Karen Fernie

Studies on the removal of a bovine spongiform encephalopathy-derived agent by processes used in the manufacture of human immunoglobulin

Background and Objectives There is still uncertainty over how the agent of variant Creutzfeld‐Jakob disease (vCJD) would partition during the manufacture of plasma derivatives. In this study, a BSE‐derived agent was used as a vCJD model to determine the extent to which infectivity could be removed by selected steps used in the manufacture of intravenous immunoglobulin (IVIG).

Observations on thermostable subpopulations of the unconventional agents that cause transmissible degenerative encephalopathies.

When scrapie agent is exposed to partially inactivating autoclave cycles, the fraction of infectivity that survives remains thermostable during relatively long periods of autoclaving. This resistant subpopulation can also be differentiated from the main population by its prolonged incubation periods in assay animals, compared with control material. Stabilisation of this subpopulation may occur through the smearing and drying of infected tissue that can occur prior to autoclaving, in which the disease-specific form of PrP protein (PrP(Sc)) could become rapidly heat-fixed. This may paradoxically be what protects this fraction of PrP(Sc) from further inactivation during autoclaving. Data are presented showing that the thermostability acquired by the resistant subpopulation is a stable characteristic; autoclaving for a second time results in very little further loss of infectivity. These observations suggest that inactivation procedures that do not involve rapid and effective fixation of PrP(Sc) may be better candidates for dealing effectively with scrapie-like agents.

Thermostability of mouse-passaged BSE and scrapie is independent of host PrP genotype: implications for the nature of the causal agents

Five experimentally maintained strains of scrapie and BSE agents have been passaged in two PrP genotypes of mice. Brain macerates were autoclaved at 126 degrees C and the levels of surviving infectivity were measured by titration. There was a large difference in the survival properties of transmissible spongiform encephalopathy (TSE) infectivity between TSE strains. PrP genotype had little effect. Phenotypic properties of the TSE strains were not affected with the exception that with one strain (ME7), incubation periods of the heated sample were longer than the controls given equivalent doses. It is concluded that PrP is probably not responsible for differences in thermostability between strains. More likely, a host-independent molecule which differs in covalent structure between strains accounts for these properties.

Exposure to autoclaving or sodium hydroxide extends the dose-response curve of the 263K strain of scrapie agent in hamsters

An analysis was made of incubation period data from experiments in which samples of brain-tissue infected with the 263K strain of scrapie agent were injected intracerebrally into hamsters following exposure of the tissue to autoclaving or sodium hydroxide. Where there was survival of infectivity, this often produced extended mean incubation periods compared with the maximal incubation periods in controls injected with untreated agent. These results confirmed that, after chemical or physical treatment, infectivity titre should not be calculated by comparing the incubation period from a single dilution-group against a standard dose-response curve for untreated agent.

Comparative studies on the thermostability of five strains of transmissible‐spongiform‐encephalopathy agent

The causal infectious agents of TSEs (transmissible spongiform encephalopathies or prion diseases) are renowned for their resistance to complete inactivation. Survival of TSE infectivity after autoclaving potentially compromises many procedures where TSE infectivity may be present, including surgical instrument sterilization. In the present study, the heat inactivation properties of five different TSE agents were tested in a variety of experiments by exposing them to a range of heat inactivation conditions. Although TSE infectivity was reduced after heating to 200 °C in a hot air oven, substantial amounts of infectivity remained. Unlike wet heat inactivation, no TSE strain‐dependent differences were observed in the reduction in the amounts of infectivity produced by dry heat inactivation. However, the incubation periods of mice infected with one dry heated TSE strain, ME7, were substantially prolonged, whereas there was little or no effect for two other TSE models. Varying autoclaving conditions for three TSE strains between 132 and 138 °C, and times of exposure between 30 and 120 min, had little or no effect on the recovery of TSE infectivity. The results illustrate the limitations of TSE agent inactivation using heat‐based methods. The results support the hypothesis that the structures of TSE agents are stabilized during heat‐inactivation procedures, rendering them much more refractory to inactivation. This may occur through dehydration of the causal agents, specifically through the removal of the water of solvation from agent structures and hence stabilize interactions between prion protein and TSE agent‐specific ligands.

Inactivation of the 22A strain of scrapie agent by autoclaving in sodium hydroxide.

Samples of macerated mouse-brain infected with the 22A strain of scrapie agent were subjected to gravity-displacement autoclaving at 121 degrees C for 30 minutes in the presence of 2 M sodium hydroxide. No infectivity was detectable by mouse bioassay in samples which were either held for an hour at room temperature before autoclaving, or autoclaved immediately after adding the hydroxide. In contrast, all of the mice injected with a control sample, held for an hour in distilled water before autoclaving, developed scrapie. If adopted as a routine decontamination procedure it is recommended that hydroxide-treated waste should be autoclaved in sealed heat-resistant containers which can withstand the pressures involved. This would make the procedure safer for the operator, and avoid autoclave damage through exposure to sodium hydroxide.

The effect of formic acid on BSE and scrapie infectivity in fixed and unfixed brain-tissue

Brain-tissue infected with scrapie-like agents remains infectious after histological fixation, and represents a source of occupational exposure. Infectivity titres in formol or paraformaldehyde-lysine-periodate (PLP)-fixed mouse-brains infected with the 301V strain of BSE agent were reduced by around six logs following treatment with formic acid. After PLP fixation and formic acid treatment, no infectivity was detectable in mouse-brain infected with the 87V strain of scrapie agent. Similarly-treated mouse-brain infected with the ME7 strain of scrapie agent showed a titre loss of approximately 5 logs. No infectivity was detectable in PLP-fixed, BSE-infected bovine brain after formic acid treatment, but this was an unreliable result. With unfixed brain homogenates, formic acid reduced the titre of ME7 by > or = 5.0 logs; technical problems limited the measured loss of BSE infectivity to > or = 1.0 logs. These studies confirm that formic acid treatment during fixation of brain-tissue significantly reduces the infectivity titres of scrapie-like agents, thus reducing the level of any occupational exposure.

SURVIVAL OF SCRAPIE AGENT AFTER EXPOSURE TO SODIUM DODECYL SULPHATE AND HEAT

Fifty mg aliquots of macerated mouse-brain infected with the 22A strain of scrapie agent were treated by exposing them without mechanical mixing to (a) distilled water for 2 h, (b) 5% sodium dodecyl sulphate (SDS) for 2 h, (c) autoclaving at 121 degrees C for 15 min in distilled water, (d) autoclaving at 121 degrees C for 15 min in 5% SDS, or (e) boiling in 5% SDS for 15 min. Prior to injection into mice, all samples were washed by a procedure that is described and was shown not to reduce infectivity titres. Although the infectivity titre of the sample that was autoclaved in SDS was reduced considerably, infectivity was present in all of the samples exposed to cold or hot SDS.

The effect of dry heat on the ME7 strain of mouse-passaged scrapie agent.

Partial survival of lyophilized scrapie agent has been reported previously following exposure to dry heat at 360 degrees C for 1 h, and led to speculation that scrapie-like agents might not be completely inactivated by incineration. However, it is known that dried infectivity is more difficult to inactivate by heat than that in hydrated samples. In this present study it was shown that the infectivity in macerates of mouse-brain infected with the ME7 strain of scrapie agent was not completely inactivated by exposure to dry heat at temperatures up to 180 degrees C for 1 h but the titre of surviving infectivity reduced progressively as the temperature was increased. No infectivity was recovered after a 1 h exposure at 200 degrees C. These data suggest that scrapie-like agents are unlikely to survive incineration.

Limited efficacy of steam sterilization to inactivate vCJD infectivity

BACKGROUND The transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt-Jakob Disease (vCJD) raised concerns about potential secondary transmissions due to the resistance of the agents causing transmissible spongiform encephalopathies (TSEs), sometimes known as prions, to commonly used methods of sterilization, notably steam sterilization (or autoclaving). It has been suggested that surgical instruments and other medical devices might retain sufficient infected tissue debris after cleaning and steam sterilization to infect patients on whom they are subsequently used. AIM To determine whether concerns about the lack of efficacy of steam sterilization of vCJD were justified. METHODS The reduction in infectivity of brain macerates of vCJD brain after steam sterilization using the standard temperatures and time recommended for autoclaving in UK hospitals (134-137°C for 3 min) was measured. FINDINGS Reductions in titre of 10(2.3) to >10(3.6) ID(50) were found. In three of four samples, infectivity was recovered after steam sterilization. CONCLUSION As noted previously, TSE strains derived from BSE sources appear to be more resistant to steam sterilization and other forms of heat inactivation than other TSE sources.