Screening for TORCH Antibodies in Croatian Childbearing-Aged Women, 2014–2023

TORCH infections usually result in mild maternal morbidity, but may cause severe congenital abnormalities. Therefore, it is important to detect maternal infections, monitor the fetus after the disease has been recognized, and define the seronegative women who are at risk of primary infection during pregnancy. From 2014 to 2023, serum samples from 1032 childbearing-aged and pregnant women (16–45 years) were tested for IgM/IgG antibodies to the most common TORCH pathogens: Toxoplasma gondii, rubella virus (RUBV), cytomegalovirus (CMV), and herpes simplex viruses (HSV-1 and HSV-2). The overall IgG seroprevalence rates were 20.1% for T. gondii, 91.3% for RUBV, 70.5% for CMV, 66.8% for HSV-1, and 3.5% for HSV-2. Only HSV-2 seroprevalence was age-related, with a significant progressive increase in seropositivity from 0% in those aged less than 26 years to 9.3% in those older than 40 years. The seroprevalence of T. gondii was higher in residents of suburban/rural areas than in residents of urban areas (27.4% vs. 17.1%). In addition, participants from continental regions were more often toxoplasma-seropositive than those from coastal regions (22.2% vs. 15.3%). HSV-1 seroprevalence was also higher in suburban/rural areas (71.7% vs. 64.7%). Obstetric history was not associated with TORCH seropositivity. Univariate and multivariate risk analysis showed that suburban/rural areas of residence and continental geographic regions were significant risk factors for T. gondii seroprevalence. Furthermore, suburban/rural area of residence was a significant risk factor for HSV-1 seroprevalence, while older age was a significant risk factor for HSV-2 seroprevalence. A declining trend in the seroprevalence of all TORCH pathogens was observed compared to previous Croatian studies (2005–2011). Similarly, the proportion of women simultaneously IgG-seropositive to two or three pathogens decreased over time. The maternal serology before pregnancy could potentially reduce the burden of congenital TORCH infections.


Introduction
TORCH-Toxoplasma gondii, others (Treponema pallidum, varicella-zoster virus, parvovirus B19, etc.), rubella virus (RUBV), cytomegalovirus (CMV), and herpes simplex viruses type 1 and 2 (HSV-1, HSV-2)-encompasses some of the most prevalent pathogens associated with congenital abnormalities.The majority of TORCH infections result in mild maternal morbidity, but have severe fetal consequences, and treatment of the mother's illness often does not affect the fetal outcome.Therefore, it is important for clinicians to identify maternal infections and monitor the fetus once the disease has been identified [1].In addition, it is important to define the seronegative women who are at risk of primary infection during pregnancy [2].
Toxoplasmosis is a parasitic infection caused by the protozoon T. gondii.The majority of immunocompetent individuals do not develop symptoms or might have nonspecific flu-like symptoms and lymphadenopathy [3].Congenital toxoplasmosis is a complication of a primary maternal T. gondii infection during pregnancy.The mother's immune system, the virulence of the strain, the parasite load, and the gestational age at which the mother was infected all affect the severity of a newborn or fetal disease [4].The risk of T. gondii transmission increases with increasing gestational age, but the disease severity decreases [5].The spectrum of clinical manifestations of congenital toxoplasmosis varies from mild symptoms to severe consequences, such as chorioretinitis, hydrocephalus, microcephaly, mental retardation, and even death [6].
Rubella is a viral disease caused by RUBV.It is a highly contagious but generally mild and in most cases self-limiting disease.However, maternal RUBV infection during the first trimester of pregnancy can cause congenital rubella syndrome (CRS).CRS represents a global public health concern, with more than 100,000 estimated annual reported cases worldwide.The risk of transplacental transmission depends on the time of infection.If a maternal infection occurs within the first 12 weeks of gestation, up to 85% of newborns will have congenital defects compared to 50% in maternal infections within 13 to 16 weeks of gestation and 25% in maternal infections during the second part of the second trimester [7].Congenital heart diseases, cataracts, hearing impairment, and developmental delay are common congenital abnormalities associated with CRS [8].
CMV is a widely distributed human beta-herpesvirus.CMV commonly causes asymptomatic or mild mononucleosis-like disease in immunocompetent children and adults; however, congenital CMV infection is a public health problem, affecting 0.67% of live births [9,10].Congenital infection may occur in primary or recurrent CMV infection (reactivation or reinfection with a different viral strain).Although mostly asymptomatic, primary maternal CMV infection in pregnancy poses the highest risk of transplacental transmission (30-35%) compared to 1.1-1.7%for non-primary infections.Fetal abnormalities include intrauterine growth restriction, intracranial calcifications, microcephaly, ventriculomegaly, chorioretinitis, and hepatomegaly [11].
HSV-1 and HSV-2 are among the most widely distributed viruses worldwide.HSV-1 predominates in orofacial lesions, while HSV-2 mainly causes genital herpes.Nevertheless, both these viruses can infect orofacial areas and the genital tract.For HSV infections that occur in the last trimester of pregnancy, the risk of neonatal infection ranges from 30% to 50%, while the risk for early pregnancy infections is only 1% [12].Several forms of neonatal infections can be identified based on the time of maternal infection: intrauterine infections (5% of cases), postnatal infections (10% of cases), and perinatal infections (85% of cases) [13].Spontaneous abortion, intrauterine growth retardation, preterm birth, and congenital and neonatal HSV infections are all linked to genital herpes infections during pregnancy.HSV infections in neonates infected intrapartum or postnatally can manifest as disease localized to the skin, eye, and/or mouth, HSV encephalitis, or disseminated HSV infection, with mortality rates of more than 80% in untreated patients [12].

Study Participants
The study included 1032 childbearing-aged women (16-45 years; Figure 1) tested at the Croatian Institute of Public Health, the largest public health institute in the country.All women tested for TORCH pathogens consecutively from January 2014 to December 2023 were included.All of the participants were of Croatian nationality and there were no migrants tested.For this study, participants were classified according to age (a five-year age group), area of residence (urban or suburban/rural), geographic region (continental or coastal), and obstetric history (non-pregnant, normal pregnancy, unfavorable obstetric history: previous spontaneous abortions, children with congenital malformations, infertility).
as disease localized to the skin, eye, and/or mouth, HSV encephalitis, or disseminated HSV infection, with mortality rates of more than 80% in untreated patients [12].

Study Participants
The study included 1032 childbearing-aged women (16-45 years; Figure 1) tested at the Croatian Institute of Public Health, the largest public health institute in the country.All women tested for TORCH pathogens consecutively from January 2014 to December 2023 were included.All of the participants were of Croatian nationality and there were no migrants tested.For this study, participants were classified according to age (a five-year age group), area of residence (urban or suburban/rural), geographic region (continental or coastal), and obstetric history (non-pregnant, normal pregnancy, unfavorable obstetric history: previous spontaneous abortions, children with congenital malformations, infertility).

Statistical Analysis
Differences between groups of categorical variables were assessed using chi-squared and Fisher's exact tests.Odds ratios (ORs) and adjusted odds ratios (AORs) ± 95% confidence intervals (CIs) were used to assess the univariate and multivariate association of positive serological tests and explanatory variables (age, area of residence, geographic region, and obstetric history).Statistical analysis was performed using STATA/MP 17.0 for Windows (StataCorp LLC, Lakeway Drive, College Station, TX, USA).The level of statistical significance was set at p < 0.05.Positive, borderline, negative ELFA = enzyme-linked fluorescence assay, ELISA = enzyme-linked immunosorbent assay; NTU = Novatec units, AI = avidity index, IU/mL = international units/mL, VE = Virotech units.

Characteristics of Study Participants
Mean participant age was 31.9 ± 5.1 years.The area of residence was urban for 725 (70.3%) of participants and suburban/rural for 307 (29.7%) of participants.The geographic region of residence was continental for 724 (70.2%) and coastal for 308 (29.8%) participants.Regarding obstetric history, 271 (26.3%) of women were not pregnant, 608 (58.9%) had a normal pregnancy and 153 (14.8%) reported an unfavorable obstetric history.
Seroepidemiologic studies in Romania showed regional differences in the prevalence of T. gondii IgG antibodies, ranging from 38.24% in Bucharest to 55.8% in western Romania [28][29][30][31][32][33][34].In addition, significant regional differences in seropositivity within France were observed, from 19.1% in Grand Est to 35.1% in Occitanie, while the highest seroprevalence of 50.7% was detected in overseas departments (Guadeloupe, French Guyana, La Réunion, Martinique and Mayotte combined) [26].Studies from Italy showed similar results.From 2013 to 2017, a seroprevalence study on toxoplasmosis was conducted in childbearing-aged women from Siena (Tuscany, central Italy) and Bari (Apulia, southern Italy) and pregnant women in Bari (2016-2017).The prevalence of seropositive childbearing-aged women in Bari was significantly higher than in Siena (22.4% vs. 12.4%), while a low prevalence (13.8%) was observed among the pregnant women tested [35].
The toxoplasma IgG seroprevalence rates differed regionally in Croatia as well, with significantly higher seropositivity in continental (22.2%) than in coastal regions (15.3%).Eating undercooked pork is one of the main sources of toxoplasmosis [36].The production and consumption of numerous traditional pork dishes in continental regions may be the explanation for a higher toxoplasma seroprevalence than in coastal regions.
Numerous studies have shown that toxoplasma seropositivity is age-related, with higher seropositivity as age increases [19,26,38].In contrast, no significant differences in IgG prevalence between age groups were observed in our study, ranging from 18.8% to 25.6% with a reverse U-shaped seroprevalence curve.Seroprevalence was lowest in the age group of 31 to 35 years.In a 2005-2009 Croatian study, these differences were significant and showed a similar seroprevalence curve with the lowest seroprevalence in the 26-to 35-year group [15].
Our study found significantly higher toxoplasma seropositivity among residents of suburban and rural areas (27.4%) compared to urban areas (17.1%).Significantly higher seroprevalence in rural areas was also observed in a Romanian study (46 vs. 36%) [31], while it was of borderline significance in a Serbian study (22.5 vs. 11.5%)[22].In Slovakia, women who lived in Bratislava's surroundings had a considerably higher seropositivity rate (63.5%) than those who lived in Bratislava (36.6%) [39].In addition to the consumption of pork, more frequent contact with cats, a definite host of T. gondii, in rural areas is the probable reason for the differences in the seroprevalence rates, since cat ownership was confirmed to be associated with toxoplasma seroprevalence [25].
A meta-analysis of RUBV prevalence in childbearing-aged and pregnant women that included the period between 2000 and 2016 found a pooled global RUBV seropositivity of 90.7%.When considering subpopulation groups, a seropositivity pooled estimate was 90.6% in pregnant women and 90.5% in childbearing-aged women, with no mention of ongoing pregnancy [40].In the Croatian childbearing-aged and pregnant women tested in this study, the seroprevalence of RUBV was 91.3%, which is lower than the 94.6% in 2005-2009 [15].Other European countries showed a seropositivity rate of 93.6% in Ireland [41], 94.4% in Norway [42], 93.4-97.7% in Spain [43][44][45], and 95.8% in Sweden [46].In the United Kingdom, seroprevalence in Liverpool was lower (93.7%)than average for the northwestern region (96.3%)[47].Italian studies showed lower seropositivity rates of 85.8% in Messina (2006Messina ( -2007) ) [48], 88.6% in Tuscany and 84.3% in Apulia (2014-2016) [49] and 81.2% in Palermo (2012-2022) [18].Similar seroprevalence of 83.5% was also found in Portugal [50].Although data on the rubella vaccination status of the participants included in this study were not available, data on measles-mumps-rubella (MMR) vaccine coverage in Croatia from the Reference Center for Epidemiology, Croatian Institute of Public Health showed a decline in the past decade.MMR primo vaccination and revaccination rates in 2013 and 2023 were 93.9/97.1% and 90.1/90.1%,respectively, which impacted the lower seroprevalence rate detected in childbearing-aged women tested in this study.
In Romania, 94.1% of fertile women tested from 2008 to 2010 were rubella IgGseropositive, while seropositivity was lower (91.5%) in those tested from 2015 to 2018 [51].A decline in RUBV seropositivity in Croatia is a result of a decrease in immunization coverage, which was also observed in Serbia (92.9% seropositive) [52].
In our study, no significant differences were observed in RUBV seropositivity between age groups (81.3-95.4%),area of residence, geographic region, or obstetric history.Similarly, seroprevalence was stable in different age groups in Romania.In addition, there was no significant difference in seroprevalence among urban and rural populations [51].In contrast, the highest proportion of seronegative women was found in the youngest age group (15-20 years, 18.7%) compared to 1.9% in the 30-to 35-year age group in northwest England [47].Similarly, the proportion of RUBV-seronegative women declined with increasing maternal age in Spain [53].
In our study, no significant differences in CMV IgG seropositivity were observed among age groups, although seroprevalence rates were lower in groups aged up to 25 years (62.5% and 66.7%, respectively) compared to groups aged above 26 years (69.7-72.4%).Stable but higher seroprevalence rates were also found in Croatia in 2005-2009 [15].Similarly, no significant differences in seropositivity by age group were found in Italy [55].In addition, no correlation between seroprevalence and maternal age was observed in one Polish study [61].However, in another study from Poland, seroprevalence differed significantly between age-stratified groups, with the highest IgG prevalence in women above 36 years of age (76.2%) compared to 58.5-66.0% in younger women [62].Moreover, in the United Kingdom, a significant increase in CMV seroprevalence was observed with maternal age from 50.9% to 75.5% [57].
In contrast to Romanian studies, which found higher CMV seropositivity in pregnant women residing in rural areas [65,66], our study found no difference in seroprevalence between residents of suburban/rural and urban areas (73.9% vs. 69.1%).
Our study found no difference in either HSV-1 or HSV-2 IgG seroprevalence among residents of continental and coastal regions.However, higher HSV-1 seropositivity was observed in women from suburban/rural areas (71.7%) than women from urban areas (64.7%).The higher HSV-1 seroprevalence in rural areas could be attributed to the lifestyle of the rural population, typically living in large households with many children and relatives.Since primary infections mainly occur in preschool-and school-aged children, close contact with these cohorts is associated with an increased risk of HSV-1 transmission [2].
Our study showed a significant increase in HSV-2 seropositivity with age.All women aged less than 25 years were HSV-2 IgG-seronegative, while a progressive increase in seroprevalence was observed starting with the 26-to 30-year age group, from 1.7 to 9.3%.Higher seropositivity in older groups reflects longer and cumulative exposure to the virus.Similar to our results, an increase in HSV-2 seropositivity with age was observed in many studies [67,72,74].No association of HSV-2 seroprevalence with age was found in Romania, with peak seroprevalence (18.3%) between 30 and 34 years of age and a slight decrease thereafter [75].
While obstetric history was not associated with HSV-1 or HSV-2 seroprevalence in Croatian childbearing-aged or pregnant women, a history of abortion was associated with HSV-2 seropositivity in German and Hungarian studies [76,77].
A Romanian study analyzed the simultaneous seroprevalence of TORCH pathogens in childbearing-aged women.Similar to each TORCH pathogen, the proportion of women simultaneously IgG-seropositive decreased over time: T. gondii-CMV 41.4% vs. 36.1%,T. gondii-RUBV 41.8% vs. 35.7%,CMV-RUBV 88.9% vs. 83.6%,and T. gondii-CMV-RUBV 39.6% vs. 33.2%[78].Simultaneous seropositivity to two or three TORCH pathogens in participants included in our study was generally low (T.gondii-RUBV 18.4%, T. gondii-CMV 14.1%, T. gondii-RUBV-CMV 13.1%) and moderate for RUBV-CMV (64.2%).In a previous Croatian study, simultaneous seropositivity was not analyzed; therefore, it was not possible to compare seroprevalence trends over time.When comparing women from urban and suburban-rural areas, simultaneous seroprevalence was higher in suburban and rural regions, which is in line with the results from Romania [78].
One recently published study analyzed the impact of latent CMV infections on spontaneous abortion history and pregnancy outcomes.In healthy women, latent CMV infection does not affect the risk of complications, while borderline-significant higher prevalence of miscarriage history was observed in women with latent CMV infection [79].Furthermore, the observed differences between the rate of pregnancy complications in groups of pregnant women with and without latent T. gondii infection were not significant [30].In our study, data on pregnancy complications and outcomes were not available, which is one of the limitations of the study.
In addition, a limitation of this study that needs to be addressed is the small number of participants in the youngest (≤25 years) and oldest (>40 years) age groups, which should be considered when interpreting the results.

Conclusions
Based on the decline in seroprevalence rates observed in many European countries, a similar trend was expected in Croatia.The results of this study confirmed this hypothesis, showing a decrease in TORCH-seroprevalence rates in Croatian childbearing-aged women in 2014-2023 compared to 2005-2009.
Information on TORCH serostatus in childbearing-aged women is important to define seronegative women who are at risk of primary infections during pregnancy.Although cases of rubella reinfections in previously vaccinated seropositive women during pregnancy are reported in the literature, CRS is rarely recorded [80,81].In addition, CMV reactivation or reinfection during pregnancy can lead to transient viremia and fetal infection, but such infections tend to be less severe and newborns are usually asymptomatic [82,83].Congenital toxoplasmosis as a result of reinfection in immunocompetent pregnant women or reactivation in pregnant women with altered immune status is exceptional [84].Since rubella is a vaccine-preventable disease, serological testing of childbearing-aged women is encouraged with the aim of vaccinating seronegative individuals before pregnancy.
The results of our study also impact other population groups, especially the immunocompromised, who are at risk of toxoplasmosis, HSV, and CMV infection.Similarly to the observed declining seroprevalence in childbearing-aged women, decreasing seroprevalence and increased susceptibility to infections probably occur in this population, which should be kept in mind.Therefore, the present results also highlight the need to monitor these pathogens in other high-risk population groups as well.

Figure 1 .
Figure 1.Distribution of study participants by age.

Figure 1 .
Figure 1.Distribution of study participants by age.

Table 1 .
Serology methods used for detection of TORCH antibodies.

Table 3 .
Prevalence of rubella virus antibodies.

Table 5 .
Prevalence of herpes simplex virus type 1 antibodies.

Table 6 .
Prevalence of herpes simplex virus type 2 antibodies.

Table 7 .
Simultaneous IgG seroprevalence of two TORCH pathogens.

Table 8 .
Simultaneous IgG seroprevalence of three TORCH pathogens.

Table 9 .
Univariate and multivariate risk for Toxoplasma gondii IgG seropositivity.

Table 10 .
Univariate and multivariate risk for rubella virus IgG seropositivity.

Table 11 .
Univariate and multivariate risk for cytomegalovirus IgG seropositivity.

Table 12 .
Univariate and multivariate risk for herpes simplex virus type 1 IgG seropositivity.

Table 13 .
Univariate and multivariate risk for herpes simplex virus type 2 IgG seropositivity.