Disinfection

S. Specter D. Jeffries
Virucidal agents may inactivate viruses either because of their physical or chemical properties. These agents generally are effective on viruses outside of their host cells. The ability of these agents to inactivate virus is very closely associated with the conditions under which they are used. Depending on the agent, this may include temperature, pH, moisture content of the sample, concentration and composition of the virus, concentration of the agent, and time of exposure. Thus, viruses kept at room temperature will become inactivated more rapidly than those held at 4~ Likewise some viruses maintained at neutral pH may be inactivated less rapidly than at either lower or higher pH (Lancz 1976), other viruses such as enteroviruses may be stable at highly acidic pH. Some viruses are also known to lose infectivity as the sample in which they are collected dries (Parker et al 1944). Thus, virus maintained in mucus containing secretions on an environmental surface is likely to retain infectivity longer than the same amount of virus in saliva, since the latter will dry more quickly (Dunham 1977). An important factor in the effectiveness of disinfection is the virus titer present at the outset of addition of the disinfectant. The loss of infectivity over time is such that if a sample that is treated with a virucidal agent is held for 15 min it may lose 50% of its infectivity, after 30 min 90% is lost, and by 60 min there is a 99% loss. If a sample contains 1000 infectious particles per milliliter at outset, after 15 min 0.1 ml will contain approximately 5 infectious units, whereas after 60 min 0.1 ml Virology Methods Manual ISBN 0-12-465330-8 has a 10% chance of containing infectious material. By contrast, if the original concentration of virus is 106 particles per milliliter, then even a 99% inactivation after 1 h may leave sufficient infectious material to cause infection; i.e. 1000 infectious units will be present in 0.1 ml. The ability of various agents to inactivate viruses is also dependent on the composition of the virus. Many viruses contain a lipid envelope that is sensitive to lipid solvents such as ether, chloroform or detergents, whereas naked (non-enveloped) viruses are resistant to such agents (Dunham 1977). Finally, the nature of the disinfection agents can determine the extent of inactivation. This will depend upon whether the agent is physical or chemical and if it is organic or inorganic. It should be noted that other organic material in solutions containing viruses may inhibit the virucidal effects of many disinfectants. Wiedbrauk and Johnston (1993) have delineated which compounds are particularly useful for viruses that are commonly encountered both in the laboratory and on environmental surfaces. Inactivation of commonly used disinfectants is shown in Table 18.1. The methods for testing virucidal agents are well delineated (Koski and Chen 1977).

Inorganic chemical agents

The most commonly used inorganic disinfectants are halophilic compounds, including Copyright 9 1996 Academic Press Ltd All rights of reproduction in any form reserved

Virology methods manual

Table 18.1. Inactivation of commonly used disinfectants

Protein

Hard Water

Detergents

Hypochlorites Phenolics Alcohols* Formaldehyde Glutaraldehyde

. . . . . .

++++ + + + +
, . . , . . m

+ + + + +
.

C C -..

* Alcohols are extremely poor in penetrating proteinaceous material and are, therefore, of very limited use as disinfectants. C, Cationic detergents chlorine and bromine (Brown et a11963; Clarke and Kabler 1964; Lund 1964). The most readily available antiviral for use is common household bleach (sodium hypochlorite), which usually can be used as a 10% solution (the solution must contain 10,000 ppm available chlorine), to inactivate many enveloped and naked viruses. The bleach is capable of destroying nucleic acids, making it a universally effective disinfectant for viruses. Thus, it is the recommended disinfectant for routine use in the clinical laboratory, the physician's office, and any setting where concerns about viral contamination exist. Its ability to inactivate nucleic acids makes household bleach an ideal agent for decontamination of a laboratory where polymerase chain reactions are performed, and carry over of nucleic acids is a concern (Prince and Andrews 1992). Chlorine and bromine are useful for the inactivation of viruses in swimming pools when used at about 0.5 parts per million. Iodine also can be used as a disinfectant of contaminated water supplies (Dunham and MacNeal 1944; Kabler 1962). Ozone is considered to be safer and more effective than chlorine for the disinfection of water, but is not practical for widespread use due to cost (Sliter 1974). Similarly, hydrogen peroxide can be used to inactivate viruses. However, the presence of other organic materials in fluids will compete with the viruses and block the effects of the H202. Acid solutions, such as 1 N HCI, and alkaline compounds, such as 2% NaOH are also useful disinfectants for inactivation of viruses. Metals and metal salts also have antiviral properties. Copper salts, mercury, potassium 354 permanganate and silver nitrate have been used as virucidal agents (Dunham 1977).

Organic chemical agents

The list of organic compounds with virucidal activity is quite long, and includes quaternary ammonium compounds, alcohols, aldehydes, ether, glycerol, beta-propiolactone, ethylene oxide, digestive enzymes and a preparation known as liquor antisepticus. Benzalkonium chloride (Zephiran) has been demonstrated to be useful for the disinfection of skin. It is the preparation of choice for cleansing animal bites, due to its ability to inactivate rabiesvirus when used at a 1:1000 dilution. By contrast this compound is not highly effective against naked viruses. O-phenylphenol is likewise active against enveloped but not naked viruses (Klein and Deforest 1963). Diethyl ether is also an effective agent for inactivating lipid containing viruses, showing virucidal activity at concentrations of 310% for these viruses. However, ether is not effective against poliovirus (a non-enveloped virus), even at 95% concentration. Seventy percent ethanol is an effective disinfectant for naked and enveloped viruses, but can be decreased in activity by substances that precipitate alcohol. This can be overcome by the addition of 0.005 N NaOH to the alcohol solution. While lower concentrations of ethanol may be effectively virucidal, the higher concentration is more effective, with most virus destroyed within a few minutes by 70%. Methanol also may be used to inactivate virus, C h a p t e r 18

Organic disinfection agents

but is considerably less effective than ethanol (Cox et al 1947). However, alcohols penetrate organic matter rather poorly and are not effective for disinfection of such materials. Furthermore, the inactivation of HIV and other viruses by alcohols is slow and the alcohol is likely to evaporate, if used for surface decontamination, before the virus is inactivated (Hanson et al 1989). Liquor antisepticus is a compound described in the National Formulary that can be used as a mouthwash (National Formulary 1965). Its principal ingredient is ethyl alcohol (28.5% by volume). Formaldehyde is an excellent virucidal agent, and can be used to inactivate viruses as a 0.3% solution (Dunham 1977; Wiedbrauk and Johnston 1993). Similarly gluteraldehyde is very useful for cold sterilization and a 2% solution will inactivate most virus in 10 min when buffered with sodium bicarbonate at pH 7.5-8.5. Beta-propiolactone is an effective virucidal agent, even when virus is in plasma, that has low toxicity in mammalian systems. It has been used in the preparation of vaccines, such as the Merieux rabies vaccine. Ionizing radiation, including X-rays, gamma rays, high energy electrons, deuterons and alpha particles, is capable of inactivating viruses. This is effected by the damaging of viral nucleic acids. However, radical scavengers are able to inhibit the effects of ionizing radiation. Viruses have different levels of resistance to ultraviolet and gamma radiation, depending on the target size of the nucleic acid. Many of the doses necessary for inactivation of mammalian viruses are listed by McCrea (1960). Ionizing radiation has been used for many years for sterilization of disposable medical supplies, and has become more popular of late for more common consumable items including food. Filters may also be used for the sterilization of liquids or air. While most common filters that are used to remove bacteria from solutions do not remove viruses, small pore filters can be used to hold back viruses. This practice is most commonly used for filtering air in biological safety cabinets or 'clean rooms' using hepa-filters. The use of such filters is generally reserved for areas where tissue culture is performed, to provide sterile air, or in Biosafety level 3 and 4 rooms, to remove the risk of contamination of the environment when pathogenic viruses are studied.

Physical agents
Elevated temperature has a distinct antiviral effect, increasing the rate of virus inactivation. The inactivation rate due to high temperature varies for different viruses and can be changed greatly by the pH of a solution. The presence of a protein stabilizer such as serum decreases the ability of heat to inactivate virus in a suspension. Most viruses lose some infectivity when heated at 56~ for 30 min. All viruses are destroyed by appropriate autoclaving, 121~ under 15 psi pressure for 15 min. Ultraviolet radiation is also a highly effective virucidal physical agent, as long as it is of suitable wavelength and intensity. Wavelengths of approximately 250 nm have been demonstrated to be effective against influenza virus (Dunham 1977). The effectiveness is significantly diminished when the virus suspension is moved away from the source of radiation. In addition, ultraviolet rays are more effective through air than water, and particulate matter (dust particles or salt crystals) can further decrease effectiveness.

Summary
There are a wide variety of methods that can be used for sterilization, disinfection and antisepsis. When possible, the recommended method for sterilization is steam heating using an autoclave. An alternative effective method of limiting contamination is to use disposable sterile items (needles, syringes, pipettes, tubes, flasks, etc.) that come in a sealed sterile container. Such items, if not autoclavable, can be sterilized using y-irradiation or ethylene oxide gas after packaging, to achieve sterilization. The recommended disinfectants for decontamination of surfaces or solutions containing viruses are bleach or gluteraldehyde. Instruments that are reused should be cleaned with detergents that will remove organic matter, prior to disinfection with these substances, as such material promotes survival of viruses.

Disinfection

355

Virology methods manual

References
Brown JR, McLean DM, Nixon MC (1963) Can J Pub Hlth 54: 267-270. Clarke NA, Kabler PW (1954) Am J Hyg 59: 119127. Cox HR, van der Scheer J, Aiston S, Bohnel E (1947) J Immunol 56: 149-166. Dunham WB (1977)In: Disinfection, Sterilization and Preservation, 2nd Edn, Block SS (Ed.) Lea and Febiger, Philadelphia, pp. 426-441 Dunham WB, MacNeal WJ (1944) J Immunol 49: 123-128. Hanson PJV, Gor D, Jeffries DJ, Collins JV (1989) Brit Med J 298: 862-864. Kabler PW (1962) Ann Rev Microbiol 16: 127-140. Klein, M Deforest A (1963) Soap Chem Spec 39: 7072. Koski TA, Chen JHS (1977) In: Disinfection, Sterilization and Preservation 2nd Edn, Block SS (Ed.) Lea and Febiger, Philadelphia, pp. 116-134. Lancz GJ (1976) Virology 75: 488-491. Lund E (1964) Am J Hyg 80: 1-10. McCrea JF (1960) Ann NY Acad Sci 83: 692-705. National Formulary, 12th Ed. (NF XlI) (1965) American Pharmaceutical Assoc, Washington, pp. 3637. Parker ER, Dunham WB, MacNeal WJ (1944) J Lab Clin Med 29: 37-42. Prince AM, Andrus L (1992) BioTechniques 12: 358360. Sliter JT (1974) J Water Pollut Control Fed 46: 4-6. Wiedbrauk DL, Johnston SLG (1993) Manual of Clinical Virology, Raven Press, New York, 273 pp.

356

Chapter 18