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Prions and the Oral Cavity
1 Infection Research Group, Glasgow Dental Hospital & School, 378 Sauchiehall Street, Glasgow G2 3JZ, Scotland, UK; Correspondence: *corresponding author, a.smith{at}dental.gla.ac.uk
Prion diseases have recently emerged as a significant challenge to health-care workers, including those involved in dentistry. Abnormal prion proteins are resistant to complete inactivation by conventional sterilization techniques. In the last decade, a new form of prion disease emerged in the UK, termed "variant CJD", thought to be acquired by consumption of bovine spongiform encephalopathy-contaminated food products. At present, CJD is an invariably fatal disease with no immediate prospect of treatment or vaccination. Of concern with the variant form of CJD, unlike the more classic forms of the disease, is the appearance of significant levels of infectivity outside the central nervous system. This raises concerns for the potential transmission of prion proteins via surgical procedures from individuals in the asymptomatic stage of the disease. This article reviews the existing knowledge base on the nature of prions, their distribution in oral tissues, and the implications for dental treatment.
Key Words: prions • oral cavity • dentistry • infection control
In 1996, the CJD Surveillance Unit (Edinburgh, UK) reported a series of 10 patients with a novel form of Creutzfeldt-Jakob Disease (CJD) and suggested that the most likely explanation was human exposure to bovine spongiform encephalopathy (BSE). These patients were younger than those with sporadic CJD, displaying prominent early psychiatric and behavioral manifestations, followed by ataxia and other movement disorders. Pathological examination of the CNS showed widespread plaques of abnormal prion protein (PrPsc) which were structurally different from those found in other forms of transmissible spongiform encephalopathies (TSEs). Subsequent cases have exhibited a consistent clinical and pathological phenotype; this disorder is now termed "variant CJD" (vCJD). The transmission of TSEs from human to human (via cannibalism in Kuru, and by medical and surgical procedures in iatrogenic CJD) has been recognized for many years, and the identification of vCJD triggered research into the potential risks of further iatrogenic transmission in a wide range of health-care interventions, including dental procedures. Much work has focused, quite rightly, on tissues that contain the highest levels of detectable activity, such as the central nervous system and lymphoreticular tissues. The route of entry of infectious material in the BSE epidemic in cattle and the Kuru epidemic in humans has been via the oral cavity, yet relatively little work has been performed on the distribution of prion proteins in this site. Oral health-care interventions account for millions of treatment episodes throughout the world, with many permutations for potential cross-infection. This paper reviews the distribution of prion proteins in oral tissues on the basis of data from animal and human experiments and outlines current issues over possible risks of iatrogenic transmission via dental treatment.
TSEs (also known as prion diseases) occur naturally in animals and humans (Prusiner, 2001). All have long incubation periods of months to years, leading to death over a short period after the onset of neurological disease. None evokes a host immune response, and all share a common non-inflammatory pathologic process in the central nervous system, with vacuolation of the grey matter (hence "spongiform" encephalopathy). Infectivity is highest in brain tissue and may also be present in some peripheral tissues (particularly lymphoid tissues), but is generally absent from body fluids, including saliva (Brown et al., 1994). The infectious agents are unique in containing no detectable nucleic acids, and are composed of an abnormal isoform of a host membrane sialoglycoprotein called "prion protein" (PrP). Some TSE agents have spread across species barriers (vCJD), some have reached epidemic proportions by entering the food chain (BSE and Kuru), others have been transmitted by inheritance of mutations in the PrP gene (familial CJD), and others have been transmitted iatrogenically by implantation or injection of contaminated material such as dura mater or pituitary material, or by contaminated neurosurgical instruments. The transmissible agent in TSEs is resistant to conventional forms of decontamination, and most measures which reduce levels of infectivity are detrimental to metal surfaces.
NATURE OF THE INFECTIOUS AGENT
PrPc is rich in
Distribution of Prion Proteins in the Trigeminal Nerve, Tooth Pulp, Gingival/Salivary Glands, and Tongue of Animal Models Several workers have demonstrated immunoreactivity to PrPsc in the trigeminal ganglion and peripheral nerves (Table 1  ). For example, intraperitoneal challenge with the 263K scrapie strain in Syrian hamsters resulted in the appearance of infectivity in trigeminal ganglion, dental pulpal tissue, and gingival tissue (Ingrosso et al., 1999). Similarly, infectivity has been reported in the trigeminal ganglion during pre-clinical phases of the disease in cattle exposed orally to BSE agent (Wells et al., 1998) and in the trigeminal ganglion of sheep and hamsters with experimental scrapie (Groschup et al., 1999). The latter demonstrated deposition of neuronal PrPsc as intracytoplasmic granular inclusions in ganglion cells. Fine granular immunostaining could also be seen in peripheral nerves adjacent to the ganglia, in a pattern suggesting peri-/adaxonal PrPsc deposition. The authors suggested that the pattern of the adaxonal deposits implied accumulation in the Schwann cell nodal processes and in the inner loop of Schwann, although the exact site of deposition or transport of PrPsc in peripheral nerve fibers remains to be established. The normal cellular isoform of PrP has been shown to move by rapid anterograde axonal transport (Borchelt et al., 1994). In contrast, some workers have failed to detect immunoreactivity in the dorsal root ganglia of mice infected intraperitoneally with a mouse-adapted CJD strain (Muramoto et al., 1993).
Infectivity has been demonstrated in the salivary glands of scrapie-affected goats (Pattison and Millson, 1962) and mice (Eklund et al., 1967; Sakaguchi et al., 1993). The observation of scrapie agent in the salivary glands of mice has prompted the suggestion that saliva may play a role in the lateral transmission of scrapie in mice. However, transmission between mice does not appear to be a hazard during experimental scrapie studies from saliva or any other source. Studies on the agent of another TSE, transmissible mink encephalopathy, have demonstrated that submandibular salivary tissue can contain infectious material up to 103 LD50 per gram of tissue after subcutaneous inoculation of the TME agent (Hadlow et al., 1987). However, PrPsc has not been detected consistently in murine salivary gland tissue (Muramoto et al., 1993), possibly due to accidental inclusion or exclusion of salivary gland lymph nodes during the dissection process. Similarly, in a study of scrapie-infected sheep, no infectivity was discovered in saliva or salivary glands assayed by a mouse intra-cerebral bioassay (Hadlow et al., 1982). In mice infected orally or via intraperitoneal injection with sheep scrapie (murine C506M3 strain) and the murine 6PB1 BSE strain, submandibular lymph nodes demonstrated immunoreactivity, although no PrPsc could be detected in palatine lymphoid formations (Maignien et al., 1999). After oral infection by both agents, the submandibular lymph nodes became positive before the CNS (Maignien et al., 1999). Recent work (Bartz et al., 2003) in hamsters has shown that intra-tongue inoculation of transmissible mink encephalopathy (TME) agent was 100,000-fold more efficient in transmitting infection than the oral (ingestion) route. PrPsc was detected in both the tongue and submandibular lymph nodes 1-2 wks after tongue inoculation, prior to deposition in the hypoglossal nucleus. The same group showed PrPsc in tongues following intra-cerebral inoculation, demonstrating that prions can also travel from the brain to the tongue (Bartz et al., 2003).
Animal Studies of the Transmission of Prions via the Dental Route More recently, inoculation of the scrapie strain 263K into the pulpal cavity of the mandibular lower incisor of 6 Syrian hamsters caused all of the animals to develop scrapie disease, with spread of the agent to the trigeminal ganglion on the inoculated side (Ingrosso et al., 1999).
The different types of human TSEs are summarized in Table 2 . The first patient known to have vCJD died in 1995. To date (August 4, 2003), there have been 139 "definite" and "probable" vCJD cases in the UK. Recent analysis of the mortality trend in vCJD suggests that, in 2002, the previously increasing trend is slowing (Andrews et al., 2003). However, it is still too early to know whether this trend will be sustained or to forecast the ultimate size of the epidemic.
Laboratory experiments provide compelling evidence that the causative agents of vCJD and BSE have a common origin (Bruce et al.,1997). The glycosylation patterns of PrPsc, susceptibility studies in mice, and patterns of disease in brain tissue from vCJD patients and BSE are similar, but are distinct from the patterns associated with scrapie, sCJD, and fCJD. There is also a genetic predisposition to the disease, since all vCJD patients analyzed to date are homozygous for methionine at codon 129 in the prion protein gene. It is widely accepted that vCJD crossed to the human population through consumption of bovine products from animals with BSE, which entered the human food chain in the 1980s and early 1990s. Cases of BSE have not been confined to the UK, and cases have been reported throughout Europe. Cases of vCJD have been detected in France, Italy, the USA, and Canada. This highlights the potential spread of this disease and raises the possibility of vCJD arising in other countries. In vCJD, unlike other types of human TSEs, infectivity is present in tissues outside the central nervous system, principally the lymphoreticular system, for some time prior to the onset of clinical signs and symptoms (Hilton et al.,1998). This raises the real possibility that patients with vCJD may receive dental treatment at a pre-symptomatic stage of their disease. The potential theoretical risk of iatrogenic transmission during dental treatment can be much reduced if all re-usable instruments are cleaned and sterilized to a high standard.
Variables that may influence the risk of developing CJD include genetic susceptibility, incubation period, infective dose, and route of exposure. In cases of iatrogenic CJD, where the infectious agent is introduced into, or near, the brain, the incubation period is typically measured in months. After peripheral or oral exposure, incubation periods range from at least 4 years to over 40 years, with a mean of 10-15 years (Brown et al., 2000). Relatively little is known about the molecular state of the protein that constitutes the self-propagating infectious unit. A single infectious unit corresponds to approximately 105 PrPsc molecules (Bolton et al., 1982). However, it is unknown whether this large aggregate is necessary for infectivity, or whether a single molecule alone is infectious. Of additional concern is the observation that transmission could be caused by infective tissue merely coming into contact with that of another individual (via contaminated surgical instruments) rather than requiring any actual transfer of tissue (Zobeley et al., 1999). The pathogenesis of prion diseases varies with different host species and prion strain combinations. It is often difficult to extrapolate the animal experimental data directly to humans. However, in vCJD, post mortem studies have shown that abnormal prion protein is accumulated in CNS, ocular, and lymphoreticular tissues, including the tonsils, spleen, and appendix. Infectivity has been demonstrated in autopsied tonsil and spleen tissues at levels around 2 logs lower than in the brain (Bruce et al., 2001). It is likely that asymptomatic patients who are developing vCJD will have potentially infective tissues in the pre-clinical phase of their illness, raising the theoretical risk of iatrogenic transmission of vCJD. Currently, it is assumed that CNS, posterior orbit, and lymphoreticular tissues are, in descending order of risk, the tissues most likely to be infective. The key issues, therefore, relate to the specific infectivity of various tissues at different stages of the incubation period and the transfer of infectivity by different routes. There is no current evidence to suggest that vCJD has been caused by iatrogenic transmission, but this remains a possibility in view of the potentially long incubation period. The infective dose required to cause vCJD is unknown. It seems likely that the oral tissues containing the highest infectivity will be located in the oral lymphoreticular region. Within the oral cavity, the largest collections of lymphoid tissues are located in the submucous tissue of the posterior third of the tongue, termed the "lingual tonsil". While these tissues are likely to contain levels of infectivity of 105 logID50/g, they are rarely traumatized during routine dental procedures.
The infectious agents of TSEs (prions) are very resistant to the usual techniques for inactivating conventional pathogens. Ionizing, ultraviolet, and microwave radiations have little effect on prion proteins (Taylor, 1999). Prions are resistant to most disinfectants used in dental practice, though concentrated bleach appears to achieve inactivation of all strains. Prions are also heat-resistant, and autoclaving at 134°C for 18 min does not achieve complete inactivation. The scrapie agent is more resistant to inactivation by autoclaving when infected tissue becomes dried onto glass or metal surfaces or following prior fixation of tissue in ethanol or formaldehyde. The cleanability of dental instruments is a key factor in attempts to reduce levels of adherent tissue on used instruments. Many dental instruments are difficult to clean by virtue of their small size and intricate design—for example, endodontic files (Smith et al., 2002). Consideration should be given to classifying such instruments as "single use only" and not attempting to decontaminate them for use on future patients. Significantly more work remains to be done to reduce risks of cross-infection by improving instrument design and cleaning methods.
To date, conventional dental treatment has no proven association with transmission of any form of CJD, although oral surgical procedures involving dura mater carry a risk (Marx and Carlson, 1991). A preliminary analysis of previous dental treatment in vCJD cases (Ward H and Will RG, personal communication) has not revealed any consistent pattern suggesting past dental treatment as a risk factor for vCJD, but collecting appropriate data is difficult. For cases of sCJD, fCJD, and iCJD, any levels of PrPsc in oral tissues are likely to be at very low levels compared with the CNS. There are no current published data for levels of PrPsc in oral tissues of vCJD cases, but evidence from studies investigating other peripheral tissues suggests that it is likely to be low (< 104 ID50/g).
Case Reports
Case Control Studies in sCJD
Infectivity in Human Trigeminal Nerve and Oral Tissues
One study has investigated the levels of prion protein in pulpal tissue from eight patients with sCJD (Blanquet-Grossard et al., 2000). By Western blotting, using a specific monoclonal antibody, this group was unable to detect any prion protein in pulpal tissue. However, the authors suggested that since the method used was relatively insensitive, the potential for transmission of CJD via dental procedures, although low, could not be dismissed. These workers calculated that 1 g of sCJD-infected pulp would be expected to contain 40 log10 LD50/g of infectivity, compared with 108-9 LD50/g of infectivity in brain tissue.
Implications for Dental Treatment The situation is less clear-cut for treatment of healthy patients deemed to be "at risk" of developing CJD. These include recipients of contaminated dura mater grafts or relatives of patients with fCJD, for which there is significant variation between different sets of official recommendations, from stringent decontamination protocols for instruments, including extended periods of autoclaving in a vacuum autoclave (ACDP/SEAC, 1998), to no special precautions at all (WHO, 2000). There is no evidence that TSEs can be transmitted by aerosol routes or contaminated surfaces; therefore, the only components of infection control procedures that merit special consideration are those relating to re-usable instruments. Routine surgery protocols for surgery surface and environmental cleaning, which are used to limit transmission of blood-borne viruses, are sufficient for treatment of known, suspected, or "at-risk" cases. At the time of writing, there is no evidence in humans to suggest that oral tissues carry high levels of PrPsc (see note below). Since it is not possible to identify patients who may be asymptomatic carriers of PrPsc, particularly vCJD, the most important recommendation is to ensure that the quality of pre-sterilization cleaning of instruments used on all patients is of the highest standard, and that instruments which cannot be reliably cleaned are treated as single-use.
The last five years have seen the emergence of a new human TSE termed "variant CJD", which results from exposure to the BSE agent. This has been accompanied by a wealth of scientific data on the responsible infectious agents called prions. However, data concerning the distribution of PrPsc and infectivity in the oral cavity are sparse. The available animal model evidence illustrates a potential for transmission via the dental route, but differences in patterns of disease and in strains of the agent make it difficult to extrapolate these findings directly to humans. Data concerning the distribution of PrPsc and infectivity in the human oral cavity are urgently required to determine the risk, if any, of cross-infection during common dental procedures. NOTE: Following acceptance of this paper, the department of health (UK) has published revised guidance for healthcare and laboratory workers on safe working with transmissible spongiform encephalopathies (TSEs) such as Creutzfeldt-Jakob Disease (CJD). This guidance replaces the edition issued in March, 1998. There are many changes which are of interest and significance to dental practitioners. Of great importance is the recognition of the low risk of transmission of infection from dental instruments provided that optimal standards of infection control and decontamination are maintained. In practice, this means that instruments used on all patients in the "at-risk" category can be treated in the same manner as those for all other patients. This recommendation is also extended to routine dentistry in confirmed or suspected CJD, cases negating the requirement to destroy or quarantine instruments. These guidelines re-inforce the notion that any patient or their relatives may be treated in general dental practice (in the same manner as any member of the general public). These guidelines are published online at www.doh.gov.uk/cjd/tseguidance.
Received for publication November 14, 2002. Revision received July 18, 2003. Accepted for publication July 23, 2003.
Journal of Dental Research, Vol. 82, No. 10,
769-775 (2003) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
-helical structures, but the disease-associated isoform PrPsc is composed mainly of β-pleated sheet and is postulated to act as a conformational template that promotes the conversion of PrPc to further PrPsc. PrPsc is toxic to neural cells, disrupting function and leading to vacuolation and cell death. Prion replication, with recruitment of PrPc into the aggregated PrPsc isoform, may be initiated by a pathogenic mutation (resulting in a PrPc predisposed to form PrPsc) in inherited prion diseases known as familial CJD (fCJD), by exposure to a seed of PrPsc in acquired disease, namely, iatrogenic CJD (iCJD) or variant CJD (vCJD), or as a result of the spontaneous conversion of PrPc to PrPsc (and subsequent formation of aggregated material) as a rare event in sporadic CJD (sCJD). 
