Associated Antenatal Health Risk Factors with Incidence of Toxoplasmosis in Egyptian Pregnant Women

MATERIALS AND METHODS

Study design and area: This cross-section study was performed between November, 2016 and June, 2017. The study participants were those all pregnant women who attended antenatal services at Antenatal Health Units in Giza Governorate, Egypt during the sample collection period. Pregnant women who were critically ill, unable to communicate and those who were not willing to provide vital information and blood samples were excluded from the study.

Sample size determination and sampling technique: The statistical formula for sample size calculation was considered, the average sero-prevalence of T. gondii was considered as 45% from literature and so the sample size was calculated as 380 and increased it to 388. A total of 388 blood specimens from pregnant women were collected as follows: 158 women in the first trimester of pregnancy with history of normal delivery and abortion were 111 and 47, respectively, 121 in the second and 109 in the third trimester. The age of the study participants ranged from 18-44 years.

Data collection: Information on socio-demographic data, history suggestive of congenital toxoplasmosis (abortion, a child with congenital anomaly) and history of exposure for the possible associated factors were recorded from all women enrolled in the study using a structured questionnaire. A full verbal explanation about the study was given by the investigators to all voluntary participants.

Laboratory investigations
ELISA test: The IgM and IgG antibodies against Toxoplasma gondii in the 388 collected serum samples were determined. The IgM antibodies were quantitatively determined by commercially available kits (SERION ELISA classic Toxoplasma gondii IgM, Order Nr.:ESR110M) according to the manufacturer's instructions. While IgG was quantitatively determined by the conventional ELISA at a concentration of 20 μL mL^–1 coating buffer, pH 9.6, the whole soluble tachyzoites antigen was prepared⁸ and the test procedure was done according to the methods described by Shaapan et al.⁹. The absorbance of the controls and samples was determined at 450 nm. Positive and negative control sera were included in each run and the cut off value was calculated as the mean OD of the positive controls+3 standard deviations while samples were considered positive if OD value is above cut off value¹⁰.

Avidity ELISA: The avidity of T. gondii-specific IgG antibodies was determined as previously described by Hedman et al.¹¹, with some modifications, the 96 well microtiter plates (Nunc, Denmark) were coated with whole soluble tachyzoites antigen (5 μg mL^–1) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. Plates were washed for three times with PBST (PBS, 0.05% tween20) and then sera diluted in PBST (1:200) were added in duplicate rows (row A and row B). After incubation for 1 h at 37°C, row B was washed three times with PBST and row A was washed three times with PBST containing 6 M urea and a fourth time with PBST. Then anti-human IgG conjugated with horseradish peroxidase (Dako, Denmark) at the dilution of 1/1000 in PBST was added and incubated for 1 h, followed by addition of the ortho-phenylene-diamine (OPD), (Merck, Germany) substrate. The reaction was stopped and the absorbance (Abs) was read by an automated ELISA reader (BIOTEC, LX800, USA) at 492 nm. Avidity index (AI; %) was calculated as the result of Abs of wells washed with PBST containing urea (U+), divided by the Abs of wells washed with PBST (U−) and multiplied with 100, based on the equation:

Data analysis and interpretation: Using Statistical Package for the Social Sciences (SPSS version 20.0) the groups were characterized according to the target variables using descriptive statistical analysis methods. Prevalence of toxoplasmosis was defined as the percentage of positive cases for serological tests. Associations between toxoplasmosis and possible risk factors were tested with Chi-square test.

Ethical considerations: The study was ethically cleared by ethical review board of National Research Centre (No. 0226 at May, 2017). The study subjects were informed about the study and written informed consents were obtained from all of the participants before collecting blood samples. Participation in the study was on voluntary basis and study subjects were free to withdraw from the study before and after collection of blood samples without losing any of the benefits they were supposed to obtain from the hospital.

RESULTS

Socio-demographic characteristics: The average age of the woman who took part in this investigation was 27±3.4. Majority of the study participants, 270(69.6%) were rural residents. Regarding their educational status, 119 (30.7%) of the respondents had a secondary school certificate. Occupation wise, the majority of the 262 (67.5%) was housewives (Table 1).

History of exposures to different T. gondii infection risk factors: Comparable results of sero-positivity were obtained among 2nd and 3rd trimesters with prevalence of 33.1 and 35.8%, respectively, 24.0% positive cases were from multigravida category, 42.5% of women with history of abortion were positive for Toxoplasma, 35.4% of pregnant women who had cats in their households and 27.4% in close contact with livestock were found to be positive for T. gondii. About 71.6% of those who took immunosuppressive drugs, 34.5% of women using river or well water as a source of drinking water, 24.4% had a habit of eating raw or undercooked vegetables and a blood transfusion history cases were 30.8% positive for infection by T. gondii (Table 2).

Total prevalence of T. gondii: The total sero-prevalence of T. gondii through the investigated pregnant women was 20.4% (79/388). The acute infection group I revealed presence of both toxoplasma IgM and IgG antibodies and had a low IgG avidity index (AI<50) included 28 women (7.2%) while the chronic infection group III showed negative IgM and positive IgG with high IgG avidity index (AI>50) included 36 women (9.3%) (Table 3).

Table 1:	Seroprevalence of T. gondii infection in relation to socio-demographic characteristics among examined pregnant women (N = 388)

Table 2:	Seroprevalence of T. gondii infection in relation to exposure to different risk factors of T. gondii infection among examined pregnant women (N = 388)

Table 3:	Detection of specific T. gondii serological markers in sera of the investigated pregnant and aborted women
P: Pregnant women in the 2nd and 3rd trimester, A: Pregnant women in the 1st trimester with history of abortion, AV: IgG Avidity

DISCUSSION

The chance of acquiring acute infection with T. gondii is high during pregnancy and the infection would have potential tragic outcomes for the mother, the fetus and newborn despite the fact that it can be prevented¹². This investigated demonstrated that overall prevalence of toxoplasmosis in examined pregnant women was 20.4%. Nearly the same prevalence (21.3%) was reported in Almadinah Almunawwarah, KSA¹³ and among Portuguese population (22%)¹⁴. Higher prevalence was reported by several researchers; 83.6% in Jimma town, Southwestern Ethiopia¹⁵, 43.6% in Khartoum State, Sudan¹⁶ and 38.8% in the south western region of Saudi Arabia¹⁷. On the other hand, lower seroprevalence was recorded by several investigators; 18.1% in South Africa¹⁸ and 18.5% at FelegeHiwot Referral Hospital, Bahir Dar town, northwest Ethiopia¹⁹.

In the concurrent work results showed that 35.4% of positive Toxoplasma sera had anti-toxoplasma IgM and IgG and low IgG avidity index (AI>50) (Acute infection group I) and 45.6% had negative anti-toxoplasma IgM and positive IgG and high IgG avidity (AI>50) (Chronic infection group III). Some parallel studies; reported that 80% sera with acute toxoplasmosis showed low avidity levels and 96% sera in chronic phase of infection showed high avidity index in Qom Province, Iran²⁰, in Saudi Arabia it was reported that 6 (18.8%) of 32 pregnant women with T. gondii IgM seropositive samples had high IgG avidity and 26 (81.3%) had low IgG avidity indicating acute toxoplasmosis²¹.

The findings of the present study showed significant association between T. gondii positive cases and age of the study participants above 30 years. Results that have been reported from Jimma town, Ethiopia and Brazil have shown statistically significant association between age of pregnant women and seroprevalence of T. gondii infection^15,22.

Current results showed that 25.9 and 7.6% of positive toxoplasma cases were from rural and urban settings, respectively with significant difference. Residence was found to be significant in rural areas (p = 0.001)¹⁶. In relation to histories of abortion, there was significant association between occurrences of previous abortions (42.5%) and seroprevalence for toxoplasmosis in the investigated positive cases (17.3%). These results agree with those from Sudan and India^16,23.

Concerning risk factors; in our study, 71.6% of the positive Toxoplasma cases taking immunosuppressive drugs were positive for Toxoplasma. Similar results were obtained from Saudi Arabia²⁴ and Sri Lankan²⁵ (non-immune women).

In the present study animal rearing accounted for 27.4% of pregnant women who were in close contact with animals and there is a considerable relation between T. gondii infection and presence of domestic cats at home (35.4%). This finding is in agreement with studies reported from Ethiopia and Taiwan^15,26. This differences and variations among reported studies could be the hazard of contracting T. gondii infection might not just be the presence of cats in the households, but it could be a contact of cats' fecal material while gardening. The absence of a statistically significant relationship between the sero-prevalence of Toxoplasma infection and some potential factors (blood transfusion, consumption of raw/undercooked vegetable and drinking water source) does not mean that they have no influence on the transmission of toxoplasmosis²⁷. However, it may suggested that such factors play a limited role in the study area for the transmission of the parasite in the studied subjects.

CONCLUSION

It can be concluded that the sero-prevalence of T. gondii infection among the pregnant women in Giza governorate was relatively high. This finding revealed that exposure to T. gondii infection may increase the risk of premature termination of pregnancy. In this study, several risk factors that could lead to an increase in the prospect of T. gondii infection in the pregnant women we redetected, including age, rural residence, an increase in gravidity and gestational age, intake of immunosuppressive drugs, history of previous abortion and contact with cat and farm animals. Therefore, implementation of regular serological testing during pregnancy, health education towards avoiding contact with cats fecal material during cleaning and gardening should be emphasized by health extension workers and other public health professionals for prevention of the disease.

SIGNIFICANCE STATEMENT

This study introduce good diagnostic efficacy for detection of toxoplasmosis in pregnant women with differentiation between the recent acute and chronic infected cases using the IgG avidity test is beneficial in differentiating recent from old Toxoplasma infection. So, the authors suggest screening of T. gondii infection for all pregnant women by detection IgG, IgM antibodies and additionally advice apply IgG avidity test (AV) to avoid risk of premature termination of pregnancy and also to look any risk factors related to the infection.

ACKNOWLEDGMENT

The authors are grateful to Department of Zoonotic Diseases, National Research Centre, for providing Toxoplasma antigen through successive mice-mice tachyzoite passage to be used in ELISA technique.