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Virucidal effect of certain chemical contraceptives on Type 2 herpesvirus
Virucidal effect of certain chemical contraceptives on Type 2 herpesvirus BALWANT
SINGH,
BOSKO JOHN
POSTIC, C.
PH.D M.D.
CUTLER,
M.D.
P~nn~yi~~arCa
Pittsburgh,
THE REPORTED CASES of gonorrhea in the United States have tripled during the last 15 years, and it is believed that they represent only a fraction of the total cases which occur in this country. There has also been a concurrent rise in the incidence of several other genital infections caused by various pathogens including herChlamydia, Mycoplasma, pesvirus, cytomegalovirus, and Trichomonas. Frequently, two or more of these agents are recovered simultaneously f’rom patients attending venereal disease clinics.’ Various nongonococcal genital infections are found much more often among sexually active younger women. In England, where statistics are available on the incidence of nongonococcal genitourinary infections, nonspecific. more frequent than urethritis in men is no\\ gonorrhea.’ Public health has long been concerned with the control of syphilis and gonorrhea as venereal disease, but has given little attention to other genital infections,
From the Graduati of Pitt,sbwgh.
Rewind
for
Accosted
Juw
School of Publk
publication
April
29,
Health,
such as herpes genitatis which was recognized and described as a venereal disease as early as 1885.” The incidence of herpes genitalis is fw greater than gmerally realized. The m?jority of patients with primar! herpes ilifections are teen-age girls and unmarried women. Seventy per cent or more of infected wmcn may have no symptoms. In the female subject, the cervix appears to be the chief site of infection and when the disease is limited to the cervix there may be little or no s~I~iptot~iatolog~.~ Male subjects may also harbor the virus asymptomatically which can be isolated from the male urethra as well as from the semen.‘. ’ This asymptomatic carriage of the virus tn members of both sexes contributes significantly to spread of the disease. During the last ten years, considerable literature has accumulated suggesting an asswiation of Type 2 herpes simplex virus infe&ms of lhc genital tract and the development of cervical cancer. Several stud& ha\,e also shown a significant association of genital herpes tvith other l,enereal infections, indicating its importance in venereal disease control programs. In view of the growing concern with increasing frequencies of sexually transmitted diseases, as \beli as a research project was uncterunplanned pregnancies, taken at the L-niversity of Pittsburgh to ident.ify vaginal contraceptive preparations with a powntial for topical prophytaxis against genitzdi infections. Various types of chemical contraceptives, creams, foams, jellies, vaginal suppositories, and foaming tablets marketed in the United States and other countries fLr intravaginal IIW
Unz~wr~i~~~
1976.
18, 1976.
Reprint wqwsts: Dr. B&want Sirtgh, Graduate School 01 Public Health, Vnizwsity (f Pittsburgh, Pittsburgh, Pwnsylnania 15261. 422
Virucidal effect of chemical contraceptives
comraceptive (10 per cent solution) and after IO minutes serial dilutions were inwulawxl in Ver0 cells from 1O+$to 1O-*
IWaterial and methods Virus strain. Type 2 hwpesCrus ( I%-Pl0, of 0ctoher 8. 1970) was obtained frotn Dr. \villiatn E. RawIs (Departtnent of L’irology and Epidemiology, Baylor L’rtiversity (:oltege of Medicine, Houston, Texas). 0ne additional passage was made in human embrwnic fibrobfast (HEF’) ceil culture in our laboratory. .&f&r 48 hours of incubation, the virus harvest was prepared, and atnpttles containitq the inf.ectious suspension were stored at about -6.5’ (1. ‘i.his stock contained IO”.~’ tissue cuhure inf&tious doses (TCiD5J per 0. I ml. Virus assay. The ‘lXXD5,1 of virus was detertnined itt human etnbryonic fibrobLtsts (HEF) and Vet-o cells. The human cell w~ture was prepared from a locally derived celi swairt.k The HI3 were grown in medium consisting of’ Hanks balanced salt solution (HESS) suppletnwted I+ith 0.5 per cent tactalbutnin hydrolyLate 2 tn11. I--~qlu~itmine, Eagle’s vitamin mixture, antibiotics, and .? per cent itiacti~ated (56’ C. fw Z%O itlit~ut~s) newbwn
423
424
Singh,
Postic,
and Cutler
Results The effect of virus treatment with 10 per cent solutions of two different chemical contraceptives in sholvn in Table I. The infectivity titration of’ virus without treatment was 106,” and 1 06.’ per 0.1 ml. in Vero cells and HEF, respectively. .4fter treatment with these contraceptives, the virus was inactivated, and the virucidal effect of these preparations is based on this evidence. No cytopathic effect was seen in Vero and HEF cell culture tubes inoculated with contraceptivctreated virus suspensions diluted with lo-’ to 1 OA’ and lo-’ to 10e9 virus. respectively. The ceil culture tubes inoculated with varying concentrations of contraceptive solution alone showed chemical toxicity up to dilutions of lo-’ for Vet-o cells and 1OY’ for HEF cell cultures, respectivelv. W’hen the chemical toxicity of contraceptives is considerecl, the TCIDS,, per 0.1 ml. of residual virus after treatment II-as estimated to be ~2.5 logs in Vero cells and ~3.5 logs for HEF cell culture tubes (Table I). The results of virus assay in rveanling mice both with and without treatment are shown in Table Il. The intracerebral LDSO per 0.03 ml. of virus prior to treatment was 5.3 but considerably less after treatment with IO per cent solutions of various chemical contraceptives. ‘I’he 50 per cent lethal dose of residual virus after treatment with contraceptives (Conceptrol, Cooper creme, Preceptin gel, Lorphyn jelly, and Milex crescent ,jelly) ranged from <0.5 to S.5 logs. Mice in control groups injected with serial dilutions of various contraceptive solutions alone (0.03 ml. per mouse) died after inoculation with the IO-’ dilution, except for those inoculated with Milex crescent jelly which was lethal in all dilutions from 1W to lo-‘. The death patterns due to acute toxicity of contraceptive and virus infection were quite different. The major proportion of deaths due to contraceptives occurred within 26 to 48 hours whereas deaths due to herpesvirus were noted after five days.
Comment The virucidal effects of several chemical contraceptives investigated are reported in this study. Both tissue culture techniques and the mouse inoculation method were usecl for the quantitation of residual viral infectivity. It was observed that Type 2 herpes simplex virus containing the original infectious suspension of 6 to 7 logs (TCIDS,, per 0.1 ml.) was inactivated upon contact
REFERENCES
1. Wentworth, B. B., Benin, P. A., Holmes, K. K., Gutman, L., Wiesner, P., and Alexander, E. R.: Isolation of viruses,
with several chemical contraceptives, even when 10 1~1 cent solutions of these contraceptives were used. Flqissu(, culture tubes of Vero cells and HEF inoculated with virus treated with 10 per cent solutions of rht: contraceptives tested did not she\% any cytopathic eff’e<,t attributable to the residual infectious inoculum. It ha5 been reported by other. investigators that ether-sensitive viruses (the arboviruses. the herpes group, and the myxoviruses and related viruses) are inactivated by benzalkonium chloride and related qumxnary ammonium germicides. ‘0 The virucidal effect of’ chemical contraceptives observed in this study is most likely due to quaternary and other surfactant mmpounds present in these products. Three possible factors co~11d interftire with the observation of a virucidal cf’fcct in assa) systems. These are effects of. serun~ proteins, pH, and temperature. Howe\fer, their influence \vas minimized bv appropriate experimental conditions and controls. -1’hus. that observed reduction in virus infectivitv amounting to several logs ‘0 jvas not ~LW 10 an) factor other tliai1 the Grucidal effect of chemi< al contracepti\-es. -r. assure optimal conditions f’or the nieasurenicnt of infectivity. the dilutions 01’ \.irus b&n-e and after treatment with contraceptives l\ere prepared in diluent containing .Y per cetit inactivated calf serum. lrrespec tive of any stabilizer present in the diluent, the treatment with contraceptives was effective in inactivating the virus, and no cytopathic effect \\.as observed in the inoculated cell culture tubes beyond that attributed to the toxicit>, of these contraceptives up to clilutions of 10-’ and 1 O-” for both Vera cells and HEF, thy latter being somewhat more vulnerable. Thcr&)rc. the virus assay for infectivity of’ residual virus after treatment was carriecl out 1~1 inOculatinCg IO-” to I O-’ in \‘ero cells and lo- to IOY’ dilutions in culture of HEF and the TCIDS,, was calculated accorclinglv. Since the original suspension titered IO” to 107 of infectious virus per 0. 1 ml., the I ,OOO- to 1O.OOO-f’olcl decrease in infectivitl occurring af’ter coiitact with chemical contraccpti\,es indicates a substantial virucidal effect. These experimental finclings encourage furthe studies to determine the potential usefulness of At-mical contraceptives in preventing sexually transmitted herpesvirus infection. The authors Irish technical assistance.
to thank
Mrs.
Mary
White
for
bacteria and other organisms f’rom venereal disease clinic patients: Methodology and products associated with multiple isolations, Health kdb, Sci. 10: 75, 1973.
Virucidal
&‘ilkox, K. R.: .A world wide view of venereal disease, BI-. J. Vetter. Dis. 48: 163, 1972. t!nna, P. Ci.: Herpes progenitahs especially in women, J. C:utan. \.ener. Dis. 1: 321. 1883. A. J., and Nail, Z. M.: The .,Josey. W. E., Nahmias, epidemiology of. Type 2 (gektal) herpes simplex virus inf’ertion, Obstet. Gwec~~l. Survey 27: 295. 1972.
effect
of chemical
contraceNives
425
SINGH,
BOSKO JOHN
POSTIC, C.
PH.D M.D.
CUTLER,
M.D.
P~nn~yi~~arCa
Pittsburgh,
THE REPORTED CASES of gonorrhea in the United States have tripled during the last 15 years, and it is believed that they represent only a fraction of the total cases which occur in this country. There has also been a concurrent rise in the incidence of several other genital infections caused by various pathogens including herChlamydia, Mycoplasma, pesvirus, cytomegalovirus, and Trichomonas. Frequently, two or more of these agents are recovered simultaneously f’rom patients attending venereal disease clinics.’ Various nongonococcal genital infections are found much more often among sexually active younger women. In England, where statistics are available on the incidence of nongonococcal genitourinary infections, nonspecific. more frequent than urethritis in men is no\\ gonorrhea.’ Public health has long been concerned with the control of syphilis and gonorrhea as venereal disease, but has given little attention to other genital infections,
From the Graduati of Pitt,sbwgh.
Rewind
for
Accosted
Juw
School of Publk
publication
April
29,
Health,
such as herpes genitatis which was recognized and described as a venereal disease as early as 1885.” The incidence of herpes genitalis is fw greater than gmerally realized. The m?jority of patients with primar! herpes ilifections are teen-age girls and unmarried women. Seventy per cent or more of infected wmcn may have no symptoms. In the female subject, the cervix appears to be the chief site of infection and when the disease is limited to the cervix there may be little or no s~I~iptot~iatolog~.~ Male subjects may also harbor the virus asymptomatically which can be isolated from the male urethra as well as from the semen.‘. ’ This asymptomatic carriage of the virus tn members of both sexes contributes significantly to spread of the disease. During the last ten years, considerable literature has accumulated suggesting an asswiation of Type 2 herpes simplex virus infe&ms of lhc genital tract and the development of cervical cancer. Several stud& ha\,e also shown a significant association of genital herpes tvith other l,enereal infections, indicating its importance in venereal disease control programs. In view of the growing concern with increasing frequencies of sexually transmitted diseases, as \beli as a research project was uncterunplanned pregnancies, taken at the L-niversity of Pittsburgh to ident.ify vaginal contraceptive preparations with a powntial for topical prophytaxis against genitzdi infections. Various types of chemical contraceptives, creams, foams, jellies, vaginal suppositories, and foaming tablets marketed in the United States and other countries fLr intravaginal IIW
Unz~wr~i~~~
1976.
18, 1976.
Reprint wqwsts: Dr. B&want Sirtgh, Graduate School 01 Public Health, Vnizwsity (f Pittsburgh, Pittsburgh, Pwnsylnania 15261. 422
Virucidal effect of chemical contraceptives
comraceptive (10 per cent solution) and after IO minutes serial dilutions were inwulawxl in Ver0 cells from 1O+$to 1O-*
IWaterial and methods Virus strain. Type 2 hwpesCrus ( I%-Pl0, of 0ctoher 8. 1970) was obtained frotn Dr. \villiatn E. RawIs (Departtnent of L’irology and Epidemiology, Baylor L’rtiversity (:oltege of Medicine, Houston, Texas). 0ne additional passage was made in human embrwnic fibrobfast (HEF’) ceil culture in our laboratory. .&f&r 48 hours of incubation, the virus harvest was prepared, and atnpttles containitq the inf.ectious suspension were stored at about -6.5’ (1. ‘i.his stock contained IO”.~’ tissue cuhure inf&tious doses (TCiD5J per 0. I ml. Virus assay. The ‘lXXD5,1 of virus was detertnined itt human etnbryonic fibrobLtsts (HEF) and Vet-o cells. The human cell w~ture was prepared from a locally derived celi swairt.k The HI3 were grown in medium consisting of’ Hanks balanced salt solution (HESS) suppletnwted I+ith 0.5 per cent tactalbutnin hydrolyLate 2 tn11. I--~qlu~itmine, Eagle’s vitamin mixture, antibiotics, and .? per cent itiacti~ated (56’ C. fw Z%O itlit~ut~s) newbwn
423
424
Singh,
Postic,
and Cutler
Results The effect of virus treatment with 10 per cent solutions of two different chemical contraceptives in sholvn in Table I. The infectivity titration of’ virus without treatment was 106,” and 1 06.’ per 0.1 ml. in Vero cells and HEF, respectively. .4fter treatment with these contraceptives, the virus was inactivated, and the virucidal effect of these preparations is based on this evidence. No cytopathic effect was seen in Vero and HEF cell culture tubes inoculated with contraceptivctreated virus suspensions diluted with lo-’ to 1 OA’ and lo-’ to 10e9 virus. respectively. The ceil culture tubes inoculated with varying concentrations of contraceptive solution alone showed chemical toxicity up to dilutions of lo-’ for Vet-o cells and 1OY’ for HEF cell cultures, respectivelv. W’hen the chemical toxicity of contraceptives is considerecl, the TCIDS,, per 0.1 ml. of residual virus after treatment II-as estimated to be ~2.5 logs in Vero cells and ~3.5 logs for HEF cell culture tubes (Table I). The results of virus assay in rveanling mice both with and without treatment are shown in Table Il. The intracerebral LDSO per 0.03 ml. of virus prior to treatment was 5.3 but considerably less after treatment with IO per cent solutions of various chemical contraceptives. ‘I’he 50 per cent lethal dose of residual virus after treatment with contraceptives (Conceptrol, Cooper creme, Preceptin gel, Lorphyn jelly, and Milex crescent ,jelly) ranged from <0.5 to S.5 logs. Mice in control groups injected with serial dilutions of various contraceptive solutions alone (0.03 ml. per mouse) died after inoculation with the IO-’ dilution, except for those inoculated with Milex crescent jelly which was lethal in all dilutions from 1W to lo-‘. The death patterns due to acute toxicity of contraceptive and virus infection were quite different. The major proportion of deaths due to contraceptives occurred within 26 to 48 hours whereas deaths due to herpesvirus were noted after five days.
Comment The virucidal effects of several chemical contraceptives investigated are reported in this study. Both tissue culture techniques and the mouse inoculation method were usecl for the quantitation of residual viral infectivity. It was observed that Type 2 herpes simplex virus containing the original infectious suspension of 6 to 7 logs (TCIDS,, per 0.1 ml.) was inactivated upon contact
REFERENCES
1. Wentworth, B. B., Benin, P. A., Holmes, K. K., Gutman, L., Wiesner, P., and Alexander, E. R.: Isolation of viruses,
with several chemical contraceptives, even when 10 1~1 cent solutions of these contraceptives were used. Flqissu(, culture tubes of Vero cells and HEF inoculated with virus treated with 10 per cent solutions of rht: contraceptives tested did not she\% any cytopathic eff’e<,t attributable to the residual infectious inoculum. It ha5 been reported by other. investigators that ether-sensitive viruses (the arboviruses. the herpes group, and the myxoviruses and related viruses) are inactivated by benzalkonium chloride and related qumxnary ammonium germicides. ‘0 The virucidal effect of’ chemical contraceptives observed in this study is most likely due to quaternary and other surfactant mmpounds present in these products. Three possible factors co~11d interftire with the observation of a virucidal cf’fcct in assa) systems. These are effects of. serun~ proteins, pH, and temperature. Howe\fer, their influence \vas minimized bv appropriate experimental conditions and controls. -1’hus. that observed reduction in virus infectivitv amounting to several logs ‘0 jvas not ~LW 10 an) factor other tliai1 the Grucidal effect of chemi< al contracepti\-es. -r. assure optimal conditions f’or the nieasurenicnt of infectivity. the dilutions 01’ \.irus b&n-e and after treatment with contraceptives l\ere prepared in diluent containing .Y per cetit inactivated calf serum. lrrespec tive of any stabilizer present in the diluent, the treatment with contraceptives was effective in inactivating the virus, and no cytopathic effect \\.as observed in the inoculated cell culture tubes beyond that attributed to the toxicit>, of these contraceptives up to clilutions of 10-’ and 1 O-” for both Vera cells and HEF, thy latter being somewhat more vulnerable. Thcr&)rc. the virus assay for infectivity of’ residual virus after treatment was carriecl out 1~1 inOculatinCg IO-” to I O-’ in \‘ero cells and lo- to IOY’ dilutions in culture of HEF and the TCIDS,, was calculated accorclinglv. Since the original suspension titered IO” to 107 of infectious virus per 0. 1 ml., the I ,OOO- to 1O.OOO-f’olcl decrease in infectivitl occurring af’ter coiitact with chemical contraccpti\,es indicates a substantial virucidal effect. These experimental finclings encourage furthe studies to determine the potential usefulness of At-mical contraceptives in preventing sexually transmitted herpesvirus infection. The authors Irish technical assistance.
to thank
Mrs.
Mary
White
for
bacteria and other organisms f’rom venereal disease clinic patients: Methodology and products associated with multiple isolations, Health kdb, Sci. 10: 75, 1973.
Virucidal
&‘ilkox, K. R.: .A world wide view of venereal disease, BI-. J. Vetter. Dis. 48: 163, 1972. t!nna, P. Ci.: Herpes progenitahs especially in women, J. C:utan. \.ener. Dis. 1: 321. 1883. A. J., and Nail, Z. M.: The .,Josey. W. E., Nahmias, epidemiology of. Type 2 (gektal) herpes simplex virus inf’ertion, Obstet. Gwec~~l. Survey 27: 295. 1972.
effect
of chemical
contraceNives
425