New prion-related disorders have emerged over the past 20 years, of which the most notable in the human context is variant Creutzfeldt-Jakob disease (CJD). This disorder is a challenge to medical and public health professionals seeking early detection and diagnosis, provision of therapy, and support for persons affected and a better understanding of transmission risks. The risk of iatrogenic transmission of the disease remains a significant threat, given the well documented cases of CJD transmission via surgery, organ transplantation, and blood transfusion. This review discusses our current understanding of the prevalence of variant CJD, the distribution of tissue infectivity, and new methods for the decontamination of surgical instruments. A comparison of emerging technologies is provided on the basis of our current perception of surgical risk to identify methods that are likely to provide sufficient safety margins and to stimulate debate about the standards needed to protect against variant CJD and CJD transmission.
Creutzfeldt-Jakob disease (CJD) is one of a family of related neurodegenerative disorders, the transmissible spongiform encephalopathies (TSEs), which present as familial, sporadic, or iatrogenic disease. A variant form of the disease emerged in the United Kingdom during the 1990s, probably because of consumption of bovine spongiform encephalopathy (BSE)—infected meat products. This form of the disease, presenting in a younger age group with a wider tissue distribution and greater resistance to inactivation than previously observed, has caused additional health care—related questions and problems.
The transmissible agent of TSEs is hypothesized to be a unique, “protein only” infectious entity, termed a prion, although this hypothesis is not universally accepted . TSEs are often characterized by the formation of protease-resistant forms (prion protein [PrP]Sc or PrPres) of the normal cellular prion protein (PrPC) as a result of mutation or sporadically by an as-yet-undefined mechanism or by “infection” following exposure to exogenous misfolded forms of the protein. The precise relationship between PrPSc and prion infectivity requires clarification; however, it is a commonly used surrogate marker for infectivity in many TSE models. The exceptional stability of prions, particularly in variant CJD, means that they are inherently less susceptible to standard methods of sterilization. Iatrogenic transmission of variant CJD via surgery, contaminated medical products, transfusion, tissue implantation, and transplantation, is, therefore, an ongoing health care concern.
Defining the level of risk of iatrogenic variant CJD transmission is complicated. Although undoubtedly low, the potential for transmission is affected by the prevalence of the disease in the population, the type and frequency of invasive procedure involving potentially infectious tissues, and the effectiveness of strategies for instrument management. In addition, decontamination methods or donor screening and selection may also affect the potential for transmission. Historical secondary transmission of CJD provides some information about potential risks, but this may be incomplete when the potential for variant CJD transmission is considered. Our current understanding of the issues involved is discussed below.
Variant CJD: The End of the Problem or a Hidden Epidemic?
The historical forms of CJD show a remarkably consistent incidence throughout the world. In contrast, to date, variant CJD has been confined to a limited number of countries, with 161 definite or probable cases in the United Kingdom up to the end of April 2006 and indigenous cases reported in France (13 cases), Ireland (2 cases), Italy, The Netherlands, Portugal, and Spain; additional cases have been reported in Canada, Japan, Ireland, the United States, and Saudi Arabia, where the disease is believed to have been first acquired in the United Kingdom or from meat imported from the United Kingdom. Since the early 1990s, estimates of the likely size of the variant CJD outbreak have been consistently revised downward, and current incidence rates suggest that the initial phase of infection is decreasing, with the eventual number of cases estimated to be <500 . This decrease has to be qualified by the observation that all patients with clinical variant CJD to date have been homozygous for methionine at the PRNP human codon 129. It now seems probable that all 3 genotypes (PRNP-Val/Val, PRNP-Meth/Val, and PRNP-Meth/Meth) are susceptible to disease, although uncertainties remain about the incubation period and clinical manifestation [3–5].
Drawing firm conclusions regarding future disease patterns is further complicated by the absence of an adequate preclinical diagnostic test. A retrospective tonsil and appendix study detected 3 of 12,674 appendix samples that had lymphoreticular accumulation of PrPSc, which is equal to 237 cases per 1 million persons in the United Kingdom (95% CI, 49–692 cases per 1 million persons) . Interpretation of these data is further complicated by the different pattern of PrPSc staining in 2 of the 3 cases with positive results, which have now been shown to be PRNP-Val/Val-129 . From animal studies, it seems unlikely that these individuals would go on to develop the disease in the course of their normal lifespan , but this remains uncertain. Pre- or subclinical incubation of the disease may pose a cross-infection risk to individuals with more-susceptible PRNP genotypes acquired by means of transplant, transfusion, and surgery. In transgenic animals with a human PRNP-Val/Val-129 genotype, there is evidence that the BSE or variant CJD agent generates a novel phenotype resembling sporadic CJD . The manifestation of future cases of BSE-derived human TSE disease may, therefore, be more difficult to identify, even if such persons present clinically.
A Reappraisal of the Relative Risks of Iatrogenic CJD Transmission on the Basis of Tissue Distribution
The transmission of various types of CJD via contaminated food, medical products, and instruments is now well documented (table 1). These transmissions can be broadly grouped into diet-related infection (kuru and BSE or variant CJD) and iatrogenic disease (derived from tissue transplants, pituitary hormones, blood, and contaminated instruments). A common theme emerges from these cases with respect to extended incubation periods (up to 40 years) and the influence of PRNP codon 129 homozygosity. Most cases of transmission have involved high-titer tissues from the CNS or with neuronal lineage. The exceptions to this are cases of variant CJD and include 2 identified clinical cases acquired by means of blood transfusion [10, 11] and a third case exhibiting detectable PrPSc in the spleen of a patient who died of other causes . Here, the assumed low titer in the blood product is counteracted by the quantity of blood product transferred from donor to patient. In the absence of effective prion disinfection, these transmissions imply that all procedures contacting variant CJD—infected blood potentially carry a small risk of variant CJD transmission. This contrasts directly with sporadic CJD, for which no evidence of transmission by blood transfusion has been identified . Because our understanding of iatrogenic transmission risks is based on historical types of CJD, the extended tissue distribution of variant CJD infectivity means that other potential routes of infection also need to be considered, as evidenced by blood transfusion.
The distribution of infectivity in tissues and organs has been examined in a number of studies using tissues from patients with cases of sporadic, variant, and iatrogenic cases of CJD. Comparison of which tissues are most infectious (table 2) is complicated by the variation in results obtained by individual groups and the relative methods used. In vivo assays (e.g., bioassay) are widely accepted as being the most sensitive method for detecting TSE infectivity in tissues. Variant CJD bioassays detected infectivity in brain, spleen, and tonsil, but not in buffy coat or plasma . Although it is the most sensitive method available, the limits of detection have been estimated to be ∼104.5 human intracerebral ID50 per g of tissue. This is caused by a species barrier to infection in the mouse strain (RIII) that is used and by the restricted inoculation volumes that are possible . To date, transgenic animals have not demonstrated significantly greater sensitivity. The failure of buffy coat and plasma to transmit disease in this study demonstrates that bioassays can produce a potentially misleading picture of transmission risks, given the evidence of blood transfusion—related transmission. Enhanced Western blot detection methods, such as phosphotungstic acid precipitation , may approach the sensitivity of bioassay and provide comparable data, given the caveat that they detect variant CJD PrPSc and not variant CJD infectivity directly. The distribution of both infectivity and PrPSc as a surrogate marker, however, shows that the variant CJD agent routinely accumulates to appreciably higher levels in a wider range of tissues than historical forms of CJD and is associated to a greater extent with both lymphoid tissues and peripheral nerves.
Classification of particular types of surgery into risk categories for variant CJD transmission would probably be consistent with previous assessments . However, variant CJD transmission presents an elevated risk, compared with historical forms of CJD, because of the wider range of tissues showing detectable PrPSc and the potential for higher levels of infectivity. Thus, surgical operations that involve contact with the spleen, lymph nodes, eye tissues (particularly retina and optic nerve ), and intestine (terminal ileum ) may prove to be high risk factors for variant CJD transmission, compared with their status as low risk factors for classical CJD . Although the risk of transmission of variant CJD via dentistry is also considered to be very low  and there is no evidence of high levels of PrPSc in dental-related variant CJD tissues , a variety of animal studies indicate that there may be a risk of transmission through trauma of the oral cavity, irrespective of any exposure through ingestion of infectious material . The relevance of these dental infection models to human disease remains to be clarified and is the subject of ongoing research.
Further uncertainty about transmission risks arises from the observation that the patterns of tissue distribution for all forms of CJD may alter with concomitant infection, as has been demonstrated in chronic inflammation model studies in the mouse . Extrapolation to a patient with chronic inflammation suggests that CJD infectivity may, in general, be redistributed to organs (e.g., spleen, kidney, liver, and pancreas) that would not normally be considered at risk and may be more reminiscent of the tissue distribution observed in variant CJD. Because many people undergoing surgical operations experience an associated inflammatory disease, this could, again, increase the overall likelihood of transmission.
Decontamination and Inactivation of Human TSE Agents
Given the uncertainties regarding incidence of future disease, infectivity of tissues, and transmission routes, tighter control of prion disinfection at all levels may be the best option for controlling iatrogenic transfer. The results of disinfection studies reviewed previously  have highlighted the absence of practical methods to achieve the log-reduction values in PrP that would normally be considered standard for other infectious agents. There has been some rationalization of challenge study design, but there are considerable difficulties in trying to compare methods of inactivation on the basis of studies that employ different TSE agents, animal models, and infection routes. Interpretation of the resulting data becomes more critical as different inactivation methodologies emerge on the market that have prion-reduction claims and as implementation is requested by commercial companies.
Bioassay currently represents the most stringent test available and, as such, offers the greatest confidence in implementing prion reduction methods (table 3). Most studies describe the use of a scrapie agent (263K or Rocky Mountain laboratory strain) attached to surgical steel to model contamination of instruments with a TSE agent. This model, replicating the increased resistance of steel-bound prions to inactivation and the direct implantation of the coated surface, has been proposed as representing the worst-case scenario . Studies reported to date have used scrapie agents routinely and a single investigation has used sporadic CJD , but none have employed either the variant CJD or BSE agent. Titration of infectivity, which forms the basis for determining log reduction in most disinfection studies, has only been reported in a single wire study  and has shown infectivity over a limited range of dilutions, compared with direct intracerebral inoculation. This probably relates to the quantity of infectivity that can be introduced by the wire method. Thus, although the presentation of prions on wires shifts the profile of the titration curve, possibly by altering prion clearance from the brain or by presenting the prion in a more efficient way to propagate infection, detection of infectivity decreases rapidly after the first few log dilutions. For lower titer agents, such as BSE and/or variant CJD or sporadic CJD agents (108–9 infectious units per g of brain tissue, compared with 1010–11 infectious units per g of brain tissue for many scrapie strains), this means that a significant log reduction in infectivity cannot be demonstrated and, arguably, may not provide sufficient evidence of decontamination. The decontamination of wires may also provide a false safeguard in that it does not demonstrate that the prion is rendered noninfective, only that it is removed from the implanted surface. Deposition of infectious prions onto other surfaces (e.g., plastics and rubbers) that are in contact with the patient remains an issue, as does the disposal of large volumes of potentially infectious wash solutions. The biological relevance of scrapie agents to (variant) CJD may also prove to be a critical factor in defining how effective a process would be in limiting iatrogenic transmission, given the very different stabilities of TSE agents to inactivation and the unique properties of human strains [31, 33]. The specific properties of BSE and variant CJD have been addressed in vivo in only 1 published study using direct intracerebral inoculation, rather than a wire implant . The only other human strain (sporadic CJD) used in vivo has highlighted the potential risks of extrapolating from scrapie strains, suggesting that for acidic SDS, sporadic CJD in human brain is 100,000-fold more difficult to inactivate than Sc237 in Syrian hamster brain . Aside from the TSE strain, the use of different animal models and challenge methods make the comparison of results difficult. The use of transgenic models (e.g., Tga20 mice  and Tg23372 mice ) gives a more rapid assessment of infectivity, but overall sensitivity is similar to that for wild-type mice or hamsters. The absence of studies (except ) directly comparing the inactivation of different types of TSE agent in their respective animal hosts makes it very difficult to evaluate which model is predictive of being able to prevent CJD transmission.
Various decontamination studies show complete elimination of detectable TSE infectivity and approach or exceed a 6-log reduction, which would represent a valuable target. A number of proprietary wash products, including Hamo 100 (Steris) and Klenzyme (Steris) , have proved to be effective in eliminating scrapie infectivity from the steel surfaces with efficacy similar to the World Health Organization (WHO) recommended treatments of 1N NaOH and 20,000 parts per million active chlorine (table 4). This has been extended in 1 study  to show apparent elimination of both scrapie Sc237 and sporadic CJD from surgical steel surfaces. There are discrepancies between studies; for example, Environ LpH (Steris) is completely effective in one study  but ineffective in a second study . Whether this is because of specific properties of the 2 different scrapie strains used, differences in experimental design, or use and/or formulation of the product remains to be determined. The additive properties of certain cleaners with a gas-phase inactivation method (e.g., vapor-phase hydrogen peroxide ) may also prove to be useful, particularly with respect to endoscopes or other sensitive equipment. A number of experimental treatments [27–31] hold promise for the future. Work on prion-reduction filtration also offers the potential to reduce the risks of transmission by transfusion, but these approaches and related issues have been reviewed elsewhere .
New Solutions Versus OLD: Where to Set the Standards for Surgical Instrument Reprocessing
Existing regulations and recommendations describe a hierarchical system for managing human TSE risks associated with surgical instruments (table 4). Instrument management forms a key component, with instruments that have contact with “high infectivity” tissues in patients with suspected or confirmed CJD and that are treated as single use (i.e., they are incinerated after 1 use). Quarantine is recommended for instruments used to treat patients with suspected CJD or patients with neurological symptoms awaiting diagnosis. When instruments need to be reprocessed, they can be decontaminated according to the WHO guidelines. Twenty thousand parts per million active chlorine is effective, whereas 1N NaOH provides a significant reduction in titer level either alone or when instruments are autoclaved under these conditions. These treatments are damaging to instruments, potentially hazardous to operators, and clearly are not practical for routine decontamination of the vast majority of surgical instruments . The question then arises as to which of the many proposed solutions to CJD decontamination should be implemented in health care facilities to supplement existing measures and provide a practical solution.
Currently, none of the methods offer safeguards that are sufficient to advocate their use in place of the front-line instrument management systems employed to reduce risks associated with known or suspected cases of variant CJD. Although there are products independently certified for use by a European Community Notified Body (CE Marked) (e.g., Hamo 100 Prion Inactivating Detergent [Steris] and Prionzyme-M [Genencor]) that have been shown to reduce the levels of prion infectivity, their use remains at the discretion of individual users and is subject to local or national restrictions. The 2 products mentioned above both operate under relatively mild alkaline conditions, clearly representing an improvement over existing recommended conditions with respect to instrument damage and user and environmental safety. The procedure for the formal recommendation of such technologies remains unclear. Setting an appropriate standard for new products is complex, and a number of conflicting factors needs to be taken into consideration. Defining a standard will require finding a balance between demonstrating a high overall reduction (>6 log) of infectivity, use of high-titer scrapie agents versus lower-titer but more biologically relevant BSE and/or variant CJD agents, and the reproducibility and practicality of available models. Studies demonstrating that there may be dangers in extrapolating from prion inactivation results for scrapie agents to CJD agents [30, 33] must be included in this evaluation. For the provision of public health, it would be appropriate to expect a standard based on an in vivo study that was performed using a TSE strain directly relevant to human disease in a model with no species barrier and that, ideally, demonstrated a >6-log reduction. However, there may be a pragmatic argument for the rapid introduction of products that have demonstrated significant reductions in prion infectivity in other models, on the basis that they may be more effective than the current practice.
All forms of invasive procedures remain low risks for iatrogenic transmission, even in countries with known endogenous cases of variant CJD. However, the possibility of endemic iatrogenic variant CJD being potentially self-sustaining means that the development and implementation of improved disinfection methods remains a high priority to augment control measures already in place. A number of disinfection methods have shown potential to be translated into clinical use. Those that are finally adopted are likely to be those that are robustly effective and can be implemented into existing work flows simply and safely, with minimal additional cost and environmental impact. Given the caveat that standardization and guidance is urgently required, there is considerable optimism that we are approaching a time when the residual risks of CJD transmission will be minimized by the introduction of efficient prion decontamination technologies.
We acknowledge Dr. Pat Cane and Prof. Phil Marsh for their critical reading of the manuscript. This manuscript is dedicated to the late Dr. Andrew Robinson, a well respected colleague and good friend.
Financial support. UK Department of Health; the European Union; and the UK Department of Environment, Food, and Rural Affairs.
Potential conflicts of interest. Previous studies and ongoing work within the authors' laboratories on protease-based decontamination of prions are funded by Genencor International. The authors receive no personal financial benefit from any of the ongoing work or future sales of products arising from it.