Infection control
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| Surgical instrument reprocessing has been cited as a risk factor for nosocomial transmission of so-called transmissible spongiform encephalop-athies (TSEs) such as variant Creutzfeldt-Jakob disease (vCJD). This factor has brought to the forefront the importance of using proper washing and sterilization techniques for reprocessing instruments. So, which cleaning processes have demonstrated efficacy against prion proteins, the causative agents of TSEs? The author of a recently completed study on this topic analyzes the choices. |
A 2005 study, Effective Cleaning Processes and Efficacy Against Prions, presents my research on the efficacy of cleaning processes in washer-disinfectors and addresses the issue of prion contamination and decontamination, respectively. Prion proteins are the causative agents of the so-called transmissible spongiform encephalopathies (TSEs) such as human Creutzfeldt-Jakob disease (CJD), scrapie of sheep, and bovine spongiform encephalopathy (BSE).
In the wake of a BSE epidemic in England, this disease has been transferred to humans via contaminated bovine tissue to give rise to a new human TSE called variant CJD or simply, vCJD. Unlike with CJD, the accumulation of infectious prion protein (PrPSc) in vCJD is not restricted to the brain.
Prions can be found not only in the lymph system, but also in muscle and blood.
Because of this ubiquitous distribution of the infectious agent and the long incubation time of the disease, surgical instrument reprocessing has been identified as a risk factor for nosocomial transmission of vCJD.
Commonly, surgical instruments are cleaned automatically in washer-disinfectors. Detergents used in this process, can be relatively mild, having 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 40–50 C or 60–70 C. respectively.
Plan of attack
The study first addressed cleaning efficacy by using three different test models:
1 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 washer-disinfector). The analysis was performed by visual inspection/photography.
2 Washing experiments in a washer-disinfector using porous sintered stainless steel discs contaminated with coagulating artificial blood. The analysis was accomplished by weighing these process challenge devices (PCDs) before and after the cleaning process.
3 Washing experiments in a washer-disinfector using hemostatic forceps of which the hinges were contaminated with coagulating sheep blood traced with the short-lived radioisotope Technetium 90. The analysis was accomplished by measuring residual radioactivity after the cleaning process.
All three test-methods provided comparable results that can be summarized as follows:
- The alkaline detergent deconex 28 Alka One, with a working solution pH of around 11, provided the best cleaning result when the temperature plateau of the cleaning step was set at 90 C.
- Processes with a 70 C and a 55 C plateau gave inferior results, with the 55 C process being the worst. This means that the hitherto used alkaline cleaning processes did not exploit the full potential of the detergents.
The study not only focused on the performance of alkaline processes with the described test methods, but also a new two-component cleaning system in the neutral-pH range.
This system, composed of a base detergent called Deconex Twin Basic and an enzyme preparation called Deconex Twin Zyme (TZ), demonstrated a cleaning performance similar to a 90 C alkaline process. The temperature plateau of this new enzymatic two-component process, however, was 55 C.
A prerequisite for using such a process is the ability of the washer-disinfector to inject two detergents at the same washing stage (based on the availability of a dosing pump and capabilities of the control software).
The second part of the study looked at the effect of the same detergents on the infectious prion protein. 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 hamsters infected with scrapie 263K.
The second consisted of stainless steel wires, contaminated with infectious brain extract, that were subjected to a decontamination process mimicking cleaning in a washer-disinfector.
In the case of the in vitro suspension assay, the effect of detergent treatment was assessed by the proteinase K/Western Blot method.
This method makes use of the fact that the PrPSc is resistant to proteinase K (PK) while the noninfectious prion protein is degraded by PK.
If by some effective treatment, e.g., with detergent, the structure of PrPSc is being altered in a way that the molecule becomes sensitive to PK, this modified or destabilized prion protein won’t be infectious anymore.
In brief, the in vitro suspension assay looks at PK-sensitivity of prion protein in detergent-treated infectious brain homogenates.
The results of the in vitro experiments were as follows:
1 Treatment of the brain extract with the alkaline detergent Deconex 28 Alka One either at 0.5 percent (pH 11.1)/10 minutes/70 C or at 1 percent (pH 11.5) /10 minutes/55 C rendered the infectious PrPSc sensitive to PK.
2 Treatment with the lower concentration (0.5 percent) at the lower temperature (55 C) could not render PrPSc sensitive to PK.
3 If the alkaline detergent at the destabilizing concentration (1 percent) and temperature (55 C) was combined with the enzyme preparation of the two-component neutral detergent system, PrPSc was already degraded before PK treatment.
Thus, the proteases in the enzyme preparation had the same effect as PK.
In essence, choosing the right combination of pH and temperature is the key to successful destabilization of the prion protein. 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. Additionally, to the in vitro experiments, I described infection assays using the hamster scrapie model. For these experiments, very small stainless steel wires were contaminated with infectious brain homogenate.
Decontamination of the wires was carried out in a specially constructed apparatus incorporating a spray system mimicking a washer-disinfector.
After decontamination, wires were implanted into the brain of healthy hamsters. The number of days after implantation, during which a hamster was living without symptoms of the TSE disease, was taken as a measure for the reduction of infectious entities on the wire surface.
If insufficiently decontaminated wires are implanted, hamsters show the disease after about 80 days.
When wires were implanted after decontamination with deconex 28 Alka One either at 0.5 percent/10 minutes/70 C or at 1 percent/10 minutes/55 C, all the hamsters 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 that can be allocated to the described decontamination processes with Deconex 28 Alka One is greater than 106.
The important message derived from the study can be described in three points:
1 The same process parameters that will provide the highest efficacy of alkaline cleaning of surgical instruments will also favor destabilization of the prion protein as well as its removal from stainless steel surfaces.
2 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.
3 These processes can routinely be used in every washer-disinfector for cleaning all alkali-tolerant instruments. They are in fact used already in many European hospitals.
Urs Rosenberg is a product manager at Borer Chemie AG in Zuchwil, Switzerland.
For more detailed information on the study, visit www.sonitol.com.
Fig. 1 Cleaning trials
The bars indicate blood residues as radioactive counts per tray after cleaning, top tray left and bottom tray right.
| Top Tray | Bottom Tay | |
| A | 129 | 71 |
| B | 70 | 39 |
| C | 27 | 24 |
| D | 40 | 27 |
The following processes were investigated:
A. Deconex 28 Alka One (0.3%/55 C/10 min)* pH 11.1
B. Deconex 28 Alka One (0.3%/70 C/8 min)* pH 11.1
C. Deconex 28 Alka One (0.3%/90 C/5 min)* pH 11.1
D. Deconex Twin Basic + Zyme (0.3+0.2%/55 C /10 min)* pH 7.9
- Although the plateau times were different for the different processes, the overall cleaning time was the same for all processes.
Fig. 2 Cleaning results and prion residues
TOSI—Immersion in water bath
(Photos of TOSI PCDs at the end of the experiment.)
PCDs were placed in a beaker containing demineralized water at time point 0. The detergent(s) were added during each run once a temperature of 30 C had been reached. The detergent concentrations and solution pH values were as follows:
Test 1: 0.3% 28AO in demineralized water: pH = 11.62 and increasing temperature up to 80 C
Test 2: 0.3% 28AO in demineralized water: pH = 11.62 and increasing temperature up to 70 C
Test 3: 0.3% 28AO in demineralized water: pH = 11.62 and increasing temperature up to 65 C
Test 4: 0.3% 28AO in demineralized water: pH = 11.62 and increasing temperature up to 60 C
Test 5: 0.3% 28AO in demineralized water: pH = 11.62 and increasing temperature up to 55 C
Test 6: 0.3% 28AO in demineralized water: pH = 11.62 and increasing temperature up to 50 C
Test 7: 0.3% TB/0.2% TZ in demineralized water: pH = 10.19 and increasing temp up to 52 C
Results of prion residue testing
Photo of (qualitative Western Blot) a black band on track that had not been treated with proteinase K (-) attests to the presence of prion protein.
In vitro treatment of hamster brain extracts containing infectious prion protein PrPSc.
1. Deconex 28 AO (0.5%/70 C/10 min), pH 11.1
2. Deconex 28 AO (1%/ 55 C/10 min), pH 11.5
3. Deconex 28 AO + TZ (1%+0.3%,/55 C/10 min), pH 11.5
4. Deconex 28 AO (0.5%/55 C/10 min), pH 11.0
5. Potassium hydroxide (0.056%/70 C/10 min), pH 12.0
6. Phosphate buffer (PBS) (70 C/10 min), pH 7.4
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).
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