J. Parasitol., 95(2), 2009, pp. 271–274 ᭧ American Society of Parasitologists 2009

SEROEPIDEMIOLOGY OF TOXOPLASMA GONDII INFECTION IN PREGNANT WOMEN IN RURAL ,

C. Alvarado-Esquivel, A. Torres-Castorena*, O. Liesenfeld†, C. R. Garcı´a-Lo´ pez‡, S. Estrada-Martı´nez§, A. Sifuentes-A´ lvarez, J. F. Marsal-Herna´ndez, R. Esquivel-Cruz࿣, F. Sandoval-Herrera*, J. A. Castan˜ eda*, and J. P. Dubey#¶ Faculty of Medicine, Jua´rez University of Durango State, Avenida Universidad S/N esquina Fanny Anitua, 34000 Durango, Dgo, Mexico. e-mail: [email protected]

ABSTRACT: The epidemiology of Toxoplasma gondii infection in pregnant women in rural Mexico is largely unknown. The sero- epidemiology of T. gondii infection in 439 pregnant women from 9 communities in rural Durango State, Mexico was investigated. Using commercial enzyme-linked immunoassays, sera were tested for T. gondii IgG, IgM, and avidity antibodies. Prevalences of T. gondii IgG antibodies in the communities varied from 0% to 20%. Overall, 36 (8.2%) of the 439 women had IgG T. gondii antibodies. Ten (2.3%) women had also T. gondii IgM antibodies; IgG avidity was high in all IgM-positive women, suggesting chronic infection. None of the women, however, had delivered a known T. gondii-infected child. The seroprevalence was signif- icantly higher (P Ͻ 0.05) in women from low socio-economic conditions (14%) than in those with higher socio-economic status (6.6%). Multivariate analysis showed that T. gondii infection was associated with soil floors at home (adjusted OR ϭ 2.89; 95% CI: 1.12–7.49). This is the first epidemiological study of T. gondii infection in pregnant women in rural Mexico.

Toxoplasma gondii has a worldwide distribution. Most T. women, of all ages and at any stage of pregnancy, residing in rural gondii infections are asymptomatic and, in some individuals, a Durango State. mild disease including cervical lymphadenopathy or ocular Epidemiological data problems may occur (Montoya and Liesenfeld, 2004; Hill et al., Epidemiological data, including socio-demographic, behavioral, and 2005). Primary infection during pregnancy may result in severe clinical characteristics, were obtained from all pregnant women. Socio- damage to the fetus (Montoya and Liesenfeld, 2004; Rorman demographic characteristics included age, birth place, place of residen- et al., 2006). Epidemiological studies on T. gondii infection in cy, occupation, and educational and socio-economic levels. Behavioral pregnant women have focused largely on urban populations. characteristics included animal contacts, removal of cat excrement, for- The epidemiology of T. gondii infection in pregnant women in eign travel, contact with soil (gardening or agriculture), eating outside of the home, consumption of raw or undercooked meat (pork, lamb, rural Mexico is essentially unknown. We recently reported se- beef, goat, boar, chicken, turkey, rabbit, deer, squirrel, horse, fish, snake, roprevalences of 14.8% to 35.8% T. gondii antibodies among or other), consumption of dried or cured meat (chorizo, ham, sausages, inhabitants of 3 rural communities in Durango, Mexico (Alva- or salami), consumption of unpasteurized milk, milk products, or water, rado-Esquivel et al., 2008). In the present study, we report se- and unwashed raw vegetable or fruit consumption. Clinical character- istics encompassed gestational age, number of pregnancies and abor- roprevalence and associated epidemiological characteristics of tions, and blood transfusions or transplants, as well as impairment of T. gondii infection in pregnant women from wider rural areas memory, reflexes, hearing, or visual status. Clinical data were obtained in Durango State, Mexico. during a direct interview with the participants.

Serological tests MATERIALS AND METHODS Sera of all pregnant women were assayed for T. gondii antibodies Study population and study design using commercially available enzyme immunoassay kits. IgG antibodies were assayed using a ‘‘Toxoplasma IgG’’ kit (Diagnostic Automation From August 2007 to February 2008 we studied pregnant women of Inc., Calabasas, California). Sera positive for T. gondii IgG antibodies 9 rural communities (5 de mayo, Colonia Hidalgo, Guadalupe Victoria, were further tested for T. gondii IgM antibodies with a ‘‘Toxoplasma Santa Clara, Vicente Guerrero, Canatla´n, , Santiago Papas- IgM’’ kit (Diagnostic Automation Inc.) and T. gondii IgG avidity kit quiaro, and Rodeo) in Durango State, Mexico. All pregnant women (VIDAS Toxo IgG Avidity, BioMe´rieux, Marcy L’Etoile, France) to attending public prenatal care clinics in the communities were invited help determine the timing of infection, e.g., low-avidity antibodies are to participate. Inclusion criteria for the study subjects were pregnant often associated with recently acquired infection (Petersen et al., 2005). All 3 tests were performed following the instructions of the manufac- turers. A positive IgG test with a negative IgM test, and a positive IgM Received 21 August 2008; revised 29 September 2008; accepted 6 test with a high IgG avidity, was interpreted as a latent infection. October 2008. * Mexican Social Security Institute, Avenida Normal, Predio Canoas Ethical aspects #200, 34000, , Durango, Mexico. This study was approved by the ethical committee of the General † Institute for Microbiology and Hygiene, Campus Benjamin Franklin, Hospital of the Mexican Social Security Institute in Durango City, Mex- Charite´ Medical School, Hindenburgdamm 27, D-12203 Berlin, Ger- ico. The purpose and procedures of the study were explained to all many. pregnant women, and written, informed consent was obtained from all ‡ Hospital of , Dgo. Domicilio Conocido, Santiago of them. Papasquiaro, Durango, Me´xico. § Institute for Scientific Research, Jua´rez University of Durango State, Statistical analysis Avenida Universidad S/N esquina Fanny Anitua, 34000 Durango City, Durango, Mexico. The statistical analysis was performed using Epi Info version 3.3.2 ࿣ Health Center of Santa Clara, Dgo, Domicilio Conocido, Santa Clara, (CDC, Atlanta, Georgia) and SPSS version 13.0 (SPSS Inc., Chicago, Durango, Me´xico. Illinois). We used the Yates corrected ␹2 and the Fisher’s exact test # United States Department of Agriculture, Agricultural Research Ser- (when values were Ͻ5) for comparison of frequencies among groups. vice, Animal and Natural Resources Institute, Animal Parasitic Dis- Bivariate and multivariate analyses were used to assess the association eases Laboratory, Building 1001, Beltsville, Maryland 20705-2350. between the characteristics of the women and T. gondii seropositivity. ¶ To whom correspondence should be addressed. As a criterion for inclusion of variables in the multivariate analysis, we

271 272 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 2, APRIL 2009

TABLE I. Selected general characteristics of pregnant women in rural communities of Durango, Mexico.

Communities with low Communities with high seroprevalence (Ͻ8.2%) seroprevalence (Ͼ8.2%) All communities Prevalence of T. gondii Prevalence of T. gondii Prevalence of T. gondii No. of No. of No. of infection infection infection women women women Characteristic tested* No. % tested* No. % tested* No. %

Age groups (years) 20 or less 86 3 3.5 47 5 10.6 133 8 6.0 21–30 158 8 5.1 66 12 18.2 224 20 8.9 Ͼ30 51 2 3.9 25 6 24 76 8 10.5 Education No education 5 0 0 4 1 25 9 1 11.1 Education 290 13 4.5 135 22 16.3 425 35 8.2 Occupation Unemployed† 253 13 5.1 127 21 16.5 380 34 8.9§ Employed‡ 39 0 0 10 0 0 49 0 0.0 Socio-economic level Low 40 4 10 60 10 16.7 100 14 14§ Medium 253 9 3.6 81 13 16 334 22 6.6

* Pregnant women for which data were available. † Unemployed ϭ no occupation, student, or housewife. ‡ Employed ϭ employee, professional, business, or other. § Statistically significant difference among the groups, P Ͻ 0.05. considered variables with a P-value Յ0.25 obtained in the bivariate pregnancies at the time they were included in the study. Un- analysis in order to allow interaction among variables. Adjusted odd employed women, students, or housewives had a significantly ratio (OR) and 95% confidence interval (CI) were calculated by mul- Ͻ tivariate analysis using multiple, unconditional logistic regression. A P- higher (P 0.05) seroprevalence (8.9%) than employed women value Ͻ0.05 was considered statistically significant. (0%). Similarly, women from a low socio-economic level had a significantly higher (P Ͻ 0.05) seroprevalence (14%) than RESULTS women from medium socio-economic conditions (6.6%). Se- roprevalence showed a slight, but not significant, increase with Seroprevalence age in communities with high seropositivity and in the whole In total, 439 pregnant women consented to be included in population. No significant differences in age, education, and this project. Of these women, 36 (8.2%) had IgG antibodies occupation were observed in women among low- and high- and 10 (2.3%) also had IgM antibodies; all 10 women with prevalence communities. However, a significantly higher (P Ͻ IgM antibodies also had high (0.587; 0.528; 0.587; 0.715; 0.34; 0.001) number of women with low socio-economic status 0.4; 0.522; 0.373; 0.719; 0.723) IgG avidity, indicating latent (42.6%) were observed in high-prevalence communities than in T. gondii infection. Seroprevalences in individual communities low-prevalence (13.7%) communities. General socio-demo- ranged from 0% to 20%: (1) 5 de mayo 16.7% (3/18); (2) Co- graphic characteristics of the 439 rural pregnant women, and lonia Hidalgo 0% (0/8); (3) Guadalupe Victoria 6.5% (3/46); seroprevalences, are shown in Table I. (4) Santa Clara 20% (1/5); (5) Vicente Guerrero 2% (1/49); (6) Canatla´n 5.7% (7/123); (7) Nuevo Ideal 7.7% (1/13); (8) San- tiago Papasquiaro 15.8% (19/120); and (9) Rodeo 1.8% (1/57). Behavioral characteristics associated with seropositivity Based on the mean seroprevalence (8.2%) found in women of In the bivariate analysis, several variables were identified as all communities, and for comparison purposes, we decided to possible risk factors associated with T. gondii infection in wom- analyze variables in women in 3 groups of communities, name- en of communities with low prevalence, communities with high Ͻ ly those with low seroprevalence ( 8.2%), with high seroprev- prevalence, or all communities together. Other factors such as Ͼ alence ( 8.2%), and all communities together. cats at home; rearing animals in general, raising chickens, birds, dogs, and pigs; and consumption of raw cow milk and meat Socio-demographic characteristics associated with from wild animals (squirrel, deer, boar, snake, cougar, skunk, seropositivity ostrich, badger) were significantly higher (P Ͻ 0.05) in women Most women studied were born in Mexico (99.8%); only 1 of communities with a high prevalence of T. gondii infection was born in the United States (0.2%). The mean age of the than those in communities with low prevalence. As seen in pregnant women was 24.5 yr (range 13 to 42 yr). Most women Table II, living in a house with a soil floor (adjusted OR ϭ were in their last (42.2%), or their second (38.0%), trimester. 2.89; 95% CI: 1.12–7.49) increased the probability of higher T. In addition, the majority of women (61.1%) had had 2 or more gondii seropositivity. ALVARADO-ESQUIVEL ET AL.—TOXOPLASMOSIS IN PREGNANT WOMEN IN RURAL MEXICO 273

TABLE II. Multivariate analysis of selected characteristics of rural pregnant women and their association with T. gondii infection.

Communities with low Communities with high seroprevalence (Ͻ8.2%) seroprevalence (Ͼ8.2%) All communities Adjusted Adjusted Adjusted 95% Confi- odds 95% Confidence odds 95% Confidence odds dence Characteristic* ratio† interval P value ratio† interval P value ratio† interval P value

Cats at home 2.42 0.69–8.52 0.16 0.63 0.23–1.75 0.38 1.18 0.56–2.51 0.65 Cleaning of cat feces 2 0.58–6.87 0.26 2.19 0.81–5.95 0.12 2.04 0.98–4.25 0.05 Raising animals‡ 1.38 0.34–5.56 0.64 4.51 0.53–37.95 0.16 2.98 0.99–8.98 0.05 Raw cow milk consumption 0.79 0.23–2.64 0.7 2.11 0.42–10.46 0.35 2.02 0.87–4.72 0.1 Untreated water consumption 1.87 0.56–6.23 0.3 1.86 0.67–5.09 0.22 1.55 0.75–3.22 0.23 Wild-animal meat consumption 1.86 0.50–6.90 0.35 0.74 0.24–2.19 0.58 1.19 0.53–2.64 0.66 Soil floors at home 2.41 0.46–12.68 0.29 3.59 0.99–13.06 0.05 2.89 1.12–7.49 0.02

* The variables included were those with a P Ͻ 0.25 obtained in the bivariate analysis. † Adjusted by age and the other characteristics included in this Table. ‡ This variable represents the raising of any kind of animals.

Clinical characteristics associated with seropositivity cow milk consumption), and wild-animal meat consumption. Frequencies of other characteristics such as age, education, and Impairments in memory, reflexes, hearing, and vision, and occupation were not significantly different among the commu- abortion occurred with comparable frequencies in pregnant nities. women residing in low- and high-seroprevalence communities. We further analyzed characteristics of the women associated Women with a history of abortion had a significantly higher seroprevalence of T. gondii IgG; this was the case in both the with T. gondii seropositivity. We observed that seroprevalence Ͻ whole population (P ϭ 0.0006) and in women living in com- of T. gondii infection was significantly higher (P 0.05) in munities with high seroprevalence (P ϭ 0.006), but not in those unemployed women, students, or housewives than in employed living in communities with low seroprevalence. In communities women. Similarly, women with a low socio-economic level with low seroprevalence, the frequency of infection was signif- showed a significantly higher seroprevalence than those with a Ͻ icantly higher (P ϭ 0.02) in pregnant women suffering from medium socio-economic level (P 0.05). We are not aware of visual impairment than in pregnant women without this health similar results in other studies on rural pregnant women. Nearly problem. Blood transfusion or transplant history did not show all unemployed women in this study were housewives, and the any association with T. gondii seroprevalence. higher seroprevalence in this group than in employed women could be explained by the higher exposure to cat excrement. DISCUSSION The higher exposure to raw meat is explained by their cooking activity and poorer hygiene (raising animals and poor sanitary Seroprevalence in pregnant women from rural Mexico in the facilities at home). present study (8.2%) is comparable with a 6.1% seroprevalence Multivariate analysis showed that the variable of a soil floor of T. gondii antibodies in a population of pregnant women at- at home (adjusted OR ϭ 2.89; 95% CI: 1.12–7.49) was asso- tending a public hospital for prenatal care in Durango City (Al- ciated with T. gondii seropositivity, supporting other findings varado-Esquivel et al., 2006). In the present study, we observed in pregnant urban women (Alvarado-Esquivel et al., 2006). In clear differences in seroprevalences of infection among differ- the present study, contact with cats, untreated water consump- ent rural communities. Epidemiologic variables (environmental, tion, or contact with soil (gardening, agriculture) did not show socio-economic, and behavioral) likely contribute to these dif- any association with T. gondii seropositivity. ferences in prevalence. Low seroprevalences may be found in Toxoplasma gondii dry climates (Montoya and Liesenfeld, 2004) and high alti- may cause abortion and loss of vision tudes, as shown in studies in humans (Hershey and McGregor, (Montoya and Liesenfeld, 2004; Pleyer et al., 2007), and these 1987; Rai et al., 1994). In an attempt to determine the causes symptoms were associated with T. gondii seropositivity in the for differences in seroprevalences among communities in this present study. However, these associations were not seen equal- study, we analyzed environmental characteristics such as alti- ly among the communities. In 2 previous studies, researchers tude, climate, annual mean temperature, annual mean rainfall, found T. gondii seroprevalences of 44.9% in women with ha- and presence of wild felines among the communities. However, bitual abortions from central Mexico (Galva´n-Ramirez et al., none of these factors appeared to have influenced the seroprev- 1995) and 47% in women with spontaneous abortions from the alence (data not shown). We then analyzed whether socio-de- south of Mexico (Zavala-Velazquez et al., 1989). Although T. mographic and behavioral characteristics of women differed in gondii may sometimes cause spontaneous abortion, especially communities with low and high seroprevalences. In contrast during the first trimester, there is no evidence that it causes with low-seroprevalence communities, high-seroprevalence habitual abortion (Dubey and Beattie, 1988). Further large- communities showed significantly higher frequencies of women scale, controlled studies are needed in Mexico before drawing from low socio-economic status who had animal contacts (cats any conclusions regarding cause–effect associations between T. at home, raising animals), poor cooking hygiene practices (raw gondii seropositivity and abortion and a loss of vision. 274 THE JOURNAL OF PARASITOLOGY, VOL. 95, NO. 2, APRIL 2009

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