Title: Increased incidence of traffic accidents in RhD-negative, Toxoplasma gondii-infected military drivers revealed by a prospective cohort study
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1 Author's response to reviews Title: Increased incidence of traffic accidents in RhD-negative, Toxoplasma gondii-infected military drivers revealed by a prospective cohort study Authors: Jaroslav Flegr (flegr@cesnet.cz) Jiri Klose (jiri.klose@uvn.cz) Martina Novotna (vitakova@seznam.cz) Miroslava Berenreitterová (mircik@centrum.cz) jan Havlicek (jan.havlicek@fhs.cuni.cz) Version: 3 Date: 9 January 2009 Author's response to reviews: see over
2 Dear Mr. Todd Enclosed please find a corrected version of the manuscript entitled Increased incidence of traffic accidents in RhD-negative, Toxoplasma gondii-infected military drivers revealed by a prospective cohort study Based on the recommendations of the referees we have made many changes in the text, figure and included one new table of results. Our responses to referees and the changes done in the corrected version of the manuscript are listed in the following List of changes. I hope that our manuscript in the new form will be acceptable to be published in BMC Infectious Diseases. Yours sincerely Jaroslav Flegr List of changes Reviewer I report Title: Increased incidence of traffic accidents in RhD-negative, Toxoplasma gondii-infected military drivers revealed by a prospective cohort study Version: 2 Date: 4 December 2008 Reviewer: Eleni Petridou Reviewer's report: The authors test an intriguing hypothesis related to intrinsic (RhD) and extrinsic (Toxoplasma infection) factors may impact on reflexes and human behavior and eventually contribute to the causation of road traffic injuries among male military conscripts. The paper suffers mainly in the description of the methodology followed, the statistical analyses undertaken and the presentation of the results. Major Compulsory Revisions 1. The missing Methods section should be clearly described and inserted as distinct entities in both the abstract and the main text. The current format is rather unconventional, hinders the reviewing assessment and is confusing for the Reader. Corrected. Information should be given on individuals who did not agree to participate and whether their psychological profile was different from those who did. Due to ethical and/or legislative reasons, we were not allowed to analyze or publish any data of subjects who did not agree to participate in the research. An anecdotal observation (increased frequency of traffic accidents in 20% of drivers who did not agree to participate), however, suggests that this subpopulation differs in both psychological profile and probability of traffic accident.
3 Also make sure that you do not intermingle results with background knowledge (e.g. An original case control study [7] has shown...of the latent infection ] Corrected 2. Operational definitions should be given e.g the authors test for anti-toxo seropositivity, which is not equivalent with changed reaction time due to CNS damage. Mention about the possibility of false positive results and cross reaction with other antibodies, such as anti-nuclear should be made. In this case, it may at least in some instances- be a link between other undiagnosed pathological conditions that might affect metabolism and hence CNS reflexes. We included following operational definition: For the purposes of this study, the subjects with negative results of IgM ELISA (positivity index < 0.9) and absorbance in IgG ELISA > 0.250, i.e. approximately 10 IU/ml, were considered latent toxoplasmosis-positive while subjects with absorbance in IgG ELISA < were considered latent toxoplasmosis negative. The probability of false positive results is very low. Three independent studies performed on patients diagnosed with ACUTE toxoplasmosis on the basis of both serology and clinical symptoms 2-14 years before the study onset and on young women with latent toxoplasmosis showed that the psychological changes and reaction times of infected subjects increased with the length of the Toxoplasma infection. This suggests that the toxoplasmosis, rather than other undiagnosed pathological conditions affect CNS reflexes. We included following sentence in the Method section: The probability of false positive results of two independent serological tests is rather low, however, theoretically, the population of subjects tested negative in serological test could be contaminated with unknown (but probably very low) number of immunocompromised subjects [18]. 3. Furthermore, it is reasonable to assume that not all toxo-infected individuals will have CNS involvement and that staging of the disease along with severity of CNS or parts of the brain involved should be taken into account. To this end, further work up i.e. MRI should be made available. From technical reasons, it is not possible to use more discriminating method or expensive method like MRI in a large prospective study. In the present, however, we run a case-control study on two populations (40 patients with schizophrenia and 40 controls). Preliminary analyses show a significant difference in MRI and FDG PET patterns between Toxoplasmafree and Toxoplasma-infected subjects in both populations. 4. It is true that several diseases or conditions have been linked to RhD grouping; the authors should provide a rational explanation about the underlying mechanism linking RhD grouping with CNS involvement among those infected with toxo.
4 We included following paragraph into the Discussion: The absence of data on RHD genotype of subjects makes any speculation on the mechanism of RhD-positivity protection very difficult. However, the results of previous studies [9,10] and current knowledge about localization and probable function of RH proteins [11,12] suggest that the ion pumps RhD and RhCE coded in RH locus and localized on erythrocytes membrane are involved in regulation of an ion balance in some critical compartment of neural or muscular tissue. Such function could be important especially for subject handicapped by presence of cysts of Toxoplasma in neural and muscular tissues. 5. Control for confounding: variables: it is not made clear which variables have been taken into account, even the most basic ones e.g. driving experience of the victims or age (not a standard deviation of the mean age is provided to check the range of the variable). Reliable information on driving experience were not available. To our knowledge, there are, however, no reasons to expect that experienced drivers have different representation in four groups of subjects (Toxo-infected RhD-positive, Toxo-positive RhD-negative, Toxo-free RhD-positive and Toxo-free RhD-negative drivers). Therefore, absence of such covariates in our model could only increase probability of type II error, i.e. probability of a false negative result of the study. The only covariate that can be suspected to covary both with toxoplasmosis status and probability of the traffic accident was the age of a driver. This confounding was included in loglinear analysis (as indicated in the new version of a paper). Following information was added into the Methods section of the corrected version of the paper: All variables including covariates which entered into each analyzes are specified in results section. 6. Completeness and validity of registration of the outcome variable should be mentioned. We included following information into the Method section: According to military legislation all traffic accidents including material damage and/or injuries should must be reported to the military police. Of course, some less serious accidents might stay unreported, however, according to experience of military traffic experts completeness of military records is much higher than in civic sphere (personal communication). 7. Describe the limitations ELISA method to assess toxo seropositivity (Clin Microbiol Infect. 2007;13: 40-47) and that it can refer only to immunocompetent individuals (Lancet 2004: 363; ).
5 The first author of the former paper is Petr Kodym who is also the head of the lab in which all our sera are tested for latent toxoplasmosis. We (and even more a personnel of this reference laboratory) are aware of possible limitations of ELISA and CFT methods used in our study. In the corrected version of the paper we specified in more details our general diagnostic strategy and indicated possible problems with immunocompromised subjects: For the purposes of this study, the subjects with negative results of IgM ELISA (positivity index < 0.9) and absorbance in IgG ELISA > 0.250, i.e. approximately 10 IU/ml, were considered latent toxoplasmosis-positive while subjects with absorbance in IgG ELISA < were considered latent toxoplasmosis negative. Less than ten subjects with different diagnosis obtained with CFT and IgG ELISA tests or with suspected acute toxoplasmosis were excluded from the present study. Theoretically, the population of subjects tested negative in serological test could be contaminated with unknown (but probably very low) number of immunocompromised subjects [18]. 8. A cross tabulation table of the study sample describing the variables used in the analysis should be presented along with the table describing the results of the logistic analysis. The results should be presented thereafter on the basis of the data shown in the tables. We included a new table showing the age and number of subjects in particular categories. The presentation of results of logistic regression would need a separate table (one for each analysis). We believe that presentation of results directly in the text is more economic and probably also more clear way for presentation of this kind of data. 9. Major discrepancies are noted between the data analysed and the results presented/discussed e.g. from the analysis it seems that toxo positivity is a risk factor only among RhD negative individuals, further on the authors, however, generalize the finding to the total study subjects. The statement in the abstract RhD positive subjects, especially Rhd heterozygotes are protected against latent toxo-induced impairment of reaction times does not seem to satisfy dose response criteria of causality. We agree with the reviewer that the best performance of RhD positive heterozygotes is very difficult to explain. A previous case-control study performed on large sample of blood donors showed that RhD positive heterozygotes are resistant to pathological effect of toxoplasmosis while RhD positive homozygotes are resistant only temporarily, their psychomotor performance decreases with length of the infection. (The psychomotor performance of RhD negative homozygotes decreases immediately after the infection.) The best performance of heterozygotes in different genes (and therefore the impossibility to apply the dose response criteria of causality) is relatively common phenomenon, however, the d allele of RHD gene is usually expected to code the (inactive) RhD molecule with large part of the aminoacid chain missing. One can expect that decreased (possibly optimal) density of RhD molecules on the surface of erythrocytes can be more easily (and more economically) achieved by downregulation of the expression of RhD molecule than by spreading of RHD gene variant with the deletion. Theoretically, the RhD molecule could have two different functions for which the optimal density of molecule on the erythrocyte surface differs. Anyway, we are afraid that such superdominace effect of RHD gene cannot be explained before a physiological role of RhD molecule is revealed.
6 In our present study we have no data concerning RHD genotype of conscripts. Therefore, the enigma of the best performance of RhD positive heterozygotes (as well as a similar enigma of better performance of Toxoplasma-infected than Toxoplasma-free heterozygotes) is not discussed. 10. Toxo prevalence and RhD phenotyping of the Czech population should be, at least presented in comparison to the ones of the present study before the results were generalized to the general population. We included following paragraph into the corrected version of the paper: In the general male Czech population of similar age the seroprevalence of Toxoplasma infection should be approximately 28.7 and frequency of RhD-negative subjects should be approximately 18%. It must be stressed out, however, that the seroprevalence of toxoplasmosis varies from 7-90% in different regions of the Czech Republic [8319]. We have currently no explanation for significantly higher frequency of RhD negative subjects in our sample of ethnically highly homogenous Czech population. 11. The paragraph pertaining to limitations of the study is missing. We included following paragraph into the corrected version of the paper: Major limitation of present study is an absence of data on RHD genotype of subjects. A published case-control study performed on large sample of blood donors showed that RhDpositive heterozygotes are resistant to pathological effect of toxoplasmosis while RhDpositive homozygotes are resistant only temporarily their psychomotor performance decreases with length of the infection. The psychomotor performance of RhD-negative homozygotes decreases immediately after the infection. Our population of RhD-positive drivers contains both RhD-positive heterozygotes and RhD-positive homozygotes (some of them with relatively low concentration of anti-toxoplasma antibodies and therefore relatively old infection). The contamination of the accident protected RhD population by unknown number of non-protected RhD-homozygotes might decrease the power of our tests and undervalue the strength of the observed effects. Minor Essential Revisions 1. Missing labels on the measurements shown in the figure are noted. Check for number consistencies between the text and the Figure (2460/2462, 706/709). The labels (number of RhD-positive/RhD-negative subjects with particular anti-toxoplasma antibody titers) is described in the legend. The errors in Figure have been corrected. 2. The background section should be shortened and the hypotheses under investigation (primary and secondary) more clearly described (.. or lower probability of being enrolled in prospective case control studies). To make the hypotheses more clear, we changed Therefore, it was impossible to tell whether Toxoplasma-infected subjects had a higher risk of traffic accidents or whether Toxoplasma-free subjects had a higher chance to be included
7 into the control sample, for example because of their higher superego strength and lower suspiciousness [13]. to Therefore, it was impossible to tell whether Toxoplasma-infected subjects had a higher risk of traffic accidents or whether Toxoplasma-free subjects had a higher chance to be involved into epidemiological surveys used for estimation of prevalence of toxoplasmosis in general population (the value used for controls in case control studies), for example because of their higher superego strength and lower suspiciousness [13]. and we also changed 1) to test whether Toxoplasma-infected subjects have a higher risk of traffic accidents or a lower probability of being enrolled as controls in prospective case-control studies and to 1) to test whether Toxoplasma-infected subjects have a higher risk of traffic accidents or a lower probability of being enrolled in epidemiological surveys. We tried to remove all unnecessary information from the Background section of the paper. Now, the Background of this (multidisciplinary) paper has 625 words, which is compatible with a usual format of BMC Infectious Disease articles. 3. Try to use conventional symbols for the presentation of the statistical results e.g. avoid t3886, ORrange etc Corrected. 4. Grammatical, spelling errors, formatting and fonts should be double checked. Done Discretionary Revisions These are recommendations for improvement which the author can choose to ignore. For example clarifications, data that would be useful but not essential. Level of interest: An article of importance in its field Quality of written English: Acceptable Statistical review: Yes, and I have assessed the statistics in my report. Declaration of competing interests: I declare that I have no competing interests. Reviewer's II report Title: Increased incidence of traffic accidents in RhD-negative, Toxoplasma gondii-infected military drivers revealed by a prospective cohort study Version: 2 Date: 9 December 2008 Reviewer: I.Cüneyt Cuneyt Balcioglu
8 Reviewer's report: This is a well-written, interesting article with significant results in the field of Parasitology. It may help encourage further studies on this field as well. Level of interest: An article whose findings are important to those with closely related research interests Quality of written English: Acceptable Statistical review: Yes, but I do not feel adequately qualified to assess the statistics. Declaration of competing interests: I declare that I have no competing interests.
O R I G I N A L A R T I C L E
Neuroendocrinology Letters Volume 29 No. 4 2008 Neurophysiological effect of the Rh factor. Protective role of the RhD molecule against Toxoplasma-induced impairment of reaction times in women Jaroslav
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