SEROPREVALENCE OF TOXOPLASMA GONDII AMONG SCHIZOPHRENICS AT HOSPITAL KAJANG

Studies have found a high seroprevalence of T. gondii in schizophrenia patients than controls. This casecontrol study was done to determine the serprevalen ce of Toxoplasma gondii and to identify its risk factors among schizophrenia patients at Hospital Kajang. Ei hty-eight (88) schizophrenia patients and 88 nonpsychiatric controls were examined for the presence of anti-Toxoplasma IgG and IgM antibodies by Enzyme Linked Immunosorbent Assay (ELISA). Question naires were used to collect socio-demographic and behavioral data among the respondents. From the results, the seroprevalence of antiT. gondii IgG antibodies was significantly higher (51.0%) in schi zophrenia patients than in controls (30.7%); (OR = 2.01; 95% CI: 2.16-3.01; p = 0.023. There was no signific ant association between the socio-demographic facto rs and toxoplasmosis. Beef consumption (p = 0.004, OR = 3.852, CI: 1.550-9.569), pork consumption (p<0.001, OR = 13.089, 95%CI: 4.730-36.219) and ris ky cat contact (p = 0.047, OR = 4.061, 95% CI: 1.985-16.745), were found to be significantly assoc iated with T. gondii infection. Our results show that there is a relationship between toxoplasmosis and s chizophrenia.


INTRODUCTION
Toxoplasma gondii is an intracellular protozoan parasite in the phylum Apicomplexa. Cats are the only definitive hosts. T. gondii however, also infects varieties of intermediate hosts, including humans (Dubey and Jones, 2008). Infection with T. gondii occurs pre-natally or post-natally. After birth, humans are generally infected with T. gondii after the ingestion of oocysts in contaminated soil or water, usually with cat feces. Infection also occurs by the ingestion of tissue cysts in undercooked meat . Congenital infection also occurs and can be very fatal to the fetus (Edelhofer and Prossinger, 2010).
Schizophrenia is a complex chronic neuropsychiatric disease of the central nervous system, believed to have multiple etiologies (Torrey et al., 2007). Latent toxoplasmosis occurs when the bradyzoite or cyst form inhabits the brain when in the brain, the parasitecan then institute infection within the Central Nervous System (CNS), manipulate the host behavior and can cause neurological and psychiatric symptoms in some infected individuals (Brown and Derkits, 2010). From a recent research carried out by Hamidinejat et al. (2010) in Iran,

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AJID it was concluded that T. gondii plays a role in the etiology of schizophrenia.
The aims of this study were to determine the seroprevalence of Toxoplasma gondii and associated environmental risk factors in schizophrenia patients and non-psychiatric controls, thereby providing an important preliminary data in Malaysia.

MATERIALS AND METHODS
The study was conducted at Hospital Kajang. It is a Government-funded district Hospital, founded in 1889 and it is located in the eastern part of Kajang town in the district of Hulu Langat in Selangor, Malaysia. Its land measures 16 acres and is situated about 30 km northeast of Kuala Lumpur. This matched case-control study was conducted in 2 populations: Eighty-eight (88) schizophrenia patients recruited from the Psychiatry department and 88 nonpsychiatric controls from the Medical and orthopedic department. Each case was matched to a control by age gender and race (Alvarado-Esquivel et al., 2011a).
The inclusion criteria included immunocompetent patients aged 18 to 60 years that were not presented with any neurological diseases. Diagnosis were done by psychiatrists and confirmed by Diagnostic and Statistical Manual of Mental Disorders, fourth edition (DSM-IV) (First et al., 1977). The mean age for both groups was 39.42 (range 18-60) years (Alvarado-Esquivel et al., 2012).
Objectives and procedures of the research were explained to the subjects. Since this study involves invasive procedures, written consent form was filled up by respondents before the blood sample was collected. Approval was granted by the ethical committee, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia and The Medical Research Ethics Committee, Malaysia.
Socio-demographic and behavioral characteristics of the participants were collected through the aid of a standardized self-administered questionnaire. We categorized Socio-demographic data as age, gender, birthplace, urban or rural dwelling, educational level, socioeconomic status and occupation (Goodwin et al., 2011). Putative risk factors comprised cat contacts, contact with cat feces; meat consumption (pork, beef, goat, sheep, mutton, chicken, or other); degree of meat cooking; consumption of untreated water, unwashed raw vegetables, fruits and contact with soil (gardening or agriculture) (Leweke et al., 2004). Serum samples were obtained by the centrifugation (at 3000 rpm for 10 min) of blood collected from the participants and kept at-20°C pending ELISA tests. (Bouhamdan et al., 2010) Serum samples were assayed for anti T. gondii IgG antibodies with a commercially available enzyme immunoassay; Anti-Toxoplasma IgG EIA well (RADIM, Italy) and microtiter plate reader (450-630 nm, DYNEX, MRX) was used to read the optical density of the plates. The samples considered positive were further tested for anti-T. gondii IgM antibodies by a commercially available enzyme immunoassay; Anti-Toxoplasma IgM EIA well (RADIM, Italy). All tests were performed following the instructions of the manufacturer.

Statistical Analysis
SPSS version 20.0 was used in analysis. McNemar chi-square analysis was performed to compare the seroprevalence of anti-Toxoplasma IgG and anti-Toxoplasma IgM antibodies among schizophrenia patients and controls. Odds Ratio (OR) and 95% confidence interval were calculated by multivariate analysis using conditional logistic regression, thus determining the level of risk involved in the study putative environmental risk factors and the subsequent association with T. gondii seropositivity. A value of p<0.05 was considered statistically significant.

RESULTS
Individuals with schizophrenia had increased levels of serum IgG antibodies to T. gondii (51%), compared with controls (30.7%). The difference in the seroprevalences was significant (OR = 2.01; 95% CI: 2.16-3.01; p = 0.023) as shown in Table 1. IgM antibodies to T. gondii, both cases and controls had the same level of seropositivity of 1.1% (p =1.00). For the sociodemographic characteristics, no significant relationship was found among the study variables (p>0.05), as shown in Table 2.
From the results of the logistic regression conducted, (   Table 3. Logistic regression analysis of environmental factors associated with T. gondii infection among study respondents 95% C.I.

DISCUSSION
Our results showed a higher seroprevalence of antiT.gondii IgG antibodies in schizophrenia patients than in controls matched by age, gender and race, thus confirming results by Alvarado-Esquivel et al. (2011b); Alipour et al. (2011) andHamidinejat et al. (2010). IgG antibodies were not significantly different between the groups due to the fact that IgM antibodies become negative within 4-12 weeks and are indicators of a recent infection.
The positive association between T. gondii infection and meat consumption from this study result confirms results from studies done in Mexico by Alvarado-Esquivel et al. (2011a); In China by Wang et al. (2006) and in the United States by Torrey et al. (2012). The prevalence of meat-related factors highlights the significance of cooking meat well (>67°C). Chickens bred for commercial purposes are possibly not included in the transmission of T. gondii because methods applied in the modern breeding process decrease soil contact, the chickens are usually kept in its frozen state before consumption (Cavalcante et al., 2006;Dubey et al., 2007).
From our results, there is a positive association between cat contact and T. gondii infection. This suggests that infection may be acquired by cleaning cat excrement containing the parasites. Association between T. gondii seropositivity and cat contact was also found in studies done by Alvarado-Esquivel et al. (2011b) among low Toxoplasma seroprevalence population of Mexico, in which from the bivariate analysis of identified behavioral characteristics, Science Publications AJID cleaning cat excrement was significantly associated with T. gondii infection (p = 0.001) and other study behavioral characteristics including meat consumption (raw or undercooked meat), consumption of unpasteurized milk, fruits, vegetables, contact with soil and types of floors at home did not show any association with T. gondii infection. Our results also confirms those done in Malaysia by Sinniah et al. (1984) and Nissapatorn et al. (2003).
Toxoplasma gondii infection was also found to be associated with pork consumption (p<0.001). From studies done by Alvarado-Esquivel et al. (2012) on a subset of Mexican patients with work accidents and low socioeconomic background, multivariate analysis showed a significant association of T.gondii to ham consumption (OR = 0.16; 95%CI: 0.05-0.51; p = 0.002). As food safety has become a focal issue in food production and marketing worldwide, consumers demand not only high quality foods, but also safe foods.

CONCLUSION
Our results confirm recent findings that Toxoplasma gondii infection is significantly associated with schizophrenia. Our result indicates that toxoplasmosis might be a very serious but ignored public health problem. This study advanced further weight on the hypothesis that T. gondii is a risk factor for schizophrenia. The results also show that T. gondii is significantly associated with beef consumption, pork consumption and risky cat contact. Our limitation included a relatively lower sample size, which was as a result of limited logistics. Nonetheless, this finding is essential as a preliminary data in Malaysia in establishing an association between T. gondii and schizophrenia.
We recommend multicenter studies to outline a significant association and establish a more clearer relationship between T. gondii infection and schizophrenia patients.

ACKNOWLEDGEMENT
The researchers wish to thank Research University Grants (RUGS, UPM) for a research grant for this project. We express our deep thanks to The Medical Research Ethics Committee, Ministry of Health, Malaysia; The Director and staff of Hospital Kajang and the Staff of Department of Psychiatry, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia at Hospital Kajang.

Conflict of Interest
No conflict of Interest to declare for this study.