Herpes Simplex Virus Seroprevalence among Pregnant Finnish Women and Their Spouses—A Six-Year Follow-Up Cohort Study

The aim was to evaluate the herpes simplex virus (HSV) seroprevalence and seroconversion among 285 pregnant women and their 120 male spouses in Finland during a six-year follow-up (FU) between 1998–2008. We also studied the effect of sexual habits, pregnancy, and other demographic factors on the acquisition of HSV infection. Combined HSV-1 and HSV-2-IgG antibodies were assessed in the first baseline serum samples with an indirect enzyme immunoassay method. The individuals with seronegative or borderline HSV serology at baseline were additionally tested using their latest FU serum sample available. The overall HSV seroprevalence during the FU was 58.9% (168/285) among the women and 53.3% (64/120) among their spouses. The seroconversion rate was 11.4% (15/132) and 12.5% (8/64) among women and their spouses, respectively. Both spouses were HSV seropositive in 39.2% (47/120). To determine the HSV-2 seroprevalence, we also tested all HSV-seropositive participants using HSV-2-specific antigen. HSV-2 seropositivity was detected in 10.9% (44/405) of the participants. The age (p = 0.006) and history of genital warts (p = 0.006) of the women were associated with combined HSV-1 and/or HSV-2 seropositivity, while a younger age was related to HSV seroconversion (p = 0.023). Among the male spouses, HSV seropositivity was associated with the practice of oral sex (p = 0.033). To conclude, women of childbearing age acquire primary HSV infections and the presence of HSV in oral epithelium is common among HSV-seropositive individuals.


Introduction
The herpes simplex virus type 1 (HSV-1) infection is transmitted usually via saliva and occurs most commonly in the head and neck region, while HSV-2 infects mostly the genital region and is sexually transmitted [1]. However, HSV-2 can also cause oral infections and HSV-1 genital infections [2,3]. Infection with the HSV (both HSV-1 and -2) is lifelong and in the oral region the latent HSV in the trigeminal ganglion can reactivate after various time intervals. During the active infection, HSV shedding can be detected in saliva and HSV DNA can be found in exfoliated oral mucosal cells of which gingival sulcular epithelium is the most studied [4]. Asymptomatic shedding of oral HSV-1 can happen at least monthly in ≥70% of the population or even more regularly [5]. In cross-sectional surveys, oral HSV shedding is detectable in 2-5% among adults [6].
The aim of the present study was to evaluate HSV seroprevalence and seroconversion (combined HSV-1 and HSV-2) among young pregnant women and their spouses during the six-year follow-up (FU) utilizing the Finnish Family HPV Study cohort [15][16][17][18][19][20]. Additionally, we were interested on the proportion of HSV-2 positivity among those who tested HSV seropositive. Furthermore, we also analyzed the association of HSV seropositivity in those who had previously tested HSV-1 DNA positive in their oral swab specimens as described earlier [21,22]. The association of pregnancy and demographic factors i.e., sexual habits with HSV seroprevalence, was also assessed.

Study Population and Clinical Samples
This study is part of the Finnish Family HPV Study which is a longitudinal cohort study of the University of Turku/Turku University Hospital, Finland, as described [15][16][17][18][19][20]. Originally, 329 families were recruited between the years 1998-2002, comprising of 329 women and their 139 male spouses and 331 neonates. An original aim of the Finnish Family HPV Study was to understand the mother-child transmission of HPV. Thus, not all of the fathersto-be participated in the study. The inclusion criteria for a parent were baseline sampling at recruitment (36 weeks of their index pregnancy) and an informed consent to participate. This study was approved by the Research Ethics Committee of Turku University Hospital (#3/1998, #2/2006, 45/180/2010 and TO7/008/2014).
In the present sub-study, the prevalence of HSV antibodies among the women and their spouses at the entry of the study was analyzed. In total, serum samples were available from 285 women (mean age 25.5, range 18-38 years) and their 120 spouses (mean age 28.7, range 19-46 years). The HSV seroconversion during the six-year FU was assessed by selecting the last FU sample available for an additional HSV testing from all HSVseronegative individuals and those with a borderline result at baseline. The mean timespan of the last FU sample was 57.3 (range 6.7-94.5) and 47.8 (range 5.0-91.5) months for the women and their spouses, respectively. In total, 132 women and 64 spouses were retested.
The demographic data recorded at 2 months after delivery including tobacco and alcohol use and sexual habits were available from the questionnaires as described earlier [15][16][17][18][19][20].

Indirect Enzyme Immunoassay (EIA)
Combined IgG antibodies against herpes simplex viruses (HSV-1, HSV-2) were analyzed with indirect enzyme immunoassay (EIA), as previously described [13,23]. Briefly, the antigens were purified from HSV-1-(strain F) and HSV-2 (strain G)-infected Vero cell cultures (American Type Culture Collection, Manassas, VA, USA) [13]. Polystyrene strips (Thermo) were coated with pooled HSV-1 and HSV-2 envelope antigens in phosphate buffered saline. Serum samples were tested as duplicates and incubated for 2 h at 37 • C, after which horseradish peroxidase (HRP)-conjugated anti-human-IgG (dilution 1:16,000, DAKO, Jena, Germany) was added. Then, tetramethylbenzidine was added and the reaction was stopped with 0.2 N sulfuric acid. The optical absorbance was measured at 450 nm by a BEP III analyzer (Siemens, Munich, Germany). The results were determined by comparing the average absorbance of specimens to negative (<1 EIA-units; EIU) and positive (100 EIU) controls. Sera with ≥10 EIU were scored positive, those with 5-9 EIU as borderline, and those with ≤4 EIU as negative. The seroconversion was defined as acquisition of detectable HSV-IgG antibodies.
The sera which scored positive in the HSV type-common EIA test (marked in the manuscript as HSV seropositive ones) were further studied for HSV-2 type-specific antibodies using HerpeSelect 2 ELISA IgG kit (Focus Diagnostics, Cypress, CA, USA). The assay was performed according to the manufacturer's instructions and the results were evaluated as index values relative to the kit cut-off calibrator, as recommended by the manufacturer.

Patient and Public Involvement
The study was based on anonymized patient records and the results were not disseminated to the participants. The participants were not involved in setting the research question, study design, study methods or in how the study was conducted.

Statistics
Statistical analyses were carried out using SPSS ® (SPSS for Windows, version 24.0.0.1, SPSS Inc., Chicago, IL, USA) and STATA (STATA/SE 14.1, StataCorp, College Station, TX, USA). Frequencies were analyzed using the χ 2 -test. Differences in the means of continuous variables were analyzed using the Mann-Whitney test or the Kruskal-Wallis test for two and multiple independent samples, respectively. ANOVA was used to derive the means (and their SD) for the continuous variables.
Oral HSV DNA status based on oral brush samples has been published earlier [21,22]. This previously published HSV DNA data was used here in the statistical analysis to disclose the possible association between the HSV serology and the presence of intraoral HSV DNA.
The age (p = 0.006) and positive history of genital warts (p = 0.006) of the women were significantly associated with HSV seropositivity. In addition, younger age was associated with increased susceptibility to HSV seroconversion among women (p = 0.023) (Supplementary Table S1). Self-reported high frequency of oral sex was associated with the HSV seropositivity among the male spouses (p = 0.033) (Supplementary Table S2). HSV seropositivity was not associated with education, number of sexual partners, sexual debut age, practicing anal sex, other sexually transmitted infections or the use of tobacco or alcohol (Tables S1 and S2).

Discussion
The present study showed that women of childbearing age acquire primary HSV infections in Finland. The HSV seroprevalence (combined for HSV-1 and HSV-2 as given in the methods) was over 50% in our study cohort. HSV seropositivity increased with age among the women, but not among their spouses. These findings are in agreement with previous studies on HSV seroprevalence in developed countries in the 21st century. A previous study reported an HSV seroprevalence of 54.3% among Finnish women giving birth at Turku University Hospital during one month in the year 2000 [13]. The most recent study on pregnant women (average age 28.5 years) in Finland showed that HSV-1 seroprevalence had further declined from 69.5% (in 1992) to 45% (in 2012) (p < 0.001) [11]. In Europe, HSV seroprevalence varies widely, for example, HSV-1 and HSV-2 seropositivity were 52% and 13% in Finland [9], 84% and 24% in Bulgaria [9] and 90.4% and 9.3% in Poland [24]. In all studied European countries [9] as well as in the USA [10] the probability for HSV carriage increased with age.
In our cohort, 11.4% (15/132) of the women and 12.5% (8/64) of their spouses seroconverted during the FU, which is consistent with the estimated overall annual rate of transmission; 4-5% per annum [6]. Younger age for women increased the probability for HSV seroconversion. These results imply that women at childbearing age acquire primary HSV infections in Finland. In a previous 20-month FU study on seronegative non-pregnant women (aged 18-30 years), in a different population, the primary HSV infection occurred in 5.3% (183/3438) of the participants [26]. The younger (18-22 years) women were more likely to experience HSV-1 seroconversion [26]. However, another Finnish study on pregnant women (median age 30 years) with two serial samplings during the pregnancy showed no cases with primary HSV infection, leading to the assumption that the risk for viral infections during pregnancy cannot be generalized [13].
Our results showed that women were slightly more often HSV seropositive than their spouses (the test detecting both HSV-1 and HSV-2 antibodies). In line with our results, HSV-1 seroprevalence is also slightly more common in women than men in the US [10] and in Northern Europe (4/7 countries) [9,24].
We have previously analyzed the presence of HSV-1 and HSV-2 DNA in oral brush samples in this cohort, as described earlier [21,22]. Of the HSV-seropositive women, 18.5% (31/168), and 20.3% (13/64) of their HSV-seropositive spouses were oral HSV-1 DNA positive with PCR during the FU, but none had tested HSV-2 DNA positive [21,22]. Two of the 15 seroconverted women (13.3%) and none of the seroconverted spouses had oral HSV-1 DNA detectable during the FU. For example, one seroconverted woman had the HSV antibody level of 70 EIU at the 36-month FU visit and HSV-1 DNA was detected at 222 copies/sample at the 24-month FU visit. Her mean HSV copy number values varied from 13 to 772 HSV DNA copies per sample at different follow-up time points [21]. Interestingly, her spouse was also HSV-1 DNA positive at the same FU visit [22]. He was HSV seronegative at baseline, but had a borderline HSV antibody level (9 EIU) at the 36-month FU visit. Furthermore, his oral brush samples at the 2-and 24-month FU visits tested HSV-1 DNA positive with 16 and 15 DNA copies, respectively.
We can speculate that the HSV-1 shedding among the HSV-1 carriers is even more common, as shedding of the HSV is likely to happen between the FU visits. Furthermore, HSV shedding would be easier to detect in saliva than in epithelial brush samples alone. Our results agree with previous studies, showing that approximately 20-30% of the HSV-1 seropositive individuals have recurrent intraoral HSV-1 infections 1-4 times annually, especially after stressful events [5]. In addition, oral shedding is mostly subclinical and some 70% of the population shed HSV-1 asymptomatically at least once a month, regardless of the seropositivity [5].
Of the HSV-seronegative women, 2.7% (3/112) and 7.4% (4/54) of their spouses had previously yielded a positive HSV DNA result in their oral sample. Intraoral HSV shedding was also found among seronegative individuals in other studies [5]. Both viral culture and PCR might be more sensitive to detect HSV than immunologic assays based on viral antibody detection. Thus, HSV infection could be more prevalent in the population than indicated by the HSV seroprevalence data alone [5].
In our study, the high frequency (self-reported in the questionnaire with the scale: frequently/sometimes/never) of oral sex was statistically significantly associated with the HSV seropositivity in the male spouses, but not among the women. The history of genital warts was associated with the women's HSV seropositivity, which might partly indicate the sexual habits of the women. Thus, we can speculate that sexual habits influence the acquisition of HSV infection among young women and men in Finland. Earlier studies showed that HSV seroprevalence was higher among sexual risk behavior groups [6,7] increasing along with the number of sexual partners [10,31]. Early age for first sexual intercourse has been associated with HSV-1 seropositivity among young individuals [32], which was, however, not shown in the present study. The overall small fraction of HSV-2 seropositive individuals in Finland was evident also in our HSV antibody results. The majority of the HSV-2 seropositive participants harbored the HSV-2 antibodies already upon entry to our study. We found no HSV-2 DNA in the oral samples of the women or the men in our earlier studies [21,22], which supports the previous observations of HSV-2 lesions being rare in the orofacial area [33].
In conclusion, the HSV seroprevalence was nearly 50% among healthy, young Finnish parents close to the birth of their offspring. Oral HSV-1 shedding is common among HSVseropositive individuals and contributes to the potential risk of transmission. Increased frequency of oral sex might increase the risk for HSV infection.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/microorganisms10081506/s1, Table S1: Key demographic characteristics of the 285 women stratified according to herpes simplex virus (HSV) serology; Table S2: Key demographic characteristics of the 120 male spouses* according to their herpes simplex virus (HSV) serology.
Author Contributions: J.L. contributed to the study concept, design, analyses, and drafting of the manuscript supervised by the co-authors. S.S. and V.H. contributed to the concept, design, analyses and revision of the manuscript. T.V., J.R. and K.K. critically revised and approved the final version for publication. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.