Seroprevalence and molecular detection of Toxoplasma gondii in patients with obsessive-compulsive disorder: a case-control study in northern Iran

Abstract

Background

The infectious hypothesis of obsessive-compulsive disorder (OCD) has gained traction, with Toxoplasma gondii (T. gondii) proposed as a potential risk factor due to its neurotropic properties and ability to alter neurotransmitter systems. However, epidemiological evidence for an association remains inconsistent. This study aimed to investigate the potential association between acute and chronic T. gondii infection and OCD in a region with high toxoplasmosis prevalence.

Methods

A case-control study was conducted involving 120 OCD patients and 135 healthy controls matched for age and gender in Mazandaran Province, Iran. Serum samples were tested for anti-T. gondii immunoglobulin (Ig) G and IgM antibodies using a commercial ELISA kit. Additionally, buffy coat samples were analyzed for T. gondii DNA using a nested-PCR assay targeting the 529 bp RE gene. We employed descriptive statistics to summarize demographic and clinical variables, used the Chi-square test for categorical data, and conducted independent-samples t-tests for continuous variables. Additionally, we applied logistic regression to evaluate the association between T. gondii infection and OCD, adjusting for confounding factors, with significance set at P < 0.05.

Results

The seroprevalence of anti-T. gondii IgG was not significantly different between OCD patients (55.0%, 66/120) and healthy controls (52.6%, 71/135) (OR: 1.10; 95% CI: 0.67–1.81, P = 0.70). All participants were negative for IgM. Molecular detection by PCR was positive in 5.8% (7/120) of patients and 3.7% (5/135) of controls, a non-significant difference (OR: 1.33; 95% CI: 0.44–4.08, P = 0.62). A significant correlation was observed between increasing age and IgG seropositivity in both groups (P < 0.05).

Conclusion

This study found no significant association between latent T. gondii infection and OCD. The seroprevalence and molecular evidence do not support a direct causal role for T. gondii in OCD pathogenesis within the studied population. Further longitudinal research is needed to investigate this relationship.

Introduction

Obsessive-compulsive disorder (OCD) is a chronic, disabling psychiatric illness characterized by the presence of intrusive, unwanted, and distressing thoughts (obsessions) and repetitive, ritualistic behaviors or mental acts (compulsions) that patients feel compelled to perform in order to alleviate anxiety. Globally, OCD affects nearly 2–3% of the population, ranking among the most common psychiatric disorders and exerting a profound impact on daily functioning, social relationships, and occupational productivity [1, 2]. Importantly, the disorder has been recognized by the World Health Organization as one of the leading causes of non-fatal disease burden among young adults, highlighting not only its clinical significance but also its socio-economic implications [3].

Although the etiology of OCD has traditionally been conceptualized within the framework of neurobiological models, it is increasingly acknowledged that such models alone cannot sufficiently explain the heterogeneity of clinical presentation and treatment response [4]. Neuroimaging studies have consistently implicated dysfunction of the cortico-striato-thalamo-cortical (CSTC) circuits, while neurochemical models emphasize serotonergic and dopaminergic dysregulation, and more recently, glutamatergic imbalance [5, 6]. Nevertheless, despite the substantial progress in elucidating these neurobiological underpinnings, the precise mechanisms remain incompletely understood, suggesting that additional environmental and immunological contributors must be considered [7].

In this regard, the infectious and immune-mediated hypotheses of OCD have attracted growing attention over the past two decades. Perhaps the most notable example is the paradigm of pediatric autoimmune neuropsychiatric disorders associated with streptococcal infections (PANDAS), which posits that post-streptococcal autoimmune processes can target basal ganglia circuits, resulting in the abrupt onset of obsessive-compulsive symptoms in children [8]. This model, although not without controversy, has provided a conceptual framework linking microbial pathogens, immune dysregulation, and psychiatric symptomatology. Importantly, it has encouraged researchers to examine whether other pathogens with neurotropic potential might similarly contribute to the onset or exacerbation of OCD in susceptible individuals [9].

Among such pathogens, Toxoplasma gondii (T. gondii), an obligate intracellular protozoan parasite with a global prevalence estimated at one-third of the world’s population, has emerged as a particularly intriguing candidate. T. gondii is transmitted primarily through ingestion of oocysts from contaminated food or water, or tissue cysts present in undercooked meat. Once ingested, the parasite establishes chronic, latent infection by encysting in neural and muscular tissue, where it can persist for the lifetime of the host [10]. Notably, the parasite possesses a unique capacity to modulate host immune responses, alter neurotransmitter metabolism, and induce subtle but chronic neuroinflammatory processes. Collectively, these features provide biologically plausible mechanisms through which T. gondii might influence the pathophysiology of psychiatric disorders, including OCD.

Experimental evidence supports this notion. For example, T. gondii has been shown to encode tyrosine hydroxylase-like enzymes capable of catalyzing dopamine synthesis, thereby directly interfering with dopaminergic transmission—a system strongly implicated in OCD pathophysiology [11]. Furthermore, latent toxoplasmosis has been associated with microglial activation and increased production of pro-inflammatory cytokines, which in turn may disrupt glutamatergic and serotonergic signaling, both of which are relevant to OCD [12, 13]. Importantly, animal studies have demonstrated that infected rodents exhibit behavioral alterations such as reduced aversion to predator odor, a phenomenon sometimes interpreted as “parasitic manipulation,” which underscores the parasite’s ability to alter host behavior at a fundamental level [14].

Epidemiological investigations into the relationship between T. gondii and psychiatric disorders have yielded intriguing but inconsistent findings. The most robust evidence exists for schizophrenia, where multiple meta-analyses have confirmed an association between latent toxoplasmosis and increased risk of illness [15]. By contrast, the evidence for a link between T. gondii and OCD remains mixed, with both positive and null associations reported in the literature [16, 17]. A systematic review and meta-analysis highlighted this inconsistency, concluding that while the evidence does not definitively support a causal relationship, the hypothesis remains biologically plausible and warrants further investigation [18].

Mazandaran Province in northern Iran offers a particularly relevant setting for such an investigation, as the prevalence of toxoplasmosis is relatively high due to dietary habits and environmental factors, including widespread exposure to undercooked meat and close contact with domestic cats. Against this backdrop, the present study was designed to evaluate whether chronic T. gondii infection is associated with OCD in this region. By employing both serological (ELISA) and molecular (nested-PCR) diagnostic approaches in a case-control design, this study aims not only to clarify the potential link between T. gondii and OCD but also to contribute to the broader discourse on infectious hypotheses of psychiatric disorders. Importantly, by integrating clinical, epidemiological, and biological perspectives, we seek to provide a nuanced understanding of this complex relationship, thereby offering insights that may ultimately inform prevention and therapeutic strategies.

Subjects and methods

Study design and participants

The present investigation was designed as a case–control study, carefully structured to examine the potential association between acute and chronic T. gondii infection and OCD. Importantly, the study was conducted in Mazandaran Province, northern Iran, during the years 2021–2022, a region where the background prevalence of toxoplasmosis is comparatively high owing to dietary habits, environmental exposures, and cultural practices. A total of 255 participants were enrolled, consisting of 120 patients diagnosed with OCD and 135 healthy controls. Patients were recruited from psychiatric outpatient clinics affiliated with Mazandaran University of Medical Sciences, whereas controls were selected from the general community through advertisement and volunteer participation. Obsessive-compulsive symptoms were assessed using the Persian-language version of Obsessive-Compulsive Inventory-Revised (OCI-R), a validated 18-item self-report measure [19]. Matching was undertaken for age and gender to minimize confounding. The study protocol was reviewed and approved by the Ethics Committee of Mazandaran University of Medical Sciences (approval code: IR.MAZUMS.REC.1401.387). Written informed consent was obtained from all participants prior to enrollment, in accordance with the principles of the Declaration of Helsinki. Confidentiality of data was strictly preserved, and participants were informed of their right to withdraw at any stage without prejudice.

Inclusion and exclusion criteria

Inclusion criteria for the case group comprised: (i) diagnosis of OCD according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), confirmed by two board-certified psychiatrists; (ii) age between 18 and 65 years; and (iii) provision of written informed consent. Exclusion criteria were carefully applied to avoid confounding influences, and included: (i) history of major psychiatric comorbidities such as schizophrenia or bipolar disorder; (ii) chronic systemic illnesses such as diabetes mellitus or autoimmune diseases; (iii) pregnancy or lactation; (iv) current or recent use of immunosuppressive medications; and (v) refusal to participate or inadequate blood sampling. The control group was required to have no personal or family history of OCD or other major psychiatric disorders, and to meet the same exclusion criteria applied to patients.

Data collection and sampling

Demographic and clinical information was systematically collected using a structured questionnaire. Variables included age, gender, place of residence (urban versus rural), educational level, occupation, and history of contact with domestic animals (particularly cats). Clinical characteristics of OCD patients, such as age of onset, duration of illness, and treatment history, were also recorded. The Yale–Brown Obsessive-Compulsive Scale (Y-BOCS) was administered to assess symptom severity, providing a quantitative measure to characterize the patient cohort. Blood samples were collected from participants and divided into two portions: one designated for serum separation and the other for buffy coat isolation in tubes containing k2-EDTA as anticoagulant. The serum was separated from whole blood, while buffy coats were obtained by centrifuging anticoagulant-treated blood. All samples were stored at -70 °C until further analysis.

Serological assay

The presence of anti-T. gondii IgG and IgM antibodies was determined using commercially available enzyme-linked immunosorbent assay (ELISA) kits (PishtazTeb, Iran), according to the kit instructions. Antibody levels were quantified in units per milliliter (U/ml) and interpreted using defined thresholds. For IgG, values below 8 U/ml were considered negative, 8 to 11 U/ml were suspicious, and values above 11 U/ml were positive. Similarly, for IgM, the negative, suspicious, and positive ranges were set at < 0.8 U/ml, 0.8–1.1 U/ml, and > 1.1 U/ml, respectively. The assay’s performance was robust, with sensitivity and specificity rates exceeding 95%. Importantly, IgG positivity was considered evidence of past or latent infection, while IgM positivity would indicate acute or recent infection. All assays were performed in duplicate, and laboratory personnel were blinded to case–control status to minimize bias.

PCR assay

To complement the serological data, molecular detection of T. gondii DNA was performed on buffy coat samples. Genomic DNA was extracted using a standardized commercial kit (DynaBio™, Takapouzist Co, Iran), following the manufacturer’s instructions. Nested polymerase chain reaction (PCR) targeting the highly repetitive 529 bp RE gene fragment was employed for its high sensitivity and specificity.

For the primary reaction, conventional PCR was performed. The primer sequences were: Forward (F) 5′- CGCTGCAGGGAGGAAGACGAAAGTTG-3′ and Reverse (R) 5′- CGCTGCAGACACAGTGCATCTGGATT-3′. The PCR master mix (12.5 µl; Fermentas) was mixed with 4 µl of the extracted DNA template. The final reaction volume of 25 µl was achieved by adding 7.3 µl of distilled water and 0.6 µl of each primer at a concentration of 1 pmol/µl. Amplification was carried out in a thermocycler (Corbett Research, Sydney, Australia) with the following cycling conditions: initial denaturation at 94 °C for 3 min (1 cycle); followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 20 s; and a final extension at 72 °C for 7 min (1 cycle).

Subsequently, PCR products were evaluated by electrophoresis on a 1% (w/v) agarose gel in Tris-borate-EDTA buffer at 85 V for 25 min and visualized under UV transillumination after staining with SafeView™ DNA Stain (Applied Biological Materials, Inc.). Positive and negative controls were included in every PCR run to ensure reliability and exclude contamination.

Statistical analysis

Data were analyzed using SPSS software version 26 (IBM Corp., Armonk, NY, USA). Descriptive statistics (mean ± standard deviation, frequency, and percentage) were calculated to summarize demographic and clinical variables. Group comparisons were conducted using Chi-square tests or Fisher’s exact test, as appropriate, for categorical variables, and independent-samples t-tests for continuous variables. Logistic regression analyses were employed to calculate odds ratios (ORs) with 95% confidence intervals (CIs), thereby estimating the strength of association between T. gondii infection and OCD after adjustment for potential confounders (age and gender). A two-tailed P-value less than 0.05 was considered statistically significant.

Results

Participant characteristics

A total of 255 individuals participated in the study, which included 120 patients with OCD (comprising 65.8% females and 34.2% males) and 135 healthy controls (63.7% females and 36.3% males). The mean age of the OCD patients was 38.6 ± 12.4 years, while the mean age for the controls was 37.9 ± 11.7 years; this age difference was not statistically significant (P = 0.62). The two groups were similar in terms of gender distribution, residential status, and occupational categories, indicating a good baseline match (see Table 1). Further stratified analysis revealed a significant positive correlation between age and IgG seropositivity in both groups (P < 0.05). However, no significant associations were found related to gender, place of residence, or occupaton (Table 2).

Table 1.

Comparison of baseline characteristic in the OCD patients and controls [n (%)]

Variables	OCD patients (n = 120)		P value	Healthy control (n = 135)		P value
	IgG positive (%)	IgG negative (%)		IgG positive (%)	IgG negative (%)
Gender
Male	23 (56.1)	18 (43.9)	0.86	29 (50.9)	28 (49.1)	0.73
Female	43 (54.4)	36 (34.8)		42 (53.8)	36 (46.2)
Age group
≤ 30	8 (24.2)	25 (75.8)	0.001*	7 (31.8)	15 (68.2)	0.04*
31–40	18 (64.3)	10 (35.7)		15 (42.9)	50 (57.1)
41–50	18 (69.2)	8 (30.8)		14 (56)	11 (44)
51–60	12 (57.1)	9 (42.9)		21 (61.8)	13 (38.2)
> 60	10 (83.3)	2 (16.7)		14 (73.7)	5 (26.3)
Residential place
Urban	42 (51.2)	40 (48.8)	0.22	43 (55.1)	35 (44.9)	0.49
Rural	24 (63.2)	14 (36.8)		28 (49.1)	29 (50.9)
Job
Self-Employed	22 (44)	28 (56)	0.08	34 (55.7)	27 (44.3)	0.55
Unemployed	3 (37.5)	5 (62.5)		4 (57.1)	3 (42.9)
Homemaker	34 (65.4)	18 (34.6)		24 (45.3)	29 (54.7)
Employee	7 (70)	3 (30)		9 (64.3)	5 (35.7)

*Significant at level of 0.05, Values are reported as frequency (percent)

Table 2.

Demographic and laboratory findings in the OCD patients and controls

Variable	Patients (n = 120)	Controls (n = 135)	P-value	OR (95% CI)
Mean age (years)	38.6 ± 12.4	37.9 ± 11.7	0.62	–
Female (%)	65.8	63.7	0.74	–
IgG positive (%)	55.0 (66/120)	52.6 (71/135)	0.70	1.10 (0.67–1.81)
IgM positive (%)	0 (0/120)	0 (0/135)	–	–
PCR positive (%)	5.8 (7/120)	3.7 (5/135)	0.62	1.33 (0.44–4.08)

Serological findings

As shown in Tables 1 and 2, anti-T. gondii IgG antibodies were present in 55% (66 out of 120) of OCD patients and 52.6% (71 out of 135) of controls, indicating no statistically significant difference between the two groups (χ² = 0.15, P = 0.70), with an odds ratio (OR) of 1.10 (95% CI: 0.67–1.81). Additionally, no participants in either group tested positive for anti-T. gondii IgM antibodies.

PCR findings

In terms of PCR findings, nested-PCR analysis aimed at detecting the 529 bp RE gene of T. gondii identified parasite DNA in 7 (5.8%) OCD patients and 5 (3.7%) controls. Although the prevalence of T. gondii DNA was slightly higher in the OCD group, this difference was not statistically significant (P = 0.62), resulting in an OR of 1.33 (95% CI: 0.44–4.08). The concordance between the serological and molecular results was generally high; however, a small number of individuals (7/120) presented discordant results, characterized by being PCR-positive but seronegative for antibodies.

Discussion

This case-control study examines the potential link between T. gondii infection and OCD. While T. gondii has been associated with other psychiatric conditions, its role in OCD remains understudied. The research compares the presence of T. gondii antibodies and DNA in individuals with OCD against a healthy control group to determine if an association exists.

The principal finding of the present investigation is the absence of a statistically significant association between T. gondii infection and OCD in a population sample from Mazandaran Province, northern Iran. Importantly, this observation converges with several previous studies that similarly failed to demonstrate a robust link between latent toxoplasmosis and OCD [12, 13], while simultaneously diverging from other investigations that reported significantly elevated seroprevalence among OCD patients [14, 15]. The inconsistency across studies underscores the complexity of disentangling infectious hypotheses of psychiatric disease and highlights the need for more comprehensive, methodologically rigorous research in this domain.

From a mechanistic perspective, several biologically plausible pathways could potentially explain an association between T. gondii and OCD, even if our findings do not provide direct support. First, the parasite has been shown to encode tyrosine hydroxylase-like enzymes capable of catalyzing dopamine synthesis [16]. As dopaminergic hyperactivity, particularly within the cortico-striato-thalamo-cortical circuits, is considered a hallmark of OCD pathophysiology [17], it is tempting to speculate that chronic infection might subtly perturb dopaminergic signaling, thereby influencing symptom expression. Second, latent T. gondii infection has been associated with persistent, low-grade neuroinflammation, characterized by microglial activation and increased production of pro-inflammatory cytokines such as interleukin-6 and tumor necrosis factor-α [18, 20]. Such immunological perturbations could interfere with glutamatergic and serotonergic neurotransmission, both of which have been repeatedly implicated in OCD [21]. Third, by analogy with PANDAS, it is conceivable that cross-reactive immune responses triggered by the parasite might target basal ganglia circuits, thereby eliciting or exacerbating obsessive-compulsive symptomatology [22].

Nevertheless, the absence of significant differences between OCD patients and controls in our study cannot be dismissed lightly. Rather, it suggests that if T. gondii does play a role in OCD, such effects may be modest, context-dependent, or confined to subgroups of particularly susceptible individuals. For example, genetic polymorphisms in immune-related genes could modulate host susceptibility to the neuropsychiatric consequences of infection [23]. Likewise, environmental factors, such as co-infections, nutritional deficiencies, or psychosocial stress, may interact synergistically with latent toxoplasmosis to influence OCD risk. These considerations underscore the importance of adopting a multifactorial framework when investigating the etiology of complex psychiatric disorders.

The significant correlation we observed between age and IgG seropositivity across groups is consistent with the well-established cumulative nature of T. gondii exposure [24]. This finding not only validates the reliability of our serological data but also emphasizes the importance of considering age as a confounder in future epidemiological studies. Interestingly, while some previous investigations have reported sex-related differences in seroprevalence or behavioral outcomes of latent toxoplasmosis [25], our data revealed no such associations. Similarly, occupational status and place of residence did not emerge as significant predictors of infection, suggesting that exposure in this region may be relatively uniform across demographic strata.

The strengths of our study include the combined use of serological and molecular diagnostics, which helps reduce the risk of misclassification. Additionally, our recruitment from a region with high endemicity of toxoplasmosis enhances the likelihood of identifying potential associations. We also applied standardized diagnostic criteria for OCD to ensure consistency in our findings.

Importantly, our findings must be situated within the broader discourse on infectious and immune-mediated hypotheses of psychiatric disorders. Although the evidence for T. gondii and OCD remains inconclusive, the hypothesis is far from refuted. Indeed, meta-analytic reviews have emphasized that heterogeneity in study populations, diagnostic methods, and sample sizes likely contribute to conflicting results [26]. Furthermore, the growing body of literature linking latent toxoplasmosis to other psychiatric conditions—including schizophrenia, bipolar disorder, and suicidal behavior—reinforces the need for continued investigation [27, 28]. Notably, it is entirely plausible that the parasite’s influence on human behavior is subtle, exerting incremental effects that may only manifest in the presence of additional biological or environmental risk factors.

Limitations

This study aimed to explore the relationship between Toxoplasma infection and OCD; however, further research is necessary to better understand this connection. Conducting a cohort study could provide valuable insights into how Toxoplasma exposure influences outcomes over time. Moreover, a larger sample size would allow for a more thorough examination of different subgroups within the study, meaning that caution should be exercised when interpreting the results. Since this research was conducted at a single center, future studies incorporating multiple centers would improve the generalizability of the findings.

Conclusion

In summary, this case–control study conducted in Mazandaran Province, Iran, found no statistically significant association between T. gondii infection and obsessive-compulsive disorder (OCD). While IgG seropositivity and PCR positivity were slightly higher in patients than in controls, these differences were not significant. Notably, the correlation between age and IgG positivity underscores the cumulative nature of exposure, consistent with global epidemiological trends. Overall, our findings indicate that if T. gondii plays a role in OCD pathogenesis, its effects are likely modest and context-dependent, potentially limited to vulnerable subgroups. Future research should continue exploring this avenue by utilizing longitudinal, multicenter designs, advanced molecular and immunological biomarkers, and neuroimaging techniques.

Abbreviations

OCD

Obsessive-compulsive disorder

T. gondii

Toxoplasma gondii

ELISA

Enzyme-linked immunosorbent assay

IgG

Immunoglobulin G

IgM

Immunoglobulin M

OR

Odds ratios

PCR

Polymerase chain reaction

Author contributions

MF and SHH conceptualized the manuscript. MMA and MF conducted data analysis. MMO and MF provided critical revisions. MMA and SHH collected samples and completed questionnaires. All authors equally contributed to writing from their perspectives, editing the manuscript, and approving the final manuscript.

Funding

The study was funded by Mazandaran University of Medical Science, Project No IR.MAZUMS.REC.1401.387. No external funding was received.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Human ethics approval and consent to participate

This study was ethically approved by the Ethics Committee of Mazandaran University of Medical Sciences (IR.MAZUMS.REC.1401.387), and all procedures adhered to ethical rules and principles set forth in the Declaration of Helsinki. Also, Informed consent was obtained from all the participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.