Determine the IgG and IgM antibodies created against T. gondii infections using the ELISA method in diabetic pregnant women in compared with non diabetic pregnant women in Sanandaj, Kurdistan, west of Iran

Background: Toxoplasma gondii is an opportunistic parasitic protozoan, which is a causative agent of serious complications such as abortion or fetal still birth in pregnant women. The purpose of this study was evaluating the seroprevalence of toxoplasmosis and risk factors in diabetic pregnant women (case) in comparison with non-diabetic pregnant women (control) from Sanandaj, Kurdistan, west of Iran. Methods: In this case-control study Toxoplasma gondii antibodies were tested in 91 cases as well as 90 controls from referred the Toohid hospital diabetes center using ELISA (enzyme-linked immunosorbent assay) method during June 2018 to October 2019 in Sanandaj, Kurdistan, west of Iran. The collected data were analyzed by SPSS 19 and using Chi-Square and Fisher tests. Results: The seropositivity for anti-Toxoplasma IgG in case group was 33% and 15.6% in the control group (p = 0.006) showing a statistically signicant difference between groups, while for anti-Toxoplasma IgM (p = 0.31) showing no statistically signicant differences between two groups. A signicant relationship was observed between age (p = 0.024) and abortion history (p = 0.05) with anti-Toxoplasma IgG antibodies in cases and controls. Conclusion: The results of current study indicated statistically signicant relationship between diabetes and toxoplasmosis. The abortion history and increase of age was found to be a major T. gondii infection risk factor.


Background
Toxoplasmosis is a zoonotic disease caused by an obligate intracellular protozoan parasite called Toxoplasma gondii, with a worldwide distribution. Felids are the de nitive hosts of the parasite, while many other mammals and birds serve as intermediate hosts (1). Toxoplasma gondii has different forms of tachyzoite, oocyst and tissue cyst. In cat epithelial cells, the parasite sexual produces millions of oocysts which are released in the feces (2). T. gondii is transmitted through contaminated food, meat, water and the placenta during pregnancy. Blood transfusion and organ transplantation have also been mentioned as possible infection risk factors (3). This parasite can lead to chronic infection in individuals, serious problems and death in immune compromised patients, hydrocephalus in congenitally infected newborns, intrauterine fetal death, and spontaneous abortion during pregnancy (4).Several studies found a positive correlation between toxoplasmosis with schizophrenia (5), and depression (6).
About 20% to 90% of the world's adult population in different regions is reported to have had contact with the parasite but only about 10% of infected individuals develop clinical signs and symptoms (7).
High prevalence of the infection has been reported among pregnant women from different foci: in Africa, the prevalence varies between 50%-70% (8), in Europe, between 9%-67% (9), whereas in Asian countries indicated between 0.8% to 28.3% (10).Seropositivity of T. gondii among Iranian women was 48% -74.6% in the northern area (11), 33% -44% in the northwest (12), 22% -37% in the southwest (13), and 41.4% in central parts of Iran (14). Overall, there are different reports and several hypotheses demonstrating that toxoplasmosis increases susceptibility to acquiring diabetes and, on the other hand, diabetic patients are more susceptible to a variety of infections such as toxoplasmosis (15)(16).WHO (World Health Organization) de nes diabetes as a chronic disease when the pancreas does not produce enough insulin, or when the body cannot effectively use insulin. GDM (Gestational diabetes mellitus) is a subtype of DM and is de ned as glucose intolerance with onset or rst recognition during pregnancy (17). There are serologic techniques to measure the level of T.gondii antibodies in humans and animals. ELISA is a most common standard method among serologic methods which is for examining anti-T. gondii antibodies. This method is easy to perform, widely available, and has high sensitivity and speci city (18).The aim of this study was to determine the IgG and IgM antibodies created against T. gondii infections using the ELISA method in diabetic pregnant women in compared with non-diabetic pregnant women in, Kurdistan, Sanandaj, west of Iran.

Methods
In this case-control study totally 181 blood samples were collected who 91 cases were registered as diabetic pregnant women(case) and 90 individuals were collected as non-diabetic pregnant women(control) referred Toohid hospital diabetes center during the period of June 2018 to October 2019 in Sanandaj, Kurdistan, west of Iran. Five mL of blood was taken from each person. Then, samples were centrifuged at 3000 rpm for 10 minutes. The sera were separated and kept at -20 ºC until the assay. All subjects completed a questionnaire including demographic information and risk factors.

Questionnaire
The applied questionnaire anchored in demographic data and risk factors including age, education, contact to animals, contact to cats and soil, consumption of half-cooked, raw and frosty meat, blood transfusion, gestational age, history of abortion, use of well and pipe water, residence area and the number of parity was prepared before the collection of blood samples and evaluated in case and control groups.
ELISA Test Serum samples were tested for level of anti-toxoplasma IgM and IgG antibodies according to the manufacturer's instructions ELISA kit (Pishtaz Teb Co, Iran). Based on the ELISA kit, If IgG and IgM antibodies concentration was > 1.1 were de ned as positive results, and < 0.9 were de ned as negative results.

Statistical analysis
The results of this study were analyzed using SPSS software (version 19). Determination of statistical signi cance was performed by Chi-square and Fisher tests to compare the seroprevalence values. P ≤ 0.05 was considered in a signi cant level.

Results
In this study a total of 181 diabetic and non-diabetic pregnant women age ranging from 17 to 40 years had participated. The mean age was 32.89 ± 4. Sociodemographic data of patients and controls are shown in Table 1, which showed patients consuming frosty meat were signi cantly higher (p = 0.024) than those of the control group (16.1%, vs. 6.7% respectively). Although no signi cant relationship was found between level of education in two groups (p = 0.59) but there was an increase in elementary phase in case group 24(26.4%) versus control group 11(12.2%).Distribution of the patients and control studied and their correlation with T. gondii seroprevalence was shown in Table 2; anti-T. gondii IgG antibodies were found in 30 (33%) of the cases and in 14 (15.6%) of the control group. The seroprevalence of T. gondii infection was signi cantly higher in cases than in controls (p = 0.006). Anti-T. gondii IgM antibodies were found in 6(6.6%) and 3(3.3%) in case and control groups respectively. There was no statistically signi cant difference in the frequency of IgM seropositivity among cases and controls (p = 0.31). Table 3 revealed signi cant correlation was found between age (p = 0.024) and abortion history (p = 0.05) with IgG seropositivity anti-T. gondi in two groups. We found no statistically signi cant relationship between all characteristics and risk factors and the IgM T. gondii antibodies.   (20) and Yemen (21.1%) (21) but lower than the results of studies from Egypt (45%) (22) and turkey (53%) (23). In addition, our study showed that the prevalence rate of IgM T. gondii antibody in diabetic pregnant women was 6.6% which was in harmony with those the results reported from Brazil (5.3%) (24), higher than as reported by Iran (2.7%) (25), and lower than as reported from Saudi Arabia (12%) (26).These differences in studies may be due the existence of variations in cultures and traditions between different nations as well as different food habits (27).Toxoplasmosis may occur in diabetic patients due acquired acute infection, or they may suffer from the spread of an active latent infection. (28).
seroprevalence of T. gondii infection was signi cantly higher in cases than control group (p = 0.06). It has been found that diabetic patients were more susceptible to have toxoplasmosis than control group which was in line with the results reported from china (20), and Egypt (22), However it was in contrast to the results performed on Iran (29). In this study there was a signi cant higher prevalence of seropositivity (IgG) between age group above 30 years old for the case and control groups (76.7% and 42.9%) respectively (p = 0.027). Similarly was reported from previous studies in geographical settings. (19,30).It could be explained that these women are exposed to the risk factors for a longer period of time than other women (31).
In current study we found signi cant relationship between the seroprevalence of history of abortion and T. gondii infection (p = 0.05).The obtained results of some of studies were opposite with our results (26, 32) but a study in north of Iran (33) reported the frequency of anti-Toxoplasma IgG (34.21%) in pregnant women with a history of abortion. Although the previous studies have shown that living in rural areas had a higher risk of Toxoplasmosis than in urban areas (34), we found no statistical difference in seroprevalence between rural and urban areas (p = 0. 66) which agreed with what has been found with the other reports (12,35).
High seroprevalence among individual from rural areas may be due poor socio-economic conditions and poor health.
In our study there was an increasing pattern of infection rates with level of education in elementary phase in case versus control group (43.3% v 21.4%) but the trend was not statistically signi cant(p = 0. 44). Other studies con rmed our ndings (36)(37). Contact with cats is often accepted as risk factors for infection with toxoplasmosis (25).In present study no signi cant relationship was found between the seroprevalence of T. gondii infection and contact to cats (p = 0.99), animals (p = 0.99), and soil (p = 0.49) which was similar to other studies (22,31). This could be explained due the climate of Kurdistan province is cold and this temperature is not suitable for the sporulation and survival of toxoplasma oocysts.
Several outbreaks and epidemiological studies have indicated the consumption of undercooked or raw meat as a source of toxoplasmosis (38). Although no correlation was found in this study(p = 0.37), agreement with previous reports . (39),reported an association of raw meat consumption with Toxoplasma in Cameroon and Japan (40)(41).
In this study no signi cant relationship between toxoplasmosis and gestational age (p = 0.64) was found but IgG in third trimesters of pregnancy in case group was more prevalent than in its similar subgroup control group (43.3% v 28.6%) , which was compatible to other studies (36,42). If the mother is infected to T. gondii during the second and third trimester of pregnancy, the probability of prenatal transmission will be increased to 80%. Early detection and speci c treatment of mothers can reduce the risk of fetal infection by up to 50%. (42).Contaminated drinking water is also a potential source of T. gondii infection (25).In our study, no association between the sources of drinking water and Toxoplasma infection was observed(p = 0.99).
The results a study in Nigeria was inconsistent with the present investigation (43) but other studies con rmed our founding (20,32). Blood transfusion as well as number of parity can also be a source of infection.The present study showed no apparent relationship between IgG-Toxoplasma infection and blood transfusion (p = 0.54) and number of parity (p = 0.99).Other studies agreement with our results (25,44).

Conclusion
The study results showed a statistically signi cantly higher proportion (33%) of seropositivity anti-Toxoplasma IgG antibodies among diabetes pregnant women compared to (15.6%) the control group. Also results of this study showed that 67% of pregnant women in sanandaj city did not have immunity against toxoplasmosis, therefore they are at risk of congenital toxoplasmosis for their fetuses and it is require awareness on the disease and its transmission, designing the preventive measures and care to avoid parasite exposure during their pregnancies.

Declarations
• Ethics approval and consent to participate * Statistically significant at P < 0.05