Seroprevalence and Vaginal Shedding of Herpes Simplex Virus Type 2 in Pregnant Adolescents and Young Women from Morelos, Mexico

Adolescents and young people are particularly vulnerable to contracting STIs, including HSV-2; furthermore, vaginal shedding of HSV-2 during pregnancy can cause vertical transmission and neonatal herpes. To evaluate the seroprevalence of HSV-2 and vaginal HSV-2 shedding in adolescent and young pregnant women, a cross-sectional study was carried out in 496 pregnant women—adolescents and young women. Venous blood and vaginal exudate samples were taken. The seroprevalence of HSV-2 was determined by ELISA and Western blot. Vaginal HSV-2 shedding was assessed by qPCR of the HSV-2 UL30 gene. The seroprevalence of HSV-2 in the study population was 8.5% (95% CI 6–11), of which 38.1% had vaginal HSV-2 shedding (95% CI 22–53). Young women presented a higher seroprevalence of HSV-2 (12.1%) than adolescents (4.3%), OR = 3.4, 95% CI 1.59–7.23. Frequent alcohol consumption was significantly associated with HSV-2 seroprevalence, OR = 2.9, 95% CI 1.27–6.99. Vaginal HSV-2 shedding is highest in the third trimester of pregnancy, but this difference is not significant. The seroprevalence of HSV-2 in adolescents and young women is similar to that previously reported in other studies. However, the proportion of women with vaginal shedding of HSV-2 is higher during the third trimester of pregnancy, increasing the risk of vertical transmission.


Introduction
Herpes simplex virus type 2 (HSV-2) is the main causative agent of genital herpes [1]. This infection remains for life and may present periodic reactivations [2]. HSV-2 is widely distributed worldwide, and WHO estimates indicate that HSV-2 affects approximately 13% of people aged 15-49 worldwide (2016 data) [1]. Typically, women are more susceptible than men [3], and pregnant women are more susceptible than non-pregnant women [4,5]. The greatest number of new infections occur during adolescence because adolescents are at high risk of contracting sexually transmitted infections (STIs), due to their behavioral and biological characteristics [6]. Adolescents have risky sexual behaviors, such as multiple sexual partners or lack of condom use [7,8]. Moreover, adolescent women produce less cervical mucus and have low estrogen levels, low vaginal pH, and cervical ectopia [9]. This epithelium is more susceptible to pathogens, unlike the squamous epithelium that lines the vagina and cervix in adult women [9].
HSV-2 infection is difficult to detect because new cases and reactivations are usually asymptomatic [10]. Like all herpes viruses, HSV-2 infection is characterized by cycling through latent and active (or lytic) stages. The active stage is the most important for viral production (shedding) and transmission to new hosts. During pregnancy, HSV-2 reactivation is common, and approximately 5% of pregnant women can transmit the infection, which occurs when the newborn has been exposed to HSV-2 in the genital tract during delivery, causing neonatal herpes [11,12]. Untreated local infection in the newborn can progress to disseminated disease, causing infections of the mouth, eyes, skin, central nervous system, and neonatal abnormalities [11,13].
In Mexico, the seroprevalence of HSV-2 has been reported in women of reproductive age [7,8,[14][15][16]. Moreover, the seroprevalence was 14% among pregnant women, higher among pregnant women aged 31 years (26.4%) than among women under 20 years of age (8.6%) [15]. However, data on the percentage of women who vaginally secrete HSV-2 are insufficient, particularly during pregnancy. Therefore, we evaluated the seroprevalence and vaginal shedding of HSV-2 in adolescents and young pregnant women. A high proportion of women in the last trimester of pregnancy were found to have vaginal shedding of HSV-2, which could cause neonatal herpes.

Study Population
A cross-sectional study was carried out in adolescent and young women (10-19 years for adolescents and 20-24 years for young people) who were pregnant and attending health services for pregnancy care in Morelos, Mexico (Yautepec and Cuernavaca Health Centers), during the period from October 2018 to March 2020.
Pregnant women up to 24 years of age were invited to participate, signed the consent/informed assent letter, and answered a questionnaire about sociodemographic, clinical, and sexual behavior characteristics. In addition, they provided vaginal exudate and blood samples. Women who consumed antibiotics for at least two weeks before sampling were excluded from the study.

Ethical Considerations
The protocol was approved by the Ethics, Biosafety, and Research committees of the National Institute of Public Health (Project CI 1529). To obtain informed consent and/or assent, prospective participants were given a written letter explaining the study and detailing the implications of their participation. Likewise, to guarantee confidentiality and anonymity, each participant was identified by a code, omitting her name at all times and without requesting binding data.

Vaginal and Blood Samples Collection
Vaginal exudate was collected with a Dacron swab, which was inserted through the vagina consecutively no deeper than 2 cm, to avoid reaching the cervical region. The swab was placed in a vial with preservation buffer (Sure Path) and stored at −20 • C until processing.
A venous blood sample was also collected and centrifuged at 3500 rpm for 6 min to obtain the serum and prepare aliquots. The samples were stored at −20 • C until processing.

Vaginal HSV-2 Shedding
Vaginal shedding of HSV-2 was assessed in women who tested seropositive for HSV-2. DNA was extracted from 400 µL of vaginal exudate using the Quick-DNA Miniprep Plus kit (Zymo Research ® , Irvine, CA, USA). The obtained DNA was quantified in a NanoDrop™ 2000 Spectrophotometer (Thermo Fisher Scientific ® , Waltham, MA, USA) and processed by qPCR, to amplify a 151 bp fragment of the UL30 gene from HSV-2 and a 140 bp fragment of the human β-globin gene as a control; each gene was amplified separately.
Each 25 µL qPCR contained 1X Maxima SYBR Green/ROX qPCR Master Mix 1, 0.3 mM of each primer, 1 µL of template DNA (≤500 ng), and 10 µL of nuclease-free water. The amplification was carried out in a CFX96TM Real Time PCR Detection System (BIO RAD ® , Hercules, USA as follows: a cycle of 95 • C for 10 min, 40 cycles of 95 • C for 15 s, annealing temperature (as shown in Table 1) for 30 s and 72 • C for 30 s, and a dissociation cycle of 95 • C at 63 • C, with fluorescence measurements every 2 • C. Each sample was processed in triplicate for both genes, and were considered positive if Ct was >32. The standardization and validation methodology for the relative quantification of the UL30 gene (HSV-2) and the human β-globin gene in anal and genital samples was carried out according to Vergara-Ortega et al. [17].

Statistical Analysis
Definition of variables: The variable "educative level" was divided into three categories-No studies/elementary school, middle school, and high school/university. "Vaginal lesions" refers reported vaginal blisters, ulcers, warts, or pimples at some time in life. "Smoking" refers to having smoked 100 or more cigarettes. "Alcohol consumption" was defined as the consumption of 5 drinks or more weekly. "Drug use" is defined as drugs having been used at least once during their lifetime. Clinical signs or symptoms considered were threatened abortion, dyspareunia, vaginal discharge, and urinary tract infection. The "use of antibiotics" refers to antibiotics taken orally or vaginally prior to the month before the study period. "Hormonal contraceptives" include pills, injections, patches, or morning-after pills. "Frequency of sex" refers to the number of times sexual intercourse was performed in a given period. "Oral sex" referred to oral sex that was received, given, or both.
A descriptive analysis was performed for the demographic, clinical, and sexual behavior variables. Means were determined for the quantitative variables, and frequencies were determined for the qualitative variables, both with 95% confidence intervals. The Shapiro Wilk test was applied to variables to verify the normality of the data. Subsequently, a bivariate analysis was carried out to identify the factors associated with the presence of antibodies against HSV-2. Finally, a multivariate logistic regression analysis was performed from the statistically significant variables (p < 0.05), and adjustment was made with the variables "Age", "Trimester", "Alcohol consumption", "Antibiotic use", "frequency of sex" and frequency of "condom use", starting from a saturated model until the most parsimonious model was obtained. Statistical analyses were performed using STATA 14.0 software.

Characteristics of Study Population
The current study included 496 women with a mean age of 19.7 ± 2.9 years, of whom 46.8% were adolescents. Almost half of the pregnant women had a high school education, 26.2% smoked or used to smoke, 43.5% used alcohol occasionally, and 26.2% consumed illicit drugs at some time. A total of 28.0% of the women were in the first trimester of pregnancy, 42.7% in the second trimester, and 29.2% in the third trimester. More than half of the women mentioned some clinical sign related to an STI, 20% reported a history of genital lesions, 10% reported miscarriage or stillbirth, and 26.4% used some form of antibiotic immediately preceding the month before the study period. Almost half of the women considered contracting an STI unlikely. The average number of lifetime sexual partners was 2, 14.9% practiced anal sex, and 38.2% practiced oral sex. A total of 12.9% of women used hormonal contraceptives, and more than half had used a condom at some time in their lives. The demographic, clinical, and sexual behavior characteristics of the pregnant women are shown in Table 2.

Asociated Factors with Seroprevalence of HSV-2
The sociodemographic, clinical, and sexual behavior characteristics of pregnant adolescent and young women, as well as their association with HSV-2 seroprevalence, are shown in Table 3. The seroprevalence of HSV-2 was 3.4 times higher in young women (12.1%) than in adolescents (4.3%). Women with a higher frequency of alcohol consumption had a 2.9 times higher probability of being seropositive for HSV-2. Pregnant women with a higher educative level had the highest HSV-2 seroprevalence, but this difference was not statistically significant. In relation to clinical variables, women who used antibiotics in the last month had a 1.9 times higher probability of being seropositive for HSV-2, showing marginal significance (p = 0.07). Women with a history of genital lesions were 1.7 times more likely to have antibodies against HSV-2, but the difference was not statistically significant. Finally, none of the sexual behavior variables were statistically significant.

Discussion
The seroprevalence of IgG antibodies against HSV-2 found among the 496 pregnant women included in this study was 4.3-8.5% among adolescents and 12.1% among young women. These data are similar to those previously reported. Worldwide, the estimated prevalence is 16.3%. However, among females in the Americas in 2016, it was 7.8% and 14.4% for adolescents and young women, respectively [18]. In Mexico, the HSV-2 seroprevalence ranges from 2.4% to 25%. A study conducted in pregnant and postpartum women from 4 different hospitals in Morelos, Mexico (2006-2009) reported a seroprevalence against HSV-2 of 14.5%, being lower among women 20 years of age or younger than among women 31 years of age or older, with values of 8.6% and 26%, respectively [15]. The national seroprevalence among Mexican men aged 15 to 49 years in 2012 was 9.9%, with a seroprevalence of 2.4% in adolescents and 11.7% in young men, while in women, it was 12.2% [19]. Another study conducted among women seen at public gynecology and colposcopy services, whose mean age was 46 years, reported an HSV-2 seroprevalence of 25% [14]. This report is the first in Mexico focused on HSV-2 in pregnant adolescents and young women, and is the first to include vaginal shedding of HSV-2 during pregnancy.
On the other hand, the incidence trend of HSV-2 is increasing in Mexico. Data from the Morbidity Yearbooks of the General Directorate of Epidemiology of the Ministry of Health of Mexico from 2011 to 2021 [20] show that, in 2011, the incidence in women was 2.69 cases/100,000 and was higher among women aged 20 to 24 years (5.47 cases/100,000), while in 2021, the incidence increased to 6.89 and 18.73 cases/100,000, respectively ( Figure 2). In the last 10 years, the incidence of HSV-2 has tripled among Mexican women. Women ages 20-24 had an average annual increase of 1.21 cases of genital herpes, more than double the average annual increase for women of 0.48 cases. The slopes are shown in Figure 2. Therefore, young women present a greater possibility of transmitting neonatal herpes during pregnancy.
Risk factors that have been previously associated with HSV-2 infection in women have been age, region, level of urbanization, sexual debut age, educative level, and previous history of abortion [16,20]. However, in this study, only age, alcohol consumption, and antibiotic use were found to be associated with HSV-2 seroprevalence.
In this study, we found that pregnant young women had a higher seroprevalence of HSV-2 than pregnant adolescents, probably due to a longer exposure time to the virus [21]; furthermore, IgG antibodies against HSV-2 indicate past exposure and an immunologic memory of infection [22]. Alcohol consumption was associated with a higher seroprevalence of HSV-2, probably due to the microenvironment of adolescents, which in turn influences their group of possible sexual partners and the probability that these partners are HSV-2 positive [23]. Excessive alcohol consumption is associated with behavioral changes; women who drink before having sexual relations could have risky sexual behaviors, which increases the risk of infection with HSV-2 and other STIs [24]. Interventions to reduce alcohol consumption in adolescents and young women could reduce risky sexual practices and HSV-2 transmission. Risk factors that have been previously associated with HSV-2 infection in women have been age, region, level of urbanization, sexual debut age, educative level, and previous history of abortion [16,20]. However, in this study, only age, alcohol consumption, and antibiotic use were found to be associated with HSV-2 seroprevalence.
In this study, we found that pregnant young women had a higher seroprevalence of HSV-2 than pregnant adolescents, probably due to a longer exposure time to the virus [21]; furthermore, IgG antibodies against HSV-2 indicate past exposure and an immunologic memory of infection [22]. Alcohol consumption was associated with a higher seroprevalence of HSV-2, probably due to the microenvironment of adolescents, which in turn influences their group of possible sexual partners and the probability that these partners are HSV-2 positive [23]. Excessive alcohol consumption is associated with behavioral changes; women who drink before having sexual relations could have risky sexual behaviors, which increases the risk of infection with HSV-2 and other STIs [24]. Interventions to reduce alcohol consumption in adolescents and young women could reduce risky sexual practices and HSV-2 transmission.
The use of antibiotics was also found to be associated (without adjusting for a higher seroprevalence), probably due to the high prevalence of urinary tract infections in the population, since women are more susceptible to urinary tract infections due to the shorter urethra, the greater proximity of the anus with the vagina, and the easier entry of pathogenic microorganisms through sexual activity and pregnancy per se [25]. Changes in the vaginal microbiota may also be associated with HSV-2 seropositive women, but this issue needs to be investigated.
Regarding educative level, young women with a high school or university education had the highest HSV-2 seroprevalence compared to those in primary or secondary education, with no statistically significant difference; however, a protective association with educative level has been reported: the higher the level of education, the lower the probability that women will acquire STIs such as HIV and HSV-2 [26,27]. Young women who attend school longer have a lower risk of acquiring HIV and HSV-2 infection due to the structure of their sexual network [27], since the number of sexual partners and the age difference between their potential partners are reduced [28]. Although this study found data contrary to what has been reported, the relationship between educative level and HSV-2 The use of antibiotics was also found to be associated (without adjusting for a higher seroprevalence), probably due to the high prevalence of urinary tract infections in the population, since women are more susceptible to urinary tract infections due to the shorter urethra, the greater proximity of the anus with the vagina, and the easier entry of pathogenic microorganisms through sexual activity and pregnancy per se [25]. Changes in the vaginal microbiota may also be associated with HSV-2 seropositive women, but this issue needs to be investigated.
Regarding educative level, young women with a high school or university education had the highest HSV-2 seroprevalence compared to those in primary or secondary education, with no statistically significant difference; however, a protective association with educative level has been reported: the higher the level of education, the lower the probability that women will acquire STIs such as HIV and HSV-2 [26,27]. Young women who attend school longer have a lower risk of acquiring HIV and HSV-2 infection due to the structure of their sexual network [27], since the number of sexual partners and the age difference between their potential partners are reduced [28]. Although this study found data contrary to what has been reported, the relationship between educative level and HSV-2 seroprevalence in young women may be due more to the age of the women than to their educative level.
Interestingly, although HSV-2 seroprevalence did not significantly differ by gestational age, we found a higher seroprevalence among women in the third trimester of pregnancy. This increase in HSV-2 seroprevalence in the last trimester may be due to a primary infection during the first trimester of pregnancy; the evidence shows that the risk of seroconversion to HSV-2 during pregnancy is 1-5%, but can reach 20% in serodiscordant couples, since, during pregnancy, the main risk factors for primary HSV-2 infection are other STIs, length of relationship, and partner's history of herpes [29]. However, the study did not track or determine HSV-2 viral detection in seronegative women, which limited detection of the women's seroconversion rate.
Furthermore, we observed a higher percentage of women with vaginal shedding of HSV-2 among women in the third trimester of pregnancy, which increases the risk of vertical transmission and the development of neonatal herpes. Neonatal herpes is usually acquired during the intrapartum period by exposure of the fetus to the virus in the maternal genital tract [30]. This transmission can occur even in the absence of lesions, due to subclinical viral shedding [31,32]. Therefore, to prevent the transmission of HSV-2 to the newborn, the timely detection of viral excretion is recommended through laboratory confirmation using HSV-2 antibody detection techniques and molecular detection techniques in vaginal swabs in women with suspected vaginal infection [31][32][33].
Unfortunately, in Mexico, neonatal herpes is not a mandatory reporting disease, so there are no reports on this infection in newborns. Preventing transmission to the newborn by reducing the late-pregnancy acquisition of infection in the mother and altering obstetric management may be the most likely approach to reduce neonatal herpes [30]. However, while the chances of a pregnant woman having an active genital lesion at the time of delivery can be reduced, asymptomatic excretion cannot always be prevented, and the risk of mother-to-child transmission persists [34,35]. Therefore, close monitoring is necessary during pregnancy in adolescents and young women to avoid the development of neonatal herpes.
Among the limitations of this study, ELISA was not used to detect the vaginal dissemination of HSV-2 in women who were seronegative for the virus, and women with primary infection who had not yet generated antibodies may have not been identified. In addition, newborns born to women with vaginal HSV-2 shedding were not followed up for vertical transmission and possible neonatal herpes.

Conclusions
The seroprevalence of HSV-2 in adolescents and young women in Mexico is similar to that previously reported. However, the proportion of women with vaginal shedding of HSV-2 is higher during the third trimester of pregnancy, increasing the risk of vertical transmission. Close surveillance is needed during adolescent and young pregnancies to prevent the development of neonatal herpes. Institutional Review Board Statement: All study procedures were reviewed and approved by the Bioethics and Research Committees of the National Institute of Public Health (permit CI 1519).

Informed Consent Statement:
Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.