mm2 or less
on stainless steel. This technique has been used on instruments.
(Richardson, Jones, Baxter et al., 2004).
Magnetic acoustic resonance spectrometry (MARS) can detect 10-20
pg/mm2 on steel, glass and quartz. This is a surface technique
presently evaluated only in model systems.
Several other novel technologies are being developed which in theory
can detect single protein aggregates.
Requirements of a prion inactivating agent or process
for use in the decontamination cycle
Before a product or process can be considered for use in a conventional
decontamination process, the following questions need to be addressed.
1. Is this product compatible with our decontamination equipment?
2. Has this product been validated for use with typical decontamination
protocols?
3. Does the product leave residues on processed equipment? Has the
toxicity of possible residues been established in relevant tissues?
4. What range of medical equipment and construction materials are
compatible with the product?
5.
What is the in-use stability of the product? Are formulation
characteristics lost over time (i.e. pH change, reduced detergency) and
what effect will this have on activity (this would require capacity tests to
have been done)?
6. How robust is product efficacy to variations in practice, e.g. type of
water used, dilution, and temperature?
7. Has the product been tested under typical conditions of soiling (which
would include dried tissue) using HTM 2030 approved soils?
21
10.
Incorporation of Prion Inactivating Technologies into the
Decontamination Cycle
There are a number of factors to consider when looking at this issue.
Compatibility with current equipment
The current operating protocols of a SSD, and more importantly the current
types of washer disinfectors being used must be taken into account. The first
question that needs to be addressed with regard to incorporation of chemical
agents is will the manufacturers of both the “new technology” and the washer
disinfectors actually work together for compatibility issues.
In respect of “manual cleaning” there are very few instruments solely being
cleaned using manual cleaning currently within SSDs, the process having
virtually been eliminated with the introduction of validated automated cleaning
and disinfection cycles. This needs to remain the situation. There is no place
in the modern decontamination cycle for pre-soaking of instruments in trays.
Where any manual procedure is involved, for example, in flexible endoscope
reprocessing, it must be followed by processing in a washer disinfector such
as an automated endoscope reprocessor. Universal precautions should apply
and all equipment should be processed via a validated, automated process.
New processes will require close monitoring and recording. Estates and
Engineering staff, together with local Infection Control personnel need to be
involved from the outset at a local level when introducing new systems. Clear
understanding and thorough knowledge of the process is key to a successful
system.
Wet (moist) versus dry
The issues around wet (moist) versus dry need to be considered although
there is some evidence to prove that spraying instruments with for example an
enzymatic solution will aid the cleaning process. Orthopaedic instrumentation
is a good example of where this type of system is being used at present. One
solution to this problem, relevant to ophthalmic and ENT surgery, has been
proposed (Crispin, Ridgway, and personal communication). This is to use a
purpose designed, validated bench-top washer disinfector (e.g. Dawmed) in
the theatre area to process the instruments immediately after surgery.
Instruments (in their trays) would be tracked through the washer disinfector in
closed containers, and then tracked to the SSD for inspection packing and
sterilization by autoclaving. Such a system would solve the need to ensure
that instruments such as phaecoemulsifiers did not dry out between
completion of surgery and commencing the decontamination process.
Synergy
When two or more processes are utilised to inactivate or remove prions, it is
unclear whether the log10 reductions in TSE infectivity of the constituent parts
are cumulative or not (synergy). DH risk assessments assume that multiple
steps accumulate with the previous step, for example, a 2 log10 from the
22
washer disinfector stage combined with a 2 log10 reduction after autoclaving
should in theory lead to an overall 4 to 5 log10 reduction. There is to our
knowledge no scientific evidence to support this theory, which would appear
to be fundamental to confirm the overall efficacy of the decontamination cycle.
Further, it is not known what contribution the introduction of novel
interventions, for example chemicals with a prion inactivating profile, will be.
The potential problem of antagonism by components of the decontamination
cycle has also not been addressed.
Once technologies have been proven within an automated process
incorporation into an operational SSD would be comparatively straightforward.
The concerns are –
• What happens if something goes wrong?
• Can we afford for it to go wrong?
• How will every cycle and instrument cleanliness be monitored?
• Who will validate the systems?
Trial sites will be needed to look at the implications, practicalities and
improvements with any introduction of a new technology. The Institute of
Decontamination Science (IDSc) as the Professional Body for the
decontamination speciality should be closely involved with disinfectant
suppliers and washer disinfector manufacturers in any trials. The IDSc can
use the technology, monitor the process, and gather and provide data
together with contributing to the ongoing discussions.
11.
Coatings and barriers
A simple, cheap and easy to use disposable barrier for protecting tonometer
heads has been developed and is scheduled to enter a full-scale clinical trial
later this year. Research on diamond-like coatings has started, whilst
research on photocatalytic coatings has shown them to be effective in
principle.
12.
23
Conclusions and recommendations for future research
•
In vitro models versus biochemical assays of infectivity: research
into the use of assays not requiring live animal assays, but relevant to
human infection by prions is to be encouraged. The relationship
between protein missfolding amplification and TSE infectivity and the
possible role for assaying TSE infectivity also requires further study.
•
Inoculation of animal models: presently, the most standardised
method for inoculation of animal brains that closest resembles surgery
involves material bound to stainless steel wires. Further work is
required to confirm the dynamics of material transferred in this manner,
and the use of different materials, which may be used in the
manufacture of reusable instruments. However, the low volumes of
inoculums that can be transferred by this method may limit its
sensitivity.
•
End-point determination: there can be no qualitative measure of
sterility. All that can be demonstrated is the amount of infectivity
removed or destroyed to the limit of detection of the assay (clearance).
Risk assessment should estimate the clearance to be achieved.
The correct end-point for assays of products or procedures against
TSEs in animals is the detection of TSE specific pathological changes,
irrespective of whether or not the animals show clinical signs of
infection.
24
•
Animal models: the choice of animal model is constrained by those
available and existing knowledge about them. In selecting model TSE
systems for use in evaluating TSE inactivation experiments, it is
important to ensure that the systems to be modelled are all included.
TSE models most likely to have high resistance to the technology
under test should be used. The range of inactivation properties of TSEs
to different mechanisms of inactivation requires further examination.
•
Inactivation of prions: the evaluation of any method (chemical or
physical) intended for prion decontamination must include
consideration of efficacy, compatibility and safety aspects. In addition,
biological (agent strain, inoculation route, host genotype, tissue),
dynamics, and kinetics (time, concentration temperature and pH) must
be considered when reviewing the activity of technologies for prion
decontamination.
•
Physical inactivation of prions: data reviewed has demonstrated that
BSE and related TSE sources have higher thermostability properties
than other TSE sources. In particular substantial infectivity may remain
after autoclaving. The addition of hydroxide to the autoclaving process
does not appear to damage good quality stainless steel instruments,
but does remove all infectivity. This technology requires further
investigation to confirm efficacy and practicality. Further work is
required to examine the relationship between high pH and heat and to
determine whether partial inactivation at higher pH may stabilise the
TSE agents. Methods using detergent inactivation under mild alkaline
conditions, possibly combined with protease digestion may be worth
further investigation.
•
Chemical inactivation: these agents must be evaluated against the
background of thorough pre-cleaning. Animal models used must not be
restricted to the use of hamster adapted scrapie prions and should be
expanded to include BSE derived strains.
•
Protein detection: Ninhydrin has little value in being able to detect low
levels of protein on surfaces that would be required for consideration of
prions and should not be used for monitoring decontamination of
reusable instruments. Further work is needed to determine a method
that combines both optimal sensitivity with practicality of use in the
SSD setting as it is clear that visual inspection is not appropriate.
•
Synergy: there is an urgent requirement for DH to seek tenders for
research into the question of synergy or antagonism by the
components of the decontamination cycle. Such studies should include
relevant animal models as well as using markers for detecting residual
protein, and should involve a standardised protocol to allow direct
comparison between groups. Such a standardised system will also
facilitate studies on the efficacy of novel interventions into the
decontamination cycle.
•
Incorporation
of
prion
inactivating
agents
into
the
decontamination cycle: it is essential that any process deemed to be
effective in the deactivation or removal of prions is fully evaluated for
use within the sterile services setting before becoming widely
recommended.
13.
Conflicts of interest
Dr Geoff Ridgway is a consultant to the Infection and Blood Policy Unit at the
Department of Health. He has also acted as a consultant to Eschmann
Equipment, Dawmed, The Steris Corporation and Advanced Sterilization
Products (a Division of Johnson and Johnson) for which services he has
received remuneration. He is a member of the Rapid Review Panel.
Prof Robert Baxter consults with Plasma-Etch Inc (Carson City, Nevada). He
does not receive remuneration for this role. Edinburgh University holds
patents on RF gas plasma decontamination technology.
Prof Stephen Denyer received financial support for his Research Unit from
The Steris Corporation and Advanced Sterilization Products (a Division of
Johnson and Johnson). He is a member of the Rapid Review Panel.
Dr G McDonnell is a member of the Association of British Healthcare
Industries (ABHI) and an employee for Steris Ltd.
Prof Robert A Somerville has recorded no conflict of interest.
Dr John Stephenson has recorded no conflict of interest.
Mr Nigel Tomlinson has recorded no conflict of interest.
Dr James Walker is employed by the Health Protection Agency (HPA). The
HPA generated the intellectual property associated with the product
Prionzyme that is being commercialised by Genencor International. HPA has
carried out research on a commercial basis for Genencor International. No
members of the group receive any financial benefit from license fees or
25
royalties. The HPA are currently testing the TSO3 Ozone Sterilizer in an
ongoing study that is funded by the Department of Health.
Mr Martin Williams has recorded no conflict of interest.
14.
Acknowledgements
Dr Geoff Ridgway (Chair)
Prof Robert Baxter
Prof Stephen Denyer
Dr G McDonnell
Prof Robert A Somerville
Dr John Stephenson
Mr Nigel Tomlinson
Dr James Walker
Mr Martin Williams
New Technologies Working Group membership
Dr Geoff Ridgway (Chair)
Mr Nigel Tomlinson
Mr Ken Holmes
Ms Sally Wellsteed
Dr Peter Bennett
Dr John Stephenson
Mr John SP Lumley
Prof Don J Jeffries
Dr Sarah Senior
Dr Mike Painter
Dr Jimmy Walker
Mr Robert Parkes
Mr Allan Hidderley
Prof David Perrett
Prof Stephen Denyer
Ms Melanie van Limborgh
Mr Martin Williams
Prof Robert Baxter
Prof Robert Somerville
15.
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