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. 2018 Mar 8;42(2):177–181. doi: 10.1007/s12639-018-0979-x

Serological and molecular diagnosis of Toxoplasma gondii in patients with schizophrenia

Adel Ebrahimzadeh 1, Mehdi Khoshsima Shahraki 2, Azad Mohammadi 1,✉
PMCID: PMC5962488  PMID: 29844620

Abstract

Schizophrenia is a persistent neuropsychiatric syndrome of uncertain source. Toxoplasmosis is the most prevalent parasitic protozoan infecting one-third of the worldwide human population. Infectious agents such as toxoplasma are the probable cause of schizophrenia. This study was aimed to evaluate the association between schizophrenia and toxoplasmosis using SAG1 and B1 Target gene. During February to December 2016, 92 patients with schizophrenia are imported in our study. All cases were assessed by serological (IgG and IgM antibodies) and molecular examinations. ELISA was performed by Commercial kits according to manufactures procedure. DNA was extracted and nested PCR was done using two pairs of primers. From 92 patients, 59 (64.13%) cases were positive for toxoplasmosis by serological examinations (14 samples positive for IgM and IgG, 40 samples positive for only IgG and 5 samples Positive for only IgM) and 58 (63.04%) were positive by Nested PCR technique. Based on the nested PCR method, 68.47 and 47.82% of samples were positive by B1 and SAG1 genes, respectively. Our results showed the importance of use both serological and molecular diagnostic methods for accurate recognition of T. gondii in patients with schizophrenia. Moreover our results indicated that B1 gene is more sensitive than SAG1 gene.

Keywords: Toxoplasmosis, Schizophrenia, ELISA, Nested PCR, Iran

Introduction

Schizophrenia is a class of pervasive neuropsychiatric complaints with ambiguous source and approximate that 1% of United States and European populations is affected (Berkovitch et al. 2017). The results of some studies recommended that genetic components are may play a role in etiology of schizophrenia (Buka et al. 2001). But recently, many extensive researches on different populations have failed to classify schizophrenia-causing genes (Leweke et al. 2004) and suggest numerous environmental reasons such as winter and spring birth, urbanity, difficulties during work and Perinatal and postnatal infections (Susser et al. 1998; Torrey and Yolken 2003; Mortensen et al. 2007). Epidemiological results indicated that infections in central nervous system in congenital period in humans, such as rubella, herpes simplex, polio, varicella zoster virus and toxoplasmosis might be associated to the risk of schizophrenia (Flegr et al. 2003; Brown et al. 2005; Amminger et al. 2007).

Toxoplasma gondii is a food borne parasite that infects a wide range of warm-blooded animals, such as cats, livestock, and humans. Cats are the definitive host for T. gondii and humans are being infected by ingesting cat feces or raw meat with cysts (Dubey and Beattie 1988). Toxoplasmosis in human is subclinical and the cyst frequently develops within the CNS. Reactivation and replication of T. gondii in brain in immuno-competent patients may cause of neurological symptoms in some patients (Flegr et al. 1996; Holliman 1997). In an animal model study in rat, toxoplasma could infect 30% of microglial cells and 10% of neurons and astrocytes (da Silva and Langoni 2009). According to these findings, it’s suggested that there is a strong association between toxoplasmosis and schizophrenia. The rates of infection in different foci of the world were calculated from 0 to 72% (Aliabadi et al. 2017). Seroprevalence rate of toxoplasmosis in Iran is range from 33 to 45% in healthy populations and from 43 to 56% in immunocompromised patients (Ahmadpour et al. 2014; Daryani et al. 2014; Teimouri et al. 2016).

Additionally, Different studies have demonstrated significant association between seroprevalence rates of toxoplasmosis and schizophrenia (Lori et al. 2017; Yuksel et al. 2017). Diagnosis of T. gondii is crucially important in these patients and there are several different serological techniques such as Sabin–Feldman and ELISA tests for diagnosis of toxoplasmosis (Sabin and Feldman 1948; Goldstein et al. 2008). Furthermore molecular methods indicated that are sensitive to detect T. gondii DNA in clinical samples (Naessens et al. 1999). Several studies indicated that multi-copy genes for diagnosis of T. gondii DNA are much more suitable and sensitive than single-copy gene. SAG1, B1, RE, ITS and other targets are previously worked and B1 indicated that is a sensitive gene by many studies (Farhadi et al. 2017; Santoro et al. 2017). So selection of the target gene for molecular tests plays a key role in sensitivity of method. Therefore, this study was aimed to evaluate the serological and molecular methods to diagnosis of toxoplasmosis in patients with schizophrenia and two target genes are compared to assess their sensitivity.

Materials and methods

Patients and sampling

In this study 92 patients with schizophrenia During February to December 2016 were selected from among patients who applied to the psychiatry ward of Baharan hospital in Zahedan city. Current work was confirmed by the Research Ethical Committee of Zahedan University of Medical Sciences, Iran. All patients were provided written informed consent after explanation of the study purpose and procedures. Schizophrenia is diagnosed clinically according to the DSM-IV (USA, 2004). Five milliliters of Venus blood samples were gathered from each patient and control group. Blood samples were centrifuged at 2500 rpm for 5 min, serums were separated and stored in − 20 °C until to use. Other parts of blood were used for DNA extraction.

Serological examinations

IgG and IgM antibodies were analyzed by the enzyme-linked immunosorbent assay, with a commercial ELISA kit (DIA.PRO DiagnosticBioprobes Srl, Italy) according to the detailed by manufacturer’s protocol. The sensitivity and specificity of the kits that estimated by manufacturer were more than 98%.

DNA extraction and polymerase chain reaction

DNA extraction was performed on 200 µl of whole blood sample using DNA extraction kit (Bioneer, Korea), according to the manufacturer’s instructions. The samples were analyzed to detect the presence of T. gondii DNA using PCR with the four primers (2 external and 2 internal primers) of the SAG1 gens with five 27-bp repeat sequences 35–190 bp in the genome of T. gondii (23- sag1- Soldati D, Boothroyd). Polymerase chain reaction was performed for first step by 2 µl of DNA, 1.5 mM of MgCl2, 250 µM of dNTPs, 1 U/µl of Top DNA polymerase, 10 mM Tris–HCL, 1 µl of each primers and distilled Water was added to reach final volume to the 20 µl. The amplification procedure was conducted with 30 cycles and the protocol started by 5 min for 94 °C of initial denaturation and followed by 1 min for 94 °C, 1 min for 57 °C and 20 s for 72 °C, finally followed by 5 min of final extension for 72 °C. Two samples with and without of DNA of T. gondii (RH strain) were utilized as positive and negative controls to recognize false-positive results. Electrophoresis on agarose gel 2% and SYBR_ Safe stain were used to analysis of First step PCR products. Diagnosis was confirmed by a nested-PCR targeting SAG1 and B1 genes and the primers are presented in Table 1.

Table 1.

The primes of T. gondii in Nested PCR technique on SAG1 gene

Genes Primers
SAG1 External primers 5′-GTT CTA ACC ACG CAC CCT GAG-3′
5′-AAG AGT GGG AGG CTC TGT GA-3′
Internal primers 5′-CAA TGT GCA CCT GTA GGA AGC-3′
5′-GTG GTT CTC CGT CGG TGT GAG-3′
B1 External primers 5′-GTT CTA ACC ACG CAC CCT GAG-3′
5′-AAG AGT GGG AGG CTC TGT GA-3′
Internal primers 5′-CAA TGT GCA CCT GTA GGA AGC-3′
5′-GTG GTT CTC CGT CGG TGT GAG-3′
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Statistical analysis

Statistical analysis was done using Chi squared test, t test and analysis of variance (ANOVA). We considered P < .005 as a significant value and Kappa test is used for the comparison between tests and two DNA targets.

Results

From 92 sample in our study, 14 (15.21%) were positive for IgM and IgG, 40 (43.47%) were positive for IgG and negative for IgM, 5 (5.43%) were positive for IgM and negative for IgG and 33 (35.86%) for negative for both IgG and IgM antibodies by ELISA techniques (Table 2).

Table 2.

ELISA values for T. gondii in blood samples in patients with schizophrenia

Antibodies IgM Total P value Kappa
Positive Negative
IgG Positive 14 (15.21%) 40 (43.47%) 54 (58.69%) .135 .041
Negative 5 (5.43%) 33 (35.86%) 38 (41.30%)
Total 19 (20.65%) 73 (81.11%) 92 (100%)
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Table 3 reports results from nested PCR analysis. Based on SAG1 gene results, From 14 positive cases in acute phase (IgG and IgM positive); 9 samples were positive by Nested PCR method. Moreover, 20 samples from negative group (IgG and IgM negative), 25 samples from chronic group (IgG positive, IgM negative) and 4 samples from pseudo-positive group (IgG negative, IgM positive) was positive by molecular techniques. Based on B1 gene, From 14 positive cases in acute phase (IgG and IgM positive); 9 samples were positive by Nested PCR method. Moreover, 22 samples from negative group (IgG and IgM negative), 27 samples from chronic group (IgG positive, IgM negative) and 3 samples from pseudo-positive group (IgG negative, IgM positive) was positive by molecular techniques (Figs. 1, 2).

Table 3.

The results of Nested PCR based on the serological groups in patients with schizophrenia

Genes Results IgM+, IgG+ IgM−, IgG− IgM+, IgG− IgM−, IgG+ P value Kappa
SAG1 Positive 9 (64.28%) 20 (60.60%) 4 (80%) 25 (62.50%) .119 .014
Negative 5 (35.72%) 13 (39.40%) 1 (20%) 15 (37.5%)
Total 14 (100%) 33 (100%) 5 (100%) 40 (100%)
B1 Positive 9 (64.28%) 22 (68.75%) 3 (75%) 27 (67.52%) .081 .074
Negative 5 (35.72%) 10 (31.25%) 1 (25%) 13 (32.5)
Total 14 (100%) 32 (100%) 4 (100%) 40 (100%)
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Fig. 1.

Fig. 1

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Electrophoretic pattern of the nested PCR on SAG1 gene, from T. gondii. M: 100 bp ladder; line 1: Positive control; line 2: Negative control; lines 3–10 are corresponds to the cases samples

Fig. 2.

Fig. 2

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Electrophoretic pattern of the nested PCR on B1 gene, from T. gondii. M: 100 bp ladder; line 1: Positive control; line 2: Negative control; lines 3–10 are corresponds to the cases samples

Discussion

In this study the relation between schizophrenia and T. gondii infection is evaluated. Many studies was accomplished in recent years have reported prevalence values of toxoplasmosis in Iran ranging from 18 to 68% in different areas (Teimouri et al. 2016) and important risk factors for infection include close contact to cat and use of foods polluted with oocyst or tissue cyst of parasite. T. gondii has been stated as one of the cause of schizophrenia (Webster et al. 2016) and there is no data about the prevalence of toxoplasmosis in these kinds of patients. From 92 patients with schizophrenia entered in our study, 59 (64.13%) cases were positive for Toxoplasmosis by serological examinations. In 1953, in Poland, Kozar et al. informed that 495 (52%) of 961 patients with schizophrenia were positive for T. gondii antibodies in comparison with 170 (25%) of 681 healthy controls (Kozar 1953).

In a similar study in Mexico, Roch and Varela indicated that 86% patients with schizophrenia and 30% of health controls was positive for toxoplasmosis (Roch and Varela 1966). In both studies there were not any significant differences between case and control groups. Of course the differences between the two groups in some studies in Iran (Daryani et al. 2010); China (Cong et al. 2005); Germany; Northern Mixican City (Yolken et al. 2001; Alvarado-Esquivel et al. 2006); and in Turkey (Cetinkaya et al. 2007) were statistically significant. Fuller Torrey et al., in a meta-analysis study indicated that the T. gondii antibodies (IgG or IgM) in patients with schizophrenia significantly higher than health control individuals with an OR of 2.73. This finding was extracted from different studies carried out over 5 decades in 17 countries by using of different serological methods (Fuller Torrey et al. 2007). Consequently; this recommends that T gondii could play an important role in the development of schizophrenia. The high seroprevalence detected in the current study was proved by molecular technique. Indeed, nested PCR was more sensitive than ELISA and 20 samples that were negative by serologic examinations; be positive for parasite DNA. The diagnosis of toxoplasmosis by serological techniques is more sensitive than molecular methods.

This showed that the presence of anti-Toxoplasma antibodies was not a sufficient criterion for recognizing toxoplasmosis. Therefore, some patients will be incorrectly recognized as being infected and undergo unnecessary anti parasitic treatment. Our results showed the importance of use both serological and molecular diagnostic methods for accurate recognition of T. gondii in patients with schizophrenia.

In current work, SAG1 and B1 as target genes were used by nested PCR techniques and our findings indicated that B1 is more sensitive than SAG1 gene. This results suggested that, using of a sensitive marker can heighten the sensitivity of a technique and recommended that B1 gene and other genetic targets such as 529 bp repeated elements, ITS will be evaluate in future to reach an acceptable sensitivity.

In conclusion, our results indicated that patients with schizophrenia are at high risk for toxoplasmosis. Therefore, in patients with schizophrenia, prevention and screening programs and so treatment of toxoplasmosis should be given more consideration. In future, supplementary Studies should be undertaken on other organisms such as Neospora caninum and Hammondi hammondi, which are closely associated to T. gondii and have serologically cross reaction. Moreover it’s needed to design some cohort studies to better define the relationship between T. gondii infection and schizophrenia.

Acknowledgements

The authors acknowledge the Parasitology lab staff of Zahedan University of Medical Sciences for financial support (Grant No. 123471).

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

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