SEROPREVALENCE OF INFECTION WITH TOXOPLASMA GONDII AMONG THE RESIDENTS OF SOUTH BACKA DISTRICT, SERBIA

Ivana Hrnjakovic Cvjetkovic1,2 *, Vesna Milosevic1,2, Tamas Petrovic3, Dusan Petric4, Radovanov Jelena1, Kovacevic Gordana1, Patic Aleksandra,2, Nikolic Natasa1,2, Nela Dopudj1, Sandra Stefan2,5, Dejan Cvjetkovic2,5 1Institute of Public Health of Vojvodina, Novi Sad, Serbia11 2University of Novi Sad, Faculty of Medicine Novi Sad, Novi Sad, Serbia 3Scientifi c Veterinary Institute ̋Novi Sad ̋, Novi Sad, Serbia 4University of Novi Sad, Faculty of Agriculture, Novi Sad, Serbia 5University of Novi Sad, Faculty of Medicine, Clinical Centre of Vojvodina, Novi Sad, Serbia

females. Possible recent infection within the last 12 months was found in 1.70% (192/11288) patients. Possible acute infection or false -positive IgM result was detected in 1.31% (148/11288) patients. Past infection was found in 2173/11288 (19.25%) patients. Equivocal results were found in 1.48% of samples (167/11288). Th e lowest frequency of anti-toxoplasma antibodies was detected in pre-school children 13.07% (97/742) and the highest in persons older than 65 years 60.18% (65/108). Comparing the results of the research done from 2014 to 2018 with the data from 1989, a signifi cant decline of seroprevalence in general population and women of generative age was found.

INTRODUCTION
Toxoplasma gondii is a ubiquitous coccidian protozoan that can infect a wide range of warm-blooded animals, including humans. Th e life cycle of this obligate intracellular parasite is complex. Domestic and wild members of the family Felidae are defi nitive host where it multiplies in the small intestine (sexual cycle). Humans and warm-blooded animals, including most livestock are intermediate hosts, where it forms a cyst in muscular and neural tissues. T. gondii causes zoonotic, cosmopolitan disease Toxoplasmosis. For immunocompetent humans it is a well-adapted parasite that causes asymptomatic infection in 10 -20% cases according to studies conducted in USA and Europe. However, in congenitally infected infants and immunocompromised patients it can cause a serious life threatening disease.

MATERIAL AND METHODS
Th e prevalence of anti-toxoplasma antibodies was determined retrospectively in 11,288 persons from South Bačka District, during a 5-year period (from January 2014 to December 2018). Th e analysed group included 9,818 females (aged 1 to 85) and 1470 males (aged 0 to 88). Among females, 9,113 (92.82%) were childbearing women (defi ned in this paper as 19-45 year-olds). Th e serum samples were analysed for toxoplasma specifi c immunoglobulin M (IgM) and G (IgG) antibodies, by ELISA IgG and ELISA IgM test (Euroimmun, Lübeck, Germany). Th e testing was performed on the automated device Euroimmun Analyzer I-2P at the Institute of Public Health of Vojvodina, Center of Virology. For determination of IgG avidity commercial ELISA using urea as a denaturing factor was carried out according to manufacturer's instruction (Euroimmun, Lübeck, Germany). A relative avidity index (RAI) was calculated and expressed as a percentile dividing the OD values with and without urea treatment.  Possible recent infection within the last 12 months was found in 1.70% (192/11288) patients in which both IgG and IgM antibodies were detected. In 2,173 (19.25%) patients a serologic profi le consistent with a past infection was found (only anti-toxoplasma IgG antibodies were positive). Th e results of Toxoplasma gondii seroprevalence in population of South Backa District by age group are shown in Table 2.

RESULTS
In the youngest group (0-3 years) seroprevalence varied from 9% (in 2015) to 18% (in 2016). In the eldest group seroprevalence varied from 47% (in 2016) to 68% (in 2014). We found that the seroprevalence for Toxoplasma gondii signifi cantly increased with age.
In the population of South Backa District seroprevalence in childbearing women was 20.04% (1826/9113). Out of 9,113 childbearing women 101 (1.1%) had serological profi le consistent with possible recent infection within the last 12 months. In 20 childbearing women avidity of IgG was determined and RIA values were in the range from 61.2% to 97% indicating past toxoplasma infection in all tested childbearing women.

DISCUSSION
Compared to other countries, a moderate seroprevalence (22.26%) for anti-Toxoplasma specifi c antibodies was found in our study in South Backa District. Th e highest seroprevalences of toxoplasmosis were detected in tropical areas in Africa (14%-78%), Brazil (75%) and Ethiopia (73%). Th e lowest seroprevalences of toxoplasmosis were observed in hot and dry area in Asia (Foroutan-Rad M, 2016). In Chinese blood donors the overall IgG seroprevalence of T. gondii infection was low 6.26% (Wang et al., 2018). Seroprevalence of 24.2% (123/508), similar to that detected in our study, was found in a healthy population of Slovakia (Studeničová et al., 2006). In a study of Coroiu and co-workers (2009), the seroprevalence for T. gondii in general population of north-western and central Romania were higher, amounting 59.4% (686/1155). In serological study conducted in Germany seroprevalence of 55% (3602/6564) was found (Wilking et al., 2016). Th e results of many studies of Toxoplasma gondii infection in humans showed diff erent rates of seroprevalence depending on geographical location, environment, socio-economic, culinary habits and applied diagnostic methods.
In the study conducted in 1989, we tested 1000 serum samples of person from South Backa District applying immunofl uorescent test for anti-toxoplas-ma IgG and IgM antibodies (Hrnjakovic Cvjetkovic, 1989). Comparing the data of these two studies we found that seroprevalence was decreasing from 30.70% in 1989 to 22.26 % in 2018 (p<0.0001). Generally, a decrease in the number of seropositive humans and animals had been detected in many European countries and USA over the last few decades. In the study comparing the U.S. prevalence of T. gondii infection in humans during period 1988-1994 and 1999-2004, a decrease from 14.1% to 9.0% in 6 to 49 year-olds was found (Jones et al., 2007). Th e T. gondii overall seroprevalence decreased from 47% in 1979/1980 to 22% (95% CI 20% to 24%) in the Portuguese population in 2013 (Gargaté et al, 2016). In our study seroprevalence increased with age from 15% in the age group of 0 to 3 year-olds to 60% in the age group > 65 years like in other similar studies (Wilking et al., 2016;Studeničová et al., 2006;Gargaté et al., 2016).
Seroprevalence in childbearing women found in this study was 20.04% (1826/9113) and it diff ers from older data from our country. Among women of reproductive age, in the area of Belgrade, during the 1988-1991 period, the rate of infection with Toxoplasma gondii was 77.4% (Bobić et al., 1998). In women of childbearing age in the period from 1990 to 2000 seroprevalence were 11% in Norway, 14% in Sweden and 8% in East England. (Tenter et al., 2000;Petersson et al., 2000) Comparing seroprevalence in childbearing women in 2014-2018 (20.04%) with seroprevalence in 1989 (33.48%) (Hrnjakovic Cvjetković, 1989), we found that seroprevalence in South Backa District signifi cantly decreased over time. Th e similar trend was noticed in the studies on seroprevalence of childbearing women from Portugal (Gargaté et al., 2016).
Possible recent infection within the last 12 months of 1.1% was determined in this study of 9,113 women of childbearing age. In the study of Mumcuoglu et al. (2014) 0.9% out of 4,758 pregnant women had the serological profi le consistent with possible recent infection. In the study of Stojanovic (1998), acute toxoplasma infection was registered in 0.61% of 1,266 pregnant women in Timok region in 1998.
Due to acquired data on seroprevalence, out of 22.26% in this study, South Backa District is listed among regions of moderate seroprevalence in respect of toxoplasmosis. In the time period from 2016 to 2018, seroprevalence of toxoplasmosis declined signifi cantly in comparison to its values in 2014. Comparing the results of the research performed from 2014 to 2018 with the data from 1989, there is a signifi cant decline of seroprevalence in general population and women at generative age, which is in compliance with the studies conducted in the USA and the EU.

ACKNOWLEDGEMENT
Th is work is conducted within the projects TR31084 and III43007 funded by the Serbian Ministry of Education, Science and Technological development