New Method for Evaluation of Virucidal Activity of Antiseptics and Disinfectants

Abstract

Counting culturable viruses adsorbed to cellulose nitrate filters (the VIRADEN method) is proposed as a simple procedure for the evaluation of the virucidal activity of antiseptics and disinfectants. The virucidal activities of two different doses of iodine, chlorine, glutaraldehyde, and chlorhexidine digluconate on poliovirus 1 were tested with a standardized procedure and with the VIRADEN method. The two procedures assayed provided similar results.

The determination of the antimicrobial activity of disinfectants and antiseptics is a complex procedure (1, 2). Published reports have shown that in suspension test methods for bactericidal activity of disinfectants, significant differences in microbicidal effects occurred both within and between laboratories (4). One of the reasons for the differences could be the relative complexity of the procedure. The determination of the virucidal activity of disinfectants and antiseptics is even more complex (3, 6). Therefore, virucidal tests must use the simplest methods possible, since methodological complexity will add extra variation to the results even when using well-standardized methods. In most tests, to stop the inactivating action of the chemicals, it is necessary to dilute the mixture to prevent damaging the cell culture on which the viruses are counted after inactivation. This approach requires the use of high virus concentrations in the mixture or the testing of great volumes of mixture, or both. Using high virus concentrations has two drawbacks. First, high virus concentrations are very unlikely to reflect the real-world situation. Second, they can cause virus aggregation, which has been a cause of concern among different authors studying the virucidal activity of disinfectants and antiseptics (8, 12, 13). Furthermore, testing large volumes of mixture is expensive in terms of both materials and labor.

We have recently described a method that we have called VIRADEN (for virus adsorption enumeration) (9, 10), which is based on the direct enumeration of viruses adsorbed into nitrate-acetate cellulose membranes. The characteristics and performance of the method make it a priori most suitable for testing the virucidal activity of disinfectants and antiseptics.

To assess it, we chose different chemicals used either as disinfectants (glutaraldehyde and chlorine) or as antiseptics (iodine and chlorhexidine digluconate). Glutaraldehyde, chlorine, and iodine are known to have virucidal activity (11), whereas chlorhexidine digluconate (Hibitane) (11) is inactive for naked viruses. The inactivation was determined in parallel with the standard methods used for this purpose and with the method modified by counting the viruses with the VIRADEN method. In order to compare the methods all the inactivation experiments were performed as indicated by the present standard methods also.

Viruses, cells, and media.

All experiments were performed with the attenuated poliovirus type 1 strain Lsc-2ab. Viruses were propagated on the buffalo green monkey kidney continuous cell line (BGM). The cells were grown in Eagle's minimum essential medium (MEM autopow; ICN Biomedicals Inc., Aurora. Ohio) containing 5% fetal bovine serum, 2 mM l-glutamine, 26.8 mM NaHCO₃, 100 U of penicillin ml⁻¹, and 100 μg of streptomycin ml⁻¹.

The overlay medium used for the standard plaque assay was Eagle's minimum essential medium supplemented with 2% fetal bovine serum, 2 mM l-glutamine, 26.8 mM NaHCO₃, 100 U of penicillin ml⁻¹, and 100 μg of streptomycin ml⁻¹. The overlay medium was double concentrated and mixed with an equal volume of 2% purified agar (Oxoid, Hampshire, United Kingdom).

The overlay medium used for the VIRADEN method was supplemented with extra antibiotics: 50 μg of gentamicin ml⁻¹, 50 μg of nystatin ml⁻¹, and 20 μg of ceftazidime ml⁻¹ as described previously (10).

Enumeration of viruses. (i) VIRADEN.

Viruses were enumerated by the VIRADEN method as previously described (10). Briefly, the virus suspension was amended by adding MgCl₂ · 6H₂O to a final concentration of 0.05 M MgCl₂. The viral suspension was then filtered through a 47-mm-diameter 3-μm-pore-size cellulose nitrate filter as indicated, at a flow rate never exceeding 200 ml per min. When the entire sample had been filtered, the membrane filter was washed by passing 100 ml of sterile 0.05 M MgCl₂ through it.

Finally the viruses adsorbed on the membrane were counted on a BGM cell monolayer as follows. The growth medium in a 60-mm-diameter petri dish with a confluent BGM cell monolayer was discarded. Then, 100 μl of a suspension of BGM cells in minimum essential medium supplemented with antibiotics containing 1.75 × 10⁷± 0.25 × 10⁷ (mean ± standard deviation) cells per ml was placed in the center of the petri dish. The membrane with the adsorbed viruses was then carefully placed upside down on top of the cell suspension and the cell monolayer, and 5 ml of the overlay medium was poured slowly onto the center of the membrane filter and spread all over the plate. Then, as with the standard plaque assay, the agar was allowed to set and the petri dishes were incubated at 37°C in the presence of 5% CO₂ at a relative humidity of more than 80% for 48 to 96 h. Then the agar and the membrane were simultaneously removed, and the monolayer on the petri dish was stained with 0.1% crystal violet.

(ii) Standard plaque assay.

Virus suspensions were titrated by the plaque assay method on confluent monolayers of BGM cells as described elsewhere (5).

Assessment of the virucidal activity of chemicals.

The disinfectants were first diluted to twice the test concentration. Then 10 ml of this solution was added to 9 ml of sterile distilled water. The pH of the solution was measured and adjusted to between 6 and 7. The reaction tube was placed in a water bath set at 22 ± 2^oC (mean ± standard deviation) and left for about 3 to 5 min before 1 ml of the virus suspension containing approximately 10⁶ PFU per ml was added. After the addition of the virus suspension, the contents of the tubes were gently mixed. The contact time was measured from this point. At the desired time interval, 1 ml of the reaction suspension was removed with a sterile pipette and immediately transferred to either 10 or 100 ml of sterile distilled water or phosphate-buffered saline (PBS), with or without a neutralizing agent depending on the disinfectant or antiseptic tested. The 10-ml tubes and the 100-ml bottles were stored at 4°C until tested.

The concentrations of the disinfectants and antiseptics tested and the contact times are indicated in Fig. 1, 2, and 3. At each contact time two 1-ml aliquots were removed and processed as follows. For iodine and chlorine, one 1-ml aliquot was added to 9 ml of sterile water and the other was added to 9 ml of PBS. All tubes contained 20 μl of 10% sodium thiosulfate solution. For glutaraldehyde and chlorhexidine digluconate, one 1-ml aliquot was added to 100 ml of sterile water and the other was added to 100 ml of PBS, and serial 10-fold dilutions were performed. Then, the viruses in the PBS were counted directly on BGM monolayers using the standard monolayer method and the viruses in the water were counted by the VIRADEN method.

FIG. 1.

Kinetics of inactivation of poliovirus type 1 measured by counting viruses by the standard plaque assay (⧫; solid line) and VIRADEN (●; broken line). Shown are graphs for dilutions of 1/5,000 (A) and 1/1,000 (B) of a 100-ml solution containing 2 g of iodine, 2.5 g of potassium iodide, and 50% ethanol.

FIG. 2.

Kinetics of inactivation of poliovirus type 1 measured by counting viruses by the standard plaque assay (⧫; solid line) and VIRADEN (●; broken line) in solutions of 1 mg (A) and 2 mg (B) of chlorine per liter prepared from a 3% commercial chlorine solution (bleach).

FIG. 3.

Kinetics of inactivation of poliovirus type 1 measured by counting viruses by the standard plaque assay (⧫; solid line) and VIRADEN (●; broken line) in 0.1% (A) and 0.05% (B) glutaraldehyde prepared from a standard 25% glutaraldehyde solution.

None of the disinfectants or antiseptics used that might be totally or partially retained into the membrane by adsorption had any effect on the BGM cells applied according to the VIRADEN method.

Chlorhexidine digluconate had no effect on the number of polioviruses tested with either of the procedures. It is well known that it has no virucidal activity on naked viruses (11). In contrast, glutaraldehyde, chlorine, and iodine did inactivate viruses (Fig. 1, 2, and 3). The observed inactivations at the different concentrations of the three chemicals were similar regardless of the methods used to measure the inactivation. Taking into consideration the reported difficulties of the repeatability and reproducibility of tests for the determination of both bactericidal (4) and virucidal (7) activities of disinfectants and antiseptics, the similarity of the results reported here and obtained by two different methods is remarkable.

Although the two methods provide similar results and consequently similar evaluations of the virucidal activity of the disinfectants, the VIRADEN method approach offers clear advantages over the standard method. Indeed, it can be applied to initially low titer virus suspensions (10), which are probably much closer to the natural conditions than the high viral titers that have to be evaluated with the standard procedures. It also allows the testing of greater volumes with much less effort, in terms of both material and labor costs.

Besides its application in suspended tests, we can foresee the use of the VIRADEN method for testing disinfectants and antiseptics on viruses already adsorbed onto the membranes.

This work was funded by Generalitat de Catalunya (1995SGR00415 and Centre de Referència en Biotecnologia).