Toxoplasma gondii infection and insomnia: A case control seroprevalence study.

We determined the association between Toxoplasma gondii (T. gondii) infection and insomnia. Through an age-and gender-matched case-control study, 577 people with insomnia (cases) and 577 people without insomnia (controls) were tested for anti-T. gondii IgG and IgM antibodies using commercially available enzyme-immunoassays. Anti-T. gondii IgG antibodies were found in 71 (12.3%) of 577 individuals with insomnia and in 46 (8.0%) of 577 controls (OR = 1.62; 95% CI: 1.09-2.39; P = 0.01). Men with insomnia had a higher (16/73: 21.9%) seroprevalence of T. gondii infection than men without insomnia (5/73: 6.8%) (OR: 3.81; 95% CI: 1.31-11.06; P = 0.009). The rate of high (>150 IU/ml) anti-T. gondii IgG antibody levels in cases was higher than the one in controls (OR = 2.21; 95% CI: 1.13-4.31; P = 0.01). Men with insomnia had a higher (8/73: 11.0%) rate of high anti-T. gondii IgG antibody levels than men without insomnia (0/73: 0.0%) (P = 0.006). The rate of high anti-T. gondii IgG antibody levels in cases >50 years old (11/180: 6.1%) was higher than that (3/180: 1.7%) in controls of the same age group (OR: 3.84; 95% CI: 1.05-14.00; P = 0.05). No difference in the rate of IgM seropositivity between cases and controls was found (OR = 1.33; 95% CI: 0.57-3.11; P = 0.50). Results of this seroepidemiology study suggest that infection with T. gondii is associated with insomnia. Men older than 50 years with T. gondii exposure might be prone to insomnia. Further research to confirm the association between seropositivity and serointensity to T. gondii and insomnia is needed.

Introduction

Toxoplasma gondii (T. gondii) is an obligate intracellular parasite [1]. Toxoplasmosis, the disease caused by T. gondii, is a zoonosis with medical and veterinary importance worldwide [2]. T. gondii infects one third of the global human population [3]. Foodborne transmission of T. gondii in humans occurs mainly by eating undercooked meat, especially pork, lamb, and wild game meat from T. gondii-infected animals, and ingestion of raw fruits and vegetables contaminated with soil containing cat feces [4]. Transmission of T. gondii may also occur by blood transfusion, tissue transplants, or ingestion of unpasteurized milk [5]. In addition, T. gondii can be transmitted vertically during pregnancy [6]. Infections with T. gondii may result in a clinical spectrum ranging from a completely asymptomatic infection to multi-organ involvement [7]. Some patients with toxoplasmosis present cervical lymphadenopathy or ocular disease, whereas a reactivation of latent disease in immunocompromised patients can cause life-threatening encephalitis [8]. Chronic toxoplasmosis might be associated with a vast array of neuropsychiatric symptoms [9]. Seroprevalence of T. gondii infection has been linked to mixed anxiety and depressive disorder [10], schizophrenia [11, 12], depression [13, 14], and suicide behavior [14, 15]. Few studies have shown a link between T. gondii infection and insomnia. However, results of such studies are controversial. In a study of students, a positive association between seropositivity to T. gondii and insomnia was found in women but not in men [16]. In two further studies, researchers found that T. gondii IgG seropositivity and serointensity were not associated with insomnia [17, 18]. T. gondii can disseminate to brain in infected hosts [3], and we hypothesize that T. gondii can involve brain structures related with sleep leading to sleep problems as insomnia. Several neurotransmitters are involved in sleep including for instance, dopamine and gamma-aminobutyric acid (GABA) [19], and T. gondii infections impact on these two neurotransmitters. T. gondii infections alter the dopamine metabolism [20], and GABAergic synapses and signaling in the central nervous system [21]. Therefore, this study was aimed to determine the association between T. gondii exposure and insomnia in a sample of people in Durango, Mexico.

Materials and methods

Study design and study population

Through an age- and gender-matched case-control study, 577 people with insomnia (cases) and 577 people without insomnia (controls) were examined for the presence of T. gondii exposure. This study was performed in the northern Mexican state of Durango from 2014 to 2019. Cases and controls were individuals attending public health care institutions for medical consultations or laboratory tests. Inclusion criteria for cases were people with insomnia, aged ≥18 years, with informed consent. Gender, occupation, and socioeconomic status were not restrictive criteria for enrollment of cases or controls. The Diagnostic and Statistical Manual of Mental Disorders, Fifth edition (DSM-5) defines insomnia as a predominant complain of dissatisfaction with sleep quantity or quality, associated with one (or more) of the following symptoms: difficulty initiating sleep, difficulty maintaining sleep, and early-morning awakening with inability to return to sleep [22]. Inclusion criteria for enrollment of controls were people without insomnia, aged ≥18 years, with informed consent. Of the 577 cases, 505 (87.5%) were women and 72 (12.5%) were men. They were 18–80 years old (mean: 43.63 ± 12.55). Whereas of the 577 controls, 503 (87.2%) were women and 74 (12.8%) were men. They were 18–81 years old (mean: 43.63 ± 12.56). There were no differences in age (P = 0.99) or gender (P = 0.93) among cases and controls.

Laboratory tests

Five ml of venous blood was drawn from each participant. After blood clotting, blood samples were centrifuged. Serum was transferred into microtubes and kept at -20°C until assayed. All samples were analyzed for anti-T. gondii IgG antibodies using the commercially available enzyme-linked immunoassay “Toxoplasma gondii IgG” kit (Diagnostic Automation/Cortez Diagnostics, Inc., Woodland Hills, California. USA). This test allows detection and quantification of anti-T. gondii IgG antibodies. A cut-off of 8 IU/ml was used to determine seropositivity. Samples with results just below 8 IU/ml (grey zone) or clearly lower were considered as negatives. Serum samples with anti-T. gondii IgG antibodies were further analyzed for detection of anti-T. gondii IgM antibodies by the commercially available enzyme-linked immunoassay “Toxoplasma gondii IgM” kit (Diagnostic Automation/Cortez Diagnostics, Inc.). Sera were analyzed every few months during the study period. Positive and negative controls were included in each run. All immunoassays were carried out as described in the manufacturer’s instructions. Laboratory tests were performed blindly, the analyst did not have information about the history of insomnia in participants during the analysis of samples.

Statistical analysis

Data analysis was performed using the software SPSS Statistics version 15. A sample size of 577 cases and 577 controls was calculated using the following parameters: a reference seroprevalence of 6.1% [23] as the expected frequency of exposure in controls, a two-sided confidence level of 95%, a power of 90%, a 1:1 proportion of cases and controls, and an odds ratio of 2.0. The student’s t-test was used to assess age matching. To compare the frequencies among the groups the Pearson’s chi-square test or the Fisher’s exact test (when a value was below 5) were used. Multivariate analysis with adjustment by age, sex, education, and residence to further assess the association between T. gondii infection and insomnia was performed. Odd ratios (OR) and corresponding 95% confidence intervals (CI) were calculated. A statistical P value of ≤ 0.05 was considered significant for all comparisons.

Ethical aspects

The Ethical Committees of the Institute of Security and Social Services for the State Workers and the General Hospital of the Secretary of Health in Durango City, Mexico approved this project (Approval No. 449/015). A written informed consent from each participant was obtained.

Results

Anti-T. gondii IgG antibodies were found in 71 (12.3%) of 577 individuals with insomnia and in 46 (8.0%) of 577 controls. A statistically significant difference (OR = 1.62; 95% CI: 1.09–2.39; P = 0.01) in anti-T. gondii IgG seroprevalence between cases and controls was found. A stratification by age and sex and seroprevalence of T. gondii infection in individuals with and without insomnia is shown in Table 1. Stratification by sex showed that men with insomnia had a significantly higher (16/73: 21.9%) seroprevalence of T. gondii infection than men without insomnia (5/73: 6.8%) (OR: 3.81; 95% CI: 1.31–11.06; P = 0.009). Whereas stratification by age groups showed that seroprevalence of T. gondii infection in cases was similar to controls regardless their age groups. Of the 71 anti-T. gondii IgG positive cases, 28 (39.4%) had anti-T. gondii IgG antibody levels higher than 150 IU/ml, 6 (8.5%) between 100 IU/ml and 150 IU/ml, and 37 (52.1%) between 8 to 99 IU/ml. On the other hand, of the 46 anti-T. gondii IgG positive controls, 13 (28.2%) had anti-T. gondii IgG antibody levels higher than 150 IU/ml, 5 (10.9%) between 100 IU/ml and 150 IU/ml, and 28 (60.9%) between 8 to 99 IU/ml. A statistically significant difference (OR = 2.21; 95% CI: 1.13–4.31; P = 0.01) in the frequency of individuals with high (>150 IU/ml) anti-T. gondii IgG antibody levels between cases and controls was found. A stratification by sex and age groups and the rates of high (>150 IU/ml) anti-T. gondii IgG antibody levels in cases and controls is shown in Table 2. Stratification by sex showed that men with insomnia had a significantly higher (8/73: 11.0%) rate of high anti-T. gondii IgG antibody levels than men without insomnia (0/73: 0.0%) (P = 0.006). Whereas stratification by age groups showed that the rate of high anti-T. gondii IgG antibody levels in cases >50 years old (11/180: 6.1%) was higher than that (3/180: 1.7%) in controls of the same age group (OR: 3.84; 95% CI: 1.05–14.00; P = 0.05). Multivariate analysis with adjustment by age, sex, education, and residence showed that T. gondii infection was positively associated with insomnia (OR = 1.57; 95% CI: 1.06–2.34; P = 0.02). Of the 71 anti-T. gondii IgG antibody seropositive cases, 21 (29.6%) were also positive for anti-T. gondii IgM antibodies. Whereas, of the 46 anti-T. gondii IgG antibody seropositive controls, 11 (23.9%) were also positive for anti-T. gondii IgM antibodies. No difference in the rate of IgM seropositivity between cases and controls was found (OR = 1.33; 95% CI: 0.57–3.11; P = 0.50).

Table 1

Stratification by sociodemographic variables in cases and controls and IgG seropositivity rates to T. gondii.

	Cases			Controls
		Seropositivity			Seropositivity			95%
	No.	to T. gondii		No.	to T. gondii		Odds	confidence	P
Characteristics	tested	No.	%	tested	No.	%	ratio	interval	value
Sex
Male	73	16	21.9	73	5	6.8	3.81	1.31–11.06	0.009
Female	504	55	10.9	504	41	8.1	1.38	0.9–2.1	0.13
Age (years)
≤30	99	9	9.1	99	5	5.1	1.80	0.6–5.8	0.26
31–50	298	37	12.4	298	27	9.1	1.42	0.8–2.4	0.18
>50	180	25	13.9	180	14	7.8	1.91	0.9–3.8	0.06
Educational level
No education	11	5	45.5	8	3	37.5	1.38	0.2–8.9	0.72
1 to 6 years	149	24	16.1	104	15	14.4	1.13	0.5–2.2	0.71
7–12 years	261	27	10.3	303	18	5.9	1.80	0.9–3.3	0.05
>12 years	154	15	9.7	157	9	5.7	1.77	0.7–4.1	0.18
Residence area
Urban	362	32	8.8	467	28	6.0	1.5	0.8–2.5	0.11
Suburban	108	16	14.8	49	6	12.2	1.2	0.4–3.4	0.66
Rural	105	23	21.9	56	12	21.4	1.0	0.4–2.2	0.94
Total	577	71	12.3	577	46	8	1.62	1.0–2.3	0.01

Table 2

Stratification by sex and age in cases and controls and rates of high (≥150 IU/ml) anti-T. gondii IgG antibody levels.

	Cases			Controls
		≥150 IU/ml			≥150 IU/ml
		anti-T. gondii			anti-T. gondii
	No.	IgG levels		No.	IgG levels		Odds	confidence	P
Characteristics	tested	No.	%	tested	No.	%	ratio	interval	value
Sex
Male	73	8	11.0	73	0	0.0	--	---	0.006
Female	504	20	4.0	504	13	2.6	1.56	0.76–3.17	0.21
Age (years)
≤30	99	4	4.0	99	1	1.0	4.12	0.45–37.60	0.36
31–50	298	13	4.4	298	9	3.0	1.46	0.61–3.48	0.38
>50	180	11	6.1	180	3	1.7	3.84	1.05–14.00	0.05
All	577	28	4.9	577	13	2.3	2.21	1.13–4.31	0.01

The dataset of the study that includes all the data used to obtain the results and conclusions of the study is available (S1 File).

Discussion

There are only few studies that have assessed the epidemiological link between T. gondii infection and insomnia. Results of such studies are controversial and there is currently scanty information as to state whether T. gondii exposure leads to insomnia. Therefore, in this age- and gender-matched case-control study we attempted to determine the association between T. gondii exposure and insomnia in a sample of people in the northern Mexican state of Durango. We observed a significantly higher seroprevalence of T. gondii infection in individuals with insomnia than in individuals without insomnia. In addition, we found a significantly higher rate of high anti-T. gondii IgG antibody levels in cases than in controls. Therefore, our results suggest that T. gondii seropositivity and serointensity are positively associated with insomnia. The association between T. gondii infection and insomnia remained significant after multivariable analysis with adjustment for age, sex, education, and residence. However, the association of T. gondii seropositivity and insomnia was observed in men but not in women, and the association between T. gondii serointensity and insomnia was found only in men and in individuals aged >50 years. The positive association between T. gondii seropositivity and insomnia found in our study contrast with that found in a previous study of 200 students in Iran, where investigators observed a positive association between T. gondii seroprevalence and insomnia in women but not in men [16]. It is not clear why the association between seropositivity and insomnia was found only in men in our study. It is unknown whether men are more vulnerable than women to the clinical effects of T. gondii. It is possible that factors associated with T. gondii exposure in men were different from those in women in our study. Further research to determine the association between sociodemographic, clinical, and behavioral factors and T. gondii exposure in men with insomnia is needed. On the other hand, results of our study also contrast with the findings of three more studies. In a study of 311 older adults recruited in a psychiatric institute and clinic in Pittsburg, USA, researchers found no association between insomnia and T. gondii seroprevalence and serointensity [17]. In a study of 2031 Old Order Amish individuals recruited in Lancaster County, USA, researchers found no association between T. gondii seropositivity and serointensity and insomnia [18]. However, a secondary analysis identified, after adjustment by age group, a statistical trend towards shorter sleep duration in seropositive men [18]. In a survey of 833 Old Order Amish participants, T. gondii seropositives reported less sleep problems and less daytime problems due to poor sleep, and higher T. gondii titers were associated with longer sleep duration, earlier bedtime, and earlier mid-sleep time [24]. Difference in the association between T. gondii exposure and insomnia among the studies might be due to differences in the characteristics of the study populations. We studied people enrolled at several health care institutions whereas researchers of other studies enrolled students [16], older adults [17], and Old Order Amish individuals [18, 24]. In addition, there are differences in the sample size and study design among the studies. We used an age- and gender-matched case-control study design and studied 577 cases and 577 controls whereas in another study 180 people suffering from insomnia and 131 normal sleeping participants with no differences in age, gender and race were studied [17]. The study of students was cross-sectional and had 200 participants [16]; and the studies of Old Order Amish were cross-sectional and included between 833 [24] and 2031 participants [18]. Risk factors for T. gondii exposure and host factors might be different among the study populations. In addition, differences in the virulence of T. gondii strains among infected hosts might exist. In an experimental mouse model of chronic T. gondii infection, investigators found that infected mice exhibited chronic sleep–wake alterations over months, including a marked increase in time spent awake [25]. Further research to determine the association between insomnia and T. gondii seropositivity and serointensity are needed. The presence of anti-T. gondii IgM antibody was determined only in anti-T. gondii IgG positive samples. The presence of IgM without IgG is not a reliable result because enzyme immune assays for anti-T. gondii IgM antibodies have high numbers of false-positive results [26]. We did not obtain information about specific sleep variables. Studies to determine an association between T. gondii infection and specific clinical variables of insomnia are needed. We did not screen for neuropsychiatric or neurodegenerative disorders among participants. Further studies with information about sleep clinical variables and the presence of neuropsychiatric or neurodegenerative disorders among participants are needed. Results of our study do not mean that T. gondii infection leads to insomnia. Only longitudinal studies may help to determine the direction of the association.

Conclusions

Results of this age- and gender-matched case-control study suggest that infection with T. gondii is associated with insomnia. Men older than 50 years with T. gondii exposure might be prone to insomnia. Further research to confirm the association between seropositivity and serointensity to T. gondii and insomnia is needed.

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19 Apr 2022

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Reviewer #1: The topic is interesting but the manuscript needs major revision.

The approval ethical number needs to add.

The authors only checked the serum samples with anti-T. gondii IgG antibodies for analyze and detection of anti-T. gondii IgM antibodies so may be lost the samples with IgM positive and IgG negative.

The data of table 1 is the positive of T. gondii is related to IgG or IgM results.

There is not any tables about IgM results.

Is T. gondii related to the kind of symptoms: 1-difficulty initiating sleep, 2- difficulty maintaining sleep and 3- early-morning awakening with inability to return to sleep, or no?

The duration of this study from 2014-2019 is very wide period and this may be affected the age grouping.

All sera that collected during 5 years were evaluated for IgG and IgM against T. gondii in the same time or no.

Reviewer #2: The article submitted for publication by C . Alvarado-Esquivel and colleagues deals with the association between Toxoplasma infection and insomnia. It reports the findings of a case control study performed on 577 patients with insomnia and 577 control subjects in Mexico, and concludes to the presence of association.

Introduction

l.88 : « responsible » there is no link of causality that has been clearly demonstrated, only statistical association

l90-95 : As mentioned by the authors several studies have already looked into this association with conflicting results, however article by Corona C should be cited and discussed (Toxoplasma gondii IgG associations with sleep-wake problems, sleep duration and timing, Pteridines. 2019 Feb; 30(1): 1–9.). Furthermore we believe that the only fundamental study in a mice model should also be included and discussed (Dupont et al, Chronic Toxoplasma gondii infection and sleep-wake alterations in mice, CNS Neurosci Ther. 2021 Aug;27(8):895-907).

Methods

The heterogeneity of symptoms associated with insomnia should also be commented on in the discussion.

Selection of controls: it is known that T. gondii seroprevalence is strongly influenced by education or residence? Thus are controls similar to cases on these parameters? If no information available, this should be discussed. Furthermore it is known that sleep is an index of brain functions as sleep alteration are usually observed in several neuropsychiatric or neurodegenerative disorders even before clinical neurological manifestations. If controls/cases were selected when attending medical institution, did these patients were screened for these disorders associated with sleep alterations? this should also be discussed.

Age groups are not described in the methods, we believe that analyzing the effects of age as a continuous variable should be better thant categorizing by arbitrary age groups.

Laboratory tests

Were the analyses performed blindly as regards to the staus of cases/controls ? Was there a grey zone in the assay used ? if so how was it further processed ?

Discussion

Reference 18 does not conclude to an association however, there is statistical trend toward shorter sleep duration in seropositive men. This should be added to the discussion, in line with results from Dupont et al, Chronic Toxoplasma gondii infection and sleep-wake alterations in mice, CNS Neurosci Ther. 2021 Aug;27(8):895-907.

Furthermore, it should be clearly stated that this study does not allow to conclude that T. gondii leads to insomnia. Statistics do only point towards an association without precluding the direction of the association, all the most if patients were not selected as free of neuropsychiatric or degenerative disorders. Cinetics is of utmost importance and cinetics cannot be performed when using only one serum.

Reviewer #3: Dear respect Editor:

The manuscript entitled: “Toxoplasma gondii infection and insomnia: a case control seroprevalence study” addresses a highly interesting topic. The study presents remarkable information. The methodology and design of this work is appropriate. I suggest that the authors could enhance the usefulness of this work for the community if they redesign the tables.

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12 May 2022

Durango, Dgo. Mexico. May 4, 2022.

Dear Editor,

Please find attached a revised version of our manuscript that has been modified according to the reviewers’ comments. In addition, please find below our response to each of the reviewers’ comments on a point-by-point basis. The revised manuscript meets PLOS ONE’s style requirements. The authors have declared that no competing interests exist. The dataset of the study that includes all the data used to obtain the results and conclusions of the study is available (Supplementary file 1).

We appreciate the valuable comments of the reviewers and we hope the revised manuscript may have more success for publication in the journal Plos One.

Kind regards,

Dr. Cosme Alvarado-Esquivel.

Laboratorio de Investigación Biomédica

Facultad de Medicina y Nutrición

Avenida Universidad S/N.

34000 Durango, Dgo. Mexico.

Tel/Fax.: 0052 618 8 271200

Email: alvaradocosme@yahoo.com

RESPONSE TO THE REVIEWERS’ COMMENTS

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Thank you for your valuable comments for improving our manuscript.

Reviewers' comments:

Reviewer #1:

1. The approval ethical number needs to add.

The approval ethical number was added (line 129).

2. The authors only checked the serum samples with anti-T. gondii IgG antibodies for analyze and detection of anti-T. gondii IgM antibodies so may be lost the samples with IgM positive and IgG negative.

The presence of anti-T. gondii IgM antibody was determined only in anti-T. gondii IgG positive samples. The presence of IgM without IgG is not a reliable result because enzyme immune assays for anti-T. gondii IgM antibodies have high numbers of false-positive results. This information was added to the Discussion section (lines 221-224).

3. The data of table 1 is the positive of T. gondii is related to IgG or IgM results.

It is related to IgG antibodies. This information was added to the Title of Table 1.

4. There is not any tables about IgM results.

Since there was no difference in the frequency of IgM antibodies among groups, no further analysis of IgM results in a Table was performed.

5. Is T. gondii related to the kind of symptoms: 1-difficulty initiating sleep, 2- difficulty maintaining sleep and 3- early-morning awakening with inability to return to sleep, or no?

We did not obtain this information. Studies to determine an association between T. gondii infection and specific symptoms of insomnia are needed. This information was added to the Discussion section (lines 224-226).

6. The duration of this study from 2014-2019 is very wide period and this may be affected the age grouping.

We further evaluate the association between T. gondii seropositivity and insomnia by adjustment for age. This information was added to the Materials and methods section (lines 121-123), Results section (lines 155-157), and Discussion section (lines 183-185).

7. All sera that collected during 5 years were evaluated for IgG and IgM against T. gondii in the same time or no.

Sera were analyzed every few months during the study period. This information was added to the Materials and methods section (line 108).

Thank you for your valuable comments for improving our manuscript.

Reviewer #2:

1. Introduction

l.88 : « responsible » there is no link of causality that has been clearly demonstrated, only statistical association

The word “responsible” was deleted. The sentence was rewritten as follows: ”Chronic toxoplasmosis might be associated with a…” (line 62).

2. l90-95: As mentioned by the authors several studies have already looked into this association with conflicting results, however article by Corona C should be cited and discussed (Toxoplasma gondii IgG associations with sleep-wake problems, sleep duration and timing, Pteridines. 2019 Feb; 30(1): 1–9.). Furthermore we believe that the only fundamental study in a mice model should also be included and discussed (Dupont et al, Chronic Toxoplasma gondii infection and sleep-wake alterations in mice, CNS Neurosci Ther. 2021 Aug;27(8):895-907).

Our results were contrasted with those reported by Corona et al, and Dupont et al, (lines 203-206, and lines 218-220, respectively).

3. Methods

The heterogeneity of symptoms associated with insomnia should also be commented on in the discussion.

We did not obtain this information. Studies to determine an association between T. gondii infection and specific symptoms of insomnia are needed. This information was added to the Discussion section (lines 224-226).

4. Selection of controls: it is known that T. gondii seroprevalence is strongly influenced by education or residence? Thus are controls similar to cases on these parameters? If no information available, this should be discussed.

Information about education and residence in participants was added to Table 1. We further evaluate the association between T. gondii seropositivity and insomnia by adjustment for age, education and residence. This information was added to the Materials and methods section (lines 121-123), Results section (lines 155-157), and Discussion section (lines 183-185).

5. Furthermore it is known that sleep is an index of brain functions as sleep alteration are usually observed in several neuropsychiatric or neurodegenerative disorders even before clinical neurological manifestations. If controls/cases were selected when attending medical institution, did these patients were screened for these disorders associated with sleep alterations? this should also be discussed.

This was a limitation of the study. We did not screen for neuropsychiatric or neurodegenerative disorders among participants. Further studies with information about sleep clinical variables and the presence of neuropsychiatric or neurodegenerative disorders among participants are needed (lines 226-229).

6. Age groups are not described in the methods, we believe that analyzing the effects of age as a continuous variable should be better thant categorizing by arbitrary age groups.

We further evaluated the association between T. gondii seropositivity and insomnia by adjustment for age. This information was added to the Materials and methods section (lines 121-123), Results section (lines 155-157), and Discussion section (lines 183-185).

7. Laboratory tests

Were the analyses performed blindly as regards to the staus of cases/controls ?

Laboratory tests were performed blindly, the analyst did not have information about the history of insomnia in participants during the analysis of samples. This information was added to the Materials and methods section (lines 110-112).

8. Was there a grey zone in the assay used ? if so how was it further processed ?

Samples with results just below 8 IU/ml (grey zone) or clearly lower were considered as negatives (lines 104-105).

9. Discussion

Reference 18 does not conclude to an association however, there is statistical trend toward shorter sleep duration in seropositive men. This should be added to the discussion, in line with results from Dupont et al, Chronic Toxoplasma gondii infection and sleep-wake alterations in mice, CNS Neurosci Ther. 2021 Aug;27(8):895-907.

Information about the secondary analysis (reference 18) that showed a trend toward shorter sleep duration in seropositive men was added in the Discussion section (lines 201-203).

10. Furthermore, it should be clearly stated that this study does not allow to conclude that T. gondii leads to insomnia. Statistics do only point towards an association without precluding the direction of the association, all the most if patients were not selected as free of neuropsychiatric or degenerative disorders. Cinetics is of utmost importance and cinetics cannot be performed when using only one serum.

We added the following information: Results of our study do not mean that T. gondii infection leads to insomnia. Only longitudinal studies may help to determine the direction of the association (lines 229-231).

Thank you for your valuable comments for improving our manuscript.

Reviewer #3:

1. I suggest that the authors could enhance the usefulness of this work for the community if they redesign the tables.

Table 1 was modified. Information about sociodemographic variables was added.

Thank you for your valuable comments for improving our manuscript.

Submitted filename: Response to reviewers Tg-Insomnia.docx

Click here for additional data file.

24 May 2022

Toxoplasma gondii infection and insomnia: a case control seroprevalence study

PONE-D-22-07752R1

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31 May 2022

PONE-D-22-07752R1

Toxoplasma gondii infection and insomnia: a case control seroprevalence study

Dear Dr. Alvarado-Esquivel:

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