Seroprevalence and Risk Factors of Toxoplasma gondii Infection in Pregnant Women from Western Romania

Abstract

Seroprevalence and risk factors of Toxoplasma gondii infection were assessed among pregnant women in Western Romania. T. gondii seroprevalence was evaluated in 208 pregnant women by demonstration of serum antibodies using the commercial Vitros anti-Toxoplasma immunoglobulin G (IgG) and IgM assays. A questionnaire was administered to obtain information regarding the risk factors associated with T. gondii seropositivity. Chi-squared tests, Fisher exact test, and Stata 9.2 (Statacorp, Texas) were used to evaluate differences between T. gondii positive and negative women. T. gondii antibodies were demonstrated in 116 (55.8%) of 208 pregnant women. Lower level of education and working with meat were found to be risk factors for T. gondii seropositivity. Pet owners (cats and/or dogs) had a higher T. gondii seroprevalence than those who did not report having any pet (p = 0.032). Women with ≥4 live births were more frequently T. gondii seropositive than those without previous births (p < 0.002). Women with histories of spontaneous abortions were more frequently T. gondii seropositive than those without such a history (p = 0.036). Our results indicate a high prevalence of T. gondii antibodies in pregnant women in Romania. Risk factors for T. gondii past infection were being in the older age group, working with meat, having pets, a lower level of education, higher gravidity, and history of spontaneous abortions. This survey provided the first data regarding risk factors for T. gondii infection in pregnant women from Western Romania.

Introduction

T oxoplasma gondii is a protozoan parasite that has the capacity to cross the placenta and infect the fetus especially if pregnant women are primarily infected during pregnancy (Montoya and Remington 2008). In congenitally infected infants, the parasite can have devastating effects, including ocular and central nervous system disease (Olariu et al. 2019). Owing to the severe complications T. gondii may cause in infected infants, it is important to survey its prevalence (Pappas et al. 2009).

T. gondii is mainly acquired through ingestion of tissue cysts in undercooked meat of infected animals or oocysts, excreted by infected cats, that contaminate food, soil, and water (Montoya and Remington 2008, Petersen et al. 2010). T. gondii prevalence varies between countries or between different groups in the same area (Pappas et al. 2009, Robert-Gangneux and Dardé 2012). In pregnant women, T. gondii seroprevalence varies from 4.5% in Vietnam, Asia (Smit et al. 2019), to 92.5% in Ghana, Africa (Ayi et al. 2009). In Europe, the prevalence of T. gondii antibodies in pregnant women varies between 9% in the United Kingdom and Norway (Nash et al. 2005, Findal et al. 2015) to 60.4% in Turkey (Harma et al. 2004).

In Romania, there is still limited information regarding T. gondii seroprevalence in pregnant women (Dubey et al. 2014). Moreover, no studies regarding risk factors associated with T. gondii infection in Romanian pregnant women has been published in the international literature. We have previously reported that in Western Romania, the prevalence of T. gondii antibodies in women of reproductive age and in the adult population was 57.6% and 64.8%, respectively (Olariu et al. 2008, 2015).

We, therefore, undertook a study to evaluate the seroprevalence and risk factors of T. gondii in pregnant women from Western Romania.

Materials and Methods

Serum samples were tested for T. gondii antibodies using the Vitros anti-Toxoplasma immunoglobulin G (IgG) and IgM assays, designed for the Vitros ECiQ immunodiagnostic system (Ortho-Clinical Diagnostics, NJ). Interpretation of Vitros results was based on the manufacturer's criteria, as follows: for T. gondii IgG antibodies: <4.00 IU/mL, negative; ≥4.00–7.99 IU/mL, borderline; and ≥8.00 IU/mL, positive. For T. gondii IgM antibodies, the ratio was classified as follows: <0.80 IU/mL, negative; ≥0.80–1.19 IU/mL, borderline; and ≥1.20 IU/mL, positive (Kasper et al. 2009). The Vitros test kits including positive and negative controls were used according to the manufacturer's protocol. For the purposes of this study, borderline results for T. gondii IgM and IgG were reported as negative. The Vitros IgG assay parameter has a sensitivity of 95.0% and a specificity of 100.0% compared with the Sabin Feldman dye test. The Vitros IgM assay parameter has a sensitivity of 92% and specificity of 82% compared with the immunoglobulin-M immunosorbent agglutination assay (IgM ISAGA) test. The combination of the Vitros IgG and IgM assays demonstrated a sensitivity and a specificity of 100% for the successful detection of all acute T. gondii infections by comparison with the Sabin Feldman dye test as the reference test (Kasper et al. 2009).

We investigated sera of 208 consecutive pregnant women attending the Gynecology and Obstetrics unit of the County Clinical Emergency Hospital in Timisoara, Romania, and in whom laboratory tests have been performed at the Clinical Laboratory of the same University Hospital. Women were enrolled regardless of their clinical status/pregnancy stage and history. Serum samples were collected, from February 2013 to June 2013, from these women residing in four counties located in Western Romania (Timis County, Caras-Severin County, Hunedoara County and Arad County), with a total population of 1,763,142. Serum samples were obtained by venipuncture and were kept at −20°C until use.

A questionnaire interview for pregnant women was carried out to obtain information regarding the risk factors associated with T. gondii infection, including age, residential area, history of eating unwashed fruits/vegetables, gardening or contact with soil, drinking untreated water, and unpasteurized milk, washing hands before handling meat and keeping pets, including cats and/or dogs. Women were also questioned regarding their exposure to raw meat, whether they had a history of preparation of foods with raw meat and had eaten raw or undercooked meat. Additional data were acquired regarding occupation, educational level (elementary school, gymnasium, high school, and college/University), the number of previous live births, and spontaneous abortions.

The survey questionnaire was created with input from physicians, veterinarians, and clinical laboratory specialists at the Municipal and County Clinical Emergency Hospitals in Timisoara, Romania.

Data were compiled in a Microsoft Excel database, version 2011 (Microsoft Corp., Redmond, WA). Means, standard deviations, and proportions are presented. Chi-squared tests or Fisher exact test, as appropriate, was used to evaluate differences between T. gondii positive and T. gondii negative women with respect to different characteristics. Crude odds ratios (ORs) were calculated for all variables to identify risk factors associated with T. gondii antibodies. All analyses were conducted with Stata 9.2 (Statacorp, Texas). A probability level of p < 0.05 was considered to indicate statistical significance.

This study was approved by the Victor Babes University Ethics Committee and informed consent was obtained from all women. For participants aged <18 years, the informed consent was provided by their parents or legal guardian. Participation in the study was voluntary and individuals were informed about the purpose of this study.

Results

Among 208 pregnant women, aged 12–41 years (mean = 27.1 years), T. gondii IgG or IgM antibodies were demonstrated in 116 (55.8%) women and their presence tended to increase with age: 51.8% (14/27) in those aged 12–20 years, 52.8% (66/125) in those aged 21–30 years, and 64.3% (36/56) in those aged 31–41 years. Both, T. gondii IgG and IgM antibodies were demonstrated in 2 women, and IgG antibodies alone in 114 women. The prevalence of IgG antibodies was slightly higher in women from rural areas (57.3%, 51/89) than in those from urban regions (54.6%, 65/119).

Risk factors for seropositivity were analyzed in 190 of 208 women who accepted to participate in the interview. No significant difference in seroprevalence was found between subjects with and without exposure to the risk factors studied.

However, T. gondii seroprevalence tended to decrease with increasing level of education from 100% in those who graduated primary/elementary classes only to 57.1% in those with gymnasium, 47.1% in those with high school, and 52.5% in those with University/College (Table 1).

Table 1.

Risk Factors for Toxoplasmosis in Pregnant Women Ascertained by Questionnaire

Potential exposure to risk factor	Toxoplasma gondii seropositive women		T. gondii seronegative women		Odds ratio (95% CI)	p
Potential exposure to risk factor	Exposed	Not exposed	Exposed	Not exposed	Odds ratio (95% CI)	p
Education level
College/university	31	—	28	—	1 (Ref.)	—
High school	33	—	37	—	0.80 (0.40–1.61)	0.543
Gymnasium	28	—	21	—	1.20 (0.56–2.59)	0.634
Primary education	10	—	0	—	NA	0.005
Living in house vs. apartment	70	32	49	37	1.65 (0.90–3.02)	0.099
Eating raw and/or undercooked meat vs. meat well done	45	59	37	48	0.97 (0.55–1.77)	0.971
Working with meat/occupation—housewives/working in food departments vs. all other professions	32	63	13	54	2.11 (1.01–4.38)	0.046
Own cat(s)	26	67	21	57	1.05 (0.53–2.07)	0.880
Own dog(s)	64	34	44	40	1.71 (0.93–3.13)	0.077
Own pets: cat(s) and/or dog(s)	67	29	45	38	1.95 (1.05–3.59)	0.032
Contact with soil	56	48	46	36	0.76 (0.51–1.64)	0.760
Unwashed hands before meal	47	57	42	43	0.84 (0.47–1.50)	0.564
Eating unwashed vegetables and fruits	31	71	32	51	0.69 (0.38–1.29)	0.245
Drink untreated water (well and river/lake)	36	68	34	50	0.78 (0.43–1.41)	0.409
Drink only bottled water vs. other types	39	65	38	46	0.72 (0.40–1.30)	0.284
Drink unpasteurized milk (goat and sheep)	4	95	8	65	0.34 (0.09–1.20)	0.079
Spontaneous abortions	24	73	10	71	2.33 (1.05–5.15)	0.036
No. of live births
0	—	8	—	8	1 (Ref.)	—
1	31	—	37	—	0.84 (0.29–2.42)	0.751
2	23	—	19	—	1.21 (0.39–3.74)	0.747
3	9	—	13	—	0.69 (0.19–2.47)	0.582
≥4	26	—	4	—	6.50 (1.61–26.10)	0.007

CI, confidence interval; NA, not applicable; Ref., reference.

No significant difference in the seroprevalence of T. gondii infection was found between the pregnant women with and without exposure to raw or undercooked meat. However, working with meat appeared to be a risk factor: housewives and those working in restaurants or food stores had a higher T. gondii seroprevalence (71.1%; 32/45) than those having different jobs (53.8%; 63/117; p = 0.046) (Table 1).

Drinking untreated water and unpasteurized milk was not associated with T. gondii infection in pregnant women (p = 0.409 and p = 0.079, respectively). No difference regarding T. gondii seroprevalence was observed between cat owners and those who did not report having cats. Interestingly, 64 (58.2%) of 108 dog owners were T. gondii infected versus 34 (45.9%) of 74 who did not report having dogs (p = 0.077). Those with two dogs were 3.13 times more likely to be positive (OR = 3.13, 95% confidence interval: 1.24–7.91) than those without dogs (p = 0.010) (data not shown). Among those reporting to have any pet (cat and/or dog), 59.8% were seropositive compared with 43.3% of those who did not report having any pet (p = 0.032) (Table 1).

Women with ≥4 live births were more frequently seropositive than those without such a history (p = 0.007). Women with histories of spontaneous abortions were significantly more frequently seropositive with T. gondii than those without such a history. Of women with demonstrated T. gondii antibodies, 24.7% (24/97) had reportedly had spontaneous abortions compared with 12.3% (10/81) of those in whom specific antibodies were not detected (p = 0.036) (Table 1).

Discussion

Our results revealed that 55.8% of pregnant women residing in Western Romania had T. gondii IgM and/or IgG antibodies. This seroprevalence is significantly higher than those reported in pregnant women by other European investigators: 9.3% in Norway (Findal et al. 2015), 17.9% in Italy (Puccio et al. 2014), 21.9% in Portugal (Lobo et al. 2017), and 26.7% in Spain (Ramos et al. 2011). However, our findings were only slightly higher than the seroprevalence of 48.6% reported in Albania (Maggi et al. 2009) and slightly lower than the seroprevalence of 58% reported in Hungary (Szénási et al. 2005) or seroprevalence of 60.4% reported in Turkey (Harma et al. 2004).

Significant differences in the seroprevalence were found between countries and between areas or groups within the same region (Pappas et al. 2009, Peyron et al. 2016). Local rates of T. gondii seropositivity may be explained by differences in environmental exposure and life style habits (Peyron et al. 2016).

In this study, the presence of T. gondii antibodies appeared to increase with age, possibility due to longer exposure to the risk factors related to T. gondii infection (Peyron et al. 2016).

T. gondii seroprevalence decreased in our study with increasing level of education from 100% in those who graduated primary/elementary classes only, to 57.1% in those with gymnasium, 47.1% in those with high school, and 52.5% in those with University/College. It has previously been demonstrated that the educational level is an important risk factor for the occurrence of T. gondii infection, and women with low educational status had higher prevalence of T. gondii antibodies (Agmas et al. 2015). The low level of education was often linked to low socioeconomic status and has been associated with low awareness of toxoplasmosis and an increased risk for exposure (Peyron et al. 2016). In general, people with a higher educational level have more knowledge about this infection and the methods of its prevention.

In this study, T. gondii seroprevalence was not found to be associated with consumption of raw or undercooked meat. However, working with meat appeared to be a risk factor. Housewives and those working in restaurants or food stores had a higher T. gondii seroprevalence (71.1%) and tended to be more exposed to infection than those with different jobs (53.8%; p = 0.046). Working with meat was previously shown to be associated with T. gondii. In a U.S. study, Jones et al. (2009) suggested that persons working with meat could inadvertently ingest undercooked meat or taste dishes before the meat was fully cooked or could inadvertently ingest undercooked meat from their hands. In a survey of pregnant women, the authors found that many women were not aware of the risk of toxoplasmosis associated with undercooked meat (Jones et al. 2009).

Contact with cats did not appear to significantly increase the risk for T. gondii in our study group. Interesting, dog owners tended to be more frequently seropositive than those who did not report having dogs. Moreover, the risk associated with exposure to dogs was higher in respondents who had two or more dogs. Frenkel et al. (2003) have shown that dogs may act as possible mechanical carriers of T. gondii oocysts and their fur as a source of infection of young children. In addition, women keeping any pet, cats, and/or dogs were more frequently seropositive than those without any pet (p = 0.032). This may explain the high T. gondii seroprevalence in our younger age group (12–20 years), and suggest that in this area, the infection probably occurred during childhood and due to high exposure to environmental condition. Previous studies demonstrated that environmental exposure is an important risk factor for T. gondii infection. Contact with soil (Cook et al. 2000, Egorov et al. 2018) or contaminated water (Bowie et al. 1997) were found important risk factors for human toxoplasmosis. Because only infected cats can shed oocysts that could contaminate the environment, the prevalence of T. gondii infection in cats may play an important role in the spread of T. gondii infection. In Central and North-Western Romania, 47% of the cats tested positive for T. gondii antibodies and the infection was found common in household cats in Romania and especially in those with outdoor access (Györke et al. 2011). We have also reported a seroprevalence of 70% for T. gondii in cats in Western Romania, which may contribute to the high seroprevalence of T. gondii among pregnant women in our study (Darabus et al. 2011).

In this study, women with a higher number of live births (>4) were more frequently seropositive than those with <4 live births (p = 0.007). Similarly, higher gravidity was previously found as an independent risk factor for congenital toxoplasmosis (Jones et al. 2009, Peyron et al. 2016) and this may be correlated with higher age. A statistical association with toxoplasmosis was found by previous authors when women indicated a previous pregnancy with an approximately two times higher risk for previously pregnant compared with nulliparous women (Avelino et al. 2004). This greater vulnerability of pregnant women to the parasite was interpreted due to alterations in the immune mechanisms inherent to gestation, resulting from suppression of immune response because of the necessity of tolerance to the fetus and/or as a consequence of hormone imbalances characteristic of the gestational condition (Avelino et al. 2004).

Our results revealed that women with a history of spontaneous abortion were more frequently seropositive than those without such a history (p = 0.036). Previous investigators (Amin et al. 2012, Wam et al. 2016) reported that a considerable proportion of spontaneous abortions can be attributed to T. gondii (Amin et al. 2012) and Wam et al. (2016) showed that T. gondii exposure may constitute a significant risk factor for stillbirths, abortions, or congenital defects during pregnancy.

Conclusions

This study provided new epidemiological data regarding toxoplasmosis in pregnant women from Romania. Our results showed a high prevalence of T. gondii among pregnant women in Western Romania. Age, educational status, working with meat, and presence of pets at home were identified as possible associated risk factors of T. gondii infection. Pet owners, particularly pregnant women, should take necessary preventive measures such as proper disposal of cat feces and keep hygiene to avoid T. gondii infection.

Footnotes

Disclaimer

Parts of this study were presented at the 92nd Annual Meeting of the American Society of Parasitologists, San Antonio, TX, June 27 to July 1, 2017, and at XXI FIGO World Congress of Gynecology and Obstetrics, Vancouver, Canada, October 4–9, 2015.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding was received for this article.

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