Seroprevalence of Toxoplasma gondii Infection among Pregnant Women Attending Antenatal Care in Asmara, Eritrea: Preliminary Report

The majority of human infections with Toxoplasma produce no symptoms, but in congenitally infected children can cause devastating effects including eye blindness, brain damage, or miscarriage. Transmission to the fetus occurs predominantly in women who acquire their primary infection during gestation. The study aimed to determine the seroprevalence of T. gondii among pregnant women attending Antenatal Care (ANC) in different sites of Asmara, Eritrea, and to identify possible risk factors associated with Toxoplasma infection among pregnant women attending the antenatal center. The study has followed a cross-sectional laboratory-based survey. The data were collected from 210 pregnant women in four health facilities. Voluntary sampling technique and a structured questionnaire were used to collect associate factors and socio-demographic information. Cobas e411 clinical chemistry Analyzers was used to test the blood serum for IgG and IgM antibodies. Epi-Info (V 7.0) was used for data entry and SPSS (V 20.0) was used for data analysis.


Introduction
Toxoplasma gondii is a microscopic zoonotic and an obligated intracellular protozoan parasite that can infect most warm-blooded animals and causes a disease called toxoplasmosis [1]. It was originally described in a North African rodent called the gundi, from which derives its speci c name [2,3]. Humans can become infected by ingestion of oocysts released in cat feces; by ingestion of raw or undercooked meat or drinking unpasteurized milk containing T. gondii tissue cysts [1,4]. Transplacental congenital transmission of tachyzoites, blood transfusion or organ transplantation are very rarely [5,6]. All stages can be infectious to human [2]. In chronically infected women; infection can be reactivated because of their immuno-compromised state [7]. Recently, in immuno-competent individuals more virulent strains that cause severe symptoms have been reported [8].
The diagnosis relies on either indirect serological tests or direct detection of the parasite or by PCR.
Serologic tests that indicating recent or past infection are most effective in immune-competent adults.
These tests include: ECLIA, ELISA, IFA, LA tests and complement xation to detect IgG and IgM antibodies [9]. The presence of a high IgM titer in the absence of signi cant IgG titers indicates early stages of primary infection. A negative IgM titer is helpful for ruling out recent infection. However, a positive IgM test should be veri ed in a reference laboratory [3,10]. Current procedures allow diagnosis of an active infection of the fetus in utero by means of PCR of amniotic uid [11].
Children over 5 years of age with severe acute toxoplasmosis or active chorioretinitis is treated with pyrimethamine. Treatment of pregnant women are controversial but treatment with spiramycin is still advocated [12]. Current evidence is insu cient to con rm that treating seroconvert during pregnancy prevents fetal infection [11].
It is noted that no study on the seroprevalence of Toxoplasma gandii in general and/or speci c target groups has been done in Eritrea. None is known on what are the associated risk factors for the spread of the disease and its causes. Therefore, the purpose of this study was to estimate the seroprevalence of T. gandii and its associated risk factors among pregnant women attending Antenatal Care (ANC) in Asmara City, Eritrea.

Materials And Methods
Study Area: The study was conducted in Asmara, Eritrea at all the health facilities that provide maternal health services, including delivery, one hospital and other three health facilities were selected for the study to represent the city.
Eritrea is situated in the horn of Africa bordered by the Sudan, Djibouti, Ethiopia and the Red Sea with 1,212 Km seashore. It covers an area of 124,000 sq. km and is divided into three topographic areas (the Western Lowlands, the Central Highlands and the Coastal Plains). Administratively, it is divided into 6 regions or zones, namely Southern Red Sea Zone, Northern Red Sea Zone, Anseba, Gash-Barka, Southern Zone and Maekel (Central Zone). The total population are about 6.1 million [13]. Asmara is in central region (Zoba Maekel), country's capital city, is the targeted area of the study.
Study Design, Study Population and Sample Size: The study was designed as cross-sectional laboratory based study. That comprises all pregnant women living in the Catchment area of Asmara administration, in the reproductive age group of 16 to 45 years [14,15].
The data was collected between July and August 2016 from 210 pregnant women (in four health facilities) whom attending their ANC follow up. Voluntary sampling technique, was implored following their consents and agreements to participate in the research study.

Data Collection Tools and Techniques:
A structured questionnaire was used to collect the pregnant women demographic information and the other associated risk factors in the study. Pregnant women were interviewed by the enumerators which were health professionals who works in the facilities where the collection had been conducted.

Collection of sample and Laboratory investigation:
A three ml venous blood sample was collected from study participants using standard procedures on Lithium heparin container and was stored in freezing condition (-20 0 c) before analysis. Serum was prepared by centrifugation of blood at 2500 rpm for 3 minutes.
The electrochemiluminescence immunoassay (ECLIA) was used for in vitro quantitative determination of IgG and IgM antibodies to Toxoplasma gondii in human serum on Elecsys and Cobas e411 immunoassay analyzers.

Quality control (QC):
For QC; Preci Control was used. Controls for the various concentration ranges were run every day when the test in use and following each calibration. Based on the measurements of cal-1 and cal-2 the cut-off was calculated automatically.

Data entry and Data Analysis:
Questionnaire completeness checking process was followed by data entry into computers using Epi-Info (V 7.0), free software package developed by CDC. To describe the data in terms of frequency and proportion SPSS (V 20.0) computer program was used [16]. To describe the age of participants, which is continuous variable, average and standard deviation were used [17]. In this study, the chi-squared test has been applied to screen for an association between IgG and IgM antibodies seropositive results and the several risk factors included in the study [18]. Ethical Issues; Informed Consent and Con dentiality: Research approval for the study was obtained from Research and Ethic Committee, Faculty of Medical Laboratory Sciences, University of Gezira. Based on the request of Faculty of Medical Laboratory Sciences, University of Gezira, the Health Research Proposal Review and Ethical Committees, MOH Eritrea had accepted and gave a permission to conduct the research on the speci ed sites. Ethical Permission was obtained from the Ministry of Health, Zoba Makel.
Participants were briefed about the aim of the study and requested their willingness to participate in the study. The interview was carried out face-to-face to obtain information and were kindly asked for their enthusiastic participation and sign on the prepared consent form.

Results
Socio-demographic characteristics of study population: Overall 210 samples of pregnant women were considered from Edagahmus mini Hospital, Akria Health Center, Vilajo/ BMCH Community Hospital and Godief Health Center each constituting 80 (38%), 50 (24%), 41 (20%) and 39 (19%) respectively. Majority of the pregnant women were in the age group of 25 to 34 years representing 60%, in the age groups of 15-24 years and 35-44 years were 27% and 13%, respectively. The mean and median age was comparable with 27 and 26 years, respectively with variation of 5 years by SD. Only 10% of the women were reported to be not married being divorced, widowed or separated while the remaining 90% were married. Equal to 98% of the participants were from Tigrigna ethnic group. 70% of the pregnant women were noted to reach secondary level while the rest 16% and 14% were reported to reach elementary and post-secondary educational level respectively. 74% were housewife, and the remaining 14% and 12% women were engaged in daily workers and other type jobs.
Majority of the pregnant women were at their 2nd trimester which accounts 42%; while the remaining 37% and 21%were in their 3rd and 1st trimesters respectively (Table 1).  antibodies. Chi-square (X 2 ) was done to assess between seropositive Toxoplasma and the factors, but no signi cant association was observed.  Based on a different research study, possible risk factors were identi ed and included in this research study. And these are: source of water, use of raw meat, soil contact and availability of the domestic animal in general and presence of cat in particular. In addition to that three (3) possible health factors history of abortion, HIV status and presence of mentally abnormal child were also included (Tables 5 and  6). Chi-square showed some signi cant association between seropositive Toxoplasma and soil contact, but no signi cant association was obtained for most other factors.

Discussion
Several research studies has noted, toxoplasmosis is food-borne infectious which is leading cause of death ranked in third after salmonellosis and listeriosis [19]. Though it is highly neglected, congenital toxoplasmosis particularly is a major problem in most communities with a high prevalence of T. gondii infection. Epidemiological studies reporting prevalence of T. gondii infection in pregnant women across the world suggest considerable variation between countries ranging from 9-67% in Europe and reaching as high as 92.5% in African countries [6]. Thus, conducting a study of the seroepidemiology of this infection among females of childbearing age could provide appropriate approaches which could help to design the preventive measures [3].
The present study showed a seroprevalence of anti-T. gondii IgG antibody of 53.6% among 210 pregnant women in Asmara. Even though these pregnant women were not at risk for toxoplasmosis, because they have already developed IgG antibodies, the remaining 46.4% are at risk of the infection. Also the seroprevalence of toxoplasmosis IgM antibody was noted to be 2.9%, which is lower than many countries. In Saudi Arabia, the prevalence of T. gondii during pregnancy for IgG and IgM antibodies were 32.5% and 6.4% respectively [14].
As it was indicated, the study was done in pregnant women in Asmara. Asmara is the capital city of Eritrea where majority of its habitants have better access to different social service such access to clean drinking water, hygiene and sanitation, education and health [15]. Had it been in other rural parts of the countries where access to social services is relatively low, the seroprevalence would have been skyrocketed.
Though, the result revealed that there was no association between seropositivity of toxoplasma antibody and different age groups, the seropositivity rate in 35-44 years age group was obviously higher than other two groups. Despite small variation in age group, the result is in line with studies from other countries [20,24]. However, in the comparative study done between pregnant women in Malaysian and Myanmar has identi ed that Toxoplasma seropositive was found to have an association with aged 30 years and above [18]. The same Malaysian study conducted among the pregnant women has also identi ed that mothers' education, gestational age, gravida, disease awareness, obstetrics history, having contact with cats or soil, consumption of undercooked meat, unpasteurized milk or untreated water have a relation with toxoplasmosis. Though this is the rst study ever done on Toxoplasmosis in the country, the result showed that the seroprevalence of Toxoplasma infection was considerably high (53.6%) using IgG antibody. When IgM antibody was considered, the disease prevalence was noted to be 2.9%. It has been noted that the areas of the study was in Asmara where the exposure to risk factors is relatively minimal. Thus, even considering this, the result was higher which requests an attention. Though some of the risk factors had included in the study, test result has showed that except soil contact, none of them had any signi cant association with the disease pattern.

Conclusion
The absence of a statistically signi cant relationship between the prevalence of Toxoplasma infection and many of the factors included in this study, does not necessarily mean that these factors have no in uence on the transmission of toxoplasmosis. This could be due the test method where it demands higher number of disease cases. Probably a lager sample size would have revealed signi cant relationships. However, in this case some of the cases were lower when results were further segment among the various socio-demographic characters and causal factors.

Declarations
Con ict of interest: We declare that we have no con ict of interest.
Ethical Issues: In this study, ethical considerations have been fully observed.