# Investigation of Anti-Toxocara and Anti-Toxoplasma Antibodies in Patients with Schizophrenia Disorder

Authors: Shahram Khademvatan; Niloufar Khajeddin; Sakineh Izadi; Elham Yousefi
Journal: Schizophrenia Research and Treatment
 (2014)

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## Abstract

Objective. The aim of the present study was to examine the relationship between Toxoplasma gondii and Toxocara spp. infections in patients with schizophrenia disorder. Method. A total of 100 patients with schizophrenia disorder and 95 healthy individuals participated in the study. Participants were tested for the presence of anti-T. gondii and anti-Toxocara spp. antibodies by ELISA and Western blotting. Data were analyzed using Chi-square test and Fisher9s exact test. Results. There were no differences in T. gondii IgG seroprevalence between patients with schizophrenia and healthy individuals (P
=
0.1), but there were differences in seroprevalence between males and females with schizophrenia (P
=
0.009). In contrast, Toxocara spp. IgG seroprevalence was greater in patients with schizophrenia disorder than in healthy individuals (P
=
0.02), but there were no differences in seroprevalence between men and women with schizophrenia (P
=
0.5). Finally, there were no differences in seroprevalence of T. gondii or Toxocara spp. IgG among different subtypes of schizophrenia, various age groups, residential area, or clinical course of treatment (P
>
0.05). Conclusion. The present study suggests that patients with schizophrenia disorder are at elevated risk of Toxocara spp. infection. Moreover, contamination with T. gondii is a risk factor for schizophrenia in women.


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## Body

## 1. Introduction

Schizophrenia is a severe, disabling mental disorder with a devastating impact on patients, their family, and society [1]. Schizophrenia is a heterogeneous disorder characterized by a range of clinical features such as positive and negative symptoms [2], including a reduction in patients’ health-related quality of life [3]. The prevalence of schizophrenia has been reported to be 1% of the adult population [4], developing in late adolescence or early adulthood, and most patients suffer from the disease throughout their lifetime [1].Toxocariasis is a helminthozoonosis, caused by the Ascaridida nematodes,Toxocara canis andToxocara cati [5]. Dogs and cats are the definitive hosts ofT. canis andT. cati, respectively, although other mammals, such as humans and rodents, can be infected [6]. Humans are infected by ingestion of infectious eggs [6], often as a result of direct contact with pets or consumption of contaminated vegetables or undercooked meat [7].Toxocara infection is also often transmitted by contact with the soil. Young children, people who live in rural areas, and people with soil-related occupation are at increased risk for toxocariasis [8–10]. The presence ofToxocaraspp. larvae in the central nervous system may cause some neurological and psychiatric disorders including schizophrenia. Kaplan et al. [11] found more seroprevalence ofToxocara spp. infection in patients with schizophrenia than in healthy individuals (45.9% versus 2%). Further, in the study conducted by El-Sayed and Ismail [12], toxocariasis was detected in 23.3% of patients with schizophrenia disorder compared with that in 2.2% of healthy controls. In another study by Alvarado-Esquivel [13], 4.7% of psychiatric inpatients were positive for anti-Toxocara IgG antibodies compared with 1.1% of healthy controls. The difference was statistically significant.Another parasite that is thought to be associated with schizophrenia isToxoplasma gondii. Toxoplasmosis is one of the most common parasitic infections in human and other warm-blooded vertebrates including birds, livestock, and marine mammals [14, 15]. Humans usually become infected by consumption of undercooked meat, unwashed or poorly washed vegetables, or contaminated drinking water [16]. After a short phase of acute toxoplasmosis, the infection becomes latent and becomes encysted in the central nervous system and in muscle tissues, potentially for the life of the infected host [17, 18]. The parasite has the ability to alter the behavior of the host to increase transmission [19]. Some studies have found changes in infected individuals’ personality characteristics [20–24].There is much evidence to suggest that schizophrenia disorder is associated with toxoplasmosis [25–32]. Daryani et al. [33] and Hamidinejat et al. [34] from Iran also observed that higher titers ofT. gondii IgG antibody was positively correlated with schizophrenia [33–35]. Some results however challenge the plausibility of this association [36, 37]. Because of these controversial findings and little knowledge about the prevalence ofToxocara spp. infection in patients with schizophrenia, the present study aimed to determine the seroprevalence ofToxocara spp. andToxoplasma infections in patients with schizophrenia disorder and to compare it with healthy controls in Ahvaz, Southwest Iran.


## 2. Methods

Research was conducted over a period of 12 months from 2012 to 2013. A total of 100 patients diagnosed as having schizophrenia disorder and 95 matched healthy controls participated in the study. The patients had been admitted into Golestan hospital affiliated to Jundishapur University of Medical Sciences in Ahvaz, Iran. The diagnosis of schizophrenia disorder was performed by two psychiatrists following the DSM-IV-TR criteria. The control group consisted of blood donors who were tested at the laboratory in Jundishapur University of Medical Sciences. The controls were assessed by both a clinical psychologist and a psychiatrist and had no history of schizophrenia disorder. Neither of the 2 groups were immunodeficient nor were any other major psychiatric disorders or neurological diseases presentable.To participate, subjects had to agree to comply with the requirements of study, and after describing the procedures and purposes of the study, written informed consents were obtained. The hospital’s institutional review board approved all procedures.A 5 mL blood sample was taken from each subject for serological analysis. Each subject was also asked to complete a questionnaire to obtain demographic data about ethnicity, gender, age, level of education obtained, marital status, and employment.


### 2.1. Serological Tests

Sera were separated by sample centrifugation at 3000 rpm for 5 min and then kept at −20°C until analysis. Anti-Toxoplasma antibodies (IgG and IgM) concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (IgG and IgM: Trinity Biotech Captia, USA). In addition, all sera were examined byToxocara IgG-ELISA test (IBL, International Gmbh, Hamburg, Germany) andToxocara Western blot test (LDBIO Diagnostics, Lyon, France) for confirmation.


### 2.2. Statistical Analysis

Data were analyzed using Chi-square test, Fisher’s exact test, and Student’st-test. Odds ratios (OR) with 95% confidence intervals (CI) were also determined. P
<
0.05 was considered significant. Statistical analyses were carried out using SPSS v16 software.


## 2.1. Serological Tests

Sera were separated by sample centrifugation at 3000 rpm for 5 min and then kept at −20°C until analysis. Anti-Toxoplasma antibodies (IgG and IgM) concentrations were measured by enzyme-linked immunosorbent assay (ELISA) (IgG and IgM: Trinity Biotech Captia, USA). In addition, all sera were examined byToxocara IgG-ELISA test (IBL, International Gmbh, Hamburg, Germany) andToxocara Western blot test (LDBIO Diagnostics, Lyon, France) for confirmation.


## 2.2. Statistical Analysis

Data were analyzed using Chi-square test, Fisher’s exact test, and Student’st-test. Odds ratios (OR) with 95% confidence intervals (CI) were also determined. P
<
0.05 was considered significant. Statistical analyses were carried out using SPSS v16 software.


## 3. Results

Demographic characteristics (sex, residence, marital status, level of education obtained, ethnicity, and age) of both patients and healthy controls are detailed in Table1. Patient and control group seroprevalence for bothT. gondii andToxocara was evaluated. Anti-Toxplasma antibodies were detected in 34% of patients with schizophrenia disorder and 47.39% of healthy individuals (P
>
0.05) (Table 2). Anti-Toxoplasma IgM antibodies were positive in 4 patients and 2 healthy individuals (P
>
0.05).Table 1
Demographic characteristics of the patient and control groups.
Feature
Frequency
Patients’ group
Control group
Total
T.  gondii
Positive
34 (34%)
45 (47.36%)
79 (40.51%)
Negative
66 (66%)
50 (52.63%)
116 (59.48%)
Toxocara
Positive
14 (14%)
4 (4.39%)
18 (9.23%)
Negative
86 (86%)
91 (95.78%)
177 (90.76%)
Sex
Female
35 (35%)
43 (45.74%)
78 (40.20%)
Male
65 (65%)
51 (54.25%)
116 (59.79%)
Residence
Urban
76 (76%)
89 (93.98%)
165 (84.61%)
Rural
24 (24%)
6 (6.31%)
30 (15.38%)
Marital status
Single
64 (64%)
77 (81.05%)
141 (72.30%)
Married
25 (25%)
18 (18.94%)
43 (22.05%)
Divorced/widowed
11 (11%)
0 (0)
11 (5.64%)
Level of  education
Grade school
74 (74%)
0 (0)
74 (37.94%)
12 years/high school
18 (18%)
9 (9.47%)
27 (13.84%)
University degree
8 (8%)
86 (90.52%)
94 (48.20%)
Ethnicity
Fars
46 (48.42%)
15 (17.85%)
61 (34.07%)
Arab
15 (15.78%)
33 (39.28%)
48 (26.81%)
Lor
19 (20%)
34 (40.47%)
53 (29.60%)
Others
15 (15.78%)
2 (2.38%)
17 (9.49%)
Age (years)
<20
0 (0)
4 (4.21%)
4 (2.05%)
20–29
32 (32%)
83 (87.36%)
115 (58.97%)
30–39
34 (34%)
7 (7.36%)
41 (21.02%)
40–49
23 (23%)
0 (0)
23 (11.79%)
>50
11 (11%)
1 (1.05%)
12 (6.15%)Table 2
Seropositivity ofToxoplasma gondii and Toxocara antibodies in patients with schizophrenia disorder and healthy controls.
Patients’ group
Healthy
Sig
OR
CI95
N (%)
N (%)
Toxoplasma gondii
34/100 (34%)
45/95 (47.36%)
0.08
0.57
0.32–1.02
Toxocara
14/100 (14%)
4/95 (4.3%)
0.02
0.27
0.08–0.8Seroprevalence of anti-Toxocara IgG were higher in schizophrenia patients (14%) than in healthy controls (4.3%) (P
=
0.02; Table 2). Western blot analysis confirmedToxocara spp. infection, with low molecular weight bands (24–35 kDa) that are specific forToxocara IgG present from all positive sera.With respect to schizophrenia subtypes, anti-T. gondii antibodies were present in 35.82% (24 of 67) of patients with paranoid type of schizophrenia disorder, 42.85% (3 of 7) of patients with catatonic type, 33.3% (6 of 18) of patients with undifferentiated type, and 33.3% (1 of 3) of patients with residual type. None of the patients with disorganized type were seropositive. The difference in infection rate among the subtypes was not statistically significant (P
=
0.9, Table 3). Anti-Toxocara antibodies were detected in 1.56% (10 of 64) of patients with paranoid type of schizophrenia disorder, 14.28% (1 of 7) of patients with catatonic type, 11.11% (2 of 18) of patients with undifferentiated type, and 20% (1 of 5) patients with disorganized type. None of the patients with residual type were seropositive. The difference in infection rate among the subtypes was not statistically significant (P
=
0.5, Table 3). Serological analyses also confirmed that there were no significant increases in anti-Toxocara and anti-Toxocara antibody levels in patients with first-episode Schizophrenia disorder compared with those in patients with recurrent episodes (P
>
0.05, Table 4).Table 3
The distribution ofToxoplasma gondii and Toxocara antibodies in patients with different subtypes of schizophrenia disorder.
Subtypes of schizophrenia
T. gondii
N (%)
P value
Toxocara
N (%)
P value
Paranoid type
24/67 (35.82%)
0.9
10/64 (1.56%)
0.5
Catatonic type
3/7 (42.85%)
1/7 (14.28%)
Residual type
1/3 (33.3%)
0/3 (0)
Undifferentiated type
6/18 (33.3%)
2/18 (11.11%)
Disorganized type
0/5 (0%)
1/5 (20%)Table 4
The distribution ofToxoplasma gondii and Toxocara antibodies in patients with schizophrenia based on clinical course of treatment.
Clinical course
N (%)
P value
OR
CI95
Toxoplasma gondii
First episode
13/30 (43.33%)
0.1
2.08
0.84–5.12
Recurrent episodes
18/67 (26.86%)
Toxocara
First episode
8/30 (26.66%)
0.06
0.22
0.06–0.75
Recurrent episodes
5/30 (16.66%)The seroprevalence of anti-T. gondii IgG antibodies in male and female patients with schizophrenia disorder was 24.61% and 51.42%, respectively, (P
=
0.009, Table 5). Anti-Toxocara antibodies were detected in 51.42% of females and 17.14% of males with schizophrenia disorder (nonsignificant). The seroprevalence ofT. gondii infection in patients living in urban and rural areas was 21.05% and 16.66%, respectively, (nonsignificant, P
=
0.7). Seroprevalence ofToxocara also was not different between patients living in urban and rural areas (Table 6).Table 5
The seroprevalence of anti-T. gondii IgG antibodies in patients with schizophrenia disorder based on gender.
Patients’ group
N (%)
Sig
OR
CI
Male
Female
Toxoplasma  gondii
16/65 (24.61%)
18/35 (51.42%)
0.009
0.3
0.12–0.73
Toxocara
8/65 (12.30%)
6/35 (17.14%)
0.5
1.47
0.46–4.65Table 6
The distribution ofToxoplasma gondiiand Toxocaraantibodies in patients with schizophrenia disorder based on their residential area.
Patients' group
N (%)
Sig
OR
CI
Urban
Rural
Toxoplasma  gondii
16/76 (21.05%)
4/24 (16.66%)
0.7
1.36
0.39–4.7
Toxocara
6/76 (7.89%)
3/24 (12.5%)
0.3
1.7
0.37–7.6The participants were divided into 5 groups based on their age (<20, 20–29, 30–39, 40–49, and >50 years). The seroprevalence ofT. gondii infection in the five age groups were 0%, 32.25%, 40%, 26.08%, and 36.36%, respectively, in patients; in healthy individuals the seroprevalence was 0%, 44.57%, 71.42%, 0%, and 100%, respectively, (nonsignificant between or within age subgroups, P
>
0.05, Table 7). ForToxocara seroprevalence, there were no differences between or within age subgroups (P
>
0.05, Table 8). Anti-Toxocara antibodies were significantly more prevalent in men with schizophrenia disorder (12.30%) than in healthy men (1.92%) (P
=
0.03), but there was no difference in women (P
=
0.1).Table 7
The distribution of latent toxoplasmosis according to the age in patients with schizophrenia disorder and healthy controls.
Patients’ group
Healthy
Sig
OR
CI95
N (%)
N (%)
Age (years)
<20
0 (0%)
0/4 (0%)
20–29
10/32 (32.25%)
37/83 (44.57%)
0.1
2.9
0.68–12.51
30–39
14/34 (41.17%)
5/7 (71.42%)
0.1
0.2
0.04–1.5
40–49
6/23 (26.08%)
0/0 (0%)
>50
4/11 (36.36%)
1/1 (100%)
0.4
0.3
0.1–0.7Table 8
The distribution of toxocariasis according to the age in patients with schizophrenia disorder and healthy controls.
Patients’ group
Healthy
Sig
OR
CI95
N (%)
N (%)
Age (years)
<20
0 (0%)
0 (0%)
20–29
4/32 (12.5%)
3/83 (4.81%)
0.1
2.9
0.68–12.5
30–39
6/34 (17.14%)
1/7 (14.28%)
0.6
1.2
0.12–12.2
40–49
2/23 (8.69%)
0/0 (0%)
>50
2/11 (18.18%)
0/1 (0%)
0.8
0.81
0.61–1.08


## 4. Discussion

The present study was conducted to investigate associations between schizophrenia disorder and parasitic infections, toxocariasis and toxoplasmosis. Because of poor hygiene, the prevalence ofToxocara spp. is higher in most localities of Iran than any elsewhere in the world. Sharif et al. in northern Iran, Arbabi and Hooshyar in central Iran, Sadjjadi et al. in southern Iran, and Khademvatan et al. in Southwest Iran reported 44%, 13.3%, 52.8%, and 45%, respectively, [6, 38–40]. With prevalence of approximately 50% in Iran, toxoplasmosis continues to be a public health problem [41]. The present study is the first to report toxocariasis in patients with schizophrenia in the Iranian population. The association betweenToxoplasma gondii and schizophrenia also has received little attention in Iran.The results of the present study show that there were significant differences between patients with schizophrenia disorder and healthy controls in seropositivity ofToxocara. We replicate the findings of the study conducted by Kaplan et al. [11] and El-Sayed and Ismail [12] that found thatToxocara spp. infection is related to schizophrenia disorder. Similarly, Alvarado-Esquivel [13] also reported thatToxocara infection is more frequent in patients with schizophrenia.These results may be related to the fact that patients with schizophrenia have inadequate hygiene and self-care skills, and they have a greater tendency to eat inappropriate things [12]. Considering the correlation between lifestyle andToxocara infection, and thatToxocara spp. is common in areas with low hygiene [11], abnormal behaviors and poor personal hygiene observed in patients with schizophrenia expose them to toxocariasis.We considered gender differences in seroprevalence ofToxocara spp. infection. Although anti-toxocariasis IgG antibodies were significantly more prevalent in men with schizophrenia than in healthy men, no difference was found in seroprevalence ofToxocara infection between patients and healthy women. This may be related to the role of gender in the experience of illness, treatment, and recovery of schizophrenia disorder. Women with schizophrenia disorder have better global outcomes than men [42]; hence, it is less probable that women with schizophrenia are exposed to toxocariasis.In the present study, there were no differences in the prevalence ofToxoplasma IgG seropositivity between patients with schizophrenia and healthy subjects. The finding is consistent with the result of the study conducted by Saraei-Sahnesaraei et al. [36] and Xiao et al. [37]. However, these results are in contrast to intervention studies [43, 44] and some direct studies [25–31, 34, 35] that support the link between toxoplasmosis and schizophrenia disorder.The reason for the difference in these findings may relate to the various genotypes ofT. gondii that vary in prevalence geographically [36] and have distinct neuropathogenic potential [34, 35]. It also may be due to the timing and route of infection, varying degrees of pathogenicity among infecting organisms, or heterogeneous etiology of schizophrenia disorder [45].There are a number of challenges to these types of epidemiological studies, including the relative insensitivity of some serological assays [46], arbitrary cutoff selection, classification by percentile ranges, grouping results as low, intermediate, or high, and analyzing antibody levels as a continuous rather than dichotomous or categorical variable [27].In addition, for the purpose of respecting patients’ rights, only those who are able and willing to complete the informed consent form are able to participate in the studies. Previous research suggests that clinical symptoms of infected schizophrenic patients are more severe than those of noninfected patients [47], therefore reducing the probability of incorporating infected schizophrenic patients into studies.In terms of gender difference, we found differences between male and female patients with schizophrenia disorder (P
=
0.009). Similar results, that is, higher seropositivity in schizophrenic women than in schizophrenic men, were also reported by Dickerson et al. [48].The present finding is in contrast with the studies conducted by Alvarado-Esquivel et al. [49], Yuksel [50], and Xiao et al. [37], which demonstrated that seroprevalence of anti-T. gondii IgG is not different between men and women with schizophrenia disorder. On the other hand, Lindová et al. [51] reported higher seropositivity in male than in female schizophrenic patients.Higher burdens in women may be explained on the basis of animal studies. Spleens of male mice produce higher level of interferon-gamma (IFN) in the early stages ofToxoplasma infection than those of female mice. High levels of IFN and tumor necrosis factor-alpha help male mice to respond toT. gondii infection more rapidly and to control the parasite multiplication [52].Regarding the residential area, some previous studies found that the risks forToxocara andToxoplasma infections are higher in rural areas than in urban areas [50, 53–55]. Our study failed to show any differences between residential areas in the prevalence of infections, consistent with some other reports [12, 37].Finally we did not find any significant differences in prevalence oftoxoplasmosis andtoxocariasis infections across various age subgroups, subtypes of schizophrenia, or between patients with first-episode schizophrenia disorder and those with recurrent episode schizophrenia.The screening forToxocara spp. antibodies depends on ELISA and Western blotting, which are the most common methods for immunodiagnosis of toxocariasis. Western blotting is more sensitive and specific than other available tests for diagnosing toxocariasis, capable of detecting low levels ofToxocara antibodies [56].


## 5. Conclusion

In conclusion, our research suggests that patients with schizophrenia disorder are at an elevated risk forToxocara spp. infection. Moreover, contamination withT. gondii should be considered as one of the risk factors for schizophrenia disorder in women.



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