Infection Control Today: case study

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, andat the same time, also explored the issue of prion contamination anddecontamination.

Bovine spongiform encephalopathy (BSE), also known as Mad Cow disease orScrapie in animals and variant Creutzfeldt Jakob disease (vCJD) in humans, iscaused by prion proteins which are a so-called transmissible spongiformencephalopathies (TSEs).

Originating from English stock animals, the BSE crises spread into WesternEurope, and today there have been verified cases in the United States and inCanada.

Contaminated bovine tissue BSE have transferred to humans, creating a newhuman TSE called variant CJD or simply vCJD. The accumulation pattern of infectious prion protein (PrPSc) in vCJD isdifferent 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 thelong incubation time of the disease, reprocessing of surgical instrument hasbeen identified as a risk factor for nosocomial transmission of vCJD. Researchhas shown that the agent of the vCJD disease, an infectious prion protein, isextremly resistant to todays sterilization methods; therefore, the argument,It does not matter if instruments are 100 percent clean, as they will besterilized, is definitely no longer valid. Today, we understand the washingprocess is fully as important as the sterilization process.

Historically, instrument washers were developed from commercial dishwashertechnology and adapted to todays sciencebased requirements. Todaysstandard washing processes require increasingly sophisticated soaps.Detergents, which are used in these processes, can be relatively mild, with a pHin the neutral range or they may be more aggressive, with values in the alkalinerange of the pH scale. Depending on the type of detergent, the cleaningtemperature is usually chosen at around 40 degrees Celsius to 50 degrees C, or60 degrees C to 70 degrees C, respectively. A number of hospitals and surgicalcenters are also using an enzymatic approach for instrument reprocessing.

The early prion inactivation approach, using a high concentration of sodiumhydroxide solution or sodium hypochlorite combined with long hold times, isgenerally lethal for medical instruments and washers. Recently, researchers havebeen looking for less destructive methods to decontaminate medical devicespotentially contaminated with prions.

In his study, Rosenberg addressed the following three questions:

• Which process sequence assures the best cleaning results, using analkaline cleaning method?

• How can the effectiveness of neutral or mild cleaning processes beenhanced?

• Is it possible to achieve efficacy against prions under routineprocess conditions, and does a good cleaning performance also mean good efficacyagainst prions?

Rosenberg studied washing efficacy by using three different test models:

• Washing experiments in a washer, using porous sintered stainlesssteel discs (spongelike, three-dimensional structures) contaminated withcoagulating 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 witha coagulating artificial blood under conditions that simulate a process in awasher-disinfector, e.g. temperature profile (no isothermal conditions, heatingrate comparable to that of a WD). Analysis was by visual inspection/photography.

• Washing experiments in a WD using Crile clamps (haemostaticforceps). The hinges of these instruments were soiled with coagulating sheepblood traced with the short-lived radioisotope Technetium 90. Analysis was accomplished by measuring residualradioactivity after the cleaning process.

The results of all three test methods were comparable:

• The alkaline detergent deconex 28 ALKA ONE with a working solutionpH of around 11 provided the best cleaning results when the temperature plateauof the cleaning step was set at 90 degrees C.

Processes with a 70 degrees C temperature plateau provided inferiorresults and even worse results for processes with 55 degrees C. This means thatthese two cleaning processes (see Figure 1) did not reach the full potential ofthe detergent.

In addition to the performance of alkaline cleaning processes, Rosenberg alsostudied a two-component cleaning system in the neutral pH-range with the sametest methods (see Figure 2a and 2b).

This detergent consisting of two components, a base detergent, deconex TWINBASIC, and a enzyme preparation, deconex TWIN ZYME, demonstrated a cleaningperformance similar to a 90 degree C alkaline process. The temperature plateauof this new enzymatic, two-component process, however, was only at 55 degrees C.This test required a double injection system for detergents which allowsinjecting two detergents during the same wash cycle.

Rosenberg also studied the effect of the same detergents on the infectiousprion proteins. The experimental approach was twofold. First, different concentrations ofdetergent at different temperatures (in vitro suspension assay) were left to acton the prions in a brain extract of test animal infected with Scrapie 263K. Thesecond consisted of stainless steel wires, contaminated with infectious brainextract, that were subjected to a decontamination process resembling cleaningconditions in a washer-disinfector.

In case of the in vitro suspension assay, the effect of detergent treatmentwas assessed by the proteinase K/Western Blot method (see Figure 3). The extracts thus treated were separated on an acrylamide gel afterundergoing no further treatment () and after additional treatment withproteinase K (+). Western blot was then performed for immunological detection ofprion protein (black bands).

This method showed that the infectious prion protein (PrPSc) resistant toproteinase K (PK) while the cellular (non-infectious) prion protein (PrPC) isdegraded by PK. In a treatment (e.g., with detergent) the structure of PrPSc isbeing altered in such a way that the molecule becomes sensitive to PK, themodified or destabilized prion protein is no longer infectious. In brief, the invitro suspension assay looks at PK-sensitivity of prion protein indetergent-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 ALKAONE 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 toproteinase K.

If the alkaline detergent deconex 28 ALKA ONE at the destabilizingconcentration (1.0 percent) and temperature (55 degrees C) was combined with theenzyme preparation of the two-component neutral detergent system, PrPSc wasalready 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 atthe lower temperature (55 degrees C) could not render PrPSc sensitive to PK.This proved the key to successful destabilization of the prion protein is inchoosing the right combination of pH (concentration of detergent) andtemperature. A high pH allows for a lower temperature and a high temperature allows for alower pH. However, there seems to be a lower limit of alkalinity concerning theprion destabilizing effect. For the formulated detergent deconex 28 ALKA ONEthis 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 ismore effective than raw material.

In addition to the in vitro experiments, Rosenberg described infection assaysusing the animal Scrapie model test. For these experiments, very small stainlesssteel wires were contaminated with infectious brain homogenate. A uniquelyconstructed apparatus functioning like a spray system in an instrument washerserved to decontaminate the wires. After decontamination, these wires wereimplanted into the brain of healthy test animals. The number of days afterimplantation, during which a test animal was living without symptoms of the TSEdisease, was taken as a measure for the reduction of infectious entities on thewire surface. Every test animal with non-decontaminated implants showed thedisease after about 80 days. All test animals with implants afterdecontamination with deconex 28 ALKA ONE either at 0.5 percent/10 minutes/ 70degrees C or at 1.0 percent/10 minutes/55 degrees C, survived for more than 277days without any symptoms of the disease.

Based on infection assays with a dilution series of brain homogenate, thereduction factor which can be allocated to the described decontaminationprocesses with deconex 28 ALKA ONE, is greater than 106. Rosenbergs study can be described in three points. The same processparameters that will provide the highest alkaline cleaning results of surgicalinstruments will also destabilize prion protein and help to remove them fromstainless steel surfaces. Simplified, a process with 0.5 percent deconex 28 Alka One and 10 minutecleaning at 70 degrees C or even better 90 degrees C (5 minute hold time) can beused as a routine measure. In addition to its efficacy against prions, italso produces excellent cleaning results. The described and effective processesare not restricted to only very specific applications (e.g., decontamination ofinstruments for brain surgery after an intervention in a CJD patient).

These processes can now routinely be used in every washer-disinfector forcleaning all alkali-tolerant instruments. They are in fact used already in manyEuropean hospitals. So far, no investigation has yet been able to show the efficacyagainst prions of a routine cleaning process at a neutral, mildly alkaline pHor enzymatic approach. In contrast, efficacy against prions could beexpected only from an alkaline process using a formulated detergent with a highpH value (greater than 11) and at a high temperature setting (greater than 70degrees C).

For reprints of the 13-page Rosenberg study, go to: www.sonitol.com 

Dieter Rudin is president of Sonitol, Inc.

Reference:

1. Rosenberg U. Effective cleaning processes and efficacy against prions. ZentralSterilization. 2005; 13 (4): 258-270.