Methodology for HIV disinfectant testing

Journal

of Hospital

infection

(1995)

Methodology

30 (Supplement),

for HIV

383-388

disinfectant

testing

J. van Bueren Laboratory

of Hospital 61 Colindale

Infection, Avenue,

Central Public Health Laboratory, London NW9 5HT, UK

Summary:

Due to the variation in protocols from studies by different workers for the inactivation of HIV by chemical disinfectants, only limited comparisons of the results can be made. These variations include those which apply to disinfectant testing in general, such as the level of organic load and the form of neutralization of the disinfectant, and those which apply particularly to HIV inactivation, such as the method used to detect infectious virus. Our suspension and carrier tests to assess the efficacy of chemical disinfectants against HIV are described and problems with the interpretation and applicability of the results are discussed. Keywords:

Disinfection;

HIV.

Introduction

HIV is capable of surviving outside the human body in wet or dried blood and body fluids for several days or weeks.’ Spillages of, or contamination by, all blood and body fluids may be infectious, so that it is important to have adequate methods of decontamination. Heat disinfection is the recommended method for inactivation of microorganisms but, where the use of heat is not possible, chemical disinfectants are required.2*3 Assessment of the virucidal activity of chemical disinfectants may be either in a suspension test, where the virus is kept in a liquid suspension, or in a carrier test, where the virus is dried onto an inanimate surface. The suspension test is suited to the screening of large numbers of products whilst the carrier test may be more representative of in-use conditions, although there are a number of limitations, such as the choice of carrier and the method chosen to inoculate and sample the contaminated surface. The results from HIV disinfection studies by different workers are often difficult to compare due to the variations in methodology. Variations may be those common to all microbiological disinfection tests, such as the level of organic load and the form of neutralization, or those which are more specific to the virus, such as the method used to detect, residual infectivity. Test

medium

The suspension medium and the level of organic loading may have a marked effect on the efficacy of a disinfectant. Disinfectant tests can be performed 0195-6701/95/060383+06

D 1995 The Hospital

fOS.OO/O

383

Infection

Society

384

J. van Bueren

under clean or dirty conditions. A level of 10% serum is often considered as a dirty test, but the percentage of blood and body fluids during practical disinfection may be considerably higher, despite recommendations for cleaning prior to disinfection.3-6 High levels of organic matter may have a substantial effect on the efficacy of a disinfectant and in-use conditions may be represented more closely by testing in the presence of a high percentage of proteinaceous material. Blood is the most common medium in which high levels of virus are likely to be found in z&o. For technical reasons coagulated blood cannot be used for testing and a large clot could significantly protect the virus from a reagent.

Measurement

of residual

virus

The method used for detecting virus after exposure to the disinfectant is also of importance. Early studies by other workers used reverse transcriptase as a marker of residual virus, but this is an indirect measurement of growth and a relatively insensitive assay compared to direct assessments of infectivity.7 In our method we regularly screened all cultures for a cytopathic effect (CPE) as a marker of infectivity. Cultures that were not clearly CPE positive after three weeks’ incubation were examined for an increase in the level of p24 core antigen compared to the initial level as an indication of growth of the virus. Viral titre was measured by using a TCIDSO endpoint dilution.’

Neutralization

of the disinfectant

Chemical disinfectants may be neutralized in a variety of ways. Dilution of the disinfectant test mixture to a non-toxic level, ultracentrifugation of the test mixture to pellet virus, or the addition of a chemical neutralizer are some of the methods which have been used. Disadvantages of the former method are that the virus is also diluted, often as much as lOOOfold, and that a low concentration of disinfectant remains in contact with the virus after the exposure period and throughout the duration of the experiment. When using this method, the initial viral titre must be high in order to show a 3-4 log reduction in titre due to the activity of the disinfectant. Our method used chemical neutralizers to prevent further activity of the disinfectant immediately following the exposure period. Effective neutralizers for many disinfectants are already published.“” Serum is also a recognized neutralizer and can demonstrate how readily a disinfectant may be inactivated by serum in Go. We tested the effectiveness of fetal calf serum (FCS) as a neutralizer against all disinfectants since it has the added advantage of being a component of the viral growth medium, avoiding the introduction of potentially cytotoxic chemical neutralizers into the reaction mixture.

Methodology

for HIV disinfectant Virus

testing

385

stock

Cell free virus (CFV) and cell associated virus (CAV) were prepared by centrifugation of cultures of the RF strain of HIV-l, maintained by continuous culture in MT-4 cells as described previously.” The standard growth medium was complete medium consisting of RPM1 1640 supplemented with 10% heat inactivated FCS, 5 mmol-i HEPES and 50 mg L-l gentamicin. Initial studies” tested the efficacy of disinfectants against both CAV and CFV, but as a time-saving measure it was decided subsequently to test CFV only (unpublished results).

Virucidal

suspension

test

The method is a slight modification of Bueren et al.” Virus was suspended in test medium containing the required level of serum (FCS). The product under test was added and, following the exposure period, neutralizer was added to prevent further action of the disinfectant. After addition of a cell suspension, the samples were mixed for 1 h to allow the virus to adsorb to the cells. The mixture was centrifuged and the pelleted cells were resuspended in fresh medium to remove the disinfectant and neutralizer before adding to the culture plate and incubating for three weeks. This protocol allows the disinfectant to be assessed against HIV suspended in high concentrations of serum. Nevertheless, a test using virus suspended in blood is more representative of in-use conditions and for this the method had to be modified slightly. Fresh human blood was used as the test medium. Following neutralization of the disinfectant, the red blood cells were removed rapidly by centrifuging the disinfectant/neutralizer mixture in a microfuge. The supernatant was added to the cell suspension and the method continued as described above. This procedure was necessary to avoid the red blood cells both interfering with the cytotoxicity assay and masking the CPE. Although the use of blood as a test medium was possible in the absence of a disinfectant, the practicalities of testing various chemicals in high quantities of blood were soon realized and in some cases it was impossible (unpublished results). Two controls were included in each test; an untreated control where the disinfectant was substituted by complete medium determined loss of viral activity during the experimental procedure. An adsorption control was also included to test for loss of virus due to residual disinfectant/neutralizer activity during the 1 h adsorption stage. Cytotoxicity

assay

During the 1 h adsorption of the virus onto the cells, the disinfectant was present at a dilution one-tenth that of the test concentration. The purpose of the neutralizer was to prevent toxicity of the disinfectant to the cells

386

J. van Bueren

during this stage. The effectiveness of different neutralizers in combination with disinfectants was assessed in a cytotoxicity assay prior to testing the virucidal efficacy of the product. The method was an adaptation of a calorimetric assay for the growth and survival of cells12 and has been described in full earlier.” The standard suspension protocol was followed with complete medium substituting for virus. After overnight incubation of the final suspension a yellow dye, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), was added to the cells and the plates were incubated for a further 4 h. MTT is cleaved by metabolically active cells to form a blue formazan product. After the formation of the blue crystals, the reaction was stopped and the crystals dissolved. The plates were read on a plate reader and the mean value for each set of test conditions was compared as a percentage of the untreated control cells which represented 100% metabolic activity. Values below 90% of the controls indicated a loss of metabolic activity and were considered to be cytotoxic. Virucidal

carrier

test

This protocol was based on the glass cover slip method of Hanson et ~1.‘~ modified from the methods of Lloyd-Evans et aZ.14and Tyler and Ayliffe” and has been described in full previously.” Stock virus or lo-fold virus dilutions were pipetted onto sterile glass cover slips and allowed to dry at the back of a class I safety cabinet in the presence of silica gel. Once dry, the cover slips were transferred aseptically to tissue culture plates and disinfectant was added to each of the test wells. Immediately following the exposure period the disinfectant was removed rapidly by aspiration and replaced by a suspension of cells. Each test condition was repeated four times and two controls were included in each experiment. The viral titre prior to drying was determined by titration of the stock virus suspension (suspension control). Once the dried cover slips had been transferred to tissue culture plates, loss of virus due to drying alone was determined by the immediate addition of the cell suspension (dried control). Discussion

The results from our studies on the efficacy of alcohols against HIV have been described previously” and some of the problems associated with such tests have also been discussed. In a suspension test, HIV in both 10 and 100% (neat) serum was inactivated rapidly by 70% ethano1.r’ Similarly, 70% alcohol rapidly inactivated virus in 10% serum dried onto glass cover slips. However, when the organic load was increased to 100% serum, it took 4-10 min for complete inactivation of dried virus, demonstrating the slow penetration of serum by alcohols. The results from studies testing hypochlorite-releasing agents against HIV suspended in serum or blood showed marked differences in the rate

Methodology

for HIV disinfectant

testing

387

of inactivation in a suspension test, dependent on the level and the type of organic load (unpublished results). CFV suspended in complete medium containing 10% serum was inactivated within 30 s at 100 ppm available chlorine in the disinfectant test mixture. In a suspension of 80% serum, the maximum concentration of available chlorine had to be increased to 500 ppm for complete inactivation to occur between 1 and 2 min. However, in the presence of 80% blood, a concentration of 100 ppm available chlorine in the disinfectant test mixture was unable to inactivate 3.75 log TCIDS,-‘mL of virus. Testing in 80% serum involved a lo-fold dilution of disinfectant on its addition to the disinfectant test mixture. Thus, a concentration of 1000 ppm available chlorine in the disinfectant test mixture represented a concentration of 10 000 ppm available chlorine in the stock preparation of disinfectant prior to addition to the virus. The recommended concentration for disinfection of blood spillages is 10 000 ppm available chlorine3 and these results demonstrate the importance of thorough cleaning to remove organic material before disinfection. These two studies demonstrate some of the general problems associated with disinfectant testing such as the choice of test and the level and type of organic load. In addition, the interpretation of the results of studies by different workers is complicated further by considerable differences in methodology, such as the use of neutralizers and toxicity controls, the method used for measuring residual virus and often the wide variations in the exposure time of the virus to the disinfectant. Some of the problems described above would be overcome by the wider use of standardized test methods. At present, there are no UK protocols for virucidal disinfectant testing and no internationally agreed standards for defining disinfectant efficacy, although a few countries do have their own standard virucidal test method (the AFNOR test in France, the DVV tests in Germany and the EPA dried carrier test in the USA) and discussions are underway to provide a standard European virucidal test. However, the methods for the inactivation of HIV by chemical disinfectants are complex. They require specialist laboratory facilities and expertise and the safety implications of such work must be considered. The costs for such tests are therefore high and it is neither cost-effective nor necessary to test all disinfectants against this virus. Nevertheless, products claiming efficacy specifically against HIV must be tested. But in the absence of standardized test methods the user must also consider carefully both the applicability of the test and the parameters chosen, such as the level of protein loading and the neutralization of the product after the test period.

This research was funded by the Department The views expressed in this paper are those funding bodies.

of Health and the Health and Safety Executive. of the author and not necessarily those of the

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of disinfectants:

preliminary