New Biocides Development - ACS Publications - American Chemical

Chapter 3

Disinfection of Prions 1,2

1,2,6

1,2,7

Kurt Giles , Surachai Supattapone , David Peretz , David V. Glidden , Henry Baron , and Stanley B. Prusiner 3

5

Downloaded by UNIV OF GUELPH LIBRARY on August 6, 2012 | http://pubs.acs.org Publication Date: September 7, 2007 | doi: 10.1021/bk-2007-0967.ch003

1

1,2,4

2

Institute for Neurodegenerative Diseases and Departments of Neurology, EpidemioIogy, and Biostatistics, and Biochemistry and Biophysics, University of California at San Francisco, San Francisco, CA 94143 Biosecurity Alliance Foundation, Paris, France Current address: Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755 Current address: Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608 3

4

5

6

7

Prions are unprecedented transmissible pathogenic agents that cause a group of fatal neurodegenerative diseases, including Creutzfeldt-Jakob disease (CJD) and kuru in humans, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in deer and elk, and scrapie in sheep and goats. Prions are resistant to standard disinfection and sterilization procedures validated against viruses and bacteria. There are well documented cases of iatrogenic prion transmission from surgical instruments and cadaveric tissue. The lack of noncorrosive procedures that inactivate prions is a cause for concern for hospital infection-control departments. A novel method to inactivate human prions completely, using sodium dodecyl sulfate at acidic pH (acidic SDS), is reported here. Prion inactivation was demonstrated on both brain homogenate and on the surface of contaminated surgical stainless steel, the latter of which proved significantly more resistant to inactivation.

52

© 2007 American Chemical Society

In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007.

53 Prion diseases represent a novel paradigm of infection since the causative agent is devoid of a nucleic acid genome, and is composed solely of an abnormal conformational isoform of the host prion protein (PrP). The abnormal isoform, termed P r P (after scrapie, the prototypic prion disease), serves as a template to recruit molecules of the normal cellular isoform (PrP ) to replicate its misfolded conformation. Prion diseases are therefore disorders of protein conformation involving template-assisted replication, resulting in P r P accumulation in the brain, which invariably causes neuronal dysfunction, degeneration, and death. The most remarkable feature that distinguishes prion diseases from viral, bacterial, fungal, and parasitic disorders is that prion diseases can manifest as infectious, inherited, or sporadic illnesses. In all cases, infectious prions composed of P r P molecules are generated in the brains of afflicted individuals. When prions are passaged into the brain of a different host species, a "transmission barrier," related primarily, but not exclusively, to differences in PrP sequence, is responsible for inefficient infection . The most notorious example of cross-species transmission is the appearance of variant (v) CJD, in teenagers and young adults in the United Kingdom, from consumption of beef infected with B S E prions . In the U S A , there is growing concern that a similar epidemic could occur from the rapid and continuing spread of C W D in both wild and farmed deer and elk, and the possible transmission to humans . A basic understanding of prion biology is essential to the establishment of sound, intelligent, and effective biosafety principles and measures. If prionrelated risk is evaluated from the perspective of the virologist, ignoring the unique properties of prions, uninformed and perhaps harmful decisions, based on erroneous assumptions, may result. Sc

c


Sc

Sc

1-3

4

5

Resistance to inactivation The unusual stability of the infectious agent responsible for scrapie in sheep was first noticed in the 1930s. A significant proportion of sheep inoculated against looping-ill virus, with a specific batch of inoculum, began to develop scrapie approximately two years later. The inoculum was prepared from pooled sheep brains, spinal cords, and spleens, treated with formalin and certified free of any detectable living virus . Concurrently, transmission of sheep scrapie to goats was experimentally demonstrated by inracerebral inoculation of scrapieinfected C N S tissue . It was therefore concluded that the batch of looping-ill inoculum contained tissue from sheep with pre-symptomatic scrapie, and that the infectious agent was unusually resistant to inactivation . Further experiments on the resistance of the scrapie agent to both ionizing and ultraviolet radiation only served to deepen its mysterious nature ' . 6

7

6

8

9


54 The differences in the inactivation profiles of prions and viruses provided the first clues that the scrapie agent was not a slow virus as had been widely thought . As preparations from Syrian hamster brain were progressively enriched for scrapie infectivity, procedures that modified proteins were found to inactivate the samples, while those that modified nucleic acids had no effect. The results of numerous studies designed to probe the molecular nature of the scrapie agent and define conditions for inactivation concluded that protein denaturants were effective at reducing infectivity titers but that complete inactivation required extremely harsh conditions . 10,

11

1 2


1 3 , 1 4

Recommendations for prion inactivation Currently recommended protocols for prion decontamination include either: (i) >2% available chlorine of sodium hypochlorite for 2 h; (ii) 2 N sodium hydroxide for 1 h; or (iii) autoclaving at 134 °C for 4.5 h " . Both sodium hypochlorite and sodium hydroxide are corrosive at the concentrations required to inactivate prions and have been reported to inactivate C J D prions incompletely under some conditions . Autoclaving is time-consuming, cannot be used with heat-sensitive or complex materials, and generates dried macerated tissue, in which prions are likely to be more resistant to inactivation. Moreover, these recommendations are largely based on the inactivation of rodent prions in crude brain suspensions. As described below, human prions are substantially more resistant to inactivation than hamster prions. Additionally, prions bound to stainless steel are more resistant to inactivation than those in tissue suspensions. Because of the foregoing limitations, surgical or dental equipment used in operations on patients suspected to have C J D is generally discarded. 14

19,

18

2 0

Iatrogenic transmission The need for a noncorrosive denaturant for prions is clearly illustrated by the cases of iatrogenic CJD in which prion-contaminated neurosurgical equipment seems to have spread prions from one patient to another ' . This concern has been heightened in Britain, where the finding of high titers of vCJD prions in lymphoid tissues worries authorities that any surgical procedure could result in the spread of prions from one patient to another . The case of a 69year-old man with vCJD, who received a blood transfusion from a young donor who eventually died of vCJD, suggests that the transfused blood contained vCJD prions . Subsequently, a second older patient who also received a blood transfusion from a young donor who died of vCJD was reported . While this 21

2 3 , 2 5

2 6

21


24

55 patient died before developing clinical signs of vCJD, the patient did have prions in his lymphoid tissues. A third case of iatrogenic transmission from trans fusion was recently reported, although the complete details have not yet been released . These cases accentuate the urgent need for an effective protocol to inactivate prions as well as to identify prion-infected specimens before they are given as therapeutics as in the case of blood . A recent study of -12,000 appendices taken at routine operation found three contained P r P based on immunohistochemical (IHC) analyses . The authors argued that this finding suggests as many as 4,000 people in the U K may be carrying prions in their lymphoid tissues. Our recent study comparing the efficacy of IHC to the conformation-dependent immunoassay (CDI) suggests that IHC may underestimate prion disease by a factor of 5 . Whether the number of people in the U K thought to be harboring prions in their lymphoid tissues is closer to 20,000 is unknown. A practical method for routine prion decontamination of all surgical instruments is urgently needed to prevent cases of iatrogenic CJD. 28

2 9

Sc


30

3 1

Prion inactivation studies Dendrimers and SDS In the course of studies on the expression of foreign PrP genes in prioninfected cultured cells, branched polyamines, or "dendrimers," were shown to render P r P susceptible to proteolytic degradation . This enhanced susceptibility to degradation could be mimicked in vitro by incubating prions at pH -3.5 with polyamine dendrimers . Intrigued by the ability of weak acids such as acetic acid (AcOH), in combination with dendrimers, to render prions susceptible to proteolytic degradation, we decided to explore the effect of a variety of protein denaturants on prion stability under weakly acidic conditions. O f all the detergents and chaotropes examined, SDS combined with A c O H proved to be the most potent reagent for inactivation of prions. This observation was unexpected since SDS at neutral pH exhibits only a modest ability to inactivate prions in our experience . The experiences of others are also noteworthy: 3% SDS at neutral p H is reported to have destroyed prion infectivity in brain homogenates when samples were boiled or autoclaved " . However, prion infectivity in macerated brain samples survived boiling for 15 min in 5% SDS at neutral pH . These findings suggest that SDS solutions at neutral pH, even when exposed to high temperatures, cannot be used for complete inactivation of prion infectivity. Sc

3 2

33

34

35

37

3 8


56


Selection of prion strain Our initial inactivation studies were performed on the Sc237 prion strain in Syrian hamster (SHa) brain homogenates for three reasons: (1) SHa prions are the most well characterized with respect to physical properties; (2) the titers of prions in brain are 10- to 1,000-fold higher than those found in other species; and (3) high-titer samples produce disease in 70 days in hamsters and 45 days in Tg mice overexpressing SHaPrP. These high titers of prions create a broad dynamic range over which measurements can be made and the short incubation times create the most sensitive system available for evaluating low levels of infectivity.

Acidic SDS denatures PrP

Sc

Sc

To examine whether solutions of 1 to 4% SDS could denature P r P more effectively at different p H values, 50 m M sodium acetate and Tris acetate buffers were used to maintain the pH between 3.5 and 10.0 for homogenates prepared from the brains of Syrian hamsters infected with Sc237 prions. Our studies demonstrated that aqueous solutions of >1% SDS could denature P r P completely at pH values 10.0, as judged by immunoblotting for PrP after the samples were subjected to limited proteolysis (Figure 1). Acidic solutions other than A c O H were found to enable SDS to denature P r P in Sc237-infected brain homogenate . However the type of detergent required was much more specific: upon testing a wide range of different detergents, only alkyl sulfates with alkyl chains of 9 to 12 carbon atoms and alkyl sulfonates with alkyl chains of 10 to 13 carbon atoms were effective at denaturing prions . Sc

Sc

39

39

Measuring prion inactivation by bioassay Sc

To determine whether denaturation of P r P caused by acidic SDS correlates with a reduction in prion infectivity, Sc237-infected brain homogenates were incubated for 2 h with different buffers at various temperatures and inoculated intracerebrally into Syrian hamsters. Inoculation of an infectious brain homogenate at neutral p H without detergent caused disease in all animals in 84 ± 0.4 d. Animals inoculated with samples exposed to either 0.5% A c O H or 1% SDS alone for 2 h at room temperature developed disease with similar incubation periods. In contrast, exposure of the inoculum to a solution of 1% SDS and 0.5% A c O H for 2 h at room temperature prolonged the median incubation time to 200 ± 2.3 d . 3 9


57


pH

# & # # & < p 9 ? < P ^

1%NP40Z 1%SDS ~

2% SDS

Z

4% SDS

Z

Figure 1. Western blot of prion-infected brain homogenate treated with detergents at various pH. Samples ofl% hamster brain homogenate containing Sc237prions were incubatedfor 15 min at 37°C with the indicated detergent and concentration at the indicated pH. Molecular masses based on migration of protein standards are 30 kDa (top) and 27 kDa (bottom).


58 The prolonged incubation times found after acidic SDS treatment of hamster brain homogenates indicate that the prion titers were reduced by a factor of ~10 , based on standard curves that relate the incubation time to the dose of prions in the inoculum ' . However, since all of the hamsters used for bioassay eventually developed disease, the prions were not completely inactivated. Although the data argue that SDS and dilute acid act synergistically to dimin ish prion infectivity, complete inactivation necessitated modification of the protocol . 7

40

4 l


39

Transgenic mice in prion research While these initial studies proved promising, concerns remained that extensive bioassays in hamsters would be complicated by the relatively short lifespan of these animals ; therefore transgenic (Tg) mice expressing high levels of SHaPrP, designated Tg7 mice, were employed for subsequent studies . To make these studies more clinically relevant, crude brain homogenates, rather than homogenates pre-cleared by low-speed centrifugation, were used. A 10% (w/v) Sc237-infected brain homogenate was serially diluted, and each dilution was inoculated into Tg7 mice. Kaplan-Meier analysis was applied to the data to determine the relationship between titer and incubation period (Figure 2A). As the infected brain homogenate is diluted, the median incubation period lengthens and the proportion of mice remaining disease-free increases. 4 2

2

43

Acidic SDS at elevated temperatures In an attempt to destroy the residual prion infectivity found by bioassay in Syrian hamsters , concentrations of SDS were increased to 2% and A c O H to 1%, and the treatment temperature raised to 65°C. Inoculation of a control Sc237-infected brain homogenate at neutral p H without detergent produced disease in Tg7 mice with a median incubation time of 46 d (Table 1). Although uninoculated Tg7 mice remained healthy for 500 d, some animals can develop a non-transmissible neuromyopathy after this time . Since mice are inoculated at 8-10 weeks of age, bioassays in Tg7 mice were terminated after 400 days to exclude the possibility of illness not caused by prions. After exposure of Sc237infected brain homogenates to 2% SDS and 1% A c O H at 65°C for 30 min, the inoculum produced prion disease in some of the mice, with significantly prolonged incubation times. Increasing the exposure time of the inoculum to 2 h or 18 h resulted in all animals remaining disease-free 400 days after inoculation. 39

4 4


In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007. 269 ± 3.2

82 ± 0.7

1.00

0.74

Wire

52 ± 0.3

>400

IP

0.00

1.00

Prop.

0.32

0.00

0.00

1.00

Prop.

>500

266 ± 8.5

131 ± 0 . 7

>500

IP

0.74

0.26

0.00

1.00

Prop.

Homogenate

Wire

>500

354 ± 1 . 6

215 ± 0 . 9

>500

IP

0.56

0.14

0.00

1.00

Prop.

Human sCJD prions in Tg23372 mice

1.00 0.75 >400 >400 1.00 >500 0.75 >500 Median incubation period (IP) ± standard error, in days, and proportion of animals remaining disease-free (Prop.) were calculated using Kaplan-Meier analysis.

>400

2% S D S 1 % A c O H , 18 h

>400

2% S D S 1% A c O H , 2 h

46 ± 0.2

Positive control

2% SDS 1% AcOH, 30 min

>400

Negative control

IP

Homogenate

Hamster Sc237 prions in Tg7 mice

Table 1. Inactivation of prions by 2% SDS + 1% AcOH at 65°C



9

io-

CL

1

10" 10-

2

10-3

5

g-

10-7

8

10-

10"

Incubation period (days)

10

io-

10"

11

Dilution of 10% brain homogenate:

g

8 8

g> t

. r\




4

10-2

Q.

Figure 2. Kaplan-Meier survival curves for 10-fold serially diluted prion-infected brain homogenate. Combined data of three independent experiments for each dilution, each inoculated into five (Sc237) or four (sCJD) mice. (A) Sc2'37 prions bioassayed in Tg7 mice. (B) sCJD prions bioassayed in Tg22372 mice.

10"

1

10"

3

-s

10-

1 0

Q. 2

o

o

T3 C

10"

8 /

6

10-

7

10"«

10 -9

brain homogenate:

Dilution of 10%

d)

i8

B


62 Inactivation of human prions by acidic SDS Because different prion strains exhibit distinct resistances to inactivation by chaotropic salts and heat , the inactivation of sporadic (s) CJD prions by acidic SDS was investigated. Brain homogenates were prepared from an sCJD patient homozygous for methionine at polymorphic residue 129, who carried no mutations in the PRNP gene, and whose unglycosylated proteinase K (PK)resistant band migrated at 21 kDa on SDS-PAGE . These human prions, designated s C J D ( M M l ) , is the most common form of CJD . To ensure the highest degree of sensitivity, a line of Tg mice expressing a chimeric human/mouse PrP gene, denoted Tg22372, were utilized; these mice are the most susceptible line for human s C J D ( M M l ) prions . It is noteworthy that like the human P r P inoculum, the PrP transgene in Tg22372 mice encodes methionine at position 129. Serially diluted brain homogenate containing s C J D ( M M l ) prions were inoculated into these mice and a similar relationship was deduced between the length of the incubation time and the size of the inoculum dose (Figure 2B). Prion disinfection studies were conducted on both intracerebrally inoculated brain homogenate and implanted wires that had been coated with the homogenate. Sporadic C J D ( M M l ) prions in brain homogenates were treated with 2% SDS and 1% A c O H at 65°C (Table 1). Acidic SDS treatment for 30 min at 65°C prolonged the incubation times from 131 ± 0.7 d to 266 ± 8.5 d. Increasing the time of exposure (2 h or 18 h) to acidic SDS at 65°C lengthened the incubation time and increased the proportion of Tg mice remaining disease-free. 4 5 - 4 9

5 0


5 0 , 5 1

52

Sc

Acidic SDS inactivates prions bound to steel wire To study the inactivation of prions on surfaces, stainless steel wires were soaked in 10% brain homogenates containing Sc237 or sCJD prions for 16 h at room temperature. The infectivity before and after disinfection procedures was demonstrated by subsequent implantation of the wire into the brain of a reporter animal . This system has become a model of choice to assess the inactivation of 53

39, 54-56

prions ' Wires coated with Sc237 prions were implanted into the parietal lobes of Tg7 mice and produced disease in 52 ± 0.3 d. Treatment of the wires at 65°C for 30 min with 2% SDS and 1% A c O H increased the incubation time to 82 ± 0.7 d after implantation; treatment at 65°C for 2 h further lengthened the incubation time, but most of the Tg7 mice still developed disease in fewer than 400 d. Only treatment at 65°C for 18 h resulted in all animals remaining disease-free at 400 days (Table 1). Similarly inactivation of prions from sCJD-coated steel wires resulted in prolonged incubation times, but significant infectivity remained (Table 1).


63


Acidic SDS and autoclaving abolish prion infectivity While exposure of Sc237 and s C J D ( M M l ) prions to 2% SDS and 1% A c O H at 65°C for 2 h was sufficient to destroy more than 99.99% of the infectivity in brain homogenates, complete inactivation was not achieved (Table 1). To determine i f autoclaving for a brief time in the presence of acidic SDS could eliminate prion infectivity, Sc237 and s C J D ( M M l ) prions in brain homogenates and on wire surfaces were exposed to 121°C in the presence and absence of acidic SDS (Table 2). Neither Sc237 nor s C J D ( M M l ) prions in brain homogenates or on wire surfaces were detectable by bioassay in Tg mice after exposure to 121 °C for 15 min in the presence of 2% SDS and 1% A c O H (Table 2). To quantify the reduction in prion titer from these procedures, Cox proportional-hazards model based on the serial dilution data (Figure 2) was employed to derive partial-likelihood ratios based 95% confidence intervals. Treatment of sCJD prions in brain homogenate for 30 min at 121°C (Table 2) results in a 1 0 reduction (lower 95% confidence interval, 10 ). The incubation periods for prion-coated wires cannot be converted into titers since the prions seem to be bound tightly to the surface of the wire and it is not possible to remove them for measurement. 68

53

5 7

Comparison of human and hamster prions Exposure of prion infected brain homogenates to acidic SDS resulted in large differences in the level of inactivation for the two prion strains studied (Tables 1 and 2; Figure 3). Because the titer of Sc237 prions is -1000-fold higher than sCJD prions in brain homogenates, data were analyzed by a stratified Cox regression in order to quantify differences between hamster and human prions. Taking the ratios of coefficients in the Cox model, it is possible to relate the effect of an inactivation procedure to an approximately equivalent dilution. Applying the stratified Cox regression to the 30-min exposure to acidic SDS at 65°C, a 1 0 reduction in infectivity for Sc237 prions and a 10 reduction for sCJD prions were estimated. The difference in inactivation between the two prion strains is 1 0 (95% confidence intervals, 10 - 1 0 ) . This analysis argues that sCJD prions in human brain homogenates are more than 100,000 times more resistant to inactivation than Sc237 prions in SHa brain homogenates. 5 8

90

38

52

37

68

Stabilities of various prion strains Prion strains, distinguished by incubation times, distribution of neuronal vacuolation, and patterns of P r P deposition, are enciphered in the conformation Sc




(0

Q. Q.

o" —^

TJ

(D

o" 0)

r—H

c cr 13 O

Proportion disease-free



Figure 3. Kaplan-Meier survival curves for prion infected brain homogenate before (solid line) and after (dashed line) inactivation with acidic SDS (2% SDS, 1% AcOH) treatmentfor 30 min at 65 °C. (A) Sc23 7prions bioassayed in Tg7 mice. (B) sCJD prions bioassayed in Tg22372 mice. gj

Q_ O

o

o

T3 C

CD CO CO CD CO

I

B


In New Biocides Development; Zhu, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 2007. >400 >400 >400 >400

Untreated, 15 min

Untreated, 30 min

Untreated, 2 h

2% S D S 1% AcOH, 15 min

2% S D S 1% AcOH, 30 min 1.00

1.00

0.88

0.88

>400

>400

>400

>400

62 ± 0.3 160 ± 7 . 3

Wire

0.27

IP

0.00

Prop. 146 ± 0.4

>500

1.00

>500

>500

1.00

1.00

>500

0.80

221 ± 1 . 0

0.00 0.00

IP

Prop.

0.88

1.00

1.00

1.00

0.00

0.00

Prop.

Homogenate

Wire

>500

>500

414 ± 1 5

>500

>500

207 ± 1 . 1

IP

1.00

1.00

0.27

1.00

0.78

0.00

Prop.

Human sCJD prions in Tg23372 mice

1.00 >500 2% S D S 1% AcOH, 2 h >400 >500 1.00 >400 1.00 1.00 Median incubation period (IP) ± standard error, in days, and proportion of animals remaining disease-free (Prop.) were calculated using Kaplan-Meier analysis.

46 ± 0.2 344 ± 2 0

Positive control

IP

Homogenate

Hamster Sc237 prions in Tg7 mice

Table 2. Inactivation of prions by 2% SDS +1% AcOH at 121°C


67 Sc 4 7 , 5 9 , 6 0

of P r P and display distinct conformational stabilities as reflected in their resistance to inactivation by chaotropes and heat . These and other findings argue that the conformation of PrP , which reflects both the strain and the PrP sequence, determines the stability of the prion. Conformational stability profiles, as measured by sensitivity to denaturation by GdnHCl gave halfmaximal (Gdn ) values of 1.5 M and 1.8 M for Sc237 p r i o n s , compared to a G d n value of 1.8 M for s C J D ( M M l ) prions . However, a greater than 100,000-fold difference was demonstrated in the susceptibility of these strains to inactivation by acidic SDS . It therefore not possible to extrapolate findings based on biophysical measurements to inactivation studies. Both the human vCJD strain and the mouse 301V strain are considered to represent the same strain as BSE, from which they were derived . However, they differ in their primary sequence: B S E is a bovine protein, vCJD is a human protein and 301V is a mouse protein, and they would not be expected to have the same inactivation characteristics. It is therefore essential that prion disinfection procedures are validated against the prion strain and species they are aimed to protect against. 4 7 , 4 9 ,

6 1 , 6 2

Sc

46,4?

1/2

63

1/2

39


6 4

4 9 , 6 5

Mechanism of acidic SDS inactivation Sc

Exposure to acidic SDS appears to denature PrP : while SDS between p H 4.5 and pH 10.0 at room temperature is a poor denaturant, it becomes an excellent denaturant at p H 10 (Figure 1). In our initial experiments, denaturation of P r P was measured by the loss of PK-resistant PrP and a decrement in prion infectivity. Immunodetection of PK-resistant PrP using Western blotting has a dynamic range of ~ 100-fold while bioassays measure prions over a ~10 -fold range as described above. In determining the mechanism of acidic SDS-mediated denaturation of PrP , immunoassays are useful but only prolonged bioassays are adequate to assess whether or not complete inactivation of prion infectivity has been achieved. Sc

9

Sc

Prion inactivation with acidic SDS From the foregoing studies, acidic SDS provides for the first time a means of completely inactivating human prions under relatively gentle conditions. s C J D ( M M l ) prions on steel wires were not completely inactivated by autoclaving at 121 °C in the absence of acidic SDS (Table 2); this contrasts with Sc237 prions that were completely inactivated by extended autoclaving alone (Table 2). It is noteworthy that the prion-coated wires were not subjected to any procedures that might reduce the level of prions, such as washing, shaking,


68 scrubbing, or sonicating. Such cleaning procedures are known to substantially reduce the titers of many different pathogens . The data presented here document the efficacy of acidic SDS combined with autoclaving for complete inactivation of human and hamster prions in brain homogenates and on the surface of steel wires (Table 2). Acidic SDS combined with autoclaving should supplant routine autoclaving used to sterilize medical and dental equipment. For equipment such as fiber optic instruments that cannot be autoclaved, submerging such equipment in acidic SDS at 65°C will substantially reduce prion infectivity but not completely eliminate it (Table 1). The results of inactivation studies described here argue that acidic SDS combined with autoclaving be applied immediately for sterilization of surgical and dental instruments. Because SDS is both a detergent and a protein denaturant, it should prove especially apt for sterilizing instruments with complex shapes, serrations, locks, bores, and crevices. In addition to inactivating prions on the surfaces of surgical and dental instruments and diagnostic equipment, acidic SDS may prove useful in the sterilization of ophthalmologic instruments as well as the cleaning of operating theaters and diagnostic suites. Acidic SDS is likely to find use in the cleansing and sterilization of equipment used in the production of biopharmaceuticals. Additionally, acidic SDS might be applied in the cleaning of abattoirs, meatprocessing plants, butcher shops, kitchens, and wherever mammalian products are prepared for human consumption. Acidic SDS might also be used on equipment employed in the rendering of offal. Because of its noncorrosive nature and ability to denature prions rapidly at relatively low temperatures, acidic SDS may be suitable for skin and wound cleansing.


17

In summary, it is important to recognize that procedures routinely used in medical and dental settings do not inactivate prions. That being the case, it may be prudent to institute acidic SDS protocols as configured in the studies presented here. Acidic SDS combined with autoclaving completely inactivated prion infectivity, even on steel surfaces. Inactivation of human and hamster prions mediated by acidic SDS occurs rapidly and can be achieved without boiling or autoclaving. Acidic SDS is noncorrosive and offers other practical advantages that make it suitable for widespread use.

References 1.

Pattison, I. H . , Experiments with scrapie with special reference to the nature of the agent and the pathology of the disease. In Slow, Latent and Temperate Virus Infections, NINDB Monograph 2, Gajdusek, D. C ., Gibbs, C. J., Jr.; Alpers, M. P., Eds. U.S. Government Printing: Washington, D.C., 1965; pp 249-257.


69 2.

3.


4.

5.

6. 7. 8. 9. 10. 11.

12.

13.

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