Seroprevalence of Infections with TORCH Agents in Romania: A Systematic Review

Maternal–fetal infectious pathology—notably the TORCH panel (Toxoplasma gondii, rubella, Cytomegalovirus, and herpes simplex viruses)—critically impacts maternal and neonatal health. This review collates data on the seroprevalence of IgG and IgM antibodies against TORCH agents in Romanian women, aiming to discern regional and population differences and identify risk factors. Twenty studies were included in the review, revealing variable seroprevalence rates across the country. Regions such as Moldavia and Banat showed higher anti-T. gondii IgG seroprevalence rates than Bihor, with notable declines in Banat. Rural, older, and multiparous women showed elevated T. gondii IgG rates. Anti-rubella vaccine introduction significantly reduced the prevalence of anti-rubella IgG antibodies, but recent vaccination coverage decreases raise concerns. CMV and HSV seroprevalence varied geographically, with rural areas generally showing higher CMV rates and HSV influenced by factors like education level and number of sexual partners. Concurrent seroprevalence of multiple TORCH components in some cases underscores potential common risk factors. This study highlights the importance of continuous monitoring and preventive measures such as vaccinations and awareness campaigns to mitigate the health impact on the pregnant population.


Introduction
Maternal-fetal infectious pathology is a crucial subject in the fields of obstetricsgynecology and neonatology, as it directly impacts the health and wellbeing of both mother and child [1]. These infections are more frequent in immunodeficient patient groups, such as those with organ transplants, HIV infection, and other immunocompromising diseases [2,3]. This research emphasizes the significance of serological testing for infectious agents encompassed in the TORCH panel, which includes Toxoplasma gondii (T. gondii), rubella virus, Cytomegalovirus (CMV), and herpes simplex viruses (HSVs) type 1 and 2. These tests are particularly relevant for women who are attempting to conceive, those who are already pregnant and, in certain situations, their newborns [4].
Serological analyses for these infectious agents can identify infections with microorganisms known to negatively affect the course of a pregnancy, the health of the developing fetus, and the subsequent wellbeing of the newborn. Early detection of these infections is
The seroprevalence of HSV-1 in Europe varies, ranging from 52% in the United Kingdom to 84% in Bulgaria [42]. A meta-analysis published in 2020 reported a mean seroprevalence of anti-HSV-1 IgG of 69.5% among women, and by region 78.7% in Eastern Europe, 77.2% in Southern Europe, 61.6% in Western Europe, and 57.7% in Northern Europe [43].
A meta-analysis study performed using European studies reported a mean seroprevalence of anti-HSV-2 IgG of 14.0% in women, and by region the seroprevalence was 9.6% in Eastern Europe, 12.2% in Southern Europe, 13.2% in Western Europe, and 13.5% in Northern Europe [47].

Aims and Scope of this Review
To date, for the majority of EU countries, there is sparse population-based prevalence data of TORCH infections in women. Moreover, as far as we are aware, these prevalence rates have rarely been summarized at the global, regional, or national levels. We could not find any systematic review or meta-analysis of the prevalence rates of these infections in Romania. However, there is a growing interest in the benefits of TORCH panel antibody screening in pregnant women, given the low number of acute infections detected. Consequently, our objective was to review and synthesize the known data about the seroprevalence of IgG and IgM antibodies against agents of the TORCH complex in women from Romania, and to highlight the differences in different population groups within the country. Also, this review aimed to analyze the impact of various risk factors on the seroprevalence of these antibodies, which could improve existing prevention strategies. Additionally, we evaluated the geographical distribution and population-specific prevalence rates of TORCH agents in Romania, identifying significant trends or patterns in the data.
The current review can provide a comprehensive overview of the seroprevalence of TORCH agents in Romania, which can assist in future research, public health initiatives, and clinical practice.

Methods
The systematic review methodology was in accordance with the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines.
The inclusion criteria for studies used in this review were as follows: • Studies that report the seroprevalence of at least one TORCH agent (T. gondii, Rubella virus, CMV, and HSV-1/2) in the general population of Romania or a specific population group within Romania, particularly women. • Studies published in the English or Romanian language. • Studies with available data on sample size, age range, and seroprevalence rates for each TORCH agent. • Studies published up to February 2023.
The exclusion criteria used were as follows: • Studies published in languages other than English or Romanian. • Articles that were duplicates, editorials, or opinion pieces without original data.

•
Studies without human subjects.
To investigate the seroprevalence of infections with TORCH agents in Romania, we conducted a systematic review of relevant studies using the following search strategy: We searched the following electronic databases: PubMed, Google Scholar, Scopus, and ISI Web of Science. The search phrases used were in the form "Agent" AND "seroprevalence" AND "women" AND "Romania", where "Agent" was replaced with "Toxoplasma"/"Rubella"/"CMV"/"HSV". Another strategy was to search for the diseases produced by these agents, e.g., "toxoplasmosis". The search was limited to studies published in the English or Romanian language up to February 2023. The search yielded a total of 153 titles (41 T. gondii + 58 rubella + 28 CMV + 26 herpes simplex). After excluding the non-human studies and articles by design, 83 studies remained. After excluding 12 duplicates, we screened the abstracts of the remaining 71 studies. After reading the abstracts and full texts, we excluded 39 additional studies that did not report the prevalence. Finally, we included 32 articles in our review, and we extracted relevant data from each study, including sample size and seroprevalence rates for IgG and IgM antibodies against agents of the TORCH complex. This selection process is presented in the PRISMA flow diagram ( Figure 1).

Quality Assessment
This review was founded upon observational studies, inherently carrying a risk of bias. Factors that may compromise the comparability and transferability of the study outcomes include discrepancies in the study populations, variations in sample size, divergent observation periods, distinct demographic regions, and disparate research methodologies.
Furthermore, our search for eligible studies was restricted to the PubMed, Google Scholar, ISI Web of Science, and Scopus databases, potentially neglecting relevant investigations available in alternative databases. Due to these considerations, this systematic review is not exempt from bias, rendering the interpretation of its findings constrained.

Quality Assessment
This review was founded upon observational studies, inherently carrying a risk of bias. Factors that may compromise the comparability and transferability of the study outcomes include discrepancies in the study populations, variations in sample size, divergent observation periods, distinct demographic regions, and disparate research methodologies.
Furthermore, our search for eligible studies was restricted to the PubMed, Google Scholar, ISI Web of Science, and Scopus databases, potentially neglecting relevant investigations available in alternative databases. Due to these considerations, this systematic review is not exempt from bias, rendering the interpretation of its findings constrained.

Results
In Romania, the majority of studies about the seroprevalence of antibodies against TORCH agents were performed after 1989 [48,49], immediately after the reagents needed to detect the antibodies became available after the fall of communism in Romania [50][51][52] The seroprevalence in the Romanian general population varied from region to region. A large study performed in 2009 on healthy people from 11 counties from northwestern and central Romania [53] reported that the seropositivity rate was 59.48% in the general popula-Microorganisms 2023, 11, 2120 6 of 28 tion, while in western Romania a seroprevalence rate of 64.8% was reported in 2015 [54]. In healthy blood donors-a group that can estimate the true population prevalence of anti-T. gondii IgG antibodies-Lupu et al. [55] reported a seroprevalence of anti-T. gondii IgG of 45.9% in western Romania in 2022.
In pregnant women, we found one study performed in 1998 reporting a seropositivity for T. gondii IgG of 43.9% in northeast Romania [50], while in central Romania in 2008 the seroprevalence was 39.0% in a study performed in 510 pregnant women [56] and 38.24% [57] in 2018 in another study from the Muntenia region. In western Romania, the seroprevalence was 55.8% [58]. A study by Motoi [60].
In women of reproductive age, a study performed in 2021 in northeast Romania (Moldavia region) found a seroprevalence of anti-T. gondii IgG of 33.84% [61]. In southeast Romania (Dobrogea region), the seroprevalence was 33.16% [14]. In a sample of 105 women aged between 16 and 35 years from northwest and central Romania, the seroprevalence was determined to be 51.43% [53]. In western Romania, the seroprevalence rate was reported at 57.6% in 2008 [62], 55.55% in 2015 [54], and 41.16% in 2020 [63]. In the same region, a descending trend of seroprevalence was reported from 43.7% in 2008-2010 to 38.8% in 2015-2018 [30]. Future studies should confirm this descending trend. In reproductiveage female blood donors from western Romania in 2022, a seroprevalence rate of 43.2% was reported [55]. In the Bihor region, a lower seroprevalence of 36.48% was reported in 2022 [64].
A study performed in northwest Romania in children with mental retardation and visual pathology showed a seroprevalence of IgG antibodies of 66.4% and 37.4%, respectively, compared with the seroprevalence rate of only 9.3% in healthy children [50]. In a sample of 15 children from southeast Romania, the seroprevalence was 0% for IgM antibodies and 33.33% for IgG [14]. Another study from western Romania reported a seroprevalence rate of 16.66% in children with pathologies unrelated to Toxoplasma infection [65]. These seroprevalence figures in children suggest Toxoplasma exposure since childhood, since the screening should not be limited to pregnant women but extended to the child population.
However, these data must be interpreted with caution, as the study groups used were from different institutions: one from a public hospital and one from a private laboratory. People addressing these two types of institutions may have different population structure in terms of age and education level, for example, and previously a higher seroprevalence of anti-Toxoplasma IgG antibodies was reported in women of reproductive age with lower educational status [66].

Risk Factors
Regarding the sex prevalence, there were no significant differences between males (60.4%) and females (68.5%) [54]. The seroprevalence of IgG antibodies generally increased with age, as showed by Olariu et al. [62], higher in women aged over 40 years (71.4%) compared with young women under 20 years of age (33.3%). This difference was confirmed by studies performed 7 years later [54] and 12 years later in the Banat region by Motoi et al. and Mihu et al.,respectively [59,63,64]. The estimated annual infection risk associated with age showed high susceptibility from childhood to adolescence (3-4.5% annual infection risk), a stable level for adults (1-2%), and then an increase up to 5.5% for individuals over 65 years old [53]. A recent study performed on healthy blood donors [55] showed that seroprevalence also increased with age, from 32.6% in the 18-25 years age group to 67.6% in the 56-63 years age group. The seroprevalence increased as the education level decreased. The multivariate analysis detected significant associations between seroprevalence and owning pets, age, and education level [55].
There are more possible explanations for the observed decrease in the seroprevalence of T. gondii infection. Cat owners, especially in urban areas, feed them with canned cat food, which is sterilized. Fewer cats are allowed to hunt rodents, which are the source of infection with Toxoplasma [67]. The raw meat that is commercially available is generally Toxoplasma-free, as the meat is deep-frozen during transportation and the cold inactivates Toxoplasma [55,68]. A study from Romania performed on pregnant women found a significant association between toxoplasmosis and the consumption of undercooked meat (p < 0.05) [57].
Vegetables are generally washed, which may decrease contamination with oocysts. Lastly, drinking water quality has improved, and more people consume bottled drinking water, which is free of oocysts [68]. Rugina et al. (2015) performed a multivariate analysis in a study on women from the Dobrogea region that showed risk ratios of 3.27 for consumption of raw/uncooked meat, 2.47 for unpasteurized milk, and 2.23 for tasting meat during cooking [14].
Furthermore, variations in prevalence were noted in relation to the residential area. The prevalence in urban areas was lower (55.3%) compared with rural areas (76.9%) [54]-a difference also noted by Mihu et al. (36% vs. 46%) [63,64] (Figure 2). annual infection risk), a stable level for adults (1-2%), and then an increase up to 5.5% for individuals over 65 years old [53]. A recent study performed on healthy blood donors [55] showed that seroprevalence also increased with age, from 32.6% in the 18-25 years age group to 67.6% in the 56-63 years age group. The seroprevalence increased as the education level decreased. The multivariate analysis detected significant associations between seroprevalence and owning pets, age, and education level [55].
There are more possible explanations for the observed decrease in the seroprevalence of T. gondii infection. Cat owners, especially in urban areas, feed them with canned cat food, which is sterilized. Fewer cats are allowed to hunt rodents, which are the source of infection with Toxoplasma [67]. The raw meat that is commercially available is generally Toxoplasma-free, as the meat is deep-frozen during transportation and the cold inactivates Toxoplasma [55,68]. A study from Romania performed on pregnant women found a significant association between toxoplasmosis and the consumption of undercooked meat (p < 0.05) [57].
Vegetables are generally washed, which may decrease contamination with oocysts. Lastly, drinking water quality has improved, and more people consume bottled drinking water, which is free of oocysts [68]. Rugina et al. (2015) performed a multivariate analysis in a study on women from the Dobrogea region that showed risk ratios of 3.27 for consumption of raw/uncooked meat, 2.47 for unpasteurized milk, and 2.23 for tasting meat during cooking [14].
Furthermore, variations in prevalence were noted in relation to the residential area. The prevalence in urban areas was lower (55.3%) compared with rural areas (76.  The seroprevalence of T. gondii was observed to be higher among pet owners (specifically, cats and/or dogs) compared to individuals without pets (p = 0.032) [58]. Also, females with a history of four or more live births exhibited a higher frequency of T. gondii seropositivity than those who had no previous births (p < 0.002) [58]. The seroprevalence of T. gondii was observed to be higher among pet owners (specifically, cats and/or dogs) compared to individuals without pets (p = 0.032) [58]. Also, females with a history of four or more live births exhibited a higher frequency of T. gondii seropositivity than those who had no previous births (p < 0.002) [58].

Anti-Toxoplasma gondii IgM
In the Banat region (Table 1), a prevalence of anti-T. gondii IgM antibodies of 0.821% was reported between 2008 and 2010, which increased in 2015-2018 to 1.054% [30]. In a study from the Bihor area that included pregnant women addressed to the infectious disease clinics [60], there was an extremely high prevalence of 14.6% of anti-T. gondii IgM, but there was a strong selection bias, as these pregnant women were referred because they presented clinical signs of toxoplasmosis. Recently, Mihu et al. [66] reported a prevalence of IgM antibodies of 8.90% in females of reproductive age. The prevalence of anti-T. gondii IgM antibodies could not be related to age group (p = 0.0997), place of residence (p = 0.3105), or year of study (p = 0.4410) [60]. 57.6% anti-T. gondii IgG, tested through latex agglutination.
Seroprevalence increased as age increased from 33.33% in the <20 years age group to 71.4% in the ≥40 years age group. The seroprevalence was higher in rural areas (70%) compared with urban areas (48.1%).
Banat region. In the Moldavia region, a study reported a seroprevalence of 7.28% of anti-T. gondii IgM [61]. Of the 15 IgM-positive women, 10 were in the 25-34 years age group, 4 in the 35-44 years age group, and 1 in the 15-19 years age group [61].
In the Dobrogea region, a seroprevalence of 2.46% of IgM antibodies was reported in women [14]. A study from the Transilvania region [56] reported a seroprevalence of IgM antibodies of 1.76% in pregnant women between 2005 and 2007. The method used to detect IgM antibodies was manual ELISA. Acute toxoplasmosis infection was predominantly observed during the first trimester of pregnancy, accounting for 66.66% of cases and representing the primary cause of abortion. The annual infection risk for women aged 20-33 years in this geographic region was determined to be 0.67%. Around 4% of cases displayed IgM persistence for over a year, while an additional 7% exhibited both positive IgM and positive IgG, suggesting a potential persistence in 11% of cases [56].
As for the Muntenia region, a study performed on women in Bucharest found a seroprevalence of 17.65% for anti-T. gondii IgM And 38.24% for anti-T. gondii IgG [57]. Out of the 30 women who were positive for both IgM and IgG, only 13.3% presented all indications (positive specific IgM and IgA antibodies, low IgG avidity, and positive PCR) for maternally acquired T. gondii infection during pregnancy, posing a risk of congenital transmission.
In interpreting the results of the studies regarding IgM antibodies, we must consider that acute toxoplasmosis can result in the persistence of anti-T. gondii IgM antibodies for several years, so the IgM-positive patients cannot be regarded as acute toxoplasmosis cases.

Anti-Rubella IgG
The prevalence of anti-rubella IgG antibodies was heavily influenced by the introduction of anti-rubella vaccines. The bivalent measles-rubella vaccine was first administered in Romania in 1998 to girls aged 15-18 years (born between 1980 and 1983); the introduction of this vaccine occurred following a measles outbreak. In April 2003, rubella vaccination was introduced for 13-14-year-old girls (eighth-grade students) during school-based campaigns. In May 2004, the monovalent measles vaccine was replaced in the national vaccination program with the trivalent measles-mumps-rubella (MMR) vaccine for children aged 12-15 months, with plans to introduce it to first-grade children (7 years old) in 2005. During this period, rubella vaccination continued for eighth-grade girls (13-14 years old) until 2008 [70,71].
The first study that we found reporting anti-rubella IgG prevalence in Romania was from 1989 (Table 2), performed on 5030 women aged 15-40 years, tested by hemagglutination inhibition reaction. In this study, 76.7% of sera were reactive for IgG antibodies [49]. Between 2002 and 2003, a large rubella outbreak of 115,000 cases was reported in Romania, with an incidence rate of 531/100,000 inhabitants and 150 CRS cases [72]. In 2008, two studies on men, women of childbearing age, and pregnant women [30,73] from the Banat region (Table 2)

Anti-Rubella IgM
The studies reporting anti-rubella IgM antibodies date as early as 2003, when the National Institute of Public Health published the results of a large rubella outbreak that comprised 115,000 cases [72]. The prevalence reported among the whole country's population at that time was 0.531%. In the same period, another study [74] from the Moldavia region reported a peak of incidence of 10.525% in urban areas and children under 14. Another rubella outbreak that occurred between 2011 and 2012 had a prevalence of 0.029% in a nationwide study [25]. In the same study, from 6182 anti-rubella-IgM-positive cases analyzed, only 28 cases of congenital rubella syndrome were identified, resulting in 11 neonatal deaths and 1 stillbirth. A study in the Banat region from 2008 to 2010 reported a prevalence of 0.479% of acute infection with rubella vs. 0.273% between 2015 and 2018 in women of childbearing age [30], showing a more than twofold decrease in prevalence. During 2019-2020, the Eurostat website reports a marked decrease in acute rubella, with only four cases in Romania [75], corresponding to a notification rate of 0.2 per 1.000.000 people, and reaching 0 reported cases during 2021-2022 [76].

Anti-CMV IgG
We found two studies from the Banat region from 1990 [52] and 1993 [51] analyzing pregnant women as well as children, which reported a prevalence of 51%/45% (Table 3). Interestingly, more recent studies, such as that of Gorun et al. from 2020, reported a high prevalence of CMV immunization of 94.6% during 2008-2010 and 91.8% during 2015-2018 [29]; these results are supported by a study on women of childbearing age by Mocanu et al. from 2021 [30], in the absence of immunization by vaccine, as there is no approved vaccine to date. Seroprevalence was found to be higher among women in rural areas compared to those in urban areas [29]. These findings indicate that the western region of Romania has a low risk profile for primary CMV infection during pregnancy, as a significant number of women are seropositive [29]. The development of an anti-CMV vaccine was considered to be a priority by the National Vaccine Program Office of the USA [77].

Anti-CMV IgM
A study from 2006 in women and children from Craiova, Oltenia region showed a prevalence of anti-CMV IgM antibodies of 0.592% [26]. In a study from the Banat area on women of childbearing age, the prevalence was 0.342% in 2008-2010, compared with 0.291% between 2015 and 2018 [30]. We can observe a descending trend of acute infection with CMV in the Banat region.

Anti-HSV IgG
We could only find two studies performed between 2004 and 2005 in the Bucharest area, Muntenia region that assessed the prevalence of IgG antibodies against HSV-1 and HSV-2 in pregnant and non-pregnant women, as well as in men. The prevalence of anti-HSV-1 IgG (87.3%/87.2%) was much higher than that for HSV-2 in both studies (15.1%/15.2%) ( Table 4). Seroprevalence was found to be higher among women in rural areas compared to those in urban areas.
The findings indicate that the western region of Romania has a low risk profile for primary CMV infection during pregnancy, as a significant number of women are seropositive.
The first study was performed on 452 pregnant women aged 15-39 years. The prevalence rates were 87.3% for HSV-1 and 15.1% for HSV-2. The prevalence of HSV-2 seropositivity was influenced by the level of education, being 6.28 times higher in elementary-school graduates and 2.26 times higher in high-school graduates compared with college graduates. Also, the history of sexual partners was directly correlated with the HSV-2 prevalence, which was found to be 2.43 times higher in individuals with two or three partners and 4.26 times higher in those with more than three partners compared to those with one sexual partner [78].
HSV-2 IgG seroprevalence was lower (11.0%) in female teenagers vs. 38.3% in adult women. Also, HSV-2 IgG seroprevalence was increased in women compared with men (17.0% vs. 10.8%). Other risk factors for HSV-2 infection, in addition to female sex and older age, included lower educational status, higher number of sexual partners, and history of genital vesicles [79].

Simultaneous Seroprevalence of IgG Antibodies against TORCH Agents
Simultaneous seroprevalence was observed for IgG antibodies against TORCH agents. A study by Mocanu et al in two separate periods (2008-2010 and 2015-2018) [8] showed that association of IgG anti-T. gondii + IgG anti-CMV was detected in 41.4%/36.1% of cases, while that between IgG anti-T. gondii + IgG anti-rubella in was detected 41.8%/35.7 of cases. Furthermore, the association of IgG anti-T. gondii + IgG anti-CMV + IgG anti-Rubella was 39.6% vs. 33.2%.

Demographic Distribution of Infections with TORCH Agents
The prevalence of antibodies against TORCH agents varied between different regions of Romania. The studies covered various regions of Romania, including the whole country, the Moldavia region, the Oltenia region, and the Banat region. This provides a comprehensive overview of prevalence of TORCH complex infections in different parts of Romania.
In western Romania, we found multiple studies investigating the seroprevalence of anti-T. gondii IgG antibodies.
Olariu et al. [62] found a seroprevalence of 57.6% in 2008 in a study performed on 184 consecutive women of childbearing age who presented to a public hospital in Timisoara, Romania. Seroprevalence increased as age increased, from 33.33% in the <20 years age group to 71.4% in the ≥40 years age group. The seroprevalence was higher in rural areas (70%) compared with urban areas (48.1%). The Banat region had different seroprevalence rates, with a study conducted in 2015 showing a rate of 55.55% [54] in women of childbearing age, while other studies conducted in 2020 and later found rates between 38.81% and 55.8% [58,59,63,64].
There were several studies from the Banat region of Romania that compared the seroprevalence of anti-T. gondii IgG levels in 2008-2010 with 2015-2018-one performed on pregnant women [59] and another on women of childbearing age [30]. The decrease in prevalence was from 43.79% to 38.81% in the first study [59] and from 43.7% to 38.8% in the second study [30]. The descending trend was maintained for associations between seroprevalence of IgG to multiple TORCH agents.
In western Romania, we found multiple studies investigating the seroprevalence of anti-T. gondii IgG antibodies.
Olariu et al. [62] found a seroprevalence of 57.6% in 2008 in a study performed on 184 consecutive women of childbearing age who presented to a public hospital in Timisoara, Romania. Seroprevalence increased as age increased, from 33.33% in the <20 yearsage group to 71.4% in the ≥40 years age group. The seroprevalence was higher in rural areas (70%) compared with urban areas (48.1%). The Banat region had different seroprevalence rates, with a study conducted in 2015 showing a rate of 55.55% [54] in women of childbearing age, while other studies conducted in 2020 and later found rates between 38.81% and 55.8% [58,59,63,64].
There were several studies from the Banat region of Romania that compared the seroprevalence of anti-T. gondii IgG levels in 2008-2010 with 2015-2018-one performed on pregnant women [59] and another on women of childbearing age [30]. The decrease in prevalence was from 43.79% to 38.81% in the first study [59] and from 43.7% to 38.8% in the second study [30]. The descending trend was maintained for associations between seroprevalence of IgG to multiple TORCH agents.
All of these studies were performed on consecutive patients referred to public hospitals who could be considered to be representative of the population of women of childbearing age. We found another study performed on female blood donors of reproductive age, who are even more suitable as a representative sample of the population, which reported a seroprevalence of 43.2% in female blood donors aged 18-45 years [55].
In the Bihor area, one study reported a low prevalence of 25.4% in pregnant women who were referred to infectious disease clinics [60]. Another study from the same area showed a seroprevalence of 36.48% [64]. These findings suggest that the Bihor area may have a lower prevalence of IgG antibodies compared to the Banat region.
In a broader study performed in northwestern and central Romania, the seroprevalence in the general population was 59.48%, higher in rural areas (63.68%) compared to urban settings (55.12%), with similar rates for both males (60.49%) and females (58.87%) [53].
Also, in the Muntenia region, there was a study on 170 pregnant women that detected anti-T. gondii IgG in 38.24% of participants and anti-T. gondii IgM in 17.65% of the tested women [57].
In summary, there is considerable variation in the seroprevalence of T. gondii in different regions of Romania. The Moldavia region and the Banat region show higher seroprevalence rates compared to the Bihor area. Rural areas generally show higher seroprevalence rates compared to urban areas in the Banat and Bihor areas [30,59,60,64].
We can observe that IgG seroprevalence rates are not very high; conversely, this means that the percentage of non-immunized women is around 60-70%. Therefore, pregnant women can be at risk of acquiring acute toxoplasmosis during gestation, due to the urge of large-scale testing for anti-T. gondii antibodies and finding mechanisms to allow for the screening of all pregnant women.

Distribution of Rubella Infection in Romania
Two studies conducted on men, women of childbearing age, and pregnant women from the Banat region (Table 2, Figure 4) revealed prevalence rates of anti-rubella IgG antibodies of 94.1%/94.1% between 2008 and 2010, and of 91.4%/91.5% between 2015 and 2018 [30,73]. Also, in the Muntenia region, there was a study on 170 pregnant women that detected anti-T. gondii IgG in 38.24% of participants and anti-T. gondii IgM in 17.65% of the tested women [57].
In summary, there is considerable variation in the seroprevalence of T. gondii in different regions of Romania. The Moldavia region and the Banat region show higher seroprevalence rates compared to the Bihor area. Rural areas generally show higher seroprevalence rates compared to urban areas in the Banat and Bihor areas [30,59,60,64].
We can observe that IgG seroprevalence rates are not very high; conversely, this means that the percentage of non-immunized women is around 60-70%. Therefore, pregnant women can be at risk of acquiring acute toxoplasmosis during gestation, due to the urge of large-scale testing for anti-T. gondii antibodies and finding mechanisms to allow for the screening of all pregnant women.

Distribution of Rubella Infection in Romania
Two studies conducted on men, women of childbearing age, and pregnant women from the Banat region (Table 2, Figure 4) revealed prevalence rates of anti-rubella IgG antibodies of 94.1%/94.1% between 2008 and 2010, and of 91.4%/91.5% between 2015 and 2018 [30,73].

IgG seroprevalence
IgM seroprevalence The prevalence of anti-rubella IgM varied significantly between different regions, with 0.531% in the whole country [72] and as high as 1.563% in Botosani County and 0.327% in Iasi County [74] in the Moldavia region.

Distribution of CMV Infection in Romania
Based on the data collected from the studies (Table 3, Figure 5), it appears that the prevalence of cytomegalovirus (CMV) infection in Romania varies depending on the region and population studied ( Figure 5). In the Banat region, studies have found a preva- The prevalence of anti-rubella IgM varied significantly between different regions, with 0.531% in the whole country [72] and as high as 1.563% in Botosani County and 0.327% in Iasi County [74] in the Moldavia region.

Distribution of CMV Infection in Romania
Based on the data collected from the studies (Table 3, Figure 5), it appears that the prevalence of cytomegalovirus (CMV) infection in Romania varies depending on the region and population studied ( Figure 5). In the Banat region, studies have found a prevalence of anti-CMV IgG ranging from 45% to 94.7% [29,30]. One study conducted in Craiova, Oltenia region found a prevalence of anti-CMV IgM antibodies of 0.592% [26].

Distribution of HSV Infection in Romania
We could only find two studies from 2010 that investigated the seroprevalence of HSV IgG antibodies among young individuals in Bucharest, Muntenia region. The results revealed higher HSV-2 rates linked to lower educational attainment and increased number of sexual partners. These factors may contribute to a greater risk of HSV-2 transmission within this population.

Limitations
Our review has some important limitations, in addition to those discussed in the "Quality assessment" section. The studies presented have limited comparability, because of the heterogeneity in the studied groups.
The differences in seroprevalence rates can be explained by confounding due to variables that were unaccounted for, or due to differences between the studied groups. The data were collected from different testing centers and in different time periods. There are significant differences in the populations of individuals who present to public healthcare facilities-where sometimes the tests are conducted for free, based on health insurance-and private testing facilities where people must pay for the tests (selection bias). The differences in population structure on variables like age, socioeconomic status, education level, and other unaccounted variables may confound the studies results.
We did not analyze other risk factors linked to toxoplasmosis (such as raw/undercooked meat consumption) that were analyzed in other studies [14]. The seroprevalence of IgM antibodies can be higher in patients who have been referred to the laboratory for confirmation of clinical signs of toxoplasmosis [14]. Also, the studies were performed in different periods, and it is possible that the seroprevalence changes over time.
The laboratory methods used to detect antibodies also differed between the studies, and serological tests may have different sensitivities and specificities. Several studies used latex agglutination to detect the antibodies [55,62], while other studies used im-

Distribution of HSV Infection in Romania
We could only find two studies from 2010 that investigated the seroprevalence of HSV IgG antibodies among young individuals in Bucharest, Muntenia region. The results revealed higher HSV-2 rates linked to lower educational attainment and increased number of sexual partners. These factors may contribute to a greater risk of HSV-2 transmission within this population.

Limitations
Our review has some important limitations, in addition to those discussed in the "Quality assessment" section. The studies presented have limited comparability, because of the heterogeneity in the studied groups.
The differences in seroprevalence rates can be explained by confounding due to variables that were unaccounted for, or due to differences between the studied groups. The data were collected from different testing centers and in different time periods. There are significant differences in the populations of individuals who present to public healthcare facilities-where sometimes the tests are conducted for free, based on health insuranceand private testing facilities where people must pay for the tests (selection bias). The differences in population structure on variables like age, socioeconomic status, education level, and other unaccounted variables may confound the studies' results.
We did not analyze other risk factors linked to toxoplasmosis (such as raw/undercooked meat consumption) that were analyzed in other studies [14]. The seroprevalence of IgM antibodies can be higher in patients who have been referred to the laboratory for confirmation of clinical signs of toxoplasmosis [14]. Also, the studies were performed in different periods, and it is possible that the seroprevalence changes over time.
The laboratory methods used to detect antibodies also differed between the studies, and serological tests may have different sensitivities and specificities. Several studies used latex agglutination to detect the antibodies [55,62], while other studies used immunoenzymatic methods [30,61]. The manual assays have their drawbacks because of timing, contamination risks, and environmental conditions that influence the speed of enzymatic reactions. Modern laboratories use automated analyzers that use thermostats, run internal controls, and adjust the readings to achieve accurate results [80].

Future Directions
This review brings together prevalence data of infections with agents included in the TORCH complex. These data can offer support for developing national strategies for preventing congenital infections. Inclusion of TORCH antibody tests in the national screening protocols would allow for the early detection of these infections and improve mother-child healthcare, because in Romania there are many mothers who cannot afford proper pregnancy monitoring, which contributes to the high rate of infant mortality [81].

Conclusions
This study examined the seroprevalence of antibodies against TORCH agents in Romania, uncovering variations by region, age, and demographic factors, with high prevalence of chronic toxoplasmosis (59.48%) in Moldavia and Banat, as well as in the whole of northwestern and central Romania [53], and with anti-T. gondii IgG antibodies more prevalent in rural areas and among individuals aged over 40 years. It also highlights the impact of the anti-rubella vaccine on anti-rubella IgG antibodies prevalence, although there has been a recent decrease in vaccination coverage. Instances of simultaneous seroprevalence against multiple TORCH agents were noted, suggesting common risk factors. The findings emphasize the need for ongoing monitoring and targeted interventions, such as vaccination and educational campaigns specifically focusing on pregnant women, to manage the diverse landscape of TORCH infections in Romania. Institutional Review Board Statement: This study was approved by the Committee of Ethics and Academic and Scientific Deontology, Craiova, Romania (approval no. 84/16.09.2020). The access to the database for the purposes of this study was approved by the Ethics Committee of the Clinical County Emergency Hospital of Craiova, Romania.
Informed Consent Statement: Because we are a teaching hospital, all patients admitted to our hospital signed a written consent form by which they agreed that their medical data could be used in scientific studies.

Data Availability Statement:
The data presented in this study are available upon request from the corresponding author. The data are not publicly available due to the patient personal data protection policy of the university and hospital.

Conflicts of Interest:
The authors declare no conflict of interest.