Toxoplasma gondii infection : seroprevalence and associated risk factors among primary schoolchildren in Lagos City , Southern Nigeria

Introduction: Toxoplasma gondii infection has been described as the most widespread zoonotic infection of humans and other animals. Information concerning T. gondii infection among schoolchildren is unavailable in Lagos City, Nigeria. Methods: This crosssectional study investigated the seroprevalence and risk factors associated with T. gondii infection among primary schoolchildren (PSC) from a community located in the center of Lagos, southern Nigeria, from November 2013 to March 2014. A total of 382 PSC were screened for the presence of sera anti-T. gondii antibodies using a latex agglutination test (TOXO Test-MT, Tokyo, Japan). A cutoff titer of ≥ 1:32 was considered positive, while titers ≥ 1:1,024 indicated high responders. Questionnaires were also used to obtain data on possible risk factors from parents/guardians. Results: The overall seroprevalence was 24% (91/382), and 83.5% (76/91) of seropositive PSC were classifi ed as high responders. Among the risk factors tested, including contact with cats and soil, consumption of raw meat and vegetables, and drinking unboiled water, none showed statistical signifi cance after multivariate adjustment. No associations were observed among age, gender, body mass index (BMI), and parents’ occupation/educational level. Conclusions: The fi ndings in this study show evidence of active infection, and hence, there is need for urgent preventive measures in this city. Further investigation is required to clarify the transmission routes. Policy makers also need to initiate prevention and control programs to protect pregnant women and immunocompromised patients in particular because they are more severely affected by T. gondii infection.

Toxoplasma gondii is an intracellular protozoan parasite with a widespread distribution.T. gondii has a large variety of hosts, including humans and most warm-blooded species, but it completes its sexual cycle and produces oocysts only in the intestinal tracts of felids (1) .Worldwide, over 6 billion people have been infected with T. gondii (2) .Although it is a cosmopolitan infection, the disease appears to be overshadowed in the tropics, especially in Sub-Saharan Africa (SSA), by other endemic diseases such as malaria and human immunodefi ciency virus (HIV) (3) .
Humans acquire T. gondii infection through oral ingestion of food or water contaminated with oocysts from cat feces, ingestion of cysts contained in raw or undercooked meat, transplacental transmission of tachyzoites from the infected mother to her fetus, and tachyzoites and tissue cyst acquisition via solid organ transplantation (1) (4) .Post infection, adults and children with normal immune function remain asymptomatic or develop only mild symptoms, such as fever and malaise, although up to 10% will present with cervical lymphadenopathy or ocular disease (1) .Of note, substantial evidence indicates that T. gondii has emerged as an interesting candidate as a possible cause of some cases of schizophrenia (5) .
Although the clinical presentation of toxoplasmosis is benign in an immunocompetent population, in immunocompromised persons, such as HIV patients, toxoplasmosis is the most frequent severe neurologic infection.Toxoplasmic encephalitis (TE)

METHODS
due to reactivation of a latent infection may lead to severe cerebral lesions in acquired immunodeficiency syndrome (AIDS) patients (6) (7) .Children and young adults can also suffer from toxoplasmic chorioretinitis, which is a common late manifestation of congenital infection or acute infection (1) .In pregnant women, when infection is transmitted to the fetus, it may lead to mental retardation, blindness, epilepsy and death (1) .The severity of the outcomes for newborns depends on the gestational stage during pregnancy when the infection occurred; early maternal infection (in the fi rst trimester) can cause more signifi cant congenital toxoplasmosis in newborns (1) (8) .T. gondii is a member of the TORCH group of infections (which includes rubella, cytomegalovirus, and herpes viruses) that can cause congenital abnormalities and even death in fetuses (1) (9) .
The detection of the T. gondii parasite itself is very diffi cult; hence, the diagnosis of human toxoplasmosis is typically based on serological detection of antibodies against T. gondii using latex agglutination (LA) testing, enzyme-linked immunosorbent assay (ELISA), and/or indirect fl uorescent antibody testing (IFAT).Latex agglutination testing is better adapted for developing countries where rural districts often lack the equipment needed for ELISA (10) (11) (12) .Moreover, LA is also simpler and easier to perform, and it shows high qualitative agreement with the Sabin-Feldman dye test, which represents the gold standard (13) .
Seroprevalence rates of T. gondii have been shown to vary from 0% in Eskimos (14) to 93% in Ghanaians (15) .In recent studies, these rates have been reported to be 70% in Indonesia, 81% in Ethiopia, 52% in Brazil, 10.8% in the U.S., and 13.2% in Korea (16) (17) (18) (19) (20) .Although these studies targeted different populations and used different immunological assays, the widespread prevalence of this infection is indisputable.
In Africa, the majority of surveys have been carried out on immunocompetent adults, followed by women of reproductive age and then on HIV/AIDS patients (21) ; however, studies concerning the seroprevalence of T. gondii in primary schoolchildren (PSC) are limited (22) .Furthermore, few studies have been performed in countries such as Somalia, East Africa, Madagascar and Swaziland (12) (23) (24) .In Nigeria, information on the seroprevalence of T. gondii among PSC is unavailable.
Toxoplasmosis has been described as an environmental disease because transmission has been shown to be promoted by poor environmental sanitation, overcrowding, poverty, certain eating habits and poor hygiene (25) (26) .
This cross-sectional study was therefore designed to investigate the seroprevalence and risk factors associated with T. gondii infection in PSC, with the aim of understanding the fundamental epidemiology and environmental risks of T. gondii infection in Lagos City, Nigeria, which is the largest city in SSA.
Primary schools located in a community of the Mainland Local Government Area (LGA), which is located within the center of Lagos City, Southern Nigeria, were selected.These schools lie within latitude 6 ᴼ 28' and 6 ᴼ 29' and longitude 3 ᴼ 12' and 3 ᴼ 13'.This community has an estimated population of approximately 100,000 inhabitants and is located partly on land and partly on the Lagos lagoon.The major occupation is fi shing and trading, especially among individuals living on the lagoon.This community suffers from serious environmental and infrastructural defi ciencies, including inadequate access roads, schools, health care facilities and housing; however, many foods that are consumed daily, such as vegetables and fi shes, are transported from this community to various areas within Lagos (27) .This cross-sectional study was carried out between November 2013 and March 2014 and was conducted in the only three government-built primary schools, namely Adekunle Anglican Church Primary School, Ayetoro African Church Primary School and Makoko Anglican Church Primary School.
Schoolchildren attending these three government-built primary schools were involved in the study.A medical laboratory scientist collected blood specimens after verifying the informed consent forms from the pupils.Attached to the consent forms were structured questionnaires designed to capture basic demographic data regarding age, gender, parental educational level and occupation.Height and weight were recorded by the health workers using standard calibrated instruments.The children were divided into three age groups to maintain a suffi cient sample size in the analysis: Group 1, < 10 years; Group 2, 10-12 years; and Group 3, > 12 years.Parental occupation was also categorized into three groups: Group 1, unemployed; Group 2, unskilled workers; and Group 3, skilled workers.
Different risk factors in the environment were also investigated in the study, including contact with cats, cat feeding practices, contact with soil, drinking unsafe water, eating raw (unwashed) vegetables, and consuming raw or undercooked meat, and were used in the multivariate analysis.Approximately 3-5ml of blood was drawn from each child by venipuncture, and the sera were collected and then stored at -20°C until use.All of the sera were screened for anti-T.gondii antibodies using a commercial test kit based on LA (TOXO Test-MT, Eiken Co. Ltd, Tokyo, Japan), according to manufacturer's instructions.A cutoff titer of 1:32 or higher was considered positive, while titers equal to or greater than 1:1,024 indicated high responders (22) (28) .
Evaluation of the associations between demographic characteristics and T. gondii infection was performed by a chi-squared test to compare the proportions of infection based on gender, age group, body mass index (BMI), and parental occupation and educational level.The log-binomial regression model was applied to investigate multiple environmental risk factors associated with T. gondii infection.The prevalence ratio, which is considered a less biased indicator than the odds ratio, was generated from the log-binomial models.All of the statistical analyses were conducted using SAS Version 9.3 (SAS Institute, Inc., Cary, NC, USA).The α-level was set at 0.05, and a p value less than 0.05 was regarded as statistically signifi cant.

Ethical considerations
Prior to commencement of the study, permission was obtained from the local education authority of the Local Government Area (Mainland LGA).Approval was also obtained  In total, 382 schoolchildren were enrolled in the present study, of which 195 were females and 187 were males, with an average age of 10.8 years.As shown in Table 1, the overall seroprevalence of T. gondii was 24% (91/382), while the highresponder rate was 20% (76/382).There was an increasing pattern of infection and high-responder status with increasing age, although this trend was not statistically significant.No significant associations were found between infection and gender, BMI, or parents' occupation/educational level (Table 1).There was high awareness of cats serving as reservoirs of some diseases (80.7%), but little was known about T. gondii (35%), as shown in Table 2.Among the symptoms presented, children with general body weakness showed higher infection rates compared with their non-weak counterparts, while headache and ocular discomfort showed no such relationships (Table 2).Table 3 shows the results of the multivariate analysis.Overall, no statistically signifi cant risk factors were identifi ed in the models.On univariate analysis, raw meat consumption showed higher risk [adjusted prevalence ratio (APR)=1.31]and contact with soil showed lower risk (APR=0.66)for T. gondii infection.Socioeconomic and demographic factors did not show a signifi cant association with T. gondii infection, including gender, age, BMI, or parental occupation/educational level.None of these factors was signifi cant after the univariate and multivariate analyses in this study (Table 3).

DISCUSSION
The LA test employed in the present study has shown 96.3% sensitivity and 97.1% specifi city (29) and ease-of-use in realworld contexts; it also has a high qualitative agreement with the Sabin-Feldman dye test, which represents the gold standard (13) .Moreover, the LA test has been widely used in remote or rural areas in many developing countries (12) (22) (30) .The community studied is located within the heart of Lagos City, the largest city in Nigeria, and its climatic and living conditions favor many parasites.PSC are a highly vulnerable population for T. gondii infection due to their habits of playing in water, soil, with pets (cats and dogs), and eating raw and unwashed foods (22) .This age group also lacks good personal hygiene.The results obtained from this study can be used to assess whether T. gondii threatens the well-being of these children, they can also be used as a reference in determining the need to plan primary prevention actions for pregnant women and people with immunosuppression.Intervention in adjacent areas in Lagos may also be required because many of the foods consumed daily, such as vegetables and fi shes, are transported from this community to various districts in Lagos (27) (31) .
The results from this study showed that 24% of PSC were infected with T. gondii.This seroprevalence was lower than previous prevalence values recorded in Brazil (32) (46.4%),São Tome and Principe (22) (63.1%),Somalia (24) (37.5%), and Madagascar (23) (36.3%), although it was higher than the prevalence values reported in Swaziland (12) (8%) and China (33) (15.13%).Variations in prevalence may also occur between regions of the same country, such as the prevalence values of 17.5% (34) and 46.4% (32) that have both been reported in Brazil.These differences could be attributable to variations in culture and tradition among different ethnicities as well as different food habits (22) (28) (35) .
Previous studies have indicated that the parent's occupation is a factor that infl uences the likelihood that a PSC will be infected with T. gondii (22) (28) (36) .Generally, children whose parents' occupations are categorized as unskilled workers tend to be infected more frequently, which could be due to the parents' lacks of suffi cient knowledge of personal hygiene.However, in this study, there was no signifi cant association between parents' occupation/educational level and infection.An alternative explanation may be that people acquire T. gondii infection through contaminated environments in the community, regardless of their occupation/educational level.In this study, no gender difference was found associated with the seroprevalence of T. gondii, supporting observations made elsewhere (34) (35) (37) .
High LA titers of sera anti-Toxoplasma antibodies are regarded as an indicator of either acute or new infection with T. gondii (38) .In present study, among seropositive PSC, 86% (42/49) of boys and 81% (34/42) of girls showed high LA titers.This result indicates that many of the children in this community were recently infected with T. gondii, which could have occurred through risk factors such as playing in soil, playing with pets (cats and dogs), eating raw and unwashed foods, eating raw/ undercooked meat or drinking of unsafe water (4) .
Substantial studies have indicated that the seroprevalence of T. gondii infection increases with age (10) (39) , and one hypothesis suggests that this outcome is the result of an increasing number of exposure years as the child grows (40) .There was an increasing pattern of infection rates with age in this study as well, but the trend was not statistically signifi cant, which may have been due to the small sample numbers in some age groups making it diffi cult to demonstrate a difference.
Among the risk factors analyzed in this study, playing/ contact with soil showed lower risk (APR=0.66)after the multivariate adjustment to evaluate for associations between multiple environmental risk factors and T. gondii infection.This route of transmission could explain the similar incidence of seropositivity between boys and girls in this community because both genders are equally exposed to contact with soil.This infection route corroborates the fi ndings of previous studies in urban Brazil (41) .
Contact with domestic cats is one of the generally accepted risk factors for infection with toxoplasmosis (1) , although no association was observed in this study, in agreement with previous studies (3) (22) (28) (38) .The way a cat's litter box is cleaned seems to account for exposure to the parasite rather than the mere presence of cats in the household; in our study, most cat owners used gloves while cleaning the litter box.The lack of an association between cat ownership and infection could also suggest that environmental contamination by cats is so widespread that cat ownership has minimal effects on the level of exposure to T. gondii (39) .
Drinking contaminated water is also a potential source of T. gondii infection (36) .A study in Nigeria showed that the seroprevalence rate was higher in pregnant women who drank well water compared with women using packaged water (42) .However, in our study, 49.1% of the children reported the use of unsafe water, and no association between the source of drinking water and Toxoplasma infection was found.Nevertheless, waterborne outbreaks of toxoplasmosis have been reported in Brazil (43) .The consumption of raw meat also did not seem to be a signifi cant risk factor in this community because meat is traditionally well cooked.However, eating raw and unwashed vegetables can serve as a route of infection, although it was not statistically signifi cant in this study.
Substantial evidence has established an association between T. gondii infection and headaches (44) ; however, in our study, no association was observed.Ocular disease has also been reported to be linked with T. gondii infection, particularly in the U.S. (45) .In our study, visual discomfort showed no association with infection.This lack of an association might be due to an insuffi cient sample size; hence, there is a need for further large-scale investigations to answer this question.Of note, our study showed an association between T. gondii infection and general body weakness, although the association was not statistically signifi cant.This weakness could also be due to the other pathogenic infections that are prevalent in Nigeria (46) ; therefore, more investigation is required in this area.Given the moderate seroprevalence recorded in present study, there is a need for preventive measures against T. gondii in Lagos, Nigeria.Although infection is generally asymptomatic in affected children, in some regions, more aggressive disease strains lead to eye damage.The prevalence recorded in this study is an indication of the potential risks that pregnant women and immunocompromised patients are exposed to.Because general environmental contamination control is very diffi cult to achieve, strategies should be focused on these two sub-populations that are at higher risk of severe disease.Preventing contamination by cat feces, particularly in children's playgrounds, and good personal hygiene, such as regular hand washing, are essential steps in the control of T. gondii infection.Our results may also be of use to policy makers for the initiation of programs that can lead to the prevention and control of toxoplasmosis in Lagos specifi cally and in Nigeria as a whole.

TABLE 1 -Demographic characteristics and infection rates of Toxoplasma gondii among primary schoolchildren in Lagos City, Southern Nigeria.
teachers of each of the selected schools and from parents or guardians of the children.Meetings were held to explain to the teachers and pupils the objectives and protocol of the study, emphasizing that participation was voluntary and that withdrawal was allowed without any reason required.Signed or thumb-printed consent was obtained from parents/guardians on behalf of their children/wards before the commencement of sample collection.The guidelines laid down for procedures involving human subjects in the Declaration of Helsinki were strictly adhered to, and approval for this study was granted by the Institutional Review Board (IRB) of the Nigerian Institute of Medical Research (NIMR) [Project No.: IRB/13/225].

TABLE 3 -Log-binomial regression model analysis of Toxoplasma gondii infection and associated risk factors among primary children in Lagos City, Southern Nigeria.
Gyang VP et al. -Toxoplasma in Nigerian schoolchildren