Toxoplasma gondii infection and liver disease: a case-control study in a northern Mexican population.

BACKGROUND: Infection with the protozoan parasite Toxoplasma gondii may cause liver disease. However, the impact of the infection in patients suffering from liver disease is unknown. Therefore, through a case-control study design, 75 adult liver disease patients attending a public hospital in Durango City, Mexico, and 150 controls from the general population of the same region matched by gender, age, and residence were examined with enzyme-linked immunoassays for the presence of anti-Toxoplasma IgG and anti-Toxoplasma IgM antibodies. Socio-demographic, clinical and behavioral characteristics from the study subjects were obtained.
RESULTS: Seroprevalence of anti-Toxoplasma IgG antibodies and IgG titers did not differ significantly in patients (10/75; 13.3%) and controls (16/150; 10.7%). Two (2.7%) patients and 5 (3.3%) controls had anti-Toxoplasma IgM antibodies (P = 0.57). Seropositivity to Toxoplasma did not show any association with the diagnosis of liver disease. In contrast, seropositivity to Toxoplasma in patients was associated with consumption of venison and quail meat. Toxoplasma seropositivity was more frequent in patients with reflex impairment (27.8%) than in patients without this impairment (8.8%) (P = 0.05). Multivariate analysis showed that Toxoplasma seropositivity in patients was associated with consumption of sheep meat (OR = 8.69; 95% CI: 1.02-73.71; P = 0.04) and rabbit meat (OR = 4.61; 95% CI: 1.06-19.98; P = 0.04).
CONCLUSIONS: Seropositivity to Toxoplasma was comparable among liver disease patients and controls. Further studies with larger sample sizes are needed to elucidate the association of Toxoplasma with liver disease. Consumption of venison, and rabbit, sheep, and quail meats may warrant further investigation.

Background

Human infection with the protozoan parasite Toxoplasma gondii occurs worldwide [1,2]. Major routes of T. gondii infections include ingesting food or water that is contaminated with oocysts shed by cats or by eating undercooked or raw meat containing tissue cysts [2-4]. The clinical spectrum of T. gondii infection varies from an asymptomatic state to severe illness. The parasite can affect the host's lymph nodes, eyes, central nervous system, liver, and heart [3,5,6]. In liver, the parasite has been associated with a number of pathological changes including hepatomegaly, granuloma, hepatitis, and necrosis [7-14]. In addition, an epidemiological study has reported an association of T. gondii infection with liver cirrhosis [15]. However, epidemiological studies on the association of infection with T. gondii and liver disease are scarce, and have not been performed in Mexico. Therefore, we performed a case-control study in Northern Mexico to determine the seroprevalence of T. gondii infection and anti-T. gondii IgG levels in adult patients with liver disease attending the Department of Gastroenterology in a secondary-care public hospital in Durango City. Furthermore, we investigated socio-demographic, clinical, and behavioral characteristics associated with T. gondii seropositivity in these patients.

Methods

Study design and study populations

Through a case-control study design, we studied the association of liver disease with infection with T. gondii in adult patients and control subjects in Durango City, Mexico from January 2009 to December 2010.

Liver disease patients

Seventy five outpatients attended in the Gastroenterology Department of a public secondary-care hospital (Mexican Social Security Institute) in Durango City, Mexico were enrolled in the study. Forty seven patients were male and twenty eight were female. The mean age of the patients was 58.65 ± 14.41 years (range: 22-85 years). All patients resided in Durango State. Patients suffered from liver cirrhosis (n = 67), steatosis (n = 4), chronic hepatitis (n = 2), acute hepatitis (n = 1), and amoebic liver abscess (n = 1). The etiology of liver cirrhosis was alcohol consumption in 35 patients, hepatitis C virus in 4 patients, and unknown in 28 patients.

Control subjects

One hundred and fifty control subjects matched with patients by age, gender, and residence were included in the study. The mean age in controls was 58.68 ± 14.35 (range: 22-86) and comparable with that in patients (P = 0.99). Control subjects were obtained from the general population of Durango City, Mexico.

Ethical aspects

This study was approved by the Institutional Ethical Committee of the Mexican Social Security Institute. The purpose and procedures of the study were explained to all participants, and a written informed consent was obtained from all of them.

Socio-demographic, clinical and behavioral data

We explored socio-demographic, clinical and behavioral characteristics of the participants with the aid of a standardized questionnaire. Socio-demographic data including age, gender, birthplace, residence area, educational level, occupation, and socio-economic level were obtained from all participants. Clinical data explored in patients included type and duration of liver disease, clinical response to treatment, presence of concomitant diseases, presence or history of lymphadenopathy, frequent headache, impairments in memory, reflexes, hearing and vision, blood transfusion, transplant or surgery history. Behavioral data included animal contacts, contact with cat feces, foreign travel, kind of meat consumption (pork, beef, goat, sheep, boar, chicken, turkey, pigeon, rabbit, deer, squirrel, horse, opossum, or other), consumption of raw or undercooked meat, unpasteurized milk, dried or cured meat (chorizo, ham, sausages or salami), consumption of unwashed raw vegetables, fruits, or untreated water, frequency of eating away from home (in restaurants or fast food outlets), contact with soil (gardening or agriculture), and types of floors at home.

Laboratory tests

Serum samples of participants were obtained and kept frozen at -20°C until analyzed. Sera were analyzed by qualitative and quantitative methods for anti-T. gondii IgG antibodies with the commercially available enzyme immunoassay kit "Toxoplasma IgG" (International Immuno-Diagnostics, Foster City, California). Anti-T. gondii IgG antibody levels were expressed as International Units (IU)/ml, and a result equal or greater than 8 IU/ml was considered positive. In addition, sera positive for anti-T. gondii IgG antibodies were further analyzed for anti-T. gondii IgM antibodies by the commercially available enzyme immunoassay "Toxoplasma IgM" kit (International Immuno-Diagnostics). All tests were performed following the instructions of the manufacturer.

Statistical Analysis

Results were analyzed with the aid of Epi Info version 3.5.1 and SPSS 15.0 (SPSS Inc. Chicago, Illinois). Age among the groups was compared by the student's t test. For comparison of the frequencies among groups, the Yates corrected or, when indicated, the Fisher exact test, were used. Bivariate and multivariate analyses were used to assess the association between subject's characteristics and T. gondii infection. Variables were included in the multivariate analysis if they had a P value equal or less than 0.25 in the bivariate analysis. Odd ratio (OR) and 95% confidence interval (CI) were calculated by multivariate analysis using multiple, unconditional, logistic regression. When a cell in the 2 × 2 contingency table had a value of zero, the odds ratio was calculated by adding 0.5 to all table cells [16]. A P value less than 0.05 was considered statistically significant.

Results

Anti-T. gondii IgG antibodies were found in 10 (13.3%) of 75 patients and in 16 (10.7%) of 150 controls (P = 0.71). Of the 10 anti-T. gondii IgG positive patients, 6 (8.0%) had IgG levels higher than 150 IU/ml, and 4 (5.3%) between 8 to 99 IU/ml. In comparison, of the 16 anti-T. gondii IgG positive controls, 9 (6.0%) had IgG levels higher than 150 IU/ml, 2 (1.3%) between 100 to 150 IU/ml, and 5 (3.3%) between 8 to 99 IU/ml. Anti-T. gondii IgG levels were comparable among patients and controls (P = 0.60). Anti-T. gondii IgM antibodies were found in 2 patients and in 5 controls (2.7% vs 3.3%, respectively; P = 0.57). The socio-demographic characteristics among seropositive and seronegative patients were not significantly different (Table 1). Seropositivity to T. gondii was significantly higher in patients with an occupation of truck driver than those with other occupations (3/3: 100% vs 8/65: 12.3%; P = 0.003). The type, duration and clinical response to treatment of liver disease did not show any association with the seroprevalence and levels of anti-T. gondii IgG (Table 2). The frequency of T. gondii seropositivity was higher in patients with reflex impairment (27.8%) than patients without this impairment (8.8%) (P = 0.05). Patients with a history of abdominal hernia repair had a significantly higher seroprevalence of T. gondii infection than those without this history (3/5: 60% vs 7/70: 10%, respectively; P = 0.01). In contrast, no statistically significant differences were observed among T. gondii positive and T. gondii negative patients in the frequencies of other clinical characteristics including concomitant diseases, frequent headaches, presence or history of lymphadenopathy, blood transfusion, or transplant, and impairments in memory, hearing or vision (Table 3).

Table 1

Socio-demographic characteristics of the patients and seropositivity to T. gondii.

			Prevalence of T. gondii infection
Characteristic	No.	%	No.	%	P value
Gender
Male	47	62.7	8	17.0	0.19
Female	28	37.3	2	7.1
Age groups (years)
30 or less	3	4.0	0	0.0
31-50	17	22.7	2	11.8	0.56
51-70	41	54.7	6	14.6
>70	14	18.7	2	14.3
Residence place
Durango City	75	100.0	10	13.3
Birth place
Durango State	67	89.3	9	13.4	0.71
Other Mexican State	8	10.7	1	12.5
Residence area
Urban	49	65.3	5	10.2	0.2
Suburban	1	1.3	0	0.0
Rural	25	33.3	5	20.0
Socio-economic level
Low	50	72.5	8	16.0	0.44
Medium	19	27.5	2	10.5
Educational level
No education	6	8.0	1	16.7	0.58
Up to 6 years	63	84.0	8	12.7
7-12 years	6	8.0	1	16.7
Occupation
No laborerb	26	34.7	1	3.8	0.07
Laborerc	49	65.3	9	18.4

bNon laborer = none occupation, student or housewife.

cLaborer = Employee, business, agriculture, construction worker, driver or other.

Table 2

Bivariate analysis of liver disease characteristics in patients and seropositivity to T. gondii infection.

	No. of subjects tested	Prevalence of T. gondii infection		P value	Anti-T. gondii IgG levels >150 IU/ml
Characteristic		No.	%		No.	%
Diagnosis
Acute hepatitis	1	0	0.0		-
Chronic hepatitis	2	0	0.0		-
Cirrhosis	67	9	13.4	0.89	5	55.6
Steatosis	4	1	25.0		1	100
Amoebic abscess	1	0	0.0		-
Alcohol related disease
Yes	35	6	17.1	0.28	3	50
No	40	4	10.0		3	75
Duration of disease
Less than 1 year	30	5	16.7	0.35	3	60
1 to 5 years	34	4	11.8		3	75
More than 5 years	11	1	9.1		1	100
Treatment response
Good	50	8	16.0		4	50
Regular	4	1	25.0		1	100
Bad	3	1	33.3	0.44	1	100

Table 3

Bivariate analysis of clinical data and infection with T. gondii in patients.

	No. of subjects tested	Prevalence of T. gondii infection		P value
Characteristic		No.	%
Concomitant disease
Yes	49	5	10.2	0.22
No	26	5	19.2
Lymphadenopathy ever
Yes	11	1	9.1	0.54
No	64	9	14.1
Headache frequently
Yes	25	3	12	0.55
No	50	7	14
Blood transfusión
Yes	50	6	12	0.44
No	25	4	16
Transplantation
Yes	4	0	0	0.55
No	71	10	14.1
Surgery ever
Yes	42	7	16.7	0.27
No	33	3	9.1
Memory impairment
Yes	32	4	12.5	0.56
No	43	6	14
Reflex impairment
Yes	18	5	27.8	0.05
No	57	5	8.8
Hearing impairment
Yes	48	6	12.5	0.51
No	27	4	14.8
Visual impairment
Yes	27	3	11.1	0.48
No	48	7	14.6

Bivariate analysis showed a number of behavioral characteristics with a P value equal or less than 0.25 including cats at home, raising animals, traveling abroad, consumption of sheep, chicken, turkey, pigeon, rabbit, deer, squirrel, quail, skunk and armadillo meats, consumption of raw milk, ham, unwashed raw fruits, and untreated water, soil contact and soil floors at home. Multivariate analysis of these behavioral characteristics showed that consumption of sheep meat (OR = 8.69; 95% CI: 1.02-73.71; P = 0.04), rabbit meat (OR = 4.61; 95% CI: 1.06-19.98; P = 0.04), venison (OR = 40.46; 95% CI: 2.25-725.75; P < 0.01), and quail meat (OR = 38.50; 95% CI: 1.70-871.99; P < 0.01) were significantly associated with T. gondii infection in patients (Table 4). Other behavioral characteristics did not show an association with T. gondii infection. Raw data of patients and controls are included in additional files [additional file 1-cases and additional file 2-controls, respectively].

Table 4

Multivariate analysis of selected characteristics of patients and their association with T. gondii infection.

	Raw numbers
Characteristic	Yes	No	Odds ratio	95% Confidence interval	P value
Cats at home	7/41	3/34	1.99	0.45 - 8.81	0.36
Raising animals	8/48	2/27	2.53	0.48 - 13.37	0.27
Traveling abroad	6/34	4/41	2.24	0.54 - 9.23	0.26
Sheep meat consumption	9/43	1/32	8.69	1.02 - 73.71	0.04
Chicken meat consumptiona	9/74	1/1	0.04	0.001-1.27	0.01
Turkey meat consumption	9/51	1/24	5.97	0.68 - 51.93	0.10
Pigeon meat consumption	3/12	7/63	2.42	0.46 - 12.69	0.29
Rabbit meat consumption	7/31	3/44	4.61	1.06 - 19.98	0.04
Venison consumptiona	10/36	0/39	40.46	2.25-725.75	<0.01
Squirrel meat consumption	5/24	5/51	2.46	0.59 - 10.29	0.21
Quail meat consumptiona	2/2	8/73	38.50	1.70-871.99	<0.01
Skunk meat consumption	2/5	8/70	3.11	0.43 - 22.39	0.25
Armadillo meat consumption	1/2	9/73	7.11	0.08-566.52	0.05
Raw milk consumption	6/31	4/44	2.26	0.56 - 8.99	0.24
Ham consumption	7/63	3/12	0.46	0.09 - 2.21	0.33
Unwashed raw fruits	5/24	5/51	2.4	0.60 - 9.58	0.21
Untreated water	9/47	1/28	6.73	0.75 - 60.49	0.08
Soil contact	9/53	1/22	3.86	0.45 - 33.17	0.21
Soil floor at home	4/16	6/59	2.35	0.51 - 10.72	0.26

aOdd ratios for these characteristics were calculated by adding 0.5 to each cell of the 2 × 2 table.

Discussion

In this seroprevalence case-control study, we found a comparable frequency of anti-T. gondii IgG and IgM antibodies in liver disease patients and controls. Similarly, levels of anti-T. gondii IgG antibodies were comparable among these groups indicating that T. gondii infection is not likely to substantially contribute to the etiology of liver disease in our patient population. We are not aware of previous reports about the association of T. gondii infection in liver disease patients in Mexico, and reports in other countries are scarce. Our results conflict with those reported in a Turkish study where researchers found an association of T. gondii infection with liver cirrhosis [15]. Most of our patients suffered from liver cirrhosis but we did not find any association between seropositivity to T. gondii and cirrhosis. Similarly, the comparable seroprevalence of T. gondii infection in patients and controls differs from those reported in an Egyptian study where researchers found a 65.5% seroprevalence of T. gondii antibodies in patients with acute and chronic hepatic diseases against a 27% seroprevalence found in controls [17]. Certainly, differences in the characteristics of the studies might explain the differences in the seroprevalences including the use of different laboratory methods and matching procedures, difference in ages of participants and proportions of controls and patients.

None of the socio-demographic characteristics and diagnosis of liver disease associated with T. gondii seropositivity in our patients. Concerning behavioral characteristics, it was noteworthy that there was an association between T. gondii seropositivity and sheep meat consumption (OR = 8.69; 95% CI: 1.02-73.71; P = 0.04). Infections with T. gondii have been reported in sheep [18]. In addition, viable T. gondii has been found in lambs destined for meat consumption in the USA [19], and ovine meat consumed in France [20]. It will therefore be of interest to examine the seroprevalence of T. gondii infection in sheep in Durango. In a recent study in the USA, elevated risk of recent T. gondii infection was associated with eating rare lamb [21]. Even frozen lamb meat has been associated with acute T. gondii infection in Brazil [22]. The association of T. gondii infection and consumption of sheep meat in our patients was unexpected since lamb meat consumption was negatively associated with T. gondii infection in a previous study in psychiatric patients in Durango [23]. We are not aware of any previous report about a positive association of T. gondii infection and consumption of sheep meat in Mexico. Remarkably, consumption of rabbit meat was also associated with T. gondii infection in patients (OR = 4.61; 95% CI: 1.06-19.98; P = 0.04). Infections with T. gondii in rabbits have been reported in several countries [24-26]. Antibodies against T. gondii were found in 77 (26.9%) of 286 domestic rabbits from three rabbit farms in Mexico [25]. However, the seroprevalence of T. gondii infection in rabbits in Durango is unknown. To the best of our knowledge there is not any previous report about the association of T. gondii infection and consumption of rabbit meat. In the present study, we also found an association between T. gondii seropositivity and consumption of quail meat (P < 0.01). Experimental infections with T. gondii in bobwhite [27] and Japanese quail [28] have been reported. We are not aware of any previous epidemiological report about the association of T. gondii infection and consumption of quail meat. More expectedly, we observed an association between T. gondii seropositivity and consumption of venison (P = < 0.01). Infections with T. gondii have been reported in deer [29,30]. Consumption of undercooked or uncooked venison has been linked to ocular toxoplasmosis in deer hunters [31]. Interestingly, toxoplasmosis with liver involvement has been reported in deer hunters who had eaten undercooked venison [32].

The frequency of T. gondii seropositivity was higher in patients with reflex impairment (27.8%) than patients without this impairment (8.8%), and this difference showed a borderline significance (P = 0.05). In a previous study in patients with visual impairment in Durango, we found that patients with reflex impairment had a significantly higher frequency of T. gondii infection than those with normal reflexes [33]. Reflex impairment might contribute to reducing the quality of life in T. gondii infected patients.

Conclusions

Seropositivity to T. gondii was comparable among liver disease patients and controls. Further studies with larger sample sizes are needed to elucidate the association of T. gondii with liver disease. Consumption of venison, and rabbit, sheep, and quail meats may warrant further investigation.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CAE conceived and designed the study protocol, participated in the coordination and management of the study, applied the questionnaires, performed the laboratory tests and data analysis, and wrote the manuscript. JLTB and MFMS obtained clinical data, applied the questionnaires and performed the data analysis. SEM performed the statistical analysis. OL performed the data analysis, and wrote the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by Fondos Mixtos Durango-Consejo Nacional de Ciencia y Tecnología, Mexico. Grant No. 66718.

Additional file 1

Patients. Raw data of patients suffering from liver diseases.

Click here for file

Additional file 2

Controls. Raw data of controls of patients suffering from liver diseases.

Click here for file