Unveiling the Potential of Enzymatic Detergents to Deactivate Infectious Prions

Abstract

When infectious misfolded proteins self-propagate, they cause transmissible spongiform encephalopathies (TSEs) or prion diseases. TSEs are rare, progressive neurodegenerative diseases with long incubation times and are always fatal. Iatrogenic transmission of these diseases is a major concern for human health, and existing methods of decontamination are either ineffective or require caustic chemical treatment followed by extended steam sterilization cycles. Research was undertaken to explore using enzymatic detergents to decontaminate prion-laden surgical devices, equipment, and stainless-steel tools using existing healthcare facility protocols, including cleaning followed by steam or low-temperature sterilization. Several formulations of enzymatic detergents were used to clean stainless steel wires contaminated with infected hamster brain homogenate. Buffering the solutions to achieve a final pH between 8.5 and 9 when diluted, followed by sonication at 45 to 60°C, was effective in rendering prions undetectable in Western blot images. Subsequent sterilization in an autoclave improved the results, causing further prion degradation. Protein misfolding cyclic amplification showed that adding a four-minute prevacuum auto-clave cycle produced a less than 5-log to 6-log reduction in infectious prion proteins using a multienzymatic detergent and a 6-log reduction using a protease enzyme detergent. Increasing the autoclave cycle to 18 minutes generated a consistent 6-log reduction for both formulations, which is the accepted benchmark for effective sterilization.

The spread of infections in healthcare settings is a serious problem. According to the Centers for Disease Control and Prevention (CDC), approximately one in 31 patients contracts a healthcare-associated infection every day.¹ Transmission can occur through contact with contaminated surgical tools and devices; therefore, cleaning and sterilizing anything that can be a vector for infection is critical. The CDC guidelines strongly recommend that staff clean medical instruments with water and either detergent or enzymatic cleaners that have been diluted appropriately and then rinse the instruments with sterile water. Before sterilizing critical medical or surgical devices, ensuring that compatible chemicals are used during cleaning is crucial.²

Unfortunately, even when protocols are followed, certain infectious agents resist decontamination. This is especially true for infectious agents that cause prion diseases, which are also known as transmissible spongiform encephalopathies (TSEs).

Prion diseases occur when prion proteins become misfolded and replicate rapidly. These neurodegenerative diseases include Creutzfeldt-Jakob disease (CJD), which can develop spontaneously in older adults or be genetically inherited, and bovine spongiform encephalopathy (BSE; also referred to as mad cow disease), which causes the human TSE equivalent of variant CJD.^3,4 Evidence suggests that prions also cause more common neurodegenerative diseases, including Alzheimer’s, Parkinson’s disease, and Lou Gehrig’s disease.⁵

Exposure to human prion diseases has also been linked to transmission via blood transfusions, contaminated surgical tools, pituitary-derived human growth hormone injections, and contaminated brain electrodes.⁶ Considering only reported incidents of unsuccessful decontamination of reusable surgical instruments, disinfection without sterilization often is the culprit, and eye surgery is one of the riskiest procedures.⁷ A 2020 literature review suggested that one in 30,000 corneal transplants will be performed using a cornea infected with undiagnosed CJD.⁸ Although documented cases of iatrogenic transmission of prion diseases are rare, these conditions are progressive, incurable, and invariably fatal.⁹ An effective method to decontaminate medical equipment could save hundreds of lives.

TSEs typically are confirmed postmortem, creating a considerable obstacle to eliminating iatrogenic transmission of TSEs. Although brain biopsies can identify CJD, this is a high-risk procedure that can cause brain damage. Imaging and blood tests can rule out other conditions or suggest the presence of prion disease; however, no quick, reliable screening process is available to warn medical staff that these agents might be present. If healthcare facilities suspect that a patient might be infected with a prion disease, they are forced to take extreme measures, including incinerating all surgical devices used on the patient.^10–12

Cleaning and Sterilization to Deactivate Prions

Standard decontamination protocols are ineffective against prions. Manual or automated cleaning, even when combined with pretreatment with enzymatic or nonenzymatic detergents and steam or low-temperature sterilization, is insufficient to remove or inactivate prions from contaminated instruments. Detectable residual contamination remains on instruments after sterilization and can be deposited onto control surfaces.¹³ In one case, TSEs were transmitted to patients when implanted electrodes previously used on a patient with CJD were cleaned with benzene and disinfected with a solution of alcohol and formaldehyde.⁶

Researchers have reported success in deactivating prions using commercial highly alkaline cleaners supplemented with enzymes such as protease and surfactants.^14,15 The only techniques proven to inactivate diseased prions involved strong concentrations of sodium hypochlorite (NaOCl; also known as bleach). Various studies on scrapie, BSE, and CJD showed that immersion in NaOCl for at least one-half hour, followed by autoclaving at 120°C to 136°C, was effective.^16–19 NaOCl without autoclaving reduced infectivity to undetectable levels but did not completely inactivate prions.

Another problem is that many chemicals used to disinfect medical instruments are carcinogenic or caustic. Benzene, formaldehyde, and NaOCl place medical staff and patients at risk, especially if not handled properly. NaOCl produces toxic gas and can damage autoclaves. Sterilizer manufacturers may specify avoiding bleach on items to be autoclaved.²⁰ Highly alkaline cleaners and lengthy autoclave cycles often damage instruments or reduce their functional lifespan. Certain instruments cannot withstand the high autoclave temperatures required to inactivate prions. Safer alternatives are needed to achieve the goal of infection prevention without unnecessary exposure to toxic chemicals.

Promise of Enzymes

Enzymes offer another route to prion decontamination. A keratinase enzyme from Bacillus licheniformis, combined with a biosurfactant, degraded scrapie prions to undetectable levels in Western blot images after 10 minutes at 65°C.²¹ Other experiments showed that extracts from Parmelia sulcata, Cladonia rangiferina, and Lobaria pulmonaria, but not those from similar lichen species, can degrade prions in TSE-infected animals.²² Although these studies demonstrated success in the laboratory, researchers have not been able to isolate the lichen enzymes responsible for destroying prions and develop a formulation that is effective in the healthcare setting.

Commercially available enzymatic detergents have the potential to be both safe and effective. Specific enzymes of interest include protease, lipase, amylase, and cellulases (or combinations thereof). In the current work, experiments were performed to test the hypothesis that this class of cleaners can degrade prions and determine the necessary experimental conditions and postprocessing treatment to achieve desired results.

Materials and Methods

Range-Finding Experiments

The experiments were performed in the Prion Research Lab at the United States Geological Survey National Wildlife Health Center in Madison, WI. The first research stage involved identifying enzymatic detergents and treatment parameters that would lead to the greatest reduction of infectious prions, thereby limiting the experimental conditions to those that are feasible for practical use in healthcare settings. Variables included dilution of the concentrated detergents with either tap or reverse osmosis (RO) water, buffering to pH ranging from 8 to 11, and various temperatures (22–60°C) and contact times (20–30 min) for soaking or sonication treatment.

The experiments tested several enzymatic detergents using unbuffered enzymatic formulations with approximately neutral pH values as positive controls. Negative controls included a detergent without enzymes, water with a pH buffer, and plain water. All detergents were diluted to a concentration of 2 oz/gal. The proprietary formulations used in these experiments contained a higher concentration of protease than that present in commercially available enzymatic detergents.

The general protocol for these experiments was to add tissue from hamster brains infected with misfolded prion proteins to the prepared formulations according to standard laboratory practices, follow the decontamination procedure (soaking or sonication), and then conduct a Western blot analysis to detect any remaining hamster agent. Results from these experiments were considered positive if no staining was present on the Western blot image (interpreted as the absence of prion protein). A negative result (i.e., visible bands on Western blot image) was interpreted as the prion protein being present.

PMCA Experiments

Three enzymatic solutions were subjected to protein misfolding cyclic amplification (PMCA) testing, which is a more quantitative and sensitive detection method, using the ideal parameters discovered in Western blot testing. PMCA is an amplification technique that multiplies misfolded prions in solution with normal prions, resulting in propagation of the prion disease.²³ It uses high-energy cyclic ultrasound to break up fibril aggregates, thereby increasing conversion potential. Each cycle of PMCA increases the number of misfolded prions exponentially, allowing detection of misfolded prions in samples that initially contained too little of the infected material to be detectable.²⁴

Experiments have shown that infectious prion proteins strongly adhere to stainless steel substrates.²⁵ To replicate stainless steel surgical equipment and tools, a 0.25-mm orthodontic ligature wire was cut into 5-mm segments and sterilized using a steam autoclave cycle. This method was designed to determine the potential for sterilization to meet the expected outcome of a 6-log reduction of prion material from contaminated stainless steel wires.

Orthodontic wires were exposed to 500 μl of infectious or normal 10% (w/v) hamster brain homogenate. Serial dilutions were used to achieve a 10^–10 concentration of infectious 10% (w/v) hamster brain homogenate to create positive controls for PMCA analysis.

Three wires were placed in each tube in an incubator/shaker and set to 37°C and 700 rpm for 2 hours to ensure wires were contaminated. Wires were visually inspected to ensure they were not adhering to one another and were covered entirely with inoculation liquid. After incubation, wires were transferred to filter paper in a petri dish (while ensuring wires were not touching) and left to dry for at least one hour before treatment. Stainless steel forceps were used to handle the wires and sterilized with ethanol followed by flame between individual wires.

To conduct PMCA, 40 μl normal hamster substrate (perfused hamster brain tissue in PMCA buffer) was added to the wells of a 96-well PMCA plate. Wires from the test preparations, including the treated wires and positive and negative controls, were placed individually into respective wells. The plates were incubated at 37°C, suspended in water above a sonicating plate, and subjected to 96 cycles of sonic energy. An aliquot from each sample well was removed for proteinase K digestion and Western blot analysis (as described above).

Two to four rounds of PMCA were run for each experiment. Subsequent rounds of PMCA were run by setting up a duplicate PMCA plate with 36 μl brain homogenate per well. A 4-μl sample from the previous PMCA round was added to the respective well on the new plate, which was incubated and sonicated as above. Log reductions were calculated by comparing the detection of 10-fold serial dilution positive controls in the current and previous rounds.

Results and Discussion

Western Blot Test Results and Relevant Parameters

The Western blot test data were analyzed using univariate or multivariate logistic regression, where lack of detection was considered a success and detection considered a failure. To address the variability in sample sizes for the various parameters, initial pH was binned into four categories: unbuffered, 8 to 8.5, 9 to 9.5, and 10 to 11. Three logistic regression models kept certain variables constant while comparing across additional variables of interest.

These experiments evaluated three detergent formulations. The positive control (C1) contains surfactants but no enzymes. The enzymatic cleaners were E1, which includes a protease enzyme, and E3, which is a multienzymatic formulation with protease, lipase, and amylase.

Model 1 evaluated the effectiveness of formulation (C1, E1, or E3), treatment time (20 or 30 min), treatment type (soak versus sonication), and pH (unbuffered, 8–8.5, 9–9.5, or 10–11), with RO water and 60°C water bath temperature held constant.

Model 2 evaluated the effectiveness of formulation (C1, E1, or E3), treatment type (soak versus sonication), water type (RO versus tap water used in diluting the cleaning solution), water bath temperature (22°C, 45°C, or 60°C), and pH (unbuffered or 9–9.5), with a treatment time of 30 minutes. The maximum treatment temperature of 60°C was chosen because exposure to 65°C or greater denatures the enzymes in the enzymatic detergents.

Model 3 evaluated the effectiveness of formulation (C1, E1, or E3), treatment time (20 or 30 min soak or sonication), pH (unbuffered or 9–9.5), and the addition of a 20-minute gravity displacement autoclave treatment with solutions prepared with RO water and held in a 60°C water bath.

For all models, results were considered significant at P < 0.05. Table 1 summarizes the results, demonstrating that formulation, pH, water type, and water temperature affected the success rate in removing prion material from samples. The highest success rates were seen with the enzymatic detergents diluted with RO water in a mildly alkaline solution with a pH between 9 and 10 and soaked or sonicated at 60°C for at least 20 minutes. Under these conditions, the best-performing formulation was E1 (87% efficacy), followed by E3 (72% efficacy). The nonenzymatic detergent C1 was only 7% effective. Adding an autoclave cycle boosted the success rate for all formulations.

Table 1.

Multivariate analysis results for all three models evaluating the parameters in the range-finding experiments. Significant effects were evaluated by the likelihood ratio chi-squared (χ²) test and reported with their associated degrees of freedom. *Significant at P < 0.05.

Model 1 test results (Table 2) demonstrated that success, which was defined as prions being undetected in more than 75% of test runs, was achieved only for pH of 9 to 9.5 or 10 to 11. None of the unbuffered formulations or those buffered to a pH of 8 to 8.5 were effective. Figure 1 shows a sample Western blot image for this model. As seen in the image, prions are undetected in the enzymatic formulations buffered to a pH of 9 to 10.

Table 2.

Western blot test results for range-finding experiments from model 1, where samples were prepared with reverse osmosis water heated to 60°C for the soak or sonication treatment. No. indicates the number of times each sample was run. Success was measured by the number of runs in which no prions were detected following treatment. The best results are highlighted by boldface type. C1 is a nonenzymatic detergent, while E1 and E3 are single-enzyme and multienzyme formulations, respectively.

Figure 1.

Western blot image from the range-finding experiments for model 1. Prions were detected in the positive and negative controls and the nonenzymatic detergent C1 at all pH values. No prions were detected when using enzymatic detergent E1 at a pH of at least 9.5 or E3 at a pH of at least 9.

Table 3 lists results from model 2. Success was observed only for enzymatic formulations using RO-filtered water and soaked at 60°C. E1 performed better than E3.

Table 3.

Western blot test results for range-finding experiments from model 2, where treatment time for soak and sonication was 30 minutes. No. indicates the number of times each sample was run. Success was measured by the number of runs in which no prions were detected following treatment. The best results are highlighted by boldface type. C1 is a nonenzymatic detergent, while E1 and E3 are single-enzyme and multienzyme formulations, respectively. Abbreviation used: RO, reverse osmosis.

Table 4 shows the results from model 3. The buffered enzymatic formulations E1 and E3 were 100% successful with or without an autoclave cycle, while C1 was successful only with buffering and autoclaving.

Table 4.

Western blot test results for range-finding experiments from model 3. Model 3 added a 20-minute gravity displacement autoclave cycle to the preferred treatment process, where the solutions were prepared with reverse osmosis water and sonicated in a 60°C water bath. No. indicates the number of times each sample was run. Success was measured by the number of runs in which no prions were detected following treatment. The best results are highlighted by boldface type. C1 is a nonenzymatic detergent, while E1 and E3 are single-enzyme and multienzyme formulations, respectively.

PMCA Test Results

The cleaning process was tested on 117 contaminated steel wires. Results showed that using enzymatic detergents and adding an autoclave cycle consistently reduced prion concentrations below the 10^–4 serial dilution, indicating a 4-log reduction or better (99.99% effective at removing prion material). However, the E1 formulation more consistently generated a 6-log reduction (99.9999% effective), with two-thirds of the experiments showing a 6-log decrease on all tested samples—even those with a 4-minute prevacuum autoclave exposure time. Auto-clave cycles of at least 18 minutes were more likely to generate a 5-log reduction or better in prion concentrations, with 97% success (defined as a 5-log reduction or better) for both formulations at 18 and 20 minutes compared with 53% for both formulations at shorter autoclave times. Table 5 summarizes the results.

Table 5.

Prion-contaminated wires were treated with enzymatic detergents E1 (single-enzyme) and E3 (multienzyme) and exposed to vacuum autoclave cycles. Protein misfolding cyclic amplification (PMCA) test results estimated the log reduction of prion material by identifying the lowest 10-fold serial dilution positive control present on the round of PMCA in which the sample was first detected (shown as greater than the reduction if never detected in three rounds). The ≥ symbol indicates that some wires achieved 6-log reduction and others in the series achieved even greater reduction.

PMCA experiments are considerably more sensitive than simple Western blot analysis, as the assay amplifies the protein. However, the platform is prone to false positives and inconsistencies between runs.²⁶ Thus, although the results of these experiments presented efficacy measures for the cleaning processes and enzymatic solutions tested, readers are advised to interpret the results conservatively. After additional platforms for quantifying prion proteins are developed, future work should confirm and better quantify the log-reduction potential of enzymatic cleaners for use on stainless steel instruments.

Of note regarding pH as a binned factor: Although the buffer pH was recorded for all experiments, the active pH of the solution was not consistently measured during cleaning. However, based on spot measurements, the active pH was between 1.2 and 1.5 units lower than the buffered pH, and this effect was more noteworthy for formulation E3 than for E1. In the healthcare setting, the buffer pH is likely more important than the active pH; therefore, we do not believe that this lack of data affects the ability to report on or interpret these results.

Conclusion

General Recommendations

If the conditions are right, properly formulated enzymatic detergents can effectively degrade infectious prions, achieving a 5-log reduction or better in prion concentration. This research has shown that optimizing cleaning parameters is necessary for effective decontamination. The enzymes in the tested formulations were most active when concentrated detergents were buffered to a mildly alkaline pH between 9 and 10 and temperatures were elevated slightly. Thus, recommended best practices include preparing cleaning solutions with a buffer solution of pH more than 9 for these specific enzymatic cleaners, then soaking or sonicating for at least 20 minutes at 60°C. Sonicating is more effective than soaking.

In addition, the results showed that RO water is essential for diluting cleaning solutions. This is likely because tap water contains impurities such as chlorine, heavy metals, dissolved solids, and other organic and inorganic contaminants. Any purified water probably would produce results similar to those using RO water.

This research demonstrated that lengthy autoclaving cycles are unnecessary to deactivate prions. A steam autoclave cycle of 18 to 20 minutes will consistently remove at least 99.99% of prion contaminants (4-log reduction) and often can achieve and even surpass a 6-log reduction, which is the accepted norm for effective sterilization. An autoclave cycle as short as four minutes can achieve similar results when the detergent type and processing parameters are optimized. Autoclaving with prevacuum rather than gravity displacement only, consistent with standard sterilization protocols for healthcare applications, is necessary to achieve reliable results.

The recommended combination of parameters falls within the scope of a U.S. patent filed by one of the authors (M.F.) and published in 2020.²⁷

Effectiveness of Enzymatic Detergents

An enzymatic cleaning process that deactivates prion contamination without damaging stainless steel instruments will greatly benefit healthcare facilities and improve safety for both staff and patients. It can replace incineration or strong bleach solutions to decontaminate instruments suspected of being exposed to infectious prions.

Although multienzymatic formulations are useful for removing a wide range of contaminants from surgical instruments, formulations with protease alone, when prepared and processed according to the recommendations above and containing high enough protease concentrations, are the most effective for removing infectious prion proteins. This decontamination protocol is also recommended for use in patients with other protein-misfolding diseases, including Alzheimer’s, Lou Gehrig’s disease, and Parkinson’s disease.

Acknowledgments

The authors recognize Chris Johnson, United States Geological Survey (USGS), for initiating the collaboration and exploring potential additional enzymes that may be effective against prion proteins. The authors thank Christina M. Carlson, USGS, for directing the research; Jay Schneider, USGS, for conducting the experiments; and Katherine L.D. Richgels, USGS, for supporting the project. The authors also thank Julia Freer Goldstein and Michael Polozani for reviewing the technical data and preparing the manuscript for publication.