Harold Common

Romain Rousseau

Sophie Putman

Henri Migaud

Julien Girard

Prof Julien Girard

High-level judo practice after hip resurfacing

Keywords: hip resurfacing arthroplasty, judo, return to sport

Background and hypothesis:

The ability to participate in sports after hip arthroplasty is increasingly being requested by patients. The possibilities of participating in sports such as judo after hip arthroplasty have not been explored sufficiently even though these sports are associated with a high risk of loosening, dislocation or revision. The aim of this study was to evaluate the return to judo after hip resurfacing arthroplasty (HRA) in terms of time and level of practice in an expert population and to determine the surgical complication rate.

Materials and Methods:

This was a retrospective observational study of all licensed judo practitioners at an expert level (≥ black belt 3rd Dan) who underwent HRA. Patients filled out a sport-specific questionnaire that captured their level of judo practice (international, national, regional, recreational), type of practice (teaching, technical recreational, competitive recreational), dan grade, weekly volume of practice and time elapsed after surgery before resuming judo. The features of the return to judo were analyzed (technical, ground, combat).

Results: Sixty-seven HRA were implanted in 60 patients: 11 were international judokas, 3 were national, 8 were regional and 38 were recreational. The case series consisted of 18 patients who were 3 rd Dan, 18 were 4 th Dan, 7 were 5 th Dan, 5 were 6 th Dan and 12 were 7 th Dan. The average follow-up was 65.3 months (range 9.9-11.9). All the clinical scores pointed to clinically and statistically significant improvement. Of the 60 patients, 53 had resumed judo at the final assessment (90%) after a mean of 4 months (2-7) for technical judo, 6 months (3-10) for ground judo and 7 months (3-12) for combat judo. Of those who did not resume their judo practice, 3 patients voluntarily changed sports, 2 had psychological apprehension and 2 had residual groin pain. The weekly volume of practice preoperatively was 0.9 hours (0-5) and postoperatively it reached 5 hours (2-18) (p < 0.001).

Conclusion:

HRA in a population of high-level judokas allows for return to sport practice without shortterm complications such as fracture, dislocation or loosening.

INTRODUCTION

The primary goal of total hip arthroplasty (THA) is to restore function and eliminate pain [START_REF] Laupacis | The effect of elective total hip replacement on health-related quality of life[END_REF][START_REF] Lavigne | Return to sport after hip resurfacing or total hip arthroplasty: a randomized study[END_REF]. Currently, the functional demands of patients have been increasing with greater desire to participate in physical activities and/or sports [START_REF] Laupacis | The effect of elective total hip replacement on health-related quality of life[END_REF][START_REF] Crowninshield | Changing demographics of patients with total joint replacement[END_REF]. The progress in surgical technique and perioperative care along with the introduction of innovative implants have helped to satisfy this demand [START_REF] Bedard | Are Trends in Total Hip Arthroplasty Bearing Surface Continuing to Change? 2007-2015 Usage in a Large Database Cohort[END_REF][START_REF] Van Driessche | Short-term comparison of postural effects of three minimally invasive hip approaches in primary total hip arthroplasty: Direct anterior, posterolateral and Röttinger[END_REF][START_REF] Kehlet | Fast-track hip and knee arthroplasty[END_REF]. Use of large-diameter heads, optimization of tribological properties and head-neck ratio along with the availability of short stems are some of the technological improvements that have contributed to reducing the dislocation rate, implant wear, and prosthetic impingement and to preserving the bone stock [START_REF] Daniel | Results of Birmingham hip resurfacing at 12 to 15 years: a single-surgeon series[END_REF]. Hip resurfacing arthroplasty (HRA) is an alternative to conventional THA that falls within the scope of these objectives: negligible risk of instability, preservation of the hip's proprioception and three-dimensional anatomy [START_REF] Daniel | Results of Birmingham hip resurfacing at 12 to 15 years: a single-surgeon series[END_REF]. This surgical technique is aimed at a specific population of younger patients who suffer from disabling primary hip osteoarthritis [START_REF] Lavigne | Return to sport after hip resurfacing or total hip arthroplasty: a randomized study[END_REF][START_REF] Daniel | Results of Birmingham hip resurfacing at 12 to 15 years: a single-surgeon series[END_REF][START_REF] Fouilleron | Running activity after hip resurfacing arthroplasty: a prospective study[END_REF][START_REF] Girard | Can patients return to high-impact physical activities after hip resurfacing? A prospective study[END_REF][START_REF] Girard | High-impact sport after hip resurfacing: The Ironman triathlon[END_REF][START_REF] Krantz | Hip resurfacing in patients under thirty years old: an attractive option for young and active patients[END_REF].

Judo is a pivot-contact combat sport. The goal is to throw the opponent during the standing combat phase or to immobilize, strangle or place the athlete in an arm lock during the ground combat phase. This requires considerable hip functional capacity in a joint that is flexible, stable and powerful [START_REF] Lefevre | Return to judo after joint replacement[END_REF]. Hip osteoarthritis in judokas can be treated by THA while keeping in mind that certain projection techniques could increase the risk of periprosthetic fracture or dislocation [START_REF] Lefevre | Return to judo after joint replacement[END_REF][START_REF] Kujala | Acute injuries in soccer, ice hockey, volleyball, basketball, judo, and karate: analysis of national registry data[END_REF]. We have also wondered about the implant's survival in the context of intensive judo practice [START_REF] Schmalzried | The John Charnley Award. Wear is a function of use, not time[END_REF]. HRA appears to meet the patient's functional objectives; however, the implant survival in judokas and return to sport have never been described in the literature.

The main goal of this study was to evaluate the return to judo after HRA in terms of time and level of practice in an expert population (minimum black belt 3rd Dan). The secondary goal was to determine the surgical revision rate. We hypothesized that HRA would allow return to judo practice at a comparable level to the one prior to the disabling osteoarthritis phase.

MATERIALS AND METHODS

Patients:

This was a single-center retrospective observational study of all licensed judo practitioners who practiced at an expert level (≥ black belt, 3 rd Dan) and who underwent an HRA procedure by a single surgeon (JG) between March 2009 and April 2018. During the inclusion period, this surgeon did 2257 HRA procedures, 67 of which met the inclusion criteria. The included patients' data was exported to a study-specific registry where the sport practiced was always recorded. The indication for HRA was made based on well-known and validated criteria (less than 65 years of age, no osteoporosis, more than 2/3 viable femoral head volume, no leg length discrepancy [START_REF] Fouilleron | Running activity after hip resurfacing arthroplasty: a prospective study[END_REF][START_REF] Girard | Can patients return to high-impact physical activities after hip resurfacing? A prospective study[END_REF]).

The resurfacing implants used were the Conserve® Plus Resurfacing Hip System (Wright Medical Technology, Arlington, TN, USA) or the BIRMINGHAM HIP™ Resurfacing System (BHR™) (Smith & Nephew, Hull, UK) with hybrid fixation (cemented femoral implant and cementless cup). The surgical technique consisted of a posterolateral approach without circumferential capsulotomy with reattachment of the external rotators and preservation of the neck proprioceptors [START_REF] Girard | Le resurfacage de hanche[END_REF].

The case series consisted of 60 patients (3 women, 57 men) who underwent 67 HRA procedures (37 with the Conserve Plus and 30 with the BHR, Table 1) at a mean age of 45 years . The right hip was operated 40 times and the left hip 27 times. None of the patients were lost to follow-up. The hip osteoarthritis was primary in most cases (44 HRA, 66%) and was secondary to femoroacetabular impingement (11 HRA, 16%), dysplasia (6 HRA), osteonecrosis (3 HRA), osteochondritis (2 HRA) or osteochondromatosis (1 HRA) in the other patients. The mean diameter of the femoral implant was 54 mm (46-60).

Fourteen patients were national or international judokas, 8 were national or regional and 38 were recreational. In terms of ranking, 43 patients were 5 th Dan or less, while 17 were higher (5 were 6 th Dan and 12 were 7 th Dan). At the time of surgery, 48 patients had stopped doing judo because of hip pain. The other 19 patients had reduced their practice and made adjustments.

The average follow-up was 65.3 months (range 9.9-11.9). Full weight bearing was allowed immediately after the HRA procedure. Postoperative thromboprophylaxis was prescribed for 6 weeks along with 15 rehabilitation sessions to prevent muscle atrophy in these athletic patients. Return to sport was allowed in the 2 nd month after the clinical follow-up visit that confirmed good muscular and joint conditions.

During the standard annual follow-up visit, all patients were reviewed in person and filled out a sport-specific questionnaire (Appendix 1) capturing their level of judo practice before the surgery (international, national, regional, recreational), type of practice before surgery (teaching, technical recreational, competitive recreational), ranking (dan grade), number of hours of judo practice per week and the time elapsed after HRA before judo activities were resumed. The features of the return to sport were analyzed (technical, ground, combat).

The clinical outcomes were based on the Merle Postel d'Aubigné score [START_REF] Merle | Numerical classification of the function of the hip[END_REF], the Harris Hip Score [START_REF] Harris | Femoral component loosening using contemporary techniques of femoral cement fixation[END_REF] and the Oxford-12 HIP score [START_REF] Delaunay | Cross-cultural adaptations of the Oxford-12 HIP score to the French speaking population[END_REF], along with the satisfaction level reported at the last follow-up visit. The joint range of motion was evaluated clinically.

Statistics

The quantitative parameters were summarized by their mean and standard deviation values or the median and interquartile intervals. Normal distribution of the data was verified graphically and with the Shapiro Wilk test. The qualitative parameters were summarized by counts and percentages. The preoperative parameters were compared to the follow-up parameters by the Chi 2 test or Fisher test for the qualitative variables and Student's t test or Mann-Whitney test for the quantitative variables. Significance threshold was set at 5%. The statistical analysis was done using SPSS software (Version 22.0, IBM Corp, Armonk, NY, USA).

RESULTS

All the functional outcomes showed a clinically and statistically significant improvement (Table 2). The mean flexion at the review was 135° (110-150°). Of the 60 patients, 53 had resumed judo at the final assessment (90%) after a mean of 4 months (2-7) for technical judo, 6 months (3-10) for ground judo and 7 months (3-12) for combat judo. Of those who did not resume their judo practice, 3 patients voluntarily changed sports, 2 had psychological apprehension and 2 had residual groin pain. No mechanical or implant-related issue was found for the residual pain in these latter 2 patients. In terms of satisfaction, 54 patients were very satisfied with the surgery, 2 were satisfied, 2 were somewhat satisfied and 2 were dissatisfied. The two patients who were dissatisfied were the same ones who had not resumed their judo practice because of residual pain.

The weekly volume of practice increased from 0.9 hours (0-5) preoperatively to 5 hours (2-18) (p < 0.001) postoperatively. We could not compare the outcomes between patients of different rankings (5th dan or higher versus lower ranks) because of the limited number of patients.

Only one patient required an early revision to drain a hematoma. At the final review (mean 5.4 years' follow-up), there were no implant dislocations or revisions.

DISCUSSION

Return to sport after THA is increasingly being requested by younger patients, especially for impact or contact-pivot sports [START_REF] Ritter | Life expectancy after total hip arthroplasty[END_REF]. Resuming judo practice in patients operated because of end-stage hip osteoarthritis is a challenging goal. In fact, this requires an implant that drastically reduces the risk of dislocation and periprosthetic fracture that could occur during a fall. This case series confirms our hypothesis that HRA allow return to judo in 90% of highgrade judokas after a mean of 6.7 years.

Some studies have been done on return to sport after HRA in other sports. Girard et al. [START_REF] Girard | High-impact sport after hip resurfacing: The Ironman triathlon[END_REF] reported on 48 patients who did long-distance triathlons at 4.7 years of follow-up. With 94% returning to sport and 28 patients having competed in an Ironman (longest triathlon distance), the authors confirm the possibility of returning to high-impact sports in a young population (44.8 years). In that study, the time before resuming a non-weight bearing activity was significantly shorter (6.2 weeks for swimming and 5.4 weeks for cycling) than a weightbearing activity (16.1 weeks for running). In another prospective study [START_REF] Girard | Can patients return to high-impact physical activities after hip resurfacing? A prospective study[END_REF], return to weight bearing sports after HRA was possible in 94% of patients after a mean of 4 months. Sandiford et al. [START_REF] Sandiford | Return to sporting activity after Birmingham hip resurfacing arthroplasty: Midterm results[END_REF] concluded that 3 months after HRA was sufficient to return to the same level of practice as before the surgery, with no significant difference in the revision rate relative to the sedentary population.

In a prospective randomized study comparing return to sport after HRA and THA, Lavigne et al. [START_REF] Lavigne | Return to sport after hip resurfacing or total hip arthroplasty: a randomized study[END_REF] showed significantly better return to high-and medium-impact sports after HRA, with no difference in the revision rate between the two groups at 1 year of follow-up. This was confirmed in a study of 235 THA by Chatterji et al. [START_REF] Chatterji | Effect of total hip arthroplasty on recreational and sporting activity[END_REF] who reported that patients changed their sports activities to low-impact sports such as walking, swimming, and cycling. The practice of judo after THA was analyzed by Lefevre et al. [START_REF] Lefevre | Return to judo after joint replacement[END_REF] in 27 patients who were able to resume their practice after a mean of 4.1 months. This was an expert population (6 th dan) with a mean age of 63 years at the time of surgery; 76% of them were able to resume judo after the surgery at a minimum of the demonstration level. No dislocation or periprosthetic fracture occurred. None of the patients had resumed competitive judo and 2 patients (7%) required revision after 6 and 9 years, respectively, due to implant loosening. This cast doubt on THA survival in this category of patients and the possibility of returning to competitive sports.

Patients who practice judo require a large movement amplitude and excellent proprioception.

It is interesting to note that the motor function of the patients' lower limb after HRA can be superimposed to that of healthy patients (given the preservation of the proprioceptors in the femoral neck) [START_REF] Szymanski | Functional performance after hip resurfacing or total hip replacement: a comparative assessment with non-operated subjects[END_REF]. Also, impact sports such as judo require restoring the hip's original 3D biomechanics, including preserving the femoral offset to recover the moment arm of the abductors and leg length. These two biomechanical parameters are automatically restored after HRA since the resected bone is replaced by the acetabular and femoral implants [START_REF] Girard | Biomechanical reconstruction of the hip: a randomised study comparing total hip resurfacing and total hip arthroplasty[END_REF][START_REF] Mont | Gait analysis of patients with resurfacing hip arthroplasty compared with hip osteoarthritis and standard total hip arthroplasty[END_REF][START_REF] Nantel | Gait patterns after total hip arthroplasty and surface replacement arthroplasty[END_REF].

The bearing used in THA can tolerate impact sports without risk of head or liner fracture [START_REF] Migaud | Do the reasons for ceramic-on-ceramic revisions differ from other bearings in total hip arthroplasty?[END_REF].

The metal-on-metal bearing generates metal debris, but only at very low levels during the practice of high-impact sports and has no clinical or biological consequences [START_REF] Girard | Can patients return to high-impact physical activities after hip resurfacing? A prospective study[END_REF][START_REF] Girard | High-impact sport after hip resurfacing: The Ironman triathlon[END_REF].

However, a longer-term follow-up of this series of cases is needed.

The current study has several limitations. This was a retrospective study with a relatively small population. However, its retrospective nature does not appear to be a limiting factor because these high-level judokas have extensive knowledge of their practice and are fully able to report their activity level. Also, our study included a unique population -judokas practicing at a minimum of 3rd dan preoperatively -with no patients lost to follow-up. The specific nature of this population would make it impossible to do a prospective study.

In our study, no revisions were needed, and no dislocations or fractures occurred. Our study confirms the possibility of sports participation after HRA as recently shown by the American Association of Hip and Knee Surgeons for return to sport after HRA [START_REF] Swanson | Activity recommendations after total hip and knee arthroplasty: a survey of the American Association for Hip and Knee Surgeons[END_REF]. This absence of complications despite doing a high-impact sport indicates that restoring optimal 3D biomechanics and using an appropriate surgical technique allow return to sports. However, the level of judo practice in this study is a selection bias that cannot be ignored. In fact, the technical mastery of this highly ranked population likely contributes to fewer falls or complications and to rapid resumption of judo practice.

Conclusion

This study confirms the possibility of returning to pivot sports such as judo in a population of high-level judokas with high technical mastery after HRA surgery without short-term complications such as fracture, dislocation or loosening. A long-term follow-up is needed to compare the survivorship in this population to a population practicing low-impact sports.

Conflict of interest:

J. Girard


M Sarrazin
email: margaux.sarrazin@gmail.com

Keywords: OEdèmes inflammatoires, sclérose, pémétrexed, toxicité cutanée Inflammatory edema, sclerosis, pemetrexed, skin toxicity

Introduction. -Le pémétrexed est une chimiothérapie de la classe des anti-folates fréquemment prescrite en oncologie thoracique. Nous rapportons un cas de toxicité cutanée rare au pémétrexed à type d'oedèmes inflammatoires et sclérodermiformes des membres inférieurs. Observation. -Un homme de 63 ans était traité par pémétrexed pour un adénocarcinome pulmonaire métastatique. Quatorze mois après l'initiation de la chimiothérapie, apparaissaient des oedèmes inflammatoires et scléreux des membres inférieurs gênant la flexion des genoux. Une origine cardiaque, rénale, hépatique, thrombotique et infectieuse des oedèmes était écartée. La responsabilité du pémétrexed était alors évoquée et ce traitement suspendu. Un dermocorticoïde de classe très forte était prescrit, permettant une rémission partielle des symptômes. En accord avec les oncologues, un traitement par nivolumab était introduit en relais du pémétrexed. Discussion. -Les oedèmes inflammatoires et scléreux des membres inférieurs sont un effet secondaire rare du pémétrexed mais classiquement décrit. La prise de dexaméthasone la veille, le jour et le lendemain de la perfusion permettrait d'en limiter l'apparition. L'arrêt de la chimiothérapie n'est pas systématique et dépend de la balance entre l'impact fonctionnel des lésions et les alternatives thérapeutiques sur le plan oncologique.

Le pémétrexed (ALIMTA®) est une chimiothérapie appartenant à la classe des antifolates. Il est indiqué en première intention dans le mésothéliome malin non opérable en association avec le cisplatine [START_REF] Hazarika | Pemetrexed in malignant pleural mesothelioma[END_REF][START_REF] Vogelzang | Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma[END_REF]. Il est également indiqué en première ligne dans le cancer bronchique non à petite cellule, non épidermoïde, localement avancé ou métastatique [START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF][START_REF] Cohen | Approval summary: pemetrexed maintenance therapy of advanced/metastatic nonsquamous, non-small cell lung cancer[END_REF].

En prévention de ces effets secondaires, une supplémentation en acide folique et en vitamine B12 est indispensable [START_REF] Vogelzang | Phase III study of pemetrexed in combination with cisplatin versus cisplatin alone in patients with malignant pleural mesothelioma[END_REF][START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF][START_REF] Villela | Pemetrexed, a novel antifolate therapeutic alternative for cancer chemotherapy[END_REF]. La prise de dexaméthasone la veille, le jour et le lendemain du traitement limiterait les effets indésirables cutanés [START_REF] Hazarika | Pemetrexed in malignant pleural mesothelioma[END_REF][START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF]. Les oedèmes inflammatoires et scléreux des membres inférieurs sont des manifestations cutanées rares de la toxicité du pémétrexed.

Observation

Un homme de 63 ans était suivi en pneumologie pour un adénocarcinome pulmonaire tubulo-

Discussion

Les toxicités cutanées du pémétrexed sont fréquentes, pouvant concerner jusqu'à 17% des patients dans les études de phase III [START_REF] Cohen | FDA drug approval summary: pemetrexed for injection (Alimta) for the treatment of non-small cell lung cancer[END_REF]. Elles peuvent se manifester par un exanthème maculopapuleux [START_REF] Piérard-Franchimont | Revisiting cutaneous adverse reactions to pemetrexed[END_REF], une éruption pustuleuse [START_REF] Bracke | Acute generalized exanthematous pustulosis after pemetrexed, and recurrence after re-introduction[END_REF], des vascularites urticariennes [START_REF] Lopes | Pemetrexed-associated urticarial vasculitis[END_REF], une nécrolyse épidermique toxique [START_REF] Tummino | Severe cutaneous toxicity after Pemetrexed as second line treatment for a refractory non small cell lung cancer[END_REF], ou un eczéma craquelé diffus [START_REF] Frouin | Asteatotic eczema ('eczema craquelé') with histopathological interface dermatitis: a new cutaneous reaction following pemetrexed[END_REF]. L'apparition d'oedèmes inflammatoires et sclérodermiformes des membres inférieurs sous pémétrexed est une toxicité cutanée plus rare du pémétrexed, pouvant atteindre 4,5 à 13% des patients traités selon les études [START_REF] Hanna | Randomized phase III trial of pemetrexed versus docetaxel in patients with non-small-cell lung cancer previously treated with chemotherapy[END_REF][START_REF] Eguia | Skin toxicities compromise prolonged pemetrexed treatment[END_REF]. Les facteurs de risque identifiés sont le sexe féminin et le nombre important de cycles de chimiothérapie [START_REF] Eguia | Skin toxicities compromise prolonged pemetrexed treatment[END_REF]. Le délai de survenue est très variable pouvant aller de 1 à 23 cycles de pémétrexed [START_REF] Eguia | Skin toxicities compromise prolonged pemetrexed treatment[END_REF][START_REF] Corbaux | Pemetrexedinduced scleroderma-like changes in the lower legs[END_REF]. Le délai moyen entre la première administration du médicament et la survenue des symptômes est de 8,3 cycles, soit environ 25 semaines. Le délai médian est de 6 cycles (Tableau).

Les lésions sont initialement des oedèmes inflammatoires des membres inférieurs, le plus souvent bilatéraux et symétriques, sans fièvre ni altération de l'état général. L'évolution est marquée par l'apparition d'une sclérose hyperpigmentée et engainante de la peau pouvant limiter les amplitudes articulaires et générer d'importantes douleurs [START_REF] Angelo | A case series of doselimiting peripheral edema observed in patients treated with pemetrexed[END_REF]. Plus rarement, l'atteinte peut être plus diffuse et peut concerner les paupières, les membres supérieurs, le thorax voire la plèvre. La fièvre peut être présente chez 10,7% des patients (Tableau). En La prise en charge thérapeutique n'est pas codifiée et dépend de la sévérité des symptômes et de leur retentissement sur la qualité de vie. La décision doit être, dans tous les cas, pluridisciplinaire. L'arrêt du traitement n'est pas systématique mais il concernait 12 patients sur les 28 cas décrits dans la littérature (Tableau). Les auteurs rapportaient une administration locale ou orale de corticostéroïdes, dans respectivement 39.3% (11/28) et 28.6% (8/28) des cas publiés. La prise orale de corticostéroïdes semble permettre une régression plus fréquente des symptômes qu'avec les dermocorticoïdes, respectivement 100% de régression (6/6) versus 75.0% (6/8), ainsi qu'un meilleur maintien dans le temps du pémétrexed, respectivement 66.7% (4/6) de maintien du pémétrexed avec les corticoïdes oraux versus 42.9% (3/7) avec les corticoïdes topiques [START_REF] Eguia | Skin toxicities compromise prolonged pemetrexed treatment[END_REF][START_REF] Angelo | A case series of doselimiting peripheral edema observed in patients treated with pemetrexed[END_REF][START_REF] Katsenos | Pemetrexed-induced cellulitis: a rare toxicity in nonsmall cell lung cancer treatment[END_REF][START_REF] Galetta | Peripheral skin edema as unusual toxicity in three patients with advanced non-small cell lung cancer treated with pemetrexed alone or in combination with cisplatin[END_REF][START_REF] Kastalli | Eyelid and feet edema induced by pemetrexed[END_REF]. Cependant, le choix du mode d'administration du corticoïde doit se faire également en fonction des comorbidités du patient. L'efficacité des corticoïdes serait liée à l'induction d'une vasoconstriction des vaisseaux dermiques, une diminution de la fuite capillaire et une limitation de la prolifération kératinocytaire et fibroblastique. En association aux corticoïdes, la pose de bande de contention élastique à haut niveau de pression trouve également sa place dans la prise en charge symptomatique.

En conclusion, les oedèmes inflammatoires et scléreux des membres inférieurs sont un effet indésirable rare et parfois méconnu du pémétrexed. La poursuite ou l'arrêt de la chimiothérapie nécessite une discussion collégiale entre le dermatologue et l'oncologue.

Conflits d'intérêts : aucun

Tableau : Résumé des cas d'oedèmes inflammatoires sclérodermiformes sous pemetrexed de la littérature. 1

  acineux. Il avait pour autres antécédents une hémochromatose, une péritonite appendiculaire et une thrombose récente d'un cathéter veineux. Il n'avait pas d'antécédent d'insuffisance veineuse des membres inférieurs. Ses traitements médicamenteux comprenaient pegfilgrastim (NEULASTA®) et tinzaparine sodique (INNOHEP®). L'adénocarcinome pulmonaire avait été traité par lobectomie inférieure gauche suivie d'une chimiothérapie adjuvante par pémétrexed et cisplatine pendant 2 mois. Trois ans après la chirurgie, l'évolution était marquée par une progression métastatique pleurale et péri-oesophagienne traitée par bevacizumab en association avec le pémétrexed et le cisplatine. Après 4 mois d'association efficace, le pémétrexed était poursuivi en monothérapie. En prévention des effets secondaires, une supplémentation en folates et vitamine B12 était maintenue tout le long du traitement. Quatorze mois après l'introduction du pémétrexed, apparaissaient des oedèmes inflammatoires bilatéraux des membres inférieurs persistants malgré un traitement antibiotique par amoxicilline plus acide clavulanique et motivant ainsi un avis dermatologique (Fig.1,2). Le patient était en bon état général et apyrétique. Les oedèmes étaient infiltrés et scléreux par endroit, chauds et douloureux, étendus de la malléole jusque mi-cuisse, bilatéraux, symétriques et prenant le godet. Le signe du canyon n'était pas observé. Il n'existait pas de signe d'insuffisance veineuse chronique (ni varicosité, ni dermite ocre, ni atrophie blanche…). La flexion des genoux était limitée en raison des douleurs et de la sclérose cutanée. Les diagnostics évoqués étaient en première intention des oedèmes inflammatoires et scléreux induits par le pémétrexed, mais aussi une fasciite à éosinophiles ou une lipodermatosclérose en poussée inflammatoire. Le bilan biologique était sans particularité, éliminant une origine rénale ou hépatique aux oedèmes. L'hémogramme était normal (en particulier absence d'éosinophilie sanguine). Il n'y avait pas de syndrome inflammatoire. L'échographie-doppler des membres inférieurs ne mettait pas en évidence de signe de thrombose récente ou d'insuffisance veineuse sur les axes jambiers. L'imagerie par résonnance magnétique (IRM) des membres inférieurs montrait une anomalie de signal circonférentiel sous-cutanée des membres inférieurs évoquant des phénomènes oedémateux ou lymphoedémateux non spécifiques, sans anomalie des masses musculaires ou des fascias. Une biopsie cutanée elliptique au bistouri était réalisée mettant en évidence une légère spongiose ainsi qu'un discret infiltrat inflammatoire macrophagique et lymphocytaire périvasculaire dermique superficiel, sans éosinophiles. Le diagnostic retenu était celui d'oedèmes inflammatoires et scléreux induits par le pémétrexed. Le traitement par pémétrexed était arrêté en raison du retentissement fonctionnel important et en accord avec l'oncologue. Un dermocorticoı̈de de classe très forte était applique ́ quotidiennement sur les membres inférieurs permettant une évolution favorable des symptômes à 3 mois. Par la suite, un traitement par nivolumab était introduit par l'oncologue pour la prise en charge du cancer pulmonaire.

  première intention, une origine thrombotique, rénale, cardiaque et hépatique doivent être éliminées. Une échographie doppler du système veineux, une échographie cardiaque transthoracique et une IRM des membres inférieurs permettent d'éliminer une thrombose veineuse, une insuffisance cardiaque et une atteinte des fascias. L'origine infectieuse bactérienne est éliminée devant le caractère bilatéral des oedèmes et l'absence de franc syndrome inflammatoire biologique. Il faut également évoquer une infection cutanée à germe opportunistes dans le contexte d'immunodépression. Le tableau est cliniquement très proche de la lipodermatosclérose d'origine veineuse ou lymphatique en poussée inflammatoire, Cependant, aucun lien entre ces 2 pathologies n'a été identifié. La physiopathologie de ce phénomène est méconnue et ne semble pas être liée à une propriété pharmacologique propre des anti-folates car il n'existe pas d'effet de classe. Cet effet indésirable ne serait pas lié à un effet cytotoxique direct du pémétrexed sur les kératinocytes ; l'analyse histologique de la biopsie cutanée ne mettait d'ailleurs pas en évidence de nécrose kératinocytaire, dans notre observation. Le pémétrexed pourrait plutôt induire une inflammation systémique non spécifique responsable d'un syndrome de fuite capillaire, comme décrit avec le docétaxel [12,14,15]. Il ne s'agit pas d'un effet spécifique du pémétrexed, car des cas d'oedème scléreux des membres inférieurs ont été décrits avec d'autres chimiothérapies, tel que la gemcitabine ou les taxanes [16,17].

  ND : non documenté ; 2 H : homme, F : femme ; 3 CBNPC : cancer bronchique non à petite cellule ; 4 CBA : cancer broncho-alvéolaire ; 5 ADK : adénocarcinome ; 6 MI : membres inférieurs, MS : membres supérieurs, 7 DC : dermocorticoïdes Figure 1. OEdèmes inflammatoires des membres inférieurs.Figure 2. Aspect scléreux et infiltré des oedèmes.

	14	H/62/CBA H/59 F/66/CBNPC 2,5 mois (3 cycles 7 4 environ)	ND ND ND 1	MI MI MI	ND 1 DC 7 et DC 7	Arrêt Arrêt Poursuite	Persistance Régression complète Régression	[24] [19] [28]
					cortisone per		complète
	Patient Sexe/ Age (années)/ Type de 15 F/65 16 H/66 tumeur 2,3,4,5 1 H/73/CBNPC 14 mois (environ 20 Nombre de cycles de pémétrexed avant les 2 lésions 3 17 F/60/ADK 7 cycles) 2 H/67/CBA ND 1 18 H/66/CBNPC 2 3 H/66/CBNPC 6 4 70/F/CBNPC 2 19 F/61/ND 7 5 H/67/ADK 21 20 F/39/ND 13 6 F/67/ADK 2 21 H/70/ND 13 7 H/83/CB 1 22 H/63/ND 14 8 F/36/CBNPC 2 9 F/70/ADK 4 23 H/74/ND 6 10 F/61/ADK endométrial 1 24 F/49/ND 8 25 H/63/ADK 23 11 H/57/CBA 16 12 H/65/CBNPC 2 26 H/75/CBNPC 11 mois (16 cycles environ) 27 F/57/CBNPC 14 mois (20 cycles	Fièvre Localisation de l'oedème 6 ND MI ND MI Non MI ND Paupières et Oui MS et MI MI Non ND 1 Non MI Non MI ND Paupières et Non MI MI ND Généralisé : ND MI paupières, MI, plèvre ND MI Oui MI ND Généralisé : Non MI paupières, MI, MI plèvre ND MI Non MI ND ND 1 ND Paupières, Non MI thorax, MI, Oui MI MS ND MI ND MI	Traitement os Cortisone per os Cortisone per DC 7 os Cortisone per prednisone os DC 7 0,5mg/kg/j 14j DC 7 DC 7 Aucun ND 1 Traitement prednisone 20mg pendant symptomatique Traitement 5jours DC 7 symptomatique Traitement DC 7 symptomatique Cortisone per os Traitement DC 7 et cortisone per symptomatique Aucun ND 1 os ND 1 ND 1 DC 7 DC 7	Modification de dose ou arrêt du pémétrexed Arrêt Diminution des doses Arrêt, relais nivolumab Régression partielle Notre cas Évolution de la Références Régression [19] sclérose complète Régression [19] complète Poursuite Régression [20] Arrêt Régression partielle [12] complète Arrêt, progression du Régression [25] Arrêt Persistance cancer complère [13] Arrêt Persistance Poursuite Persistance [26] [13] Arrêt Régression [14] Poursuite Persistance [26] complète Diminution dose 375mg/m 2 Régression [14] complète Poursuite Persistance [26] Poursuite Décès dans les [21] Arrêt Régression [26] suites du cancer Poursuite complète Régression partielle [22] Diminution dose à 75% Régression [18] Arrêt Régression [26] complète Arrêt Régression partielle [23] complète Poursuite [26] Poursuite Persistance [27] Régression complète [24] Diminution dose à 75% Persistance Poursuite Régression [28] [24] complète ND Régression partielle [28]
		environ)


Abdou Malik Da Silva

Sandra Courquet

Francis Raoul

Dominique Rieffel

Patrick Giraudoux

Laurence Millon

Jenny Knapp
email: jknapp@univ-fcomte.fr

Abdou Malik

Da Silva

Assessment of the exposure to Echinococcus multilocularis associated with carnivore faeces using real-time quantitative PCR and flotation technique assays

Keywords: Echinococcus multilocularis, Egg, Definitive host, Copro-qPCR diagnostic, Flotation, Spatial distribution

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

Introduction

The larval stage of the cestode Echinococcus multilocularis is responsible for alveolar echinococcosis (AE), an expanding, potentially lethal, zoonosis that occurs only in the northern hemisphere, including large areas of Asia, Europe, and North America [START_REF] Deplazes | Global distribution of alveolar and cystic echinococcosis[END_REF]. The lifecycle is mainly sylvatic, based on predator-prey interactions between wild and domestic carnivores acting as definitive hosts, and small rodents as intermediate hosts. The adult worms live in the small intestine of carnivores and produce the infectious form at sexual maturity, the egg, eliminated into the environment with faeces during the patent period. Development of the larval stage of the parasite following the ingestion of eggs causes AE in humans. In Europe, E. multilocularis eggs have only rarely been identified in dog and cat faeces [START_REF] Dyachenko | Echinococcus multilocularis infections in domestic dogs and cats from Germany and other European countries[END_REF][START_REF] Deplazes | Role of pet dogs and cats in the transmission of helminthic zoonoses in Europe, with a focus on echinococcosis and toxocarosis[END_REF][START_REF] Umhang | Echinococcus multilocularis in dogs from two French endemic areas: No evidence of infection but hazardous deworming practices[END_REF]Umhang et al., , 2014[START_REF] Umhang | Echinococcus multilocularis detection in the intestines and feces of free-ranging domestic cats (Felis s. catus) and European wildcats (Felis s. silvestris) from northeastern France[END_REF]Knapp et al., 2016a). The red fox (Vulpes vulpes) has been identified as the main definitive host of E. multilocularis, in which the parasite has experimentally shown a high reproduction rate and high biotic potential, whereas domestic carnivores, notably the cat, likely excrete few or no eggs [START_REF] Kapel | Reproductive potential of Echinococcus multilocularis in experimentally infected foxes, dogs, raccoon dogs and cats[END_REF][START_REF] Hegglin | Control of Echinococcus multilocularis: strategies, feasibility and cost-benefit analyses[END_REF][START_REF] Umhang | Echinococcus multilocularis detection in the intestines and feces of free-ranging domestic cats (Felis s. catus) and European wildcats (Felis s. silvestris) from northeastern France[END_REF][START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF].

The use of carnivore stools in ecological investigations on E. multilocularis transmission seems appropriate, as stool contamination is representative of environmental contamination by the infectious form of the parasite and it is the source of human exposure [START_REF] Conraths | Echinococcus multilocularis: Epidemiology, surveillance and state-of-the-art diagnostics from a veterinary public health perspective[END_REF][START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF]. In addition, the spatial location of the faeces containing infective E. multilocularis eggs is a key parameter for studying areas of risk and understanding the transmission processes to the intermediate hosts and humans, to propose appropriate prevention or control measures [START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF]. Moreover, the analysis of faecal samples is a non-invasive approach, allowing the preservation of animal populations, essential when studying pets and ethically more acceptable for wild animals.

The spatial distribution of carnivore faeces and E. multilocularis prevalence appear heterogeneous and aggregate in micro-foci over large areas including historically and newly endemic rural and urban sites. The reasons are in part linked to the defecation behaviour of the definitive hosts. For instance, micro-local hotspots of high E. multilocularis prevalence in fox faeces were detected in different rural situations in a study carried out in an eastern region of France [START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF]. Moreover, at a local level, E. multilocularis-positive faeces were found in 19/94 sampled kitchen gardens studied [START_REF] Umhang | A flotation/sieving method to detect Echinococcus multilocularis and Toxocara spp. eggs in soil by real-time PCR[END_REF]. In a North American urban setting, E. multilocularis prevalence in the faeces of coyotes varied significantly across the study area [START_REF] Liccioli | Spatial heterogeneity and temporal variations in Echinococcus multilocularis infections in wild hosts in a North American urban setting[END_REF]. In a rural context in China, E. multilocularis environmental contamination via dog faeces was focused where dogs defecated inside villages [START_REF] Vaniscotte | Role of dog behaviour and environmental fecal contamination in transmission of Echinococcus multilocularis in Tibetan communities[END_REF]. Given the close proximity of humans and the main definitive host of E. multilocularis in rural settings, this environmental overdispersal can potentially be of major public health importance. However, E. multilocularis prevalence is often assessed by detecting E. multilocularis DNA, not necessarily eggs. Furthermore, the sporadic copro-sample collection may lead to failure to detect the parasite and a misestimation of environmental contamination by E. multilocularis via carnivore faeces.

The development and optimization of multiplex real-time PCR techniques, based on the amplification of specific mitochondrial DNA targets, has resulted in the very sensitive and rapid detection of host species and zoonotic parasites such as E. multilocularis in faeces collected from the environment [START_REF] Farrell | Dietary separation of sympatric carnivores identified by molecular analysis of scats[END_REF]Nonaka et al., 2009;[START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF]Knapp et al., , 2016b)). Molecular tools (PCR or real-time quantitative PCR (qPCR)) detect the genetic material in faeces not only from eggs but also from cells or tissue fragments shed by the worm present in the digestive tract of the infected animal [START_REF] Al-Sabi | Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes[END_REF] and other traces of the parasite in transit. The feeding behaviour and coprophagia common in certain carnivore species such as dogs may explain the innocuous passage of parasitic elements, and thus the detection of parasitic DNA, in the digestive system of these animals [START_REF] Frenkel | An apparent role of dogs in the transmission of Toxoplasma gondii. The probable importance of xenosmophilia[END_REF][START_REF] Fahrion | Toxocara eggs shed by dogs and cats and their molecular and morphometric species-specific identification: is the finding of T. cati eggs shed by dogs of epidemiological relevance?[END_REF]Nijsse et al., 2015).

Echinococcus multilocularis DNA may also originate directly from the digested metacestode due to predation of infected rodents [START_REF] Umhang | Echinococcus multilocularis detection in the intestines and feces of free-ranging domestic cats (Felis s. catus) and European wildcats (Felis s. silvestris) from northeastern France[END_REF]. These events can all potentially lead to overestimation, to an unknown extent, of the parasitic prevalence in faeces by molecular detection techniques. Thus, the detection of eggs in stool material is still a key step in the assessment of the environmental exposure of intermediate hosts or humans to E. multilocularis.

Eggs may be concentrated by flotation techniques and identified at the family level (Taeniidae) based on their morphological characteristics [START_REF] Mathis | An improved test system for PCR-based specific detection of Echinococcus multilocularis eggs[END_REF][START_REF] Dryden | Comparison of common fecal flotation techniques for the recovery of parasite eggs and oocysts[END_REF] ,and PCR on isolated egg DNA remains the method of choice for the species-level diagnosis [START_REF] Gasser | Rapid sequencing of rDNA from single worms and eggs of parasitic helminths[END_REF].

This work aimed to determine the percentage of faeces containing E. multilocularis eggs in a rural town and its surroundings and whether this level of precision in relevant in assessing exposure of the intermediate hosts to E. multilocularis. For this purpose, we periodically collected carnivore faeces in order to obtain suitable samples for parasite detection. We developed a combined molecular and microscopic approach to investigate exposure of potential hosts to E. multilocularis in the environment from field-collected carnivore faeces. This strategy is first based on the screening of E. multilocularis DNA in faeces, using qPCR and then the identification of E. multilocularis eggs in positive qPCR samples by a flotation technique, confirmed by specific PCR.

Then, carnivore defecation patterns were spatialized to compare the local distribution of E. multilocularis DNA and eggs in contaminated faeces.

Materials and methods

Copro-sampling

Faeces were collected every 2 months between March 2017 and November 2018 in a village and its surroundings in the eastern part of France (006°26'46"E, 46°56'53"N), in order to regularly obtain fresh material. This site was located in an historic endemic area for E. multilocularis [START_REF] Combes | Westward spread of Echinococcus multilocularis in foxes, France, 2005-2010[END_REF]Piarroux et al., 2013Piarroux et al., , 2015)). The studied area has a surface of 5.28 km 2 , with a density of 25 inhabitants per km 2 , and is located at an average altitude of 971 m above sea level in a predominantly agricultural landscape in which the proportion of permanent grassland to total land is 52% (see the 2010 General Agriculture Census (RGA) from the Ministry of Agriculture, France, http://recensement-agricole.agriculture.gouv.fr/). It has a continental climate, with long, cold, snowy winters. Average temperatures range from -2.2 to 15.1°C with average rainfall of 1,317 mm/year (www.meteofrance.com). All sampled carnivore faeces were collected from the study area access roads within the administrative limits (along a predetermined circuit of 25 km, Fig. 1). Fox, dog, cat, mustelid or undetermined faeces were initially identified in the field by observing their physical characteristics (shape, size, composition). All sampled faeces were geolocated with a Global Positioning System (GPS) tracking device, with a precision of ± 10 m. Those were collected in an individual plastic bags and were stored for 7 days at -80°C for decontamination to prevent human infection, and then at -20°C before laboratory analyses.

Faecal DNA extraction and qPCR protocols for carnivore host faecal test and E. multilocularis diagnostic tests

Faecal DNA was extracted from a 0.5 g sample [START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF] and purified using the QIAmp Fast DNA Stool Kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations, performed on a QIAcube automaton (Qiagen, Hilden, Germany), a robotic workstation for automated purification of DNA.

A host faecal qPCR test was performed on the copro-DNA to identify the carnivore host.

The test was carried out as described above and was based on amplification of a fragment of the cytB gene for Vulpes vulpes (fox), Canis familiaris (dog), and Felis catus (cat) (Knapp et al., 2016b). This molecular identification of host species used as reference was compared with the identification from morphological criteria (described above) in order to verify the accuracy of faecal identification in the field. DNA extracts were tested in an E. multilocularis-specific qPCR for the presence of E. multilocularis DNA, in duplicate, based on the amplification of a portion of the mitochondrial rrnL gene [START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF](Knapp et al., , 2016b) ) (rrn-E. multilocularis forward primer 5'-CTGTGATCTTGGTGTAGTAGTTGAGATTT-3', rrn-E. multilocularis reverse primer 5'-GGGGTCAATCACAACAACCC-3', rrn-E. multilocularis probe 5'-FAM-TGGTCTGTTCGACCTTTTTAGCCTCCAT-TAMRA -3') for PCR products of 202 bp.

The presence of inhibitory factors was tested in the two previous qPCRs using the Alea qPCR control tool (Alea forward primer 5'-CCTAAAAATGTCTATGATTGGTCCACTA-3', Alea reverse primer 5'-GGGAGTACCTTGCCATACAAAATT-3', Alea probe 5'-VIC-TTAAATCAACTCCTAAATCCGCGCGATAGG-TAMRA-3') at a concentration of 6.10 -6 ng/µl, corresponding to 1,000 plasmid copies/µl of the internal control (Knapp et al., 2016b).

DNA-free extraction controls (extraction from only kit reagents), PCR-positive (DNA extracts from E. multilocularis worms) and -negative (ultra-pure water instead of DNA extract) controls were used to ensure there was no contamination during the DNA extraction and the qPCR steps.

The qPCRs were performed for 45 cycles and reactions with quantitative cycle (Cq) numbers < 45 cycles were considered to be positive for E. multilocularis by qPCR and Cq < 30 for the species detected among the three targeted carnivore species in the faecal qPCR. The presence of PCR inhibitors was noted on the basis of the duplex E. multilocularis /Alea qPCR results, considering a Cq > 37 as the threshold beyond which the PCR inhibitors interfered with the PCR process (Knapp et al., 2016b). All samples were tested in duplicate by qPCR.

Isolation of parasite eggs from faeces

All positive faeces from the E. multilocularis-specific qPCR screening and a portion of randomly selected negative faeces, as controls, (n = 25) were subjected to a sucrose flotation technique as described in [START_REF] Matsuo | Modified sugar centrifugal flotation technique for recovering Echinococcus multilocularis eggs from soil[END_REF] and [START_REF] Escotte-Binet | A rapid and sensitive method to detect Toxoplasma gondii oocysts in soil samples[END_REF] to concentrate and isolate the parasite eggs, based on differential specific gravity (SG) between the eggs and particles. Briefly, the protocol consists of dispersing 5 g of faeces in 20 ml of 0.1% Tween 80 by vortexing to separate the eggs from the organic particles. After a centrifugation step (2,500 g, 10 min), the pellet is resuspended in distilled water and deposited on a sucrose solution (SG = 1.27) to float the eggs. Then the supernatant is concentrated in a volume of < 1 ml after centrifugation.

Microscopic observation and sampling of individual E. multilocularis eggs

All flotation supernatants were examined using a Leica DMI3000-B micromanipulator (Leica Microsystems, Wetzlar, Germany) equipped with an inverted microscope to identify taeniid eggs at a 400X magnification. The observed taeniid eggs were picked individually with micropipettes integrated into the micromanipulator and put in approximately 100 µl of distilled water in individual microtubes. For each positive flotation sample, DNA from up to five individual taeniid eggs was extracted using the QIAmp DNA Mini Kit (Qiagen, Hilden, Germany) in an elution volume of 50 µl following the manufacturer's instructions. However, the lysis step was extended from 1-3 h to overnight. The resulting DNA was used to confirm the E. multilocularis egg identification by two PCRs: the E. multilocularis-specific qPCR described above and the E. multilocularis-specific PCR EMH15/H17, confirmed by electrophoresis. The E. multilocularisspecific PCR EMH15/H17 is based on the amplification of a portion of the mitochondrial 12S rRNA gene [START_REF] Dinkel | Detection of Echinococcus multilocularis in the definitive host: coprodiagnosis by PCR as an alternative to necropsy[END_REF][START_REF] Stieger | Spatial and temporal aspects of urban transmission of Echinococcus multilocularis[END_REF][START_REF] Georges | Usefulness of PCR analysis for diagnosis of alveolar echinococcosis with unusual localizations: two case studies[END_REF]) (EM-H15, forward primer: 5'-CCATATTACAACAATATTCCTATC-3', EM-H17, reverse primer: 5'-GTGAGTGATTCTTGTTAGGGGAAG-3') for an expected PCR product size of 200 bp. Then, several PCR products (n = 6) were randomly selected and sequenced by the Sanger method to confirm the E. multilocularis diagnosis [START_REF] Sanger | DNA sequencing with chain-terminating inhibitors[END_REF]. The nucleotide sequences obtained were characterized by comparison with an E. multilocularis nucleotide sequence reference in GenBank using the online genetic databases and the Basic Local Alignment Search Tool (BLAST) available on the NCBI website.

In addition, for each negative flotation sample, the supernatants were extracted and subjected to E. multilocularis-specific qPCR to confirm the microscopic observations (see Fig. 2 for a synopsis of the complete protocol).

Determination of flotation technique sensitivity

The effectiveness of the sucrose flotation technique followed by microscopic observation and detection with E. multilocularis-specific qPCR (described above) was evaluated by testing it in duplicate series of faecal-free E. multilocularis DNA samples (5 g) spiked with 10 E. multilocularis eggs (n=3), five E. multilocularis eggs (n=3), three E. multilocularis eggs (n=3) and one E. multilocularis egg (n=3). These spiked samples were frozen at -80°C for 7 days before testing. The faecal samples were obtained from the colon of captive foxes exhibiting no worms of the Taenia genus nor E. multilocularis at the experimental station of the ANSES laboratory, France.

Echinococcus multilocularis eggs were obtained from a faecal sample from the colon of a naturally infected fox and confirmed with E. multilocularis-specific qPCR.

Statistical and spatial data analysis

Echinococcus multilocularis faecal prevalence between the various carnivore species was compared using the Fisher's exact test. The non-parametric Mann-Whitney U Test was used to compare the Cq values between E. multilocularis qPCR-positive faeces, with or without E. multilocularis eggs detected. A Receiver Operating Characteristic (ROC) curve analysis was used to estimate the ability of the E. multilocularis-specific qPCR (E. multilocularis DNA screening in faeces) to differentiate faeces containing E. multilocularis eggs or not. An optimal threshold value was determined by maximizing the Youden index. Faeces with a Cq higher than the optimal threshold value were classified as E. multilocularis egg-negative, whereas samples with a Cq lower than the optimal threshold value were classified as E. multilocularis egg-positive [START_REF] Fluss | Estimation of the Youden index and its associated cutoff Point[END_REF]. ROC, area under ROC curve (AUC) and the optimal threshold value were calculated using the pROC R package [START_REF] Robin | pROC: an open-source package for R and S+ to analyze and compare ROC curves[END_REF].

For statistical tests, differences were considered significant for P ≤ 0.05.

The spatial distributions of carnivore faeces and E. multilocularis faecal occurrence were mapped on orthophotos (BD Ortho version 2 of the 'Institut Géographique National', France, with a 50 cm resolution as background).

The randomness of the distribution of the E. multilocularis faecal prevalence was tested using a spatial scan statistic developed by Kulldorff and implemented in SatScan v9.6 software [START_REF] Kulldorff | Spatial disease clusters: detection and inference[END_REF]. We tested the null hypothesis that the number of faeces containing E. multilocularis DNA or eggs follows a binomial distribution along the predetermined circuit. In SatScan, we used the Bernoulli Model, with the cases defined as faeces which were found to contain E. multilocularis DNA or eggs and the controls as negative faeces. Kulldorff's method places a circular window on the map and moves the circle centre over each point location so that the window includes different sets of neighbouring points at different positions. At each point location, the radius of the circle increases continuously from zero to a maximum radius, defined by the user and defaulted in this study to a radius containing at most 50% of the total number of points.

Potential clusters were detected by calculating a likelihood ratio for each circle, comparing an expected number of cases (under the null hypothesis) with the observed number of cases in and outside the window. The most likely cluster is the circle with the maximum likelihood ratio among all radius circles at all possible locations.

Computations were performed and graphical displays were generated using R 3.5.1 (R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria, https://www.R-project.org) with the packages foreign 0.8-70 (R Core Team (2017). Foreign: Read Data Stored by 'Minitab, 'S', 'SAS', 'SPSS', 'Stata', 'Systat', 'Weka', 'dBase',…, https://CRAN.R-project.org/package=foreign), rgdal 1.3-9 (Bivand, R., Keitt, T., Rowlingson, B., Pebesma, E., Sumner, M., Hijmans, R., Rouault, E., Warmerdam, F., Ooms, J., Rundel, C., 2019. rgdal: Bindings for the "Geospatial" data abstraction library) and the geographical information system Quantum GIS 3.2.3-Bonn (QGIS Development Team, 2018.

QGIS Geographic Information System, http://www.qgis.com). Kulldorff's statistics were computed using SatScan v9.6 March 2018 (https://www.satscan.org).

Data accessibility

Raw data are available in Mendeley Data, DOI: https://dx.doi.org/10.17632/bhzccxfy9c.3.

Results

Host species identity of faeces collected in the field

We collected 814 faecal samples from March 2017 to November 2018. In total, 763 faecal samples (93.7% of the sampling) were identified by the host faecal qPCR test, including 278 fox faeces (34.15%), 431 dog faeces (52.95%), and 54 cat faeces (6.63%).

The morphological assessment made on fox, dog and cat faeces was confirmed by species qPCRs for 68%, 86% and 52% of the copro-samples, respectively (Table 1). For mustelid faeces identified on morphological criteria, 56% and 28% of samples were reassigned to fox and dog stools, respectively, by molecular analysis. Most of the undetermined faeces in the field were identified by molecular assessment (87.5%).

The host faecal test was unsuccessful for 51 faecal samples for which identification was made based on morphological criteria. This affected 31 fox, 14 dog, one cat, four mustelid, and one undetermined stools (Table 1).

Only four samples (0.5% of all samples analysed) contained PCR inhibitors based on duplex E. multilocularis/Alea qPCR results.

Echinococcus multilocularis in carnivore hosts

Validation of the sucrose flotation protocol for frozen E. multilocularis eggs

When testing naive frozen copro-samples of 5 g spiked with 10 E. multilocularis eggs, a microscopic observation and a positive E. multilocularis qPCR signal were obtained for all samples after the sucrose flotation technique, corresponding to 100% sensitivity (Table 2). Average Cq values ranged from 30.73 ±0.08 for 10 eggs to 37.72±0.16 for one egg. When using five eggs, then one egg, sensitivity decreased from 66.7% to 33.3% for the E. multilocularis qPCR.

Echinococcus multilocularis DNA and egg prevalence in carnivore faeces

We detected E. multilocularis DNA in 96 faeces samples (11.8% of the collection, 95% Confidence interval (CI): 9.7-14.3%). Echinococcus multilocularis DNA was detected significantly more frequently in fox faeces (25.2%, 95% CI: 20.3-30.8%) than cat faeces (9.3%, 95% CI: 3.5-21.1%) or dog faeces (3.5%, 95% CI: 2.0-5.8%) (Table 3, Fisher's exact test, P < 0.0001).

Among the 96 carnivore faecal samples containing E. multilocularis DNA, 32 (33.3%) were found to contain E. multilocularis eggs, based on the sucrose flotation technique. The identification of E. multilocularis was confirmed by E. multilocularis specific qPCR performed on DNA extracts from five individual taeniid eggs isolated from each flotation-positive faecal sample, for which between one and five were positive, with a mean Cq of 34.2 ± 1.46 (n = 52). All selected PCR EMH15/H17 products (n = 6, PCR EMH15/H17 positive from E. multilocularis eggs isolated from one dog, one cat, one undetermined host, and three fox stools) were confirmed to be E. multilocularis, with 100% identity to the E. multilocularis reference sequence (GenBank accession number MK116545.1).

Eggs were observed mostly in fox faeces (n = 27/70), one of five cat faeces, one of 15 dog faeces, and three of six qPCR-undetermined host faeces (Table 3, Fisher's exact test, P < 0.0001).

Those three faecal samples were identified morphologically as two fox and one dog stool.

All selected E. multilocularis qPCR-negative faeces (n = 25) did not contain E. multilocularis eggs, as their flotation supernatants were also negative by E. multilocularis qPCR, except for one sample that had an average Cq of 37.34.

Relationship between the E. multilocularis-specific qPCRs for E. multilocularis egg-positive and egg-negative faeces

Comparison of the Cq values obtained from E. multilocularis-specific qPCR performed on carnivore faeces showed that, among positive faeces, the median Cq of the faeces not containing E. multilocularis eggs was higher than that of the faeces containing E. multilocularis eggs: 38.92 versus 36.9. The distributions of Cq values between E. multilocularis qPCR-positive faeces with or without E. multilocularis eggs differed significantly (Fig. 3A, non-parametric Mann-Whitney test, W= 1511, P < 0.001). All but two faecal samples with a Cq less than 33.62 which had taeniid eggs observed under a microscope, confirmed E. multilocularis by E. multilocularis-specific qPCR and PCR (Fig. 3A). However, the ROC curve showed that the Cq values had only a weak ability to differentiate samples containing E. multilocularis eggs and those without (AUC = 0.73, 95% CI: 0.614-0.847). Beyond the optimal threshold Cq of 38.36, corresponding to a sensitivity of 81% (95% CI: 66-94%) and a specificity of 69% (95% CI: 58-80%), most of the samples were negative by flotation (88%) (Fig. 3B).

Spatial distribution of E. multilocularis DNA and eggs via carnivore faeces

Domestic carnivore faeces (in particular, dog) were more prominent downtown than outside the village (Fig. 4C). Fox faeces were mostly found outside the village but some were also found inside (Fig. 4B).

Kulldorff's statistics showed two significant spatial clusters for E. multilocularis DNApositive faeces and E. multilocularis egg-positive faeces for all carnivores pooled, located in the same areas in both cases. The E. multilocularis DNA faecal prevalence was clearly overdispersed, with a high-rate cluster (P =1.4×10 -6 ) located in the western part of the site but near the centre and a low-rate cluster (P = 0.02) in the eastern part (Fig. 4Aa). The faecal prevalence of E. multilocularis eggs showed significant clusters (high rate: Pp =10 -9 , low rate: P = 4.75×10 -3 ) within the same geographical locations as those for E. multilocularis DNA faecal prevalence (Fig. 4Ab and4Aa).

For fox, E. multilocularis DNA-positive faeces (high rate: P = 0.01, low rate: P = 0.02) and E. multilocularis egg-positive faeces (high rate: P = 0.002, low rate: P = 0.01) were also aggregated (Fig. 4Ba and4Bb). Echinococcus multilocularis contamination in domestic carnivores was not found to be clustered, likely due to the low number of positive samples (Fig. 4C). Contamination clusters were due to the high rate of contamination in fox faeces.

Discussion

This study focused on collecting evidence of E. multilocularis eggs in carnivore faeces in which E. multilocularis DNA was first detected by E. multilocularis-specific qPCR. The E. multilocularis DNA and egg detection methods are notably effective in the patent phase of infection [START_REF] Al-Sabi | Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes[END_REF]. DNA detection is possible but remains uncertain during the pre-patent and post-patent periods due to irregular excretion of tissue or eggs [START_REF] Deplazes | Molecular tools for studies on the transmission biology of Echinococcus multilocularis[END_REF]. Here, the E. multilocularis qPCR Cq values were significantly lower for samples in which eggs were present than for flotation-negative samples, implying a larger amount of E. multilocularis DNA in E. multilocularis qPCR-positive faeces. Our results suggest that in our protocol (DNA extraction and qPCR protocol), faeces for which the E. multilocularis qPCR Cq values were less than 33.62 were likely to contain E. multilocularis eggs. The effectiveness of the key step of DNA extraction from taeniid or E. multilocularis eggs using a kit with only chemical lysis was previously demonstrated [START_REF] Štefanić | Polymerase chain reaction for detection of patent infections of Echinococcus granulosus ("sheep strain") in naturally infected dogs[END_REF][START_REF] Davidson | Echinococcus multilocularisadaptation of a worm egg isolation procedure coupled with a multiplex PCR assay to carry out large-scale screening of red foxes (Vulpes vulpes) in Norway[END_REF]Øines et al., 2014;[START_REF] Umhang | A flotation/sieving method to detect Echinococcus multilocularis and Toxocara spp. eggs in soil by real-time PCR[END_REF]. This Cq value is very close to the mean E. multilocularis qPCR Cq of a DNA extract from one E. multilocularis egg (34.2 ± 1.46). In a previous study, the detection limit of PCR targeting mitochondrial DNA (E. multilocularis, Echinococcus granulosus, and that from single eggs of other tainiids), was estimated to be 33 copies [START_REF] Trachsel | Identification of taeniid eggs in the faeces from carnivores based on multiplex PCR using targets in mitochondrial DNA[END_REF]. Conversely, no conclusion can be drawn concerning the presence/absence of E. multilocularis eggs in faeces with E. multilocularis qPCR Cq values ranging from 33.62 to 38.36, the maximum beyond which most of the samples were negative. This maximum corresponds to a mean E. multilocularis qPCR Cq of a DNA extract of faeces (500 mg) experimentally spiked with one E. multilocularis egg (37.6 ± 1.34) [START_REF] Knapp | Real time PCR to detect the environmental faecal contamination by Echinococcus multilocularis from red fox stools[END_REF]. The absence of eggs in this interval could be justified by a high concentration of E. multilocularis-free DNA. This is probably related to random sampling bias. Indeed, the E. multilocularis qPCR was performed from only 0.5 g of faeces and the flotation technique from 5 g. In cases of low-grade infection, this small subsample size would be more likely to contain no eggs. This could also explain the weak ability of the E. multilocularis qPCR to differentiate samples containing E. multilocularis eggs and those without. The E. multilocularis qPCR performed directly on carnivore faeces probably detects more E. multilocularis-free DNA than E. multilocularis eggs. It appears that flotation is necessary to assess exposure to E. multilocularis associated with carnivore faeces in the environment. The sucrose flotation method combined with microscopical observation and qPCR detection used in this study proved to have sensitivity of 100% for the detection of 10 E. multilocularis eggs in 5 g of frozen copro-samples. Additionally, the sucrose flotation method enabled the potential recovery of only one egg in 5 g of faeces. Moreover, a flotation sample with no eggs gave a mean Cq of 37.34, demonstrating the presence of trace amounts of E. multilocularis DNA in the supernatant. The choice of the sucrose solution was justified by its safety and neutrality on parasitic elements, which can facilitate the recognition of eggs under the microscope after flotation. For instance, zinc chloride (SG=1.42), although more effective in floating eggs, could potentially alter the structural integrity of oocysts [START_REF] Escotte-Binet | A rapid and sensitive method to detect Toxoplasma gondii oocysts in soil samples[END_REF] and frozen taeniid eggs (our personal observations). It is also very hazardous to human and environmental health (National Center for Biotechnology Information. PubChem Database., 2005. Zinc chloride, CID=5727. https://pubchem.ncbi.nlm.nih.gov/compound/Zinc-chloride#section=Safety-and-Hazards). On the other hand, some E. multilocularis qPCR results on isolated taeniid eggs were negative, likely related to the fact that other taeniid species can be found in carnivore intestines [START_REF] Umhang | Echinococcus multilocularis in dogs from two French endemic areas: No evidence of infection but hazardous deworming practices[END_REF][START_REF] Li | Survey on helminths in the small intestine of wild foxes in Qinghai, China[END_REF]Umhang et al., 2014[START_REF] Umhang | Echinococcus multilocularis detection in the intestines and feces of free-ranging domestic cats (Felis s. catus) and European wildcats (Felis s. silvestris) from northeastern France[END_REF][START_REF] Karamon | First report of Echinococcus multilocularis in cats in Poland: a monitoring study in cats and dogs from a rural area and animal shelter in a highly endemic region[END_REF][START_REF] Tylkowska | Presence of tapeworms (Cestoda) in Red Fox (Vulpes Vulpes) in North-western Poland, with particular emphasis on Echinococcus Multilocularis[END_REF]. The individual isolation of eggs offers the possibility to non-invasively study the genetic diversity of the parasite in the same carnivore host faeces using the tandemly repeated microsatellite marker EmsB [START_REF] Bart | EmsB, a tandem repeated multi-loci microsatellite, new tool to investigate the genetic diversity of Echinococcus multilocularis[END_REF][START_REF] Knapp | Assessment of use of microsatellite polymorphism analysis for improving spatial distribution tracking of Echinococcus multilocularis[END_REF][START_REF] Knapp | Genetic diversity of the cestode Echinococcus multilocularis in red foxes at a continental scale in Europe[END_REF]Umhang et al., 2014;[START_REF] Valot | Genomic characterization of EmsB microsatellite loci in Echinococcus multilocularis[END_REF] and avoid complex analysis of egg or DNA pools [START_REF] Carlsgart | Multiplex PCR on single unembryonated Ascaris (roundworm) eggs[END_REF].

The use of species-specific qPCRs made it possible to identify the carnivore species for 763 of the 814 faeces samples collected, for a success rate of 93.7%. According to this molecular method, dog and fox faeces were better identified in the field than cat and mustelid faeces. Our results indicate that morphological identification of copro-material remains uncertain. Confirmation by a molecular reference technique is advisable to understand the relative contribution of each definitive species to environmental contamination [START_REF] Umhang | A flotation/sieving method to detect Echinococcus multilocularis and Toxocara spp. eggs in soil by real-time PCR[END_REF][START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF]. Only 0.5% of all analysed samples contained PCR inhibitor agents. [START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF] reported a similar success rate (94.3%) but a higher rate of faeces containing inhibitors (9.7%). In our study, the frequent sampling of faeces probably allowed us to obtain fresher samples, less subject to environmental hazards and DNA degradation than copro-samples obtained by sporadic collection [START_REF] Kovach | Evaluation of fecal mtDNA analysis as a method to determine the geographic distribution of a rare lagomorph[END_REF]Nonaka et al., 2009). Improved sampling conditions and the use of specific kits for DNA extraction reduce the presence of PCR inhibitors and thus increase the sensitivity and specificity of qPCR methods for species identification from stools [START_REF] Al-Sabi | Comparative copro-diagnosis of Echinococcus multilocularis in experimentally infected foxes[END_REF]Knapp et al., 2014).

More than half of the faeces collected among domestic carnivores were from dogs versus only 7% for cats. These faeces were found mostly in the centre of the village near houses. Fox faeces accounted for 1/3 of all samples and were found in the centre and surrounding area. The presence of fox faeces in proximity to humans is not surprising, as several studies have reported that foxes approach human dwellings at night in rural areas to feed [START_REF] Janko | Space use and resting site selection of red foxes (Vulpes vulpes) living near villages and small towns in Southern Germany[END_REF][START_REF] Krauze-Gryz | The good, the bad, and the ugly: space use and intraguild interactions among three opportunistic predators-cat ( Felis catus ), dog ( Canis lupus familiaris ), and red fox ( Vulpes vulpes )under human pressure[END_REF][START_REF] Scott | Changes in the distribution of red foxes (Vulpes vulpes) in urban areas in Great Britain: findings and limitations of a media-driven nationwide survey[END_REF], which was especially noticed in the study site [START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF].

However, E. multilocularis DNA was detected in 25% of fox faeces (70/278) versus only 4% (15/431) of dog faeces and 9.3% (5/54) of cat faeces. This relatively high prevalence of E. multilocularis DNA in cat faeces was observed (4/43; 9.3%) in an endemic area in the north-eastern part of France (Knapp et al., 2016a). This observation could be due to the small number of samples collected or a possible multiple copro-sampling of the same or a few infected cats. In foxes and dogs, similar findings were reported for the same study site in 2012 and 2013 [START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF].

Indeed, E. multilocularis DNA was detected in 25.85% (61/236) of fox faeces compared with 1.46% (3/205) of dog faeces. Fox faeces collected from 94 vegetable gardens located in the northeast of France were also reported to be the most contaminated, with a rate of 35% (24/69) versus 11% (2/18) for dog faeces and 7% (6/86) for cat faeces [START_REF] Umhang | A flotation/sieving method to detect Echinococcus multilocularis and Toxocara spp. eggs in soil by real-time PCR[END_REF]. In light of the reported prevalence of E. multilocularis DNA, these studies, as well as ours, confirm the high risk of transmission of E. multilocularis, associated mainly with foxes, the role of cats and dogs being considered marginal [START_REF] Hegglin | Control of Echinococcus multilocularis: strategies, feasibility and cost-benefit analyses[END_REF]. We confirmed these hypotheses by the detection of E. multilocularis eggs after flotation, mainly in fox faeces in this study (27/32) and only in 1/15 copro-samples from dogs and 1/5 from cats. Despite a low prevalence of E. multilocularis DNA and eggs in dog faeces, the contribution of dogs in the transmission of E. multilocularis to humans may be sizable due to their proximity with them and the presence of a large amount of dog faeces downtown in the village studied (Liccioli et al., 2015a;[START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF]. Thus, the exposure risk related to domestic carnivores has to be more carefully considered.

We are planning further studies based on in vivo experiments in mice to assess the infectivity of E. multilocularis eggs originating from cat and dog faeces from our study site.

Despite the low number of faeces containing E. multilocularis eggs, their spatial distribution was also overdispersed in the study area, with the same hotspot as faeces detected with E. multilocularis DNA. Such E. multilocularis environmental contamination is likely to persist for several months. Indeed, E. multilocularis eggs can survive for more than 1 year in cold conditions [START_REF] Veit | Influence of environmental factors on the infectivity of Echinococcus multilocularis eggs[END_REF]. Our study confirms that contamination via the definitive host faeces on a local scale is generally overdispersed [START_REF] Giraudoux | Transmission ecology of Echinococcus multilocularis in wildlife: what can be learned from comparative studies and multiscale approaches?[END_REF][START_REF] Vaniscotte | Role of dog behaviour and environmental fecal contamination in transmission of Echinococcus multilocularis in Tibetan communities[END_REF][START_REF] Liccioli | Spatial heterogeneity and temporal variations in Echinococcus multilocularis infections in wild hosts in a North American urban setting[END_REF][START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF]. The activity patterns and defecation behaviour of the definitive hosts have been suggested as possible causes of such micro-local hotspots of E. multilocularis transmission to intermediate hosts and humans [START_REF] Robardet | Fox defecation behaviour in relation to spatial distribution of voles in an urbanised area: an increasing risk of transmission of Echinococcus multilocularis?[END_REF][START_REF] Giraudoux | Transmission ecosystems of Echinococcus multilocularis in China and Central Asia[END_REF]Raoul et al., 2015;[START_REF] Knapp | Rural and urban distribution of wild and domestic carnivore stools in the context of Echinococcus multilocularis environmental exposure[END_REF]. It cannot be excluded that, in the present study, only a small number of individuals deposited multiple faeces collected in the field. It is therefore possible that such spatial heterogeneity of environmental contamination by E. multilocularis was the consequence of a very few individuals in the definitive host population (notably foxes) harbouring almost the entire parasitic biomass (Raoul et al., 2001;[START_REF] Fischer | Echinococcus multilocularis infections of rural, residential and urban foxes (Vulpes vulpes) in the canton of Geneva, Switzerland[END_REF]. Individual identification of collected faeces could thus allow us to know the true E. multilocularis prevalence in a definitive host population in a non-invasive manner and to identify the contribution of each individual to the environmental contamination (Liccioli et al., 2015b).

Our data suggest that E. multilocularis DNA and/or egg detection in carnivore faeces (mainly that of foxes) is appropriate in ecological studies of E. multilocularis transmission, as their spatial distribution presented the same characteristics. According to our results, E. multilocularis egg detection from E. multilocularis-positive qPCR samples is especially useful in evaluating exposure due to infected domestic animals which have close contact with humans. Screening and flotation were performed using 0.5 and 5 g of faeces, respectively. qPCR, real-time quantitative PCR.

Fig. 1 .

 1 Fig. 1. The predetermined circuit (red dash) of carnivore faeces collection on the study site (a rural town in the eastern part of France (006°26'46"E, 46°56'53"N)).

Fig. 2 .

 2 Fig. 2. Workflow of Echinococcus multilocularis DNA and egg detection in carnivore faeces.

Fig. 3 .

 3 Fig. 3. Relationship between quantitative cycle (Cq) values obtained from Echinococcus multilocularis-specific real-time quantitative PCR (E. multilocularis qPCR) performed on carnivore faeces containing E. multilocularis eggs (E. multilocularis egg (+)) or not (E. multilocularis egg (-)). (A) Distribution of faeces containing E. multilocularis eggs, or not, according to the Cq values of E. multilocularis-specific qPCRs. N, n: sample size. Boxes represent the inter-quartile distances; thick lines, the medians; whiskers, the extremes values; crosses, the means; and circles, outliers. Statistical significance was determined using the non-parametric Mann-Whitney U Test. ***P = 0.001. (B) Receiver Operating Characteristic (ROC) curve of the E. multilocularis-specific qPCR Cq values to predict the presence/absence of E. multilocularis eggs (area under ROC curve (AUC) = 0.73). The optimal threshold by the Youden index Cq = 38.365, corresponding to a sensitivity of 81% (95% Confidence interval (CI): 66-94%) and a specificity of 69% (95% CI: 58-80%).

Fig. 4 .

 4 Fig. 4. Kulldorff statistics on the distribution of carnivore faeces in the studied site in France, from Echinococcus multilocularis DNA-positive faeces (Aa, Ba, Ca) and E. multilocularis egg-positive faeces (Ab, Bb, Cb). (A) All carnivore faeces collected; (B) fox faeces; (C) dog and cat faeces.

  Screening E. multilocularis DNA in faeces by E. multilocularis-specific qPCR E. multilocularis DNA + E. multilocularis DNA -E. multilocularis egg detection by sucrose flotation -microscopic analysisconfirmation by E. multilocularis-specific qPCR n=25 selected at random E. multilocularis egg -E. multilocularis egg + Confirmation by E. multilocularis-

Table 1 .

 1 Comparison between faeces identification based on morphological criteria (shape, size, and composition) and faeces identification based on molecular analysis (by speciesspecific real-time quantitative PCRs)Bold values indicate occurrence of faeces per species for which morphological identification was confirmed by molecular analysis.

			Molecular identification
	Morphological identification	Fox	Dog	Cat	Undetermined	Total
	Fox	220 (68.3%)	39 (12.1%)	32 (10.0%)	31 (9.6%)	322
	Dog	38 (8.7%)	374 (85.8%) 10 (2.3%)	14 (3.2%)	436
	Cat	5 (21.7%)	5 (21.7%)	12 (52.2%) 1 (4.4%)	23
	Mustelids	14 (56.0%)	7 (28.0%)	0 (0.0%)	4 (16.0%)	25
	Undetermined	1 (12.5%)	6 (75.0%)	0 (0.0%)	1 (12.5%)	8
	Total	278	431	54	51	814

Table 2 .

 2 Method sensitivities (in %) of the sucrose flotation technique combined with microscopic observation and detection by specific real-time quantitative PCR (qPCR) forEchinococcus multilocularis in frozen copro-samples (5 g). For each number of E. multilocularis eggs, three samples were used.

	Number of eggs	Microscopic observation	qPCR E. multilocularis
	0	-	00.0% (0/3)
	1	00.0% (0/3)	33.3% (1/3)
	3	00.0% (0/3)	33.3% (1/3)
	5	33.3% (1/3)	66.7% (2/3)
	10	100.0% (3/3)	100.0% (3/3)

Table 3 .

 3 Detection of Echinococcus multilocularis DNA and eggs in carnivore host faecal samples collected between March 2017 and November 2018 in a rural town in the eastern part of France. qPCR, real-time quantitative PCR; CI, Confidence Interval.

	Host identification	Fox	Dog	Cat	Undetermined	Total
	Total sampled	278	431	54	51	814
	qPCR E. multilocularis (number of positive samples)	70	15	5	6	96
	Prevalence %	25.2	3.5	9.3	11.7	11.8
	95% CI	(20.3-30.8)	(2.0-5.8)	(3.5-21.1)	(4.9-24.6)	(9.7-14.3)
	E. multilocularis egg-positive samples	27	1	1	3	32

Acknowledgements

This work was supported by the "Zones Ateliers" network (https://inee.cnrs.fr/fr/zonesateliers) and carried out in the "Zone Atelier Arc Jurassien" (http://zaaj.univ-fcomte.fr), France.

This work was also supported by the "Région Franche-Comté" (France) through the purchase of the Leica DMI3000-B micromanipulator equipment (Grant 2013 No. 903/5110/309). We would also like to thank the ''Maison des Sciences de l'Homme et de l'Environnement Claude Nicolas Ledoux" (France) for providing the Orthophotos.

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