TOXOPLASMOSIS IN PREGNANT WOMEN IN YEMEN: THE IMMUNE STATUS AND POTENTIAL RISK FACTORS

Background and aims: Toxoplasmosis is caused as a result of intracellular protozoan organism, Toxoplasma gondii, and is a widespread disease. Toxoplasmosis can progress to a serious systemic disease (congenital form); once a mother becomes infected for the first time for the period of pregnancy, a transient haematopoietic parasite can appear with focal lesions produced wi thin the placenta, thus infecting the fetus. Therefore, this cross-sectional study aimed at assessing the immune status of the pregnant woman towards toxoplasmosis, determining the prevalence of T. gondii in Amran city -Yemen, and identifying the potential risk factors that affect the pregnant woman towards toxoplasmosis. Subjects and methods: This study included 280 pregnant women who were admitted to maternity clinics at Amran General Hospital and Family Health Center in Amran City -Yemen during the period from December 2016 to June 2017. The questionnaire was used to obtain relevant demographic data and potential risk factors. Then, blood samples were collected from pregnant women and tested for identification of IgM and IgG antibodies against T. gondii using the ELISA technique. Results: The total positive rate for IgM antibodies was 3.6%, while for IgG was 27.9%. A high IgM rate equal to 5.3% was observed in the 31-35 age group, and a higher IgG rate (36.8%) was observed in the same age group. There were 69.3% of pregnant women susceptible to infection with toxoplasmosis, 2.9% recently infected, 27.1% protected, and 0.7% had secondary infection. The high rate of IgM positivity was observed in the second trimester of pregnancy (4.7%), while the lowest rate was in the third trimester of pregnancy (2%). A slight increase in the rate of IgG positivity was observed in the third trimester of pregnancy (32%). There was a significant association between contact with animals such as cats and infection with toxoplasmosis, where OR=5.4, CI=1.01-30.13, χ2=4.59, p=0.04. While there was no significant association with the other studied factors. Conclusion: Yemen has less seropositivity than other Arab and African countries as a result of variation in risk factors. Data on T. gondii infection during pregnancy is scarce in numerous countries, in particular where there is a lack of political constancy such as Yemen. The risk factors identified included proximity to the cats and domistic animals, and increased maternal age. Toxoplasmosis in pregnancy in Yemen may be a really underestimated health problem. More research is needed.


INTRODUCTION
Toxoplasma gondii (T. gondii), a widespread singlecelled parasite that is an intracellular parasite, is one of the most successful eukaryotic pathogens infecting warm-blooded vertebrates comprise humans, and is capable of causing toxoplasmosis 1,2 . The distribution and its capability to preserve a benign symbiosis with its host is a trait that allows it to be widely seen as one of the most successful parasites on Earth 2 . It is estimated that equal to a third of the world's population is carriers of toxoplasma infection, and the life cycle of this parasite participates a large role in its simplicity of transmission 3, 4 . Toxoplasmosis is found in every country and rates of seropositivity range from 10% to 90% 5, 6 . Globally, more than 6 billion people are infected with T. gondii 7 . Humans are generally infected by oral intake of food, water, or ingestion soil polluted with oocycts, or by ingestion of raw vegetables and undercooked meat contaminated with cysts of T. gondii 8 . Though T. gondii infection is very common, the clinical implication in the immunocompetent host is benign and asymptomatic and only in about 10% of cases, cervical or occipital lymphadenopathy and ocular disease may occur 9 . The significance is mainly attributable to primary infection during pregnancy that may result in congenital Toxoplasmosis, abortions, perinatal deaths and still births 5 . This comprises a wide range of symptoms, expanding from mild chorioretinitis, which can appear many years after birth, to mental retardation, miscarriage, hydrocephalus, microcephaly and seizures 10, 11 . Moreover, reactivation of latent infection in immunocompromised conditions may occur resulting in T. gondii encephalitis or disseminated infections 9,12 . Its infection induces several immunological changes in the body which are characterized by the production of the immunoglobulins IgM, IgG and IgA 13 . Detection of toxoplasmosis is at present based on serological techniques including examination of IgM and IgG antibodies, the former indicating recent infection while the latter indicating previous exposure with protective immunity 2,14 . The overall incidence rate of Toxoplasmosis in Arab countries is approximately 30% to 50%, making it among the global regions with the highest prevalence. The broad distribution of the infection is likely due to its complex transmission patterns and co-evolution of the parasite with multiple hosts 15-17 . In Yemen, data on infection among females of childbearing age or during pregnancy are limited. Moreover, few studies have been conducted recently on vulvovaginal candidiasis during pregnancy, vaccination against tetanus among pregnant women, its prevalence, potential risk factors, awareness of congenital CMV, rubella virus infection in pregnant women and markers of hepatitis B virus serum among pregnant women 18-23 . In Yemen, pregnant women are not routinely screened for T. gondii during pregnancy, and there is no follow-up. Thus, the current study was carried out to reveal the state of sero-epidemics and potential risk factors for T. gondii in pregnant women in Amran area in Yemen.

SUBJECTS AND LABORATORY METHODS
A cross-sectional study was completed during the interval from December 2016 to June 2017. Two hundred and eighty pregnant women, who were attended to the obstetrical clinics in General Amran Hospital, and Family health Centers in Amran city, Yemen, were included in this study. Inclusion criteria: Inclusion criteria included pregnant women attended to the obstetrical clinics in General Amran Hospital, and Family health Centers in Amran city. Exclusion criteria: Exclusion criteria included nonpregnant women, pregnant women with active diseases such as tuberculosis, etc., receiving treatment with immunosuppressive drugs, or suffering from immunodeficiency diseases. Data collection: A full history was taken from each studied pregnant woman, and the findings were written down in predesigned questionnaire. The collected data included: Demographic characteristics of participant, and information about the risk factors. Sample Collection: Four to five ml of venous blood samples were obtained from each pregnant into a plain tube. Then sera were separated from clotted blood and kept in freezer at (-20 o C) until tested. The samples were tested for the presence of anti-toxoplasma (IgM and IgG) antibodies by using an Enzyme Linked Immunosorbent Assay (ELISA) commercial available (Ratio Diagnostics, RD, Germany).

Ethical considerations
During data collection, pregnant women who gave their written consent for screening of their blood to detect T. gondii antibodies were selected. Brief explanation of the objective and the significance of the study were given to each participant in order to obtain verbal consent and to have the signature to avoid misunderstanding. The study proposal was evaluated and approved by the Ethics Committee of Faculty of Medicine and Health Sciences, Sana'a University with a reference number (102) dated 10-04-2016. Also, all data, including patient identification, have been kept confidential.

Statistical analysis
Data were recorded using appropriate descriptive statistics (including frequency, mean, and standard deviation). The odds ratio (OR) was used to determine the strength of the association between two events, such as positive IgG with age, gestation stages, and residence. The association between infection with toxoplasmosis with risk factors as contact with animals etc. The two events in the current study were independent if and only if OR was equal to 1. For sample constraints of odds ratio in small numbers (less than 5), Fisher's exact test was used as an alternative estimator for the association between events in the current study.

RESULTS
The study results illustrated on 5 tables:  Table 3 shows the association of Toxoplasma IgM antibodies with stages of pregnancy. The high rate of IgM positivity was observed in the second trimester of pregnancy (4.7%), while the lowest rate was in the third trimester of pregnancy (2%). Table 4 shows the association of Toxoplasma IgG antibodies with stages of pregnancy.  A slight increase in the rate of IgG positivity was observed in the third trimester of pregnancy (32%). Table 5 shows the association of toxoplasma IgG antibodies with potential risk factors. There was a significant association between contact with animals such as cats and infection with toxoplasmosis among pregnant women, where OR=5.4, CI=1.01-30.13, χ2 =4.59, p=0.04. While there was no significant association with the other studied factors.

DISCUSSION
This study showed an overall 27.9% sero-prevalence of anti-T. gondii IgG antibody among pregnant women in Amran City (Table 1).This result is within the range of seroprevalence of anti-T. gondii IgG antibody among pregnant women reported in previous studies in which they have shown a seropositivity rate in Arab region ranging from 22.9 to 58.2% 24-27 .This results was lower than that reported from Saudi Arabia, which it range from 38-61% 27-29 . While 27.9% seroprevalence of anti-T. gondii antibody was slightly higher than that reported from UAE (22.9%) 24 .
When age was considered as a factor effects the prevalence of anti-T. gondii IgG antibody among pregnant women there was slight evaluating in the prevalence of anti-T. gondii IgG antibody with increasing age of pregnant women, e.g. rate in age group 20-24 years was 23.8%, this rate rise to 36.8% in age group 30-34 years (Table 1). This is similar to the results in the North Africa region, where the seroprevalence of T. gondii is higher in older age groups compared to younger age groups 30 31 and Zemene et al., 33 findings showing that contact with animals, especially cats, is significantly associated with toxoplasmosis seropositivity. There was no association between eating uncooked or raw meat (OR rate 1.08, 95% CI=0. 13-8.99; p=0.939) and T. gondii seropositivity in the current study. This finding is contradicted by Walle et al.,study 34 in Africa where there is a strong association between eating undercooked or raw meat (OR=5.73, 95% CI=1. 35-24.39; p=0.02) and toxoplasmosis 34-37 . These results can be explained by the fact that daily consumption of raw meat is very common in many parts of Africa 38-40 . There was no significant association between residence in rural areas (OR=1.05, 95% CI=0.41 -1.6; p=0.47) and T. gondii-positive antibody (Table 5).