Efficacy Against Infectious Prions in Instrument Reprocessing
By Dieter Rudin
A2005 study by Urs Rosenberg, PhD,1 addressed the effectiveness of cleaning processes in instrument washers, and at the same time, also explored the issue of prion contamination and decontamination.
Bovine spongiform encephalopathy (BSE), also known as Mad Cow disease or Scrapie in animals and variant Creutzfeldt Jakob disease (vCJD) in humans, is caused by prion proteins which are a so-called transmissible spongiform encephalopathies (TSEs).
Originating from English stock animals, the BSE crises spread into Western Europe, and today there have been verified cases in the United States and in Canada.
Contaminated bovine tissue BSE have transferred to humans, creating a new human TSE called variant CJD or simply vCJD. The accumulation pattern of infectious prion protein (PrPSc) in vCJD is different from the one in CJD; these vCJD prion proteins are found in the brain, lymph system, muscle, and blood, as well as other parts of the body.
Because of the pervasive distribution of these infectious proteins and the long incubation time of the disease, reprocessing of surgical instrument has been identified as a risk factor for nosocomial transmission of vCJD. Research has shown that the agent of the vCJD disease, an infectious prion protein, is extremly resistant to todays sterilization methods; therefore, the argument, It does not matter if instruments are 100 percent clean, as they will be sterilized, is definitely no longer valid. Today, we understand the washing process is fully as important as the sterilization process.
Historically, instrument washers were developed from commercial dishwasher technology and adapted to todays sciencebased requirements. Todays standard washing processes require increasingly sophisticated soaps. Detergents, which are used in these processes, can be relatively mild, with a pH in the neutral range or they may be more aggressive, with values in the alkaline range of the pH scale. Depending on the type of detergent, the cleaning temperature is usually chosen at around 40 degrees Celsius to 50 degrees C, or 60 degrees C to 70 degrees C, respectively. A number of hospitals and surgical centers are also using an enzymatic approach for instrument reprocessing.
The early prion inactivation approach, using a high concentration of sodium hydroxide solution or sodium hypochlorite combined with long hold times, is generally lethal for medical instruments and washers. Recently, researchers have been looking for less destructive methods to decontaminate medical devices potentially contaminated with prions.
In his study, Rosenberg addressed the following three questions:
- Which process sequence assures the best cleaning results, using an alkaline cleaning method?
- How can the effectiveness of neutral or mild cleaning processes be enhanced?
- Is it possible to achieve efficacy against prions under routine process conditions, and does a good cleaning performance also mean good efficacy against prions?
Rosenberg studied washing efficacy by using three different test models:
- Washing experiments in a washer, using porous sintered stainless steel discs (spongelike, three-dimensional structures) contaminated with coagulating artificial blood. Analysis was accomplished by weighing these process challenge devices (PCD) before and after the cleaning process.
- Immersion experiments using stainless steel sheets contaminated with a coagulating artificial blood under conditions that simulate a process in a washer-disinfector, e.g. temperature profile (no isothermal conditions, heating rate comparable to that of a WD). Analysis was by visual inspection/photography.
- Washing experiments in a WD using Crile clamps (haemostatic forceps). The hinges of these instruments were soiled with coagulating sheep blood traced with the short-lived radioisotope Technetium 90. Analysis was accomplished by measuring residual radioactivity after the cleaning process.
The results of all three test methods were comparable:
- The alkaline detergent deconex 28 ALKA ONE with a working solution pH of around 11 provided the best cleaning results when the temperature plateau of the cleaning step was set at 90 degrees C.
- Processes with a 70 degrees C temperature plateau provided inferior results and even worse results for processes with 55 degrees C. This means that these two cleaning processes (see Figure 1) did not reach the full potential of the detergent.
In addition to the performance of alkaline cleaning processes, Rosenberg also studied a two-component cleaning system in the neutral pH-range with the same test methods (see Figure 2a and 2b).
This detergent consisting of two components, a base detergent, deconex TWIN BASIC, and a enzyme preparation, deconex TWIN ZYME, demonstrated a cleaning performance similar to a 90 degree C alkaline process. The temperature plateau of this new enzymatic, two-component process, however, was only at 55 degrees C. This test required a double injection system for detergents which allows injecting two detergents during the same wash cycle.
Rosenberg also studied the effect of the same detergents on the infectious prion proteins. The experimental approach was twofold. First, different concentrations of detergent at different temperatures (in vitro suspension assay) were left to act on the prions in a brain extract of test animal infected with Scrapie 263K. The second consisted of stainless steel wires, contaminated with infectious brain extract, that were subjected to a decontamination process resembling cleaning conditions in a washer-disinfector.
In case of the in vitro suspension assay, the effect of detergent treatment was assessed by the proteinase K/Western Blot method (see Figure 3). The extracts thus treated were separated on an acrylamide gel after undergoing no further treatment () and after additional treatment with proteinase K (+). Western blot was then performed for immunological detection of prion protein (black bands).
This method showed that the infectious prion protein (PrPSc) resistant to proteinase K (PK) while the cellular (non-infectious) prion protein (PrPC) is degraded by PK. In a treatment (e.g., with detergent) the structure of PrPSc is being altered in such a way that the molecule becomes sensitive to PK, the modified or destabilized prion protein is no longer infectious. In brief, the in vitro suspension assay looks at PK-sensitivity of prion protein in detergent-treated infectious brain homogenates.
The following are the results of the in vitro experiments:
Treatment of the brain extract with the alkaline detergent deconex 28 ALKA ONE either at 0.5 percent (pH 11.1)/10 minutes/70 degrees C or at 1.0 percent (pH 11.5)/10 minutes/ 55 degrees C rendered the infectious PrPSc sensitive to proteinase K.
If the alkaline detergent deconex 28 ALKA ONE at the destabilizing concentration (1.0 percent) and temperature (55 degrees C) was combined with the enzyme preparation of the two-component neutral detergent system, PrPSc was already degraded before PKtreatment. Thus, the proteases in the enzyme preparation had the same effect as PK. Treatment with the lower concentration (0.5 percent) of alkaline detergent at the lower temperature (55 degrees C) could not render PrPSc sensitive to PK. This proved the key to successful destabilization of the prion protein is in choosing the right combination of pH (concentration of detergent) and temperature. A high pH allows for a lower temperature and a high temperature allows for a lower pH. However, there seems to be a lower limit of alkalinity concerning the prion destabilizing effect. For the formulated detergent deconex 28 ALKA ONE this limit is at about pH 11. For pure potassium hydroxide (caustic potash), this limit seems to be above pH 12. This means that the formulated detergent is more effective than raw material.
In addition to the in vitro experiments, Rosenberg described infection assays using the animal Scrapie model test. For these experiments, very small stainless steel wires were contaminated with infectious brain homogenate. A uniquely constructed apparatus functioning like a spray system in an instrument washer served to decontaminate the wires. After decontamination, these wires were implanted into the brain of healthy test animals. The number of days after implantation, during which a test animal was living without symptoms of the TSE disease, was taken as a measure for the reduction of infectious entities on the wire surface. Every test animal with non-decontaminated implants showed the disease after about 80 days. All test animals with implants after decontamination with deconex 28 ALKA ONE either at 0.5 percent/10 minutes/ 70 degrees C or at 1.0 percent/10 minutes/55 degrees C, survived for more than 277 days without any symptoms of the disease.
Based on infection assays with a dilution series of brain homogenate, the reduction factor which can be allocated to the described decontamination processes with deconex 28 ALKA ONE, is greater than 106. Rosenbergs study can be described in three points. The same process parameters that will provide the highest alkaline cleaning results of surgical instruments will also destabilize prion protein and help to remove them from stainless steel surfaces. Simplified, a process with 0.5 percent deconex 28 Alka One and 10 minute cleaning at 70 degrees C or even better 90 degrees C (5 minute hold time) can be used as a routine measure. In addition to its efficacy against prions, it also produces excellent cleaning results. The described and effective processes are not restricted to only very specific applications (e.g., decontamination of instruments for brain surgery after an intervention in a CJD patient).
These processes can now routinely be used in every washer-disinfector for cleaning all alkali-tolerant instruments. They are in fact used already in many European hospitals. So far, no investigation has yet been able to show the efficacy against prions of a routine cleaning process at a neutral, mildly alkaline pH or enzymatic approach. In contrast, efficacy against prions could be expected only from an alkaline process using a formulated detergent with a high pH value (greater than 11) and at a high temperature setting (greater than 70 degrees C).
For reprints of the 13-page Rosenberg study, go to: www.sonitol.com
Dieter Rudin is president of Sonitol, Inc.
1. Rosenberg U. Effective cleaning processes and efficacy against prions. Zentral Sterilization. 2005; 13 (4): 258-270.