Toxoplasma gondii Infection and Suicidal Behavior in People with Alcohol Consumption

We determined the association between T. gondii infection and suicidal behavior in people with alcohol consumption. One-thousand four-hundred and twenty-three people with alcohol consumption were screened for suicidal behavior and tested for anti-T. gondii IgG and IgM antibodies using commercially available enzyme-linked immunosorbent assays. Anti-T. gondii IgG antibodies were found in 34 of 224 (15.2%) individuals with suicidal ideation and in 118 (9.8%) of 1199 individuals without suicidal ideation (OR: 1.63; 95% CI: 1.08–2.47; p = 0.01). Seropositivity to T. gondii was associated with suicidal ideation in women (OR: 2.24; 95% CI: 1.33–3.78; p = 0.001) and in individuals aged ≤30 years (OR: 2.68; 95% CI: 1.22–5.87; p = 0.01) and >50 years (OR: 2.85; 95% CI: 1.19–6.77; p = 0.01). Anti-T. gondii IgG antibodies were found in 17 of 136 (12.5%) individuals with suicide attempts and in 135 (10.5%) of 1287 individuals without suicide attempts (OR: 1.21; 95% CI: 0.71–2.08; p = 0.47). Seroprevalence of T. gondii infection was associated with suicide attempts in women (OR: 1.88; 95% CI: 0.99–3.55; p = 0.04). No association between anti-T. gondii IgM and suicidal ideation or suicide attempts was found. Results suggest that T. gondii infection is associated with suicidal behavior in people with alcohol consumption.


Introduction
Toxoplasma gondii (T. gondii) is an apicomplexan protozoan [1]. Infections with T. gondii are common in humans and animals worldwide [2]. About 30% of the world's population is infected with T. gondii [3]. Toxoplasmosis, the disease caused by T. gondii, is considered a neglected parasitic infection requiring public health action [4]. Infection with T. gondii is mainly acquired by ingestion of food or water that is contaminated with oocysts shed by cats or by eating undercooked or raw meat containing tissue cysts [5]. Clinically, infection with T. gondii is usually asymptomatic in immunocompetent individuals; however, in immunocompromised patients or an infected fetus, it may cause devasting effects [6]. Lymphadenopathy, ocular disease and encephalitis are some of the clinical features of toxoplasmosis [5]. However, T. gondii has been linked to behavioral changes in humans [7]. Infection with T. gondii has been associated with schizophrenia [8,9], mixed anxiety and depressive disorder [10], anxiety, obsessive-compulsive disorder, autism [11], and greater impulsivity and aggressiveness [12]. Several studies have linked T. gondii infection and self-harm and suicidal behaviors [13][14][15][16][17][18]. Suicide is an important public health concern that takes around 800,000 lives globally every year [19]. Alcohol consumption is a wellrecognized factor associated with suicidal behavior [20][21][22][23]. Ingestion of alcohol may occur prior to suicide, as shown in a recent study where alcohol in blood was detected in 30.2% of suicide victims [24]. There is currently no information about the link between T. gondii infection and suicidal behavior in persons with alcohol consumption. Therefore, we sought to determine the association between T. gondii exposure and suicidal behavior in a sample of people with alcohol consumption in Durango City, Mexico. In this northern Mexican city, a low (6.1%) seroprevalence of T. gondii infection in the general population has been reported [25].

Results
Of the 1423 people with alcohol consumption studied, 224 (15.7%) had a history of suicidal ideation and 1199 (84.3%) did not have a history of suicidal ideation. Anti-T. gondii IgG antibodies were found in 34 of the 224 (15.2%) individuals with suicidal ideation and in 118 (9.8%) of the 1199 individuals without suicidal ideation. Seroprevalence of T. gondii infection was significantly higher in individuals with suicidal ideation than in individuals without suicidal ideation (OR: 1.63; 95% CI: 1.08-2.47; p = 0.01). Table 1 shows a stratification by age and sex and seroprevalence of T. gondii infection in individuals with and without suicidal ideation. Stratification by sex showed that women with suicidal ideation had a significantly higher (23/167: 13.8%) seroprevalence of T. gondii infection than women without suicidal ideation (53/799: 6.6%) (OR: 2.24; 95% CI: 1.33-3.78; p = 0.001). Whereas stratification by age groups showed that seroprevalence of T. gondii infection was significantly higher in individuals aged ≤30 and >50 years with suicidal ideation than individuals of the same age groups without suicidal ideation. High (>150 IU/mL) anti-T. gondii IgG antibody levels were found in 12 (5.4%) of the 224 individuals with suicidal ideation and in 50 (4.2%) of the 1199 individuals without suicidal ideation (OR: 1.30; 95% CI: 0.68-2.48; p = 0.42). Table 2 shows a stratification by sex and age groups of the study population and the association between high (>150 IU/mL) anti-T. gondii IgG antibody levels and suicidal ideation. Stratification by sex and age groups showed no association between the rates of high levels of anti-T. gondii antibodies and suicidal ideation.
Concerning suicide attempts, of the 1423 people with alcohol consumption studied, 136 (9.6%) had a history of suicide attempts and 1287 (90.4%) did not have a history of suicide attempts. Anti-T. gondii IgG antibodies were found in 17 of the 136 (12.5%) individuals with suicide attempts and in 135 (10.5%) of the 1287 individuals without suicide attempts. Seroprevalence of T. gondii infection in individuals with suicide attempts (12.5%) was similar to that (10.5%) in individuals without suicide attempts (OR: 1.21; 95% CI: 0.71-2.08; p = 0.47). A stratification by age and sex and seroprevalence of T. gondii infection in individuals with and without suicide attempts is shown in Table 3. Women with suicide attempts had a significantly higher (13/101: 12.9%) seroprevalence of T. gondii infection than women without suicide attempts (63/865: 7.3%) (OR: 1.88; 95% CI: 0.99-3.55; p = 0.04). Stratification by age groups showed no association between the seropositivity to T. gondii and suicide attempts. Table 2. Stratification by sex and age and the association between high (>150 IU/mL) levels of anti-T. gondii IgG and suicidal ideation in people with alcohol consumption. High (>150 IU/mL) anti-T. gondii IgG antibody levels were found in 5 (3.7%) of the 136 individuals with suicide attempts and in 57 (4.4%) of the 1287 individuals without suicide attempts (OR: 0.82; 95% CI: 0.32-2.09; p = 0.82). Table 4 shows a stratification by sex and age groups of the study population and the association between high (>150 IU/mL) anti-T. gondii IgG antibody levels and suicide attempts. Stratification by sex and age groups showed no association between the rates of high levels of anti-T. gondii antibodies and suicide attempts.

History of Suicidal Ideation
Anti-T. Suicide attempters have had from 1 to 20 suicide attempts (median: 1). Table 5 shows the association between the number of suicide attempts and rates of seropositivity and high levels of anti-T. gondii IgG antibodies. Seroprevalence of T. gondii infection or the rates of high anti-T. gondii IgG antibodies did not vary with the number of suicide attempts.  Methods of suicide attempt reported by suicide attempters included wrist cuts (n = 22), hanging (n = 15), drug overdose (n = 63), and other (n = 17) (firearm, poisoning, and throwing oneself to vehicles). Seroprevalence of T. gondii infection did not vary with the method of suicide attempt (p = 0.60). In contrast, the frequency of high anti-T. gondii antibodies was significantly higher in individuals with wrist cuts (3/22: 13.6%) than in individuals who used hanging, drug overdose, and other methods for suicide attempts (1/95: 1.1%) (OR: 14.84; 95% CI: 1.46-150.46; p = 0.02). The frequency of seropositivity to IgM among individuals with anti-T. gondii IgG antibodies was similar among the groups of methods used for suicide attempts (p = 0.34).

Discussion
Alcohol consumption and T. gondii exposure have been individually associated with suicidal behavior. However, there is a lack of information about the association between T. gondii exposure and suicidal behavior in persons with alcohol consumption. In the current study, we assessed the association between T. gondii exposure and suicidal behavior in a sample of people with alcohol consumption in Durango, Mexico. We found a significantly higher seroprevalence of T. gondii infection in individuals with suicidal ideation than in individuals without suicidal ideation. The 15.7% seroprevalence of T. gondii infection found in individuals with suicidal ideation in the current study is also higher than the one (6.1%) reported in the general population of Durango City [25]. Results thus suggest that T. gondii exposure is associated with suicidal ideation in individuals with alcohol consumption. This association was observed in women and individuals aged ≤30 years and >50 years. Our results are in line with another report on the association between T. gondii exposure and suicidal behavior. In a study of children and adolescents aged 11 to 18 years with depression, investigators found that seropositivity to T. gondii was associated with suicidal ideation [26]. In contrast, our results conflict with a negative association between T. gondii exposure and suicidal ideation in psychiatric patients suffering from mental and behavioral disorders due to psychoactive substance use [27]. However, in that study, only 38 patients with alcohol use were studied. In a National Health and Nutrition Examination Survey with data collected between 2009 and 2012 that included subjects aged 20 to 80 years in the USA, researchers found no association between suicidal ideation and anti-T. gondii antibody titer [28]. Differences in the association among the studies could be due to differences in the characteristics of the study population. We studied only people with alcohol consumption whereas researchers in the American survey studied the general population. In addition, risk factors for T. gondii infection and suicidal behavior might be different among the study populations. In the present study, exposure to T. gondii was not associated with suicidal ideation in the middle age group (31-50 years). It is unclear why there was not an association in this age group. It raises the question of whether this age group could be more resistant to the possible behavioral effects induced by T. gondii than the younger and older age groups. With respect to suicide attempts, we found an association between T. gondii seropositivity and suicide attempts in women but not in men or the whole population studied. It is not clear why this association was found in women but not in men. It is possible that the greater association of T. gondii exposure and suicidal behaviors found more in women rather than in men could be due to the difference in risk factors, such as type of work, diet, contact with animals, and others among the gender groups. The 9.6% seroprevalence of T. gondii infection found in individuals with suicide attempts in the current study is slightly higher than the one (6.1%) reported in the general population of Durango City [25]. The lack of association between suicide attempts and T. gondii seropositivity found in the whole study population in this survey agrees with results found in other studies. A lack of association between T. gondii seropositivity and suicide attempts was reported in patients with mood disorders in the USA [13] and in psychiatric outpatients in Mexico [15]. In contrast, in a study in Turkey, researchers found a significantly higher rate of T. gondii seropositivity in suicide attempters than in healthy volunteers [29]. On the other hand, the association between T. gondii exposure and suicide attempts in women found in the current study agrees with that found in a study in the USA where researchers observed a positive relationship between rates of infection with T. gondii and suicide in women of postmenopausal age [14]. In a study of schizophrenic patients in Iran, investigators found a lower rate of suicide attempts in T. gondii seropositive male patients than in seronegative ones [30]. In the present study, no association between the frequency of high anti-T. gondii IgG antibody levels and suicide attempts was found. This result conflicts with those reported in other studies. An association between the frequency of high anti-T. gondii antibody levels and suicide attempts were found in psychiatric outpatients in Mexico [15], patients with recurrent mood disorders in the USA [13], and schizophrenic patients younger than 38 years in Germany [17]. The difference in the findings among the studies could be due to differences in the characteristics of the participants among the studies. It seems that the association between high anti-T. gondii antibody levels and suicide attempts is more readily observed in psychiatric patients than in people with alcohol consumption. In the current study, no association between T. gondii seropositivity or serointensity and number of suicide attempts was found. The lack of this association was also reported in a study of patients with recurrent mood disorders [13]. In contrast, a study in psychiatric outpatients found that the seroprevalence of T. gondii infection increased with the number of suicide attempts [15]. Interestingly, we found that the frequency of high anti-T. gondii antibodies was significantly higher in individuals with wrist cuts than in individuals who used other methods for suicide attempts. Further research to determine the association between T. gondii serointensity and methods for suicide attempts should be conducted.
This study has some limitations: (1) we considered a qualitative (history of alcohol consumption) rather than a quantitative (number and type of alcoholic drinks) criterium for enrollment of participants; and (2) we could not trace back the date of infection and correlated it with suicidal behavior. Therefore, further research to determine the relationship between T. gondii exposure and: (1) the quantity of alcohol consumption, and (2) the time of having a suicidal behavior episode is needed. The association of suicidal behavior and T. gondii exposure found in the present study should not be concluded as categorical due to the possible presence of interactions of different risk factors and the absence of knowledge and characteristics that would be important to determine the certainty of this epidemiological link. Additional and more complete studies to confirm the association between T. gondii exposure and suicidal behavior in alcohol consumers should be conducted.

Materials and Methods
We performed a cross-sectional study of 1423 people with alcohol consumption in Durango, Mexico from 2014 to 2019. The following inclusion criteria were established: people with a history of alcohol consumption (at least one alcoholic drink every month for the last six months) and any gender, aged ≥15 years, with informed consent. In this exploratory study, the quantity of alcohol consumption was not considered for analysis. Birthplace and residence place were not restrictive criteria for enrollment. Participants were enrolled at public health institutions (hospitals and health care units). Participation was voluntary without compensation. Data were collected and revised by our research team with the aid of the laboratory personnel of health institutions and students.
Face-to-face interviews were conducted and a questionnaire was used to record the sociodemographic and clinical data of participants. Sociodemographic data of participants are shown in Table 6. The mean age of the study population was 38.13 ± 12.99 (range 15-84) years. There were more women than men because women were more willing to participate than men. Most (1010: 71.0%) participants were healthy, 408 (28.7%) were ill, and in 5 (0.3%) no information about health status was recorded. No information about the treatment of psychiatric disorders was obtained. None of the participants reported having an alcohol use disorder. Suicidal behavior items include a history of suicidal ideation, suicide attempts, number of suicide attempts, date of last suicide attempt, and method of suicide attempt.
A blood sample from each participant was obtained. Blood was centrifuged and serum was obtained and stored at −20 • C until analyzed. Serum samples were tested for anti-T. gondii IgG antibodies using a commercially available enzyme immunoassay "Toxoplasma gondii IgG" kit (Diagnostic Automation/Cortez Diagnostics, Inc., Woodland Hills, CA, USA). Serum samples with anti-T. gondii IgG antibodies were further tested for anti-T. gondii IgM antibodies by a commercially available enzyme immunoassay, the "Toxoplasma gondii IgM" kit (Diagnostic Automation/Cortez Diagnostics, Inc.). All assays were performed following the manufacturer's instructions.
The data were analyzed with the software Epi Info version 7 and IBM SPSS Statistics version 20. The Pearson's chi-square test and the Fisher exact test (when values were less than 5) were used to compare the frequencies among the groups. The sample size (n = 941) was calculated using a population size of 100,000, a 99% confidence level, an expected frequency of T. gondii infection of 6.1% [25], and 2% of confidence limits. Therefore, the number of participants (n = 1423) was representative of the larger population from which it was collected. Odd ratios (OR) and 95% confidence intervals (CI) were calculated, and the statistical significance was set at a p value < 0.05.

Conclusions
The results of this study suggest that T. gondii seropositivity is associated with suicidal behavior in people with alcohol consumption. Results of this first study on the association between T. gondii exposure and suicidal behavior in people with alcohol consumption warrant further research.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.