WO1998032334A1

WO1998032334A1 - Decontamination using guanidine thiocyanate

Info

Publication number: WO1998032334A1
Application number: PCT/US1998/001324
Authority: WO
Inventors: Laura Manuelidis
Original assignee: Yale University
Priority date: 1997-01-27
Filing date: 1998-01-22
Publication date: 1998-07-30
Also published as: AU5963598A

Abstract

A substrate such as medical or veterinary equipment or tissue are decontaminated to remove infectious agents by contacting the substrate with an effective amount of a guanidium compound such as guanidine thiocyanate. Equipment such as surgical or dental instruments are decontaminated by immersion or wetting with a guanidium compound composition, or wiping the surfaces with a material embedded or coated with a guanidium compound composition. Potentially infective tissue and tissue products are typically decontaminated by direct application of a guanidium compound composition to the biological material. One exemplary embodiment employs solutions containing at least about 3 M guanidine thiocyanate. Compositions useful in the practice of the invention may contain adjunct ingredients such as detergents, other wetting agents, stabilizers, and the like. In some embodiments, decontamination methods of the invention include other steps such as heating, sonication, and soaking to enhance the disinfectant effect.

Description

DECONTAMINATION USING GUANIDINE THIOCYANATE

Technical Field

This invention relates to the decontamination of infectious agents causing Creutzfeidt-Jakob Disease, other transmissible encephalopathies, and die like, using guanidium compounds such as guanidine thiocyanate.

Background of the Invention

Creutzfeldt-Jakob Disease (CJD) is a rare but devastating nerurode- generative disease. Although caused by a transmissible agent, clinical symptoms may not become apparent for as long as 30 years after infection. Because white blood cells carry die infectious agent, the possibility ϋiat this disease could be transmitted through routine medical and surgical procedures was considered (1). Over the past 10 years England has witnessed an explosive epidemic infection of cows with a similar agent that induces bovine spongiform encephalopathy (BSE). The BSE agent probably originated in processed feed contaminated with the sheep scrapie agent (2) but it is biologically distinct from common scrapie agent strains in sheep (3). It was suggested that variants of the scrapie agent selected in cows might become more virulent for humans tiian commonly assumed (4). Whereas endemic scrapie infections of sheep have not been linked to human disease, several unusual human CJD cases have now been linked to the bovine infection (5,6). Prion protein (PrP) polymorphisms are sometimes considered critical for infection. However, die single PrP 129 Met-Met polymoφhism found in uiese patients is very common (38% of Caucasians), and it is unlikely diat tiiis polymoφhism is always present in me wide variety of animals that have acquired BSE. There is me unsettling possibility that me incidence of CJD may rise significantly because many individuals, including young people, have been repeatedly exposed to d is new and apparently more virulent agent strain.

There is currentiy no in vivo test to detect transmissible encephalopaϋiies during early stages of infection in any species. Nor are mere any molecular markers to distinguish agent strains that are more or less virulent for humans. Inadvertent transmissions are therefore problematic. Of especial concern is die likelihood of enhanced virulence of d e human adapted BSE agent. Many experiments have shown that once mese agents are passaged in a new host they typically become more virulent for tiiat host, yielding more rapidly progressive disease. Thus spreading infections from the known BSE- infected people, as well as from other asymptomatic infected individuals, must be considered. Asymtomatic but infected people have been used as donors for merapeutic tissue and blood products (4). Sensitive tests to detect early infections to prevent iatrogenic spread of disease will ultimately rely on d e molecular characteristics of d e infectious agent. However mese are not resolved and mere are two fundamentally different concepts in play. One predominant notion is tiiat a host protein, PrP, converts itself into an infectious protein or prion (7). The alternate view is tiiat die agent is a virus diat affects host PrP (4,8,9,10). Neidier view is experimentally proven. Although PrP is clearly involved in pamogenesis, purified PrP does not correlate with titer and has failed to transmit infectious disease. On me omer hand, almough disruption of viral particles reduces CJD titer (11), only candidate viral sequences have been defined (12).

Immediate healm-related initiatives must merefore rest on proven experimental approaches to reduce titer and tiiereby minimize the risk of contracting disease. One practical problem is decontamination of human and animal tissues. Because me agent is infectious at high dilutions, is carried in the reticulo- endo elial system, and is highly concentrated in me brain at asymptomatic stages of disease (10, 13), general and simple methods to significantly reduce tissue infectivity are needed. These memods should be applicable for routine use on all residual tissues because infectivity cannot be predicted. In a surgical setting, tissue fragments can adhere to operative instruments. Some larger instruments, such as dental drill holders, are not disposable. Even mough mese instruments can be at some distance from me operative field, tiiey are exposed to tissue microdroplets. Dental work also frequendy exposes nerve roots, and tiius dental procedures could become a common source for the spread of CJD. Additionally, the expense of discarding specialized surgical instruments after a single use may not be possible in many countries. Finally, previous and proposed slaughter of cattle to reduce BSE exposure in Europe, and e routine removal of brain and spinal cord during butchering, can lead to contamination of large facilities.

Up to the present time tiiree memods have been used to reduce instru- ment contamination and mese generally reduce titer by > 3 logs. They are i) exposure to household bleach (a procedure diat can corrode many fine instruments, mechanical parts and stainless steel); ϋ) prolonged autoclaving (a method not feasible for many large instruments or surfaces); and iii) immersion in concentrated alkaline solutions, i.e., 0.1-1M NaOH. The latter solutions are corrosive not only to instruments and surfaces, but also cause severe skin burns.

Summary of the Invention

It is an object of me invention to provide a new metiiod of decontaminating infectious agents.

It is anomer object of me invention to provide a decontamination metiiod particularly efficacious for removal of infectious agents causing Creutzfeldt-Jakob Disease and other transmissible eneephalopatiiies from a substrate. These and otiier objects are accomplished by me present invention, which provides a metiiod for decontaminating a substrate such as medical or veterinary equipment, tissue, or tissue products to remove infectious agents by contacting the substrate witii an effective amount of a guanidium compound such as guanidine thiocyanate. In a typical embodiment, medical or veterinary equipment such as surgical or dental instruments are decontaminated by immersing or wetting the equipment in a composition containing a guanidium compound, or by wiping d e surfaces of the equipment wim a material embedded or coated with a guanidium compound composition. One embodiment employs solutions containing at least about 3 M guanidine thiocyanate. In ano er embodiment, potentially infective tissue or tissue products are decontaminated by contacting diem with a composition containing from about 0.5 to about 3.5 M guanidium compound.

Methods of die invention may include other steps such as sonication and/or heating (including autoclaving) and/or soaking articles in die presence of a guanidium compound, as well as otiier steps incorporating otiier mediods of disinfection and cleaning such as treatment wim chlorine dioxide and otiier germ- killing compositions.

Guanidine tiiiocyanate, guanidine hydrochloride, and the like guanidium compound compositions used in die practice of die invention may contain other ingredients such as detergents, wetting agents, stabilizers and die like in the formulations.

Detailed Description of the Invention

Broadly speaking, this invention provides a method for decontaminating a substrate to remove infectious agents by contacting d e substrate with an effec- tive amount of a guanidium compound. Substrates include any type of surface or carrier which could provide a locus for me accumulation of viruses, bacteria, fungi, spores and the like disease-causing microorganisms and pathogens. Typical substrates include, but are not limited to, medical and veterinary equipment, especially hardware such as surgical and dental instruments, as well as potentially infective tissue and tissue products.

In the practice of the invention, a substrate to be decontaminated is contacted with an effective amount of a guanidium compound (herein occasionally referred to as Gdn). Guanidium compounds include guanidine hydrochloride, guanidine thiocyanate, related compounds, including guanidium-containing compounds and derivatives, and mixmres thereof. By "guanidine tiiiocyanate" is meant guanidine tiiiocyanate (GdnSCN), active derivatives of guanidine tiiiocyanate, guanidine isothiocyanate, active derivatives of guanidine isothiocyanate, and mixtures thereof. Correspondingly, by "guanidine hydrochloride" is meant guanidine hydrochloride (GdnHCl), active derivatives of guanidine hydrochloride, and mixtures thereof. In preferred embodiments, d e substrate is contacted with a composition containing GdnSCN; liquid compositions are particularly preferred. Liquid compositions of the invention include any type diat will dissolve, suspend, or otherwise disperse die active ingredient such as solutions, suspensions, gels, and die like. Aqueous GdnSCN, GdnHCl, or GdnSCN/GdnHCl solutions are employed in many embodiments. Though lower concentrations may be employed where substrates are soaked in guanidium compositions for extended periods, typical concentrations range between about 2.5 and 6 M. As will be illustrated more fully in the next section, compositions containing at least about 2.5 M, preferably greater tiian 3.8 M, GdnSCN are useful for certain applications.

For potentially infective biological materials such as tissue or tissue products, including blood, blood products (including serum proteins), and organs involved in transplants, lower concentrations, i.e., from about 0.5 M to about 3 M, are employed in typical embodiments. In some embodiments involving decontamination of biological material such as dierapeutic tissue and blood products, potentially transmissible agents are removed prior to extraction of medically active compounds by contact with guanidium solutions. The decontamination solution is then removed from die tissue.

Compositions useful in the memods of d e invention may contain other ingredients in die formulation, preferably those diat are inert in the sense of not bringing about a significant deactivation of die active ingredient. These include other disinfectants or detergents that have disinfectant properties that augment the active ingredient of the invention; stabilizers diat prolong die shelf-life of prod- ucts containing GdnHCl and/or GdnSCN; wetting agents and detergents that enhance or assist application and/or penetration, and/or hold active ingredient in contact wim die substrate; and die like. In an embodiment illustrated hereafter, a GdnSCN composition containing sarkosyl was employed.

The substrate is typically contacted wim GdnSCN and/or GdnHCl for such time under such conditions to effect decontamination of the surface. In most embodiments, a statistically significant decrease in viral, bacterial, and/or fungal titer, preferably by __ 3 logs, from e uwiecontaminated substrate is observed. In an embodiment described in die Examples diat follow, animal assays are employed to assess die extent of decontamination. The substrate may be immersed in or wet with die GdnSCN or other guanidium composition, or wiped with a material embedded or coated with a GdnSCN or otiier guanidium composition.

Mediods of die invention employing GdnSCN and/or GdnHCl may be combined with otiier methods of sterilization, cleansing, and sanitizing. For example, some embodiments, particularly tiiose involving die disinfection and sterilization of surgical and dental instruments, may involve a sonication and/or at least one heating step. Heating to at least about 55 ^*C, and in some cases to at least about 75'C, is preferred. Instruments may be autoclaved in the presence (if in accord with E.P.A. requirements) or absence of GdnSCN and/or GdnHCl before or after contact witii it. In one embodiment, instruments immersed in a GdnSCN composition are autoclaved to achieve effective decontamination of particularly recalcitrant and/or dangerous patiiogens. Medical or veterinary hardware may be sonicated before or after treatment with GdnSCN, or sonicated in its presence such as in an ultrasonic cleaning apparatus; this is particularly advantageous where tiiere is tissue adhering to the equipment or where vigorous washing is not possible. Washing and other disinfectants such as treatment with Clorox^® or chlorine dioxide, hydrogen peroxide, base, acid, and the like may be used in conjunction with GdnSCN and/or GdnHCl use.

It is an advantage of the invention tiiat GdnSCN and/or GdnHCl is/are particularly efficacious for removal of infectious agents causing Creutzfeldt-Jakob Disease and otiier transmissible encephalopathies from a substrate, as well as other resistant pathogens such as hepatitis B. They are also likely to be effective for other resistant pathogens. In embodiments involving the treatment of tissue or tissue products, die invention provides for extraction or separation of medically relevant biological material free of transmissible agents.

The following are presented to further illustrate and explain the present invention and should not be taken as limiting in any regard.

Examples

In studies to uncover essential molecular components of die CJD agent it was found found that concentrations of GdnHCl as low as 2.5M effectively disrupted die nucleic acids and capsid proteins of viral particles. Concomitantly, CJD titer was reduced by - 3 logs (99.7%), whereas solubilization and separation of PrP from sedimenting infectious particles did not reduce titer (11). It was therefore suggested that GdnHCl treatment might be used to reduce contamination and experiments were initiated to validate this approach. Others subsequently hypothesized that GdnHCl might be useful for dental instrument decontamination (14). More disruptive GdnSCN solutions could result in even greater levels of agent inactivation and therefore verified tiύs experimentally.

To simplify the method for clinical settings, a commercially available solution of 4M GdnSCN was used (RNA lysis buffer #40082, Perkin Elmer). This solution has a similar composition as die one we use to efficiently extract nucleic acids from more purified CJD preparations (12). It conveniently contains a detergent tiiat can aid in tissue penetration and disruption, and also has a reasonable shelf life of > 1 year. Moreover, tiiis solution can be easily modified to further enhance decontamination. Moderate heat treatment was also tested to find if agent inactivation could be augmented. The temperatures tested are easily achieved with old instrument sterilizers in dentists offices. Brain tissue from a long established CJD hamster model (serial passage 31, strain SY) was chosen because it yields reproducible titers. The very high lipid composition and complexity of brain can compromise inactivating procedures and therefore whole brain was chosen as a difficult inactivation target. Experimental groups of six hamsters each were injected with: 1) CJD homogenates in saline without inactivation, 2) CJD saline homogenates treated at 75 'C for 25 min, 3) CJD homogenates lysed in GdnSCN and 4) CJD GdnSCN homogenates treated at 75 'C for 25 min.

To maximize viral input each half of a CJD infected brain was directly homogenized in either normal saline or GdnSCN lysis buffer (10% w/v). A 100- ml aliquot of each respective homogenate was heated in a 1.5 ml polypropylene tube in a PCR machine while die parallel homogenates were kept at 22^* C. Each of the four homogenates were then diluted to 0.5 mg brain/ml and 50 ml was inoculated intracerbrally per hamster (11). The large dilution was calculated 1) to preclude any toxic effects of GdnSCN, 2) to yield incubation time titers in the linear range for saline homogenates (20), and 3) to approximate end-point LD50 determinationsn for GdnSCN samples. As summarized in Table 1 (below), hamsters inoculated with CJD brain in saline developed clinical disease by 166 days. There was a small ( ~ 3 fold or 0.55 logs) but statistically significant decrease in titer with moderate heating in saline. Typical CJD changes (10) were verified histologically in both saline groups. A far more substantial reduction in titer was found in GdnSCN treated parallel samples. Indeed, none of these animals showed any signs of disease at > 350 days post inoculation. With GdnSCN die titer was reduced by ~ 5 logs (2.3xl0⁸/gm to < lOVgm). Because the GdnSCN treatment alone was so effective, the effect of heating GdnSCN homogenates to increased inactivation could not be assessed. Nonemeless, die saline controls indicate diat heating at 75 ^*C or higher should yield even more effective inactivations, although toxic fumes at 75 ^*C could be released.

Additional experiments on infectious concentrates have shown heating at 55 'C further enhances disruption by molecular analyses, and heat increase penetration into mechanical lubricants (15). Exposure of stainless steel surgical instruments (Dumont tweezers) for 24 hours to 4M GdnSCN, moreover, showed no corrosive effects. Therefore nondisposable surgical instruments may be significantly decontaminated by immersion and sonication in me above GdnSCN solution (GdnSCN wipes for steel surfaces can be incinerated). These treatments may have universal application for die inactivation of many other infectious agents, including non-enveloped viruses diat are relatively resistant to inactivation such as hepatitis B. A recent outbreak of Hepatitis B was traced to incompletely sterilized EEG needles (see below) and perhaps could have been avoided wi die above precautions.

The outlined protocol is also based on the inability to find any reconsti- tution of CJD infectivity when more purified infectious preparations were treated with less concentrated and disruptive GdnHCl solutions (11). After dilution of GdnHCl, the reduction in titer remained. Thus washing trace GdnSCN should have few risks. Using similar GdnHCl treatment and dilution, anotiier laboratory reported diat PrP rapidly refolded into an "infectious conformation" as assessed by gel assays. However, tiiere was no data showing infectious titer was restored (16). Moreover, similar refolding experiments have been irreproducible, with PrP conversion ascribed to an artifact of incompletely denatured aggregates (17). Older studies have likewise shown reductions in scrapie titer widi otiier Gdn solutions, but the application of such treatments for decontaminating instruments and surfaces have not been previously considered.

Use of a standard solution and protocol should help eliminate variable inactivations. Although somewhat different sensitivities to die same chemical and physical treatments have been reported with different agent strains (e.g., hamster adapted scrapie and CJD), all preparations are highly sensitive to > 3M GdnSCN. Additionally, minor methodological and preparation differences can often account for vastly different claims about d e resistance of these agents. The efficacy ascribed to these agents should be viewed in die framework of conventional viral studies where absolute inactivations are rarely achieved. Indeed, glutaraldehyde- treated HIV material can be infectious (15), and after dry heat treatment of tissue products at 80^*C, die risk of Hepatitis C infections can still be as high as 10% (18). These two enveloped viruses are relatively sensitive in die viral spectrum. Furthermore, Hepatitis B, a more resistant virus, was not inactivated when dry heat sterilizers were used to decontaminate reusable subcutaneous EEG electrodes (19).

Incomplete inactivations may be due, at least in part, to inadequate tissue penetration. Penetration should be significantly enhanced with strongly disruptive GdnSCN. Nonetheless, methods of die invention are disclosed witii die recognition that absolute inactivations of CJD-like agents or other resistant viruses can not yet be guaranteed. Indeed, it is possible tiiat the incomplete but still harsh rendering process used in the production of scrapie-infected feed itself selected for more resistant agent variants causing BSE. Large BSE-infected cattle are currently being sterilized by cremation, and some cow remains have already been dumped in open pits in northern Scotland. There is also die possibility tiiat some remains may be incompletely ashed, and it may be prudent now to take added new steps to prevent die spread of residual, and perhaps more resistant agent variants to wild animals. Treatment of incompletely ashed or partially inactivated remains witii GdnSCN derivatives could help prevent further propagation of evolving virulent strains.

Table 1

Group Davs signs (SEM) Titer/gm (SEM) 1) CJD-saline 166.2 (1) 2.3x10" (2. lxlO⁷) 2) CJD-saline & heat 180.2 (0.5) 6.5xl0⁷ (2.8x10*)

3) CJD-GdnSCN >350

4) CJD-GdnSCN & heat > 350 j≤.10³

TABLE I: Days incubation to clinical signs of disease and titer per gram of brain for each experimental group. Two-tailed t-test showed significant reduc- tion in homogenate titer after moderate heating in saline (p< 10-6), with far more inactivation after GdnSCN (longer incubation time). Memods: To maximize viral input each half of a CJD infected brain was directly homogenized in either normal saline or GdnSCN lysis buffer (10% w/v). A 100 ml aliquot of each respective homogenate was heated in a 1.5 ml polypropylene tube in a PCR machine while the parallel homogenates were kept at 22' C. Each of the four homogenates were then diluted to 0.5 mg brain/ml and 50 μl was inoculated intracerebrally per hamster (11). The large dilution was calculated 1) to preclude any toxic effects of GdnSCN, 2) to yield incubation time titers in die linear range for saline homogenates (20) and 3) to approximate end-point LD50 deteπninations for GdnSCN samples. Titer calculations are based on die conservative estimate diat 1 infectious unit (IU) will produce clinical signs at 294 days, and tiius end-point determinations at 600 days yield a fraction of 1 IU, e. ., 1 of 5 animals with disease at 600 days is equivalent to 0.2 IU in the inoculum. The above description is for die purpose of teaching the person of ordinary skill in die art how to practice die present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to die skilled worker upon reading die description. It is intended, however, that all such obvious modifications and variations be included within die scope of d e present invention, which is defined by die following claims. The claims are intended to cover die claimed components and steps in any sequence which is effective to meet die objectives there intended, unless die context specifically indicates d e contrary.

The invention was made with partial support from Grants NS 12674 and NS34569 from die National Institote of Healdi. The government has certain rights in the invention.

References

1. Manuelidis, EE, Gorgacz, EJ, and Manuelidis, L: Viremia in experimental Creutzfeldt-Jakob disease. Science 1978; 200: 1069-1071.

2. Anderson, RM, et al.: Transmission dynamics and epidemiology of BSE in British cattle. Nature 1996; 382: 779-788.

3. Bruce, ME: Scrapie strain variation and mutation. Br Med Bull. 1993; 49: 822-38.

4. Manuelidis, L: The dimensions of Creutzfeldt-Jakob Disease. Transfusion 1994; 34: 915-928.

5. Will, R, et al.: A new variant of Creutzfeldt-Jakob disease in die UK. Lancet 1996; 347: 921-925.

6. Lasemezas, C, et al.: BSE transmission to macaques. Nature 1996; 381: 733-4.

7. Prusiner, SB: Molecular biology of prion diseases. Science 1991; 252: 1515- 1522.

8. Bruce, ME and Dickinson, AG: Biological evidence tiiat scrapie has an independent genome. /. Gen. Virol. 1987; 68: 79-89.

9. Diringer, H, Blode, H, and Oberdieck, U: Virus-induced amyloidosis in scrapie involves a change in covalent linkages in die preamyloid. Arch. Virol. 1991; 118: 127-31.

10. Manuelidis, L and Fritch, W: Infectivity and host responses in Creutzfeldt-Jakob Disease. Virology 1996; 215: 46-59.

11. Manuelidis, L, Sklaviadis, T, Akowitz, A, and Fritch, W: Viral particles are required for infection in neurodegenerative Creutzfeldt-Jakob disease. Proc. Natl. Acad. Sci. USA 1995; 92: 5124-5128.

12. Dron, M and Manuelidis, L: Visualization of viral candidate cDNAs in infectious brain fractions from Creutzfeldt-Jakob Disease by representational difference analysis. /. Neurovirol. 1996; 2: 240-248.

13. Xi, YG, et al.: Amphotericin B treatment dissociates in vivo replication of die scrapie agent from PrP accumulation. Nature 1992; 356: 598-601.

14. Oken, R and McGeer, P: Letter to the editor. J Dental Res. 1995; 74: 1836. 15. Lewis, DL and Arens, M: Resistance of microorganisms to disinfection in dental and medical devices. Nature Medicine 1995; 1: 956-8.

16. Bessen, RA, et al.: Non-genetic propagation of strain-specific properties of scrapie prion protein. Nature 1995; 375: 698-700.

17. Riesner, D, et al.: Disruption of prion rods generates 10-nm spherical particles having high a-helical content and lacking scrapie infectivity. . Virol. 1996; 70: 1714- 1722.

18. Rizza, CR, Fletcher, ML, and Kernoff, PB: Confirmation of viral safety of dry heated factor VD3 concentrate (8Y) prepared by Bio Products Laboratory (BPL): a report on behalf of U.K. Haemophilia Centre Directors. Br J Haematol. 1993; 84: 269-72.

19. McKinnon, D.J. Thirty new cases of Hepatitis B found. All had EEGs at six clinics, officials say. Toronto Star June 6, 1996, page SC4.

20. Manuelidis, L, Sklaviadis, T, and Manuelidis, EE: Evidence suggesting that PrP is not the infectious agent in Creutzfeldt-Jakob disease. EMBO J. 1987; 6: 341-347.

The above papers are hereby incorporated herein in dieir entireties by reference.

Claims

1. A method for decontaminating a substrate to remove infectious agents comprising contacting the substrate with an effective amount of guanidine thiocyanate.

2. A method according to claim 1 wherein die substrate comprises the surfaces of surgical or dental instruments.

3. A method according to claim 2 comprising immersing the instruments in a liquid composition containing guanidine tiiiocyanate.

4. A method according to claim 3 wherein die composition is a solution containing at least about 4 M guanidine tiiiocyanate.

5. A method according to claim 3 further comprising die step of sonicating the instruments when they are immersed in die guanidine tiiiocyanate composition.

6. A method according to claim 3 further comprising at least one autoclaving step.

7. A method according to claim 6 wherein die immersed instruments are auto- claved in de guanidine tiiiocyanate composition.

8. A method according to claim 1 wherein die substrate is wiped with a material embedded or coated witii a guanidine tiiiocyanate composition.

9. A method according to claim 8 wherein de substrate is wiped with a detergent solution after wiping with the guanidine tiiiocyanate composition.

10. A method according to claim 1 wherein the substrate is tissue which is decontaminated by pouring an excess guanidine tiiiocyanate composition on the tissue.

11. A method of decontamininating equipment, tissues or tissue samples to remove infectious agents comprising contacting said equipment, tissues or tissue samples witii a composition containing an effective amount of guanidine tiiiocyanate.

12. A method according to claim 11 wherein die composition is a solution.

13. A method according to claim 12 wherein die composition contains at least about 4 M guanidine thiocyanate.

14. A method according to claim 13 wherein die equipment comprises surgical or dental instruments tiiat are decontaminated by immersing in die guanidine tiiiocyanate composition.

15. A method according to claim 11 wherein die equipment or tissues are wiped with a material coated or embedded with the guanidine tiiiocyanate composition.

16. A method according to claim 11 further comprising the step of heating the equipment, tissues or tissue samples in die presence of die guanidine tiiiocyanate composition.

17. A method for decontaminating the surfaces of medical equipment to remove infectious agents comprising contacting the surfaces with a liquid composition containing at least about 3 M guanidine thiocyanate.

18. A metiiod according to claim 18 further comprising the step of heating die liquid composition when in contact witii die medical equipment surface.

19. A metiiod according to claim 19 wherein the composition is heated to at least about 55^* C.

20. A metiiod for decontaminating a biological sample to remove transmissible agents comprising contacting die sample witii a composition containing an effective amount of guanidine thiocyanate.

21. A method according to claim 21 wherein die sample is further extracted to obtain medically active compounds.

AMENDED CLAIMS

[received by the International Bureau on 3 June 1998 (03.06.98); original claims 18, 19 and 21 amended; remaining claims unchanged (2 pages)]

11. A method of decontamininating equipment, tissues or tissue samples to remove infectious agents comprising contacting said equipment, tissues or tissue samples with a composition containing an effective amount of guanidine thiocyanate.

12. A method according to claim 11 wherein the composition is a solution.

13. A method according to claim 12 wherein the composition contains at least about 4 M guanidine thiocyanate.

14. A method according to claim 13 wherein the equipment comprises surgical or dental instruments that are decontaminated by immersing in the guanidine thiocyanate composition.

15. A method according to claim 11 wherein the equipment or tissues are wiped with a material coated or embedded with the guanidine thiocyanate composition.

16. A method according to claim 11 further comprising the step of heating the equipment, tissues or tissue samples in the presence of the guanidine thiocyanate composition.

18. A method according to claim 17 further comprising the step of heating the liquid composition when in contact with the medical equipment surface.

19. A method according to claim 18 wherem the composition is heated to at least about 55 "C.

20. A method for decontaminating a biological sample to remove transmissible agents comprising contacting the sample with a composition containing an effective amount of guanidine thiocyanate.

21. A method according to claim 20 wherein the sample is further extracted to obtain medically active compounds.