Viruses disinfectant tests

The testing of disinfectants for virucidal activity is not an easy matter. As pointed out earlier (Chapter 3), viruses are unable to grow in artificial culture media and thus some other system, usually employing living cells, must be considered. One such example is tissue culture, but not all virus types can propagate under such circumstances and so an alternative approach has to be adopted in specific instances. The principles of such methods are given below. 

A standardized viral suspension is exposed, in the presence of yeast suspension, to appropriate dilutions of disinfectant in WHO hard water. At appropriate times, dilutions are made in inactivated horse serum and each dilution is inoculated into tissue cell culture or embryonated eggs (as appropriate for the test virus). The drop in infectivity of the treated virus is compared with that of the control (untreated) virus. 

Plaque assays, at present, apply to only a very limited number of viruses, e.g. poliovirus, herpes virus, human rotavirus. The principle ofthese assays is as follows test virus is dried on to coverslips which are immersed in various concentrations oftest disinfectant... 

For assaying herpes virus, monolayers of baby hamster kidney (BHK) cells are used. Virus titre is expressed as the number of plaque-forming units (pfu) per millilitre before and after exposure to a disinfectant, so that the virucidal efficacy of the test agent can be determined. A diagrammatic representation is given in Fig. 11.7. 

The hepatitis B virus (HB V) does not grow in tissue culture and an acceptable animal model has been found to be the chimpanzee. This is observed for clinical infection after inoculation with treated and untreated virus, care being taken in the test series that residual disinfectant is removed by adequate means before inoculation into the animal. 

Duck hepatitis B virus (DHBV) has been proposed as a possible model for the inactivation of human HBV by chemical disinfectants. The principle of the test method uses viral DNA polymerase (DNAP) as a target, total inhibition in vitro of DNAP by chemical disinfectants being predictive of inactivation of infectivity in vivo. 

Studies have demonstrated that one such method is to examine the effects of disinfectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase) activity. In essence, HIV is an RNA virus after it enters a cell the RNA is converted to DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect on T lymphocytes, and in the assay reverse transcriptase activity is determined after exposure to different concentrations of various disinfectants. However, it has been suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory alternative to tests whereby infectious HIV can be detected in systems employing fresh human peripheral blood mononuclear cells. 

Tyler R. Ayliffe G.A.J. (1987) A surface test for virucidal activity of disinfectants preliminary study with herpes virus. J Hasp Infect, 9, 22-29. 

Phenol coefficient tests were developed in the early 20th century when typhoid fever was a significant public health problem and phenolics were used to disinfect contaminated utensils and other inanimate objects. Details of such tests can be found in earlier editions of this book. However, as non-phenolic disinfectants became more widely available, tests that more closely paralleled the conditions under which disinfectants were being used (e.g. blood spills) and which included a more diverse range of microbial types (e.g. viruses, bacteria, fungi, protozoa) were developed. Evaluation of a disinfectant s efficacy was based on its ability to kill microbes, i.e. its cidal activity, under environmental conditions mimicking as closely as possible real life situations. As an essential component of each test was a final viability assay, removal or neutralization of any residual disinfectant became a significant consideration. 

With tests involving liquid systems the early growth of viable cells can be assessed by some lightscattering processes while blood culture techniques have classically used the production of C02 as an indicator of bacterial metabolism and growth. In addition, the availability of molecular techniques, such as quantitative PCR, may be useful in demonstrating the presence or growth of microorganisms that are slow or difficult to culture under usual laboratory conditions, e.g. viruses. This may obviate the need to neutralize residual disinfectant with some assays. 

The vimcidal activity of monopercitric acid (MPCA) was determined by suspension tests against both nonenveloped and enveloped viruses including VACV [120], The study showed that vimses were 99.9% inactivated by a 0.5% concentration within 30 s demonstrating MPCA as a suitable candidate for a disinfectant. Sugimoto and Toyoshima [107] reported on the inactivation of VACV by N -Cocoyl-L-Arginine Ethyl Ester, DL-Pyroglutamic Acid Salt after a 30 min exposure at room temperature. At all tested concentrations (0.025%, 0.05%, 0.1%, and 0.25%), there was greater than 90% inactivation of VACV. 

CEN Virucidal Suspension Test for Chemical Disinfectants and Antiseptics Used in Human Medicine DRAFT A suspension of the test virus, with or without a soil load, is mixed with the test agent and the mixture held at 20°C for the desired contact time. The mixture is then titrated for infectious virus. Under review by CEN/TC 216... 

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