a Laboratoire de virologie, Hôpital Broussais, Paris, France; b Unité INSERM U430 and Université Pierre et Marie Curie (Paris VI), Hôpital Broussais; c Institut Alfred Fournier, Paris, France
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Abstract |
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Introduction |
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Development of vaginal microbicides should be one of the most feasible approaches to prevention of the heterosexual transmission of HIV.2,3 An ideal vaginal microbicide with activity against HIV should also be effective against other STDs. Many international agencies, including the World Health Organization and UNAIDS, have emphasized this position,4 since the development of an effective vaccine against HIV is unlikely for several more years. The paramount importance of female-controlled methods to prevent or reduce the risk of sexually acquired HIV is highlighted by epidemiological and sociological features demonstrating that women are both the most vulnerable population to HIV infection and the most important social group capable of developing prevention strategies, particularly in developing countries.
Benzalkonium chloride (BZK) is a positively charged surface active alkylamine biocide belonging to the group of quaternary ammonium salts.5 BZK is a cationic detergent containing two localized regions, one being hydrophilic and the other hydrophobic. It acts on surface membranes as a surfactant, which reduces the interfacial tension between two immiscible phases, then disrupting membrane integrity. BZK is an efficient spermicide when used correctly during sexual intercourse,6 since it ruptures the spermatozoidal membrane.5 A concentration as low as 0.025% totally inhibits sperm motility in vitro after exposure for 30 s.7 In Europe, BZK has been approved for over the counter human use as a local contraceptive since 1970. When used in low doses (25 mg) and applied in a formulation that allows it to be discharged with semen and vaginal fluids after intercourse, no relevant side-effects have been reported.8 BZK is also an effective microbicide, which directly damages the surface envelope of many pathogens,5 including microorganisms causing STDs, such as Chlamydia trachomatis,9 and herpes virus hominis type 2 (HSV-2).10 Although the virucidal activity of BZK against HIV was reported as early as 1987,11 and later on confirmed in at least five reports,10,1215 preclinical development of BZK as a possible topical microbicide for the prevention of HIV and STD has gained little attention until now.
In the present in vitro study, we evaluated the kinetics of inactivation obtained by BZK against C. trachomatis, and against a panel of selected viruses differing in their sensitivity to physicochemical agents (enveloped and non-enveloped viruses), in the nature of their genomes (DNA and RNA viruses), and in their infection, replication and transmission strategies. This panel included representative human pathogenic viruses, as recommended to validate inactivation procedures.16
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Materials and methods |
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Assays were performed with a primary solution of BZK at a concentration of 495 mg/L (1.40 mmol/L) (Laboratoire Innothéra, Arcueil, France). Pure compound, without the presence of other substances included in commercial formulations, was used. Stock dilutions were prepared in sterile phosphate-buffered saline (PBS). The final working dilutions were obtained by mixing each stock dilution with an equal volume of each stock of tested microorganisms. Finally, the concentrations (w/v) of BZK used to evaluate the inactivation of C. trachomatis were 12.5, 25, 50, 125, 250 and 500 µg/L; those to evaluate the inactivation of the panel of viruses were 1, 1.25, 2.5, 5, 12.5, 25, 50, 125, 250 and 500 µg/L.
Infectious agents strains and cell lines
Cytomegalovirus (CMV) laboratory strain AD169 was obtained from Professor F. Brun-Vezinet (Hôpital Bichat Claude Bernard, Paris, France). Clinical isolates of HSV-2, respiratory syncytial virus (RSV) subgroup A and adenovirus (ADV) serotype 2017 were recovered from clinical specimens from inpatients of Broussais Hospital. Clinical isolates of enterovirus (ENV) and BK virus (BKV) were kindly provided by Professor F. Freymut (Centre Hospitalier et Universitaire de Caen, France). HSV-2, CMV, ADV, RSV and ENV were propagated in human diploid embryonic lung fibroblasts (MRC-5) (Eurobio, Les Ulys, France), grown to confluence on 24-well plates and used between passages 25 and 30. BKV was grown in Vero cells in the laboratory, on 24-well plates.
Monolayer cultures were grown in Eagle's minimal essential medium (MEM) containing 50 IU/mL penicillin, 50 mg/L streptomycin, and supplemented with 10% fetal bovine serum (Eurobio) and 1% l-glutamine. The plates were fed every 3 days with MEM containing 2% fetal calf serum (FCS). Wells were observed every day until the development of typical cytopathic effect. BKV, which does not give an easily identifiable cytopathic effect in Vero cells, was arbitrarily grown for 15 days.
Supernatants of HSV-2, CMV, ADV, RSV and BKV cultures were then subjected to rapid viral culture, as described previously,18 with slight modification for each virus. In brief, 500 µL of cell-free supernatant, diluted 1:200 in MEM, were plated in duplicate on to MRC-5 fibroblasts or Vero cells grown on 24-well tissue culture plates. Following centrifugation of the plates at 3500g for 45 min at 21°C, the inoculum was removed, the plates were rinsed with sterile PBS and 500 µL of fresh complete refeeding MEM containing 2% FCS was added. After a 24 h incubation for HSV-2 and CMV, a 48 h incubation for ADV and a 72 h incubation for RSV and BKV, at 37°C under 5% CO2, the monolayers were fixed with acetone and stained for viral antigens, using commercially available monoclonal antibodies [C. trachomatis: antibody against the major outer membrane protein (Dade Behring, Marburg, Germany); HSV-2: antibody against HSV-2 and HSV-1 common antigen (CHA437; Argène Biosoft, Varilhes, France); CMV: antibodies against immediate-early (E13) and early (2A2) antigens (Argène Biosoft); RSV: antibody against sub-units F0 (70 kDa) and F1 (48 kDa) of fusion protein of RVS (subgroups A and B) (18B2; Argène Biosoft); ADV: antibody against group-specific antigen (H-60) (Argène Biosoft); BKV: antibody against the T antigen (Valbiotech, Paris, France)], according to the manufacturers' instructions. The plates were viewed under a Leitz fluorescence microscope. The viral titre of the 1:200 dilution of the supernatant culture was estimated by the number of infected cells counted per well, and expressed as number of infected MRC-5 or Vero cells per mL. Supernatant virus from ENV culture was quantified by plaque-forming titration, as described previously.19 Finally, stocks of HSV-2, CMV, ADV, RSV and BKV corresponding to a viral titre of 10000 infected cells per mL, and a stock of ENV containing 10000 pfu/mL, were kept frozen at 80°C until use.
The reference serovar D strain of C. trachomatis (ATCC VR885) was propagated in cycloheximide (1 mg/L)-treated HeLa 229 cell monolayers grown in 20 mM HEPES-buffered minimum essential medium with Earle's salts (EMEM) and supplemented with 10% FCS and glucose (5 mg/mL). A stock containing 2000 inclusion forming units (ifu)/mL was kept frozen at 80°C until use.
In vitro inactivation assays
Assay system for C. trachomatis.
A 1 mL aliquot of the chlamydial suspension (2000 ifu) was added to the same volume of each of the six working dilutions of BZK. After a brief agitation, the mixture was held at 37°C for a period of 1, 10, 20, 30, 40, 50 or 60 min. A negative control consisted of 1 mL of the chlamydial suspension incubated with 1 mL of PBS for each period of incubation. For each period of incubation, a control of toxicity consisted of 1 mL of the chlamydial suspension incubated with 1 mL of PBS containing 0.1% (w/v) BZK, a concentration two-fold higher than the highest concentration of BZK used. After completion of each incubation time, the reaction mixture was centrifuged at 11000g for 15 min at room temperature. The pellet was washed with 3 mL PBS, and centrifuged again as previously. Washing was repeated twice. The resulting pellet was diluted in 300 µL of sucrosephosphate buffer; 150 µL aliquots were plated in duplicate on to HeLa 229 grown on 96-well tissue culture plates. Following centrifugation at 3700g for 1 h at 37°C, the inoculum was replaced with 200 µL of fresh EMEM with 5% FCS containing 5 mM glutamine, 100 mg/L streptomycin, 100 mg/L penicillin and 1 mg/L cycloheximide. Following 48 h incubation at 36°C under 5% CO2, the monolayers were fixed with acetone and incubated with fluorescein-labelled antibody recognizing the major outer membrane protein (Dade Behring). The number of positive intracytoplasmic inclusions of C. trachomatis was counted in each well under a Leitz fluorescence microscope (magnification x100).
Assay system for viruses.
A 500 µL aliquot of each viral stock was added to the same volume of each of the 10 working dilutions of BZK. After a brief agitation, the mixture was held at 37°C for a period of 5, 10, 20, 30, 40, 50 or 60 min. For each virus, a negative control consisted of 500 µL of the viral stock incubated with 500 µL of PBS for each period of incubation. A control for toxicity consisted of 500 µL of the viral stock incubated with 500 µl of PBS containing 0.1% (w/v) BZK, for each period of incubation. After completion of each incubation time, the reaction mixture was centrifuged at 120000g for 1 h at room temperature. The pellet was washed with 500 µL PBS, and centrifuged again at 120000g for 10 min at room temperature. A second washing was repeated similarly. The resulting pellet was diluted in 1 mL of complete MEM for the test mixture as well as for the non-BZK-containing control. For HSV-2, CMV, ADV, RSV and BKV, 500 µL aliquots were plated in duplicate on to MRC-5 fibroblasts or Vero cells grown to confluence on 24-well tissue culture plates. Following centrifugation at 3500g for 45 min at 21°C, the inoculum was replaced with 500 µL of fresh complete growth medium. After a 24 h incubation at 37°C under 5% CO2 for HSV-2 and CMV, a 48 h incubation under similar conditions for ADV and a 72 h incubation for RSV and BKV, the monolayers were fixed, and the number of infected MRC-5 fibroblasts or Vero cells was counted after staining with monoclonal antibodies to viral antigens, as described previously. The titres of virus incubated with BZK and in non-BZK-containing control were estimated by the number of infected MRC-5 or Vero cells per mL. For ENV, 500 µL aliquots were incubated in duplicate in MRC-5 fibroblasts 24-well plates, for subsequent virus assay by plaque-forming titration.
Calculation of inactivation, residual infectivity and reduction in virus infectivity.
For all viruses tested and C. trachomatis, the level of inactivation was determined at each incubation time by comparison with the non-BZK-containing control. The inactivation factor (i) was calculated as follows: i (%) = [1 X/c] x 100, where X is the relative viral titre in test mixture with BZK and c is the relative titre in non-BZK-containing control. The residual infectivity (ri) corresponded to the expression: ri (%) = [X/c] x 100. For all viruses tested, the reduction in virus infectivity, i, was defined as the log10 of the difference in viral titre between the BZK-exposed virus and non-BZK-containing control; this parameter was null if X = c; it was calculated as follows if X differs from c:
i = log10 (cX) = log10 [c(1i) x c] = log10 [i x c]. A 3.0 log10 reduction in virus infectivity was considered an acceptable criterion of significant efficacy.20
Cytotoxicity assays.
Toxicity of BZK for MRC-5 fibroblasts, Vero cells and HeLa 229 cells was based on inhibition of cell growth. The cells were seeded at 4 x 103 cells per well into 96-well microtitre plates and allowed to proliferate for 24 h in MEM containing 20% FCS. After 24 h, MEM containing different concentrations (in duplicate) of BZK was added. After 3 days' incubation at 37°C, the cell number was determined with a Coulter counter. The minimum cytotoxic concentration is expressed as the 50% cytotoxic concentration (CC50) required to reduce cell growth by 50% relative to the number of cells in the control cultures.
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Results |
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The kinetics of in vitro susceptibility of C. trachomatis, HSV-2, CMV, RSV, ADV, ENT and BKV to exposure to increasing concentrations of BZK for increasing in-cubation times are given in Table I. BZK showed marked anti-C. trachomatis activity over the entire range of concentrations tested and incubation times: >99% killing was obtained after only 1 min incubation at a concentration of 0.00125% (12.5 µg/L). For all viruses and incubation times, a concentration of 0.0001% BZK was not significant. At a concentration of 0.00125%, significant inactivation was obtained only for CMV after a 5 min incubation. HSV-2 and CMV appeared the most BZK-sensitive viruses, and showed similar inactivation curves which were clearly dose dependent and proportional to the incubation time. For both viruses, inactivation greater than 95% was obtained after a 5 min incubation at a concentration of 0.0025% (25 µg/L), and after a 50 min incubation at a concentration of 0.00125% (1.25 µg/L). RSV, another enveloped virus, appeared slightly less sensitive to BZK than HSV-2 and CMV, but its inactivation curves rejoined those of HSV-2 and CMV after a 20 min incubation period. ADV, ENV and BKV, three non-enveloped viruses, were less sensitive to BZK than the enveloped viruses. Inactivation greater than 95% of ADV was obtained only with the highest concentrations of BZK and the longest incubation times. Finally, ENT and BKV showed similar inactivation curves, BKV, however, being slightly more resistant than ENV to BZK. Even at the highest concentrations of BZK and after the longest incubation times, a significant residual infectivity was observed for both viruses.
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Discussion |
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Strong activity of BZK against enveloped viruses and C. trachomatis is probably explained by the destruction of viral or cellular envelopes by the detergent. BZK was also efficient against non-enveloped viruses, whose inactivation was observed with higher concentrations and incubation times. Although resistance or very low sensitivity to BZK of DNA (ADV) and RNA (poliovirus and rhinovirus) non-enveloped viruses incubated for 10 min with proprietary preparations containing the spermicide have been described previously, BZK was highly effective at inactivating rotavirus, a non-enveloped RNA virus.12 These findings suggest that, in addition to the destruction of the envelope, the ionic detergent BZK may also alter either viral integrity or specific virushost cell interactions.
The antichlamydial effect of BZK was demonstrated previously using an in vitro susceptibility assay and a murine model of C. trachomatis lower genital tract infection.9 In this report, BZK showed more than 99% inactivation of 5.0 x 107 ifu of C. trachomatis at a concentration of 0.004% after 15 min incubation.9 In our study, the antichlamydial activity occurred at a concentration as low as 0.00125% after only 1 min incubation with 103 ifu of C. trachomatis, a finding that clearly confirms the very high efficacy of BZK against C. trachomatis.
BZK was highly effective at inactivating HSV-2 in vitro. Similarly, Jennings & Clegg10 reported that BZK at a concentration of 0.025% inactivates within 0.5 min an HSV-2 load of 1.0 x 105 pfu/mL. However, Kawana and colleagues12 reported that HSV type 1 (HSV-1) was only partially inactivated in vitro by two different proprietary preparations containing BZK, in contrast to the complete inactivation of other enveloped viruses (rubella, measles, mumps and influenza viruses); these results suggest a partial resistance related to the envelope structure of this virus. Whether the sensitivities of HSV-1 and HSV-2 to BZK are different should be confirmed. RSV, an enveloped virus known for its low resistance to physicochemical reagents, appeared slightly less sensitive to BZK than HSV-2 and CMV. Cellular debris containing RSV are generally present in the supernatant of viral culture on MRC-5 fibroblasts, and this may account for a slightly decreased sensitivity of this virus to BZK. In the present study, the sensitivity of the three non-enveloped viruses ADV, ENV and BKV to BZK was time and dose dependent, and inversely proportional to their resistance to physicochemical agents. This confirms that BZK may be effective against non-enveloped viruses, but only after a prolonged incubation or at high concentration.
It is our hope that the current results will renew interest in the STD-prevention qualities of existing BZK-containing vaginal products. Indeed, BZK is highly bactericidal and acts against a large number of bacteria causing STDs including C. trachomatis, Haemophilus ducreyi, Treponema pallidum and Neisseria gonorroheae.5,9,21 Furthermore, BZK is also virucidal against a wide spectrum of sexually transmitted pathogenic viruses, such as HIV, CMV22 and HSV, which is also a cofactor in HIV transmission.23
It is now well established that the risk of heterosexual transmission of HIV is increased in the presence of genital ulcer diseases as well as in the presence of non-ulcerative STDs.1 These complex interrelationships between HIV infection and other STDs explain in part the heterosexual spread of HIV infection, especially in developing countries. Genital infections due to human papillomavirus are causally associated with cervical cancer, one of the most common cancers in women worldwide. In our hands, the activity of BZK on BKV at the concentrations tested appeared generally virostatic. However, considering the high concentrations of BZK in spermicidal preparations, and the prolonged duration of vaginal retention of the surfactant spermicide, whose concentrations drop significantly only 4 h after vaginal insertion,24 BZK should have an in vivo inhibitory effect on genital papillomavirus. The potent activity of BZK against enveloped viruses also makes this molecule potentially effective against hepatitis B virus, an enveloped virus frequently transmitted sexually, as well as against hepatitis C virus, another enveloped virus that could be transmitted via sexual intercourse.25 Furthermore, all the in vitro studies indicate that antimicrobial activity of BZK is generally reached with very low concentrations of the drug. At these concentrations, BZK is not absorbed through the vaginal wall, as shown by tests in women and rats,26 and it is considered to be safe when used normally, e.g. if the woman has intercourse at a frequency of less than once per day, and if the formulation allows it to be discharged naturally from the vagina after intercourse.8 However, clinical studies in humans, including a rigorous estimation of possible adverse effects of BZK on cervicovaginal mucosa of HIV-seronegative as well as HIV-seropositive women, need further evaluation, since it has been reported recently that daily applications of BZK on the vaginal epithelium in the pig-tailed macaque for 4 days were shown to produce irritation or inflammation of the genital mucosa.27
In conclusion, BZK demonstrates potent in vitro activity against the majority of microorganisms causing sexually transmitted infectious diseases, including those acting as major genital cofactors of HIV transmission and associated with high morbidity or mortality. These attributes qualify BZK as a particularly attractive candidate for microbicide development.
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Acknowledgments |
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Notes |
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References |
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Received 4 October 1999; returned 3 March 2000; revised 12 May 2000; accepted 26 May 2000