Is Toxoplasma gondii Infection Associated with Sexual Promiscuity? A Cross-Sectional Study

Round 1

Reviewer 1 Report

General comment:

• The authors have suggested there is an association between T.gondii infection and sexual promiscuity. In terms of novelty, this study serves as an additional “reinforcement” study on a previous similar study (Alvarado-Esquivel et al, 2006). However, what makes this study interesting is the big sample size available. However, the manuscript can still be further improved with the specific comments below.

 

Major comments:

• The introduction needs improvement in terms of establishing the novelty and hypothesis of your study. Firstly, the introduction fails to illustrate how the important link between T.gondii infection and sexual promiscuity is derived. More specific literature work about sexual behavior and T.gondii infection has to be carried out and established in the introduction. From your introduction, I cannot quite comprehend how you derive your hypothesis that there is an association between T.gondii infection and sexual promiscuity. This gives me the impression that authors are cherry-picking different factors e.g., sexual promiscuity, traffic accidents, impulsivity, etc, and formulate a hypothesis based on which factors give a positive association. Please improvise the introduction. Or is it due to reference number 18 ((Alvarado-Esquivel et al, 2006)? If so, then this article is not original as this association was already found. So, what is the point of your study? This has to be clear and tell readers about your purpose of conducting this study when an article has already been published on. In summary, please elaborate in your introduction why you are conducting this study based on prior literature about sexual behaviors. What is the difference between your study and previous study? I think this has to be clearly written and established in the introduction again. If not, the whole study is like a repeat of a past study.

 

• What are the T.gondii strains infecting your subjects? It is critical to inform readers what T.gondii strains were dominant in your studies as different strains may have different degrees of influence on inducing depression. Perhaps, some genotyping experiments can be carried out to determine the strain. Please include this very important piece of information in your results and discussion.

 

• In lines 71 and 84, the association was calculated based on the age band, what is the significance behind the calculation of this association? This was not mentioned or discussed especially why 31-50 years age band has a P>0.005.

 

• In line 82, what is the significance of measuring higher IgG levels? If a subject is infected based on your commercial kit (8 IU/ml), he or she is toxoplasmosis-positive. Hence, is there any significance in going deeper in establishing an association between how high IgG antibodies and sexual promiscuity? Please explain in detail and also suggest/discuss why 31-50 age band did not have a significant association. Please justify these selections in a scientific manner both in results and in materials/methods.

 

• In line 88, why was IgM measured? Please explain clearly and discuss your results.

 

• In line 111, important references are missing with regards to animal behavior and T.gondii infection. There are a few recent reviews that I recommend and hope to see included:

1. Behavioral biology of Toxoplasma gondii infection.

DOI: https://doi.org/10.1186/s13071-020-04528-x

 

2. Toxoplasma gondii: An Underestimated Threat?

DOI: https://doi.org/10.1016/j.pt.2020.08.005

 

• In line 118, the dopamine-T.gondii theory has been a hot topic in the 2010s but since then, this theory has been proven otherwise in the last couple of years by various groups such as Sibley and Vyas. In addition to the dopamine D4 receptor gene, the tyrosine hydroxylase gene ( a precursor to dopamine synthesis) has been heavily speculated to be involved in behavior change though this may be debunked (Elizabeth A Gaskell et. al, 2009, DOI: 10.1371/journal.pone.0004801). Please try to take a balanced approach in your discussion and also incorporate these authors as references as well. 2 relevant references I would like to see included:

1. Behavioral Manipulation by Toxoplasma gondii: Does Brain Residence Matter? DOI: 10.1016/j.pt.2020.12.006

 

2. Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

doi: 10.1128/IAI.02465-14

 

The first reference by Vyas group will also support your discussion on higher testosterone levels in more sexually promiscuous people and t.gondii infection.

 

• In material and methods under 4.2, key details regarding the serology tests are missing. What is the sensitivity and precision of essays? Please state these values and how will this affect your result accuracy? What is the significance of measuring IgM as well? Please elaborate.

Author Response

1. The introduction needs improvement in terms of establishing the novelty and hypothesis of your study. Firstly, the introduction fails to illustrate how the important link between T.gondii infection and sexual promiscuity is derived. More specific literature work about sexual behavior and T.gondii infection has to be carried out and established in the introduction. From your introduction, I cannot quite comprehend how you derive your hypothesis that there is an association between T.gondii infection and sexual promiscuity. This gives me the impression that authors are cherry-picking different factors e.g., sexual promiscuity, traffic accidents, impulsivity, etc, and formulate a hypothesis based on which factors give a positive association. Please improvise the introduction. Or is it due to reference number 18 ((Alvarado-Esquivel et al, 2006)? If so, then this article is not original as this association was already found. So, what is the point of your study? This has to be clear and tell readers about your purpose of conducting this study when an article has already been published on. In summary, please elaborate in your introduction why you are conducting this study based on prior literature about sexual behaviors. What is the difference between your study and previous study? I think this has to be clearly written and established in the introduction again. If not, the whole study is like a repeat of a past study.

We thank the reviewer for these comments.

 

We addressed the issues by adding the following information to the Introduction section (lines 54-80):

 

In contrast, some studies have shown a lack of association between T. gondii seropositivity and depression in pregnant women [18], completed suicide [19], and bipolar disorder [20]. Reports thus indicate that further research to elucidate the link between T. gondii infection and clinical and behavioral changes are largely needed. It is unclear whether T. gondii infection might be link to sexual behavior. We hypothesized that sexual promiscuity is associated with T. gondii infection. On the one hand, a group of researchers have hypothesized that toxoplasmosis can be a sexually transmitted infection [21]. If T. gondii can be transmitted by sexual intercourse thus persons with sexual promiscuity might have a higher risk for T. gondii infection than persons without this practice. High seroprevalence of T. gondii infection has been reported in population groups with large number of sexual partners or having a sexually transmitted infection including female sex workers [22] and people with human immunodeficiency virus infection [23]. On the other hand, high seroprevalence of infection with T. gondii is found in psychiatric patients [11, 24] and sexual promiscuity is a common behavior in patients with psychiatric illnesses. An increased in sexual activity and promiscuity have been found in patients with bipolar depression [25], depressed children [26], patients with borderline personality disorder [27], and psychiatric patients with a history of suicide attempts [28]. To the best of our knowledge there is not any case-control study that had studied the epidemiological association between T. gondii infection and sexual promiscuity. In a study of 137 psychiatric inpatients in Mexico, an association between T. gondii seropositivity and sexual promiscuity was found [24]. However, in such a study, the analysis was performed in a quite limited number of psychiatric inpatients (not in their controls), in a single hospital, and the association was obtained without any matching by age and gender. Therefore, in the present study including a large sample of participants, in several public health facilities, and with an age- and gender-matched case-control study design we aimed to determine the association between T. gondii seropositivity and sexual promiscuity.    

 

In addition, we added the following information in the Discussion section (lines 129-131):

 

However, in such a study, no stratification by age and sex was performed, and no association between sexual promiscuity and T. gondii serointensity or IgM seropositivity was assessed

 

 

2. What are the T. gondii strains infecting your subjects? It is critical to inform readers what T. gondii strains were dominant in your studies as different strains may have different degrees of influence on inducing depression. Perhaps, some genotyping experiments can be carried out to determine the strain. Please include this very important piece of information in your results and discussion.

 

We thank the reviewer for this comment.

 

We do not have information about the T. gondii strains infecting people in Durango, Mexico. We did not perform molecular analyses in our study. It is of interest to determine the T. gondii strains in people in Mexico and their association with behavioral changes including sexual promiscuity (lines 200-203).

 

 

3. In lines 71 and 84, the association was calculated based on the age band, what is the significance behind the calculation of this association? This was not mentioned or discussed especially why 31-50 years age band has a P>0.005.

 

We thank the reviewer for this comment.

 

We discussed the significance behind the calculation of the association as follows (lines 129-138):

 

In the present study, we performed a stratification by age groups in order to determine whether the observed association was uniform regardless of age of participants or whether it occurred only in a certain age group. Seropositivity to T. gondii was associated with sexual promiscuity in all age groups, whereas T. gondii serointensity was associated with sexual promiscuity in people aged ≤30 years and >50 years but not in individuals aged 31-50 years. It is unclear why the T. gondii serointensity association was not uniformly observed in all age groups. It is possible that unknown factors linked to T. gondii infection and/or sexual promiscuity present in individuals aged 31-50 years differed from those present in younger and older individuals.

 

 

4. In line 82, what is the significance of measuring higher IgG levels? If a subject is infected based on your commercial kit (8 IU/ml), he or she is toxoplasmosis-positive. Hence, is there any significance in going deeper in establishing an association between how high IgG antibodies and sexual promiscuity? Please explain in detail and also suggest/discuss why 31-50 age band did not have a significant association. Please justify these selections in a scientific manner both in results and in materials/methods.

 

We thank the reviewer for this comment.

 

Information about the meaning of high anti-T. gondii IgG antibodies was added to the Materials and Methods section as follows (lines 221-224).

 

High anti-T. gondii IgG antibody levels may indicate an acute or recent infection, or in chronic infections reactivation or reinfection. Measuring the level of IgG can provide information about an on-going or resumed replication of the pathogen [40].

 

In addition, we discussed the significance of measuring higher IgG levels as follows (lines 140-148):

 

Anti-T. gondii IgG antibodies peaks at 3 months after infection and then remains at a plateau level for six months and after one year starts to decrease [29]. A study of mice showed that increased IgG levels correlated with proliferation of tachyzoites in the brain during the chronic stage of infection suggesting that high anti-T. gondii IgG antibody levels may indicate an occurrence of cerebral tachyzoite growth in immunocompetent individuals chronically infected with T. gondii [30]. Thus, in the present study, a considerable number of participants with high anti-T. gondii IgG antibody levels and sexual promiscuity might have had a recent infection or a reactivation of the infection.

 

5. In line 88, why was IgM measured? Please explain clearly and discuss your results.

 

We thank the reviewer for this comment.

 

Information about the IgM testing was added to the Discussion section as follows (lines 148-152):

 

Anti-T. gondii IgM antibodies usually appear after one week of infection and disappear after 9 months but can remain for two years or more in some individuals [29]. Thus, the lack of association between T. gondii IgM seropositivity and sexual promiscuity found in the current study suggests that the association between T. gondii infection and sexual promiscuity occurred in individuals with chronic infections.

 

 

6. In line 111, important references are missing with regards to animal behavior and T. gondii infection. There are a few recent reviews that I recommend and hope to see included:
7. Behavioral biology of Toxoplasma gondii infection.

DOI: https://doi.org/10.1186/s13071-020-04528-x

 

2. Toxoplasma gondii: An Underestimated Threat?

DOI: https://doi.org/10.1016/j.pt.2020.08.005

 

We thank the reviewer for this comment.

 

Both interesting reviews were included (lines 152-158).

 

 

7. In line 118, the dopamine-T. gondii theory has been a hot topic in the 2010s but since then, this theory has been proven otherwise in the last couple of years by various groups such as Sibley and Vyas. In addition to the dopamine D4 receptor gene, the tyrosine hydroxylase gene ( a precursor to dopamine synthesis) has been heavily speculated to be involved in behavior change though this may be debunked (Elizabeth A Gaskell et. al, 2009, DOI: 10.1371/journal.pone.0004801). Please try to take a balanced approach in your discussion and also incorporate these authors as references as well. 2 relevant references I would like to see included:

1. Behavioral Manipulation by Toxoplasma gondii: Does Brain Residence Matter? DOI: 10.1016/j.pt.2020.12.006

 

1. Reassessment of the Role of Aromatic Amino Acid Hydroxylases and the Effect of Infection by Toxoplasma gondii on Host Dopamine

doi: 10.1128/IAI.02465-14

 

We thank the reviewer for this comment.

 

These interesting articles were included (lines 162-167, and 175-179).

 

 

8. The first reference by Vyas group will also support your discussion on higher testosterone levels in more sexually promiscuous people and t.gondii infection.

 

We thank the reviewer for this comment.

 

This interesting reference by Vyas supporting the increase in the synthesis of testosterone was included in the Discussion section (lines 162-164).

 

 

9. In material and methods under 4.2, key details regarding the serology tests are missing. What is the sensitivity and precision of essays? Please state these values and how will this affect your result accuracy? What is the significance of measuring IgM as well? Please elaborate.

 

We thank the reviewer for this comment.

 

Information about sensitivity, specificity and precision of the tests was added (lines 219-221 and 226-229).

 

Thank you for your valuable comments for improving our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript (Pathogens-1392721) submitted by Alvarado-Esquivel et al. describes the prevalence of Toxoplasma gondii infection in people in Mexico. The manuscript presents interesting results, however I have a few reservations.

Line 38: "exposed", should be "infected". The entire human population is at risk.

Line 41-43: The main route of infestation with the parasite is through undercooked or raw meat, this should be emphasized.

In my opinion, the authors draw too far-reaching conclusions. It is necessary to be careful when drawing conclusions having such large disproportions between the studied groups. In the analysis, gender should also be linked, depending on age, with the frequency of infection. Living conditions should be taken into account (is it possible to correlate urban and rural inhabitants?). All of the above can have a significant impact. It is well known that with age, a greater proportion of the population is infected by the parasite. Moreover, a greater proportion of the population is infected in rural areas than in urban areas. It is worth enriching the results with such an analysis as well as a subsequent discussion. The analysis of the results should definitely be extended to include eating habits, housing conditions, etc.

Author Response

1. Line 38: "exposed", should be "infected". The entire human population is at risk.

 

We thank the reviewer for this comment.

 

The word “exposed” was replaced with “infected” (line 36).

 

 

2. Line 41-43: The main route of infestation with the parasite is through undercooked or raw meat, this should be emphasized.

 

We thank the reviewer for this comment.

 

The route of infection dealing with consumption of undercooked or raw meat was emphasized (lines 41-42).

 

 

3. In my opinion, the authors draw too far-reaching conclusions. It is necessary to be careful when drawing conclusions having such large disproportions between the studied groups. In the analysis, gender should also be linked, depending on age, with the frequency of infection. Living conditions should be taken into account (is it possible to correlate urban and rural inhabitants?). All of the above can have a significant impact. It is well known that with age, a greater proportion of the population is infected by the parasite. Moreover, a greater proportion of the population is infected in rural areas than in urban areas. It is worth enriching the results with such an analysis as well as a subsequent discussion. The analysis of the results should definitely be extended to include eating habits, housing conditions, etc.

 

We thank the reviewer for this comment.

 

We have performed a further statistical analysis using logistic regression with adjustment for age, gender, and residence area. Information of this new analysis was added (lines 86-89 and 238-240).

 

Thank you for your valuable comments for improving our manuscript.

 

Round 2

Reviewer 1 Report

The authors have done a good job in addressing my concerns.

Author Response

Thank you for your kind comments that allowed us to greatly improve our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript (Pathogens-1392721) submitted by Alvarado-Esquivel et al. describes the association of sexual promiscuity with Toxoplasma gondii infection.

I am satisfied with the changes introduced by the authors in line with the reviewers' suggestions.

In my opinion, the obtained results could be used for a more extensive analysis, nevertheless, I would like to thank the author for additional analyzes in line with my suggestions. In the future, I advise authors to analyze data in more aspects, even combining several factors.

 

Author Response

Thank you for this comment.

 

Information about the limitations of the study including the statistical analysis was added to the Discussion section as follows (lines 205-214):

 

The present study has limitations: 1) we performed a logistic regression analysis with adjustment of only few variables, and additional research with a more extensive analysis of variables should be conducted; and 2) we did not perform molecular analyses. It is of interest to determine the T. gondii genotypes in people in Mexico. It raises the question whether any T. gondii genotype might be linked to behavioral changes including sexual promiscuity. Further research to determine the association between T. gondii infection and sexual promiscuity using a case-control study design is needed. In addition, studies to determine the seroepidemiology of T. gondii infection in people with sexual promiscuity including analysis of sociodemographic, housing, clinical and behavioral characteristics of participants should be conducted.

 

Thank you for your kind comments that allowed us to greatly improve our manuscript.

 

 

 

Author Response File: Author Response.pdf