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\begin{document}

\title{Toxoplasma gondii Infection by Serological and Molecular Methods in
Schizophrenia Patients with and without Suicide Attempts: An
Age-Sex-Matched Case-Control Study}



\author[1]{Özer Akgül}%
\author[2]{Ömer Faruk Demirel}%
\author[2]{Cana Poyraz Aksoy}%
\author[2]{Ezgi Tanrıöver Aydın}%
\author[2]{Nuray Uysal}%
\author[2]{Ersel Bulu}%
\author[3]{Burcu Sapmaz}%
\author[3]{Reyhan Çalışkan}%
\author[3]{Yaşar Ali Öner}%
\affil[1]{Affiliation not available}%
\affil[2]{Department of Psychiatry, Istanbul University – Cerrahpaşa, Cerrahpaşa School of Medicine}%
\affil[3]{Department of Medical Microbiology, Istanbul Aydin University, School of Medicine}%


\vspace{-1em}



  
  \date{May 24, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
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\endgroup





\selectlanguage{english}
\begin{abstract}
Introduction: The opinion that latent T. gondii infection is having a
broadly asymptomatic projection has now been interrogated, in specific
due to the echoed association between the latent infection and an
elevated incidence of schizophrenia or even suicide attempts.
Notwithstanding conducted studies aimed to understand this feasible link
are restricted. Methods: In the present case-control study, we focused
to illuminate the relationship between the serological and molecular
presence of T. gondii and schizophrenia with or without the suicide
attempts by comparing it with healthy individuals. A total of 237
participants (117 in schizophrenia; 120 in healthy control) were
included in this study. Results: Overall, latent T. gondii infections
were found statistically higher in 63 (53.8\%) of the 117 patients with
schizophrenia and in 33 (27.5\%) of the 120 controls (p \textless{}
0.001). In schizophrenia patients, seroprevalence T. gondii was again
found to be statistically higher in suicide attempters (59.6\%),
compared to no history of suicide attempts (48.3\%) (p \textless{}
0.05). The molecular positivity rate of T. gondii DNA was higher in the
schizophrenia group, compared to the healthy control group (p
\textless{} 0.05), whereas the history of suicide attempts was not
statistically associated (p = 0.831) with T. gondii DNA positivity by
PCR. Conclusion: This case-control study enlightens additional
demonstration to the belief that T. gondii infection would be an
underlying component for the pathophysiology of schizophrenia.
Regardless of the clarity results of this study, this supposition
warrants further endorsement.%
\end{abstract}%



\sloppy


\emph{Toxoplasma gondii} \textbf{Infection by Serological and Molecular
Methods in Schizophrenia Patients with and without Suicide Attempts: An
Age-Sex-Matched Case-Control Study}

\emph{T. gondii} \textbf{with ELISA and PCR in Schizophrenia}

\selectlanguage{ngerman}Özer AKGÜL \textsuperscript{1, *}, Ömer Faruk
DEM\selectlanguage{polish}İ\selectlanguage{english}REL\textsuperscript{2}, Cana AKSOY PORAZ \textsuperscript{2}, Ezgi
TANRI\selectlanguage{ngerman}ÖVER AYDIN \textsuperscript{2}, Nuray UYSAL \textsuperscript{2},
Ersel BULU \textsuperscript{2}, Burcu SAPMAZ \textsuperscript{1}, Reyhan
ÇALI\selectlanguage{polish}Ş\selectlanguage{english}KAN \textsuperscript{1}, Ya\selectlanguage{polish}ş\selectlanguage{english}ar Ali \selectlanguage{ngerman}ÖNER \textsuperscript{1}

\emph{Affiliations:}

Department of Medical Microbiology, Istanbul Aydin University, School of
Medicine, Istanbul, Turkey

Department of Psychiatry, Istanbul University -- Cerrahpa\selectlanguage{polish}ş\selectlanguage{english}a, Cerrahpa\selectlanguage{polish}ş\selectlanguage{english}a
School of Medicine, Istanbul, Turkey

\emph{* Corresponding Author:}\selectlanguage{ngerman}Özer AKGÜL Address: Department of Medical
Microbiology, Istanbul Aydin University, School of Medicine, 34295
Sefaköy, Istanbul, Turkey Tel: +90-532-565-9944 Fax: +90-212-425-5759
E-mail: akgulozer@hotmail.com

\emph{Toxoplasma gondii} \textbf{Infection by Serological and Molecular
Methods in Schizophrenia Patients with and without Suicide Attempts: An
Age-Sex-Matched Case-Control Study}

\textbf{Abstract}

\textbf{Introduction:} The opinion that latent \emph{T. gondii}infection
is having a broadly asymptomatic projection has now been interrogated,
in specific due to the echoed association between the latent infection
and an elevated incidence of schizophrenia or even suicide attempts.
Notwithstanding conducted studies aimed to understand this feasible link
are restricted. \textbf{Methods:} In the present case-control study, we
focused to illuminate the relationship between the serological and
molecular presence of \emph{T. gondii} and schizophrenia with or without
the suicide attempts by comparing it with healthy individuals. A total
of 237 participants (117 in schizophrenia; 120 in healthy control) were
included in this study. \textbf{Results:}Overall, latent \emph{T.
gondii} infections were found statistically higher in 63 (53.8\%) of the
117 patients with schizophrenia and in 33 (27.5\%) of the 120 controls
(p \textless{} 0.001). In schizophrenia patients, seroprevalence
\emph{T. gondii} was again found to be statistically higher in suicide
attempters (59.6\%), compared to no history of suicide attempts (48.3\%)
(p \textless{} 0.05). The molecular positivity rate of \emph{T. gondii}
DNA was higher in the schizophrenia group, compared to the healthy
control group (p \textless{} 0.05), whereas the history of suicide
attempts was not statistically associated (p = 0.831) with \emph{T.
gondii} DNA positivity by PCR.\textbf{Conclusion:} This case-control
study enlightens additional demonstration to the belief that \emph{T.
gondii} infection would be an underlying component for the
pathophysiology of schizophrenia. Regardless of the clarity results of
this study, this supposition warrants further endorsement.

\emph{Keywords:} Schizophrenia, Suicide attempt,\emph{Toxoplasma gondii}
, ELISA, PCR



\textbf{What's already known about this topic?}

\emph{Toxoplasma gondii} (\emph{T. gondii} ) is a neurotropic parasite
and its infection maybe take a key position in the risk of schizophrenia
and also suicide attempts.

\textbf{What does this article add?}

Beyond the serologically based retrospective research, \emph{T.
gondii}detection was examined by molecular methods in this case-control
study, and the relationship between \emph{T. gondii} positivity in
schizophrenia and suicide attempt risk was investigated for the first
time with the healthy control group with similar age-gender data.

\textbf{1. INTRODUCTION}

\emph{Toxoplasma gondii} (\emph{T. gondii} ) is a neurotropic parasite
affecting warm-blooded animals including humans\textsuperscript{1} and
is found in more or less 30\% of people.\textsuperscript{2} Felines are
the final host for \emph{T. gondii} , shedding in their feces up to
millions of oocyst stages daily, which become contagious in
nature.\textsuperscript{3} In people, the infection may be transmitted
by the ingestion of water or food. In this way, the majority of \emph{T.
gondii} infections remain asymptomatic or maybe produce infectious
mononucleosis in people who have an intact immune system. Severe
toxoplasmosis may develop if \emph{T. gondii} infects immunocompromised
patients such as acquired immunodeficiency syndrome
(AIDS).\textsuperscript{4} In parallel with the achievements after the
concept of microbiota, it is thought that microorganisms may be
associated with non-infectious pathological changes.

The pathophysiology of schizophrenia is tangled, and biological factors
are among the leading causes.\textsuperscript{5} Thus, studies focusing
to elucidate the possible agents in schizophrenia have been carried out.
In many studies conducted for this purpose, \emph{T. gondii} has been
evaluated as the prominent agent in this relationship due to its tropism
and persistence in the brain.\textsuperscript{6} Several reports are
describing that \emph{T. gondii} is linked to the elevated risk of mice
catching by felines,\textsuperscript{7} to modulated behavioral change
by influencing neurotransmitters in the human
brain.\textsuperscript{8}In humans, dysregulation of some
neurotransmitters is significantly related to schizophrenia. \emph{T.
gondii} can synthesize tyrosine hydrolase and thus dopamine
dysregulation may occur. Furthermore,\emph{T. gondii} also modifies the
level of a vast of other neurotransmitters, including glutamate,
serotonin, norepinephrine, and \selectlanguage{greek}γ-\selectlanguage{english}aminobutyric acid
(GABA).\textsuperscript{9} The link between schizophrenia and \emph{T.
gondii} seems to be vigorous, as evidenced by numerous seroprevalence
and neurotransmitter studies.

Worldwide, almost 800.000 people die by suicide and roughly 10 million
individuals attempt suicide annually. Suicide attempts are recognized to
be a complex mechanism, with psychological, social and biological
perspectives involved.\textsuperscript{10} In 2009, \emph{T. gondii} , a
hitherto unforeseen agent, has been associated with the suicide attempt.
The possible relationship between \emph{T. gondii} and suicide attempts
has also been shown in a current seroprevalence study. According to the
author, \emph{T. gondii} IgG positivity was statistically highly
correlated with lower agreeableness, lower conscientiousness and higher
neuroticism personality types.\textsuperscript{11} Overall,
topic-related and current studies provide considerable evidence for the
hypothesis that \emph{T. gondii} infections maybe take a key position in
the risk of the suicide attempt.\textsuperscript{12}

In this case-control study, it was aimed to elucidate the relationship
between \emph{T. gondii} , which was detected by serological and
molecular methods, and patients with schizophrenia, and the suicide
attempt by comparing it with the healthy individuals.

\textbf{2. METHODS}

In this case-control study, the detection and prevalence of \emph{T.
gondii} by serological and molecular methods in patients with
schizophrenia were compared to the healthy control.

\emph{2.1. Study design and sample size calculation}

This study was approved by the Istanbul Aydin University Clinical
Research Ethics Committee with B.30.2.AYD.0.00.00-480.2/217 protocol
number before the research. The statistical power analysis was showed
that a minimum sample of n=73 in each group would have the assumption of
95\% CI and power of 80\%. A nominal \selectlanguage{greek}α\selectlanguage{english}=0.05 and two-tailed tests were
used in the power calculations.

We recruited a total of 117 schizophrenia patients aged 18 -- 65 years
as a case group. For each schizophrenia patient, one healthy control
people were recruited. Individuals in the control group were randomly
selected from the city where the patients lived (Istanbul, Turkey) and
matched for age (\selectlanguage{ngerman}± 3 years) and gender. The workflow diagram clarifies
the design of this case-control study (Fig 1).

\emph{2.2. Study participants and sampling}

In this case-control study, a total of 237 volunteers between 18 and 65
years old were enrolled in the schizophrenia or the healthy control
groups. One hundred seventeen patients with schizophrenia who applied to
the psychiatry department of Istanbul University-Cerrahpa\selectlanguage{polish}ş\selectlanguage{english}a, Cerrahpa\selectlanguage{polish}ş\selectlanguage{english}a
Medical Faculty in Istanbul were enrolled in the schizophrenia group of
the study. Schizophrenia is diagnosed clinically according to the
Diagnostic and Statistical Manual of Mental Disorders (DSM--5). Patients
diagnosing with schizoaffective or schizophreniform disorders were
excluded from this study. Also, possible suicide attempts of the
patients with schizophrenia were determined with the Suicide Behaviors
Questionnaire-Revised (SBQ-R) and clinical interviews. The healthy
control group was consisted of 120 participants without any psychiatric
disease or family history and having similar age and gender data with
the schizophrenia group. Two venous blood samples from each volunteer
were taken by venipuncture from a single cubital region and were
collected into plain and containing dipotassium
ethylenediaminetetraacetic acid (K\textsubscript{2}EDTA) vacuum tubes.
The tubes were filled and K\textsubscript{2}EDTA tubes were immediately
mixed with gentle 180\selectlanguage{ngerman}° inversion. Serum samples were separated from
plain blood tubes by centrifugation at 2500 rpm and stored at -80°C
prior to \emph{T. gondii} IgG analysis. Whole blood samples were also
stored at -80°C for the assessment of \emph{T. gondii} DNA by molecular
methods.

\emph{2.3. Serological evaluation}

Serum samples were evaluated for \emph{T. gondii} IgG with IBL-America
Toxoplasma IgG enzyme-linked immunosorbent assay (ELISA) kit
(Minneapolis, MN, USA) following the manufacturers' instructions, for
which the sensitivity and specificity values were more than
98\%.\emph{T. gondii} IgG titers were read at optical density 450 nm,
and seropositivity was defined as a \emph{T. gondii} IgG titer equal to
or higher than 9 IU/mL.

\emph{2.4. DNA extraction}

DNA was extracted using Plus Blood Genomic DNA Purification Kit
(GeneMark Diagnostics, Carlsbad, CA, USA) according to the
manufacturers' instructions. NanoDrop 2000c spectrophotometer (Thermo
Fisher Scientific, Waltham, Massachusetts, USA) was used to measure DNA
concentration and purity at 260/280 nm in extraction samples. The
extracted DNA was stored at \selectlanguage{english}-20degC prior to testing.

\emph{2.5. Polymerase chain reaction (PCR)}

For the molecular detection of \emph{T. gondii} , the real-time PCR was
performed in the CFX96 Touch Real-Time PCR Detection System (Bio-Rad
Laboratories, Hercules, California USA) using targeted a 529 bp repeat
element and internal transcribed spacer-1 (ITS-1)
region-specific\emph{T. gondii} primers listed in Table 1. The reaction
mixture contained 15 \selectlanguage{greek}μ\selectlanguage{english}L of dH\textsubscript{2}O, 5 \selectlanguage{greek}μ\selectlanguage{english}L of 5X Hot Start
Master Mix (GMbiolab, Taiwan, China), 2 \selectlanguage{greek}μ\selectlanguage{english}L of 10 \selectlanguage{greek}μ\selectlanguage{english}mol/L forward and
reverse primer, and 3 \selectlanguage{greek}μ\selectlanguage{english}L DNA Template. The thermal cycling was performed
at 37 cycles (Enzyme activation at 95\selectlanguage{ngerman}°C for 5 minutes, denaturing at
95°C for 30 seconds, annealing at 56°C for 30 seconds and extension at
72°C for 1 minute). Melting curves were generated from 50 to 95°C and
read every 4°C for 5 seconds.

\textbf{Table 1.} Nucleotide sequences of primers used in this study\selectlanguage{english}
\begin{longtable}[]{@{}ll@{}}
\toprule
\textbf{Primer} & \textbf{Sequence (5' - 3')}\tabularnewline
\midrule
\endhead
ITS-1 Forward & ATCCCAACAGAGACACGAATT\tabularnewline
ITS-1 Reverse & ACACGTCCTTATTCTTTATTAACCA\tabularnewline
\bottomrule
\end{longtable}

\emph{2.6. Detection of Amplicon}

After completion of real-time PCR, the amplified products were confirmed
by gel electrophoresis. Five mL of each amplicon were loaded on 2\%
agarose gel containing ethidium bromide (EtBr) and SYBR\selectlanguage{ngerman}® Gold nucleic
acid gel stain (Invitrogen Life Technologies, Eugene, Oregon, USA). The
gel electrophoresis was performed at 25 V for 30 min and was visualized
by Safe Imager \textsuperscript{TM} (Invitrogen Detection Technologies,
Eugene, Oregon, USA).

\emph{2.7. Statistical analysis}

Data analyses were evaluated with the Statistical Package for the Social
Sciences (SPSS, IBM Corp.) version 25. Normal distribution and
homogeneity of the results were evaluated with Shapiro-Wilk and Levene's
test, respectively. Parametric and non-parametric test assumptions were
done by Student's t-test and one-way ANOVA, as appropriate. A
\emph{p}\textless{} 0.05 was considered to indicate a statistically
significant difference. Statistical significance is denoted by an
asterisk.

\textbf{3. RESULTS}

Sociodemographic and other characteristics are shown in Table 2. A total
of 117 patients diagnosed with schizophrenia and 120 healthy controls
matched for age and gender were included in this study (Fig 1). The mean
age was 47.51 years (SD=24.83) in suicide attempters of subdivided
schizophrenia group, 43.96 years (SD=18.33) in no history of the suicide
attempt of subdivided schizophrenia group, and 42.27 years (SD=29.11) in
the healthy control group (p=0.416). Of all participants 50.2\% of them
were male and 49.8\% were female (Table 2). As an expected and
unfortunate consequence of schizophrenia, a considerably higher
proportion of participants were unemployed (61.4\% and 65.0\% vs 20.0\%;
p = 0.018) and single (71.9\% and 58.3 vs 34.2; p = 0.043), compared to
control group. No other substantial difference was evaluated between
groups regarding the education and socioeconomic levels of the
participants (Table 2).

\textbf{Table 2.} Sociodemographic and other characteristics of the
participants\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Schizophrenia n (\%)\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Schizophrenia n (\%)\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
Healthy Control n (\%)\strut
\end{minipage} & \begin{minipage}[b]{0.16\columnwidth}\raggedright\strut
\emph{p-value}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
History of suicide attempt\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
No history of suicide attempt\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Gender\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Male\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
34 (59.6)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
32 (53.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
53 (44.2)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.619\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Female\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
23 (40.4)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
28 (46.7)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
67 (55.8)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Marital Status\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Single\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
41 (71.9)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
35 (58.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
41 (34.2)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.043}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Married\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
16 (28.1)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
25 (41.7)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
79 (65.8)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Education Level\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
None\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
8 (14.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
11 (18.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
3 (2.5)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.138\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Primary\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
27 (47.4)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
31 (51.7)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
28 (23.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Secondary\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
13 (22.8)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
12 (20.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
30 (25.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
University\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
9 (15.8)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
6 (10.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
59 (49.2)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Socioeconomic Level *\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
No income\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
11 (19.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
22 (36.7)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
7 (5.8)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.073\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Low\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
22 (38.6)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
17 (28.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
34 (28.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Intermediate\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
24 (42.1)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
19 (31.7)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
50 (41.7)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
High\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0 (0.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
2 (3.3)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
29 (24.2)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Employment\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Unemployed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
35 (61.4)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
39 (65.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
24 (20.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\textbf{0.018}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Employed\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
22 (38.6)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
21 (35.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
96 (80.0)\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Age\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Age\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Mean \selectlanguage{ngerman}± SD\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Mean \selectlanguage{ngerman}± SD\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
Mean \selectlanguage{ngerman}± SD\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
0.416\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
47.51\selectlanguage{ngerman}±24.83\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
43.96\selectlanguage{ngerman}±18.33\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
42.27\selectlanguage{ngerman}±29.11\strut
\end{minipage} & \begin{minipage}[t]{0.14\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

* Low \textless{}3000[?]; intermediate 3000 -- 5000 [?]; High \textgreater{}
5000 [?]

\emph{T. gondii} IgG antibodies were found statistically higher in 63
(53.8\%) of the 117 patients with schizophrenia and in 33 (27.5\%) of
the 120 controls (p \textless{} 0.001). When the cases included in the
schizophrenia group are examined by dividing into two sub-groups
according to their suicide attempt history, the relationship between the
history of suicide attempt and seroprevalence \emph{T. gondii} was again
found to be statistically significant (p \textless{} 0.05). In
reflection of our serological findings, it was determined that the rates
of \emph{T. gondii} seropositivity were higher in the schizophrenia
group, compared to the control group. Likewise, \emph{T. gondii} IgG
antibodies positivity was found to be more in schizophrenia patients
with a history of suicide attempt (Table 3).

The results in Table 3 also summarized that the molecular detection
of\emph{T. gondii} by PCR. The molecular positivity rate of T. gondii
was higher in schizophrenia group, compared to healthy control group (p
\textless{} 0.05), whereas history of suicide attempts was not
statistically associated (p = 0.831) with \emph{T. gondii} positivity by
PCR (Table 3). This could be attributed to the fact that the \emph{T.
gondii} is less detected by the molecular method than the serological
method. In this study, the amplified products were visualized by the
standard gel electrophoresis and Figure 2 shows a sample image of
electrophoresis.

\textbf{Table 3.} Comparison between schizophrenia and control group
for\emph{T. gondii} status\selectlanguage{english}
\begin{longtable}[]{@{}llllllll@{}}
\toprule
\begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Serological profile of \emph{T. gondii} n (\%)\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Serological profile of \emph{T. gondii} n (\%)\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\emph{p-value}\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Molecular profile of \emph{T. gondii} n (\%)\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
Molecular profile of \emph{T. gondii} n (\%)\strut
\end{minipage} & \begin{minipage}[b]{0.12\columnwidth}\raggedright\strut
\emph{p-value}\strut
\end{minipage}\tabularnewline
\midrule
\endhead
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
IgG+\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
IgG-\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PCR+\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
PCR-\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Schizophrenia\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Suicide attempt +\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
34 (59.6)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
23 (40.4)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{\textless{}0.05}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
16 (28.1)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
41 (71.9)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
0.831\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Suicide attempt -\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
29 (48.3)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
31 (51.7)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
12 (20.0)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
48 (80.0)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Total\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
63 (53.8)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
54 (46.2)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{\textless{}0.001}\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
28 (23.9)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
89 (76.1)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\textbf{\textless{}0.05}\strut
\end{minipage}\tabularnewline
\begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Healthy Control\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
Healthy Control\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
33 (27.5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
87 (72.5)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
2 (1.7)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
118 (98.3)\strut
\end{minipage} & \begin{minipage}[t]{0.10\columnwidth}\raggedright\strut
\strut
\end{minipage}\tabularnewline
\bottomrule
\end{longtable}

\textbf{4. DISCUSSION}

In this case-control study, we used serological and molecular techniques
to elucidate a possible connection between \emph{T. gondii} and
schizophrenia patients compared to healthy people. To the best of our
knowledge, this is the first case-control study conducted in
schizophrenia and healthy individuals with the diagnosis of \emph{T.
gondii} by serological and also molecular methods. Overall, \emph{T.
gondii} positivity was found more common in patients with schizophrenia
(53.8\%), compared to healthy individuals (27.5\%) by ELISA in this
study (p \textless{} 0.001).

Conventional diagnostic approaches for \emph{T. gondii} infection
include microscopical, cultural, serological, and radiological methods.
Because of the fact that natural scantness of conventional diagnostic
techniques, PCR may be adopted to diagnose \emph{T.
gondii}infection.\textsuperscript{13} As far as we know, there is only
one study\textsuperscript{14} that has limitations such as the lack of a
healthy control group and targeting the SAG1 and B1 regions in the
molecular diagnosis of \emph{T. gondii} , although it is approximately
similar in general concept to our study. Despite its limitations in this
retrospective study, the authors highlighted that the prominence to use
serological and molecular techniques for correct identification
of\emph{T. gondii} in schizophrenia patients.\textsuperscript{14}

In recent years there has been a burgeoning concern about the influence
of latent \emph{T. gondii} infection on mental disorders, especially
schizophrenia. The nature of this connection remains unclear, although
it is conceivable that they manifest causality.\textsuperscript{15} To
date, numerous studies have been carried out showing the relationship
between \emph{T. gondii} and
schizophrenia.\textsuperscript{16--19}Likewise, these studies and also
our study, a meta-analysis reveals a general significant relation having
with \emph{T. gondii} latent infection and schizophrenia. Also, this
well-designed and highly scientific paper saying that the latent
\emph{T. gondii} acts an impact on human behavior as
well.\textsuperscript{20} After two years of this meta-analysis, a
case-control study has been published to determine the association
between schizophrenia and \emph{T. gondii} conducted with 99
schizophrenia patients and 152 healthy blood donors. Although only
serological methods were used in this study and using only this analysis
has some limitations, the authors reported a positive relationship
between \emph{T. gondii} and schizophrenia.\textsuperscript{21}

In contrast to various publications that declared a significant
correlation between \emph{T. gondii} and schizophrenia, some studies
unable to show a significant
relationship.\textsuperscript{22--24}Nevertheless, it should not be
underestimated that the quality and quantity of studies showing the
percentages of \emph{T. gondii} , which has only approximately 30\%
positivity in a healthy population worldwide\textsuperscript{25}, was
found to be elevated especially in patients with schizophrenia. In this
manner, it is clear that further research is still warranted.

Suicide is a crucial emerging health problem, commonly seen in people
with mental disorders, and previous suicide attempts or even suicide
ideation are the best indicators of possible suicide
risk.\textsuperscript{26} In 2019, a meta-analysis with the aim to
determine if \emph{T. gondii} is linked with suicide attempt was
published.\textsuperscript{12} In this meta-analysis, 14 studies about
suicide attempts were included and the authors have calculated a
statistically highly significant correlation for \emph{T. gondii} IgG
antibodies with suicide attempters (OR = 1.39; 95\% CI 1.10--1.76, p =
0006). Moreover, the authors reported that latent \emph{T.
gondii}infection may play a significant role in the risk of suicide
attempts and potential agents should be evaluated
further.\textsuperscript{12}

Besides the contribution of our study with preliminary results
of\emph{T. gondii} and suicidal attempt history relation in patients
with schizophrenia, there are two major limitations in this study that
could be addressed in future further research. First, we enrolled a
relatively small sample size (in 117 patients with schizophrenia), which
would bias the results of our overall and subdivided schizophrenia group
analyses toward the null hypothesis. Second, we were unable to evaluate
the first-episode psychosis in stages of schizophrenia in the case
group. As previously noticed\textsuperscript{27}, if patients with the
first-episode psychosis of schizophrenia were possible to include in our
study, these outcomes would provide an idea or even insight into
how\emph{T. gondii} behaves during the development of this surreptitious
disease.

\textbf{5. CONCLUSION}

In conclusion, our study revealed a relation with both schizophrenia
and\emph{T. gondii} , also the suicidal attempt of these patients.
Although it is difficult to suggest a direct etiological interaction,
after this preliminary finding we can see the \emph{T. gondii} and
schizophrenia link more obvious. On the other side, there is currently
no radical treatment for latent \emph{T. gondii} and no effective
vaccines. Simultaneously with its knotty prevalence, \emph{T. gondii} is
a considerable and sketchily realized agent. Ultimately, it will be
decisive to advanced multidisciplinary study (encouraging to taken
together with medical microbiology and psychiatry) to clear insight into
the fiendishly mechanisms of neuropsychiatric disorders and to conduct
more effective and plausible studies to stiffen for this neurotrophic
links.

\textbf{Funding}

This work was supported by the Istanbul Aydin University Scientific
Research Projects Unit (2020/13).

\textbf{Conflict of Interest}

The authors have declared that there are no conflicts of interest in
relation to the subject of this study.

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\textbf{Figure 1.} Workflow diagram of patients with schizophrenia and
matched healthy individuals included in this study

\textbf{Figure 2.} PCR amplicon of T. gondii ITS-1 region identified on
2\% agarose gel electrophoresis. M, 100 bp marker; P, T. gondii positive
DNA; Lane 1, T. gondii PCR positive sample; Lane 2 -- 5, T. gondii PCR
negative samples; N, negative control.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-1/Figure-1}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-2/Figure-2}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
\end{document}

