Evaluation of the disinfectant effect of Solprogel against human immunodeficiency virus type 1 (HIV-l). A. Hernhndez, F. J. Belda, J. Dominguez,. L. Matas, M.
Journal of Hospital Infection (1996) 34, 223-228
Evaluation of the disinfectant effect of Solprogel against human immunodeficiency virus type 1 (HIV-l) A. Hernhndez, M.
Servicio
F. J. Belda, J. Dominguez, L. Matas, M. Gimenez, Caraballo, C. Ramil and V. Ausina
de Microbiologia, Hospital Universitario Germans Universidad Autdnoma de Barcelona, Spain
Received 20 March
1996; revised manuscript
Trias
i Pujol,
accepted 26 June 1996
Summary:
The antiviral activities of sodium dichloroisocyanurate (NaDCC) and a commercial product (Solprogel 2%) against human immunodeficiency virus type 1 (HIV-l) were investigated using a quantitative suspension test method. Solprogel is a compound that contains NaDCC and a biodegradable polymer of acrylic acid. Viral suspensions were prepared containing 3.2 x lo6 tissue culture infective dose 50 (TCIDSO) in culture media. Syncytium formation in the MT-2 line and HIV antigen p24 on the supernatant of the cultures were used to determine viral titre. Results indicate that satisfactory disinfection (lOOO-fold reduction in 5 min) can be achieved using NaDCC and Solprogel at concentrations of 100 and 120ppm available chlorine, respectively.
Keywords:
Disinfectant
granules;
sodium
dichloroisocyanurate;
HIV;
Sol-
progel.
Introduction
Precise information on the efficacy of chemical disinfection procedures against human immunodeficiency virus (HIV-l) is of growing interest because of the continuing rise in the number of AIDS cases, the repeated hospitalization of HIV-infected individuals and the consequent increased risk of accidental exposure to HIV-contaminated material for healthcare providers. The ability of HIV to survive at room temprature under a variety of environmental conditions has been demonstrated.’ Cell-free and cellassociated HIV cultures suspended in 10% serum have been known to remain infectious for several weeks at room temperature.2 Preliminary studies however, indicate that HIV is extremely sensitive to the action of (NaDCC) sodium hypochlorite (NaOC1).3,4 Sodium dichloroisocyanurate
Correspondence to: Dr Vicente Ausina, Trias i Pujol, Cra. de Canyet s/n 08019 0195-6701/96/110223+06
Servicio Badalona,
de Microbiologia, Barcelona, Spain.
Hospital
Universitario
0 1996 The Hospital
$12.00/O
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Germans
Infectmn
Society
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A. Hernhndez
et al.
is an organic chlorine compound containing two= N-Cl groups that hydrolyse in water to form the imino (=N-H) group and HOCl, which is the antimicrobial component. HOC1 is also the active component in bleach solutions, which have been shown capable of destroying HIV.3 Hypochlorite solutions are prepared for use in hospitals either by diluting NaOCl solutions or by dissolving NaDCC-containing tablets or granules in water to release hypochlorous acid. The method currently recommended is to prepare a solution containing 100 000 ppm Cl* by diluting household bleach to 10% strength, and then to pour the solution over a spillage and leave it for 30-60 min. This procedure has disadvantages however, because not all household bleaches contain 100 000 ppm av. Cl*. Previous studiessm7 have shown that NaDCC has many advantages over NaOCl. The NaDCC granules are quicker and easier to use because no dilutions are required, and give solutions of known available chlorine concentration. NaDCC also has less tendency to be inactivated by organic matter and is less corrosive to metals. Still newer solid compounds with decontaminating and deodorizing properties have recently appeared on the market offering additional alternatives to steam sterilization or incineration. One such product, Solprogel (Laboratorios Inibsa S.A., Barcelona, Spain), is supplied as a dry powder form that can be sprinkled on to a liquid to convert it to a granulated gel that contains 2% NaDCC. We have determined the minimum in-vitro concentration of NaDCC and the minimum time of contact needed to inactivate HIV-l for both NaDCC and Solprogel. Materials
and methods
Virus and cells The HIV-lIIIB virus strain was used. The virus was grown in MT-2 cell cultures, suspended in growth medium (GM) (RPM1 1640 supplemented with 10% heat-inactivated fetal calf serum, 1% L-glutamine, penicillin and streptomycin) and kept at 37°C in 5% CO2 in air. Cell-free virus stock was obtained by centrifuging the cultures at 830 g for 15 min and removing the supernatants for pelleting by centrifugation (100 000 g, 10 min, 4°C). Cellfree virus-containing supernatants were then frozen in aliquots at -80°C.
Virus titrations Determination of virus titre was based on infectivity assays. Briefly, lofold dilutions of each timed sample were prepared in GM; 100 l.tL of each dilution was then inoculated into each of three replicates in a 24 well microtitre plate containin O-9 mL MT-2 cells (3.0 x 10’ cell/mL) in GM. The cultures were incubated at 37°C 5% CO2 in air. GM was added to each well after three to four days. On days 4, 7 and 11, cultures were examined microscopically for cytopathic effect (syncytium formation) and
NaDCC
activity
against
HIV-l
225
culture fluid was harvested for the HIV antigen (Ag) p24 assay (NEK-060, Du Pont de Nemour, Belgium). Virus titre was calculated using the Spearman-Kgrber formula,8 and expressed as the tissue culture infective dose 50 (TCIDSO), which is the reciprocal of the highest dilution at which syncytia develop and/or HIV Ags are expressed.
Disinfectant
solutions
Solutions
of NaDCC. NaDCC granules (Delsa S.A., Barcelona, Spain) containing 60% av. Cl, were dissolved in sterile distilled water. According to the manufacturer’s data 2 g NaDCC dissolved in 120 mL water yields 10 000 ppm av. C12. Working solutions of NaDCC at 1000 and 100 ppm av. Cl2 were prepared from this initial solution. A fresh dilution of disinfectant was prepared in distilled water before each use.
Solprogel.
Solprogel (Laboratorios Inibsa S.A., Barcelona, Spain) is a dry powder containing a biodegradable polymer of acrylic acid with the empirical formula (C3H302 Na) 2.1 x (C3H402) and with a large number of bonds among its hydrophilic chains. When Solprogel comes into contact with a liquid residue, the liquid is solidified by solvation of the polymer’s hydrophilic groups; the compound also has a strong ability to expand and encapsulate. Solprogel contains NaDCC which yields Cl2 as described previously. Following the manufacturer’s instructions, 10 g/L of Solprogel was dissolved in GM containing the stock virus, to give 120 ppm av. CIZ.
Cytotoxicity
assays
Initially, the disinfectant solutions were examined for toxic effect on the target cells used to assess residual virus infectivity using a virus titration procedure. Cell viability for each sample was checked microscopically by trypan blue exclusion at 24 and 48 h, and every two or three days thereafter for a period of at least 11 days, unless clear evidence of cytotoxicity or cell lysis appeared before the end of that term.
Virus treatment Successive 10-l dilutions of stock virus in NaDCC disinfectant solutions were prepared (10 000, 1000 and 100 ppm av. Cl*). Solprogel was added to a 10-l solution of virus stock in GM. Times of contact with the virus were 5, 15 and 60 min. Once the treatment period was over, the samples were diluted in NTE buffer (10 mM Tris pH 8; 1 mM EDTA; 10 mM NaCl; pH 7.4) to neutralize the disinfectant. The Solprogel samples were homogenized in a vortex for 2-3 min and then centrifuged at 2500 g for 10 min to eliminate gel residues, and later ultracentrifuged at 100 000 g for 10 min at 4°C to pellet the viral particles. The pellets were resuspended in 1 mL GM, filtered through a 0.45 urn filter (Schleicher and Schenell, Dassal, Germany) and stored at -80°C until the test of residual infectivity could be performed in MT-2 cells.
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A. Hernhdez
et al.
Virus survival HIV survival was measured directly by infection of cell cultures (syncytium formation) and indirectly by HIV p24 Ag. Viral titres of the suspensions treated with NaDCC and Solprogel, and of untreated control solutions, were obtained by means of lo-fold dilutions (10-2-10-6) on MT-2 cells, as described above. These dilutions were inoculated in triplicate in 24 well plates, which were then incubated for 11 days at 37°C in air enriched with 5% CO,. The presence of infectious virus was determined by examining the viral cultures each three to four days for any cytopathic effect and the culture fluid was then harvested for HIV-l p24 assay. Results
After the incubation period of the cytotoxicity assay the traces of disinfectant had no significant toxic effect on the MT-2 cells (viability >85%). Virus titres for HIV-l suspensions were determined before and after treatment with either NaDCC or Solprogel. The virus titre of the stock suspension was 3.2 x lo6 TCIDSO and under the experimental conditions of this study the stock titre decreased to 6.8 x 10’ TCIDSO (Table I). Infectious virus was detected in all untreated samples but was not detected in any of the samples treated with NaDCC or Solprogel, indicating that virions present in inoculates after treatment were unable to replicate in MT-2 cells. The presence or absence of cytopathic effect coincided with positive or negative determination of Ag ~24. The titre of virus in all untreated (control) samples was 6.8 x 10’ TCID50. The titre of virus in samples treated with either NaDCC or Solprogel was ~10~. NaDCC and Solprogel at concentrations of 100 and 120 ppm of av. Cl2 respectively, achieved a thousand-fold reduction in TCID50>3 in 5 min. Discussion
HIV is a serious hazard for healthcare workers, and it is essential to determine the virucidal activities of disinfectant agents used in hospitals and laboratories under strict experimental conditions. To test the virucidal activity of one commercial biocide against HIV-l, we used a quantitative suspension test. Syncytial inhibition assays on the MT-2 line and HIV Ag p24 on the supernatant of the cultures was used to determine viral titre. Because HIV replication is highly dependent upon the ability of the cells used to grow the virus, chemical cytotoxicity can be an important source of error. Richman et aL9 pointed out that if the cells used to assay a chemically treated sample of HIV are killed by traces of the inactivating agent, the resulting absence of viral production could be interpreted as viral inactivation, whereas it would in fact be the result of cytotoxicity. Our results, however, indicate that traces of the disinfectant solutions we used had no toxic effect on MT-2 cells.
NaDCC Table
Sample*
I.
Effect on HIV-l
activity
against
HIV-l
of 5, I5 and 60 min treatments NaDCC and Solprogel
Time in contact with disinfectant (min)
PSI PS2
227
with several concentrations
p24
Antigen$
Virus titre (pg/mL)
Dilution
CPEt
10-6 10-S
neg >400
3.2 x IO6
+
10-S 10-4
neg >400
6.8 x 105
+
1o-5 10-4
-
neg >400
6.8 x lo5
+
PS4
10-s lO-4
+
neg >400
6.8 x IO5
PS5
IO-5 1o-4
+
neg >400
6.8 x 105
10-2 10-2 IO-*
-
PS3
Sl 155 60 s2
5 ii:,
s3
s4
5 15 60
10-2 lo-* lo-*
-
10-2 IO-2 10-2
-
10-2 10-2 1 o-2
-
of
neg neg neg
