Susceptibility of HIV to inactivation by disinfectants and ultraviolet light

Journal of Hospital Infection (1995) 30, 167-180

Susceptibility

of HIV to inactivation by disinfectants and ultraviolet light

J. D. Druce, Victorian

D. Jardine,

S. A. Locarnini

and C. J. Birch

Infectious Diseases Reference Laboratory, Fair-eld Hospital, Yarra Bend Road, Fairfield, Victoria 3078, Australia Accepted for publication

15 March

1995

Summary: Assays were developed to assess a variety of conditions and presentations of infectious HIV to potential inactivating sources. A range of commercially available disinfectants with active constituents including glutaraldehyde, chlorine, phenolics, alcohol, iodine and quaternary ammonium compounds was tested. In addition, U.V. light was investigated as a potential inactivating source. All products were assessed against cell-free HIV in culture medium and cell-associated HIV suspended in medium or whole human blood. All products completely inactivated cell-free HIV following a 1 min exposure. However, cell-associated HIV was more resilient, requiring exposure of 5 min or more for some disinfectants. The effectiveness of the disinfectants was further compromised in the presence of blood. Keywords: HIV,

disinfectants;

U.V. light.

Introduction

Considerable importance has been placed on the use of disinfection procedures that reliably inactivate HIV in blood or other body fluids, and material from laboratory cultures. While several studies have investigated the efficacy of inactivation of HIV through disinfection, they have often used different methods and criteria to establish virucidal activity.‘-’ In general, complete viral inactivation is assumed if a 10 OOO-fold reduction in infectious titre is obtained. However, this may not be an acceptable level of reduction for HIV, because the final titre may be considerably influenced by the environment immediately in contact with the virus during exposure to the disinfectant, and expectations among health carers is that demonstrably complete inactivation should be the minimum standard. Despite the absence of clearly defined international guidelines for the evaluation of disinfectant activity against HIV, it is possible to obtain useful data on the stability of HIV under certain experimental conditions. We

Correspondence 0195-6701/95/070167+14

to: Dr J. D. Druce. $08.00/O

0 1995 The Hospital

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therefore established suspension methods of assessment based on the protocol of Gordon et al.’ Our assays enable measurement of the effect of disinfectants (or other potential inactivators) on unprotected (cell-free) virus as well as virus that is protected from its immediate environment due to its close association with cells and cell membranes (cell-associated virus). Furthermore, the use of whole human blood to simulate a more realistic organic load on HIV survival following exposure to disinfectant can also be evaluated. Some of the difficulties associated with evaluating the antiviral activity of disinfectants include the need for removal of the cytotoxic effects of the chemical agent before virus isolation is attempted, and on acceptance of appropriate criteria for evidence of virus inactivation. By adapting the methods of Gordon et al,’ the removal of cytotoxic factors from the virus/disinfectant mixtures before attempting isolation is relatively simple and reliable. However, the criteria for virus inactivation needs an evaluation of several different factors, including the effects of increasing protein load on disinfectant efficacy. In addition, the detection of HIV may involve measuring several different markers including reverse transcriptase or p24 antigen, observing cytopathic effects (cpe) or detecting nucleic acid [polymerase chain reaction (PCR)]. Using any of these markers alone to measure infectivity can cause problems of interpretation. In our assays we used the absence of cpe and negative (or declining) p24 levels in culture supernates as indicators of virucidal activity. When establishing the assays, we used an HIV isolate that produced high titres of cell-free virus to prepare a stock virus suspension. This stock had a titre of 1 x lo6 TCIDSO mL-‘, which is greater than the titre of HIV found in most infected patients, irrespective of the stage of the disease.Hence, the disinfectants were evaluated under conditions at least as rigorous as might be found in a real-life situation. For cellassociated HIV experiments, infected cell numbers that approximated or exceeded those found in viva were used to determine efficacy.“’ The criterion for successful inactivation in our assay was complete inactivation of HIV (titre reductions up to 106). Several commercially available disinfectants were assessed. These contained varied active components, including glutaraldehyde, phenolics, iodine, chlorine, quaternary ammonium compounds and ethanol. Each was analysed under conditions in which the degree of protection afforded to the virus by contaminating biological material varied according to whether fetal calf serum, cells or whole human blood was present. Further experiments to assess HIV inactivation by U.V. light were investigated using small volumes of cell-free or cell-associated virus suspended in cell culture medium or blood and exposed to U.V. light in class II safety cabinets.

Inactivation Materials

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169

methods

Disinfectants Disinfectants were purchased ‘off the shelf’. Dilutions to the concentrations specified by the manufacturer were made in tissue-culture grade water and tested within 3 h. Products assessed were 1% glutaraldehyde (‘AIDAL’, supplied ready for use), chlorine-based solutions (‘Milton’ and ‘DET-SOL 5000’ diluted to 5000 ppm available chlorine, and ‘Domestos’, a viscous bleach containing 50 000 ppm available chlorine), 1.6% iodine (‘Sanichick’), a phenolic-based product (‘Clearsol 50’, containing 202 g L-’ alkyl phenols and xylenols and diluted 1: 50); a quaternary ammonium-based product (‘Pine-o-clean’, 1.5% w/w, diluted 1: 20); a potassium monopersulphatebased product (1 and 2% ‘Virkon’) and absolute ethanol (Rhone-Poulenc) diluted to 70% with water and also used at a 75% final (v/v) concentration (100% ethanol mixed with virus stock in the ratio of 3 :l).

Viruses HIV-l strain 228200, a primary isolate passaged twice through MT-2 cells, was used in all experiments involving cell-free virus. This isolate produces characteristic cpe associated with HIV infection, in particular multinucleated giant cells (syncytium formation). The stock of strain 228200 had a titre of 1 x lo6 TCID,, mL-‘. T lymphocytes chronically infected with the HTLVIIIa isolate (H9/HTLVIIIa cells) were used in all experiments involving cell-associated HIV. Cells The T lymphocyte line MT-2 was used in experiments involving the reisolation of HIV following its exposure to disinfectants. This cell-line supports the replication of isolate 228200 and HTLVIIIs derived from H9/ HTLVIII, cells.

Medium The medium used for maintenance and passage of MT-2 and HC)/HTLVI I IB cells was RF-lo, an RPM1 1640-based medium containing 10% fetal calf serum. All experiments involving the re-isolation of HIV following exposure to disinfectants were also carried out in cells maintained in RF-lo.

Disinfection protocols Cell-free HIV experiments. One hundred

microlitres of HIV stock was mixed with 100 pL of disinfectant (1 :l) for 1 or 5 min in polyallomer ultracentrifugation tubes (Beckman). Immediately following exposure, 10 mL of 5% sucrose in TES buffer (10 mM Tris-HCl, 1 mM EDTA and 150 MM NaCl) was added, and the contents of the tube centrifuged at 120 000 g for 60 min at 4°C in an SW 41 rotor. This procedure was designed to dilute the disinfectant and, by pelleting virus, remove factors potentially

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toxic to MT-2 cells. Untreated controls substituted for disinfectant underwent the disinfectant-treated samples. The (viral) centrifugation was resuspended in 100 PL prior to HIV isolation attempts.

in which RF-10 medium was same procedures as used for the pellet obtained following ultraof RF-10 medium immediately

Centrifugution control. To investigate possible inactivation occurring during the centrifugation step (that is, during the additional 1 h exposure to diluted disinfectant), 100 PL of virus stock was added to 10 mL of 5% sucrose containing 100 PL of disinfectant. This mixture was centrifuged as above. Cell-associated HIV experiments. A total of 100000 H9/HTLVIIIB cells suspended in 100 l.tL of either culture medium or whole unclotted human blood from an HIV seronegative donor was mixed with 100 PL of disinfectant for 1 and 5 min (RF-10 medium) or 1 and 10min (blood). Untreated cells and cells mechanically homogenized through use of 10 cycles with a Dounce homogenizer in RF-10 medium were used as untreated controls. Immediately following exposure, disinfectant was diluted by the addition of 10mL of culture medium and the mixture centrifuged at 2000 rpm for 10 min at room temperature. The pellet was resuspended in 100 PL of RF-10 medium and used as the source of HIV for isolation attempts. Surface-acting experiments. One hundred microlitres of disinfectant was placed into the wells of a 96-well microtitre tray (Linbro), and aspirated after 10 s. The residual film on the surface of the wells was then allowed to dry at room temperature for 24 h. Five microlitres of cell-free virus was then placed on the surface of the disinfectant-treated wells for 1 and 5 min. Two hundred microlitres of RF-10 medium was added, then immediately removed, and added to 10 mL of 5% sucrose in an ultracentrifuge tube. The tube was spun at 120 000 g for 60 min at 4°C in an SW 41 rotor. Isolation of HIV following exposure to disinfectants. Viral or cell membrane pellets obtained using the above procedures were resuspended in 100 pL of RF-10 medium, and 50 PL volumes inoculated into duplicate wells of a 24-well tissue culture tray (Costar) containing 100 000 MT-2 cells in 1 mL of RF-10 medium. These cultures were incubated at 37°C in 5% CO2 for seven days for experiments involving cell-free virus and 14 days for cellassociated experiments. The cells were viewed microscopically every two to three days for evidence of HIV-induced cpe. Supernates harvested at seven or 14 days were analysed for HIV p24 antigen content using a commercial kit (Organon Teknika HIV antigen EIA).

Exposure to u.v. light. Five microlitres virus suspended

in medium

of cell-free virus or cell-associated or blood was spotted on to 13 mm round plastic

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coverslips (Thermanox) and placed at three locations in a class II safety cabinet. These locations were designated location A (front centre), location B (directly under the U.V. source) and location C (front side). Samples were exposed to U.V. light for between 5 and 60min. Control coverslips on to which the same virus preparation were spotted were also placed in the cabinet at location B but were shielded from the U.V. light. The U.V. light intensity at each location was measured using a U.V. monitor (Spectroline 254 nm shortwave U.V. meter, Spectronics Corp., Westbury, NY). Following exposure to U.V. light, each coverslip was placed into the wells of tissue culture trays containing 100 000 MT-2 cells (24-well tissue culture trays, Costar). The trays were then incubated at 37°C in 5% CO* for seven days. Cells were examined microscopically at days three and seven for evidence of HIV-induced cpe. Results

Cell-free HIV Under conditions in which HIV was cell free, each product inactivated the virus within 1 min as assessed by negative cpe in cultures inoculated with treated virus (Table I). However, the day seven supernates originating from the treatment of HIV with glutaraldehyde, potassium monopersulphatebased products and ethanol, contained p24 antigen levels above the cutoff. We assumed this to be fixed, residual antigen introduced with the inoculum (see below). Following ultracentrifugation of HIV in the presence of diluted disinfectant (the centrifugation control experiment), infectious virus was not recoverable from chlorineand iodine-treated controls indicating that this dilution of disinfectant (1 :lOO) was sufficient to inactivate HIV following 60 min contact (Table I). Because the other products did not inactivate HIV under these diluted conditions, it is likely that the inactivation achieved by each of them in the experimental protocol was complete within the specified time, rather than during the centrifugation procedure. Cell-associated HIV Cell-associated HIV suspended in medium containing 10% fetal calf serum was completely inactivated by glutaraldehyde, hypochlorite, iodine, phenolics and 75% ethanol following a 1 min exposure (Table II). However, 35% ethanol, the potassium monopersulphate-based product and the quaternary ammonium compound did not completely inactivate cell-associated HIV following an exposure of up to 5 min (Table II). A 1% concentration of the monopersulphate-based product inactivated HIV after 10 min; 2% solutions failed to inactivate HIV after 1 min exposure, but no residual infectivity was observed following 5 min exposure (Table II). Elevated p24 levels were observed in the absence of cpe in glutaraldehyde, iodine, ethanol (75%) and phenolic-based compounds. When supernate from these cultures was

(1: 50)

medium cells

cytopathic

Ethanol

Phenolics 35%

I:100

0.8% 25 000 ppm

I:40

: 5

:

: : :

:

:

:

iGG

0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.21 0.16 1.95 0.00

0.07 0.06 0.11 0.61 0.00 0.00 0.00

NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG NEG

~24 Day 7

cpe Day 7

effects; 1 + , 2 + , 3 + and 4 + = 25, 50, 75 and 100% of cells infected, respectively;

Quaternary ammonium compounds 1.6% Iodine Hypochlorite 50 000 ppm

NEG, no evidence of HIV-specific exposed to suspension. p24 EIA cut-off=0.06.

Culture Control

70% Ethanol

‘Clearsol’

‘Sanichick’ ‘Domestos’

(1: 20)

5000 ppm

Hypochlorite

(1:l)

‘Milton’

‘Pine-o-clean’

0.5%

monopersulphate

Potassium

1%

‘Virkon’ 2500 ppm

0.5%

1% Glutaraldehyde

(neat)

Exposure(min)

suspended in RF-l 0 culture medium

Final concentration

HIV

‘AIDAL’

constituent

of cell-free

Active

I. Inactivation

Product

Table

( ), dilution

of product

4+

4+

4+

NEG NEG

4+

NEG

4+

4+

Centrifugation control cpe Day 7

P b 2 i-i z ft

r

2%

(1 :l)

‘Virkon’

‘Milton’

NEG, no evidence of HIV-specific cytopathic effects; dilution of product exposed to suspension. p24 EIA cut-off = 0.06. *Homogenized infectious cells were used as ‘untreated

cells*

1 +,

2+,

3+

controls’.

and 4+

75%

Ethanol

100% (3 :l)

Ethanol

I:100

=25,

25 000 ppm

35%

70% (1:l)

Ethanol

50 000 ppm

0.8%

1:40

2500 ppm

1%

0.5%

2500 ppm

0.5%

Final concentration

of cell-associated HIV

Ethanol

Phenolics

(1: 50)

‘Clearsol’

Homogenized Control cells

5000 ppm

monopersulphate

Quaternary ammonium compounds 1.6% Iodine

Hypochlorite

(1: 20)

Hypochlorite

Potassium

‘Domestos’

‘Sanichick’

‘Pine-o-clean’

1%

‘Virkon’

5000 ppm

Hypochlorite

‘DET-SOL

5000’

1% Glutaraldehyde

‘AIDAL’

constituent

Active

II. Inactivation

Product

Table

50, 75 and

10

PZG

t: NEG NEG

2 NEG NEG NEG NEG NEG NEG

I?3 1.01 nt nt

ozo 0.00 1.96 1.93 0.00 0.00 0.00 0.00 0.01 0.00 nt

oz3 0.03 0.04 nt nt nt nt nt

0.52

( ),

~24 Day 13

nt, not tested;

1% 1.50 0.00 1.53 0.00 0.00 0.01 0.01 1.74 1.78 0.24 0.00 0.00 0.00 0.36 0.11 1.99 1.99 1.64 1.52 1.97 0.00

0.78 nt nt nt

~24 Day 7

respectively;

N4E’c NEG NEG NEG

NEG NEG NEG NEG NEG nt nt nt

cw Day 13

100% of cells infected,

2G

&G NEG NEG NEG 1+ trace NEG NEG NEG NEG NEG NEG 4+ 4+ NEG NEG

IGG

10 1 5

:

10

:

NEG NEG NEG NEG NEG 2+

we Day 7

1 10

Exposure (min)

suspended in RF-10 culture medium

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passaged to fresh MT-2 cells which were incubated at 37°C for 2 h then washed and resuspended in fresh medium, there was no evidence of virus replication seven days later based on cpe and p24 levels (results not shown).

Cell-associated HIV

in blood

Under conditions in which cells chronically infected with HIV were suspended in whole human blood, HIV was completely inactivated by glutaraldehyde, iodine and 75% ethanol following 1 min exposure, and by hypochlorite following 10 min exposure. Each of the other products failed to completely inactivate cell-associated HIV in the presence of blood, despite exposures of up to 10 min (Table III).

Isolation of HIV from disinfectant-treated

surfaces

Results assessing the inactivating capability of disinfectants, applied to a surface 24 h prior to its exposure to HIV, are shown in Table IV. Iodine and one of the hypochlorites (‘Domestos’) both inactivated 5 PL of cellfree HIV (containing 5 x lo3 TCID,, of virus) following a 1 min contact. The quaternary ammonium compound and a second chlorine-based product (‘Milton’) required up to 5 min to inactivate the same volume of virus, while the phenolic compound did not inactivate the virus following 5 min exposure.

Susceptibility

of HIV to U.V. light Inactivation of HIV by U.V. light was influenced by the strength of U.V. light and the biological composition of the medium in which HIV was contained. When 5 PL of cell-free HIV containing 5 x lo3 TCID,, of virus was placed at various sites in the cabinet, inactivation following exposure to U.V. light was achieved after 5 min at a position directly under the U.V. lamp (location B) and after 10 min at the front (location A) and side (location C) (Table V). Cell-associated HIV was not completely inactivated at any position in the cabinet after 15 min exposure. When HIV in this form was placed directly under the U.V. source (location B), complete inactivation was achieved after 20 min, compared with 30 min when virus was placed at the front and side of the cabinets (locations A and C, respectively). Exposure times of up to 60 min did not completely inactivate cell-associated HIV in the presence of blood. Discussion

In general, two techniques have been used to assess virucidal disinfection, the carrier methods,4s5 and the suspension method.’ The carrier method is a modification of that used to assess bacterial disinfection. It involves a virus preparation dried on to the surface of a coverslip or alternative solid phase, which is then immersed into disinfectant. Following a specified

2%

(1 :l)

‘Virkon’

‘Milton’

50 000 ppm

75%

1:lOO

25 000 ppm

0.8%

1:40

2500 ppm

1%

0.5%

2500 ppm

0.5%

Final concentration

1: 1 10

1: 1 10

1:

l:, 1:

1 10

PSG NEG toxic toxic 2+ 1+ NEG NEG &G

2+

2

2+

NEG NEG trace NEG 2+ 2

we Day 7

suspended in blood

Exposure (min)

of cell-associated HIV

N:G NEG nt nt nt nt NEG NEG ISG

nt nt nt nt nt nt nt

respectively;

Ini6 1.77 1.64 1.50 0.02 1.78

1.77 1.77 1.77 1.77 1.76 0.65 0.89 nt

oni4 0.79 nt nt nt nt 1.58 1.07 0.00 1.72

1% 0.01 nt nt nt nt nt nt nt

0.53

~24 Day 13

nt, not tested; ( ),

0.71

NEG NEG ITO 0.01 nt I?5

~24 Day 7

we Day 13

NEG, no evidence of HIV-specific cytopathic effects; 1 +, 2+, 3+ and 4+ =25, 50, 75 and 100% of cells infected, dilution of product exposed to suspension. p24 EIA cut-off =0.06. *Homogenized infectious cells suspended in blood were used as ‘untreated controls’.

cells*

100% (3:l)

Ethanol

Ethanol

Phenolics

(1: 50)

‘Clearsol’

Homogenized Control cells

5000 ppm

Quaternary ammonium compounds 1.6% Iodine

Hypochlorite

monopersulphate

Potassium

Inactivation

WfM-I nnm

III.

Hvnnrhlnrite-

Hypochlorite

(1: 20)

constituent

1% Glutaraldehyde

Active

‘Domestos’

‘Sanichick’

‘Pine-o-clean’

1%

‘Virkon’

Product

Table

2

X

%

F it E. s =. ;

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J. D. Druce

Table IV. Inactivation Product ‘Milton’

of cell-free

HIV

applied to a surface treated with chemical agents 24 h previously

Active constituent (1:l)

Exposure (min)

Hypochlorite

‘Sanichick’

(neat)

Quaternary ammonium Iodine

‘Domestos’

(neat)

Hypochlorite

‘Pine-o-clean’

et al.

(1: 20)

‘Clearsol’ (1: 50)

Day 5

cpe

Day 7

trace NEG l%G NEG NEG NEG NEG trace NEG 1+ NEG

Phenolic

Untreated surface Control cells

cpe

Day 13 i$G

ZG NEG NEG NEG NEG 2+ :I NEG

IZG NEG NEG NEG NEG 4+ 4+ 4+ NEG

NEG, no evidence of HIV-specific cytopathic effects; 1+, 2+, 3 + and 4+ =25, 50, 75 and 100% of cells infected, respectively; trace, cytopathic effect observed in few cells; ( ), dilution of product applied to surface.

Table V. Ultraviolet

inactivation

of HIV

U.V. light intensity

in class II safety cabinet: cytopathological readings

Location A xlSOuWcm-*

Location B X 220 UW cm-’

Location C ~150uWcm-2

&G NEG

NEG NEG NEG

&G NEG

4+ 3+ NEG NEG

PZG NEG NEG

4+ 3+ NEG NEG

Cell-free suspended in medium 5 min 10min 15 min Cell-associated suspended in medium 15 min 20 min 30 min 45 min Cell-associated suspended in blood 45 min 60 min Controls 30/60 min (shielded) Control cells NEG, no evidence of HIV-specific cytopathic cells infected, respectively; nt, not tested.

4+ 4+ IGG effects; 1 +, 2 + , 3 + and 4 + = 2.5, 50, 75 and 100% of

time, the coverslip is removed and remove or dilute residual disinfectant coating then placed in direct contact with susceptible Although this method has potential benefits potential loss of virus from the surface during contact

effect

rinsed in a wash solution to the coverslip. The coverslip is cells, and isolation attempted. in terms of removing toxicity, the immersion in disinfectant

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or in the subsequent washings can lead to misleading results. Failure to isolate virus may be mistakenly attributed to disinfectant activity rather than a loss of virus through dilution during the procedure. We observed that isotonic solutions (for example local anaesthetics) were able to strip virus from the solid phase during exposure. This virus was subsequently lost during washings, prior to exposure to cells. Due to the viscosity, colour or lack of transparency of some disinfectant products, we decided not to use the carrier method in this assessment. An alternative (suspension) method, whereby virus suspended in liquid is exposed to disinfectant, has been evaluated by Gordon et al.’ Virus was exposed to disinfectant at a fixed ratio of 1: 9 for a designated contact time. Immediate dilution of the disinfectant by a factor of 1O-100, or the addition of a neutralizing agent, was then used to remove potential toxic effects of residual disinfectant prior to recovery of virus in susceptible cells. The method relies on the availability of a sufficiently high-titre virus stock so that a 10 OOO-fold reduction in virus titre can be demonstrated. We used a modification of the suspension method, whereby virus suspended in medium was exposed to an equal volume of disinfectant. This effectively dilutes the disinfectant by a factor of 2 for the period of contact, so that assessment of the disinfectant is often at a concentration half that recommended by the manufacturer. However, in cases where disinfectant is applied to a sizeable spill, our evaluation at a 1 :l dilution of the recommended concentration is not unreasonable. Following exposure of HIV to disinfectant for the appropriate contact times, subsequent contact between the two was reduced by dilution and ultracentrifugation to pellet the virus prior to inoculation of treated virus into susceptible cells. This procedure reduces the risk of transferring toxicity to the cells in which HIV recovery experiments are subsequently undertaken. However, in overcoming the problem of toxicity, the virus is exposed to a 1:lOO dilution of disinfectant for the duration of the centrifugation (1 h in the case of our cell-free experiments). This dilution is probably sufficient to halt further inactivation in most cases. However, in some cases there is a potential for further inactivation of virus, but this can be measured by pelleting untreated virus through a 1:lOO dilution of disinfectant under the same conditions of centrifugation, then assessing reduction in virus titre. Only the chlorineand iodine-based products appeared to retain activity against HIV at this dilution and duration of exposure. The inactivation of cell-free HIV by disinfectants is a measure of their direct effect on virus that is afforded no protection other than that provided by 10% fetal calf serum present in the culture medium. In these experiments HIV was inactivated within 1 min by all the disinfectants tested, including ethanol. This latter finding contradicts the results of Hanson et ~1.’ who found that HIV dried on to surfaces was not inactivated by ethanol following

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a 20 min contact. However, Martin et ~1.” found that complete inactivation was achieved using a suspension method. A more rigorous assessment of disinfectant efficacy is gained when the virus is cell associated. In the case of HIV, the virus is not thought to be infectious until it has budded from the cell although there is evidence of cell-to-cell transmission of HIV that does not appear to involve virions.““2 Nevertheless, recently budded virions or even infectious ‘cores’ are likely to be afforded some degree of protection by surrounding cell membranes, and the integrity of these membranes needs to be altered if the disinfectant is to come in contact with the virus. When cell-associated HIV was suspended in culture medium and exposed to the disinfectants, several which were active against cell-free HIV failed to completely inactivate cell-associated HIV. In particular, ethanol (35%) and the quaternary ammonium compounds did not inactivate cell-associated HIV after 5 min exposure. The potassium monopersulphate-based products did not inactivate HIV after 1 min exposure. The other disinfectants appeared to completely inactivate cell-associated HIV based on lack of cpe in cultures. However, cultures originating from glutaraldehyde, iodine, 75% ethanol and phenolic-treated virus were cpe negative but had raised p24 levels in the supernate at day seven. These levels subsequently declined over the next seven days. As the passaged supernate from these cultures with elevated p24 (but negative cpe) failed to produce an active infection, the raised p24 levels detected in the original cultures were likely due to fixed residual p24 originating from the initial exposure to disinfectant rather than a measure of actively replicating virus. When blood was combined with cell-associated HIV, the efficacy of several disinfectants was further compromised. Only glutaraldehyde, iodine and 75% ethanol completely inactivated the virus following a 1 min exposure under these conditions. One chlorine-based product (‘DET-SOL 5000’) was effective following 10 min exposure. A second chlorine-based product (‘Domestos’), could not be satisfactorily evaluated because of clumping of erythrocytes on exposure to it, and we were unable to remove residual toxicity under the test conditions used. Each of the remaining disinfectants failed to completely inactivate cell-associated HIV in the presence of blood, despite exposure times of up to 10 min. The presence of erythrocytes or associated debris did not exert any low level cytotoxic effects on MT-2 cells, as suggested by Gordon et al. in their assays.’ Our observations of productive cpe in most cultures, including those in which blood was present, suggests that interference from these potential cytotoxic factors is limited. This is most clearly seen with the untreated control, which developed cpe very quickly in the presence of maximum amounts of intact erythrocytes. The surface-protection experiments were undertaken to assess whether a disinfectant could provide a virus-free surface for 24 h. The disinfectants evaluated were those considered to be relatively non-toxic if used in an open work area. The maximum volume of virus used was 5 pL (containing

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179

5 x lo3 infectious doses), chosen to mimic an inadvertent or invisible spill or splash with virus, such as may occur in a laboratory safety cabinet. The experiments were conducted using cell-free virus. None of the products tested claimed to have anti-HIV activity under the conditions evaluated. As the conditions of the experiment were defined by the volume of virus stock added to the surface and the use of cell-free virus, extrapolation of the results to a larger spill is inappropriate, since it is likely that surface disinfectants could be diluted or not come in contact with virus contained within a larger droplet. Iodine and one of the chlorine-based products (‘Domestos’) retained surface activity sufficient to inactivate cell-free HIV following a 1 min contact period. The quaternary ammonium compound and a second chlorine-based compound (‘Milton’) also retained activity but required 5 min of contact to completely inactivate the virus contained within the 5 l.tL volume. The phenolic compound had no activity despite a 5 min contact time. Ultraviolet inactivation of HIV, like disinfectant inactivation of the virus, was influenced by the degree of protection afforded to the virus by the protein in the surrounding environment. Cell-free HIV suspended in medium was inactivated at all sites in the cabinet following 10 min of exposure. However, when the virus was cell associated, up to 30 min were required for inactivation at all sites in the cabinet. We were unable to demonstrate complete inactivation of cell-associated virus in blood, despite exposure of 1 h to the U.V. source. In conclusion, we have used a modified suspension method to assess the virucidal activity of a number of disinfectants. The method overcomes the problems of toxicity and uses complete virus inactivation as a measure of efficacy. In practice, a 106-fold reduction was achieved by exposure to several of the disinfectants tested, but the end result depended on the presence or absence of protective membranes or protein. A similar pattern of inactivation by U.V. light was also observed. On the basis of these results, laboratory and other health workers should give careful consideration to the type of disinfectant used in their work environment and, where appropriate, their approach to management of work within biological safety cabinets. References 1. Gordon V, Parry S, Bellamy K, Osborne R. Assessment of chemical disinfectants against human immunodeficiency virus: overcoming the problem of cytotoxicity and the evaluation of selected actives. J Vi& Methods 1993; 45: 247-257. 2. Hanson PJV, Gor D, Jeffries DJ, Collins JV. Chemical inactivation of HIV on surfaces. BMJ 1989; 298: 862-864. activity of disinfectants: studies with the 3. Tyler R, Ayliffe GAJ, Bradley C. Virucidal poliovirus. J Hasp Infect 1990; 15: 339-345. 4. Tyler R, Ayliffe GAJ. A surface test for virucidal activity of disinfectants: preliminary study with herpes virus. J Hosp Infect 1987; 9: 22-29. A, Viza D, Busnel RG. Chemical inactivation of human im5. Aranda-Anzaldo munodeficiency virus in vitro. J Viral Methods 1992; 37: 71-82.

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