Viral Hepatitis - part 10 ppsx

Disinfection and sterilization 805 turers submit a pre-market application that may include additional specifi c microbicidal activity data, device/ chemical compatibility data and detailed instructions to the user regarding the ‘safe and effective use’ of the product. The FDA also regulates all sterilization devices such as ethylene oxide, ozone and hydrogen peroxide gas plasma sterilizers, steam autoclaves and dry heat ovens. In addition to pharmaceutical drugs, the FDA regulates chemical germicides formulated as antiseptics, used to inhibit or kill micro-organisms on the skin or in tissue. These types of chemical germicides are catego- rized basically by use pattern (e.g. antimicrobial hand- washes, patient preoperative skin preparations, skin wound cleansers, skin wound protectants and surgical hand scrubs) and are not regulated or registered in the same fashion that the EPA regulates and registers a disinfectant. Currently, data are not available to accurately assess the effi cacy of many of the antimicrobial antiseptic formulations on the market. Consequently, health- care workers must make product selection decisions based on information derived from the manufacturer, published studies in the literature and guidelines from expert groups. 5 The US Centers for Disease Control and Preven- tion (CDC) does not approve, regulate or test chemical germicides formulated as disinfectants or antiseptics. Rather, the CDC recommends broad strategies for the use of sterilants, disinfectants and antiseptics to prevent transmission of infections in the health-care environment. 1,6,7 Defi nitions The defi nitions of sterilization, disinfection, antisep- sis and other related terms such as decontamination and sanitization are generally accepted in the scientifi c community, but some of these terms are misused. It is important not only to understand the defi nition and implied capabilities of each procedure, but also to understand how to achieve and in some cases monitor each state. Sterilization and disinfection The term sterilization is one that students and profes- sionals have memorized and recited seemingly for ever. It can be the simplest and the most complex concept depending on how it is viewed and how it is applied. The defi nition of sterilization can change depending on the user’s vantage point. We choose to view this term somewhat like a hologram and will defi ne it in the context of: 1 the state of sterilization, 2 the procedure of sterilization, 3 the application of sterilization. Any item, device or solution is considered to be sterile when it is completely free of all living micro-organisms. This state of sterility is the objective of the sterilization procedure and, when viewed in this context, the defi - nition is categorical and absolute, i.e. an item is either sterile or it is not. A sterilization procedure is one that kills all micro-organisms, including high numbers of bacterial spores, representatives of the most resistant microbial forms. Sterilization can be accomplished by heat, ethylene oxide gas, hydrogen peroxide gas plasma, ozone or radiation (in industry). From an operational standpoint, a sterilization procedure cannot be categorically defi ned. Rather, the procedure is defi ned as a process, after which the probability of a micro-organism surviving on an item subjected to the sterilization procedures is less than one in one million (10 –6 ). This is referred to as the ‘sterility assurance level,’ and it is this approach that is used by the medical device industry to sterilize large quantities of medical devices. Some criteria used in the production and labelling of a sterile device are listed in Table 53.1. The application of sterilization principles in industry is much more sophisticated and controlled than sterilization procedures used in hospitals. However, steam autoclaves, ethylene oxide gas sterilizers, hydrogen peroxide gas plasma sterilizers and dry heat sterilization ovens used in health-care facilities have operational protocols that are validated by the manufacturer to accomplish sterilization, and all the variables that control for the inactivation of micro-organisms are either automated or built into simple controls in the devices. In addition, the sterilization cycles can be monitored with mechanical, chemical and/or biological indicators. The application of the sterilization process takes into account additional considerations. This approach in- volves the use strategy associated with a particular medical device (or medical fl uid) and the context of its degree of contact with patients. Spaulding in 1972 1 proposed that instruments and medical devices be divided into three general categories based on the theoretical risk of infection if the surfaces are contaminated at time Table 53.1 Criteria used in producing sterile devices Good manufacturing practices Use of biological indicators Validated sterilization process Sterility testing of a subsample of the batch subjected to the sterilization process Process controls Quality control of materials Post-sterilization testing of devices for function 1405130059_4_053.indd 8051405130059_4_053.indd 805 01/04/2005 11:44:0401/04/2005 11:44:04 Chapter 53806 of use. Briefl y, medical instruments or devices that are exposed to normally sterile areas of the body require sterilization; instruments or devices that touch mucous membranes may be either sterilized or disinfected; and instruments, medical equipment or environmental surfaces that touch only intact skin or come into contact with the patient only indirectly can be either cleaned and then disinfected with an intermediate-level disinfectant, sanitized with a low-level disinfectant or simply cleaned with soap and water. These instruments or other medical surfaces are termed (with respect to their need to be sterile at time of use) ‘critical’, ‘semi-critical’ or ‘non-critical’, respectively. Selection of the appropriate disinfecting procedure in the last category (non-critical) will include consideration of the nature of the surface, as well as the type and degree of contamination, as shown in Table 53.2. In the context of these categorizations, Spaulding (1972) also classifi ed chemical germicides by activity level. The activity levels are listed in Table 53.3 and are as follows. 1 High-level disinfection. This is a procedure that kills all viruses, fungi and vegetative micro-organisms but not necessarily high numbers of bacterial spores. These chemical germicides, by Spaulding’s defi nition, are those that are capable of accomplishing sterilization (e.g. killing of all microbial forms including high numbers of bacterial spores) when the contact time is relatively long (6–10 hours). When used as high-level disinfectants, the contact times are comparatively short (5–45 minutes). These chemical germicides are very potent sporicides and, in the United States, are those registered with the FDA as sterilant/disinfectants or simply, high-level disinfectants. 2 Intermediate-level disinfection. This is a procedure that kills vegetative micro-organisms including comparatively resistant vegetative microbial forms such as My- cobacterium tuberculosis. All fungi and most viruses are also killed. These chemical germicides often correspond Table 53.2 Relationship of germicide type, type of device or surface and process Type of germicide Type of device or surface Process Sterilant/disinfectant Critical (heat-sensitive rigid endoscopes) Sterilization (sporicidal chemical, prolonged contact time) Semi-critical (medical instruments, re- usable heat-sensitive anaesthesia circuits, endotracheal tubes, laryngoscope) High-level disinfection (sporicidal, chemical, short contact time) Hospital disinfectant (with label claim for tuberculocidal activity) Non-critical (medical equipment, blood- contaminated control knobs of medical equipment) Intermediate-level disinfection Hospital disinfectant/sanitizer Non-critical (environmental surfaces: exteriors of machines, fl oors, walls, other housekeeping surfaces) Low-level disinfection to soap and water washing Table 53.3 Levels of disinfectant action according to type of micro-organism Bacteria Virus Level of action Spores Mycobacterium spp.* Vegetative cells Fungi † Non-lipid, small Lipid, medium-sized High +‡ + + + + + Intermediate –§ + + + ±¶ + Low – – + ± ± + Plus sign indicates that a killing effect can be expected; a minus sign indicates little or no killing effect. Adapted from Favero and Bond. 1 *Laboratory potency tests usually employ M. tuberculosis var. bovis. † Includes asexual spores but not necessarily chlamydospores or sexual spores. ‡High-level disinfectants are chemical sterilants (sporicides); inactivation of high numbers of bacterial spores can be expected only when extended exposure times are used, e.g. 6–10 hours for sterilization vs 5–45 minutes for high-level disinfection. §Some intermediate-level disinfectants (e.g. hypochlorites) may show some sporicidal activity, while others (e.g. alcohols, phenolics) have none. ¶Some intermediate-level disinfectants (e.g. certain phenolics, isopropyl alcohol) may have limited virucidal activity, even though they readily inactivate Mycobacterium spp. 1405130059_4_053.indd 8061405130059_4_053.indd 806 01/04/2005 11:44:0401/04/2005 11:44:04 Disinfection and sterilization 807 to EPA-approved ‘hospital disinfectants’ that are also ‘tuberculocidal’. 3 Low-level disinfection. This is a procedure that kills most vegetative bacteria, some fungi, and some viruses. Comparatively resistant vegetative forms such as M. tuberculosis are not killed. These chemical germicides are often ones that are approved in the USA by EPA as hospital disinfectants or sanitizers. General use strategies of sterilization and disinfection using a variety of physical or chemical agents (with specifi c reference to hepatitis viruses) are shown in Table 53.4. 1 Spaulding’s system for classifying devices and strategies for disinfection and sterilization is quite conservative. There is a direct relationship between the degree of conservatism as expressed by the probability of a micro-organism surviving a particular procedure and the microbicidal potency of the physical or chemical germicidal agent. For example, a sterilization procedure accomplished by steam autoclaving, ethylene oxide gas, or hydrogen peroxide gas plasma sterilization, by design and defi nition, will result in a one-in-one million probability of a surviving micro-organism if the procedure Table 53.4 Some physical and chemical methods for inactivating hepatitis viruses* Class Class concentration or level Activity Sterilization Heat Moist heat (steam under pressure) 250 °F (121 °C), 15 min Prevacuum cycle 270 °F (132 °C), 5 min Dry heat 170 °C, 1 h 160 °C, 2 h 121 °C, 16 h or longer Ethylene oxide 450–500 mg/L, 55–60 °C Hydrogen peroxide gas plasma Manufacturer’s instructions Disinfection Heat Moist heat 75–100 °C High Liquid† Glutaraldehyde, aqueous‡ Variable High Ortho-phthalaldehyde 0.55% High Hydrogen peroxide, stabilized 6–10% High Formaldehyde, aqueous§ 3–8% High to intermediate Iodophors¶ 40–50 mg/L free iodine at use-dilution Intermediate Chlorine compounds** 500–5000 mg/L free available chlorine Intermediate Phenolic compounds†† 0.5–3% Intermediate Quaternary ammonium compounds‡‡ 0.1–2% Low *Adequate precleaning of surfaces is vital for any disinfecting or sterilizing procedure. Short exposure times may not be adequate to disinfect many objects, especially those that are diffi cult to clean because of narrow channels or other areas that can harbour organic material. Although alcohols (e.g. isopropanol, ethanol) have been shown to be effective in killing HBV, we do not recommend that they be used generally for this purpose due to rapid evaporation and consequent diffi culty in maintaining proper contact times. Immersion of small items in alcohols could be considered. †This list of liquid chemical germicides contains generic formulations. Other commercially available formulations based on the listed active ingredients can also be considered for use. Information in the scientifi c literature or presented at symposia or scientifi c meetings can also be considered in determining the suitability of certain formulations. The following US FDA site lists cleared formulations: http:// www.fda.gov/cdrh/ode/germlab.html. ‡Manufacturer’s instructions regarding use should be closely followed. §Because of the controversy regarding the role of formaldehyde as a potential occupational carcinogen, the use of formaldehyde is recommended only in limited circumstances under carefully controlled conditions of ventilation or vapour containment, e.g. disinfection of certain haemodialysis equipment. ¶Only those iodophors designed as hard surface disinfectants should be used, and manufacturer’s instructions regarding proper use- dilution and product stability should be closely followed. Check product label claims for demonstrated activity against Mycobacterium spp. (tuberculocidal activity) as well as a spectrum of lipid and non-lipid viruses. **See text. ††Check product label claims for demonstrated activity against Mycobacterium spp. (tuberculocidal activity) as well as a spectrum of lipid and non-lipid viruses. ‡‡Quaternary ammonium compounds are not tuberculocidal and may not have signifi cant effect against a variety of non-lipid viruses. This class of germicide is used primarily for routine housekeeping throughout health-care facilities. 1405130059_4_053.indd 8071405130059_4_053.indd 807 01/04/2005 11:44:0401/04/2005 11:44:04 Chapter 53808 had initially been challenged with 10 6 highly resistant bacterial spores. The risk of infection resulting from the use of an item that was subjected to this type of procedure, assuming that the procedure had been carried out properly, would appear to be zero. Correspondingly, the probability of contamination and the theoretical probability of infection associated with sterilization or high-, intermediate- or low-level disinfection with liquid chemical agents would increase as the overall germicidal potency of the selected germicidal agent or procedure decreased. A process of liquid chemical sterilization would, at best, be three orders of magnitude less reliable than a conventional sterilization procedure. From a practical standpoint, this means that there is a lower level of con- fi dence with such procedures, and if and when mistakes are made there is a higher chance of failure than with a sterilization procedure. When operational errors are made, the consequences are magnifi ed when a procedure of lower overall potency is used. When less reliable sterilization procedures such as this are used, they should invariably be accompanied by very precise protocols, policies and quality assurance monitoring. Decontamination Another term quite often used in health-care facilities is decontamination. A process of decontamination is one that renders a device or items safe to handle, i.e. safe in the context of being reasonably free from disease transmission risk. In many instances, this process is a sterilization procedure such as steam autoclaving, and this is often the most cost-effective way of decontaminating a device or item. Conversely, the decontamination process may be ordinary soap and water cleaning of an instrument, a device or an area. When chemical germicides are used for decontamination, they can range in activity from concentrated oxidative agents such as sodium hypochlorite, hydrogen peroxide or chlorine dioxide, which may be used to decon- taminate spills of cultured or concentrated infectious agents in research or clinical laboratories, to low-level disinfectants or sanitizers when general housekeeping of environmental surfaces is the objective. Antiseptic The term antiseptic is used to describe a substance that has antimicrobial activity and is formulated for use on or in living tissue to remove, inhibit growth of or inactivate micro-organisms. Quite often, the distinc- tion between an antiseptic and a disinfectant is not made. However, the differences between a disinfectant and an antiseptic are very great and applications are signifi cantly different. A disinfectant is a chemical germicide formulated for use solely on inanimate surfaces such as medical instruments or environmental surfaces. An antiseptic is formulated for use solely on or in living tissues. Some chemical agents such as iodophors can be used as active ingredients in chemical germicides that are formulated either as disinfectants or antiseptics. However, the precise formulations are signifi cantly different, use patterns are different and the germicidal effi cacy of each formula- tion differs substantially. Consequently, disinfectants should never be used as antiseptics and antiseptics should never be used to disinfect instruments or environmental surfaces. Factors that infl uence germicidal activity Micro-organisms vary widely in their resistance to sterilants and disinfectants. The most resistant microbial forms are bacterial spores (e.g. typically from the aerobic spore-forming genera, Bacillus and Geobacil- lus) and few, if any, other micro-organisms approach the broad resistance of these organisms to either heat, chemicals or radiation. A number of factors, some of which are associated with micro-organisms them- selves and others with the surrounding physical and chemical environment, can signifi cantly infl uence the antimicrobial effi cacy of chemical germicides. Some factors are more important than others, but all should be considered when planning sterilization and disinfection strategies for medical and surgical devices and materials. Briefl y, these factors are as follows. Type of micro-organism Bacterial spores are more resistant than mycobacteria, fungi, vegetative bacteria and viruses. Some types of viruses are more resistant to germicides than others. As a general guide, one should defi ne the state or degree of inactivation needed (i.e. sterilization or various levels of disinfection) and then choose the most appropriate germicidal agent and method of application. Number of micro-organisms All other factors being equal, the greater the number of micro-organisms on a device, the longer it takes to kill this microbial population. It is for this reason that devices, especially those that are disinfected, should be thoroughly cleaned prior to being sterilized or disinfected. 1405130059_4_053.indd 8081405130059_4_053.indd 808 01/04/2005 11:44:0401/04/2005 11:44:04 Disinfection and sterilization 809 Intrinsic resistance of micro-organisms Bacterial spores have already been mentioned, but very few species in the genera Bacillus, Geobacillus or Clostridium are actually responsible for hospital- acquired infections. However, organisms such as M. tuberculosis var. bovis and non-tuberculous mycobacteria, as well as naturally occurring gram-negative water bacteria such as Pseudomonas aeruginosa and other pseudomonads can, under some circumstances, be relatively resistant to chemical disinfectants. After bacterial spores, Mycobacterium spp. are considered one of the more resistant classes of micro-organisms. It is for this reason that chemical germicides approved as ‘tuberculocides’ are sometimes recommended for purposes of decontamination or disinfection when a higher activity germicide is sought. It is usually not a concern for transmission of M. tuberculosis (M. tuberculosis is transmitted via contaminated aerosols but not by surfaces), but rather a defi nition or specifi ca- tion that can be used to describe a germicide with a relatively broad range of germicidal activity. Resist- ance of certain non-lipid viruses is similar to that of mycobacteria. 8 Amount of organic soil present on the item to be disinfected or sterilized Blood, faeces or other organic soil may contribute to failure of a disinfecting or sterilizing procedure in three ways. Organic soil may contain large and di- verse microbial populations, may prevent penetra- tion of germicidal agents or may directly inactivate certain germicidal chemicals. This factor, perhaps even more than others, underscores the necessity of precleaning items thoroughly prior to disinfection or sterilization. Type and concentration of germicide Generally, with all other factors being constant, the higher the concentration of a germicide, the greater is its effectiveness and the exposure time necessary for disinfection or sterilization can be shorter. If a chemical agent is reused over a period of time, the product effectiveness may be reduced due to a variety of factors such as dilution or organic contamination. Time and temperature of exposure With few exceptions, the longer the exposure times to a given chemical agent, the greater is its effectiveness. An increase in temperature will signifi cantly increase germicidal effectiveness, but deterioration or evaporation of the agent along with an increase in corro- siveness may also occur. Other product- or process-related factors The presence of organic or inorganic loads, pH and the degree of hydration of biological material may signifi cantly affect the potency of certain chemical germicides. For these as well as other factors given above, care should be taken to examine closely and follow label instructions of proprietary germicides. Device-related factors The device or item being disinfected or sterilized must be physically and chemically compatible with the chosen procedure to ensure effectiveness and con- tinued function of the device or item. Also, factors such as ease of access and cleaning as well as the size of the device or item are important considerations. The manufacturer of the item being reprocessed is the best source of pertinent information in this regard. Inactivation of hepatitis viruses Germicidal activity of physical and chemical agents against the human hepatitis viruses has been diffi - cult to establish because most (HBV, HCV, HDV and HEV) have not yet been grown in tissue culture. With the exception of HAV, comparative virucidal testing, for the most part, has not been performed as it has for other types of viruses that can be conveniently cultured and tested in the laboratory. HAV HAV appears to have the same degree of resistance to chemical germicides and reagents as other picornavi- ruses. 8,9 Table 53.5 presents activity against HAV by various physical and chemical agents. 9,10 These data underscore the importance, in practice, of thorough cleaning of surfaces to remove gross organic soil and, at the same time, reduce the level of viral contamination. HBV and HCV As pointed out in other parts of this book, hepatitis B and C are diseases of major public health signifi - cance. Both viruses can be transmitted in health-care settings from patient to patient and from patient to staff member, and HBV has been shown to have an environmentally mediated mode of transmission. This is due, at least in part, to the comparatively high numbers of HBV in the blood of certain infected pa- 1405130059_4_053.indd 8091405130059_4_053.indd 809 01/04/2005 11:44:0401/04/2005 11:44:04 Chapter 53810 tients (sometimes as high as 10 8 –10 9 /mL) and also, the ability to survive for a period of time after drying. 11 However, as these viruses cannot be grown in tissue culture, data used to verify disinfection and sterilization procedures have been deduced from experiments using human volunteers, blood products that received some degree of treatment and where disease or infection was followed in human recipients, or experiments in which chimpanzees were used to determine HBV inactivation using infectivity as a crite- rion. In addition, there have been other experimental approaches to demonstrate that certain physical and chemical agents can alter the immunological reactivity of hepatitis B surface antigen (HBsAg) as well as the morphological alteration of various components of the intact virus. We have used HBsAg as a marker for HBV in order to determine the potential for environmentally mediated modes of transmission as well as inactivation capabilities of various chemical and physical agents. 12,13 Detection of HBsAg on environmental surfaces does not indicate positively the simultaneous presence of viable HBV, but it does serve as an indicator of contamination with potentially infective material. We and others have shown that HBsAg can be quantifi ed and traced in environmental surfaces as an adjunct to longitudinal or epi- demic investigations. 13–18 Because there is no evidence to suggest that the resistance level of HBV is equivalent to or even approaches the demonstrated stability of the immunological reactivity of HBsAg, we proposed in 1977 that the immunological reactivity of HBsAg is much more resistant to physical and chemical stresses than is the infectious virion. Consequently, those chemical and physical stresses that were shown to destroy the immunological reactivity of HBsAg can be assumed to be effective against HBV. We further proposed that the resistance level of HBV be considered equivalent to that of M. tuberculosis, i.e. less resistant than bacterial spores but more resistant than most micro-organisms. Subsequently, even this assumption was shown to be overly conservative when a number of intermediate- to high-level disinfectants were shown to be effective against HBV. 4 Table 53.5 Effects of chemical agents and heat on HAV viability Inoculum/exposure conditions Agent (exposure time) Result (log reduction) Tissue culture-derived HAV with 10% faeces added, dry inoculum, 1 min exposure – 2% glutaraldehyde >4 – 5000 mg/L cl 2 >4 – 0.4% quaternary ammonium compound plus 23% HCl >4 – 3000 mg/L available chlorine <1 – iodophor, 75 mg/L I 2 <1 – phenolics, with and without alcohol <1 – quaternary ammonium compounds, with and without alcohol <1 – 70% ethanol <1 – 3.5% peracetic acid <1 – 6% hydrogen peroxide <1 Tissue culture-derived HAV, room temperature, liquid inoculum – 10 mg/L available chlorine (15 min) 3 – 3 mg/L I 2 (15 min) 3 – 300 mg/L peracetic acid (15 min) <3 – alcohol (3 min) 2.25 – alcohol (12 h) 4.75 HAV + chimpanzee faeces, 18% suspension, 10 6 MID/mL, marmoset IV recovery* – 500 mg/L available chlorine (10 min) <4 – 5000 mg/L available chlorine (10 min) 4† – 75 °C wet (10 min) <5 – 75 °C wet (30 min) ≥5 – 25 °C dry, 42% RH (1 month) <5‡ Tissue culture-derived HAV – room temperature (1 week) 2 – 60 °C wet (6–12 h) >5.25 – 85 °C wet (1 min) >5.25 MID, marmoset infective doses. Modifi ed, in part, from Thraenhart 1991 (Tables 26–13, 26–14, 26–16). 9 *McCaustland KA, Bond WW, Spelbring JA, unpublished data. †104 MID per test, both animals inoculated with treated material were not infected. ‡From McCaustland et al. 10 1405130059_4_053.indd 8101405130059_4_053.indd 810 01/04/2005 11:44:0501/04/2005 11:44:05 Disinfection and sterilization 811 HBV has been shown to be inactivated by several moderately potent disinfectants, including 0.2% and 0.1% glutaraldehyde, 500 p.p.m. free chlorine from sodium hypochlorite, an iodophor disinfectant and isopropyl or ethyl alcohol. 11,19 Table 53.6 gives a summary of some of the inactivation potentials of various physical agents and germicides in tests using titred inocula and chimpanzee infectivity assays. 11,19,20 As infectivity experiments using chimpanzees are not suitable for the quantitative determination of HBV inactivation, more reliance has been placed on other avenues of experimentation. Thraenhart et al. 21,22 de- veloped a test referred to as the ‘morphologic alteration and disintegration test’ (MADT). This has been standardized and used in Germany for determining the effect of chemical germicides and physical agents on HBV. The hypothesis of MADT is that the physical destruction of intact HBV virus particles is correlated with the inactivation of infectivity using chimpanzee infectivity tests. 22 The MADT uses the human HBV but the test procedure is too complicated and cumbersome for routine use. It requires expensive equipment and personnel with considerable experience and skill in electron microscopy. The HBV suspensions required need to be highly concentrated and pure enough to allow the viral particles to be readily visualized and counted under the electron microscope. And some chemical germicides such as glutaraldehyde and alcohol act by fi xing proteins and preserving the structural integrity of the material being treated. Virus particles exposed to them may appear morphologically unal- tered, although being non-infectious. This could lead to false-negative results. Testing based on HBV polymerase inactivation has also been used to test germicides. 23 This testing method has not been widely accepted because it is based on an indirect measure of virus infectivity and requires highly purifi ed virus preparations. The sensitivity of the test is also questionable. There have been recent studies that propose the use of animal hepatitis viruses as surrogates in germicide testing protocols. The Peking duck virus resembles HBV closely in viraemia, carcinoma production, 24 and inactivation profi les. 25,26 The duck hepatitis B virus (DHBV) infects primary duck liver cells in vitro, thus making the test more cost-effective compared with the use of chimpanzees or even ducklings as experimental animals. 25 The US EPA issued guidelines in August 2000 27 on protocols for testing the effi cacy of disinfectants that use DHBV as a surrogate for HBV. Pugh and co-investigators 28 inoculated carriers with DHBV, dried them and then exposed them to specifi c germicides. After a specifi ed contact time, the disinfectant–virus mixture is eluted off the test carriers, neutralized and then serially diluted for culture in duck hepatocytes. Detection of DHBV replication is performed either by immunofl uorescence or by nucleic acid detection methods. Chan-Myers and Roberts 20 tested ortho-phthalaldehyde (OPA), for effi cacy against DHBV and bovine viral diarrhoea virus (BVDV), a surrogate for HCV. The virus cultures containing 5% horse serum as organic soil load were dried onto the bottom of petri dishes and a Table 53.6 Complete inactivation of HBV inoculum by chemicals and heat* Inoculum Treatment Reference no. Human plasma (dry) 10 min, 20 °C (all tests) 11 10 6 CID 500 mg/L available chlorine sodium hypochlorite 10 6 CID 70% isopropyl alcohol 10 6 CID 0.125% glutaraldehyde 0.44% phenol 10 6 CID 75 mg/L available iodine; iodophor 10 6 CID 2% glutaraldehyde, pH 8.6 Human plasma (liquid) 10 5 CID 5 min, 24 °C: 1% glutaraldehyde 19 2.0 × 10 5 CID 5 min, 24 °C: 0.1% glutaraldehyde 19 3.3 × 10 5 CID 2 min, 24 °C: 80% ethyl alcohol 19 10 5 CID 2 min, 98 °C 19 Surrogate viruses 25 Duck hepatitis B virus 5 min, 20 °C: 0.31% 5 log reduction CID, chimpanzee infective doses. *As measured by chimpanzee infectivity tests; titred inocula. 1405130059_4_053.indd 8111405130059_4_053.indd 811 01/04/2005 11:44:0501/04/2005 11:44:05 Chapter 53812 quantity of known concentration of OPA solution was added. After an exposure of 5 minutes at 20 ºC, the virus was recovered and titrated on monolayers of either duck hepatocyctes or bovine turbinate cells for infectivity. The results showed that dilute OPA (0.31%; the use concentration of OPA is 0.55%) completely inactivated both DHBV and BVDC in 5 minutes at 20 ºC. Testing against HCV is also diffi cult because the virus cannot be visualized or effectively grown in tissue culture. HCV replicate in Vero cells without producing any cytopathic effects. However, BVDC has some prop- erties similar to HCV and has been used in the blood product industry as a surrogate for HCV.29–31 Recent studies show that the enveloped nature of HCV makes it relatively susceptible to inactivation by phenolics and chlorine. 32 Other hepatitis viruses The effects of physical and chemical agents on other human hepatitis viruses, HDV and HEV, have not been studied extensively but, as mentioned previously, we are aware of no evidence to suggest that any of these viruses are intrinsically more resistant to physical or chemical agents than most viruses or that the general resistance levels can even approach that of bacterial spores. Consequently, we continue to propose that the resistance levels of the human hepatitis viruses that have not been studied in great detail be considered near that of M. tuberculosis var. bovis and non-lipid viruses (e.g. poliovirus), but much less than that of bacterial spores. Sterilization, disinfection and housekeeping in the laboratory Conventional sterilization procedures such as steam autoclaving, dry heat, ethylene oxide gas and hydrogen peroxide gas plasma can be relied upon to effectively inactivate all hepatitis viruses. This is also true for liquid chemical germicides used as sterilants (sporicides). Such procedures are used primarily for medical instruments that are reprocessed for use on patients in health-care facilities. It is emphasized that this class of potent chemical germicide is designed and intended for exclusive use in ‘total immersion’ reprocessing of certain heat-sensitive medical instruments and is not appropriate for use on environmental surfaces. In the context of laboratory settings where liquid chemical germicides may be used to disinfect laboratory worktops or laboratory instruments directly exposed to cultured or concentrated hepatitis viruses or human or animal source specimens containing these agents, it is recommended that chemical disinfectants in their appropriate concentrations and contact times capable of producing an intermediate level of disinfection activity should be used (e.g. oxidative or phenolic chemicals: see Table 53.4). For general housekeeping purposes such as cleaning fl oors, walls and other similar environmental surfaces in the laboratory area, any disinfectant-detergent product can be used according to the manufacturer’s instructions. In some high-risk areas such as laboratories, haemodialysis units and other spill- or splash- prone health care environments, one is confronted with the problem of decontaminating large and small blood spills, patient care equipment that becomes contaminated with blood and frequently touched instrument surfaces such as control knobs, which may play a role in environmentally mediated transmission of hepatitis B. The strategies for applying the principles of HBV inactivation vary according to the item or surface being considered, its potential role in the risk of hepatitis virus transmission and, to a certain extent, the thermal and chemical sensitivities of the surface or instrument. For example, if a signifi cant spill of blood occurred on the fl oor or a countertop in a laboratory, the objective of the procedure to inactivate HBV or other blood-borne hepatitis viruses would be one of decontamination or disinfection and not sterilization. Consequently, in such a situation we would recommend that gloves be worn and the blood spill be absorbed with disposable towels. The spill site should be cleaned of all visible blood, and then the area should be wiped down with clean towels soaked in an appropriate intermediate-level disinfectant such as a freshly made 1/100 dilution of commercially available household bleach (approximate 5–6% sodium hypochlorite depending on brand, intended minimum of 0.05% at fi nal dilution). All soiled towels should be put in a plastic bag or other leak-proof container for dis- posal. The concentration of disinfectant used depends primarily on the type of surface that is involved. For example, in the case of a direct spill on a porous surface that cannot be physically cleaned before disinfection, 0.5% sodium hypochlorite (5000 mg/L available chlorine) should be used. On the other hand, if the surface is hard and smooth and has been cleaned appropriately, then 0.05% sodium hypochlorite (500 mg/L available chlorine) is suffi cient. For commercially available chemical disinfectants, the use concentrations and instructions specifi ed by the manufacturer should be closely followed. Other types of environmental surfaces of concern include surfaces that are touched frequently, such as control knobs or panels on laboratory instruments. Ideally, gloves should be worn and manipulated in a manner 1405130059_4_053.indd 8121405130059_4_053.indd 812 01/04/2005 11:44:0501/04/2005 11:44:05 Disinfection and sterilization 813 appropriate not only to avoid skin contact with patient materials but to avoid ‘fi nger painting’ of this contamination to a variety of other frequently touched surfaces. As this ideal is seldom fully realized in a busy laboratory setting, laboratory instrument and equipment surfaces (including fi xtures such as light switches and door pulls or push plates) should be routinely cleaned and disinfected. The objective here would be to reduce the level of possible contamination to such an extent that the likelihood of disease transmission is remote. In a practical sense, this could mean that a cloth soaked in either 0.05% sodium hypochlorite or a suitable proprietary disinfectant or disinfectant/detergent could be used. In this context, the element of physical cleaning is as important, if not more important, than the choice of the disinfectant. It is not necessary, cost-effective or in many cases even feasible to attempt more powerful germicidal procedures with these types of items or surfaces. As a rule, routine daily cleaning procedures used for general microbiological laboratories can be used for laboratories in which blood specimens are processed. Obviously, special attention should be given to areas or items visibly contaminated with blood or faeces. Fur- thermore, cleaning personnel must be alerted to the potential hazards associated with blood, serum and faecal contamination. Floors and other housekeeping surfaces contaminated in this manner should be thoroughly cleaned of gross material and then treated with a detergent-disinfectant. Gloves should be worn by cleaning personnel doing these duties. However, in the case of large blood spills as mentioned above, this type of procedure may have to be augmented by specifi c site decontamination using a more potent chemical agent such as an intermediate-level disinfectant (see Table 53.4). Conclusions Strategies for disinfection and sterilization used in hospitals and other health-care institutions are based on relatively conservative criteria and do not need to be changed because of concern regarding the presence of hepatitis viruses. These viruses are inactivated by a wide variety of common physical and chemical sterilization and disinfection procedures. The resistance of individual species of hepatitis viruses to heat and chemical germicides varies, but none exceeds the resistance levels of bacterial spores or M. tuberculosis. Consequently, conventional sterilization, disinfection or decontamination procedures can be used for processing medical devices used on patients known to have viral hepatitis infection. Extraordinary procedures or formulations are not needed, nor is there an indication for the preferential use of products with specifi c label claims of effi cacy against specifi c hepatitis viruses. References 1 Favero M, Bond W. Chemical disinfection of medical surgical material. In: Block SS, ed. Disinfection, Sterilization and Preserva- tion, 5th edn. Philadelphia, PA: Lippincott, Williams & Wilkins, 2001:881–917. 2 Rutala, WA. Guideline for selection and use of disinfectants. 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Arch Virol Suppl 1993;8:133–9. 27 US Environmental Protection Agency. Scientifi c Advisory Panel (SAP) September 1997 meeting: effi cacy testing for hepatitis B type virus. Washington, DC: Antimicrobials Division, US Envi- ronmental Protection Agency, 1997. 28 Pugh C, Ijaz MK, Suchmann DB. Use of surrogate models for testing effi cacy of disinfectants against hepatitis B virus. Am J Infect Control 1999;27:375–6. 29 Borovec S, Broumis C, Adcock W, Fang R, Uren E. Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat. Biologicals 1998;26:237–44. 30 Chandra S, Cavanaugh JE, Lin CM et al. Virus reduction in the preparation of intravenous immune globulin: in vitro experiments. Transfusion 1999;39:249–57. 31 Shimizu YK, Purcell RH, Yoshikura H. Correlation between the infectivity of hepatitis C virus in vivo and its infectivity in vitro. Proc Natl Acad Sci USA 1993;91:6037–40. 32 Agolini G, Russo A, Clementi M. Effect of phenolic and chlorine disinfectants on hepatitis C virus binding and infectivity. Am J Infect Control 1999;27:236–9. 1405130059_4_053.indd 8141405130059_4_053.indd 814 01/04/2005 11:44:0501/04/2005 11:44:05 [...]... UTA-6 UHCV-11 UHCV-32 - - - - - - - - - - - + + - - + + - - + + - - + - + - + - + - + - + Ab Tet IFN-α UHCV-11 - α1 α2 α3 + + + + + + + + ISGF3 SIF-A SIF-B SIF-C (b) kDa 28.4 (c) 20.5 It was recently found that transgenic mice expressing the HCV polyprotein under the transcriptional control of an α1-antitrypsin promotor had a strong inhibition of IFN-α-induced Jak-STAT signalling in their liver cells.49... Virus infection IFN-αn IRF-3 HCV NS3/4A ISGF3 Stat1 +Stat2 +IRF-9 VAK (TBK1) p IRF-3 ATF2/ c-Jun p IRF-7 VAK(IKKε) IRF-7 NFκB IRF-7 3 3 3 3 IFNs The system is controlled by three crucial features of IFR-7: expression of the IRF-7 gene is totally dependent on IFN-α/β-induced ISGF3 signalling, the IRF-7 protein has a short half-life, and IRF-7 (as well as IRF-3) must be activated by virus-induced phosphorylation.55... anti-HBV strategies J Gastroenterol Hepatol 2002;17(Suppl.):S460–S463 8 LeGuerhier F, Pichoud C, Guerret S et al Characterization of the antiviral effect of 2’,3’-dideoxy-2’, 3’-didehydro-beta-L-5fluorocytidine in the duck hepatitis B virus infection model Antimicrob Agents Chemother 2000;44:111–22 9 Le Guerhier F, Pichoud C, Jamard C et al Antiviral activity of beta-L-2’,3’-dideoxy-2’,3’-didehydro- 5- uorocytidine... Jak-STAT signalling occurred downstream of STAT tyrosine phosphorylation and resulted in reduced upregulation of IFN-α target genes,45 as well as in an inhibition of antiviral IFN effector functions.48 Interestingly, PKR activity was not influenced by the expression of HCV proteins in these cell lines (see below) 30/03/2005 12:53:35 818 Chapter 54 Cell line (a) UTA-6 UHCV-11 UHCV-32 - - - - - - - - - -. .. A AP-GA V -D -Q -G -T-F-ACPN -V -T R -A R-K -A D T Figure 54.5 The HCV ISDR Examples of sequences of HCV amino acid residues 2209–2248 within the NS5A protein in genotype 1b-infected IFN-α non-responders and responders Amino acid residues are indicated by the standard single-letter code Dashes indicate residues identical to those in the HCV-J prototype genotype... for hepatitis B virus and Epstein-Barr virus Antimicrob Agents Chemother 1995;39:979–81 54 Condreay LD, Jansen RW, Powdrill TF et al Evaluation of the potent anti -hepatitis B virus agent (-) cis- 5- uoro- 1-[ 2-( hydroxymethyl )-1 ,3-oxathiolan-5-yl]cytosine in a novel in vivo model Antimicrob Agents Chemother 1994;38:616–19 55 Schinazi RF, Gosselin G, Faraj A et al Pure nucleoside enantiomers of beta-2’,3’-dideoxycytidine... the hepatitis B virus (HBV) and hepatitis C virus (HCV) are leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (HCC) worldwide Interferon-α (IFN-α) or pegylated IFN-α (PEG-IFN-α), combined with ribavirin in the case of chronic hepatitis C, are currently used to treat both forms of chronic viral hepatitis Sustained virological response rates, however, are limited to 3 0-4 0%... antihepadnaviral activities of combinations of penciclovir, lamivudine, and adefovir Antimicrob Agents Chemother 2000;44:551–60 29 Aguesse-Germon S, Liu SH, Chevallier M et al Inhibitory effect of 2 - uoro-5-methyl-beta-L-arabinofuranosyl-uracil on duck hepatitis B virus replication Antimicrob Agents Chemother 1998 42:369–76 30 Heijtink RA, De Wilde GA, Kruining J et al Inhibitory effect of 9(2-phosphonylmethoxyethyl)-adenine... Standard 109 108 107 A Untreated IFN-α 0 24 48 72 96 0 24 48 72 96 Time (h) HCV 28S ß-act HCV replicons/µg RNA (x108) (a) (b) 4 3 2 Untreated IFN-α 1 0 0 24 48 72 Time (h) 96 Figure 54.2 IFN-α inhibits HCV subgenomic RNA replication (a) Northern blot analysis of a Huh-7 cell line harbouring a subgenomic HCV replicon Cells were incubated for the indicated times in the absence or presence of 100 0 U/mL IFN-α... observed previously in primary duck and woodchuck hepatocytes It emphasizes that long-term antiviral treatment is required to control 824 1405130059_4_055.indd 824 01/04/2005 11:46:31 New in vitro testing systems for hepatitis B and C viruses Control 3TC (µM) β-L-Fd4C (µM) 0.1 1 0.1 1 - RC - L - CCC - - SS - Figure 55.1 HBV DNA synthesis in human primary hepatocytes and its inhibition by nucleoside . α2 α3 ++ ++ ++ ++++ + IFN-α +-+ - -+ + +- - - ++++ Cell line UTA-6 UHCV-11 UHCV-32 UHCV-11 Ab Tet (a) (c) (b) Figure 54.3 Inhibition of IFN signalling through the Jak-STAT pathway in cells expressing. IFN-αn ISGs 7 3 IRF-7 IRF-3 IRF-3 ATF2/ c-Jun VAK (TBK1) NFκB IFN-β/α1 HCV NS3/4A IFN-α1 IFN-β 3 3 3 3 p IRF-7 IRF-7 p Virus infection ISGF3 Stat1 +Stat2 +IRF-9 VAK(IKKε) IFN-αn IFN-α/β receptor Figure. features of IFR-7: expression of the IRF-7 gene is totally dependent on IFN-α/β-induced ISGF3 signalling, the IRF-7 protein has a short half-life, and IRF-7 (as well as IRF-3) must be activated