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Toxoplasma Gondii Infection and Depression: A Case–Control Seroprevalence Study

We assessed the association of Toxoplasma gondii infection and depression in a sample of psychiatric patients and control subjects without depression. We performed an age- and gender-matched case–control study of 89 patients suffering from depression attended in a public psychiatric hospital in Durango City, Mexico and 356 control subjects without depression from the general population of the same city. Participants were tested for the presence of anti-Toxoplasma IgG and IgM antibodies using enzyme-linked immunoassays. Anti-T. gondii IgG antibodies were found in 11 (12.4%) of the 89 cases and in 22 (6.2%) of the 356 controls (OR = 2.14; 95% CI: 1.00–4.59; P = 0.04). Anti-T. gondii IgM antibodies were found in four (19%) of 21 anti-T. gondii IgG seropositive controls but not in 11 anti-T. gondii IgG seropositive cases (P = 0.27). Patients aged 30 years old and younger had a significantly higher seroprevalence of T. gondii infection than controls of the same age group (P = 0.001). Results of the present study suggest a potential association between T. gondii infection and depression. Furthers studies to confirm our results and to determine the epidemiology of T. gondii in young depressed patients should be conducted.


Introduction
The coccidian parasite Toxoplasma gondii (T. gondii) causes infections all around the world [1]. Main routes of T. gondii infection are eating raw or under cooked meat containing tissue cysts, and ingestion of water contaminated with oocysts shed by cats [2,3]. Infection with T. gondii can also occur vertically [2,4]. Most infections with T. gondii are asymptomatic. However, infection with T. gondii may lead to eye, lymph node, and central nervous system disease, i.e., in congenital toxoplasmosis and in immunocompromised patients [2,5,6]. Seroprevalence of T. gondii is high in psychiatric patients in general [7,8]. A number of mental illnesses have been linked to T. gondii infection including schizophrenia [9,10], anxiety [11], and cognitive impairment [12][13][14]. There is controversy about an association of T. gondii infection and mood disorders. In a study of psychiatric patents in Cuba, researchers found the highest percentage of reactivity to the toxoplasmin intradermal test among patients with depressive mental disorders [15]. A case of depression with T. gondii seropositivity who responded to antidepressant treatment only after adequate treatment for T. gondii was reported [16]. However, in the Third National Health and Nutrition Survey in the USA, researchers did not fi nd an association between T. gondii seroprevalence and history of major depression or dysthymia [17]. On the other hand, T. gondii antibody titers have been linked to suicide attempts in patients with recurrent mood disorders [18]. Therefore, we sought to determine the association between T. gondii infection and depression in patients attended in a public psychiatric hospital in Durango City, Mexico.

Study design and patients studied
Through an age-and gender-matched case-control study design, we examined 89 psychiatric patients suffering from depression attended in a public psychiatric hospital in Durango, Mexico and 356 control subjects without depression from the general population of the same city. This study was performed from August to November 2015. Inclusion criteria for enrollment of cases were: 1) patients suffering from depression attended in the Hospital of Mental Health "Dr. Miguel Vallebueno" of the Secretary of Health in Durango City; 2) aged 17 years and older; and 3) who accepted to participate in the study. Gender was not a restrictive criterion for enrollment. Of the 89 cases, 64 (71.9%) were females and 25 (28.1%) were males. Mean age in cases was 38.65 ± 12.93 (range 17-78) years old. The diagnosis of depressive disorder was classifi ed using the International Classifi cation of Diseases (ICD-10) (http://www.who.int/classifi cations/icd/en/). Control subjects were matched with cases for gender and age. Controls were randomly selected from the general population of Durango City. Controls considered themselves as healthy without depression. Inclusion criteria for enrollment of controls were: 1) subjects of the general population of Durango City without depression; 2) aged 17 years and older; and 3) who accepted to participate in the study. Of the 356 controls, 260 (73.0%) were females and 96 (27.0%) were males. Mean age in controls was 38.66 ± 12.89 (range 17-80) years old. No statistically signifi cant differences in age (P = 0.99) and gender (P = 0.83) between cases and controls were found.

Detection of anti-T. gondii antibodies
Serum samples from participants were obtained and stored at −20 °C until analyzed. Anti-T. gondii IgG antibodies were determined in sera with the commercially available enzyme immunoassay (EIA) kit "Toxoplasma IgG" (Diagnostic Automation/Cortez Diagnostics Inc., Woodland Hills, CA, USA). Anti-T. gondii IgG antibody levels were expressed as International Units (IU)/ml, and results ≥8 IU/ml were considered positive. Seropositive for anti-T. gondii IgG antibodies were further analyzed for anti-T. gondii IgM antibodies by the commercially available EIA "Toxoplasma IgM" kit (Diagnostic Automation/Cor-tez Diagnostics Inc.). All IgG and IgM assays were performed according to the instructions of the manufacturer.

Statistical analysis
We performed the statistical analysis with the aid of the software SPSS 15.0 (SPSS Inc., Chicago, Illinois) and Epi Info 7. For calculation of the sample size, we used a 95% confi dence level, a power of 80%, a 1:4 proportion of cases and controls, a reference seroprevalence of 6.1% [19] as the expected frequency of exposure in controls, and an odds ratio of 3.3. The result of the sample size calculation was 67 cases and 268 controls. The student's t test was used to compare the age among the groups. The association of T. gondii infection and depression was assessed with the two-tailed Pearson's Chi-square test or the Fisher's exact test (when cell values were less than 5). We calculated the odds ratio (OR) and 95% confi dence interval (CI), and statistical signifi cance was set at a P value of <0.05.

Ethical aspects
This study was approved by The Ethics Committee of the General Hospital of the Secretary of Health in Durango City, Mexico. The purpose and procedures of this casecontrol study were explained to all participants, and a written informed consent was obtained from all of them.

Results
Of the 89 cases of depression included in the study, 69 (77.5%) suffered from a depressive episode (ICD-10 code F32) and 20 (22.5%) from recurrent depressive disorders (ICD-10 code F33). Table 1 shows the diagnoses of the patients studied and their correlation with T. gondii seroprevalence. Anti-T. gondii IgG antibodies were found in 11 (12.4%) of the 89 cases and in 22 (6.2%) of the 356 controls. The seroprevalence of T. gondii infection was signifi cantly higher in cases than in controls (OR = 2.14; 95% CI: 1.00-4.59; P = 0.04). Of the 11 anti-T. gondii IgG positive cases, four (36.4%) had IgG levels higher than 150 IU/ml, one (9.1%) between 100 and 150 IU/ml, and six (54.5%) between 8 and 99 IU/ml. In comparison, of the 22 anti-T. gondii IgG positive controls, 11 (50.0%) had IgG levels higher than 150 IU/ml, one (4.5%) between 100 and 150 IU/ml, and ten (45.5%) between 8 and 99 IU/ml. The frequency of high (>150 IU/ml) anti-T. gondii IgG levels was similar in cases and controls (OR = 1.47; 95% CI: 0.45-4.74; P = 0.51). Seroprevalence of T. gondii infection did not vary with the types of depression diagnoses (Table 1). Patients with a depressive episode (ICD-10 code F32) had a similar seroprevalence of T. gondii than patients with recurrent depressive disorders (ICD-10 code When results of the seroprevalence of T. gondii infection were collapsed by sex and age, we observed that seroprevalence did not vary with sex but with age groups. Patients aged 30 years old and younger had a signifi cantly higher seroprevalence of T. gondii infection than controls of the same age group (P = 0.001) ( Table 2). Seroprevalence did not vary between cases and controls in the age groups of 31-50 years and older than 50 years.

Discussion
Currently, there are fragmentary and sometimes confl icting results about the association of T. gondii infection and depression [15][16][17]. This study aimed to determine whether T. gondii seropositivity is associated with depression in Durango City, Mexico. Results of our study show that psychiatric patients suffering from depression have a signifi cantly higher seroprevalence of T. gondii infection than control subjects without depression in the general population of the same city. Thus, results of this gender-and age-matched case-control seroprevalence provide further support for the association of T. gondii infection with depression as reported in other studies. In a study of 70 women veterans, signifi cant associations between T. gondii seropositivity and the Center for Epidemiologic Studies Depression score, Profi le for Mood States -depression, and total mood disturbance score were found [20]. In a study of pregnant women in the USA, prenatal depression was associated with higher T. gondii titers in infected women [21]. In addition, the report of a 32-year-old male suffering from depression with T. gondii seropositivity who responded to antidepressant treatment only after adequate treatment for T. gondii [16] supports the potential role of T. gondii in depression. In addition, patients suffering from bipolar disorder with positive serology to T. gondii treated with antipsychotic and/or mood stabilizers having known in vitro anti-Toxoplasmic activity had less depressive episodes than patients treated with drugs having no anti-Toxoplasmic activity [22]. On the other hand, researchers did not fi nd a signifi cant OR (1.21; P = 0.28) supporting the asso- ciation of the presence of anti-T. gondii IgG antibodies and major depression in a meta-analysis [9]. Similarly, in the Third National Health and Nutrition Survey in the USA, no association between T. gondii seroprevalence and history of major depression was found [17]. These studies examined the association of T. gondii infection with major depression. However, in the present study, we examined the association of infection with mild, moderate, and severe forms of depression and did not fi nd an association of T. gondii infection with severe depression. Instead, we found an association of infection with mild depression. However, since the present study only included a limited number of cases of severe depression (n = 15), further research with a larger number of cases with severe depression to elucidate the association of this disease with infection should be conducted.
In the present study, seroprevalence of T. gondii infection was similar in male and female patients suffering from depression. In contrast, young patients (30 years old and younger) but not other age groups suffering from depression had a signifi cantly higher seroprevalence than controls of the same age group. Of interest, the seroprevalence (17.9%) found in young patients suffering from depression is remarkably high in Durango City, and seroprevalence of T. gondii infection in patients with depression did not follow the typical increase with age. On the contrary, seroprevalence decreased from 17.9% in the youngest group to 6.7% in the oldest group (51 years and older). In addition, the seroprevalence in young patients with depression is much higher than those reported in subjects of the same age in other population groups in the same city studied; for instance, seroprevalences of T. gondii infection in subjects 30 years and younger were 4.3% in workers with occupational exposure to animals [23], 5.4% in fruit and vegetable workers [24], and 8% in meat workers [25].
Of note, none of the cases seropositive to T. gondii had IgM antibodies, suggesting that depression might be associated with latent infection. It is not clear how T. gondii causes depression. Tachyzoites of T. gondii invade a number of cells in brain [26]. It is possible that T. gondii causes mood disorders through changes in the levels of brain dopamine and serotonin [21]. The T. gondii genome contains two aromatic aminoacid hydroxylases that could affect dopamine and/or serotonin biosynthesis [27]. T. gondii could cause depression by affecting the tryptophan metabolism and the hypothalamic-pituitary-adrenal axis [27].
This study has limitations. The sample size of cases with depression was small. In addition, we studied only patients who sought medical care at the Hospital of Mental Health, and thus, results cannot be extrapolated to the general population. Comparison of T. gondii seroprevalence between depressed patients and patients suffering from psychiatric diseases other than depression is still needed. Further studies with a larger sample size of cases with depression and with population groups other than psychiatric inpatients (i.e., pregnant women, general population) to determine the association of T. gondii exposure and depression should be conducted.

Conclusions
Results of the present study suggest an association between T. gondii exposure and depression. Young depressed patients had the highest seroprevalence of T. gondii infection. Further studies to confi rm our results and to determine the epidemiology of T. gondii exposure in young depressed patients should be conducted.