Seroepidemiology of Toxoplasma gondii Infection in Tepehuanos in Durango,Mexico

Abstract

The epidemiology of Toxoplasma gondii infection is known to depend on a number of behavioral factors, including contact with animals and eating habits. The epidemiology in specific populations (i.e., in indigenous ethnic groups) is not well studied. We therefore investigated the presence of anti-Toxoplasma IgG and IgM antibodies in 156 Tepehuanos, an indigenous ethnic group in Durango State in northern Mexico, using ELISAs. Of the 156 Tepehuanos (mean age 31.03±16.71 years), 35 (22.4%) had anti-Toxoplasma IgG antibodies. IgG levels of 14–99, 100–150, and >150 IU/mL were found in 15 (42.9%), 1 (2.8%), and 19 (54.3%) anti-Toxoplasma IgG-positive Tepehuanos, respectively. Also, 15 (9.6%) subjects had anti-Toxoplasma IgM antibodies. Male Tepehuanos aged 31–50 years showed a significantly higher frequency of exposure to T. gondii than female Tepehuanos of the same age. Seroprevalence of anti-T. gondii IgM antibodies was significantly higher in Tepehuanos without education than those with 7–12 years of education. Tepehuanos suffering from frequent headaches had significantly higher seroprevalences of anti-T. gondii IgG and IgM antibodies than those without this clinical feature. In addition, Tepehuanos suffering from hearing impairment had significantly higher seroprevalence of anti-T. gondii IgM antibodies than those without this clinical feature. Logistic regression analysis did not show an association of T. gondii exposure with any behavioral characteristics, including contact with cats or eating habits. The seroprevalence of T. gondii exposure in Tepehuanos is comparable to that reported in other populations in North America; however, seroprevalence in the largely rural Tepehuanos is higher than that reported in a nearby urban center. Toxoplasma may be impacting the health of Tepehuanos. This is the first report of T. gondii infection in Tepehuanos, and the results should prove useful for the design of preventive measures.

Introduction

T he parasite Toxoplasma gondii (T. gondii) is widely distributed around the world (Dubey 2010). Human infections with T. gondii may lead to serious illness affecting lymph nodes, eyes, and the central nervous system (Montoya and Liesenfeld 2004; Walker and Zunt 2005; Balasundaram et al. 2010). The main routes of T. gondii transmission in humans are ingesting food or water contaminated with oocysts shed by cats, and eating undercooked or raw meat containing tissue cysts (Montoya and Liesenfeld 2004). There is little knowledge about the epidemiology of T. gondii infection in Mexico, and we are not aware of any report in the medical literature about the epidemiology of T. gondii infection in Tepehuanos (an indigenous ethnic group in northwestern Mexico). Tepehuanos live in remote mountains in rural regions in Durango State, Mexico. Rural communities in Durango have poor health care services for the diagnosis, treatment, and prevention of infectious diseases. In fact, previous studies in Durango have shown higher frequencies of T. gondii exposure in rural populations (Alvarado-Esquivel et al. 2008,2010a) than in the urban capital city (Alvarado-Esquivel et al. 2011). Therefore, through a cross-sectional study we aimed to determine the seroprevalence of T. gondii exposure in Tepehuanos in Durango State, Mexico, and to identify the characteristics associated with Toxoplasma seropositivity.

Materials and Methods

Study design and study population

We performed a cross-sectional survey from January 2010 to March 2011 in Tepehuanos living in Durango State, Mexico. Of 1,632,934 inhabitants in Durango State, 26,453 are Tepehuanos. The great majority of Tepehuanos live in rural areas in the mountainous region in Durango State, under disadvantaged socioeconomic conditions including poor housing, food, and sanitation. Inclusion criteria for the study subjects were: (1) Tepehuano ethnicity (those who speak the Tepehuano language and identify themselves as Tepehuanos), (2) either gender, (3) 15 years and older, (4) any occupation, (5) any socioeconomic level, and (6) those that voluntarily agreed to participate.

Ethical aspects

This study was approved by the Institutional Ethical Committee of the General Hospital of Durango City of the Secretary of Health. The purpose and procedures of the study were explained to all participants. Participation in the study was voluntary without incentives. Written informed consent was obtained from all participants. Since most Tepehuanos also speak Spanish, the consent form was written in Spanish. When a Tepehuano was unable to read or understand the consent form, one or more Tepehuanos aided as interpreters. The results were given individually to the participants.

Sociodemographic, clinical, and behavioral data

We obtained the characteristics of the participants by a face-to-face interview using a standardized questionnaire. No medical terms were used in the questionnaire, and clarification of the questions was given when needed. Sociodemographic data including age, gender, birth place, residence place, residence area, educational level, occupation, and socioeconomic level were obtained from all participants. Clinical data explored included the presence of underlying diseases, presence or history of lymphadenopathy, frequent presence of headaches (occurring three or more times per week), memory, reflex, hearing, and visual impairments, and a history of surgery, blood transfusion or transplants. In women a history of miscarriage was also documented. Contributing and confounding risk factors of behavioral data included animal contacts; contact with cat excrement; foreign travel; meat consumption (pork, beef, goat, lamb, boar, chicken, turkey, pigeon, duck, rabbit, venison, squirrel, horse, opossum, or other); frequency of meat consumption; consumption of raw or undercooked meat, unpasteurized milk, or dried or cured meat (ham, sausages, salami, or chorizo); consumption of unwashed raw vegetables or fruits or untreated water; frequency of eating out of home (in restaurants or fast food outlets); contact with soil (gardening or agriculture); and the types of floors at home were obtained from all participants.

Serological examination for T. gondii antibodies

Serum samples were obtained from all participants and kept frozen at –20°C until analysis. Serum samples were analyzed by qualitative (positive or negative result) and quantitative methods for anti-T. gondii IgG antibodies with a commercially available enzyme immunoassay, the Toxoplasma IgG kit (International Immuno-Diagnostics, Foster City, CA). Anti-T. gondii IgG antibody levels were expressed as international units (IU)/mL, and a result ≥8 IU/mL was considered positive. In addition, sera positive for T. gondii IgG were further analyzed for anti-T. gondii IgM antibodies by a commercially available enzyme immunoassay, the Toxoplasma IgM kit (International Immuno-Diagnostics). All tests were performed following the instructions of the manufacturer.

Statistical analysis

The statistical analysis was performed with the aid of the software Epi Info version 3.5.1 and SPSS version 15.0. For calculation of the sample size, we used a reference seroprevalence of 23.8% (Alvarado-Esquivel et al. 2008) as the expected frequency of the factor under study, 26,453 as the size of the population from which the sample was selected, a worst acceptable result of 17.1%, and a confidence level of 95%. The result of the calculation was 154 subjects. We used the Yates' corrected chi-square test and the Fisher's exact test (when values were <5) for comparison of the frequencies among groups. Bivariate and multivariate analyses were used to assess the association between the characteristics of the subjects and T. gondii seropositivity. Variables were included in the multivariate analysis if they had a p value ≤0.25 in the bivariate analysis. Age-adjusted odds ratio (OR) and 95% confidence intervals (CI) were calculated by multivariate analysis using multiple, unconditional logistic regression. A p value <0.05 was considered statistically significant.

Results

Of 180 Tepehuanos solicited, 156 (86.7%) agreed to participate. General sociodemographic characteristics of the 156 Tepehuanos studied are shown in Table 1. Seroprevalence of T. gondii exposure tended to be higher in males than in females and was found to increase with age. Seroprevalence in male and female Tepehuanos aged 15–30, 31–50, and >50 years old were 21.1%, 40%, and 33.3%, and 19.7%, 5.9%, and 30%, respectively. Male Tepehuanos aged 31–50 years showed a significantly higher frequency of exposure to T. gondii than female Tepehuanos of the same age (6/15 versus 1/17; p=0.02). Most participants were born in Durango, and their mean age was 31.03±16.71 years (range 15–89 years). Of the 156 Tepehuanos, 35 (22.4%) had anti-T. gondii IgG antibodies, and 15 (9.6%) individuals were also positive for anti-T. gondii IgM antibodies. Of the 35 anti-T. gondii-IgG-positive participants, 19 (54.3%) had IgG levels higher than 150 IU/mL, 1 (2.8%) had a level of 100–150 IU/mL, and 15 (42.9%) had levels between 14 and 99 IU/mL. The seroprevalence of anti-T. gondii IgM antibodies was significantly higher in Tepehuanos without an education than in those with 7–12 years of education (p=0.01). The seroprevalence of anti-T. gondii IgM antibodies did not vary significantly with age.

Table 1.

Sociodemographic Characteristics of Tepehuanos and Seroprevalence of Toxoplasma gondii Infection

		Positive for anti-T. gondii IgG		Positive for anti-T. gondii IgM
Characteristic	No. of subjects tested ^a	No.	%	No.	%
Gender
Male	68	19	27.9	8	11.8
Female	88	16	18.2	7	8.0
Age groups (years)
15–30	98	20	20.4	8	8.2
31–50	32	7	21.9	5	15.7
>50	25	8	32.0	2	8.0
Birth place
Durango State	153	34	22.2	14	9.2
Other Mexican state	2	1	50.0	1	50.0
Residence place
Durango State	156	35	22.4	15	100.0
Residence area
Urban	13	1	7.7	1	7.7
Rural	141	34	24.1	14	9.9
Educational level
No education	43	13	30.2	8	18.6^d
1–6 years	31	6	19.4	3	9.7
7–12 years	80	16	20.0	4	5.0
13 or more years	1	0	0.0	0	0.0
Occupation
Non-laborer^b	86	17	19.8	6	7.0
Laborer^c	69	18	26.1	9	13.0
Socioeconomic level
Low	147	34	23.1	15	10.2
Medium	8	1	12.5	0	0.0

Participants with available data.

Non laborer=student or homemaker.

Laborer=employee, business, agriculture, factory worker, construction worker or other.

Significantly higher seroprevalence was seen in Tepehuanos without an education than in those with 7–12 years of education (p=0.01).

With respect to clinical data (Table 2), Tepehuanos suffering from frequent headaches had significantly higher seroprevalences of anti-T. gondii IgG and IgM antibodies than those without this clinical feature (p=0.03 and p=0.01, respectively). In addition, Tepehuanos suffering from hearing impairment had significantly higher seroprevalence of anti-T. gondii IgM antibodies than those without this clinical feature (p=0.02). The frequencies of other clinical characteristics, including underlying disease, presence or history of lymphadenopathy, memory, reflex, and visual impairments, surgery history, blood transfusion, and transplant history were similar among T. gondii-positive and T. gondii-negative individuals. Miscarriage history in women was not associated with T. gondii seropositivity.

Table 2.

Bivariate Analysis of Clinical Data and Infection with Toxoplasma gondii in Tepehuanos of Durango State, Mexico

		Positive for anti-T. gondii IgG		Positive for anti-T. gondii IgM
Characteristic	No. of subjects tested ^a	No.	%	No.	%
Clinical status
Healthy	107	22	20.6	8	7.5
Ill	46	13	28.3	7	15.2
Lymphadenopathy
Yes	26	5	19.2	4	15.4
No	130	30	23.1	11	8.5
Frequent headaches
Yes	65	20	30.8^b	11	16.9^c
No	91	15	16.5	4	4.4
Memory impairment
Yes	32	7	21.9	3	9.4
No	124	28	22.6	12	9.7
Reflex impairment
Yes	25	6	24	3	12.0
No	131	29	22.1	12	9.2
Hearing impairment
Yes	19	6	31.6	5	26.3^d
No	137	29	21.2	10	7.3
Visual impairment
Yes	35	11	31.4	6	17.1
No	121	24	19.8	9	7.4
Surgery
Yes	27	4	14.8	3	11.1
No	129	31	24	12	9.3
Blood transfusion
Yes	16	3	18.8	2	12.5
No	140	32	22.9	13	9.3
Transplantation
Yes	1	0	0	0	0.0
No	155	35	22.6	15	9.7
Abortions in women
Yes	14	4	28.6	3	21.4
No	33	4	12.1	12	36.4

Participants with available data.

Significantly different (p=0.03).

Significantly different (p=0.01).

Significantly different (p=0.02).

Concerning behavioral characteristics (Table 3), the bivariate analysis did not show an association of T. gondii seropositivity with any of these characteristics, including contact with cats or cleaning cat excrement; raising animals; traveling; consumption of meat; frequency of meat consumption; degree of meat cooking; consumption of raw milk, dried or cured meat, unwashed raw vegetables or fruits, or untreated water; frequency of eating out of the home; soil contact; and soil floors at home. Further analysis using logistic regression did not show an association of T. gondii exposure with behavioral characteristics.

Table 3.

Selected Putative Risk Factors for Infection with Toxoplasma gondii in Tepehuanos in Durango, Mexico

		Positive for anti-T. gondii IgG		Positive for anti-T. gondii IgM
Characteristic	No. of subjects tested ^a	No.	%	No.	%
Cats at home
Yes	80	18	22.5	6	7.5
No	76	17	22.4	9	11.8
Pork consumption
Yes	138	32	23.2	13	9.4
No	18	3	16.7	2	11.1
Beef consumption
Yes	144	32	22.2	15	10.4
No	12	3	25	0	0.0
Chicken consumption
Yes	144	33	22.9	14	9.7
No	12	2	16.7	1	8.3
Pigeon consumption
Yes	31	10	32.3	4	12.9
No	125	25	20	11	8.8
Duck consumption
Yes	11	4	36.4	1	9.1
No	145	31	21.4	14	9.7
Rabbit consumption
Yes	42	12	28.6	5	11.9
No	114	23	20.2	10	8.8
Venison consumption
Yes	129	29	22.5	14	10.9
No	27	6	22.2	1	3.7
Squirrel consumption
Yes	54	14	25.9	7	13.0
No	102	21	20.6	8	7.8
Degree of meat cooking
Raw or undercooked	11	3	27.3	2	18.2
Well done	142	32	22.5	13	9.2
Unwashed raw vegetables
Yes	62	18	29	8	12.9
No	93	17	18.3	7	7.5
Unwashed raw fruits
Yes	85	23	27.1	11	12.9
No	70	12	17.1	4	5.7
Untreated water
Yes	128	31	24.2	14	10.9
No	27	4	14.8	1	3.7
Frequency of eating out of the home
Never	33	10	30.3	6	18.2
1–10 times a year	99	24	24.2	8	8.1
>10 times a year	22	1	4.5	1	4.5
Soil contact
Yes	142	34	23.9	14	9.9
No	13	1	7.7	1	7.7
Floor at home
Ceramic or wood	21	3	14.3	2	9.5
Concrete	57	13	22.8	8	14.0
Soil	77	19	24.7	5	6.5

Participants with available data.

Discussion

The 22.4% seroprevalence of T. gondii exposure in Tepehuanos is similar to that (23.8–30.3%) found in other rural communities of Durango State (Alvarado-Esquivel et al. 2008,2010a). The seroprevalence found in Tepehuanos was also similar to the 24.6% seroprevalence recently reported in the U.S. (Jones et al. 2009). In contrast, the seroprevalence found in Tepehuanos was lower than the 60% seroprevalence reported in the Nunavik Inuit in Canada (Messier et al. 2009), and those reported in South American countries, including Argentina, Brazil, Colombia, and Venezuela (37–92%) (Tenter et al, 2000; Sroka et al. 2010; Dubey 2010). In addition, the seroprevalence in Tepehuanos was lower than the estimated mean worldwide seroprevalence (by up to one-third) (Montoya and Liesenfeld 2004; Dubey 2010). However, in the local context, the seroprevalence found in Tepehuanos was higher than the seroprevalences reported in urban populations in Durango City. In 4 previous studies, the seroprevalence of exposure to T. gondii ranged between 6.1 and 12% among pregnant women (Alvarado-Esquivel et al. 2006), healthy blood donors (Alvarado-Esquivel et al. 2007), subjects suffering from a number of underlying diseases (Alvarado-Esquivel et al. 2010b), and the general population in Durango City (Alvarado-Esquivel et al. 2011). Differences in sanitation between urban and rural communities may contribute to the marked differences seen in seroprevalences. Tepehuanos live in poverty and lack urban services, including potable water and drainage, and they live in poor housing conditions, including soil floors at home. Tepehuanos do not eat meat frequently because they cannot afford to buy it. Some Tepehuanos hunt wild animals for food, but they eat well-cooked meat. These factors, including a paucity of meat consumption and thorough cooking of meat, may contribute to the low levels of T. gondii infections seen in Tepehuanos. The seroprevalence of T. gondii exposure was higher in male Tepehuanos aged 31–50 years than in female Tepehuanos of the same age. This finding agrees with that reported in the Third National Health and Nutrition Examination Survey in the U.S., in which researchers found a significantly higher seroprevalence of T. gondii exposure in males 30–39 years old than in females of the same age (Jones et al. 2001). In our study, the difference in seroprevalence among male and female Tepehuanos aged 31–50 years might be explained by differences in occupational and behavioral characteristics. In this age group, the frequency of occupation in agriculture, consumption of meat from wild animals, and eating unwashed raw fruits were significantly (p<0.05) higher in male than in female Tepehuanos (data not shown). Better hygienic practices by female Tepehuanos than those of males may be related to a better understanding of public health messaging by female than male Tepehuanos.

IgG levels in most Tepehuanos were either low or high. Low levels of specific T. gondii IgG antibodies may be present early after exposure or in chronic infection. On the other hand, high levels of IgG may reflect a peak during acute infection, or may persist due to chronic infection. Therefore, the results of one positive serum sample only establishes that the host was infected at some time in the past (Dubey 2010). The bipolar titers seen in our study suggest that the transition from low to high (or high to low) must be quite rapid. Existing serological assays cannot determine the route of infection (oocysts versus tissue cyst consumption), but research on this topic is underway (Hill et al. 2011).

Concerning clinical characteristics, remarkably, T. gondii seropositivity was associated with frequent headaches in Tepehuanos. Headaches are a characteristic symptom of toxoplasmosis in immunocompetent (Silva et al. 2008) and immunocompromised (Uneke et al. 2005) individuals. In addition, T. gondii IgM seropositivity was associated with hearing impairment in Tepehuanos. Seropositivity to anti-T. gondii IgM antibodies has been linked to sudden deafness (Schlottmann et al. 1996; Noorbakhsh et al. 2008). This is the first time we found associations of T. gondii seropositivity with headaches and hearing impairment in a population of Durango State. It is not clear why only Tepehuanos have shown these associations, and not the other populations studied in Durango, including pregnant women (Alvarado-Esquivel et al. 2006), healthy blood donors (Alvarado-Esquivel et al. 2007), Mennonites (Alvarado-Esquivel et al. 2010a), and general populations in urban (Alvarado-Esquivel et al. 2011) and rural communities (Alvarado-Esquivel et al. 2008). Perhaps recent and symptomatic infections were more frequent in Tepehuanos than in the other populations previously studied. The higher frequency (9.6%) of IgM-positive cases seen among Tepehuanos than in other rural populations in Durango (2.2% to 3.3%) (Alvarado-Esquivel et al. 2008,2010a) supports this assumption. On the other hand, socioeconomic conditions in Tepehuanos are different from those in the other populations; Tepehuanos are largely poorer and suffer from malnutrition more commonly than other populations in Durango. This may impair host defenses against T. gondii, and thus symptoms might appear more frequently than in other populations with better nutrition. Seroprevalence of T. gondii exposure was higher in Tepehuanos with visual impairments and a history of miscarriage than those without these clinical characteristics. However, the differences did not reach statistical significance, probably as a result of the small sample size of the study.

With respect to behavioral characteristics, there was a trend toward higher seroprevalence in those who consumed venison, squirrel and pigeon meat, unwashed raw fruits and vegetables, and untreated water. Consumption of beef was not associated with T. gondii infection; this is consistent with the fact that cattle are poor hosts for toxoplasmosis (Dubey 2010; Chikweto et al. 2011). Further research to determine the main transmission routes of T. gondii infection in Tepehuanos is needed. Tepehuanos live in some of the poorest sanitation environments in Durango State. Many Tepehuanos live in houses with soil floors, and the great majority drinks untreated water. In addition, cats are frequently observed near their homes. It is likely that these factors are responsible for the increased rates of infection seen in this population.

We conclude: (1) that the seroprevalence of T. gondii in Tepehuanos is comparable to that reported in other populations in North America; however, the seroprevalence in the largely rural Tepehuanos is higher than that reported in a nearby urban center; and (2) Toxoplasma may be impacting the health of Tepehuanos. This is the first report of T. gondii infection in Tepehuanos, and the results are useful for the design of optimal preventive measures.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

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