Dr Philipp Koehler
email: philipp.koehler@uk-koeln.de

Jörg Janne

Nikolay Klimko

Keywords: Candidemia, invasive candidiasis, invasive fungal infection, outbreak, epidemiology

Background

Recent outbreaks of Candida auris further exemplify that invasive Candida infections are a substantial threat to patients and health care systems. Even short treatment delays are associated with higher mortality rates. Epidemiological shifts towards more resistant Candida spp. require careful surveillance.

Objectives

Triggered by the emergence of C. auris and by increasing antifungal resistance rates the European Confederation of Medical Mycology developed an international Candida Registry (FungiScope™ CandiReg) to allow contemporary multinational surveillance.

Methods

CandiReg serves as platform for international cooperation to enhance research regarding invasive Candida infections. CandiReg uses the General Data Protection Regulation compliant data platform ClinicalSurveys.net that holds the electronic case report forms (eCRF). Data entry is supported via an interactive macro created by the software that can be accessed via any internet browser.

Results

CandiReg provides an eCRF for invasive Candida infections that can be used for a variety of studies from cohort studies on attributable mortality to evaluations of guideline adherence, offering to the investigators of the 28 ECMM member countries the opportunity to document their cases of invasive Candida infection. CandiReg allows the monitoring of epidemiology of invasive Candida infections, including monitoring of multinational outbreaks. Here, we describe the structure and management of the CandiReg platform.

Conclusion

CandiReg supports the collection of clinical information and isolates to improve the knowledge on epidemiology and eventually to improve management of invasive Candida infections. CandiReg promotes international collaboration, improving the availability and quality of evidence on invasive Candida infection and contributes to improved patient management.

Introduction

Invasive Candida infections are among the most common bloodstream infections and are associated with high morbidity and mortality. [START_REF] Kullberg | Invasive Candidiasis[END_REF][START_REF] Wisplinghoff | Nosocomial bloodstream infections due to Candida spp. in the USA: species distribution, clinical features and antifungal susceptibilities[END_REF][START_REF] Koehler | Morbidity and mortality of candidaemia in Europe: an epidemiologic meta-analysis[END_REF] Candida species are commensals of the human gastrointestinal microbiota and skin, but may translocate to the bloodstream and cause life-threatening infections.

Invasive Candida infection (ICI) is an increasing threat to patients in the ICU, patients undergoing complicated or repeated gastrointestinal surgery and for immunocompromised patients, e.g., cancer patients, or recipients of solid organ or stem cell transplants. [START_REF] Lortholary | Worrisome trends in incidence and mortality of candidemia in intensive care units (Paris area, 2002-2010)[END_REF] In some cases, dissemination complicates acute blood stream infection with deep tissue and organ involvement. Due to novel medical and immunological interventions and treatments, an increasing number of critically ill patients are at risk ICI. Common etiological agents are Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis and Pichia kudriavzevii (formerly Candida krusei) -depending on geographical region and patients' risk groups. The emergence of C. auris, a novel, usually fluconazole-resistant and often multidrug-resistant Candida species has caused outbreaks worldwide and led to clinical alerts to U.S. and European healthcare facilities. [START_REF]healthcare facilities: global emergence of invasive infections caused by the multidrug-resistant yeast Candida auris[END_REF][START_REF]Guidance for the laboratory investigation, management and infection prevention and control for cases of Candida auris[END_REF][START_REF]Erhöhte Aufmerksamkeit bei Candida-auris-Fällen notwendig[END_REF][START_REF]Candida auris in healthcare settings -Europe -first update[END_REF] In an Indian hospital C. auris has become the second most common cause of candidemia after C. tropicalis. [START_REF] Mathur | Five-year profile of candidaemia at an Indian trauma centre: High rates of Candida auris blood stream infections[END_REF] The ECMM Candida Registry (CandiReg) was founded in January 2018, triggered by the recent C. auris candidemia outbreaks in Spain and the United Kingdom. [START_REF] Rhodes | Genomic epidemiology of the UK outbreak of the emerging human fungal pathogen Candida auris[END_REF][START_REF] Ruiz-Gaitan | An outbreak due to Candida auris with prolonged colonisation and candidaemia in a tertiary care European hospital[END_REF][START_REF] Schelenz | First hospital outbreak of the globally emerging Candida auris in a European hospital[END_REF] The main objective of CandiReg is to overcome the lack of knowledge on epidemiology, clinical course, and molecular characteristics of invasive Candida infections and to function as a platform for international multicenter studies and surveillance of multinational C. auris outbreaks. The specific objectives are to describe the incidence, to monitor trends globally and locally over time, to define patient risk groups, to assess antifungal resistance among Candida spp. causing invasive diseases worldwide, to assess attributable mortality and to assess to the cost augmentation associated with invasive Candida infection. A further goal of this platform is to set up a multinational collection of resistant Candida isolates with characterization at a molecular level, including the evaluation of resistance genes.

We herein describe how FungiScope™ CandiReg is set up, maintained, and how it can be used to investigate the occurrence of ICI and potential outbreaks in future.

Accepted Article

.

Methods

CandiReg is an international non-interventional multicenter registry project. Treating physicians and medical microbiologists alike are invited to participate in the collection of clinical data and fungal isolates from cases of candidemia and tissue invasive candidiasis. The registry was founded in January 2018 and is ongoing without a defined endpoint. CandiReg uses an electronic case report form (eCRF) using the online electronic data capture platform www.clinicalsurveys.net (in cooperation with Questback GmbH, Cologne, Germany). eCRF structure is modular and the system is programmed to adapt the eCRF by displaying or hiding items according to the documented case (e.g. candidemia vs. control patient). Case registration is on a voluntary basis. Target groups are patients with invasive candidiasis or candidemia. Export will be performed in SPSS-labelled data files in SPSS binary format. Statistical analyses will be performed with IBM SPSS Statistics software v.25.0 (IBM Corp., Armonk, NY, USA/United States).

Ethical and General Data Protection Regulation considerations

Results

Case documentation and data collection

Patients with candidemia or invasive candidiasis confirmed by culture, histopathology or microscopy can be included. A second cohort is defined by patients with signs of disseminated Candida infection without culture, histological or microscopical evidence (e.g. CT-imaging of target-lesions in liver and spleen and positive Candida antigen or Beta-D-Glucan tests in a neutropenic patient), so that chronic disseminated, culture negative candidiasis can be documented in an adjusted eCRF separable from patients with candidemia. Patients without evidence of invasive disease or those with colonization Accepted Article . only are not eligible.

The following demographic data are collected: age group at the date of diagnosis, gender, year of infection, weight, ethnicity, details on primary underlying disease or conditions, immunosuppression, further risk factors, echocardiography and ophthalmology results, and mycological procedures allowing ICI diagnosis and sites of infection (Table 1). Details on management including antifungal treatment; recording drug, dose, duration, route of administration, therapeutic drug monitoring, reason for stopping, drug-related adverse events and surgical procedures, catheter management, clearance of bloodstream infection or infected sites are documented. Treatment indication is differentiated into prophylaxis, empiric, pre-emptive and targeted treatment for ambulatory and inpatient parenteral antifungal therapy.

Response to antifungal therapy is evaluated after two and four weeks, three and six months and on the final day of observation. In addition, treatment response and outcome of the underlying disease as well as potential prolongation of hospital stay are also documented. Information on outcome includes overall mortality and attributable mortality. If available, autopsy results are recorded.

Quality Management is covered once yearly from participating centers with regard to guideline implementation and adherence (ECIL, ESCMID, / ECMM, IDSA; EQUAL Candida Score, Infectious Diseases / Microbiology consulting services; Fellow of the ECMM availability, treatment in an ECMM Excellence Centre). [START_REF] Tissot | ECIL-6 guidelines for the treatment of invasive candidiasis, aspergillosis and mucormycosis in leukemia and hematopoietic stem cell transplant patients[END_REF][START_REF] Ullmann | ESCMID* guideline for the diagnosis and management of Candida diseases 2012: adults with haematological malignancies and after haematopoietic stem cell transplantation (HCT)[END_REF][START_REF] Cuenca-Estrella | ESCMID* guideline for the diagnosis and management of Candida diseases 2012: diagnostic procedures[END_REF][START_REF] Cornely | ESCMID* guideline for the diagnosis and management of Candida diseases 2012: non-neutropenic adult patients[END_REF][START_REF] Pappas | Clinical Practice Guideline for the Management of Candidiasis: 2016 Update by the Infectious Diseases Society of America[END_REF][START_REF] Mellinghoff | EQUAL Candida Score: An ECMM score derived from current guidelines to measure QUAlity of Clinical Candidaemia Management[END_REF] Furthermore economic key figures and hospital characteristics with normal ward vs. ICU beds, candidemia per year, admissions per year, medical vs. surgical ward / ICU, consumption of antifungals in defined daily doses (DDD) are gathered. To implement health economic analyses on the incremental costs and attributable mortality analysis the study will in part use a matched case control design. Controls will be included from the same hospitals that include cases (i.e. one control per case, both from the same hospital). Controls will be matched by demographics, underlying diseases and risk factors as well as duration of hospitalization. [START_REF] Wey | Hospital-acquired candidemia. The attributable mortality and excess length of stay[END_REF][START_REF] Gudlaugsson | Attributable mortality of nosocomial candidemia, revisited[END_REF]

Isolates collection

In addition to clinical data, partners can contribute with Candida isolates considered as emergent (e.g. Candida auris) for formal species identification and susceptibility testing. Isolates will be stored and made available to all collaborating partners for developing research projects. The following laboratory-based research will be conducted: isolate identification, macro-morphology, in vitro susceptibility testing according to EUCAST, evaluation and analysis of resistance mechanisms.

Isolates are processed de-centrally at National Reference Centers or ECMM Excellence Centers, which serve as the central mycology reference laboratories. [START_REF] Hoenigl | Global guidelines and initiatives from the European Confederation of Medical Mycology to improve patient care and research worldwide: New leadership is about working together[END_REF][START_REF] Cornely | Improving outcome of fungal diseases -Guiding experts and patients towards excellence[END_REF] Species identification and

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. susceptibilities of all isolates are determined or confirmed using reference methods including mass spectrometry (MALDI-TOF MS/VITEK MS) and molecular methods (sequencing ITS, IGS or equivalent informative target). Mass-spectra and molecular data are analyzed using the MALDI-TOF libraries as well as sequencing databases such as MycoBank (http://www.mycobank.org/) and CBS-KNAW GenBank (http://www.westerdijkinstitute.nl/collections/). In addition, all isolates are stored in triplicate at -80°C for research exchange among CandiReg collaborators. Any such exchange is preceded by the contributor's approval. The only exception from this self-rule is, if a contributor cannot be reached despite all efforts. Results of these analyses are also included in CandiReg, while the isolate remains stored in that reference laboratory.

Case recruitment and data analyses and use

The main variables to be analyzed are: clinical course and features of ICI, diagnostic procedures performed to confirm the diagnosis, description of antifungal treatment regimens, guideline implementation and adherence and their efficacy and impact on patient survival. The aim is to analyze the efficacy of current recommendations for diagnosis and treatment, to inform future consensus guidelines and to develop clinical screening and diagnostic procedures.

Discussion and outlook

CandiReg serves as platform for international cooperation and studies on attributable mortality, Candida-reactive T cells, evaluations of guideline adherence [START_REF] Mellinghoff | Analyzing candidemia guideline adherence identifies opportunities for antifungal stewardship[END_REF][START_REF] Koehler | Candida-Reactive T Cells for the Diagnosis of Invasive Candida Infection-A Prospective Pilot Study[END_REF][25] , and recently the third multicenter ECMM study [START_REF] Klingspor | Invasive Candida infections in surgical patients in intensive care units: a prospective, multicentre survey initiated by the European Confederation of Medical Mycology (ECMM) (2006-2008)[END_REF][START_REF] Tortorano | Epidemiology of candidaemia in Europe: results of 28-month European Confederation of Medical Mycology (ECMM) hospital-based surveillance study[END_REF] on incremental costs associated with nosocomial invasive candidiasis in Europe, CANDIDA III was initiated taking advantage of this platform. The CANDIDA III study focuses on evaluating the attributable mortality and costs as well as diagnostic and therapeutic approaches (including prolonged hospital stay for completion of parenteral antifungal treatment) of nosocomial invasive candidiasis in Europe. As a secondary objective, it will evaluate the antifungal resistance among Candida spp. causing invasive disease across Europe. The study will use a matched case control design, which will allow the implementation of health economic analysis on the incremental costs associated with invasive Candida infections. The ECMM will use the platform for future multinational surveillance studies on invasive candidemia in Europe. Moreover, investigators maintain familiarity with the platform, which may enable more rapid case entry in case of an outbreak. To date, 265 patients have been enrolled in the register as part of five open or completed studies. [START_REF] Mellinghoff | Analyzing candidemia guideline adherence identifies opportunities for antifungal stewardship[END_REF][START_REF] Koehler | Candida-Reactive T Cells for the Diagnosis of Invasive Candida Infection-A Prospective Pilot Study[END_REF][25][START_REF] Koehler | Candida reactive T cells for the diagnosis of invasive Candida infection of the lumbar vertebral spine[END_REF] The results derived from this platform will be promoted as poster or oral presentations

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. at national and international infectious diseases, mycological and health-economic conferences.

Internationally visible publications obtained from the CandiReg registry have already been published in or will be submitted to peer-reviewed journals.

CandiReg will enhance knowledge about ICI and facilitate the analysis of epidemiological shifts and resistance trends. Currently, clinicians, microbiologists and researchers from 28 countries are involved (Figure 1). With this registry, we collect real life data with long-term observations. The registry will provide controlled or uncontrolled level II evidence and is ready in case of an outbreak situation similar to other FungiScope™ Registries. [START_REF] Seidel | FungiScope -Global Emerging Fungal Infection Registry[END_REF] There are a number of limitations of registry based studies. The retrospective acquisition of anonymized data of the individual patient reduces data quality, but is indispensable to protect privacy and comply with data protection guidelines. Also, the follow-up time of the cases may often be somewhat short. However, these registries offer the opportunity to contribute to progress in the understanding of epidemiology, pathophysiology, natural history and efficacy of treatments on a pan-European or even worldwide scale. As proven ICI is a relatively rare condition, pooling these cases and analyzing them in large cohorts can significantly improve our knowledge of ICI in real life settings and contribute to the design of clinical trials. Furthermore, they provide data unobtainable by controlled trials.

In conclusion, the CandiReg platform promotes international collaboration, increases the quality of available evidence on invasive Candida infection and can contribute to improvement of patient management. 23

CandiReg

  is approved by the local Institutional Review Board and Ethics Committee of the University Hospital Cologne (UHC) (Identifier of the University of Cologne Ethics Committee: 17-485). If needed, further local Ethics Committee approvals will be included. The study is registered at clinicaltrials.gov, identifier NCT03450005. CandiReg uses the General Data Protection Regulation (GDPR) compliant platform ClinicalSurveys.net. Data entry is carried out via any internet browser using encrypted communication. All documented data are automatically collected in a database. All Good Epidemiological Practice (GEP) requirements are met by the software.

Figure 1 .

 1 Figure 1. ECMM Member countries contributing to CandiReg, as of May 2019. The current 28 member countries are colored in blue.

Acknowledgments

The authors thank Susann Blossfeld and Joyce van den Boogaard for their administrative and technical assistance.

Axel Hamprecht has received lecture honoraria from Bruker Daltonics, BD and Thermo Fisher outside the submitted work Felix C. Koehler has received grants from the German Federal Ministry of Research and Education and non-financial support from Miltenyi Biotec GmbH, Bergisch Gladbach, Germany outside the submitted work. Oliver Kurzai has received honoraria from Basilea, BG Chemische Industrie, Heidelberg Engineering GmbH and research support from Astellas, Basilea, Gilead, MSD, Pfizer, Virotech and Fujifilm Wako outside the submitted work. Strategies, German Society for Internal Medicine (DGIM) and grants from Merck / MSD, Gilead, Pfizer, Astellas Pharma, Basilea, German Centre for Infection Research (DZIF), German Federal Ministry of Education and Research (BMBF) outside the submitted work. Alexandre Alanio has received research grants from Astellas, Gilead, Merck/MSD and received lecture honoraria from Astellas, Gilead outside the submitted work. Ana Alastruey-Izquierdo has received research grants or honoraria as a speaker or advisor from Gilead Sciences, MSD, Astellas, Pfizer, F2G, Amplix and Scynexis in the last five years outside the submitted work. Valentina Arsic Arsenijevic has participated in advisory boards and/or received research grants and speaker honoraria from from Pfizer; Astellas, Schering-Plough, MSD, and Gilead in the past five years outside the submitted work. Jean-Pierre Gangneux reports personal fees from Astellas Pharma , Gilead Sciences and Pfizer outside the submitted work. Neil A.R. Gow has received honoria for presentations and lectures from Gilead Sciences outside the submitted work. submitted work. Cornelia Lass-Flörl has received research grants from Astellas, Basilea, F2G, Gilead, is a consultant to Pfizer, Gilead and received lecture honoraria from Astellas, Basilea, Gilead, and Pfizer outside the submitted work. Emmanuel Roilides has received research grants from Pfizer, Merck Alida Fe Talento has received financial grants from Gilead Sciences Ltd., Pfizer Healthcare Ireland and MSD Ireland and received lecture honoraria from Gilead Sciences Ltd., and Pfizer Healthcare Ireland outside the submitted work. Anna Maria Tortorano received research funding from Gilead outside the submitted work. Paul E. Verweij has received research grants from F2G, Gilead Sciences, Merck/MSD, and Pfizer, is a consultant to F2G, and Scynexis, and received lecture honoraria from F2G, Gilead Sciences, Merck/MSD and Pfizer outside the submitted work. Martin Hoenigl received research funding by Gilead outside the submitted work. Oliver A. Cornely has received research grants from Actelion, Amplyx, Astellas, Basilea,

Funding Scheme

CandiReg is an academic project and funded by the European Confederation of Medical Mycology and the University of Cologne.

Author contributions

Philipp Koehler, Martin Hoenigl and Oliver A. Cornely conceived the project idea, drafted the manuscript, revised, discussed and approved the final manuscript. Maiken Cavling Arendrup, Sevtap Arikan-Akdagli, Matteo Bassetti, Stéphane Bretagne, Lena Klingspor, Katrien Lagrou, Jacques F. Meis, Riina Rautemaa-Richardson, Silke Schelenz, Axel Hamprecht, Felix C. Koehler, Oliver Kurzai, Jon Salmanton-Garcia, Jörg-Janne Vehreschild, Alexandre Alanio, Ana Alastruey-Izquierdo, Valentina Arsic Arsenijevic, Jean-Pierre Gangneux, Neil A.R. Gow, Suzana Hadina, Petr Hamal, Elizabeth Johnson, Nikolay Klimko, Cornelia Lass-Flörl, Mihai Mares, Volkan Özenci, Tamas Papp, Emmanuel Roilides, Raquel Sabino, Esther Segal, Alida Fe Talento, Anna Maria Tortorano and Paul E. Verweij revised, discussed and approved the final manuscript.

Conflicts of Interests

Philipp

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.


Charlotte Pronier

F Helene

D Celine

P Stanislas

Fabrice Carrat

PROF Vincent Thibault
email: vincent.thibault@chu-rennes.fr

Hélène Fontaine

Céline Dorival

Stanislas Pol

Genetic diversity of genotype 6 HCV infections in France: epidemiology and consequences for treatment strategy Running Title: Genotype 6 HCV diversity and treatment

Keywords:

Genetic diversity of genotype 6 HCV infections in France

epidemiology and consequences for treatment strategy

Introduction

Hepatitis C virus (HCV) is one of the major causative agents of chronic liver disease. Globally, in 2015, an estimated 71 million people were living with chronic HCV infection. The epidemic caused by HCV affects all regions, with major distribution differences between and within countries 1 . HCV is classified into at least seven major genotypes (GT1-7) further subdivided into at least 86 subtypes [START_REF] Smith | Expanded classification of hepatitis C virus into 7 genotypes and 67 subtypes: Updated criteria and genotype assignment web resource[END_REF] . A recent publication provides evidence for an additional eighth genotype [START_REF] Borgia | Identification of a novel hepatitis C virus genotype from Punjab, India: expanding classification of hepatitis C virus into 8 genotypes[END_REF] . HCV genotype 6 (GT6) is almost exclusively restricted to Southeast Asia (China, Vietnam, Cambodia and Laos) and it is occasionally found in migrant patients from endemic countries. Among the 7 HCV genotypes, GT6 exhibits the highest genetic diversity as assessed by intra-genotype pairwise distance and has been subdivided in at least 23 subtypes (a to w) [START_REF] Thong | Hepatitis C virus genotype 6: Virology, epidemiology, genetic variation and clinical implication[END_REF][START_REF] Li | An expanded taxonomy of hepatitis C virus genotype 6: Characterization of 22 new full-length viral genomes[END_REF] .

Only two studies based on different HCV infected patient populations evaluated the specific liver disease natural history induced by GT6 with divergent results [START_REF] Seto | Natural history of chronic hepatitis C: genotype 1 versus genotype 6[END_REF][START_REF] Lee | HCV genotype 6 increased the risk for hepatocellular carcinoma among Asian patients with liver cirrhosis[END_REF] . Seto et al., in a monocentric study from Hong-Kong, did not report any difference in the progression of the disease when comparing two groups of patients infected with either GT1 or GT6. By contrast, Lee et al. looking at Asian patients from the USA and Hong-Kong reported an increased risk of liver complications only in cirrhotic patients when compared to a GT1 infected control group. In summary, although based on few reports, GT6 infection natural history does not seem different to what observed with GT1.

Whatever the evolution of the disease, the current WHO strategy is however to eliminate HCV infection from the planet by 2030 and availability of highly potent direct acting antivirals (DAA) justify to treat all screened positive patients independently of the liver lesions 1 . Until recent availability of pan-genotypic antiviral drugs in developed countries, HCV genotype has been a key marker to guide the best treatment option [START_REF] Association | EASL Clinical Practice Guidelines: management of hepatitis C virus infection[END_REF] . Considering the high genetic heterogeneity within GT6, it should be pointed out that limited data are available on both in vitro and in vivo antiviral activity of DAAs on Accepted Article this genotype. Data are even scarcer when considering subtypes. Indeed, most data on DAA efficacy to date have been derived from clinical trials conducted in Western countries, where GT6-HCV is rare and only few data sets exist for countries such as Cambodia and Vietnam with higher GT6 prevalence. Few in vitro studies have clearly identified significant difference in DAA potency according to GT6 subtypes or even selection of resistance [START_REF] Cheng | In vitro antiviral activity and resistance profile characterization of the hepatitis C virus NS5a inhibitor ledipasvir[END_REF][START_REF] Pham | HCV genotype 6a escape from and resistance to velpatasvir, pibrentasvir, and sofosbuvir in robust infectious cell culture models[END_REF][START_REF] Mcphee | Impact of preexisting hepatitis c virus genotype 6 NS3, NS5A, and NS5B polymorphisms on the in vitro potency of direct-acting antiviral agents[END_REF][START_REF] Schnell | Hepatitis C virus genetic diversity by geographic region within genotype 1-6 subtypes among patients treated with glecaprevir and pibrentasvir[END_REF] . Importantly, while their data validate DAA activity on subtype 6a prototypes, efficacy on less frequent subtypes is missing despite great polymorphism on residues known to influence DAA efficacy [START_REF] Sorbo | Hepatitis C virus drug resistance associated substitutions and their clinical relevance: Update 2018[END_REF] . Thus, precise genetic characterization of HCV strains is useful to identify rare subtypes, likely those not included in large clinical trials, to detect clusters of transmission in high-risk groups or to identify polymorphisms associated with antiviral resistance.

GT6 infection rarely occurs in France and little is known about the mode of transmission of this genotype and its diversity in Western Europe. The prospective French ANRS CO22 Hepather cohort is a unique observational national multicenter setting to document, without bias, the distribution of GT6 infection in France and to study treatment response of GT6 infected patients. Through phylogenic analysis of NS3, NS5A and NS5B coding sequences, HCV-GT6 diversity was studied in patients included in the Hepather cohort and treatment efficacy was documented within each subtype.

Materials and methods

Study design and participants

The ANRS CO22 Hepather cohort ("Therapeutic options for hepatitis B and C: a French cohort") is a national multicenter prospective observational cohort study of patients with viral hepatitis B or C (this study is registered with ClinicalTrials.gov, number NCT01953458) (Refer to supplementary materials). We selected all patients with HCV genotype 6 infection who initiated treatment before December 2017 (n=38).

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HCV sequence amplification, sequencing and analysis

For all GT6 HCV infections identified in the database, HCV subtype was determined on a plasma sample drawn at inclusion. NS3, NS5A and NS5B regions were RT-PCR amplified and sequenced using previously described protocols [START_REF] Andre-Garnier | An NS5A single optimized method to determine genotype, subtype and resistance profiles of Hepatitis C strains[END_REF] . Each protein encoding sequences were aligned to HCV GT1-7 reference set downloaded from the ICTV website containing complete genomes of all subtypes described by Smith et al. and recently updated according to ICTV 2 . Based on each gene coding sequence alignments, phylogenic analyses were performed using MEGA 7.0 Software [START_REF] Kumar | MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets[END_REF] . Briefly, the evolutionary history was inferred using the Neighbor-Joining method, the evolutionary distances were computed using the Maximum Composite Likelihood method and tree robustness was assessed through 500 replicate bootstrap tests.

Protein residues known to impact drug sensitivity were reported (refer to supplementary materials).

Results

Patient characteristics

Data mining on Hepather database initially identified 38 cohort participants with GT6 infection. For 2 patients, HCV sequence determination revealed a misclassification and allowed for reclassification as genotype 1a and 3 (Fig. 1). Overall, 36 patients out of 14,603 (0.25%) were confirmed as infected with a GT6 virus (Table 1). Patients had a mean age of 59 years, 44.4% were male, most (33/36; 91.7%) were born in Southeast Asia (Cambodia, Vietnam, Laos) and the 3 remaining were European.

While transfusion could explain 16.7% of HCV transmission and injecting drug use, tattooing and piercing accounted each for 5.6% of the cases. The remaining routes of transmission were unknown, even though most patients reported longer periods than tourism travels in their country of birth. Noteworthy, 23 patients (64%) had an HBV serological profile indicating past HBV infection (anti-HBc positive). Cirrhotic patients represented 13 (36%) of the GT6 infected subjects and mild fibrosis (Metavir score  2) was documented for 16 patients (44%). Compared to the whole cohort, Accepted Article genotype 6 infected patients came more likely from Asia (p<0.0001), were more often treatmentnaïve (p=0.04) and had a higher viral load (p=0.0004).

HCV GT6 subtype distribution

Sequencing was successful for all NS3 and NS5A encoding regions while failure occurred for 7 (19%) NS5B genes (figure 1). Failure was attributed to non-optimal RT-PCR conditions and eventually lack of material to repeat the experiment. Phylogenic analysis allowed subtype determination for 32 (89%) strains while 4 sequences did not cluster with enough confidence into confirmed subtypes. Whatever analyzed gene, phylogenic analyses provided concordant classification into 10 different subtypes. A representative NS5A phylogenic tree is shown on figure 2. Among sequences that did not cluster with assigned subtypes, F4 presented similarities with JX183550 and C2 was close to KJ567649 and KJ567650. Strains A4 and H4 were more distant to any reference sequences, although some proximity was noticed with subtype 6o and 6q, respectively. The most prevalent subtypes were 6e (10; 27.8%), 6a (8; 22.2%), and 6q (4; 11.1%). Interestingly, among notified subtypes (n=20) as registered within the database, the initially determined subtype was only confirmed through sequencing for 9 (45%) samples.

Despite a large genotype diversity within each country, GT6q (n=4) was only found in patients from Cambodia where subtype 6e (n= 6; 37.5%) is also predominant. In patients from Vietnam, GT6a (n=6; 42.8%) was the most prevalent (Figure 3).

Polymorphism

GT6 polymorphisms were studied for critical residues (Resistance Associated Substitutions or RAS) known to confer resistance to currently available DAA (figure 4). For each viral target (NS3 and NS5A), a protein alignment of 76 reference sequences covering all GT6 subtypes was generated and key residues were compared to those most prevalent in genotype 1a strains. For the NS3 protein, positions 41, 43, 54, 55, 155, 156 were similar to the one found in GT1a and mostly conserved whatever the subtype (Fig. 4a). Positions 56 and 168 were also frequently conserved with only few

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sequences with a unique change either Y56F or D168E. More diversity was noted on residues 36, 80, 122 and 170, the highest number of different residues occurred on positions 36 and 80. While the most prevalent residues at RAS positions were identical to those of GT1a, position 122 was the less conserved (S122T/N).

For protein NS5A, residues 32 and 38 were identical to GT1a strain and residue 92 had the lowest variability (fig. 4b). By contrast, positions 28, 30, 58 and to a lower extent 24, 31 and 93 showed very frequent polymorphisms with up to 8 different variations for a unique position. The most prevalent changes were concomitantly found at residues M28V, H58P and Y93T. Noteworthy, S282T NS5B residue reported to confer decreased sensitivity to sofosbuvir was never found throughout analyzed Gt6-sequences and residues L320 and V321, possibly impacting sofosbuvir activity were also conserved throughout all sequences. Residue L159, located upstream, was not covered by our sequencing approach.

Real life treatment efficacy

As the cohort was initiated in 2012, several lines of treatment options have been recommended over the course of time. In our cohort, one-third (13/36) of the patients had received a previous treatment, either IFN-based for the majority (92.3%; n=12) or DAA-based for one patient (GZR/EBR) (Table 2). Among the patients who had received IFN, most (n=8; 66.7%) were relapsers and one was non-responder. Treatment was initiated for 30 patients after inclusion in the Hepather cohort, 18 were naïve and 12 were previously treated. Most patients (n= 27; 90%) received a SOF-containing DAA combination treatment, either associated with an anti-NS5A (n= 20; 66.7%), an anti-NS3 (n= 1; 3.3%) or both (n= 2; 6.7%). Sofosbuvir combined with IFN plus ribavirin was given to 4 patients and the remaining 3 patients received a Glecaprevir/Pibrentasvir combination. Median duration of DAA treatment was 12 weeks.

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All treated patients achieved SVR12. Among NS5A inhibitors, ledipasvir (n=11) was the most used, followed by daclatasvir (n=7), velpatasvir (n=4) and pibrentasvir (n=3). All cirrhotic patients, 7 treatment-experienced and 6 treatment naïve, were successfully treated. Noteworthy, no serious adverse event or treatment discontinuation was reported for treated patients even in patients with advanced liver fibrosis.

Discussion

Although awareness on hepatitis C infection as a preventable infectious disease is significantly decreasing its incidence worldwide, people who inject drugs (PWID) and migration fluxes from highly endemic countries remain major sources of new infections [START_REF] Bruggmann | Historical epidemiology of hepatitis C virus (HCV) in selected countries[END_REF] . As genotype 6 infected patients are rarely encountered in France, a systematic study based on the largest observational cohort in France (ANRS CO22 Hepather) was conducted to describe the characteristics of HCV-GT6 infected patients and the efficacy of new treatments on this specific population.

While recent DAA based treatment options appear fully active on circulating GT6 strains, a large diversity of GT6 viruses was characterized. This heterogeneity likely reflects imported cases from disparate sources, rather than infections acquired in France through one specific mode of transmission. The absence of clear transmission cluster within GT6 subtypes tends to support the hypothesis that most transmissions occurred in the country of origin rather than locally through common risk factors. Distribution of many subtypes in the Hepather cohort directly reflects the subtype distribution in the patient's country of origin [START_REF] Li | An expanded taxonomy of hepatitis C virus genotype 6: Characterization of 22 new full-length viral genomes[END_REF] . Indeed, all cases but 3 (8%) were born in GT6 endemic areas. Among the 3 patients potentially infected in France, one was through injection drug use, a second by possible nosocomial transmission and for the last one, no specific risk was found.

The identified strains for these 3 patients belonged to 2 different subtypes (o and a); the two 6o strains being not phylogenetically related. Noteworthy, beside geographic origins, Hepather GT6

Accepted Article

infected patients do not differ from those infected with other genotypes in terms of age, gender and liver disease.

The number of GT6 infected patients identified in the Hepather cohort is consistent with different prevalence studies performed in France, reporting around 0.2% of patients being infected by this genotype. As previously described, genotype characterization may sometimes be impaired by the methodological approach. Indeed, 2 out of the 38 (5.2%) patients were erroneously documented as genotype 6 infected, likely reflecting either a wrong input or a bias due to the genotyping technical approach, especially if targeting the 5'-non coding region [START_REF] Yang | Performance comparison of the Versant HCV genotype 2.0 assay (LiPA) and the Abbott Realtime HCV genotype II assay for detecting hepatitis C virus genotype 6[END_REF][START_REF] Larrat | Sequencing assays for failed genotyping with the versant hepatitis C virus genotype assay (LiPA), version 2.0[END_REF][START_REF] Némoz | Evaluation of the cobas® GT hepatitis C virus genotyping assay in G1-6 viruses including low viral loads and LiPA failures[END_REF] . Thus, the true prevalence of GT6 could potentially be slightly underestimated when the strains are misclassified as GT1 when using 5'-non coding region analysis. Using sequencing approaches based on more informative genomic regions should be promoted, particularly to document transmission in at-risk populations [START_REF] Chueca | Using NS5B sequencing for hepatitis C virus genotyping reveals discordances with commercial platforms[END_REF] . For all sequenced strains, concordant phylogenic analyses whatever the chosen region, NS3, NS5A or NS5B were obtained and most sequences clustered with GenBank available data. Routine sequencing analysis is also useful to document hypothetical new subtypes or baseline RAS that may impair treatment efficacy, particularly in difficult to treat patients. Four strains could indeed not be classified according to the most recent ICTV nomenclature and showed an important genetic distance from available classified sequences 2 . These strains presented no match either with the recently described GT6xg subtype [START_REF] Ye | Identification of a new HCV subtype 6xg among injection drug users in Kachin, Myanmar[END_REF] . Interestingly, most recent studies that reported gt6 subtype distribution from large clinical trial on new DAA did not report any "o" or "f" subtypes and no firm conclusion can be drawn from these trials on DAA efficacy on these less prevalent subtypes [START_REF] Boyd | HCV genotype 4, 5 and 6: Distribution of viral subtypes and sustained virologic response rates in clinical trials of approved direct-acting antiviral regimens[END_REF][START_REF] Hezode | Resistance analysis in patients with genotype 1-6 HCV infection treated with sofosbuvir/velpatasvir in the phase III studies[END_REF] .

Indeed, McPhee et al. report in vitro high level of resistance to velpatasvir in replicons carrying both

NS5A L28T/R30S RAS, a common motif found exclusively in subtype 6f and also found in one of our patients [START_REF] Mcphee | Impact of preexisting hepatitis c virus genotype 6 NS3, NS5A, and NS5B polymorphisms on the in vitro potency of direct-acting antiviral agents[END_REF] . We believe awareness should be promoted when treating uncommon subtypes that may not be as susceptible to the so-called "pan-genotypic" current combinations, even though current real-world efficacy data are very reassuring.

Accepted Article

Data on antiviral efficacy on GT6 natural strains are very scarce. Yet, the large heterogeneity of GT6 strains even at the DAA target protein level raises the possibility of reduced antiviral susceptibility compared to what observed on GT1 viruses. Indeed, an important polymorphism could be documented on selected reference sequences belonging to all GT6 subtypes at classical RAS positions (Figure 4). While results from large clinical trials that included few GT6 patients usually report good efficacy on this genotype, few studies, using mostly artificial chimeric constructs based on a GT6a backbone reported in vitro DAA activity. Regarding antiprotease activity, glecaprevir had similar antiviral activity compared to grazoprevir against GT 6a and 6e tested replicons, with EC 50 values of 0.86 and 0.21 nM, respectively; values similar to what observed for GT1a strains. However, selected glecaprevir RAS at position D168A/H/V reduced the susceptibility to glecaprevir by 38 to 146-fold in cells containing a genotype 6a replicon. In the same system, grazoprevir demonstrated also a 247 to 1,048-fold reduced activity [START_REF] Pham | HCV genotype 1-6 NS3 residue 80 substitutions impact protease inhibitor activity and promote viral escape[END_REF][START_REF] Jensen | Substitutions at NS3 Residue 155, 156, or 168 of Hepatitis C Virus Genotypes 2 to 6 Induce Complex Patterns of Protease Inhibitor Resistance[END_REF] . More recently, a publication looking at efficacy of a 12 week grazoprevir/elbasvir treatment, reported an SVR in only 62.9% (22/35) of a subgroup of GT6 patients, demonstrating the lack of power of this combination for this genotype [START_REF] George | Elbasvir/grazoprevir in Asia-Pacific/Russian participants with chronic hepatitis C virus genotype 1, 4, or 6 infection[END_REF] . Analysis of GT6 NS3 from different subtypes identified a rare D168E polymorphism not described as modifying anti-NS3 susceptibility and no such polymorphism was found on our patient sequences. Our findings and reports from the literature are thus quite reassuring in term of second generation anti-NS3 efficacy on all GT6 subtypes, with no evidence for polymorphism significantly modifying DAA potency [START_REF] Mcphee | Impact of preexisting hepatitis c virus genotype 6 NS3, NS5A, and NS5B polymorphisms on the in vitro potency of direct-acting antiviral agents[END_REF] .

Regarding anti-NS5A potency, daclatasvir was as effective in vitro against a GT6a hybrid replicon (EC 50 74 pM) as on a similar GT1a construct [START_REF] Wang | Comparison of daclatasvir resistance barriers on NS5A from hepatitis C virus genotypes 1 to 6: implications for cross-genotype activity[END_REF] . Except for P32L leading to a high level of resistance (EC 50 250 nM), the following RAS, Q24H, L31M, P32S and T58A/S are known to confer modest levels of resistance against daclatasvir (EC 50 2-44 nM). In our set, RAS L31M was identified in one case and T58A, contributing to lower treatment efficacy, in 5 patients. Interestingly, substitution T58A is found in all 6o subtypes from this study as in the included reference sequences and could therefore represent a specific protein signature for this subtype. However, it should be pointed out that patients presenting this T58A polymorphism and treated with a daclatasvir containing combination

Accepted Article

(n=3) did clear the virus. Ledipasvir has a potent in vitro antiviral activity against GT-1a replicons with EC 50 around 0.031 nM. However, on GT6, the study by Cheng et al. describes a comparatively lower activity of ledipasvir, with a decreasing efficacy for subtypes 6a (EC 50 of 1.1 nM) even more pronounced for GT-6e with an EC 50 of 264 nM [START_REF] Cheng | In vitro antiviral activity and resistance profile characterization of the hepatitis C virus NS5a inhibitor ledipasvir[END_REF] . Interestingly, 3 GT6e cirrhotic patients included in the Hepather cohort and treated with sofosbuvir-ledipasvir, achieved SVR. McPhee et al. also describe a double variant NS5A L28A/R30S with a more than 10,000 fold reduced sensitivity to ledipasvir [START_REF] Mcphee | Impact of preexisting hepatitis c virus genotype 6 NS3, NS5A, and NS5B polymorphisms on the in vitro potency of direct-acting antiviral agents[END_REF] . This motif was detected in one GT6f patient who had a sustained virological response to 3 months of SOF-LED + ribavirin combination treatment. Antiviral activity of pibrentasvir was unaffected against a panel of replicons integrating NS5A genes from infected patients with subtypes 6a, 6e or 6p (n=5) with an EC 50 around 0.7 pM, despite polymorphisms detected in some of these clinical samples [START_REF] Schnell | Hepatitis C virus genetic diversity by geographic region within genotype 1-6 subtypes among patients treated with glecaprevir and pibrentasvir[END_REF][START_REF] Ng | In vitro antiviral activity and resistance profile of the next-generation hepatitis C virus NS5a inhibitor pibrentasvir[END_REF] . By contrast, a slight difference in velpatasvir antiviral activity has been reported between 6a and 6e subtypes, with EC 50 of 6 and 130 pM respectively [START_REF] Cheng | GS-5816, a second generation HCV NS5a inhibitor with potent antiviral activity, broad genotypic coverage and a high resistance barrier[END_REF] . Moreover, a valine substitution at position L31 could also potentially affect velpatasvir activity on GT6a as recently demonstrated by Pham et al. [START_REF] Pham | HCV genotype 6a escape from and resistance to velpatasvir, pibrentasvir, and sofosbuvir in robust infectious cell culture models[END_REF] . Analyses performed on reference sequences and on sequences from our patients never identified such L31V polymorphism but the residue change L31M/I was found in 2 patients. Although exceptional, clinical sofosbuvir resistance involves the selection of a S282T RAS that leads to increased EC50 against the molecule but also major impairment of the virus replication fitness [START_REF] Xu | In vitro selection of resistance to sofosbuvir in HCV replicons of genotype 1 to 6[END_REF] . Whether on selected GT6 reference sequences or sequences generated from the Hepather patients, this RAS was never detected, suggesting that sofosbuvir constitutes a solid cornerstone for GT6 combination treatments. A recent single study, describing a S282T prevalence of 20.7% in a Chinese cohort infected with GT6a-HCV, should however raises our attention on the possible emergence of such variants [START_REF] Li | Naturally occurring resistance-associated variants to hepatitis C virus direct-acting antiviral agents in treatment-naive HCV genotype 6a-infected patients[END_REF] . Of note, no change was observed on residues L320 and V321, involved by some authors with decreased sensitivity to sofosbuvir. L159 was however not covered by our sequencing approach.

Accepted Article

Several conclusions can be drawn from our experience. First, the overall real life treatment efficacy was 100% in these GT6 infected patients, a higher rate than what was found in 3 studies using a SOF/LED combination approach [START_REF] Xu | In vitro selection of resistance to sofosbuvir in HCV replicons of genotype 1 to 6[END_REF][START_REF] Li | Naturally occurring resistance-associated variants to hepatitis C virus direct-acting antiviral agents in treatment-naive HCV genotype 6a-infected patients[END_REF][START_REF] Gane | Efficacy of ledipasvir and sofosbuvir, with or without ribavirin, for 12 weeks in patients with HCV genotype 3 or 6 infection[END_REF] . As this retrospective study is based on a prospective cohort, it was not possible to gather more details on viral kinetics or adverse events but the observed high SVR rate certainly validates the French successive updated recommendations to manage HCV infected patients throughout the inclusion period (2012-2017). Consequently, all patients did not receive the same combination strategy over time, which prevented us to better document treatment response according to well described SVR predictors. Second, the frequent polymorphism linked to subtype diversity had no effect on clinical treatment efficacy. This finding is quite surprising as several residue positions known to interfere with treatment efficacy in other genotypes were detected in a number of strains. As a note of caution, one should emphasize that all but one patient received at least 12 weeks of treatment. Only one patient received an 8-week combination of SOF-VEL-VOX. Twelveweek treatment duration is not anymore the standard of care and according to recent clinical trials, 8 weeks are probably enough to clear the virus in absence of cirrhosis for some combinations. Further studies are encouraged to document treatment efficacy in genotype 6 infected patients that will be eligible to 8 weeks treatment with pangenotypic combinations.

On a practical point of view, our study based on a large and diverse patient population conveys a very positive message regarding the efficacy of current treatment strategies to cure any GT6 subtype infection, at least with a 12-week regimen. Despite abundant polymorphisms on RAS, approved DAA combinations used according to current recommendations lead to very high rate of sustained viral response, 100% in our real-life cohort. With the recent availability of pangenotypic combinations, virus genotyping presents less clinical relevance in treatment naïve patients. Yet, documentation of RAS polymorphisms for rare genotypes could be relevant as little proof of evidence exists regarding efficacy of these new treatments on such strains. Moreover, sequence determination is useful to document cluster of transmissions, particularly in high-risk population. Our study demonstrates that Accepted Article most genotype 6 infections in France, also possibly in other European countries, does not result from transmissions at the country level but rather from imported cases. (plain triangles) were aligned and the evolutionary distances were computed using the maximum composite likelihood method. The evolutionary history was inferred using the Neighbor-Joining method and a bootstrap value of 500. The tree is drawn to scale indicated at the bottom left.

Accepted Article

Genotype 6 subtypes corresponding to Hepather sequences are circled and called when possible.

3 )Figure 1 :

 31 Figure 1: General study outline

Figure 2 :

 2 Figure 2: Genotype 6 NS5A coding sequence phylogenic tree

Figure 3 :

 3 Figure 3: genotype 6 subtype distribution according to country of birth. Each subtype is represented

Figure 4 :

 4 Figure 4: NS3(a) and NS5A(b) protein polymorphisms for common RAS are represented as Logo

Table 1 .

 1 Baseline characteristics of patients

	n (%)	(cohort-n=11,863)	P-value

Median RNA HCV inclusion (log IU/ml), [IQR]

	6.42 [0.81]	6.03 § [0.94]	0.0004
	†

missing in 51 patients, ‡ missing in 1,406 patients, § Missing in 906 patients Accepted ArticleTable 2 :

 2 subtype distribution and treatment regimen

		n (%)
	Determined subtype
	 6a	8 (22.2)
	 6e	10 (27.8)
	 6f	1 (2.8)
	 6h	1 (2.8)
	 6o	3 (8.3)
	 6p	2 (5.6)
	 6q	4 (11.1)
	 6r	1 (2.8)
	 6t	1 (2.8)
	 6xc	1 (2.8)
	 6?	4 (11.1)
	Treatment (n=30)
	 Interferon + ribavirin + Sofosbuvir	4 (13.

Funding

This work was supported by INSERM-ANRS (France REcherche Nord&sud Sida-vih Hepatites), ANR (Agence Nationale de la Recherche), DGS (Direction Générale de la Santé) and Gilead, Abbvie, MSD, Janssen, BMS, Roche.

Conflict of interest disclosure statement

H. Fontaine declares participation at therapeutic trials and personal fees from Roche, Janssen, Gilead, Bristol Myers Squibb, MSD and Abbvie S. Pol has received consulting and lecturing fees from Bristol-Myers Squibb, Janssen, Gilead, MSD, Abbvie and grants from Bristol-Myers Squibb, Gilead, Roche and MSD.

V. Thibault has received travel grants and lecturing fees from Bristol-Myers Squibb, Janssen, Gilead, MSD and Abbvie.

C. Pronier, C. Dorival and F. Carrat declare no conflict of interest for the presented work.


Aurélie Jayol

Nicolas Kieffer

Laurent Poirel

François Guérin

Deniz Güneser

Vincent Cattoir

Patrice Nordmann

Evaluation of the Rapid Polymyxin NP test and its industrial version for the detection of polymyxin-resistant Enterobacteriaceae

The commercial Rapid Polymyxin NP test was evaluated to detect colistin-resistant Enterobacteriaceae. A total of 223 enterobacterial isolates corresponding to 136 resistant (including 38 MCR-like producers), 19 heteroresistant, and 78 colistin-susceptible isolates were tested. The test was performed according to the manufacturer's instruction, and the color of the wells was read after 2 and 3 hours of incubation. The results were compared with those of the homemade Rapid Polymyxin NP test, and manual broth microdilution according to EUCAST guidelines was used as the reference method to determine the performance of the test. Excellent performance of the commercial Rapid Polymyxin NP test was found with a very major error rate, a major error rate, a sensitivity, and a specificity of 1.9%, 5.1%, 98.1%, and 94.9%, respectively. The performance of the homemade Polymyxin NP test was similar, with a slightly better value for the very major error (1.2 %).

Polymyxins represent one of the few remaining treatment options for treating carbapenemase-producing Enterobacteriaceae. The increasing use of polymyxins is now associated with increasing isolation of colistin-resistant isolates worldwide [START_REF] Poirel | Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes[END_REF]. Since the identification of the plasmid-mediated colistin resistance gene mcr-1, and then more recently of other MCR-like encoding genes, controlling the dissemination of colistin resistance has now become a major concern [START_REF] Poirel | Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes[END_REF]. Indeed, the fear is to isolate carbapenem-and polymyxin-resistant isolates, therefore exhibiting pandrug resistance. It becomes now mandatory to possess reliable and rapid diagnostic techniques for determining polymyxin susceptibility.

The broth microdilution (BMD) technique that is time-consuming (18 h) has been retained as the reference method for evaluating polymyxin susceptibility. A rapid test (Rapid Polymyxin NP test) has been recently developed to detect colistin resistance in Enterobacteriaceae within 2 hours (Nordmann et al., 2016a,b). This test is based on the detection of the glucose metabolism of Enterobacteriaceae upon culture and differentiates colistin-susceptible from colistin-resistant isolates by supplementing the growth medium with a given concentration of colistin. An industrial version of this test is now available (ELITechGroup, Puteaux, France). The objective of this study was to evaluate the performance of this industrial Rapid Polymyxin NP test to detect colistin resistance and heteroresistance in Enterobacteriaceae.

A collection of 233 enterobacterial isolates was tested using the homemade and the industrial Rapid Polymyxin NP test. It included 136 colistin-resistant, 19 colistin-heteroresistant [START_REF] Guerin | Cluster-dependent colistin hetero-resistance in Enterobacter cloacae complex[END_REF][START_REF] Hjort | Unstable tandem gene amplification generates heteroresistance (variation in resistance within a population) to colistin in Salmonella enterica[END_REF], and 78 colistin-susceptible isolates, belonging to various enterobacterial species. Among the 136 colistin-resistant isolates, 10 belonged to bacterial species intrinsically resistant to colistin, 120 had been characterized for their acquired mechanisms of resistance (chromosomally or plasmid encoded), and 6 isolates presented an unknown mechanism of resistance (Table 1). Overall, resistance mechanisms corresponded to substitutions or truncations in the PmrA/PmrB proteins, the PhoP/PhoQ regulatory proteins, and the MgrB protein. In addition, 38 of the resistant isolates produced the plasmid-mediated MCR-1, MCR-2, MCR-3, or MCR-4 determinants.

The homemade and industrial Polymyxin NP tests were both performed by using a 3-3.5 McFarland bacterial suspension into 0.9% NaCl as recommended (Nordmann et al., 2016a,b, and manufacturer guidelines). Results were interpreted by 2 independent readers after 2 and 3 hours of incubation at 37 °C. A positive test corresponded to an orange-to-yellow color change, whereas an orange color corresponded to a negative test.

⁎ Corresponding author. Tel.: +41-26-300-9582. E-mail address: laurent.poirel@unifr.ch (L. Poirel).

1

A c c e p t e d M a n u s c r i p t MICs of colistin were determined in parallel by BMD using cationadjusted Muller Hinton broth (Bio-Rad, Cressier, Switzerland), and Escherichia coli ATCC 25922 was used as control strain. In case of "skip wells" (likely indicating heteroresistance), MICs were determined by considering the well with the highest concentration in which growth was observed, regardless the clear wells [START_REF] Guerin | Cluster-dependent colistin hetero-resistance in Enterobacter cloacae complex[END_REF]. Isolates with MICs of colistin N2 μg/mL were considered as resistant, whereas isolates showing a MIC ≤2 μg/mL were considered as susceptible. Results of the 2 Polymyxin NP tests read after 3 hours of incubation were compared with those of the BMD. Errors were ranked as follows: very major error (VME) defined by isolates categorized as susceptible using the marketed panel but resistant by the BMD reference method (false-susceptibility result), and major error (ME) defined by isolates categorized as resistant using the marketed panel but susceptible by the BMD reference method (false-resistant result).

Among the susceptible isolates tested (n = 78), 4 ME (5.1%) were detected with both tests giving a specificity of 94.9% (74/78). Those false-positive results corresponded to isolates having MICs of colistin close to the breakpoint value (i.e., 1-2 μg/mL).

Among the resistant isolates (n = 155), 2 VME (1.2%) and 3 VME (1.9%) were detected with both the homemade and of the industrial Rapid Polymyxin NP tests, giving sensitivity rates of 98.7% (153/155) and 98.1% (152/155), respectively. Experiments were reproduced twice with distinct reading persons and results showed perfect agreement. A single E. coli with an unknown mechanism of resistance was not detected by using both tests. A single and 2 heteroresistant Enterobacter cloacae isolates were not detected with the homemade and of the industrial Rapid Polymyxin NP tests, respectively. Noteworthy, 3 heteroresistant E. cloacae isolates were detected after 2 hours of incubation with the homemade test, whereas they were detected after 3 hours of incubation with the industrial test. The performance of the homemade test was therefore quite equivalent to the industrial test for detection of colistin heteroresistance. Such detection is challenging because most of the routine methods (Etest, Vitek-2 automated system) give false-susceptible results for such isolates [START_REF] Poirel | Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes[END_REF]. Recently, an evaluation of the homemade Rapid Polymyxin NP test for detection of colistin heteroresistance in Enterobacter spp. was performed, using a collection of 25 isolates, and a low sensitivity (25%) was actually found [START_REF] Simar | Evaluation of the Rapid Polymyxin NP test for Polymyxin B resistance detection using Enterobacter cloacae and Enterobacter aerogenes isolates[END_REF]. Such difference of results remains unexplained, but it is worth highlighting that the strains tested were different. Further studies performed in different laboratories will be needed to evaluate the reliability of the Polymyxin NP tests to detect colistin heteroresistance.

Noteworthy, all the 38 isolates producing a plasmid-mediated colistin resistance determinant (MCR-1 to MCR-4) were perfectly detected by the 2 methods within 2 hours.

To conclude, the homemade and the industrial Polymyxin NP tests showed high sensitivity (98.1%-98.7%) and specificity (94.9%) for detection of colistin resistance in Enterobacteriaceae, regardless of the resistance mechanisms. Those tests therefore possess the potential to be reliable tools for screening colistin-resistant Enterobacteriaceae in clinical laboratories.

5

A c c e p t e d M a n u s c r i p t

Table 1

 1 Features of the tested strain collection and comparative results between the homemade and commercialized Rapid Polymyxin NP tests.

	Strains	Species	Homemade Rapid	Industrial Rapid	MIC of colistin	Resistance	Reference
			Polymyxin NP a	Polymyxin NP a	(μg/mL)	mechanisms
	Resistant isolates
	R1583	P. vulgaris	+	+		N16	Intrinsic	This study
	R2260	P. mirabilis	+	+		N16	Intrinsic	This study
	R2556	P. mirabilis	+	+		N16	Intrinsic	This study
	R1473	M. morganii	+	+		N16	Intrinsic	This study
	R1474	M. morganii	+	+		N16	Intrinsic	This study
	R1607	P. stuartii	+	+		N16	Intrinsic	This study
	R2593	P. rettgeri	+	+		N16	Intrinsic	This study
	R2594 R1278 R2480 FR-06 FR-07 FR-08 FR-09 FR-10 FR-11 FR-12 FR-13 FR-14 FR-15 FR-16 FR-17 FR-18 FR-19 FR-20 FR-22 FR-23 FR-24 FR-25 FR-26 FR-27 FR-28 FR-31 FR-32 FR-33 FR-34 FR-35 FR-36	P. rettgeri S. marcescens S. marcescens K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae	+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +	+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +	N16 N16 N16 32 32 128 32 16 32 16 32 32 16 128 N128 32 32 64 64 64 4 128 128 64 128 64 64 32 64 128 128 M a n u s c r i p t Intrinsic This study Intrinsic This study Intrinsic This study PmrA G53C (Nordmann et al., 2016a) PmrA G53S (Nordmann et al., 2016a) PmrA G53S (Nordmann et al., 2016a) PmrB L17Q (Nordmann et al., 2016a) PmrB T157P (Nordmann et al., 2016a) PmrB T157P (Nordmann et al., 2016a) PmrB T157P (Nordmann et al., 2016a) PmrB T157P (Nordmann et al., 2016a) PmrB T157P (Nordmann et al., 2016a) PmrB T157P (Nordmann et al., 2016a) PhoP D191Y and deletion of 25 nt (Nordmann et al., 2016a) PhoQ R16C (Nordmann et al., 2016a) MgrB W20R (Nordmann et al., 2016a) MgrB M27K (Nordmann et al., 2016a) MgrB C39Y (Nordmann et al., 2016a) MgrB I45T (Nordmann et al., 2016a) MgrB P46S (Nordmann et al., 2016a) MgrB W47R (Nordmann et al., 2016a) MgrB truncated (2 aa) (Nordmann et al., 2016a) MgrB truncated (2 aa) (Nordmann et al., 2016a) MgrB truncated (2 aa) (Nordmann et al., 2016a) MgrB truncated (27 aa) (Nordmann et al., 2016a) MgrB truncated (29 aa) (Nordmann et al., 2016a) MgrB truncated (29 aa) (Nordmann et al., 2016a) MgrB truncated (29 aa) (Nordmann et al., 2016a) MgrB truncated (29 aa) (Nordmann et al., 2016a) MgrB truncated (29 aa) (Nordmann et al., 2016a) MgrB truncated (29 aa) (Nordmann et al., 2016a)
	FR-37	K. pneumoniae	+	+		32	MgrB truncated (32 aa)	(Nordmann et al., 2016a)
	FR-38 FR-39 FR-40 FR-41 FR-42 FR-43 FR-44 FR-45 FR-46 FR-47 FR-48 FR-49 FR-50 FR-51 FR-52 FR-54 FR-56 FR-58 FR-62 FR-63 FR-67 FR-68 FR-69 FR-81 FR-84 FR-89 A c c e p t e d K. pneumoniae + + 32 K. pneumoniae + + 6 4 K. pneumoniae + + 6 4 K. pneumoniae + + 3 2 K. pneumoniae + + N128 K. pneumoniae + + 6 4 K. pneumoniae + + 6 4 K. pneumoniae + + 128 K. pneumoniae + + 6 4 K. pneumoniae + + 6 4 K. pneumoniae + + 128 K. pneumoniae + + 6 4 K. pneumoniae + + 1 6 K. pneumoniae + + 6 4 K. pneumoniae + + 128 K. pneumoniae + + 128 K. pneumoniae + + 6 4 K. pneumoniae + + 3 2 K. pneumoniae + + 6 4 K. pneumoniae + + 128 K. pneumoniae + + 128 K. pneumoniae + + 6 4 K. pneumoniae + + 128 K. pneumoniae + + 64 K. pneumoniae + + 128 K. pneumoniae + + N128	MgrB truncated (46 aa) mgrB truncated by IS1R mgrB truncated by ISEcp1/bla CTX-M-15 mgrB truncated by IS102-like mgrB truncated by IS102-like mgrB truncated by IS903b mgrB truncated by IS2 mgrB truncated by IS1R mgrB truncated by IS1R mgrB truncated by IS903b-like mgrB truncated by IS903-like mgrB truncated by IS5-like mgrB truncated by IS5-like mgrB truncated by IS5-like mgrB truncated by IS5-like mgrB truncated by ISKpn13 mgrB truncated by ISKpn26-like mgrB truncated by ISKpn26-like mgrB truncated by IS903b mgrB truncated by IS1R-like mgrB truncated by ISKpn26-like mgrB truncated by ISKpn14 mgrB truncated by IS1R Full mgrB gene deletion Deletion nt 70 mgrB and substitution nt 73 Deletion nt 23 to 33 mgrB	(Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a) (Nordmann et al., 2016a)
	FR-90	K. pneumoniae	+	+		64	Deletion nt 30 et 31 mgrB	(Nordmann et al., 2016a)
	FR-87	K. pneumoniae	+	+		16	Deletion nt 100 mgrB	(Nordmann et al., 2016a)
	FR-30	K. pneumoniae	+	+		N128	MgrB truncated (27 aa)	(Nordmann et al., 2016a)
	FR-53	K. pneumoniae	+	+		6 4	mgrB truncated by IS5-like	(Nordmann et al., 2016a)
	FR-55	K. pneumoniae	+	+		N 128	mgrB truncated by ISKpn26-like	(Nordmann et al., 2016a)
	FR-57	K. pneumoniae	+	+		128	mgrB truncated by ISKpn26-like	(Nordmann et al., 2016a)
	FR-59	K. pneumoniae	+	+		3 2	mgrB truncated by IS903B	(Nordmann et al., 2016a)
								(continued on next page)
						2

Table 1

 1 (continued)

	Strains	Species	Homemade Rapid	Industrial Rapid	MIC of colistin	Resistance	Reference
			Polymyxin NP a	Polymyxin NP a	(μg/mL)	mechanisms
	R281	E. asburiae	-	-	b1	NA	This study
	R1414	C. freundii	-	-	b1	NA	This study
	R1415	C. freundii	-	-	b1	NA	This study
	R1416	C. freundii	-	-	b1	NA	This study
	R1527	S. Concord	-	-	b1	NA	This study
	R1528	Salmonella spp.	-	-	b1	NA	This study
	R1529	S. Isangi	-	-	b1	NA	This study
	C362	K. pneumoniae	-	-	0.125	NA	This study
	C367	K. pneumoniae	-	-	0.125	NA	This study
	C370	K. pneumoniae	-	-	0.125	NA	This study
	C382	K. pneumoniae	-	-	0.125	NA	This study
	C383	K. pneumoniae	-	-	0.125	NA	This study
	C609	K. pneumoniae	-	-	0.125	NA	This study
	C643	K. pneumoniae	-	-	0.125	NA	This study

a Isolates with discordant results are shaded.

Acknowledgments

This work has been funded by the University of Fribourg and by the Swiss National Reference Center for Emerging Antibiotic Resistance, Fribourg, Switzerland. It has also been funded by the Swiss National Science Foundation (projects FNS-407240_177381 and FNS-407240_177382). We thank the following colleagues who provided colistin-resistant isolates: A. Brink, J.W. Decousser, J. Fernandez-Dominguez, J.Y. Madec, M.V. Villegas, M. Yilmaz, D.I. Andersson, and H. Nicoloff.

Conflict of Interest

None to declare.


Cristina Rivera

Caroline G L Cao

TOWARD IMPROVED COMMUNICATION IN LAPAROSCOPIC SURGERY: ACCOUNTING FOR MULTIPLE FRAMES OF REFERENCE AND MENTAL ROTATIONS

This work is an examination of barriers to communication between the attending and assisting surgeons during laparoscopic surgery, where the same image of the surgical site is viewed from different vantage points with respect to the patient. Part of the problem lies with the multiple frames of reference each surgeon holds, and the mental rotations each must perform to construct a common frame of reference for communication and collaborative work. An experiment was conducted to demonstrate the effects of display-control incongruency on the performance of an aiming task in a simulated laparoscopic environment. Aiming performance was best when the camera was oriented at 0° perspective and worsened as the angle of deviation from 0° increased. Performance was affected to a greater degree by viewing perspectives from the left of the subject than viewing perspectives from the right. Results also suggest that when surgeons are facing each other, as is the case in many laparoscopic surgeries, one surgeon's performance will be worse than the other's. The mismatched display-control perspectives are compounded by ambiguous spatial references in verbal communication. From these findings, a case can be made for the importance of vocabulary that forces a common frame of reference during laparoscopic surgery.

INTRODUCTION

In laparoscopic surgery, the surgeon does not have a direct view of the abdominal cavity, but rather an indirect view provided by a small camera inserted into the area of interest through a small incision. The camera view is displayed on one or more monitors in the operating room. Other incisions allow for the insertion of long tools to perform the surgical tasks. In most cases there are at least two surgeons working in collaboration: the "attending" who takes primary responsibility for the case, and one or more "assisting" surgeons. Sometimes the attending surgeon is an experienced surgeon, while the assisting surgeon is a novice surgeon who requires guidance from the attending throughout the procedure.

Effective communication between the attending and the assisting in these scenarios is paramount, both for the training of the novice surgeon, and for the success of the collaboration and the surgery. Verbal instruction is vital because it is the main mechanism attendings have to convey what they would like help with during the procedure. They may gesture with their hands or tools, and/or point to the monitor. However, there are certain instructions that simply must be given verbally. The quality of instruction directly affects the quality of an assisting surgeon's performance. Misunderstandings slow things down because assisting surgeons have to double-check on intentions and required actions.

PROBLEM

In laparoscopic surgery, a common misunderstanding during collaboration relates to the spatial orientation of the surgical tools or targets. This is because the display space and the workspace are separated. Due to this control-display separation, surgeons must perform mental rotations to match what they see on the screen to what they do at the site. Further, although they often have the same orientations with respect to the monitors, surgeons often have different perspectives with respect to the patient. This means that each surgeon may be required to perform additional mental rotations in order to arrive at a common frame of reference to communicate with one another. Because laparoscopic surgery imposes this higher cognitive workload on the surgeons than open surgery, it can lead to confusion and degradation in communication. While there are other factors, such as authority and stress, affecting communication between surgeons, this paper is limited to how communication is affected by the issues surrounding mental rotations and frames of reference. We describe the first in a series of experiments to investigate this problem.

Frames of reference

The idea that mismatched frames of reference (FOR) degrade navigation performance is one already proven in the field of aviation (for review, see [START_REF] Gugerty | Reference-Frame Misalignment and Cardinal Direction Judgements: Group Differences and Strategies[END_REF][START_REF] Aretz | The Design of Electronic Map Displays[END_REF]. Since many analogies can be drawn between conventional geographic navigation and navigation within the human body, it is safe to expect that FOR coping strategies in conventional navigation can help us understand FOR problems in surgery.

Logically, pilots use different vocabularies for different FOR. Aretz pointed out in his work on map displays that when pilot's FOR were egocentric, they used clock directions to describe locations. If, however, they were using a world-centered FOR, which was necessary to communicate effectively with the air traffic controllers, they used compass headings [START_REF] Aretz | The Design of Electronic Map Displays[END_REF].

As long as pilots use these vocabularies, it eliminates the question of which FOR is being employed and therefore the possibility of confusion.

Similarly, laparoscopic procedures involve more than one possible frame of reference (i.e., egocentric, or exocentric). Surgeons also have certain conventions which help eliminate ambiguity. However, visits to the OR and interviews with surgical residents have shown us that they tend to mix these vocabularies with other ways of speaking to indicate directions. For example, "cephalad" and "lateral" are sometimes used to indicate direction toward the head and side of the body, respectively. "Up" and "towards you" (or towards some other object) are also used to indicate these same directions. It is not clear how a surgeon chooses the directional labels.

Shepard and Hurwitz pointed out that the "perceptual availability" of FOR vary (1985). For example, humans are accustomed to functioning in a gravitational field which always helps them distinguish up from down. This distinction is available whether they hold an egocentric or exocentric reference frame. In this way, the perceptual availability of the egocentric and exocentric FOR are similar. This contrasts with the left-right directions, which vary depending on the reference frame (e.g. Do you mean my right or your right?) [START_REF] Shepard | Upward direction, mental rotation, and discrimination of left and right turns in maps[END_REF]. Similarly, the "availability" of the frame of reference (e.g., what makes the most sense to the speaker) may determine a surgeon's choice of words. The fact that humans can hold different frames of reference simultaneously can exacerbate the problem [START_REF] Lemay | Pointing to an Allocentric and Egocentric Remembered Target[END_REF].

Mental rotations

Contrary to the direct viewing available in open surgery, laparoscopic surgeons must deal with the frequent mismatch between the orientation of the view of the surgical site and the orientation of the physical surgical site with respect to the surgeon. The need to perform mental rotations in laparoscopic surgery is due primarily to the use of a laparoscope. When inserted into the abdominal cavity, it can be angled and rotated, with the insertion point acting as a fulcrum. The camera is often moved several times during a procedure. Therefore, surgeons must sometimes deal with a rotated view on the monitor, as well as non-canonical view when the camera is moved about its fulcrum.

It is useful then to review what is already known about visuomotor transformations in the context of surgery. We know that individuals vary in their natural spatial ability. Keehner et al. found that having less natural spatial ability affected novice laparoscopic surgeons more than experts [START_REF] Keehner | Spatial ability, experience, and skill in laparoscopic surgery[END_REF]. Since experienced surgeons perform more automatically, they may have forgotten what it was like to perform surgery when they relied more on their natural spatial talents. In other words, it is harder for the teachers to tailor the instructions when they can't remember the difficulties in learning as a student. Several studies have been conducted to better understand the visual spatial talents of surgeons. A study by Risucci suggested that these talents were higher among surgeons than the average population (2002). Further, Keehner presented results which suggested that once individuals self selected into a career in surgery, there was no difference in spatial ability between experienced and novice laparoscopic surgeons (2004).

Cooper's work on mental rotations showed that if an object was presented in an unexpected way, people do one of two things: 1) Rotate it in their heads to the familiar way or 2) Rotate the world around it until it matches their familiar orientation (1976). The question that follows from this is exactly how the mental process works for surgeons. It is possible that surgeons perform mental rotations in one of three ways: 1) They train themselves to be in a state of permanent mental rotation while performing surgical tasks; 2) They discretely perform rotations as needed; 3) They perform mental rotations as a combination of the first two -dual processing. Dual processing theory may be the most probable explanation for the problems associated with performing complex mental rotations [START_REF] Barrett | Individual Differences in Working Memory Capacity and Dual -Process Theories of the Mind[END_REF]). The interplay between the automatic and the controlled process may be the source of confusion when surgeons are performing mental rotations and trying to maintain the correct frame of reference at the same time.

Research in aviation provides insight into how human operators deal with mental rotation tasks. Certain aircraft provide pilots with the ability to select the orientation of the map displayed to them (e.g. track up vs. north up) as they fly. The problem with this, Aretz stated, is that after they have chosen an alignment, a stressful situation could cause them to forget this alignment and set a course in the wrong direction, believing the map to still be in the previous alignment (1991). If we think of the laparoscopic monitor as analogous to the map and a rotation of the camera as the selection of map orientation, we can expect a novice surgeon may forget this rotation and move his tool toward the wrong tissue or organ. This problem can manifest itself in other ways as well. For example, if a teaching surgeon indicated a particular perspective, and that perspective required a mental rotation in order for the student to understand what to do, the student would make the rotation. If while the student was in the process of making this rotation, he became distracted, he may forget which perspective he was trying to match. The student may then be forced to revert back to some more comfortable orientation while deciding how to proceed. In order to avoid re-orienting each time, he may store up each rotation in memory and call it up each time the frame of reference is changed. The question becomes what are these surgeons mental processes and how do they affect communication.

It was hypothesized that mental rotations in laparoscopic surgery degrade surgical skills performance and communication. In particular, we hypothesized that performance varies depending on the perspective and rotation of the laparoscope. To test this hypothesis, an experiment was conducted.

METHODS

Subjects. Thirty undergraduate students from Tufts

University participated this study. None had any previous experience with laparoscopic surgery.

Apparatus. A Simulab endoscopic training box was used to simulate the abdominal cavity of a patient. The task space consisted of a 4x4 inch wooden block with six nails of varying vertical heights arranged in a circle equidistant apart. The nails were numbered 1-6. On the underside of the block, wires connected each of the nails to an RCX module. Wires were also attached to the grasping part of an Endo Clinch II laparoscopic grasper and connected to the RCX. In this way, a circuit was completed each time the subject touched the grasper tip to a nail. An RCX program was created to record the time of contact (i.e., states of open or closed circuit). A 0° endoscope was used to project the image of the task space to a TV monitor that was at the subject's eye level.

Tasks. Using the grasper, subjects were asked to touch the nails in sequence as quickly and accurately as possible, under various viewing conditions.

Experimental Design. A between-subjects design was used, with 3 groups, each with 2 opposing camera perspectives (see Figure 1) and 6 camera rotations. Each group consisted of 10 subjects; each subject performed the aiming task three times under each viewing condition. The camera perspectives were counter-balanced, while the camera rotation conditions were randomized.

PERSPECTIVE

Group 1

Group 2 Group 3

90° 45° 0° -45° ROTATION -90° Figure 1. Camera perspectives.
Dependent Measures. Dwell time, movement time, and total time were measured. Dwell time was the amount of time subjects spent in contact with the nails; movement time was the amount of time they spent traveling between nails; and total time was the sum of the dwell and movement times, or the task completion time.

Analysis. ANOVAs were performed using SPSS software for the three groups of subjects with an alpha value of 0.05. Analyses were done on dwell, movement, and total times, respectively.

RESULTS AND DISCUSSION

There were significant main effects for perspective (Dwell time: p<.001, Movement time: p <.001 Total time: p <.001) (see Figure 2). Significant main effects were also found for rotation (Dwell: p<.001, Movement: p <.001, Total: p <.001). There were significant interaction effects for the two dependent variables (Dwell: p< .001, Movement: p<.001, Total: p<.001).

As expected, subjects performed best when the camera perspective was at 0° or 180° on dwell, movement, and total time. For all three time classifications, post-hoc tests showed no differences between a 45° and a -45° perspective, collapsed over camera rotations. However, there was a difference between the 90° and -90° perspectives (Dwell: p = .01, Movement: p = .02, Total: p = .02) and 0° and 180° perspectives (Dwell: p = .04, Movement: p < .001, Total: p = p < .001). Post-hoc tests revealed only one difference for rotation angles, collapsed over perspectives. The movement time was longer when the camera was rotated at -90° vs. 45°.

Figure 3 shows the graphs of movement time for individual camera perspectives as a function of camera rotations. Only movement times are shown here, although similar patterns were found for dwell time and total time.

When the camera was at 0°, directed at the workspace from the subject's viewing perspective, the performance was most stable, even across rotations. As the camera was deviated from this canonical perspective, performance began to fluctuate. At 45° and 90° perspectives, performance was similar across rotations, suggesting that these two angles were perceptually equivalent. As the camera is moved further to the right of the subject, at 180°, performance worsens. Performance at -45° and 180° are mirror images of one another. This would seem to suggest that camera deviations to the left of the subject are more detrimental to performance than deviations to the right. Deviation of 45° to the left is equivalent to the distortion caused by 180° to the right. If we think of the perspectives in pairs of opposing angles, the results become extremely useful. In the case of 90° and -90°, they represent perspectives of two different surgeons standing on opposite sides of the patient (see Figure 4). Similarly for 0° and 180°, the camera may be directed along the patient's medial-lateral axis as in Figure 5. Figure 5. Scenario of 0° and 180° perspectives for two surgeons.

If the surgeon and assisting surgeon stood on opposite sides of a patient in these scenarios, as they do in many cases, our results suggest that one of these surgeons will always be at a disadvantage. This mismatch in performance has implications for communication. If one surgeon has the more intuitive scenario, it may be difficult for him to understand the other surgeon's situation in order to provide effective directional information. Schober showed that in situations where the people performing a task did not share the same point of view, the person giving instructions tended to give them from the other person's perspective (1995). It is expected that that this sympathetic behavior is more difficult in the laparoscopic surgery environment.

Thus far, our results support the idea that laparoscopic surgery poses different challenges to different surgeons even as they work on the same case. Add to this disparity differences in experience, spatial ability, and communication styles, and it is easy to see why frustration, misunderstandings, and errors can arise in the operating room. Our next step is to use what we have learned about the differences in perspective to investigate verbal communication of directional information in the surgical environment. We will then impose a standard structure to the vocabulary found in the OR to minimize effort and maximize performance in surgeon's collaboration. It is expected that the end result of this work will help surgeons communicate more effectively in laparoscopic surgery.

  Figure 2. Dwell, movement, and total times as a function of camera perspectives.

Figure 3 .

 3 Figure 3. Movement time as a function of camera rotations for different camera perspectives.

Figure 4 .

 4 Figure 4. Scenario of 90° and -90° perspectives for two surgeons.

ACKNOWLEDGMENTS

This work was funded by the National Science Foundation under grant #0238284. The authors acknowledge A. Kogan, N. Nolan, and J. Webster for building the testing apparatus and for data collection.