Introduction
Toxoplasmosis is a zoonotic infection with a global distribution that infects humans and most warm-blooded animals. It is caused by the protozoan Toxoplasma gondii [1, 2]. The most common ways of transmission of toxoplasmosis are through eating contaminated raw or undercooked meats containing tissue cysts or water and uncooked food containing sporulated oocysts, as well as vertical transmission during pregnancy. The risk factors for toxoplasmosis include the abundance of cats in the living environment, geographical conditions, and consuming raw and undercooked meat [1, 3]. Congenital infection is one of the most serious forms of toxoplasmosis that occurs during acute toxoplasmosis in a seronegative pregnant woman [3]. Ramsar has a suitable environment for the survival and transmission of T. gondii oocyst. The aim of this study was to determine the seroprevalence of toxoplasmosis and to identify its risk factors in pregnant women who were referred to the pregnancy care center of Ramsar city.

Methods
This is an analytical-cross sectional study, which was conducted with the participation of pregnant women who were referred for common pregnancy tests to the pregnancy care center of Ramsar between October 2017 and September 2018. The sample size was calculated at 191 based on a toxoplasmosis prevalence of 60% [8], an accuracy of 0.07, and a confidence interval of 95%. The research objectives were explained to pregnant women and informed consent was obtained. Demographic and behavioral characteristics were collected through interviews. Sera were separated from the blood samples and kept at -20°C until the test was performed.
Anti-Toxoplasma IgG and IgM antibodies were tested by ELISA method, according to the manufacturer’s instructions of the kit (Euroimmun, Germany). Then, in order to determine the approximate time of toxoplasmosis infection, IgM-positive samples were tested with an IgG avidity kit (Euroimmun, Germany).
Laboratory results and questionnaire data were statistically analyzed by SPSS software, version 18, using the chi-square (χ2) and Fisher’s exact tests. A statistical significance level of 0.05 was considered.

Result 
Anti-toxoplasma IgG and IgM were found in 46.1% (88 out of 191) and 5.8% (11 out of 191) of pregnant women, respectively. Also, 4.7% (9 out of 191) of these IgM-positive pregnant women were IgG-positive. Overall, out of 22 samples suspected of acute toxoplasma infection (samples with positive or borderline IgM value), which were investigated by IgG avidity test, 91% had high and 9% had intermediate avidity and no sample had low avidity.
None of the demographic characteristics, such as age (P=0.536), occupation (P=0.802), place of residence (P=0.626), education (P=0.321), income (P=0.999), trimesters of pregnancy (P=0.983), frequency of pregnancy (P=0.206), and history of abortion (P=0.927) was correlated with toxoplasmosis. Moreover, behavioral characteristics, such as water consumption (P=0.944), contact with soil (P=0.718), presence of cats in household (P=0.77), consumption of vegetables (P=0.596), the type of meat consumed (P=0.966), and the way of meat cooking (P=0.646) had no significant correlation with toxoplasmosis seropositivity.

Discussion 
In this study, the prevalence of toxoplasmosis was estimated at 46.1%. This relatively high rate of prevalence indicates that there are suitable conditions for the transmission of toxoplasmosis in Ramsar. The results of similar studies conducted on humans and animals in neighboring environments confirm the high prevalence of toxoplasmosis in the region [8, 9]. In a study performed on the population of pregnant women in the cities of Ramsar and Tonekabon (adjoining city) in 2013, the seroprevalence of Toxoplasma infection in pregnant women was reported as 58.8% [8]. The difference in the seroprevalence of toxoplasmosis in these studies can be due to the difference in the test methods and tools used, as well as the difference in the study area, change in residence lifestyle, and the time interval between the two studies. According to various climatic and epidemiological conditions, different rates of toxoplasmosis prevalence in pregnant women have been reported from different regions of the country. So far, the lowest prevalence of toxoplasmosis in pregnant women is 11.35% in Sistan and Baluchistan [11] and the highest prevalence is reported at 75.02% in Mazandaran province [12].
In the meta-analysis of toxoplasmosis conducted in 2018 on Iranian pregnant women, the pooled seroprevalence of overall toxoplasma infection, IgG antibody, and IgM antibody was estimated at 41.3%, 39.2%, and 4%, respectively [7].
Another review article looked at the global prevalence of acute toxoplasmosis infection in pregnant women from 74 countries and estimated its prevalence at 1.1%. According to this study, the highest prevalence of acute toxoplasmosis during pregnancy in the world was reported in the Middle East and North Africa at the rate of 2.5% and the prevalence of acute infection in 9880 Iranian pregnant women was estimated at 2.6% [14]. Considering the 1,114,128 births registered in 2020 in Iran [15] and based on the estimate (2.6%), the occurrence of 28,967 cases of acute toxoplasmosis in Iranian pregnant women is expected in this year. Therefore, congenital toxoplasmosis is a serious health problem in Iran and requires preventive measures. 
In the present study, the prevalence of anti-toxoplasma IgM was estimated at 5.8%. Based on the IgG avidity test performed on suspected samples, no case of low avidity, which is representative of acute toxoplasmosis, was detected. 
In this research, none of the demographic and behavioral characteristics demonstrated a significant correlation with toxoplasma seropositivity, which is similar to the results of Hosseini et al. [8]. The results of toxoplasmosis seroepidemiological studies often are different because of the variety of transmission routes and the prevalence rate of this infection in the studied communities.
This study showed that a considerable percentage of pregnant women in Ramsar was not immune to Toxoplasma infection. Regarding the non-implementation of the mandatory screening program for pregnant women to detect toxoplasmosis in pregnancy care, it seems that increasing the knowledge of pregnant women about the transmission routes and prevention of toxoplasmosis will reduce the risk of contracting the congenital form of this infection.

Ethical Considerations

Compliance with ethical guidelines
This study was approved by the Ethics Committee of the Guilan University of Medical Sciences (Code.: IR.GUMS.REC.1396.218) and all participants included in the study signed informed consent.

Funding
This study was supported by the Guilan University of Medical Sciences (Grant No. 95051409).

Authors' contributions
Conceptualization and study design: All the authors
Acquisition, analysis, or interpretation of data: Hamzeh Shabanian, Bijan Majidi-Shad and Zahra Atrkar-Roshan; Drafting of the manuscript: Hamzeh Shabanian, Bijan Majidi-Shad, Meysam Sharifdini; Critical revision of the manuscript for important intellectual content: Bijan Majidi-Shad, Reza Jafari-Shakib, Meysam Sharifdini; Statistical analysis: Zahra Atrkar-Roshan; Financial resources: Bijan Majidi-Shad; Administrative, technical and material support: Bijan Majidi-Shad, Hamzeh Shabanian, Reza Jafari-Shakib; Study supervision: Bijan Majidi-Shad and Reza Jafari-Shakib.

Conflicts of interest
The authors declared no conflict of interest.

Acknowledgements
This study was extracted from Hamzeh Shabanian’s MSc thesis and supported by the Deputy for Research and Technology of Guilan University of Medical Sciences (Code: 95051409).
 

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