Anti-Toxoplasma gondii Antibodies: Prevalence and Risk Factors among Pregnant Women Accessing Antenatal Care in Some Primary Health Centers in Jos Metropolis, Nigeria

Ocheme Julius Okojokwu1*, Innocent Ajegba Onaji2, Entonu Elijah Entonu1, Bashiru Shafa Abubakar3, Maryam Bisola Adebayo4, Nafisa Abduljalil Adamu5, Daniel Iduh Ejembi1, Ibrahim Abubakar Yusuf4, Amos Obaje Ogaji1, Murna Ahmed Ali1, Joseph Aje Anejo-Okopi1 1Department of Microbiology, Faculty of Natural Sciences, University of Jos, Plateau State, Nigeria; 2Department of Pharmaceutical Microbiology, Faculty of Pharmaceutical Sciences, University of Jos, Plateau State, Nigeria; 3Department of Zoology, Faculty of Natural and Applied Sciences, Nasarawa State University, Keffi, Nasarawa State, Nigeria; 4Department of Microbiology, Faculty of Life Sciences, Ahmadu Bello University, Zaria, Nigeria; 5Department of Microbiology, Federal University Dutsin-Ma, Katsina State, Nigeria Email: okojokwuoj@gmail.com


Introduction
Toxoplasma gondii, which occurs worldwide, is an obligate intracellular apicomplexan protozoan parasite that is responsible for toxoplasmosis in animals and humans 1,2 . It is estimated that over one-third of the world human population is infected, with reported incidence rate of congenital toxoplasmosis ranging from 400 to 4000 cases per year 3,4 . The parasite is implicated for 1.2 million disability-adjusted life years annually 5 .
The main transmission pathway of T. gondii includes vertical transmission (from pregnant mother to fetus), eating raw or undercooked meat, and consumption of vegetables, water, and foods contaminated with oocysts of the parasites 6 . Should primary infection occurs during pregnancy, the parasite could cross the placental barrier causing congenital toxoplasmosis, which may lead to myriad of health problems such as spontaneous miscarriage, stillbirth, or congenital abnormalities such as mental retardation, hydrocephalus, intracerebral calcifications, and chorioretinitis [7][8][9][10][11] . Awoke et al. 9 reported that antenatal screening of pregnant women for T. gondii infection is based on IgG and IgM antibodies detection and is the main means of monitoring the risk for congenital toxoplasmosis.
The danger of congenital toxoplasma infection and the seriousness of fetal harm are reliant on the gestational age when the maternal infection takes place 12 . The general danger of congenital maternal infection from primary toxoplasmosis during pregnancy varies from 20% to 50% if left untreated 13 . Prevention of congenital toxoplasmosis can be achieved by identifying non-immunized women at the onset of pregnancy and enlighten them on how to prevent the infection, and by serological follow-up. Multiple testing for Toxoplasma-specific IgG and IgM helps to differentiate between acute and chronic infections 12 .
Despite the risk of vertical transmission of toxoplasmosis, serological tests for T. gondii infection are not routinely performed during pregnancy in Nigeria. Hence, prevalence and the burden of toxoplasmosis are poorly understood. The aim of this study, therefore, was to investigate the seroprevalence and risk factors of T. gondii infection by determining (1) the seroprevalence of IgG and IgM specific to T. gondii and (2) risk factors that are associated with toxoplasmosis.

Study area and population
This cross-sectional study was carried out from January to May 2019 in Jos, located between latitude 9°55'42.56''N and longitude 8°53'31.63''E, among women attending antenatal care at some Primary Health Centers (PHC) in Jos, Plateau State Nigeria.

Ethical considerations
Ethical clearance was obtained from the Ethical Review Committee of the Plateau State Specialist Hospital, Jos, Nigeria, under the number NHREC/O5/01/2010b. A signed informed consent form was obtained from each study subject at the beginning of the study. Confidentiality of patient information and samples was ensured and maintained at all times. Spent samples were appropriately autoclaved and disposed after usage.

Collection of blood sample and sociodemographic data
This research was a cross-sectional study and it was carried out within 5 months. Samples were collected from pregnant women accessing antenatal care at some (PHC Dogo Agogo, PHC Jos Township, and PHC Jos Jarawa), Jos. Information on sociodemographic as well as some risk factors of T. gondii infection was collected using a structured questionnaire. Approximately 3 ml blood samples were aspirated from each patient and coded. The samples were kept at ambient temperature to clot and were thereafter spun at 1000 g for 10 min. The sera were collected in 2-ml Eppendorf tubes and taken to Plateau State Human Virology Research Centre in an icebox where the samples were kept at −20°C until analysis 30 .

Serological testing
Testing for anti-T. gondii antibodies (IgM and IgG) were done with enzyme-linked immunosorbent assay (ELISA kit provided by Diagnostic Automation, INC USA). The kits were used following the manufacturer's instructions and guidelines.

Statistical analysis of data
Data from this study were saved on Excel ® spreadsheet and analyzed using Statistical Package for the Social Science version 23. Chi-square test was used to determine the relationships between the risk factors and seropositivity. P ≤0.05 was considered statistically significant.

Results
Overall, 182 subjects were included in this study; 84 (46.2%) had anti-Toxoplasma IgG antibody, while 2 (1.1%) were positive for IgM antibody. Two study subjects that had IgM antibody were also seropositive for anti-Toxoplasma IgG antibody representing 2.4% (2/84) of the women who were seropositive for anti-Toxoplasma IgG. Ninety-eight (98 [53.4%]) of the subjects were seronegative for both anti-Toxoplasma IgG and IgM antibodies ( Figure 1).
Although the demographic parameters show no association (P > 0.05) with the prevalence of anti-Toxoplasma antibodies, the age group 11-20 years had the highest prevalence of IgG antibody, while women within the age group 41-50 years had 0.0% prevalence of IgG antibody to T. gondii. On the other hand, all the 2 subjects that tested positive to IgM antibody were in the age bracket of 31-40 years. With respect to educational status, the seroprevalence anti-Toxoplasma IgG antibody decreased from 100% in women without formal education to 40.0% in women with tertiary level of education. The two women who were seropositive for IgM antibody had secondary level of education (Table 1). In terms of occupation of the pregnant women, majority of the women were unemployed (53.8%), while 46.2% of them were employed. The highest prevalence (66.7%) of IgG antibody was detected among students, while the least (44.2%) was detected among housewives. In similar vein, the housewives 2.3% prevalence of IgM antibody ( Figure 2).
There was statistically significant association (χ 2 = 14.324; P = 0.001) between trimester and prevalence of anti-Toxoplasma IgM antibody. None of all the other risk factors studied in the present research had association (P > 0.05) with anti-Toxoplasma antibodies ( Table 2).

Discussion
The present study is among the few works in Nigeria that explore the seroprevalence of T. gondii infection Our findings showed that the seroprevalence of anti-T. gondii antibodies in women visiting antenatal clinic in PHC in Jos was 46.2% and 1.1% for IgG and IgM, respectively. This implies that 46.2% of the study subjects had been exposed and possibly recovered from the infection, while 1.1% of the women have ongoing or current Toxoplasma infection. Hence, the finding suggests that the parasitic infection is endemic in the study area. The anti-T. gondii IgG prevalence reported that in the current study was higher than previously reported ones for Sudan (41.7%), Somalia (29.6%), and Algeria (52.2%) 32 . When compared with findings from other parts of Nigeria, the seroprevalence was higher than the 32.6% and 40.6% reported by Deji-Agboola 33 and Akinbami et al. 34 in Lagos and 29.1% obtained among pregnant women in Zaria 35 . These various findings lend credence to the fact that Toxoplasma infection varies significantly within and between countries 5,36-39 . The difference in the seroprevalence of T. gondii infection could be attributed to the variation in geographic locations of the study areas as manifested in differences in humidity and temperature. Higher temperatures and/or humid environments favor sporulation of oocysts 40,41 . Furthermore, the variation in   prevalence of T. gondii infection could be partly due to the use of different analytic techniques such as the use of ELISA versus rapid diagnostic tool and/or polymerase chain reaction. This is because these analytic techniques have different rates of sensitivity and specificity 42 . In addition, difference in socioeconomic status of the women and variation in samples could have impacted on prevalence reported in the various findings, thus bringing about the discrepancies in the reported figures. The correlation between sociodemographic characteristics of the pregnant women and prevalence of anti-T. gondii antibodies were assessed in the current study. In concordance with findings from other authors 43,44 , there was no significant association between Toxoplasma seroprevalence and increasing maternal age, but younger women (21-30 years) were found to have a relatively higher seroprevalence rate compared to older women (>30 years). This might be explained by younger rather than older women's preference for outings overeating at home. During those outings, the women are exposed to grilled meat which might be undercooked. Fruits and salads which may be contaminated with the parasite oocysts are also often eaten in such outings hence increasing the risk of infections [45][46][47] . Prevalence of anti-T. gondii antibody (IgG) in this study decreased with increasing educational level. This finding was in accordance with the study done in Debre Tabor, Northwest Ethiopia, where pregnant women who could neither read nor write had the highest toxoplasmosis infection rate compared with those with primary, secondary, and tertiary levels of education 48,49 . Illiterate women are more likely to engage in more risky unsanitary and unhygienic practices hence are more liable to be infected by T. gondii. Though significant association was not observed between prevalence of anti-Toxoplasma gondii antibodies and both occupation and educational level, the prevalence increases with decrease in educational level. This is in concordance with reported findings from Africa 50-52 .
However, there was a significant relationship between trimester of pregnancy and prevalence of anti-T. gondii IgM antibody. Women in the first trimester of pregnancy had the highest prevalence of Toxoplasma infection. Although the gravidity of the women was not established in this study, Mandour et al. 53 reported an increased percentage of multigravid women, in whom complicated pregnancy outcomes have been reported compared with primigravid females. In view of that finding, it is likely that the majority of the women in the first trimester of their pregnancy were multigravid females.
Our findings showed no association between T. gondii infection and contact with cats. This suggests that contact with cats does not translate to zoonosis, but more pertinent in causing zoonosis is the improper handling of cats' fecal matter 54 . While this finding disagrees with the reports of Lim et al. 55 and Adeniyi et al., 56 where they showed a significant association between contact with cats and seroprevalence of T. gondii infection, it is consistent with the findings of Doehring et al. 57 , Nijem and Al-Amleh, 40 and Wang et al. 58 . It was also found that though immunecompromised individuals were reported to be more prone to toxoplasmosis 59 , HIV-negative women had a higher prevalence of the infection in comparison with HIVpositive pregnant counterparts. It is likely that the use of antiretroviral therapy by HIV-positive women reduced the probability of opportunistic infections.
Two pregnant women with active/ongoing toxoplasmosis were detected. This implies that their fetuses were at risk of congenital T. gondii infection with attendant adverse and sometime fatal consequences. The presence

Conclusion and Recommendation
This study confirms prevalence of T. gondii infection among pregnant women accessing antenatal care in Jos metropolis, Plateau State, Nigeria. None of the risk factors studied with respect to IgG antibody was associated with toxoplasmosis. In spite of the absence of a significant association between possible risk factors and the infection, there is a need for the enlightenment of women, particularly pregnant women, on the risk factors and how to prevent the contraction of toxoplasmosis.