Brigitte Schlehofer

Maria Blettner

Monika Moissonnier

Isabelle Deltour

Graham G Giles

Bruce Armstrong

Jack Siemiatycki

Marie-Elise Parent

Daniel Krewski

Christoffer Johansen

Joachim Schüz
email: schuzj@iarc.fr

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Gabriele Berg-Beckhoff

Siegal Sadetzki

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Association of allergic diseases and epilepsy with risk of glioma, meningioma and acoustic neuroma: results from the INTERPHONE international case-control study

Keywords: allergies, epilepsy, brain tumours, multicenter case-control study

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

We have described this in the discussion (page 18).

Reviewer: MINOR POINTS

The authors first state that they have performed frequency matching. Then on page 14 they state "Only diagnoses that occurred up to two years prior to the tumor diagnosis (cases) or reference date (controls; date of diagnosis of corresponding case)". Here it sounds like an individual matching was performed. I do not understand this correctly. Our answer: We thank the reviewer for this comment as this was confusing. To make a long story short, controls were drawn in a frequency-matched way but post-hoc individually assigned to cases. This was necessary for the mobile phone-related approach as prevalence changed rapidly over time, and we kept the approach for all other Interphone related analyses. We added this explanation in the Methods. Our procedure for matching is described in more detail in the cited Interphone Paper ( Ref 6). Briefly: All 16 study centres selected there controls randomly from the source population. Our study-design called for controls to be individually-or frequencymatched to cases, with 1:1 matching for glioma and meningioma (except Germany which has done 1:2) and 1:2 for acoustic neuroma. Controls were matched on year of birth (within 5-year categories), sex and study region. Canada-Ottawa, Vancouver, France, Israel, Japan, new-Zealand and UK-North matched individually, the other countries used first frequency matching and conducted individual matching post-hoc. Post-hoc matched controls were only those who were interviewed as close as possible in time to the respected case interview (about 3 month) (reference date), in addition to the general matching criteria. For the analyses we stratified for age, sex and study region for different reasons depended on the topic. Therefore we used conditional logistic regression analyses for individual and post-hoc matching.

Reviewer:

The table layout of tables 1-2 is not comfortable (slashes between numbers of people, etc.). Our answer: We improved the layout of the tables. We left the slashes between cases and controls as we felt this helps for clarity, but removed them otherwise. Also we removed abbreviations and checked for consistency.

CI 95% confidence interval OR Odds ratio

Declarations

Funding:

There was no particular grant for the present work with the core writing group contributing from their regular positions (BS, MB, MM, JS), with some funding for travel and meetings provided by the International Agency for Research on Cancer (IARC/WHO). Epilepsy or epileptic seizures can occur as early symptoms of brain tumors and it has been hypothesized that seizure susceptibility increases due to interaction between tumor cell metabolism and the neuronal network [4,5].

Interphone is an international multi-center case-control study carried out in 13 countries and coordinated by IARC/WHO [6]. It focused on the association between mobile phone use and brain tumours, but data on allergic diseases and epilepsy were also collected. Here we report the results from the analysis of the pooled data set from the sixteen study centers on the associations between history of allergic diseases and epilepsy and risk of glioma, meningioma and acoustic neuroma.

Methods

The study population consists of incident, histologically or imaging-confirmed cases of glioma, meningioma and acoustic neuroma occurring between 2000 and 2004, 30-59 years old at diagnosis and their controls (sampled in frequency-matched manner and post-hoc individually assigned with one per case for glioma and meningioma, two per case for acoustic neuroma).

Controls were matched on age, sex, and study region (for details see [6,7]).

Interviews were performed by trained interviewers, mainly using a computer-assisted questionnaire, either face-to-face (93.9 % cases, 99.5 % controls) or by telephone. Proxy interviews were conducted when the participant was too ill or deceased. This was the case for 336 glioma cases, 41 meningioma cases, 3 acoustic neuroma cases and 40 controls. The interview captured information on mobile phone use, ionizing and non-ionizing medical radiation exposures, socio-demographic factors and other potential risk factors for brain tumours. In addition, a history of various physician-diagnosed medical conditions, were asked, including diagnoses of asthma, hay fever, and eczema; which are conditions thought to reflect allergic reactions. Details were asked about the age at onset of these diseases, and for eczema the age when the symptoms stopped. Similar questions were asked for epilepsy.

Statistical approaches followed the strategy of all analyses of the Interphone study (for details see [6][7]). Conditional logistic regression models were used to estimate pooled odds ratios (ORs) and their 95% confidence intervals (CIs), adjusted for education and time interval between case and respective control interviews. Analyses were performed for each brain tumour type, and for men and women separately, or -if analysis was performed for men and women combined -adjusted for gender. Subgroup analyses were done separately for highgrade (type III-IV) and low-grade (type I and II) glioma, based on ICD-O morphological codes (details see [6,7]).

Reference categories were defined as "never diagnosed with allergy" and "never diagnosed with epilepsy", respectively, as reported by the study subjects. Only diagnoses that occurred up to two years prior to the tumour diagnosis (cases) or reference date (controls; date of diagnosis of corresponding case) were included. Missing data was less than 5% for epilepsy and less than 1% for the allergies.

For each asthma, hay fever, and eczema, we created a binary variable (ever/never). We also investigated whether time since first diagnosis (< 10 years, 10 -19 years, ≥20 years) or age at onset (< 10 years, 10 -19 years, ≥20 years) was associated with the diseases. For eczema, we distinguished past and current rash. We also estimated ORs for one or more than one allergy compared with no allergy. Sensitivity analyses were performed by excluding proxy interviews and by including smoking as a potential confounder, but made no difference to the main results (data not shown).

Results

In total, the analyses included data from 2693 glioma cases (62.6% response rate), 2396 meningioma cases (76.9%) and 1102 acoustic neuroma cases (81.0%) and 6321 control subjects (44%). The distribution of cases and controls by selected demographic factors is presented in Supplementary Table 1. For all tumour types, educational level was slightly higher for controls than for cases.

For glioma, ORs below 1 were found for participants who were ever diagnosed with asthma (OR 0.73, CI 0.58-0.92), hay fever (OR 0.72, CI 0.61-0.86) or eczema (OR 0.78, CI 0.64-0.94), or "any allergy" (OR 0.71, CI 0.61-0.82) (Table 1). The result for eczema was driven by those with current rash. ORs were lowest when the allergies occurred less than ten years before glioma tumour diagnosis, and for those whose allergies started in adulthood. Subdivision into high grade and low grade glioma showed that the decrease was driven by the results for high-grade glioma (Table 1). For meningioma, no association was seen in relation to asthma (OR 0.91, CI 0.72-1.14) or to hay fever (OR 0.91, CI 0.76-1.10), but eczema showed a slightly lower risk (OR 0.84, CI 0.70-1.02), that was more pronounced for those with current rash (OR 0.76; CI 0.60-0.95) (Table 1). For both tumour types there was little difference in ORs between men and women, but for hay fever and eczema the ORs for men were somewhat lower (Table 2).

For acoustic neuroma no association was found with asthma (OR 1.02, CI 0.75-1.37), hay fever (OR 0.91, CI 0.72-1.14), or eczema (OR 1.02, CI 0.78-1.32), overall and by time since start of the allergy or by age at onset. The results were similar for men and women (Tables 1 and2).

A prior diagnosis of epilepsy was associated with an increased OR for glioma (OR 2.94, CI 1.87-4.63) and for meningioma (OR 2.12, CI 1.27-3.56) (Table1). Subgroup analyses for glioma and meningioma and epilepsy were based on small numbers (Tables 1 and2). However, for both glioma and meningioma, sex-specific analyses revealed higher risks for men than for women.

The OR was higher for low-grade glioma (OR 5.71, CI 2.48-13.1) compared with high-grade glioma (OR 2.01, CI 1.14-3.54). For both glioma and meningioma, the highest ORs were seen for adult-onset epilepsy, and for subjects whose epilepsy was diagnosed less than 10 years before the reference date. ORs were not increased for acoustic neuroma (Table 1), however, analyses were based on small numbers of subjects with epilepsy.

Discussion

These results presented were based on data from all Interphone study centres [6,7]. Some differences in results published from single or smaller groups of study centres [e.g. 3, as most recent], may be due to chance or to differences in participation rates, prevalence of specific diseases or other factors.

Allergic diseases

We found inverse associations of asthma, hay fever and eczema with risk of glioma, especially for high-grade glioma, for both men and women. Allergic diseases diagnosed closer to the diagnosis of the high-grade glioma (less than 10 years) were associated with lower ORs than those diagnosed earlier and at early ages. Our findings are consistent with previous studies, a recent meta-analysis [8] and review [9]. Overall, no association was seen between low-grade glioma, meningioma, and acoustic neuroma with any of the three allergic diseases [1].

Decreased ORs were observed, however, for low grade glioma and for meningioma for those who at time of interview reported current eczema.

Prospective studies found lower levels of total or respiratory-specific immunoglobulin IgE, a biomarker of allergy, in glioma patients, strengthening our observation of an inverse association [1,8]. The underlying biogenetic mechanism is not fully understood. The immediate hypersensitivity reactions of these three allergies are mediated by IgE, and this may be influenced by preclinical tumours. Further investigations of immunologic mechanisms, for example in the immunosurveillance system, and investigations of germline SNPs or genetic risk factors are needed for better understanding of the mechanism [2].

History of Epilepsy

In line with earlier epidemiological studies and clinical observations, we found elevated ORs of glioma and meningioma in relation to past epilepsy with the highest ORs for low-grade glioma, a finding also described by other studies [4]. No association was seen between history of epilepsy and acoustic neuroma but numbers of subjects were small. Epilepsy and epileptic seizures prior, but close to the diagnosis of glioma or meningioma are known to be important symptoms of brain tumours as an early warning sign and a prognostic factor for survival [5].

Different hypotheses concerning the epileptogenesis in tumour cells and peritumoral cells have been discussed, e.g. that an aberrant tumour cell metabolism may influence the neuronal network leading to seizures [4,10].

Strengths and Limitations

This is to our knowledge the largest ever case-control study on this topic. With all centres following the same study protocol, no compromises had to be made when pooling the data.

Participation proportions in cases (glioma 63%, meningioma 77% and acoustic neuroma 81%)

were high and the distribution of cases by sex and age was as to be expected for the respective tumours types. For glioma, proxy interviews were used for 12% of cases, but excluding them had little effect on the results. Main limitations were the low response proportion among controls and the fact that all data on medical diagnoses were based on self-reports of a physician diagnosis, leading to concerns about potential selection and recall bias.

Selection bias was of particular concern as response rates did somewhat differ by education (lower with shorter education) and prevalence of allergies may also differ by education; but in fact the association between allergies and education was weak (data not shown). Odds ratios with or without adjustment for education did hardly differ. Stratified analysis by education did not show any differences for glioma. For meningioma, the odds ratios differed slightly between groups, but were close to 1 for any educational level. Hence we conclude that the inverse association for glioma is not due to selection bias but the minor decrease in odds ratios for meningioma may well be.

Conclusions

Findings from this large-scale, international case-control study with a representative distribution of cases for the respective tumour types, add to the growing evidence that people with allergies have a lower risk of glioma than those without allergies, especially for high-grade glioma, but not for meningioma or acoustic neuroma. It also confirms the association between epilepsy and glioma and meningioma, most likely due to epilepsy being a symptom for a sizeable proportion of these tumours.

Introduction

Epidemiological studies have consistently found an inverse association between a history of allergic diseases and risk of glioma, while results were conflicting for meningioma and acoustic neuroma [1][2][3]. Underlying biological mechanisms appear to be complex; however, there is agreement that immunologic functions play an important role in the development of brain tumours, and allergic diseases probably indicate an effective immunosurveillance system [1,2].

Epilepsy or epileptic seizures can occur as early symptoms of brain tumors and it has been hypothesized that seizure susceptibility increases due to interaction between tumor cell metabolism and the neuronal network [4,5].

Interphone is an international multi-center case-control study carried out in 13 countries and coordinated by IARC/WHO [6]. It focused on the association between mobile phone use and brain tumours, but data on allergic diseases and epilepsy were also collected. Here we report the results from the analysis of the pooled data set from the sixteen study centers on the associations between history of allergic diseases and epilepsy and risk of glioma, meningioma and acoustic neuroma.

Methods

The study population consists of incident, histologically or imaging-confirmed cases of glioma, meningioma and acoustic neuroma occurring between 2000 and 2004, 30-59 years old at diagnosis and their controls (sampled in frequency-matched manner and post-hoc individually assigned with one per case for glioma and meningioma, two per case for acoustic neuroma).

Controls were matched on age, sex, and study region (for details see [6,7]).

Interviews were performed by trained interviewers, mainly using a computer-assisted questionnaire, either face-to-face (93.9 % cases, 99.5 % controls) or by telephone. Proxy interviews were conducted when the participant was too ill or deceased. This was the case for 336 glioma cases, 41 meningioma cases, 3 acoustic neuroma cases and 40 controls. The interview captured information on mobile phone use, ionizing and non-ionizing medical radiation exposures, socio-demographic factors and other potential risk factors for brain tumours. In addition, a history of various physician-diagnosed medical conditions, were asked, including diagnoses of asthma, hay fever, and eczema; which are conditions thought to reflect allergic reactions. Details were asked about the age at onset of these diseases, and for eczema the age when the symptoms stopped. Similar questions were asked for epilepsy.

Statistical approaches followed the strategy of all analyses of the Interphone study (for details see [6][7]). Conditional logistic regression models for frequency-matched data sets were used to estimate pooled odds ratios (ORs) and their 95% confidence intervals (CIs), adjusted for education and time interval between case and respective control interviews. Analyses were performed for each brain tumour type, and for men and women separately, or -if analysis was performed for men and women combined -adjusted for gender. Subgroup analyses were done separately for high-grade (type III-IV) and low-grade (type I and II) glioma, based on ICD-O morphological codes (details see [6,7]).

Reference categories were defined as "never diagnosed with allergy" and "never diagnosed with epilepsy", respectively, as reported by the study subjects. Only diagnoses that occurred up to two years prior to the tumour diagnosis (cases) or reference date (controls; date of diagnosis of corresponding case) were included. Missing data was less than 5% for epilepsy and less than 1% for the allergies.

For each asthma, hay fever, and eczema, we created a binary variable (ever/never). We also investigated whether time since first diagnosis (< 10 years, 10 -19 years, ≥20 years) or age at onset (< 10 years, 10 -19 years, ≥20 years) was associated with the diseases. For eczema, we distinguished past and current rash. We also estimated ORs for one or more than one allergy compared with no allergy. Sensitivity analyses were performed by excluding proxy interviews and by including smoking as a potential confounder, but made no difference to the main results (data not shown).

Results

In total, the analyses included data from 2693 glioma cases (62.6% response rate), 2396 meningioma cases (76.9%) and 1102 acoustic neuroma cases (81.0%) and 6321 control subjects (44%). The distribution of cases and controls by selected demographic factors is presented in Supplementary Table 1. For all tumour types, educational level was slightly higher for controls than for cases.

For glioma, ORs below 1 were found for participants who were ever diagnosed with asthma (OR 0.73, CI 0.58-0.92), hay fever (OR 0.72, CI 0.61-0.86) or eczema (OR 0.78, CI 0.64-0.94), or "any allergy" (OR 0.71, CI 0.61-0.82) (Table 1). The result for eczema was driven by those with current rash. ORs were lowest when the allergies occurred less than ten years before glioma tumour diagnosis, and for those whose allergies started in adulthood. Subdivision into high grade and low grade glioma showed that the decrease was driven by the results for high-grade glioma (Table 1). For meningioma, no association was seen in relation to asthma (OR 0.91, CI 0.72-1.14) or to hay fever (OR 0.91, CI 0.76-1.10), but eczema showed a slightly lower risk (OR 0.84, CI 0.70-1.02), that was more pronounced for those with current rash (OR 0.76; CI 0.60-0.95) (Table 1). For both tumour types there was little difference in ORs between men and women, but for hay fever and eczema the ORs for men were somewhat lower (Table 2).

For acoustic neuroma no association was found with asthma (OR 1.02, CI 0.75-1.37), hay fever (OR 0.91, CI 0.72-1.14), or eczema (OR 1.02, CI 0.78-1.32), overall and by time since start of the allergy or by age at onset. The results were similar for men and women (Tables 1 and2).

A prior diagnosis of epilepsy was associated with an increased OR for glioma (OR 2.94, CI 1.87-4.63) and for meningioma (OR 2.12, CI 1.27-3.56) (Table1). Subgroup analyses for glioma and meningioma and epilepsy were based on small numbers (Tables 1 and2). However, for both glioma and meningioma, sex-specific analyses revealed higher risks for men than for women.

The OR was higher for low-grade glioma (OR 5.71, CI 2.48-13.1) compared with high-grade glioma (OR 2.01, CI 1.14-3.54). For both glioma and meningioma, the highest ORs were seen for adult-onset epilepsy, and for subjects whose epilepsy was diagnosed less than 10 years before the reference date. ORs were not increased for acoustic neuroma (Table 1), however, analyses were based on small numbers of subjects with epilepsy.

Discussion

These results presented were based on data from all Interphone study centres [6,7]. Some differences in results published from single or smaller groups of study centres [e.g. 3, as most recent], may be due to chance or to differences in participation rates, prevalence of specific diseases or other factors.

Allergic diseases

We found inverse associations of asthma, hay fever and eczema with risk of glioma, especially for high-grade glioma, for both men and women. Allergic diseases diagnosed closer to the diagnosis of the high-grade glioma (less than 10 years) were associated with lower ORs than those diagnosed earlier and at early ages. Our findings are consistent with previous studies, a recent meta-analysis [8] and review [9]. Overall, no association was seen between low-grade glioma, meningioma, and acoustic neuroma with any of the three allergic diseases [1]. Decreased ORs were observed, however, for low grade glioma and for meningioma for those who at time of interview reported current eczema.

Prospective studies found lower levels of total or respiratory-specific immunoglobulin IgE, a biomarker of allergy, in glioma patients, strengthening our observation of an inverse association [1,8]. The underlying biogenetic mechanism is not fully understood. The immediate hypersensitivity reactions of these three allergies are mediated by IgE, and this may be influenced by preclinical tumours. Further investigations of immunologic mechanisms, for example in the immunosurveillance system, and investigations of germline SNPs or genetic risk factors are needed for better understanding of the mechanism [2].

History of Epilepsy

In line with earlier epidemiological studies and clinical observations, we found elevated ORs of glioma and meningioma in relation to past epilepsy with the highest ORs for low-grade glioma, a finding also described by other studies [4]. No association was seen between history of epilepsy and acoustic neuroma but numbers of subjects were small. Epilepsy and epileptic seizures prior, but close to the diagnosis of glioma or meningioma are known to be important symptoms of brain tumours as an early warning sign and a prognostic factor for survival [5].

Different hypotheses concerning the epileptogenesis in tumour cells and peritumoral cells have been discussed, e.g. that an aberrant tumour cell metabolism may influence the neuronal network leading to seizures [4,10].

Strengths and Limitations

This is to our knowledge the largest ever case-control study on this topic. With all centres following the same study protocol, no compromises had to be made when pooling the data.

Participation proportions in cases (glioma 63%, meningioma 77% and acoustic neuroma 81%) were high and the distribution of cases by sex and age was as to be expected for the respective tumours types. For glioma, proxy interviews were used for 12% of cases, but excluding them had little effect on the results. Main limitations were the low response proportion among controls and the fact that all data on medical diagnoses were based on self-reports of a physician diagnosis, leading to concerns about potential selection and recall bias.

Selection bias was of particular concern as response rates did somewhat differ by education (lower with shorter education) and prevalence of allergies may also differ by education; but in fact the association between allergies and education was weak (data not shown). Odds ratios with or without adjustment for education did hardly differ. Stratified analysis by education did not show any differences for glioma. For meningioma, the odds ratios differed slightly between groups, but were close to 1 for any educational level. Hence we conclude that the inverse association for glioma is not due to selection bias but the minor decrease in odds ratios for meningioma may well be.

Conclusions

Findings from this large-scale, international case-control study with a representative distribution of cases for the respective tumour types, add to the growing evidence that people with allergies have a lower risk of glioma than those without allergies, especially for high-grade glioma, but not for meningioma or acoustic neuroma. It also confirms the association between epilepsy and glioma and meningioma, most likely due to epilepsy being a symptom for a sizeable proportion of these tumours. Dear Editor, we are very grateful for the helpful comments of the reviewer. Please find our answer below.

COMMENTS TO THE AUTHOR:

Reviewer #1: EJEP-D-21-01240 The authors present a very well written manuscript. With the amount of sites and coauthors, one can only imagine the coordination work that was necessary before submission.

Reviewer:

MAJOR POINT Selection Bias

The prevalence of allergic diseases, medical diagnosis, and self-report of allergic diseases by individuals is social class-dependent in many nations of Europe. The low response in the control group (44%) was associated with oversampling of the higher social class. Thus, the control group overestimates the exposure prevalence of interest (allergic diseases). This bias may at least partly explain the odds ratio of < 1 for allergic diseases. The authors address this selection bias by statistically adjusting their analyses for education. Please explain how the authors removed the selection bias from the data. I would rather have expected a bias analysis (at least deterministic, preferably probabilistic) for the selection bias.

Our answer:

We like to thank the reviewer for raising this very important point. Indeed, we observed differences in the response rates by educational level, minor in cases and more so in controls. We observed however only weak associations between prevalence of different allergies and educational level. Taking these two together, we believe there is no major impact by selection bias.

We have investigated this in different separate analyses. As already reported, adjustment for educational level did not change any results. Stratified analyses by educational level did not change the results for glioma. For meningioma the risk differed slightly between educational levels, however was still close to 1.00 for any educational level. However, any decreased odd ratio may be explained to a small extent by selection bias.

We have described this in the discussion (page 18).

Reviewer:

MINOR POINTS

The authors first state that they have performed frequency matching. Then on page 14 they state "Only diagnoses that occurred up to two years prior to the tumor diagnosis (cases) or reference date (controls; date of diagnosis of corresponding case)". Here it sounds like an individual matching was performed. I do not understand this correctly.

Our answer:

We thank the reviewer for this comment as this was confusing. To make a long story short, controls were drawn in a frequency-matched way but post-hoc individually assigned to cases. This was necessary for the mobile phone-related approach as prevalence changed rapidly over time, and we kept the approach for all other Interphone related analyses. We added this explanation in the Methods.

Our procedure for matching is described in more detail in the cited Interphone Paper ( Ref 6). Briefly: All 16 study centres selected there controls randomly from the source population. Our study-design called for controls to be individually-or frequency-matched to cases, with 1:1

Rebuttal letter

Click here to access/download;attachment to manuscript;Response Letter Jan 2022.docx matching for glioma and meningioma (except Germany which has done 1:2) and 1:2 for acoustic neuroma. Controls were matched on year of birth (within 5-year categories), sex and study region.

Canada-Ottawa, Vancouver, France, Israel, Japan, new-Zealand and UK-North matched individually, the other countries used first frequency matching and conducted individual matching post-hoc. Post-hoc matched controls were only those who were interviewed as close as possible in time to the respected case interview (about 3 month) (reference date), in addition to the general matching criteria. For the analyses we stratified for age, sex and study region for different reasons depended on the topic.

Therefore we used conditional logistic regression analyses for individual and post-hoc matching.

Reviewer:

The table layout of tables 1-2 is not comfortable (slashes between numbers of people, etc.).

Our answer:

We improved the layout of the tables. We left the slashes between cases and controls as we felt this helps for clarity, but removed them otherwise. Also we removed abbreviations and checked for consistency.

With best regards, sincerely, Dr. Brigitte Schlehofer

  The Canadian centres in Ottawa/Vancouver were supported by a university-industry partnership grant from the Canadian Institutes of Health Research (CIHR), the latter including partial support from the Canadian Wireless Telecommunications Association. The CIHR university-industry partnerships program also includes provisions that ensure complete scientific independence of the investigators. D. Krewski is the Natural Sciences and Engineering Research Council of Canada Chair in Risk Science at the University of Ottawa.Additional funding for the study in France was provided by l'Association pour la Recherche sur le Cancer (ARC) [Contrat No. 5142] and three network operators (Orange, SFR, Bouygues Télécom). The funds provided by the operators represented 5% of the total cost of the French study and were governed by contracts guaranteeing the complete scientific independence of the investigators.The Finnish Interphone study received additional national funding from Emil Aaltonen Foundation and Academy of Finland (Grant No. 80921).The German Interphone study received additional national funding from the ''Deutsches Mobilfunkforschungsprogramm [German Mobile Phone Research Program]'' of the GermanFederal Ministry of Environment, Nuclear Safety, and Nature Protection; the Ministry of Environment and Traffic of the state of Baden-Württemberg; the Ministry of Environment of the state of North Rhine-Westphalia; and the MAIFOR Programme of the University of Mainz. , assisted by Roger Parslow (UK North); Jan Ivar Martinsen for additional programming work; Liz Findlay (UK North) who contributed a great deal to the development of materials and training of interviewers; the research assistants and interviewers in the different study centres who ensured that the study was carried out with care and consideration for the participants; the clinical practitioners, particularly neurosurgeons and ear, nose and throat surgeons, who permitted and facilitated our approaches to their patients; and the participants who gave so generously of their time. The Australian team would like to acknowledge the overall support given to study design and implementation by Associate Prof. Michael Besser and Prof. Andrew Kaye; the special support Associate Prof. Besser and Dr Paul Fagan gave to this study of acoustic neuroma. We thank also our fieldwork staff in Melbourne -Monique Kilkenny, Georgina Marr, Tracey McPhail, Fiona Phillips, Hayley Shaw, Yvonne Torn-Broers; and Sydney -Matthew Carroll, Sally Dunlop, Virginia MacDonald and Elizabeth Willowsand the many interviewers for their hard work, and the NSW and Victorian Cancer Registries for aiding case identification.

	Introduction
	Epidemiological studies have consistently found an inverse association between a history of
	allergic diseases and risk of glioma, while results were conflicting for meningioma and acoustic
	neuroma [1-3]. Underlying biological mechanisms appear to be complex; however, there is
	agreement that immunologic functions play an important role in the development of brain
	tumours, and allergic diseases probably indicate an effective immunosurveillance system [1, 2].
	The Canada -Montreal team acknowledges the diligent work of fieldwork staff including Marie-
	Claire Goulet, Sylvie Plante, Sally Campbell and the interviewer team. The following hospitals
	and physicians in Montreal permitted access to their patients: CHUM -Hôpital Notre-Dame (Dr
	Wieslaw Michel Bojanowski, Dr Jean Jacques Dufour, Dr François Lavigne, Dr Robert A.
	Moumdjian); Neurological Institute of Montreal (Dr Rolando Del Maestro, Dr Richard Leblanc);
	Hôpital du Sacré -Coeur de Montréal (Dr Marc F. Giroux); The Jewish General Hospital (Dr
	Gerard Mohr, Dr Jamie Miles Rappaport). The Canada -Ottawa centre gratefully acknowledges
	the work of the interview team, particularly Lynn Pratt and Daniel Bedard for their leading roles
	in study coordination; participating clinicians at the Ottawa Hospital included Drs. Brien Benoit,
	Martin J. Corsten, André Lamothe, William Miller, Paul F. Odell, and David Schramm. The Danish
	Interphone team likes to thank Michael Kosteljanetz (Neurosurgical Department, Neuroscience
	Centre, University Hospital of Copenhagen), Hans Skovgaard Poulsen (Department of Radiation
	Biology, Finsen Centre, University Hospital of Copenhagen) and Jens Thomsen (Department of
	Otolaryngology-Head and Neck Surgery, Gentofte Hospital, University of Copenhagen,
	Hellerup). Furthermore, we like to thank Lars H. Thomassen for skilful computer assistance. The
	Finnish Interphone team acknowledges the roles of Tiina Salminen ja Anna Lahkola in study
	coordination and research nurse Anu Outinen (STUK), neuropathologist Hannu Haapasalo MD,
	PhD (Tampere University Hospital, Dept of Pathology), chief physician Risto Sankila, MD, PhD
	(Finnish Cancer Registry), Prof. Juha Jääskeläinen (Helsinki University Hospital, Dept of
	Neurosurgery, currently Kuopio University Hospital), Prof. Matti Vapalahti (Kuopio University
	Hospital, Dept of Neurosurgery), Prof. John Koivukangas (Oulu University Hospital, Dept of
	Neurosurgery), chief physician Simo Valtanen (Turku University Hospital, Dept of
	Neurosurgery), chief physician Timo Kuurne (Tampere University Hospital, Dept of
	Neurosurgery) in collection of the data. The French Interphone team would like to thank the

The INTERPHONE study was supported by funding from the European Fifth Framework Program, 'Quality of Life and Management of Living Resources' (contract QLK4-CT-1999901563) and the International Union against Cancer (UICC). The UICC received funds for this purpose from the Mobile Manufacturers' Forum and GSM Association. Provision of funds to the INTERPHONE study investigators via the UICC was governed by agreements that guaranteed INTERPHONE's complete scientific independence. The terms of these agreements are publicly available at https://interphone.iarc.fr/funding The Australian centre was supported by the Australian National Health and Medical Research Council (EME Grant 219129) with funds originally derived from mobile phone service licence fees. Cancer Council NSW and Cancer Council Victoria provided most of the infrastructure for the project in Australia. The Canada -Montreal study was primarily funded by a grant from the Canadian Institutes of Health Research (project 15 MOP-42525). Additionally, Dr Siemiatycki's research team was partly funded by the Canada Research Chair programme and by the Guzzo-CRS Chair in Environment and Cancer. Dr Parent had a salary award from the Fonds de la recherche en santé du Québec. The Japanese Interphone study was fully funded by the Ministry of Internal Affairs and Communications of Japan. Funding in New Zealand for this project was provided by the Health Research Council of New Zealand, the Cancer Society of New Zealand, the Wellington Medical Research Foundation, the Hawke's Bay Medical Research Foundation and the Waikato Medical Research Foundation. The Swedish centre was additionally supported by the Swedish Research Council and the Swedish Cancer Society. The UK North study received additional funding from the Health and Safety Executive, the Department of Health, the Mobile Telecommunications, Health and Research (MTHR) program, and the Scottish Executive. The University of Leeds received some financial support on behalf of the 4 centres of the 'UK North Study' from the UK Network Operators (O2, Orange, T-Mobile, Vodafone, '3') under legal signed contractual agreements which guaranteed complete independence for the scientific investigators. The Southeast England Centre wishes to acknowledge additional funding from the Mobile Telecommunications, Health and Research (MTHR) programme. versions

Table 1 :

 1 Association between tumour and allergy or epilepsy, by time since start 1 and age at onset2

			Glioma	low grade glioma	high grade glioma	meningioma	acoustic neuroma
		cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI-)	cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI)
	Asthma
	Ever/never
	never	2453/2630 1.00	751/797 1.00		1678/1805 1.00		2161/2377 1.00	1001/1928 1.00
	ever

Table 2 :

 2 Association between tumour and allergy or epilepsy, by gender 1 Supplementary table: Description of the study population for the analyses of allergic conditions and epilepsy. Interphone Study Group

		glioma -men	glioma -women	meningioma -men	meningioma -women	acoustic neuroma -men	acoustic neuroma -women
		cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI)	cases/ controls	OR (95% CI)
	Asthma
	never	1477/1590 1.00	976/1040 1.00	519/575 1.00	1642/1802 1.00	488/943 1.00	513/985 1.00
	ever	127/165 0.75 (0.55-1.02) 93/138 0.71 (0.50-1.02) 45/52 1.00 (0.61-1.63)	183/211 0.88 (0.68-1.14) 47/87 1.02 (0.63-1.63) 52/117 0.95 (0.64-1.42)
	Hay Fever
	never	1360/1428 1.00	891/937 1.00	483/533 1.00	1523/1629 1.00	440/843 1.00	455/877 1.00
	ever	234/319 0.67 (0.53-0.85) 176/239 0.75 (0.58-0.98) 81/95 0.87 (0.58-1.31)	289/370 0.91 (0.74-1.12) 93/181 0.93 (0.65-1.32) 108/221 0.85 (0.62-1.16)
	Eczema
	never	1446/1536 1.00	904/955 1.00	521/552 1.00	1543/1655 1.00	473/898 1.00	481/893 1.00
	ever	150/214 0.74 (0.56-0.97) 163/221 0.80 (0.61-1.05) 44/78 0.59 (0.36-0.94)	272/348 0.91 (0.74-1.12) 62/129 1.01 (0.65-1.55) 83/204 1.04 (0.74-1.45)
	Any allergy
	none	1174/1176 1.00	729/718 1.00	423/445 1.00	1238/1286 1.00	378/708 1.00	379/685 1.00
	1 and more	400/549 0.66 (0.54-0.80) 321/440 0.75 (0.6-0.94) 137/177 0.77 (0.56-1.06)	562/701 0.88 (0.74-1.04) 153/309 0.93 (0.69-1.27) 183/406 0.87 (0.67-1.14)
	Epilepsy
	never	1493/1675 1.00	1006/1136 1.00	530/603 1.00	1746/1929 1.00	514/989 1.00	547/1068 1.00
	ever	62/20	3.71 (2.02-6.82)	39/13 2.37 (1.16-4.81) 20/5 5.46 (1.67-17.8)	41/27	1.55 (0.86-2.79)	8/11 2.23 (0.65-7.66)	9/15 1.26 (0.46-3.43)
	1 Reference category (never): no symptoms of disease up to two years before tumour diagnosis (cases) or reference date (controls); adjusted for education and time at interview;
	cases and controls answering "don't know" for a disease or had missing values in the adjustment variables were excluded from analyses

Table 1 :

 1 Association between tumour and allergy or epilepsy, by time since start 1 and age at onset2

		Glioma	low grade glioma	high grade glioma	meningioma	acoustic neuroma
	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)
	Asthma
	Ever/never

Table 2 :

 2 Association between tumour and allergy or epilepsy, by gender 1 control could be matched to more than one case2 nonparticipating not included in analyses because of incomplete data, unmatched3 in Denmark, Finland, Germany, Italy, Norway, and Sweden 4 in France, Germany, UK, Australia, Canada, Japan, and New Zealand

												acoustic. neuroma --
		glioma -men	glioma -women	meningioma -men	meningioma -women	acoustic. neuroma -men		women
		cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)	cases/ controls.	OR (95% CI-95%)
	Asthma
	never	1477/1590 1.00	976/1040 1.00	519/575 1.00	1642/1802 1.00	488/943 1.00	513/985 1.00
	ever	127/165 0.75 (0.55-1.02) 93/138 0.71 (0.50-1.02) 45/52 1.00 (0.61-1.63)	183/211 0.88 (0.68-1.14) 47/87 1.02 (0.63-1.63) 52/117 0.95 (0.64-1.42)
	Hay Fever
	never	1360/1428 1.00	891/937 1.00	483/533 1.00	1523/1629 1.00	440/843 1.00	455/877 1.00
	ever	234/319 0.67 (0.53-0.85) 176/239 0.75 (0.58-0.98) 81/95 0.87 (0.58-1.31)	289/370 0.91 (0.74-1.12) 93/181 0.93 (0.65-1.32) 108/221 0.85 (0.62-1.16)
	Eczema
	never	1446/1536 1.00	904/955 1.00	521/552 1.00	1543/1655 1.00	473/898 1.00	481/893 1.00
	ever	150/214 0.74 (0.56-0.97) 163/221 0.80 (0.61-1.05) 44/78 0.59 (0.36-0.94)	272/348 0.91 (0.74-1.12) 62/129 1.01 (0.65-1.55) 83/204 1.04 (0.74-1.45)
	Any allergy
	none	1174/1176 1.00	729/718 1.00	423/445 1.00	1238/1286 1.00	378/708 1.00	379/685 1.00
	1 and more	400/549 0.66 (0.54-0.80) 321/440 0.75 (0.6-0.94) 137/177 0.77 (0.56-1.06)	562/701 0.88 (0.74-1.04) 153/309 0.93 (0.69-1.27) 183/406 0.87 (0.67-1.14)
	Epilepsy
	never	1493/1675 1.00	1006/1136 1.00	530/603 1.00	1746/1929 1.00	514/989 1.00	547/1068 1.00
	ever	62/20	3.71 (2.02-6.82)	39/13 2.37 (1.16-4.81) 20/5 5.46 (1.67-17.8)	41/27	1.55 (0.86-2.79)	8/11 2.23 (0.65-7.66)	9/15 1.26 (0.46-3.43)
	1 Reference category (never): no symptoms of disease up to two years before tumour diagnosis (cases) or reference date (controls); adjusted for education and time at interview;
	cases and controls answering "don't know" for a disease or had missing values in the adjustment variables were excluded from analyses

Cancer & Radiation Epidemiology Unit, Gertner Institute for Epidemiology & Health Policy Research, Sheba Medical Center, Tel-Hashomer, Israel

Between first diagnosis by a physician and two years before tumour diagnosis (cases) or reference date (controls)

Acknowledgments

The authors like to thank Mr Klaus Schlaefer (German INTERPHONE team) for his contribution to this work and to INTERPHONE Germany in general, who sadly passed away before the end of this project.

The authors are grateful to Lesley Richardson (Montreal, Canadaformerly at IARC) for her major role in the coordination of the study; Emilie Combalot and Helene Tardy for their skillful data management at the coordination centre; Dr Baruch Modan (Israeldeceased) for his assistance and enthusiasm in the design and setting up of this study; James Doughty (UK North), who performed miracles implementing the CAPI in several languages and several

Acknowledgments

The authors like to thank Mr Klaus Schlaefer (German INTERPHONE team) for his contribution to this work and to INTERPHONE Germany in general, who sadly passed away before the end of this project.

The authors are grateful to Lesley Richardson (Montreal, Canadaformerly at IARC) for her major role in the coordination of the study; Emilie Combalot and Helene Tardy for their skillful data management at the coordination centre; Dr Baruch Modan (Israeldeceased) for his assistance and enthusiasm in the design and setting up of this study; James Doughty (UK North), who performed miracles implementing the CAPI in several languages and several versions, assisted by Roger Parslow (UK North); Jan Ivar Martinsen for additional programming work; Liz Findlay (UK North) who contributed a great deal to the development of materials and training of interviewers; the research assistants and interviewers in the different study centres who ensured that the study was carried out with care and consideration for the participants; the clinical practitioners, particularly neurosurgeons and ear, nose and throat surgeons, who permitted and facilitated our approaches to their patients; and the participants who gave so generously of their time. The Australian team would like to acknowledge the overall support given to study design and implementation by Associate Prof. Michael Besser and Prof. Andrew Kaye; the special support Associate Prof. Besser and Dr Paul Fagan gave to this study of acoustic neuroma. We thank also our fieldwork staff in Melbourne -Monique Kilkenny, Georgina Marr, Tracey McPhail, Fiona Phillips, Hayley Shaw, Yvonne Torn-Broers; and Sydney -Matthew Carroll, Sally Dunlop, Virginia MacDonald and Elizabeth Willowsand the many interviewers for their hard work, and the NSW and Victorian Cancer Registries for aiding case identification. The Canada -Montreal team acknowledges the diligent work of fieldwork staff including Marie-Claire Goulet, Sylvie Plante, Sally Campbell and the interviewer team. The following hospitals and physicians in Montreal permitted access to their patients: CHUM -Hôpital Notre-Dame (Dr Wieslaw Michel Bojanowski, Dr Jean Jacques Dufour, Dr François Lavigne, Dr Robert A. Moumdjian); Neurological Institute of Montreal (Dr Rolando Del Maestro, Dr Richard Leblanc); Hôpital du Sacré -Coeur de Montréal (Dr Marc F. Giroux); The Jewish General Hospital (Dr Gerard Mohr, Dr Jamie Miles Rappaport). The Canada -Ottawa centre gratefully acknowledges the work of the interview team, particularly Lynn Pratt and Daniel Bedard for their leading roles in study coordination; participating clinicians at the Ottawa Hospital included Drs. Brien Benoit, Martin J. Corsten, André Lamothe, William Miller, Paul F. Odell, and David Schramm. The Danish Interphone team likes to thank Michael Kosteljanetz (Neurosurgical Department, Neuroscience Centre, University Hospital of Copenhagen), Hans Skovgaard Poulsen (Department of Radiation Biology, Finsen Centre, University Hospital of Copenhagen) and Jens Thomsen (Department of Otolaryngology-Head and Neck Surgery, Gentofte Hospital, University of Copenhagen, Hellerup). Furthermore, we like to thank Lars H. Thomassen for skilful computer assistance. The Finnish Interphone team acknowledges the roles of Tiina Salminen ja Anna Lahkola in study coordination and research nurse Anu Outinen (STUK), neuropathologist Hannu Haapasalo MD, PhD (Tampere University Hospital, Dept of Pathology), chief physician Risto Sankila, MD, PhD (Finnish Cancer Registry), Prof. Juha Jääskeläinen (Helsinki University Hospital, Dept of Neurosurgery, currently Kuopio University Hospital), Prof. Matti Vapalahti (Kuopio University Hospital, Dept of Neurosurgery), Prof. John Koivukangas (Oulu University Hospital, Dept of Neurosurgery), chief physician Simo Valtanen (Turku University Hospital, Dept of Neurosurgery), chief physician Timo Kuurne (Tampere University Hospital, Dept of Neurosurgery) in collection of the data. The French Interphone team would like to thank the

archives, whose assistance proved essential to the success of the project. The German Interphone Group would like to thank Stephanie Estel, Marianne Brömmel, Melanie Kaiser and Anna Wilms for organizing the field phase and all our interviewers for their skilful work. We thank the clinical Interphone team for their support and collaboration (

Funding in New Zealand for this project was provided by the Health Research Council of New Zealand, the Cancer Society of New Zealand, the Wellington Medical Research Foundation, the Hawke's Bay Medical Research Foundation and the Waikato Medical Research Foundation.

The Swedish centre was additionally supported by the Swedish Research Council and the Swedish Cancer Society.

The UK North study received additional funding from the Health and Safety Executive, the Department of Health, the Mobile Telecommunications, Health and Research (MTHR) program, and the Scottish Executive. The University of Leeds received some financial support on behalf of the 4 centres of the 'UK North Study' from the UK Network Operators (O2, Orange, T-Mobile, Vodafone, '3') under legal signed contractual agreements which guaranteed complete independence for the scientific investigators.

The Southeast England Centre wishes to acknowledge additional funding from the Mobile Telecommunications, Health and Research (MTHR) programme.

Availability of data

Original data are not available as per ethical clearance and national data privacy legislations.

Code availability

Programming code of analysis used for the present paper can be obtained by contacting the corresponding author.

Availability of data

Original data are not available as per ethical clearance and national data privacy legislations.

Code availability

Programming code of analysis used for the present paper can be obtained by contacting the corresponding author.

Conflicts of interest

The authors confirm that they have no conflicts of interest.

Authors' contributions

BS, MB and JS designed and jointly led the present project and drafted the manuscript. MM carried out the analysis. All other authors were also involved in the INTERPHONE study, its design, conduct and interpretation. EC was the overall coordinator of the INTERPHONE project. All authors reviewed and approved the manuscript.

Ethics approval

IARC Ethical approval was granted on 25 November 1999 (No ERC-Project 99-010). All study centers obtained national ethical approval.

Consent to participate

All participants of the INTERPHONE study filled in written informed consent.

Consent to publication

All authors critically reviewed and approved the final version of the manuscript.

Disclaimer

Where authors are identified as personnel of IARC/WHO, the authors alone are responsible for the views expressed in this article, and they do not necessarily represent the decisions, policy, or views of IARC/WHO.

CI 95% confidence interval

OR

Odds ratio

Declarations

Funding:

There was no particular grant for the present work with the core writing group contributing from their regular positions (BS, MB, MM, JS), with some funding for travel and meetings provided by the International Agency for Research on Cancer (IARC/WHO).

Conflicts of interest

The authors confirm that they have no conflicts of interest.

Authors' contributions

BS, MB and JS designed and jointly led the present project and drafted the manuscript. MM carried out the analysis. All other authors were also involved in the INTERPHONE study, its design, conduct and interpretation. EC was the overall coordinator of the INTERPHONE project.

All authors reviewed and approved the manuscript.

Ethics approval

IARC Ethical approval was granted on 25 November 1999 (No ERC-Project 99-010). All study centers obtained national ethical approval.

Consent to participate

All participants of the INTERPHONE study filled in written informed consent.

Consent to publication

All authors critically reviewed and approved the final version of the manuscript.

Disclaimer

Where authors are identified as personnel of IARC/WHO, the authors alone are responsible for the views expressed in this article, and they do not necessarily represent the decisions, policy, or views of IARC/WHO.

Supplementary

Disclosure of potential conflicts of interest

Authors must disclose all relationships or interests that could have direct or potential influence or impart bias on the work. Although an author may not feel there is any conflict, disclosure of all relationships and interests provides a more complete and transparent process, leading to an accurate and objective assessment of the work. Awareness of real or perceived conflicts of interest is a perspective to which the readers are entitled. This is not meant to imply that a financial relationship with an organization that sponsored the research or compensation received for consultancy work is inappropriate. For examples of potential conflicts of interests that are directly or indirectly related to the research please visit:


Estelle Lebègue
email: zestelle.lebegue@univ-nantes.fr

Nazua L Costa

Ricardo O Louro

Frédéric Barrière
email: frederic.barriere@univ-rennes1.fr

Electrochemical Single Nano-Impacts of Electroactive Shewanella Oneidensis Bacteria Onto Carbon Ultramicroelectrode

Electrochemical single nano-impacts of electroactive Shewanella oneidensis bacteria at a 7 µm diameter carbon fibre ultramicroelectrode in an aqueous potassium phosphate buffer (pH = 7.2) solution containing a redox active probe (potassium ferro-or ferricyanide) is reported.

We present chronoamperometric measurements recorded at the ultramicroelectrode polarized at the potential of the steady-state current of the redox probe in solution (oxidation for K 4 Fe(CN) 6 or reduction for K 3 Fe(CN) 6 ) in the presence of bacteria. The shape of current transients associated to single bacteria nano-impacts is compared and discussed as a function of the redox probe in solution and of the ultramicroelectrode applied potential.

The rapid and selective detection of bacteria at the level of a single cell is a fundamental scientific challenge to progress toward more efficient biosensors and diagnotics. [1][2][3][4][5] To this end, fast and easy handling systems for detection of bacteria at the single cell level (the highest possible sensitivity) have become reachable with the promising electrochemistry of single nano-impacts. 3,4,[6][7][8][9][10][11] Electrochemical detection of single bacteria with the nano-impacts method has only been reported in few studies, 3,4,[12][13][14] four of which involving detection of living label-free bacteria. 4,[12][13][14] These four reports especially focus on Escherichia coli as a model bacterium and have not been extended to electroactive bacteria that perform electrode biocatalytic processes in microbial fuel cells and in other microbial bioelectrochemical systems. 15,16 Studies on extracellular electron transfer at the microorganism/electrode interface mainly involve two Gram-negative mesophilic bacteria able to electrically connect their respiratory metabolism to extracellular solid electron acceptors: Shewanella oneidensis MR-1 and Geobacter sulfurreducens. [15][16][17][18] It is well known that Shewanella oneidensis biofilms attached to graphitic carbon electrodes transfer electrons at a formal potential of about -0.2 V vs. SHE. [START_REF] Marsili | Proc. Natl. Acad. Sci[END_REF][START_REF] Baron | [END_REF][21] The electrochemical nano-impacts method is fully relevant for evaluating the electrochemical activity of a single bacteria cell at an ultramicroelectrode 22 under controlled conditions such as in the absence of a biofilm in order to minimize the effect of secreted materials. 23 For single bacteria nano-impacts electrochemical detection, the strategy is to record a chronoamperometric measurement in an aqueous solution containing a redox probe at an ultramicroelectrode polarized at the oxidation or reduction potential of the electrochemically active species and to observe a "current step" when one bacterium impacts the ultramicroelectrode, corresponding to a "blocking effect" (Fig. 1A). 4,12,13 Recent studies have shown that in these conditions (presence of an aqueous redox probe and bacteria), the response signal expected from single bacterium collision may also be a "current spike" corresponding to either the own electrochemical activity of the bacterium toward the redox probe at the ultramicroelectrode applied potential 13,14 or a "bouncing effect" of the bacterium that does not stick to the ultramicroelectrode surface (Fig. 1B). 12 The fundamental mechanism that occurs during the single bacterium nano-impact event is not fully understood and requires more in-depth studies with different bacterial strains.

Based on our expertise and knowledge in both the electrochemical nano-impacts technique [24][25][26] and electroactive bacteria studied in microbial fuel cells, 18,27,28 we present herein first results on electrochemical single collisions at a carbon fibre ultramicroelectrode of Shewanella oneidensis as a model electroactive bacteria (Fig. 1). Chronoamperometric measurements were performed in potassium phosphate buffer aqueous solution containing a redox probe (20 mM potassium ferro-or ferricyanide) in the presence of electroactive bacteria at a polarized 7 µm diameter carbon ultramicroelectrode (Fig. 1).

Experimental

Reagents. All chemicals were used as purchased without further purification. Milli-Q water (18.2 MΩ cm) was used to prepare all solutions. Potassium ferricyanide (99.0% min) and potassium ferrocyanide trihydrate (99.0% min) were obtained from Sigma Aldrich.

Potassium phosphate monobasic (99.0% min) and potassium phosphate dibasic (99.0% min) were purchased from VWR (USA).

Bacterial strain and growth conditions. Pure culture of Shewanella oneidensis MR-1 was grown aerobically in Luria Bertani (LB) medium (Sigma Aldrich). Briefly, 1 vial of culture stock was thawed and 1% of livestock cells was used to inoculate 20 mL culture medium. The cells were incubated overnight at 30 °C with shaking at 150 rpm. After 16 hours growth, the undiluted cells optical density at 600 nm (OD600) was 2.0 consistent with the exponential growth phase. 29 Live culture of S. oneidensis MR-1 grown in LB medium and culture stocks were stored at -80 °C with 50% glycerol.

Electrochemical measurements.

Electrochemical experiments were carried out at room temperature (21 ± 3 °C) with a three-electrode cell placed in a Faraday cage and an Autolab PGSTAT302N potentiostat/galvanostat (Eco Chemie B.V., Netherlands) using Nova 2.1 as electrochemical software (Metrohm). All solutions were deaerated by bubbling argon for 5 minutes before each measurement. For all recorded chronamperometric curves, the sample interval (in sampling time) was 0.1 s. A carbon fibre ultramicroelectrode (7 µm diameter) obtained from IJ Cambria Scientific Ltd. as the working electrode, a platinum wire as the counter electrode, and an Ag/AgCl, KCl 3 M reference electrode were used to perform all electrochemical measurements. Before each experiment, carbon ultramicroelectrodes were gently polished using a wetted fine grid silicon carbide paper from Struers (4000-grid SiC) and washed several times with ultrapure water. The S. oneidensis bacteria solution injected in the electrochemical cell before chronoamperometric measurements was prepared by 15 min centrifugation at 3000 rpm of 1 mL culture stock solution to remove the LB growth medium and resuspension during 15 min in an aqueous solution of 20 mM potassium phosphate buffer (pH 7.2). The bacteria solution in potassium phosphate buffer was prepared just before the electrochemical nano-impact experiments and used immediately within 1 hour maximum.

Results and Discussion

The applied potentials for the chronoamperometric measurements presented in Fig. 2 is +0.6 V vs. Ag/AgCl for K 4 Fe(CN) 6 and -0.3 V vs. Ag/AgCl for K 3 Fe(CN) 6 and correspond to the steady-state current of the aqueous redox probes at a CF UME as determined by cyclic voltammetry (Fig. S1). In the absence of bacteria in solution, no current step or spike is observed over a 200 s polarization time at either +0.6 V or -0.3 V (Fig. 2 Top, blue and orange curves respectively). After addition of S. oneidensis bacteria in solution containing K 4 Fe(CN) 6 as redox probe (Fig. 2 Bottom, blue curve), the stair step current response is detected and assigned to single bacteria nano-impacts onto the CF UME surface, which locally block the flux of Fe(CN) 6 4-to the electrode surface. The magnitude of the limiting faradic current decrease (a few percent) by the stair step (or blip, vide infra) is consistent with the relative size of Shewanella cells (commonly 1 to 2 m 2 ) to that of the electrode (ca. 38 m 2 ). Here, a current decrease in the shape of stair steps is observed and indicates that most of the bacteria that impact stick and remain on the CF UME surface after collision (except for occasional current spikes or bouncing events of single cells sometimes observed at the beginning of the chronoamperogram, e.g. at ca. 19 and 52 s on Fig. 2 Bottom, blue curve).

Note that the lower initial current recorded in the chronoamperometric curve (12.75 nA) compared with that observed in the absence of bacteria (22.30 nA) is assigned to a slight dilution of ferrocyanide after the addition of bacteria and to the variability in the surface state and geometry of the CF UME upon mechanical polishing between each measurement.

Control experiments performed at different potential values showed that the stair step current response related to discrete bacteria nano-impacts in K 4 Fe(CN) 6 aqueous redox probe solution is detected for any applied potential corresponding to the steady-state current of Fe(CN) 6 4-oxidation (E app. ≥ +0.3 V vs. Ag/AgCl) at a CF UME (Fig. S2, blue curves). These observations with S. oneidensis in K 4 Fe(CN) 6 are in agreement with previous works reporting blocking electrochemical nano-impacts of single Escherichia coli bacteria in potassium ferrocyanide aqueous solutions. 4,12,13 The concentration of S. oneidensis bacteria in the electrochemical cell is estimated at ca. 1.5 × 10 8 CFU mL -1 (250 fM) after dilution of the stock solution with OD600 = 2.0. 30 The experimental frequency f exp of single current step in the chronoamperometric curve is estimated at 0.07 ± 0.02 Hz based on three different electrochemical measurements performed in the same experimental conditions over 200 s (Fig. 2 Bottom, blue curve). This frequency value corresponds to a bacteria concentration C bac of about 16.6 pM according to Equation 1 below (in conditions where the mass transport is controlled by diffusion). 9,14

C bac = f exp /4Dr e [1]
In Equation 1, D is the diffusion coefficient of bacteria (5 × 10 -9 cm 2 s -1 ) [START_REF] Bray | Cell movements : from molecules to motility[END_REF] and r e is the ultramicroelectrode radius (r e = 3.5 µm). The bacteria concentration C bac estimated from the experimental frequency f exp of current steps (Fig. 2 Bottom, blue curve) and Equation 1 is two orders of magnitude higher than the concentration determined from OD600. First, this discrepancy may be rationalized by the fact that collision experiments do not discriminate between living and dead bacteria. 4,12,13 Therefore, it is plausible that collisions of dead cells (in addition to living ones) contribute to the higher than expected experimental collision frequency. Furthermore, it has been reported that positively charged surfaces are more favourable for the formation of catalytic biofilms of microbial fuel cell electrodes because of the electrostatic interaction with the negatively charged surface of most electroactive bacteria. [START_REF] Guo | [END_REF][33][34] In addition to the electrostatic attraction between the negatively charged surface of S. oneidensis and the positively charged CF UME surface (at E app. ≥ +0.3 V vs. Ag/AgCl) the higher frequency of current steps from single cell nano-impacts onto CF UME may also be supported by the following biological effect: S. oneidensis MR-1 is known to sense and respond to electron acceptor gradients in its environment. 35 Indeed, an increase in S.

oneidensis motility (cell swimming speed) has been reported when cells are exposed to high enough applied potentials at the working electrode of a micro-electrochemical cell (electrokinesis). [START_REF] Harris | Proc. Natl. Acad. Sci[END_REF] S. oneidensis is a very versatile microorganism that shows growth with a polar flagellum at a broad range of temperatures and pH values. [START_REF] Venkateswaran | [END_REF] With respect to motility, the electrokinesis experiments mentioned above were carried out in phosphate buffer (with a concentration in the order of 10 to 50 mM) at pH 7, [START_REF] Nealson | The Prokaryotes: Volume 6: Proteobacteria: Gamma Subclass[END_REF] quite close to our experimental conditions. Hence the results reported here on the increased attraction of S. oneidensis cells to the CF UME polarized at high enough potential may be rationalized by a combination of three effects: single collisions of dead cells (in addition to living ones), electrostatic attraction and/or electrokinesis.

Concerning the chronoamperometric measurements recorded at -0.3 V vs. Ag/AgCl with K 3 Fe(CN) 6 as aqueous redox probe in the presence of S. oneidensis bacteria (Fig. 2 Bottom, orange curve), the shape of associated single collisions is not a stair step but a current blip or spike. This trend was also observed for any applied potential corresponding to the steady-state current of Fe(CN) 6 3-reduction (E app. ≤ +0.1 V vs. Ag/AgCl) at a CF UME in K 3 Fe(CN) 6 aqueous solution containing bacteria (Fig. S2, orange curves). According to Ronspees and Thorgaard, 12 these blip-shaped current transients are associated to short bounce events onto CF UME surface as opposed to the sticking collisions and stair step current response discussed above. Indeed, in these experimental conditions, S. oneidensis bacteria single nano-impacts present spike-shaped current transients that return to the baseline current after 0.3-0.8 s. This behaviour can be explained by the inhibition of Fe(CN) 6 3-diffusive flux during the short bounce events of the bacteria at the CF UME surface. 12 Compared with the chronoamperometry experiments recorded during K 4 Fe(CN) 6 reduction and discussed above, only the polarization potential of the CF UME and the redox probe oxidation state were changed. The difference in the current transient response for S. oneidensis single collisions is most likely related to the potential applied at the CF UME rather than to the type of aqueous redox probe or to a specific intrinsic response from bacteria cells (as discussed in the next paragraph). 13,14,23,[START_REF] Wu | [END_REF] Gao et al. 13 suggested that negatively charged bacteria such as E. coli detach from the UME surface when the potential is more negative than the potential of zero charge. This suggestion is fully relevant to explain the bounce events (current spikes) observed for single nano-impacts of S. oneidensis onto CF UME polarized at -0.3 V vs.

Ag/AgCl in potassium ferricyanide aqueous solution. Indeed, it has been reported that Shewanella strains are highly negatively charged at neutral pH and hence are electrostatically repulsed by negatively charged electrode surfaces. 40 Here we find that the negatively charged S. oneidensis bacteria are repelled by the CF UME surface at applied potentials E app. ≤ +0.1 V vs. Ag/AgCl. This hypothesis is also confirmed by the lower and poorly reproducible frequency of current spikes (ranging from 0.005 to 0.075 Hz) in comparison with the frequency of current steps observed in Fig. 2 and S2 for the same S. oneidensis cells concentration. Indeed, the detection of current spikes is more difficult to observe and the frequency of events is highly variable over this time range for the same bacteria concentration in solution, in agreement with the repulsing effect of the CF UME surface on the single cells.

Hence the frequency of current spikes cannot be confidently related to the bacteria concentration in these experimental conditions.

Although it is well known that ferricyanide can be directly reduced by some bacteria 13 and that it enhances the electricity generation by S. oneidensis in microbial fuel cells [START_REF] Wu | [END_REF] we find no significant intrinsic redox activity from single S. oneidensis cells toward the aqueous redox probe in control chronoamperometry experiments (Fig. S3). This is in contrast with results reported by Gao et al. with E. coli (in 100 mM K 3 Fe(CN) 6 at +0.5 V vs. Ag/AgCl). 13 Our observations point to the attraction/repulsion of negatively charged single S. oneidensis bacteria by the polarized CF UME surface as a function of the applied potential and to an electrokinesis of the cells toward the electrode when polarized as an electron acceptor for the electroactive bacteria.

Summary

The electrochemical nano-impacts experiments of single electroactive Shewanella oneidensis bacteria at a carbon fibre ultramicroelectrode have been successfully achieved in potassium phosphate buffer solution containing an aqueous redox probe: potassium ferro-or ferricyanide. A typical stair step current response has been detected for cell nano-impacts at the potential of steady-state current of Fe(CN) 6 4-redox probe (E app ≥ +0.3 V vs. Ag/AgCl), corresponding to the sticking of single bacteria on the ultramicroelectrode surface. In contrast, we have observed current spikes for bacteria collisions in the chronoamperometric measurements recorded at the potential of steady-state current of Fe(CN) 6 3-redox probe (E app ≤ +0.1 V vs. Ag/AgCl). This has been assigned to short bounce events due to electrostatic repulsion of negatively charged Shewanella oneidensis cells by the negatively charged ultramicroelectrode surface. These first results open the way to studies with a range of other aqueous redox probes (with different redox potentials) and bacteria species.

Figure 1 .

 1 Figure 1. Schematic representation and typical electrochemical signal for single nano-impact experiments of Shewanella oneidensis bacteria recorded at a 7 μm carbon ultramicroelectrode polarized at (A) +0.6 V vs. Ag/AgCl in 20 mM potassium ferrocyanide and (B) -0.3 V vs.Ag/AgCl in 20 mM potassium ferricyanide with 20 mM potassium phosphate buffer at pH 7.2 as aqueous electrolyte.

Figure 2 .

 2 Figure 2. Chronoamperometric measurements recorded on 7 μm carbon ultramicroelectrode at +0.6 V vs. Ag/AgCl in 20 mM K 4 Fe(CN) 6 (LEFT, blue curves) and -0.3 V vs. Ag/AgCl in 20 mM K 3 Fe(CN) 6 (RIGHT, orange curves) in 20 mM potassium phosphate buffer at pH 7.2 as aqueous electrolyte under inert atmosphere (Ar) in (TOP) the absence and (BOTTOM) the presence of Shewanella oneidensis bacteria in solution.

Acknowledgments

The authors acknowledge the France-Portugal PHC PESSOA program for support, project 40814ZE.

ORCID

Estelle Lebègue: https://orcid.org/0000-0002-8610-0891 Ricardo O. Louro: https://orcid.org/0000-0002-2392-6450 Frédéric Barrière: https://orcid.org/0000-0001-5515-4080

Supplementary Material

Electrochemical Single Nano-Impacts of Electroactive Shewanella Oneidensis Bacteria Onto Carbon Ultramicroelectrode Estelle Lebègue, 1,z Nazua L. Costa, 2 Ricardo O. Louro, 2 and Frédéric Barrière 3,z 1 Université de Nantes, CNRS, CEISAM UMR 6230, F-44000, Nantes, France 2 Universidade NOVA de Lisboa, Instituto de Tecnologia Química e Biológica, António Xavier, 2780-157 Oeiras, Portugal 3 Univ Rennes, CNRS, Institut des Sciences Chimiques de Rennes -UMR 6226, F-35000, Rennes, France z estelle.lebegue@univ-nantes.fr and frederic.barriere@univ-rennes1.fr


Julien Berthe

Olivier Lejeune

Jessy Jaunat

Pierre-Yves Ancelin

Alain Devos

Nicolas Bollot

Contribution of airborne LiDAR on the mapping of vulnerability to groundwater pollution -Example of the drinking water catchment of Trépail

scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Methodology Introduction

Airborne LiDAR is a laser tool that allows the creation of a high resolution DTM of the relief under the forest. Its application concerns archaeology, geomorphology but also hydrogeology. The objective of this study is to propose a method of mapping vulnerability to pollution adapted to the karst plateau under forest.

The EPIK approach, is created and tested for karstic environment. It's a multi-criteria spatial analysis based on superimposed and weighted indices allowing the zoning of the vulnerability to pollution. This method is improved here with the lidar approach with the use of the Lidar tool which allows to better constrain each layer of spatial information.

Study area

The spring of Trépail is located in the eastern part of the Montagne de Reims. It emerges on the front of the cuesta of Ile-de-France characterized by the triptic forest, vineyard and culture.

It is a karstic spring captured for the drinking water supply of the village.

Result

The resolution of vulnerability maps to groundwater pollution is improved by the use of lidar.

It also allows the creation of new layers that would not exist without LiDAR (more spatial information) and to improve the understanding of water flow. This makes the epik method operational at the cadastral parcel scale, and therefore operational from a regulatory point of view. Thus, the Lidar allows an approach of the surface of feeding more coherent with the reality of the ground by including the impact of the ditches, by modifying the surface of the AAC and by detecting new zones of vulnerability.

Conclusion

The LiDAR data allowed to map a karstic border located on the slopes of the Montagne de Reims within the topographic watershed but also in the adjacent watersheds (hydrographic capture). Moreover, the LiDAR resolution allows to propose a vulnerability mapping at the parcel scale. As such, it can be applied to land development projects to achieve zero pollution in water.

Finally, the topographic survey by airborne LiDAR carried out by IGN, at the scale of France, whose data will be publicly available in 2026, seems to be of great interest for the creation and updating of PPCs and AACs, especially in karstic contexts.

Fig 1 :

 1 Fig 1 : operation of the airborne LiDAR

Fig 7 :

 7 Fig 7 : Mapping of vulnerabitility of water pollution with EPIK method (with LiDAR) Fig 8 : Comparison between the PPC of the Trépail drinking water source and the vulnerability maps