Prevalence and association between herpes simplex virus types 1 and 2-specific antibodies in attendees at a sexually transmitted disease clinic

RW Roesta, WI van der Meijdena, G van Dijka, J Groenb, PGH Mulderc, GMGM Verjansb,d and ADME Osterhausb

a Departments of Dermatology and Venereology,
b Virology,
c Epidemiology and Biostatistics, University Hospital Rotterdam-Dijkzigt and Erasmus University Rotterdam, The Netherlands.
d Rotterdam Eye Hospital, The Netherlands.

Wim Roest, MD, Department of Dermato-Venereology, Room 1783, Erasmus University Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands. E-mail: roest{at}derma.fgg.eur.nl

Abstract

Background Seroprevalence of herpes simplex virus type 1 (HSV-1) and HSV-2 was determined in 1993 and 1998 in a randomly selected study group of 1024 and 654 attendees, respectively, at the sexually transmitted disease (STD) clinic of the University Hospital Rotterdam-Dijkzigt, The Netherlands. Correlations of HSV-1 and HSV-2 seropositivity were investigated. The relationship between HSV-1 and HSV-2 antibodies was also studied.

Methods Data were collected in a cross-sectional study from February 1993 until February 1994 and from January 1998 until December 1998. Glycoprotein G (gG) HSV type specific serum IgG was determined.

Results Seroprevalence of HSV-1 was 68% versus 59% (1993 versus 1998, {chi}2-test P < 0.001), of HSV-2 it was 30% versus 22% (1993 versus 1998, {chi}2-test P < 0.001). Using logistic regression analyses, HSV-1 and HSV-2 seropositivity were significantly associated with age and ethnicity in both groups. In 1993, HSV-1 seropositivity also correlated with lower level of education and female gender, whereas in 1998 it correlated with ‘number of sexual partners in the past 6 months’ and ‘present diagnosis of STD’. In both groups, HSV-2 seropositivity was also more prevalent in females and related to sexual lifestyle variables. In an exposure-disease model, HSV-1 seropositivity was not correlated with HSV-2 seropositivity (odds ratio 1993 = 1.1, 95% CI : 0.8–1.7; odds ratio in 1998 = 1.0, 95% CI : 0.5–1.8).

Conclusions Seroprevalence of HSV-1 and HSV-2 is falling among STD clinic attendees in Rotterdam. A changing pattern of risk factors for HSV-1 seropositivity indicates increasing sexual transmission of HSV-1. Seropositivity for HSV-2 correlated with known risk factors. A previous HSV-1 infection does not reduce susceptibility to subsequent genital HSV-2 infections.

KEY MESSAGES

Keywords Herpes simplex virus type 1, herpes simplex virus type 2, genital herpes, epidemiology, seroprevalence, risk factors, transmission

Accepted 17 October 2000

Infection with herpes simplex virus (HSV) type 1 or 2, is the most common cause of genital ulceration in the developed world.1 Although the majority of cases are caused by HSV-2 infection, HSV-1 has been reported in an increasing number of genital herpes cases in the UK.2 Accumulating reports have provided evidence that HSV-2 seroprevalence is on the rise, particularly in the US.3 It was suggested that this increase was related to a lower rate of HSV-1 infection in adolescents.4 Considering that HSV-induced genital ulcerative disease may facilitate both transmission and acquisition of human immunodeficiency virus (HIV),57 it is not surprising that genital herpes has been labelled as ‘a persistent health care problem, which calls for continuing public awareness’.8 However, reliable data on HSV seroprevalence remain sketchy.

In the Netherlands, data on HSV-2 seroprevalence are only available for sexually transmitted disease (STD) clinic attendees in Amsterdam (1986–1988) and Groningen (1998),9,10 with a seroprevalence of 32% and 22%, respectively. It is difficult to interpret these HSV-2 seroprevalences because populations from different geographical areas were studied. Moreover, HSV-1 seroprevalence was not mentioned, despite the fact that sexual transmission of HSV-1 was suggested as increasing in recent years.11

In the present study, HSV-1 and HSV-2 seroprevalence was determined using HSV type-specific commercial tests12 in randomly selected groups of patients attending our STD clinic in 1993 and 1998. Herpes simplex virus type-specific tests (a rapid immunoblot assay [RIBA] in 1993 and an enzyme linked immunosorbent assay [ELISA] in 1998) were used and correlations between demographic characteristics and sexual behaviour studied. The relationship between HSV-1 and HSV-2 seropositivity is also described.

Methods

Study population
A representative random sample was taken retrospectively from attendees visiting the STD clinic from February 1993 until February 1994 (study group 1993) and from January 1998 until December 1998 (study group 1998). Stored serum samples were selected randomly from attendees who participated in HIV serosurveillance studies at the STD clinic. The result of the HIV test was conveyed to the patient upon request (in 1998 only). All attendees were asked to answer questions covering demographic characteristics (age, ethnic group, level of education and residence) and sexual behaviour (sexual preference, having had passive anal intercourse during the past 6 months, worked as or visits to a commercial sex worker during the past 6 months, number of partners during the past 6 months and having a history of STD). The medical history was taken by a physician after which all patients underwent a routine STD check-up with examination for gonorrhoea, Chlamydia trachomatis infection, non-specific urethritis, vaginal trichomoniasis, condylomata acuminata, pubic lice and genital scabies, syphilis and hepatitis B. In the case of genital ulcers or erosions additional testing for herpes genitalis, syphilis and chanchroid was carried out.

The local Medical Ethics Committee of the University Hospital Rotterdam-Dijkzigt approved the study protocol and informed consent was obtained from all patients.

Laboratory methods
Sera collected from February 1993 to February 1994 were examined for HSV type-specific antibodies using a rapid immunoblot assay (RIBA) (Chiron Corporation, Emeryville, CA, USA) according to the manufacturer's guidelines. Briefly, sera were incubated on strips coated with HSV type-specific glycoprotein G (gG1 and gG2)—including a positive and negative IgG control —for 4 hours at room temperature, diluted and washed three times. Bound antibodies were detected using an anti-human specific IgG conjugate. Results were scored for HSV-1 and HSV-2 according to the manufacturer's criteria.

Sera collected from January 1998 to December 1998 were examined for HSV type-specific antibodies using a commercial ELISA (Gull Inc., Murray, UT, USA), according to the manufacturer's guidelines. Briefly, diluted patients' sera were incubated with affinity purified gG1 or gG2 antigen bound to ELISA plate wells. Plates were washed and incubated with enzyme labelled anti-human IgG to detect bound antibodies. After washing and adding a chromogenic substrate (sodium azide), and stopping reagent (sodium hydroxide), specimens containing either HSV-1 or HSV-2 antibodies produced a colour endpoint reaction which was detected with a standard ELISA plate reader. Results were scored for HSV-1 and HSV-2 antibodies according to the manufacturer's criteria. The sensitivity and specificity of the RIBA versus the Gull ELISA was 99.2% versus 99.7% and 97.1% versus 96.7%, respectively.13,14

Statistical analyses
To examine the relationship between demographic and sexual behaviour variables and seropositivity for HSV-1 and HSV-2, univariate analyses were performed by calculating odds ratios (OR) and 95% CI.15

Multivariate models were constructed with ‘year of obtaining a blood sample’ (1993 versus 1998) as dependent variable. Either HSV-1 or HSV-2 serostatus and all variables from univariate analyses were included in the latter model as potential confounding factors.

To determine risk factors independently correlated with HSV-1 or HSV-2 seropositivity, data were analysed in multivariate models with HSV-1 or HSV-2 serostatus as dependent variable. All variables from univariate analyses were included in these models, except HIV serostatus because there were too few seropositive samples. The variables ‘sexual preference by gender’ and ‘having had passive anal intercourse during the past 6 months’ were combined in the analyses to estimate a possible increased risk for HSV infection because of receptive anal intercourse, stratified by gender, as has been described.9 By using stepwise backward elimination based on the likelihood-ratio test, initial predictive models were constructed with only those variables which, adjusted for each other, were significantly associated with HSV-1 or HSV-2 seropositivity. The likelihood-ratio test (P < 0.05) was used to eliminate variables from the initial models and the score statistic (P < 0.1) was used to determine whether eliminated variables could be re-entered in the reduced models. Variables significantly correlated with the dependent variable in only one year were re-introduced in the model of the other year. The remaining eliminated variables were tested either separately or all together to determine whether they could be re-entered in the reduced models.

The independent relationship between HSV-1 and HSV-2 seropositivity was studied in an exposure-disease model which was constructed following a modelling strategy described by Kleinbaum.16 The exposure variable in this model was the test result of HSV gG1, based on the assumption that HSV gG1 seropositivity reflects prior infection with HSV-1. Likewise, the disease variable was the test result for HSV gG2. To estimate the effect of HSV-1 seropositivity on HSV-2 seropositivity as accurately as possible all variables from univariate analyses were included in this model in order to adjust for possible confounding factors. Interaction terms between the exposure variable and the main effect variables from univariate analyses were included. A more parsimonious model was constructed using the stepwise backward elimination procedure as described previously. Separate models were constructed for both study groups. The goal of constructing these models was to calculate an overall estimate for the effect of HSV-1 seropositivity on the likelihood of HSV-2 seropositivity, for each year separately.

Results were analysed using SPSS 8.0 (SPSS Inc., Chicago, IL, USA). Other statistical tests were carried out as described in the text.

Results

From February 1993 to February 1994, 2701 (87%) patients at the STD clinic eligible to participate in the HIV serosurveillance study agreed to do so. From January 1998 to December 1998 the response rate was 78% (2904/3705). Details of non-participants in 1993 were reported elsewhere.17 Briefly, in multivariate analyses, male and female non-participants were more likely to be foreigners. Male non-participants were more likely to have more than one partner during the past 6 months and one or more STD.

We tested a random sample of 1024 sera (403 women, 538 heterosexual men, and 83 homo/bisexual men) collected from February 1993 to February 1994. The median age (range) of women was 27 (15–63) years, of heterosexual men 33 (17–69) years and of homosexual men 34 (17–68) years. A random sample of 654 sera (292 women, 307 heterosexual men, and 55 homo/bisexual men) collected from January 1998 to December 1998 was tested. The median age (range) of women was 27 (13–60) years, of heterosexual men 31 (16–71) years and of homosexual men 31 (18–60) years.

The overall seroprevalence of HSV-1 in 1993 versus 1998 was 68% (695/1024) versus 59% (384/654) ({chi}2-test, P < 0.001). Likewise, seroprevalence of HSV-2 was 30% (303/1024) versus 22% (141/654) ({chi}2-test, P < 0.001).

Factors associated with HSV-1 seropositivity in univariate analysis
In both study groups HSV-1 seropositivity increased strongly with age, non-Dutch ethnic background and lower level of education (Table 1Go). In 1993, HSV-1 seropositivity was significantly associated with ‘living in Rotterdam’ and there was some evidence for such an association in 1998 (OR = 1.3, 95% CI : 0.9–1.9). In 1993 ‘having had passive anal intercourse in the past 6 months’ was associated with lower HSV-1 seropositivity (OR = 0.5, 95% CI : 0.3–0.8). This effect was not observed in 1998 (OR = 1.3, 95% CI : 0.8–2.1). ‘Having worked as or visits to a commercial sex worker’ and ‘having had >4 sexual partners in the past 6 months’ increased the likelihood of being HSV-1 seropositive in both years. A significant association was also noted with ‘having a history of STD’. In both years HSV-1 seropositivity was not associated with HIV seropositivity. However, the corresponding 95% CI were wide (Table 3Go).


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Table 1 Univariate analyses of demographic and sexual behaviour profile correlated with antibody to herpes simplex virus (HSV) type 1 among attendees at a sexually transmitted disease (STD) clinic in Rotterdam, The Netherlands, 1993 versus 1998
 

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Table 3 Multiple logistic analyses for significant variables with specific antibodies to herpes simplex virus (HSV) type 1 and type 2 in 1993 compared to 1998
 
Factors associated with HSV-2 seropositivity in univariate analysis
Table 2Go shows the univariate analyses of demographic characteristics and sexual behaviour variables with HSV-2 seropositivity in 1993 and 1998. In both study groups, HSV-2 seropositivity showed a strong association with age, with peak levels among those >=35 years. In addition, HSV-2 seropositivity was related to ethnic background in both samples. Prevalence of antibody to HSV-2 increased significantly with decreasing level of education in both periods. In 1993, homo/bisexual orientation was significantly associated with higher seroprevalence of HSV-2; this was not observed in 1998. In both periods HSV-2 infection was more prevalent in females. ‘Having had passive anal intercourse in the past 6 months’ did not prove to be significantly correlated with HSV-2 seropositivity. However, other sexual behaviour variables were predictors for HSV-2 serostatus in both periods (Table 2Go). Human immunodeficiency virus serostatus was associated with HSV seropositivity in 1993 but not in 1998. In both years, HSV-1 seropositivity was significantly associated with HSV-2 seropositivity.


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Table 2 Univariate analyses of demographic and sexual behaviour profile correlated with antibody to herpes simplex virus (HSV) type 2 among attendees at a sexually transmitted disease (STD) clinic in Rotterdam, The Netherlands, 1993 versus 1998
 
To determine if HSV-1 and HSV-2 serostatus differed between 1993 and 1998 a multivariate model which adjusted for possible differences in the composition of both study groups was constructed. After correction for possible confounding factors, the seroprevalence of HSV-1 was significantly lower in 1993 compared with 1998 (OR = 0.6, 95% CI : 0.4–0.8, P = 0.001). This was also the case for the seroprevalence of HSV-2 (OR = 0.6, 95% CI : 0.4–0.7, P = 0.001) (models not shown).

Factors associated with HSV-1 seropositivity in multivariate analyses
Table 3Go presents the results of the stepwise backward logistic regression analyses for HSV-1 seropositivity. In multivariate analyses age and non-Dutch ethnic background were associated with HSV-1 seropositivity in 1993 and 1998. Lower level of education (P < 0.0001) and female gender (P < 0.01) were independently associated with HSV-1 infection in 1993 only. ‘Having had passive anal intercourse in the past 6 months’ was without an increased associated risk of HSV-1 infection. In 1993, none of the sexual behaviour variables were associated with an increased likelihood of HSV-1 infection. However, there was evidence for such an association in 1998.

A ‘z test’ was used to determine whether the differences between the coefficients of the variables ‘having had >4 partners in the past 6 months’ and ‘having a present diagnosis of STD’ changed significantly between 1993 and 1998. A borderline significant P-value (P = 0.06) was calculated for ‘having had >4 partners in the past 6 months’. The coefficients of ‘having a present diagnosis of STD’ did not change significantly between both years (P = 0.15).

Factors associated with HSV-2 seropositivity multivariate analysis
Table 3Go presents the results of the stepwise backward logistic regression analyses for HSV-2 seropositivity. In both years, increasing age and ethnic background were predictors of HSV-2 seropositivity in multivariate analyses (all variables, overall P-value < 0.01) (Table 3Go), although those of Turkish, North African and Mediterranean origin had no statistically significant increased likelihood of HSV-2 seropositivity. Lower level of education was correlated with HSV-2 seropositivity in 1993 only (P = 0.01). Female gender was associated with an increased likelihood of HSV-2 infection in both years. People involved in commercial sex in 1998 were more likely to be HSV-2 seropositive as were those ‘having had >4 partners in the past 6 months’ in 1993. A ‘history of STD’ was strongly correlated with past HSV-2 infection in both years.

Additional tests were performed to investigate whether eliminated variables could be re-introduced in the final logistic regression models for HSV-1 and HSV-2 in Table 3Go. None of the variables tested, either separately or together, had any significant effect (data not shown).

Exposure-disease model
The crude OR and the adjusted OR from the exposure-disease model describing the effect of HSV-1 seropositivity on susceptibility to genital HSV-2 infection are presented in Table 4Go. There is strong evidence that HSV-1 seropositivity is associated with HSV-2 seropositivity in 1993 (OR = 1.8, 95% CI : 1.3–2.5) and some evidence for such an association in 1998 (OR = 1.5, 95% CI : 0.9–2.5).


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Table 4 Crude odds ratio (OR) versus adjusted odds ratio for exposure of herpes simplex virus (HSV) type 1 to herpes simplex virus type 2
 
In the final model none of the interaction terms between the exposure variable (HSV-1) and the main effect variables proved to be statistically significant and they were therefore removed by the stepwise backward elimination procedure. In the final model HSV-1 seropositivity was not associated with HSV-2 seropositivity (1993: OR = 1.1, 95% CI : 0.8–1.7; 1998: OR = 1.0, 95% CI : 0.5–1.8).

Discussion

In this study, the seroprevalence of HSV-1 and HSV-2 was examined in a randomly selected group of STD clinic attendees. The independent risk factors correlated with HSV-1 and HSV-2 seropositivity were summarized.

Seroprevalence of HSV-1 was 68% in 1993 compared to 59% in 1998. For HSV-2, seroprevalences were 30% and 22%, respectively. After adjusting for confounding factors in multivariate models these differences remained highly significant (both P = 0.001). This supports the conclusion that HSV-1 and HSV-2 infections were becoming less common in our study population comparing 1993 and 1998.

This is the first study to report HSV-1 seroprevalence in The Netherlands and can therefore be used as baseline prevalence for STD clinic populations. The decrease in HSV-1 seroprevalence was most striking in individuals younger than 25 years of age (60% in 1993 versus 50% in 1998). This corroborates observations by others who have reported a decreased incidence of childhood oral HSV infection in Europe.18,19

Risk factors for HSV-1 seropositivity in The Netherlands have not been published. In 1993 and 1998 age and ethnic background were correlated with HSV-1 seropositivity. Lower level of education and female gender were only associated with HSV-1 seropositivity in 1993. These risk factors have been reported earlier as being associated with HSV-1 seropositivity in other countries.19,20

Several studies have indicated an increasing incidence of genital herpes caused by HSV-1 in the past decade.11,21,22 This trend has been attributed to ‘changes in sexual practices’ and falling rates of HSV-1 labial herpes infections in childhood. In this study, none of the sexual behaviour variables were correlated with HSV-1 seropositivity in 1993. However, in 1998 ‘having had >4 sexual partners in the past 6 months’ and ‘having a present diagnosis of STD’ were linked with HSV-1 seropositivity. These changes in risk factors support the view that sexual transmission of HSV-1 is increasing in The Netherlands.

Since the late 1970s a rise in HSV-2 seroprevalence has been observed in the general population in the US, reaching 22% for the period 1988 to 1994.3 This increasing prevalence has also been observed in both developed and developing countries.20,23 Data on HSV-2 seroprevalence in The Netherlands to date are limited to STD clinic attendees. In 1986–1988, HSV-2 seroprevalence was 32% in Amsterdam and 22% in 1998, in Groningen.9,10 These differences may either indicate that variation of HSV-2 seroprevalence in The Netherlands has a geographical pattern, or that HSV-2 infection is decreasing. The study presented here supports the latter view. In making such comparisons one has to be aware that the selected groups do not represent the general population. However, the falling rate of HSV-2 infection among a population at high risk of STD does not suggest an increasing number of HSV-2 seropositive individuals in the general population. Since HSV-2 seroprevalence can be used as a surrogate marker for sexual lifestyle,24 this downward trend—mainly in individuals younger than 25 years of age—could be an effect of ongoing national public health campaigns promoting safer sexual practices. Future studies of STD clinic attendees and in the general population may establish whether HSV-2 seroprevalence continues to decrease in The Netherlands.

Risk factors independently correlated with HSV-2 seropositivity were also determined. In 1993 and 1998 these were age, ethnic background, female gender and a history of STD. ‘Lower level of education’ and ‘number of partners in the past 6 months’ increased the likelihood of being seropositive for HSV-2 in 1993. ‘Having worked as or visits to a commercial sex worker’ had a similar effect in 1998. The risk factors reported here confirm the reported recognized risk factors for HSV-2 seropositivity.3,4,24

An important hypothesis in HSV research is that prior labial HSV-1 infection reduces the risk of genital HSV-2 infection and has an attenuating effect on the severity of HSV-2.2528 This view has been supported by the presence of cross-reactive neutralizing antibodies after HSV infection. In addition, local (cross-reactive) T cell-mediated immune responses have been shown to be important in resolving genital HSV infection.29 Nonetheless, epidemiological evidence to back up the proposition of reduced susceptibility to HSV-2 infection after an earlier labial HSV-1 infection is based on only a few small case-control studies.30,31 A large epidemiological study in which the relationship between the presence of HSV-1 and HSV-2 specific antibodies is described was published in 1999.32 In that prospective study 2393 sexually active HSV-2 seronegative individuals were examined for clinical and serological evidence of new HSV infection. Of the participants, 1508 were seropositive for HSV-1 and 885 were seronegative. It appeared that previous HSV-1 infection did not reduce the incidence of HSV-2 infection. The multivariate exposure-disease model presented in our study provides additional evidence that the risk of genital HSV-2 infection is independent of a previous HSV-1 infection. These results allow the conclusion that HSV-1 antibodies fail to protect against subsequent HSV-2 infection. However, because this conclusion contradicts one of the paradigms in HSV research, additional immunological studies are recommended to corroborate the findings reported here.

Acknowledgments

GMGMV was subsidized by ‘SWOO’ (Rotterdam Eye Hospital). The authors thank MJC Eijkemans for reviewing the statistical methods used, C Maas for providing technical assistance and RP Verkooyen for providing access to epidemiological data. This work was supported by a grant from Glaxo Wellcome.

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