Dr Paul Cottu
email: paul.cottu@curie.fr

Bruno Coudert
email: bcoudert@cgfl.fr

David Perol
email: david.perol@lyon.unicancer.fr

Anne Doly
email: anne.doly@gmail.com

Julien Manson
email: julien.manson@ght-novo.fr

Olivier Aujoulat
email: aujoulato@ghrmsa.fr

Hugues Barletta
email: dr.barletta@orange.fr

Nassera Chalabi
email: nassera.chalabi@roche.com

Laurence Samelson
email: laurence.samelon@roche.com

Xavier Pivot
email: xpivot@strasbourg.unicancer.fr

with breast cancer having received human epidermal growth factor receptor 2-targeted treatments: Results from the french personalized reimbursement model database (2011-2018)

Keywords: breast cancer, HER2, retrospective real world data study, pertuzumab, trastuzumab, trastuzumab emtansine

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

Introduction

The human epidermal growth factor receptor 2 (HER2) gene is amplified, overexpressed, or both in approximately 15% of breast cancers (BC), and is associated with aggressive disease [START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene[END_REF][START_REF] Dawood | Prognosis of women with metastatic breast cancer by HER2 status and trastuzumab treatment: an institutionalbased review[END_REF]. The first approved HER2-targeted therapy (trastuzumab, Herceptin®, a humanized monoclonal antibody that targets the extracellular domain of the HER2 receptor) has proven an outstanding clinical benefit in both advanced and early disease when combined with cytotoxic chemotherapy [START_REF] Marty | Randomized phase II trial of the efficacy and safety of trastuzumab combined with docetaxel in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer administered as first-line treatment: the M77001 study group[END_REF][START_REF] Slamon | Adjuvant trastuzumab in HER2-positive breast cancer[END_REF][START_REF] Cameron | 11 years' follow-up of trastuzumab after adjuvant chemotherapy in HER2-positive early breast cancer: final analysis of the HERceptin Adjuvant (HERA) trial[END_REF]. However, patients with HER2+ BC may still experience relapse and/or progressive disease, and new targeted therapies, i.e. pertuzumab [START_REF] Baselga | Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer[END_REF][START_REF] Swain | Pertuzumab, trastuzumab, and docetaxel in HER2-positive metastatic breast cancer[END_REF] and trastuzumab emtansine (T-DM1) [START_REF] Verma | Trastuzumab emtansine for HER2-positive advanced breast cancer[END_REF], were developed to improve patient outcomes in first-and second-line advanced or metastatic setting (mBC).

These recent changes in HER2-targeted therapies have led to a refinement of treatment strategies in HER2+ BC. Recent international guidelines have established the level of evidence and recommendations for use of new intravenous and oral HER2 targeted therapies in mBC [START_REF] Cardoso | 4th ESO-ESMO International Consensus Guidelines for Advanced Breast Cancer (ABC 4) †[END_REF], and have strengthened the recommendations for HER2+ eBC [START_REF] Burstein | Estimating the benefits of therapy for early-stage breast cancer: the St. Gallen International Consensus Guidelines for the primary therapy of early breast cancer 2019[END_REF]. Within this context, there is a real need for real-world data accurately describing the use of these treatments and its evolution over time, as well as compliance with current recommendations.

Two large ongoing French real-world studies aim to fill this gap: the Epidemiological Strategy and Medical Economics (ESME) research program compiling data on a retrospective then prospective basis in mBC patients since 2014 [START_REF] Pérol | The ongoing French metastatic breast cancer (MBC) cohort: the example-based methodology of the Epidemiological Strategy and Medical Economics (ESME)[END_REF], and the prospective 10-year CANTO (CANcer TOxicities) study focusing on the toxicity of drug and non-drug cancer therapies in patients with early BC stage (eBC) [START_REF] Vaz-Luis | UNICANCER: French prospective cohort study of treatment-related chronic toxicity in women with localised breast cancer (CANTO)[END_REF]. However, both studies are conducted mainly in comprehensive cancer centers and focus on either mBC or eBC, but not both. Within this context, the Personalized Reimbursement Model (PRM) nationwide French project involving 132 French hospitals (including cancer centers, university and community public and nonpublic hospitals) was developed by Roche, in order to generate consistent real-world data over time in patients with early or advanced breast cancer treated with trastuzumab, pertuzumab or T-DM1, and to ultimately propose new reimbursement models. The present report describes the 2011-2018 evolution of the hospital use of these drugs in HER2+ BC patients according to BC stage and metastatic treatment line.

Patients and methods

PRM is a retrospective, non-interventional study based on secondary use of patient data, conducted in French public and non-public hospitals usually dealing with patients with HER2+ BC and exposed to a treatment including trastuzumab, pertuzumab, or T-DM1 at least once since January 2011. According to French legislation, patients received written information about the study. All included patients had no objection to sharing their data. Only anonymized data were used for analysis.

Procedures

Retrospective data on eligible patients were extracted two times a year from the chemotherapy Electronic Pharmacy Record (EPR) systems of participating centers, which include all patients exposed to injectable cancer treatments. Within ~300 French centers treating BC patients [START_REF] Slamon | Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene[END_REF], random recruitment of 132 hospitals was performed after double stratification including types of hospitals (university hospital, community hospital, comprehensive cancer center, and private clinic) and French regions (N=6). Among these 132 hospitals, 120 centers participated in the present study. Eligible patients were women having received anti-HER2 therapy (princeps drugs trastuzumab, pertuzumab or T-DM1) at least once between January 1 st , 2011 and September 30 th , 2018. Use of biosimilars was not recorded per protocol. Data collection was securely performed by an health data accredited hosting provider, from hospital EPR systems in which useful data are already available. No study-related involvement from pharmacists, physicians or study nurses was necessary at any time during data extraction process.

After extraction and anonymization, data from non-eligible patients were removed from the analysis, as well as data from patients with unavailable or inconsistent BC stage(s) over time.

More specifically, the different steps between data transmission by hospitals and definition of the PRM analysis population were as follows. After files with raw data sent by the participating centers had been integrated into a dedicated database without any transformation (source files), a first data mapping was built for standardization of data formats (dates and numeric or text fields). Diagnoses, disease stages, treatments and treatment lines were then extracted and coded for each captured administration. Filters were then applied to identify inconsistent or out of scope data (patients who did not met PRM selection criteria, inadequate data such as non-chemotherapy treatments, duplicates or lack of treatment dose) before defining the eligible patient population. Using algorithms and rules validated by the independent PRM scientific committee, last controls aimed to complete disease stages on the basis of stages identified before and/or after the time of the missing information, to assign disease stages using therapeutic patterns prescribed (standard protocols for early or advanced breast cancer, immunotherapy given for metastatic disease…), and to finally validate sequential treatment lines (i.e treatment sequences). Once these rules and algorithms were applied, the last patients with missing/ incomplete/ inconsistent disease stage were excluded before defining the PRM analysis population.

All results reported here are based on the data extraction performed in September 2018.

Roche had access only to aggregated results.

Assessments

Extracted data included demographic, clinical patient characteristics (year of birth, height and weight), disease stage assessment, treatments received for eBC and mBC (treatment line, systemic and anti-HER2 treatments, dates of administration and doses), and participation in an interventional study assessing injectable cancer treatments.

Statistical methods

The representativeness of the 132 initial participating centers was ensured, overall and according to center type and geographical location, on the basis of data using the national public hospital database (https://www.atih.sante.fr/; see Supplementary material, Table S1).

Among the 60,204 BC patients with at least one infusion of chemotherapy in French hospitals in 2017 (at project implementation), 30,009 patients (49.8%) were treated in the PRM centers including 10,211 patients treated at least with an anti-HER2 drug, confirming the good representativeness of our sample.

Descriptive analyses were performed on the use of targeted treatments, including trastuzumab, pertuzumab, and T-DM1 (evolution of the use of treatments, combined therapies, and treatment duration), in the overall analysis population, and according to disease stage and treatment line. For pertuzumab and T-DM1, treatment use was analyzed according to three periods: 2011-2013 (before both treatments were available), 2014-2016 (after approval), and 2017-2018 (current period). We also analyzed the treatment duration of pertuzumab and T-DM1 according selection criteria closed to those used in CLEOPATRA and EMILIA studies in order to compare data between real-life and clinical trials data [START_REF] Baselga | Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer[END_REF][START_REF] Verma | Trastuzumab emtansine for HER2-positive advanced breast cancer[END_REF]. Successive treatment lines were also described.

Analyses were carried out using Apache Spark 2.1.0, PostgreSQL 8.5.6, and Python 2.7.5 softwares.

Results

As of September 30, 2018, data from 26,350 patients were extracted from the EPR systems of 120 centers and 21,119 of them (80%) with available and consistent BC stage(s) over time were retained for analysis.

Stages of breast cancer and treatment lines

Over the 2011-2018 period, 27,428 treatment sequences were documented for the 21,119 analyzed patients. From 2011, 16,398 (77.7%) patients (median age: 56 years; range: 21-99) received injectable treatment(s) for eBC (Figure 1). Taking into account that a single patient could present with early and then metastatic disease stages between 2011 and 2018, 6,030 (28.6%) patients were treated at least once for mBC during this period: 4,841 (80.3%) of them in first-line, and/or 2,471 (40.1%) in second-line, and/or 1,707 (28.3%) in at least third-line.

At the start of these metastatic treatment lines, the respective median ages of patients were similar: 59 years (range: 49-69), 60 years (range: 50-68), and 59 years (range: 50-62), respectively.

Treatments at early stage of breast cancer

Between 2011 and 2018, trastuzumab was combined with at least one taxane for eBC in 14,634/16,398 (89.2%) patients (with both taxane and anthracycline: 10,262/16,398, 62.6%) (Figure 2

First line therapy in metastatic breast cancer

Prior to 2014, pertuzumab was used in clinical trials in 71/1,227 (5.8%) patients having received a 1 st -line metastatic therapy. The prescription of pertuzumab increased as soon as during the 2014-2017 period until it was used in 940/1,395 (67.4%) patients between 2017 and 2018 (Figure 3). Pertuzumab-trastuzumab-taxane was the most frequently reported combination over this last period (826/1,395, 59.2%), in particular in patients younger than 70 years (644/1,016, 63.4% versus 182/379, 48.0% in older patients). From 2011-2013 to 2017-2018, the use of the trastuzumab-taxane combination decreased from 579/1,227 (47.2%) to 128//1,395 (9.2%) patients. The median treatment duration of pertuzumab was 18.1 months (IQR: 8.3-28.9; mean: 20.4±14.8 months).

Second line therapy in metastatic breast cancer

Prior to its approval in 2014, T-DM1 was used in clinical trials in 40/647 (6.2%) patients having received a second-line metastatic therapy (see Supplementary material, Figure S2). It was used in only 372/691 (53.8%) patients during the 2017-2018 period. During the same two periods, the use of trastuzumab-vinorelbine and trastuzumab-taxane combinations decreased dramatically in second line setting, from 193/647 (29.8%) to 10/691 (1.5%) patients and from 188/647 (29.1%) to 47/691 (6.7%), respectively. The median duration of T-DM1 was 7.6 months (IQR 4.1-15.8; mean: 11.5±10.6 months). After second-line T-DM1 (326/605, 53.9%), the 86 patients who received a further third-line therapy were mostly treated with trastuzumab (N=66, 76.7%). (Figure 5). Taken together, these data suggest that implementation in the routine practice of major drugs such as pertuzumab and T-DM1 is quick and efficient. However, it also appears from figures 4 and 5 that substantial subsets of patients received these drugs off label (e.g. pertuzumab after the first line), or conversely were not exposed at all. More than 35% of patients did not receive pertuzumab as first line treatment, and more than 45% of patients did not received T-DM1 as second line treatment.

Successive metastatic lines of treatment

Discussion

There is a growing need for real-world data on use of cancer treatments, notably to (i) assess compliance with recommendations, (ii) confirm in a real-life setting previous results from interventional studies, and (iii) follow the evolution of patient management over time. The PRM project, based on data from French hospitals, was thus designed to provide consistent real-world data over time on the therapeutic strategies implemented in HER2+ BC patients.

We developed an original method based on regular, automated extractions from electronic pharmacy records. Our analysis led to robust data, notably thanks to data controls and corrections performed after data extraction. The quick, automated data collection process we have developed led to project sustainability and made possible data extractions on a regular basis, thus able to generate accurate and updated analyses that can be repeatedly performed within only two months of time. Based on data extraction performed in September 2018, the present report assessed evolution in injectable cancer treatments used in hospitals according to BC stage and treatment line in 21,119 patients exposed to either trastuzumab, pertuzumab or T-DM1 at least once from 2011. Use of biosimilars was not recorded per protocol.

In patients with early breast cancer, trastuzumab was used accordingly to the French Haute Autorité de Santé and the European Medicines Agency recommendations, in combination with at least one taxane in most cases, and a median treatment duration of one year.

In patients with metastatic BC, treatment strategies have evolved considerably in France since pertuzumab (in December 2013) and T-DM1 (in July 2014) became available. The proportion of patients treated with first-line pertuzumab increased to 67% (in combination with trastuzumab and taxane: 59%) during the 2017-2018 period, as well as second-line T-DM1 (up to 57%) decreasing the use of trastuzumab-vinorelbine and trastuzumab-taxane combinations. These evolutions are in accordance with the 2017 European guidelines for mBC, prevailing at the time of data extraction [START_REF] Cardoso | 3rd ESO-ESMO International Consensus Guidelines for Advanced Breast Cancer (ABC 3)[END_REF] and suggest that physicians tend to prescribe new treatments as soon as available when possible, especially in the metastatic setting. However, our results also strikingly suggest that important proportions of patients are not given access to state-of-the-art therapies, with potential and presently non-evaluable medical consequences for individual patients. In our study, we observed the non-use of firstline pertuzumab in around 30% of patients. As drug access is fortunately not an issue in France, reasons for this underuse of key medications have to be explored. It might be hypothesized that indeed about 30% of patients are primarily resistant to conventional trastuzumab based combinations as evidenced in populations with short delay relapses in the CLEOPATRA and PERUSE studies [START_REF] Baselga | Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer[END_REF][START_REF] Bachelot | Preliminary safety and efficacy of first-line pertuzumab combined with trastuzumab and taxane therapy for HER2-positive locally recurrent or metastatic breast cancer (PERUSE)[END_REF]. Another possibility is the use of double HER2 blockade with non-approved drugs in this setting. For instance, capecitabine therapy as used in the PHEREXA trial [START_REF] Urruticoechea | Randomized Phase III Trial of Trastuzumab Plus Capecitabine With or Without Pertuzumab in Patients With Human Epidermal Growth Factor Receptor 2-Positive Metastatic Breast Cancer Who Experienced Disease Progression During or After Trastuzumab-Based Therapy[END_REF] is not recorded in our system. Finally, in some patients, chemotherapy-free regimens might have been used, according to residual side effects from prior treatments, or in combination with endocrine treatment. In addition, pertuzumabtrastuzumab-taxane was less frequently prescribed in patients over 70 years (48.0% versus 63.4% in younger patients). One likely reason for the under-use in elderly could be their frequent comorbidities and potential drug toxicities. Finally, some patients received T-DM1 in the first line setting, probably based on the non-inferiority re-analysis of the MARIANNE trial [START_REF] Perez | Trastuzumab emtansine with or without pertuzumab versus trastuzumab with taxane for human epidermal growth factor receptor 2-positive advanced breast cancer: Final results from MARIANNE[END_REF].

Treatment durations with pertuzumab and T-DM1 were described in patients with a first injection at least 18 months before data extraction, and with characteristics close to those of previous clinical trials. The respective median treatment durations were strikingly similar to the median progression-free survival observed in the CLEOPATRA study [START_REF] Baselga | Pertuzumab plus trastuzumab plus docetaxel for metastatic breast cancer[END_REF] for pertuzumab (18.0 and 18.7 months, respectively), and in the EMILIA study [START_REF] Verma | Trastuzumab emtansine for HER2-positive advanced breast cancer[END_REF] for T-DM1 (7.6 and 9.6 months, respectively), indirectly confirming the efficacy of both drugs in routine medical practice.

One limitation of the PRM project is that no clinical data, such as tumor characteristics, response to treatment or drug toxicity, are entered into the hospital EPR systems. In addition, oral cancer therapies prescribed at the hospital, as well as combined non-cancer therapies, are not tracked by hospitals. Use of biosimilars, which have now entered common practice, will be captured in future data extractions. This may lead to incomplete treatment history. To fill this gap, matching with other medical databases, such as electronic medical records, existing clinical registries or databases, or national public health databases could be an opportunity.

  ). No major changes were observed over time regarding these combinations: between 876/1,389 (63.1%) patients in 2011 and 900/1,753 (51.3%) patients in 2018 for the trastuzumab-taxane-anthracycline combination, and between 315/1,389 (22.7%) and 674/1,753 (38.4%) patients for the trastuzumab-taxane anthracycline-free combination. In adjuvant setting, median duration of trastuzumab was 54.0 weeks (interquartile range, IQR: 35.0-55.3; mean duration: 46.0±24.3 weeks).

Figures 4 and 5

 5 Figures 4 and 5 show how successive therapeutic strategies have evolved between the 2011-

Figure 1 :

 1 Figure 1: Disease stages and treatment lines in patients with HER2-positive breast

*

  Several reasons possible for a same patient A same single patient could receive different lines of treatments and could have early and then metastatic BC stages during follow-up (N=1,309).

Figure 2 :Figure 3 :Figure 4 :

 234 Figure 2: Evolution in HER2+ targeted treatments used in early stage breast cancer -2011-2018 period

Figure 5 :

 5 Figure 5: Therapeutic strategies used in patients with HER2+ metastatic breast cancer -2017-2018 period

Acknowledgements

The authors would like to thank all the centers and women participating in this project. Support funded by Roche was provided by Brice Hyaumet and Joël Da Costa from ADVANCED SCHEMA (data management, statistical analysis and global project management), and Dr. Marie-Odile BARBAZA from AUXESIA (manuscript preparation).

Funding

This work was supported by Roche. Roche was involved in the statistical analysis. All the authors participated in interpreting the study results, and in the critical revision and approval of the final version of the manuscript.

Highlights

• There is a growing need for real-world data on how cancer treatments are used • Use of therapies for early or advanced HER2+ breast cancer have changed over time • Cancer treatments were captured in electronic pharmacy systems in French hospitals

• We provide real-world data on 2011-2018 evolution in injectable HER2 treatments.

• Results suggest that improvement is necessary for an optimal use.

• Drug-related indicators will be further built for value-based pricing solutions

Conclusions

The PRM project is the first program providing consistent real-world information over time on the therapeutic strategies implemented in a large and representative population of French HER2+ BC patients. In addition to a better knowledge of present patient management and how treatments evolution are implemented in routine care, such data will be useful to build medico-economic models or new value-based pricing solutions in a real-life setting. This project is currently extended to lung cancer, and could be enlarged to other cancers and pathologies.

Conflict of interest

This work was supported by Roche. PC, BC, DP, AD, JM, OA, HB and XP received institutional funding for the study. NC and LS are Roche employees.


Dorothée Bedock

Pierre Bel Lassen

Alexis Mathian

Pauline Moreau

Julie Couffignal

Cécile Ciangura

Christine Poitou-Bernert

Anne-Caroline Jeannin

Helena Mosbah

Jehane Fadlallah

Zahir Amoura

Prof Jean-Michel Oppert
email: jean-michel.oppert@aphp.fr

Pauline Faucher

Prevalence and severity of malnutrition in hospitalized COVID-19 patients

Keywords: COVID-19, malnutrition, pneumonia, SARS-Cov2, albumin

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

Introduction

Malnutrition is known as a risk factor for severity of and mortality from viral pneumonia since the times of the 1918 influenza pandemic [START_REF] Short | Back to the Future: Lessons Learned From the 1918 Influenza Pandemic[END_REF]. Similarly, the recently described SARS-Cov2 infection (COVID- [START_REF] Arkin | Nutrition in critically ill patients with COVID-19: Challenges and special considerations[END_REF] and related pneumonia could be closely associated with malnutrition [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF]. Indeed, a number of features observed in COVID-19 patients are likely to lead to body weight loss and malnutrition [START_REF] Barazzoni | ESPEN expert statements and practical guidance for nutritional management of individuals with SARS-CoV-2 infection[END_REF][START_REF] Miller | Clinical presentation and course of COVID-19[END_REF]. These include: symptoms potentially resulting in decreased food intake such as dyspnea, dysgueusia, anosmia, anorexia, dysphagia, nausea, vomiting, diarrhea; hyper-metabolism and increased energy requirements, as observed in various types of severe infection [START_REF] Wischmeyer | Nutrition Therapy in Sepsis[END_REF]; older age with frailty and various comorbidities [START_REF] Barazzoni | ESPEN expert statements and practical guidance for nutritional management of individuals with SARS-CoV-2 infection[END_REF]; prolonged hospital stay in conventional or intensive care units [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF].

Currently, very little is known about the prevalence of malnutrition in COVID-19 patients and its potential effect on disease severity and mortality. In one study from Wuhan, China, using the widely used questionnaire-based Mini Nutritional Assessment (MNA) tool, Li et al. reported that 52.7% of hospitalized COVID-19 patients over the age of 65 years were malnourished and 27.5% of them were at risk of malnutrition [START_REF] Li | Prevalence of malnutrition and analysis of related factors in elderly patients with COVID-19 in Wuhan, China[END_REF]. In another study, Du et al.

identified a composite malnutrition score, based on albumin and cholesterol levels and lymphocyte count (Controlling Nutritional Status and Prognostic Nutritional Index), as an independent predictor of mortality in COVID-19 patients [START_REF] Chen | Comparison of the Clinical Implications among Two Different Nutritional Indices in Hospitalized Patients with COVID-19[END_REF]. There is also some evidence in recent literature of an association between low albumin levels and COVID-19 severity [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF].

Importantly, major phenotypic criteria of malnutrition such as low body mass index (BMI) or recent weight loss have not been described in this setting.

The objectives of the present study were to assess the prevalence and severity of malnutrition in hospitalized adult patients with COVID-19 admitted in a (non-intensive) medical unit. We first assessed malnutrition according to the phenotypical and biological criteria defined by the Global Leadership Initiative on Malnutrition (GLIM) [START_REF] Cederholm | GLIM criteria for the diagnosis of malnutrition -A consensus report from the global clinical nutrition community[END_REF], a consensus scheme for diagnosing malnutrition in adults in clinical settings. We then investigated the links between malnutrition and disease severity at admission. Finally, we studied the impact of malnutrition parameters on specific clinical outcomes such as transfer to an intensive care unit (ICU) or death.

Materials & methods

Study population

This observational longitudinal study included all COVID-19 adult patients admitted to the E3M Institute in Pitié-Salpétrière hospital from March 21 st 2020 to April 24 th 2020. All patients were admitted from the emergency department, medical units or intensive care units.

Confirmed SARS-Cov2 infection was defined by a positive result of real-time reversetranscriptase-polymerase-chain-reaction (RT-PCR) assay of nasal and pharyngeal swab specimens and/or evocative CT-scan lesions. All data were collected in the context of care, thus, in accordance with the French law, including the GPRD, an informed consent of the patient was not sought. Patients were informed that data collected in medical records might be used for research study in accordance to privacy rules and that they could express their refusal.

Demographics, comorbidities, laboratory values

Data on characteristics such as patient age, sex, weight and smoking status and comorbidities including diabetes and hypertension, known to have a potential impact on the prognosis of COVID-19 [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF], were extracted from the medical charts. Blood samples were collected in all patients at admission for determination of albumin, transthyretin, C-reactive protein (CRP), D-Dimers, lactate dehydrogenase and lymphocyte blood count to assess nutritional status, magnitude of inflammatory response and prognostic markers from earlier COVID-19 studies [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF].

Nutritional assessment

Height and last known weight (i.e. weight before COVID-19 within the past 6 months) data were collected by questioning patients during or shortly after their stay in hospital. Weight was measured on calibrated scales during their medical stay allowing to calculate percentage of weight loss defined as 100*(last known weight -measured weight) / last known weight. BMI was calculated as measured weight (kg) divided by height (meter) squared.

Diagnosis of malnutrition according to the GLIM criteria requires at least one etiologic criterion among reduced food intake or assimilation, inflammation or disease burden and at least one phenotypic criterion among non-volitional weight loss, low BMI and reduced muscle mass [START_REF] Cederholm | GLIM criteria for the diagnosis of malnutrition -A consensus report from the global clinical nutrition community[END_REF]. In our setting, we considered that an etiologic criterion of malnutrition was always present due to COVID-19 induced inflammation. Additionally, in most cases, reduced food intake or assimilation, the other etiologic criterion was present due to COVID-19 functional signs (anosmia, dysgueusia, nausea, vomiting, diarrhea). Phenotypic criteria were assessed using BMI and % weight loss. We also used the GLIM criteria to categorize the severity of malnutrition. We distinguished three groups of patients: i) no malnutrition; ii) moderate malnutrition defined by weight loss between 5 to 10% within the past 6 months or

Assessment of severity of COVID-19

Initial common symptoms of COVID-19 were screened including fever at home, high body temperature at admission (defined by body temperature > 37.5°C) and gastrointestinal symptoms (nausea, vomiting, diarrhea), anosmia, dysgueusia and anorexia. Oxygen intake (L/min) at admission and during hospitalization, chest CT scan grade were also recorded to assess COVID-19 respiratory severity. Transfer to an intensive care unit (ICU) from our medical unit and death were considered as the two main unfavorable clinical outcomes.

Statistical analyses

Continuous variables were expressed as mean ± SD. Categorical variables were expressed as absolute values and percentages. Continuous variable with a non-parametric distribution were log-transformed before analysis. Linear regression with analysis of variance (ANOVA) for continuous variables and Pearson's chi-square (χ2) test or Fisher's exact test for discrete variables were used to compare the different characteristics according to nutrition status.

When significant overall differences were found, Tukey's post hoc test or pairwise chi-square test with Bonferroni correction were performed for continuous or discrete variables, respectively. The links between nutritional parameters and negative outcomes of COVID-19 (transfer to ICU or death) were investigated using logistic regression in a univariate model after exclusion from the analysis of patients initially admitted from ICU. Small sampleadjusted unconditional maximal likelihood estimation (UMLE) was used to determine odds ratio and confidence interval when the number of patients was too low in the contingency table. To find out if the observed associations were independent of age and inflammation (known to be prognostic factors of COVID-19), a multivariate logistic regression analysis was performed using age and CRP as covariates. Statistical tests were considered significant if P<0.05. All statistical analyses were conducted using R studio software version 1.2.1335 (http://www.r-project.org).

Results

Prevalence of malnutrition in COVID-19 hospitalized patients

A total of 160 patients were admitted during the study period and complete nutritional data were available for 114 subjects. Subjects without complete nutritional data were older (67.5y +/-15.5 vs. 59.9 +/-15.9, p=0.006) but there was no significant difference in the prevalence of hypertension, diabetes, COPD, chronic kidney disease, coronary heart disease and active neoplasia (data not shown). Among these, 18 were transferred to our medical unit from ICU whereas 96 were admitted as a first line of care. The main characteristics of the cohort are shown in Table 1. In this sample, 47 (42.1%) patients had malnutrition according to the GLIM criteria with a prevalence of moderate and severe malnutrition of 23.7% and 18.4% respectively (Figure 1A). Prevalence of malnutrition was significantly higher in patients admitted from ICU than in the other patients (66.7% vs. 37.5% respectively, p<0.05) (Figure 1B). Age, presence of diabetes, hypertension, obesity, COPD, chronic kidney disease, coronary heart diseases and active neoplasia, were not significantly different according to nutritional status. The main nutritional parameters that differed between the 3 groups were BMI and % weight loss (p<0.01) while there was no significant difference in albumin or transthyretin levels (Table 1).

Presentation of COVID-19 and malnutrition status

Fever, one of the most common symptoms of COVID-19, was less frequently reported as an initial symptom in patients with severe malnutrition compared to those without malnutrition (61.9% vs 89.4% respectively, p < 0.05). A similar trend was observed for high body temperature at admission which was present in 23.8% of severely malnourished patients vs. 55.6% and 46.2 % in the moderate malnutrition and the group without malnutrition, respectively (p=0.08). There was no significant difference in any other common symptoms of COVID-19 including anosmia or dysgueusia, nausea, diarrhea, vomiting and respiratory symptoms. There was no significant difference in oxygen intake at day one or in the severity of the initial chest CT scan between the three groups. Pneumonia severity was also not different between the three groups. Lymphocytes count was significantly higher in the severe malnutrition group than compared with moderate malnutrition group and group without malnutrition (p<0.05); there was no difference in levels of CRP and D-dimer between the three groups.

Factors associated with negative outcomes of COVID-19

As summarized in Table 3, a lower albumin level at admission was significantly associated with a higher risk of transfer to ICU (for 10g/l of albumin, OR: 0.31 [0.1; 0.7]; p<0.01).

Importantly, in a multivariate analysis adjusted for age and initial CRP levels, this association remained significant (OR: 0.38 [0.14, 0.96]; p<0.05). Although not significant, there was a trend for a higher risk of mortality in patients with weight loss above 5% of initial weight (OR: 3.7 [1.0; 26.5], p=0.09). Nutritional status was not associated with the risk of transfer to ICU or death.

Discussion

In this study, in adult patients hospitalized for documented COVID-19 in a non-intensive medical unit from a university hospital, we found that 42.1% of patients were malnourished and 18.4% of them had severe malnutrition. When stratifying our sample based on mode of admission to our unit, as much as two thirds (66.7%) of patients admitted from ICU were malnourished and more than a third (38.9%) displayed severe malnutrition. Low albumin levels were associated with serious adverse outcomes such as transfer to ICU, independently of age and CRP.

The overall prevalence of malnutrition in the present study is very high. Poor nutritional status is an established risk factor for community-acquired pneumonia [START_REF]Risk Factors for Community-Acquired Pneumonia in Adults: A Systematic Review of Observational Studies[END_REF] and in an Italian study by Russo et al., malnutrition was an independent risk factor for hospitalization in residents of long-term care facilities with such home-acquired pneumonia [START_REF] Russo | Clinical features, therapy and outcome of patients hospitalized or not for nursing-home acquired pneumonia[END_REF]. Prevalence of malnutrition in these hospitalized elderly patients (mean age 81.5 y) was 40.4% which is comparable to our results despite a much younger age in our population. This high prevalence of malnutrition in COVID-19 patients is in the same order of magnitude as the one reported by Li et al. in hospitalized COVID-19 patients in Wuhan (52.7%) [START_REF] Li | Prevalence of malnutrition and analysis of related factors in elderly patients with COVID-19 in Wuhan, China[END_REF]. However, to compare these two results is difficult since Li. et al. studied elderly subjects and they only assessed malnutrition using the MNA tool. In addition, using MNA does not allow to specifically assess the severity of malnutrition. Moreover, although the prevalence of malnutrition in COVID-19 patients in our study is very high with about two thirds of malnourished patients among those admitted from ICU, this number could still be underestimated. Indeed, we were not able to assess muscular strength which is one of the phenotypic criteria of malnutrition according to GLIM [START_REF] Cederholm | GLIM criteria for the diagnosis of malnutrition -A consensus report from the global clinical nutrition community[END_REF]. Given the marked deterioration of general condition or performance status associated with COVID-19, it is likely that muscle strength would be decreased in this setting. In addition, prolonged stay in ICU is known to be associated with muscle wasting, also resulting in reduced muscular strength [START_REF] Flower | Muscle wasting in the critically ill patient: how to minimise subsequent disability[END_REF].

We found a significant association between hypoalbuminemia at admission and worsening of COVID-19, reflected by a necessity of transfer to the ICU. A difference of 10g/l of albumin at admission was associated with a 3-fold increased risk of transfer to ICU. Importantly, the relationship we found between low albumin and transfer to ICU was independent of CRP level. Indeed, it is well known that albumin is an important inflammation marker and is not exclusively dependent on nutritional status [START_REF] Keller | Nutritional Laboratory Markers in Malnutrition[END_REF]. In addition, high CRP has been shown to be associated with the progression of COVID-19, as expected in a context of severe infection [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF]. Moreover, we performed a complementary analysis showing that there is a significant correlation between albuminemia and recent weight loss (r=-0.19, p=0.05) (data not shown).

Altogether, this suggests that even in this highly inflammatory context, lower albumin remains a marker of weight loss and malnutrition.

The predictive role of low albumin levels regarding transfer to ICU is in line with previous findings by Liu et al. [START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF]. These authors observed in COVID-19 patients that a difference of 10g/l in baseline albumin concentration was associated with a 5-fold increased risk of acute respiratory distress syndrome and a 2-fold increased risk of mortality [START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF]. It can be hypothesized that poor nutritional status would weaken immune response of the host, as observed in other settings [START_REF] Lomax | Prebiotics, immune function, infection and inflammation: a review of the evidence[END_REF][START_REF] Yaqoob | Ageing alters the impact of nutrition on immune function[END_REF]. Furthermore, experimental studies with the influenza virus suggest that not only does malnutrition suppress immune response to the virus but it could actually facilitate the emergence of novel variants, which may display increased pathogenicity relative to the original virus strain [START_REF] Beck | Host nutritional status: the neglected virulence factor[END_REF]. Altogether, our data reinforce the importance of routine assessment of nutritional markers in COVID-19 patients as recommended by a recent statement of ESPEN [START_REF] Barazzoni | ESPEN expert statements and practical guidance for nutritional management of individuals with SARS-CoV-2 infection[END_REF].

Our data show that patients with severe malnutrition had less reported fever as part of initial symptoms and there was a trend for less frequent high body temperature at admission in severely malnourished patients. Although this result was unexpected and requires confirmation in additional studies, it was demonstrated by Kauffman et al. that severe proteincalorie malnutrition could lead to altered thermoregulation and poor febrile response [START_REF] Kauffman | Fever and malnutrition: endogenous pyrogen/interleukin-1 in malnourished patients[END_REF].

Since higher initial body temperature has been described as a pejorative factor of COVID-19 evolution in previous reports [START_REF] Wu | Risk Factors Associated With Acute Respiratory Distress Syndrome and Death in Patients With Coronavirus Disease 2019 Pneumonia in Wuhan, China[END_REF][START_REF] Liu | Analysis of factors associated with disease outcomes in hospitalized patients with 2019 novel coronavirus disease[END_REF] it is important to note that fever may be underestimated in severely malnourished patients.

An important strength of our study in COVID-19 patients is that we used the specific metrics defined by GLIM [START_REF] Cederholm | GLIM criteria for the diagnosis of malnutrition -A consensus report from the global clinical nutrition community[END_REF] for the diagnosis of malnutrition, yet difficult to collect owing to the lack of scales and in consideration of the hygienic precautions required [START_REF] Caccialanza | Early nutritional supplementation in non-critically ill patients hospitalized for the 2019 novel coronavirus disease (COVID-19): Rationale and feasibility of a shared pragmatic protocol[END_REF]. In this series of consecutive adult patients with a mean age under 60 years and with various comorbidities, we were able to collect a range of phenotypical (BMI, weight loss) and biological (albumin, CRP) parameters to describe nutritional status concurrently with clinical signs related to COVID-19. Beyond the baseline information we collected, we were able to obtain follow-up data, specifically regarding transfer to ICU or death. Our study therefore provides new nutritional knowledge in the setting of the COVID-19 and an improved description of malnutrition using objective phenotypic criteria and not limited to questionnaire data [START_REF] Li | Prevalence of malnutrition and analysis of related factors in elderly patients with COVID-19 in Wuhan, China[END_REF][START_REF] Chen | Comparison of the Clinical Implications among Two Different Nutritional Indices in Hospitalized Patients with COVID-19[END_REF]. Some limitations need to be mentioned. Our sample size possibly led to insufficient power when assessing relationships between severity of malnutrition and evolution of COVID-19.

Although we assessed in our patients a relatively large set of clinical and biological variables of interest, difficulties in obtaining such simple measures as body weight in this acute or postacute setting should not be overlooked, as emphasized recently [START_REF] Barazzoni | ESPEN expert statements and practical guidance for nutritional management of individuals with SARS-CoV-2 infection[END_REF][START_REF] Caccialanza | Early nutritional supplementation in non-critically ill patients hospitalized for the 2019 novel coronavirus disease (COVID-19): Rationale and feasibility of a shared pragmatic protocol[END_REF][START_REF] Arkin | Nutrition in critically ill patients with COVID-19: Challenges and special considerations[END_REF][START_REF] Krznarić | A simple remote nutritional screening tool and practical guidance for nutritional care in primary practice during the COVID-19 pandemic[END_REF]. Selection bias cannot be ruled out. Patients admitted to our hospital through the emergency room were either immediately transferred to intensive care when in an acute condition or transferred to our and COVID-19 could be bidirectional, as the infection can potentially lead to severe malnutrition but malnutrition may negatively impact prognosis in infected patients.

In conclusion, we observed a prevalence of malnutrition in COVID-19 patients hospitalized in non-intensive medical units as high as 42% and even higher (67%) when patients are admitted from an ICU. Low albumin levels at admission is a predictive marker of more severe outcome of the disease (transfer to ICU). These results reinforce the value of nutritional risk screening and the need of early nutritional management in COVID-19 patients, as recently emphasized by ESPEN [START_REF] Barazzoni | ESPEN expert statements and practical guidance for nutritional management of individuals with SARS-CoV-2 infection[END_REF] and others [START_REF] Caccialanza | Early nutritional supplementation in non-critically ill patients hospitalized for the 2019 novel coronavirus disease (COVID-19): Rationale and feasibility of a shared pragmatic protocol[END_REF][START_REF] Arkin | Nutrition in critically ill patients with COVID-19: Challenges and special considerations[END_REF][START_REF] Krznarić | A simple remote nutritional screening tool and practical guidance for nutritional care in primary practice during the COVID-19 pandemic[END_REF]. Further research is needed to assess the impact of nutritional care on longer term prognosis in these patients.

Figure 1 .

 1 Figure 1. Prevalence of malnutrition in hospitalized COVID-19 patients

Figure 1 ICU

 1 Figure 1

Table 1 . General and nutritional characteristics

 1

		All	No	Moderate	Severe	P
			malnutrition	Malnutrition	Malnutrition	value
	Number of patients	114	66	27	21
	General
	Age (years)	59.9 (15.9) 58.4 (15.2) 61.1 (16.6)	63.0 (17.1)	0.47
	Male, n (%)	69 (60.5)	38 (57.6)	20 (74.1)	11 (52.4)	0.23
	Hypertension, n (%)	60 (52.6)	35 (53.0)	16 (59.3)	9 (42.9)	0.53
	Diabetes, n (%)	44 (38.6)	28 (42.4)	7 (25.9)	9 (42.9)	0.30
	COPD, n (%)	8 (7)	5 (7.6)	1 (3.7)	2 (9.5)	0.69
	CKD, n (%)	23 (20.2)	13 (19.7)	5 (18.5)	5 (23.8)	0.90
	CHD, n (%)	23 (20.4)	13 (20)	4 (14.8)	6 (28.6)	0.53
	Active neoplasia, n (%)	9 (8.3)	5 (7.9)	2 (7.7)	2 (10)	0.90
	Active smoker, n (%)	7 (6.7)	4 (6.8)	1 (3.9)	2 (10.5)	0.75
	Anthropometrics
	BMI (kg/m 2 )	26.6 (5.1) 27.9 (4.9)	26.3 (4.1)	23.3 (5.3)	<0.01
	Obesity, n (%)	28 (25.0)	20 (31.2)	6 (22.2)	2 (9.5)	0.13
	Low BMI, n (%)	13 (11.6)	2 (3.1)	3 (11.1)	8 (38.1)	<0.01
	Very low BMI, n (%)	7 (6.2)	0 (0.0)	0 (0.0)	7 (33.3)	<0.01
	Weight loss (%)	4.41 (5.6) 1.07 (3.9)	6.54 (1.9)	12.2 (4.4)	<0.01
	Weight loss >5%, n (%) 47 (41.2)	2 (3.0)	25 (92.6)	20 (95.2)	<0.01
	Weight loss >10%, n (%) 18 (15.8)	0 (0.00%)	0 (0.0)	18 (85.7)	<0.01
	Biological markers
	Albumin (g/l)	30.1 (5.5) 30.4 (6.0)	30.0 (4.8)	29.3 (4.9)	0.87
	Albumin < 35 g/l, n (%) 91 (84.3)	49 (81.7)	23 (85.2)	19 (90.5)	0.68

Table 2 : Clinical presentations of COVID-19 in the three groups of patients

 2

	Results are expressed as mean (SD) for continuous data and n (%) for categorical data.
	P values shown result from analysis of variance (ANOVA) for continuous data and Chi square or Fisher's
	exact test for categorical data between the 3 categories of nutritional status.
	CRP : C-Reactive Protein

Table 3 . Factors associated with transfer to intensive care unit or death

 3 Univariate logistic regression. a small sample-adjusted UMLE was used to determine Odds ratio and confidence interval because of low n in contingency table (cf methods for explanation).

	OR : Odds Ratio
	95% CI : 95% Confidence Interval

Acknowledgments:

The authors express their thanks to all doctors, nurses, dieticians and all other staff of E3M institute at Pitié-Salpêtrière university hospital (AP-HP) that participated to the collection of data and patient care.

Funding sources

None

Authorship statement

D. Bedock, P. Bel Lassen and P. Faucher conceptualized the study; D. Bedock, P. Bel Lassen A. Mathian, P. Moreau and J. Fadlallah collected the data; P. Bel Lassen and P. Moreau analysed the data; D. Bedock, P. Bel Lassen and P. Faucher drafted the original manuscript;

J.-M. Oppert supervised the study; all authors reviewed and edited the final manuscript.

Conflict of Interest

The authors declare that they have no conflict of interest.


Dr Manon Bolzinger
email: manon.bolzinger@outlook.frnepasutiliser

Erik Estivalèzes

Adeline Gallini

Eva Polirsztok

Kariman Abelin-Genevois

Christiane Baunin

Jérôme Sales De Gauzy

Pascal Swider

Kariman Abelin- Genevois

Évaluation IRM de l'hydratation des disques intervertébraux lombaires non pathologiques dans une population pédiatrique

Résumé

Introduction : Le disque intervertébral (DIV) est formé par l'annulus fibrosus (AF) et le nucleus pulposus (NP) complexe inerte hydraté. La capacité de déformation du DIV, corrélée à celle du NP, dépend de son hydratation. Quand le DIV vieillit, son hydratation diminue, ainsi que ses capacités de déformation. Dans la scoliose idiopathique de l'adolescent, une des hypothèses étiologiques repose sur le DIV, étudié pour le diagnostic et le suivi de cette pathologie.

Hypothèse : l'hydratation du DIV dépend du genre, de l'âge et du niveau du segment rachidien dans une population pédiatrique asymptomatique. Le corollaire est d'établir un groupe contrôle de patients sains.

Matériel et méthodes : Une cohorte de patients avec une IRM rachidienne normale a été inclue : 98 patients, âge moyen 13.3 ans. L'évaluation des volumes des disques et de l'hydratation de chaque DIV a été effectuée à partir de séquences IRM T2, en utilisant un logiciel de traitement d'images validé. L'étude portait sur la colonne lombaire, de la jonction thoraco-lombaire à la jonction lombo-sacrée. Il a été admis que l'hydratation du DIV était liée au ratio des volumes NP et AF. Une analyse linéaire mixte multivariée a été utilisée pour explorer le rôle de l'âge, du genre et du niveau segmentaire, sur l'hydratation des disques.

Résultats : L'hydratation du groupe des garçons était globalement plus élevée que celle du groupe des filles sans différence significative. L'hydratation augmentait avec l'âge (+0.005 pour chaque année supplémentaire, p=0.0213). L'hydratation des DIV semblait plus importante au niveau thoraco-lombaire comparé au niveau lombaire, mais sans différence statistiquement significative. Conclusion : Nous avons établi une base de données incluant l'hydratation de disques lombaires non pathologiques à partir d'IRM, selon l'âge, le sexe et le niveau du segment, avec des intervalles de confiance à 95%.

Niveau de preuve: IV

Mots clés: Disques intervertébraux, orthopédie pédiatrique, IRM, traitement d'image.

I -Introduction

L'homéostasie du segment vertébral est associée à des transports en diffusion entre le disque intervertébral (DIV) et le corps vertébral adjacent à travers la plaque cartilagineuse [START_REF] Urban | Nutrition of the intervertebral disc[END_REF]. A l'âge adulte, le DIV est une structure avasculaire, dans laquelle les liquides passent du nucleus pulposus (NP) (complexe hydraté) vers une structure laminaire périphérique, l'annulus fibrosus (AF). La vascularisation du disque évolue progressivement avec la croissance pour s'estomper en fin de croissance du tronc à l'âge pré-adulte. L'hydratation du DIV dépend de son contenu en protéoglycannes. Dans des conditions physiologiques, des échanges de liquides et de protéines ont lieu par méchanotransduction, impliquant l'hydrostatisme et des pressions osmotiques cycliques associées au rythme nycthéméral [START_REF] Setton | Mechanobiology of the intervertebral disc and relevance to disc degeneration[END_REF][START_REF] Henriksson | Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: a study in four species[END_REF]. La composition et l'organisation de la matrice extracellulaire du DIV détermine ses propriétés biomécaniques. Elles varient selon l'âge, l'état physiologique et la situation dans le rachis. La perte d'eau est un des premiers signes de DIV pathologique [START_REF] Benneker | Correlation of radiographic and MRI parameters to morphological and biochemical assessment of intervertebral disc degeneration[END_REF].

Notre étude porte sur une population pédiatrique qui servira de contrôle pour l'étude de la scoliose idiopathique chez les enfants. Parmi les facteurs étiologiques multiples de la SIA, une des hypothèses repose sur le DIV, étudié pour le diagnostic et le suivi de cette pathologie par IRM [START_REF] Violas | A method to investigate intervertebral disc morphology from MRI in early idiopathic scoliosis: a preliminary evaluation in a group of 14 patients[END_REF][START_REF] Abelin-Genevois | Spino-pelvic alignment influences disc hydration properties after AIS surgery: a prospective MRI-based study[END_REF][START_REF] Waldenberg | MRI histogram analysis enables objective and continuous classification of intervertebral disc degeneration[END_REF]. Après une arthrodèse postérieure, la réaction des disques sous l'instrumentation soumis à des contraintes supplémentaires est liée au niveau de l'instrumentation et au nombre de niveaux fusionnés [START_REF] Green | Long-term magnetic resonance imaging follow-up demonstrates minimal transitional level lumbar disc degeneration after posterior spine fusion for adolescent idiopathic scoliosis[END_REF][START_REF] Pesenti | Evolution of adolescent idiopathic scoliosis: results of a multicenter study at 20 years' follow-up[END_REF]. Une étude pionnière [START_REF] Antoniou | The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration[END_REF] suggère une distinction en trois groupes selon la composition de la matrice extracellulaire du DIV et son hydratation: avant 5 ans, entre 25 et 40 ans, entre 40 et 80 ans. Une étude complémentaire [START_REF] Krueger | Changes in T2 relaxation times associated with maturation of the human intervertebral disk[END_REF] suggère que la maturation des DIV lombaires est en relation avec une augmentation de l'hydratation.

Notre hypothèse stipule qu'entre l'âge de 7 et 20 ans, l'hydratation d'un DIV non pathologique dépend du genre, de l'âge et du niveau du segment.

Notre but est de déterminer les relations entre l'hydratation d'un DIV lombaire, le genre, l'âge et le niveau vertébral concerné dans une population pédiatrique sans pathologie du rachis. Secondairement, nous désirons réaliser des courbes de prédictions de l'hydratation des DIV lombaires selon le genre, l'âge et le niveau rachidien concerné dans une population pédiatrique.

II -Matériel and Méthodes

Recrutement des patients

Il s'agit d'une étude rétrospective, incluant consécutivement une cohorte de 98 patients (50% de filles), d'âge moyen de 13.3 ans (DS=2.92) ayant eu une IRM montrant un rachis considéré comme normal durant deux années au CHU de Toulouse (04/2015-04/2017). Tous les patients ont fait une IRM pour un diagnostic urologique, neurologique, ou une douleur lombaire. L'étude portait sur la colonne lombaire, de la jonction thoracolombaire à la jonction lombo-sacrée. Les acquisitions ont été réalisées en séquence T2 dans le plan sagittal avec des coupes consécutives (15 à 20 coupes par patient). Le DIV était représenté sur une dizaine de coupes consécutives. La référence de la machine était Philips Achieva X-series 3.0 T. Les critères d'inclusion étaient : âge compris entre 7 et 20 ans inclus, et absence de pathologie du rachis ou de trouble de la posture rachidienne diagnostiquée sur l'IRM après lecture de l'examen par un spécialiste de l'imagerie pédiatrique.

Propriétés des volumes du disque

La détection et la segmentation automatique des DIV sur IRM demeure non totalement résolue malgré des progrès effectifs [START_REF] Zheng | Evaluation and comparison of 3D intervertebral disc localization and segmentation methods for 3D T2 MR data: a grand challenge[END_REF][START_REF] Gervais | MRI signal distribution within the intervertebral disc as a biomarker of adolescent idiopathic scoliosis and spondylolisthesis[END_REF]. Le procédé utilisé impliquait un logiciel de traitement d'image Biomechlab® précédemment validé par des études cliniques [START_REF] Violas | A method to investigate intervertebral disc morphology from MRI in early idiopathic scoliosis: a preliminary evaluation in a group of 14 patients[END_REF][START_REF] Abelin-Genevois | Spino-pelvic alignment influences disc hydration properties after AIS surgery: a prospective MRI-based study[END_REF][START_REF] Violas | Quantification of intervertebral Disc Volume Properties below spine fusion, using Magnetic resonance imaging, iin adolescent idiopathic scoliosis surgery[END_REF], développé dans l'environnement Matlab (Mathworks ®). Apres décodage automatique des images DICOM, les propriétés des volumes du disque étaient extrapolées en utilisant une détection semi-automatique des contours externes de AF et NP. Le protocole était répété sur chaque coupe sagittale par un chirurgien spécialiste. Chaque disque était représenté sur 10 coupes adjacentes en moyenne. La segmentation et la reconstruction 3D des DIV à partir de l'IRM sont représentées en figure 1. Les disques T12-L1 à L5-S1 ont été explorés. Anatomiquement, le NP est identifié comme étant un tissu proche d'un fluide visqueux, très hydraté, apparaissant en hyper signal en T2. La segmentation 2D des coupes IRM et la reconstruction 3D ont permis de définir le critère d'hydratation du disque comme étant le rapport entre le volume du NP et celui de l'AF. La teneur en eau est concentrée dans le NP qui diminue en volume lorsque le DIV se déshydrate alors que le volume du DIV reste stable. La relation entre l'hydratation du DIV et le rapport des volumes NP / DIV est anatomiquement fiable.

Modèle statistique

Pour l'analyse statistique, un modèle linéaire mixte multivarié a été utilisé pour prendre en compte la variation interindividuelle des patients. Nous désirons déterminer un modèle de l'hydratation selon le niveau rachidien (T12-L1 à L5-S1), le genre (garçon ou fille) et l'âge. Ce groupe d'âge correspond à la période d'évolutivité de la SIA [START_REF] Duval-Beaupere | Pour une théorie unique de l'évaluation des scolioses[END_REF][START_REF] Duval-Beaupère | Growth in scoliotics[END_REF]. La significativité des résultats est déterminée par p < 0.05. Un modèle statistique générique de la distribution de l'hydratation de la DIV a été proposé en fonction de l'âge, du genre et du niveau de segment. Douze modèles ont été proposés avec un intervalle de confiance de 95 %.

III -Résultats

Le tableau 1 résume les valeurs brutes des propriétés du volume du disque à partir de la segmentation des images d'IRM par niveau de segment et pour l'ensemble de la cohorte (n = 98). Les volumes moyens de DIV et de NP étaient respectivement de 8973 mm3 et 2823 mm3. L'hydratation moyenne était de 0,335. Les résultats sont illustrés à la figure 2.

Le tableau 2 résume l'analyse statistique de l'hydratation par âge, genre et segment correspondant au modèle linéaire mixte multivarié.

Le disque T12-L1 a été considéré comme une référence pour étudier l'influence du niveau segmentaire. L'hydratation des niveaux inférieurs a toujours été plus faible, différence non significative (0,1 ≤ p ≤ 0,8). De plus, l'hydratation a diminué le long de la colonne vertébrale, mais sans signification statistique (p = 0,4915).

En ce qui concerne le genre, le groupe des hommes a été pris comme référence. Le groupe des filles a montré une hydratation plus faible avec une diminution moyenne de -0,0158 mais sans signification statistique (p = 0,21).

Un modèle générique statistique de la population a été proposé à partir d'une régression linéaire avec des intervalles de confiance de 95%. Les variations de l'hydratation selon l'âge, le genre et le niveau segmentaire ont été proposées figure 3. Les modèles de régression linéaire ont montré une augmentation pour le groupe des garçons sans dépendance à l'égard du niveau de segment et légèrement supérieure à celle du groupe des filles. Pour les segments du groupe féminin, T12-L1 et L5-S1 n'ont montré aucune dépendance à l'âge et un impact très faible pour les disques L1-L2 à L4-L5.

La méthodologie a permis de proposer des intervalles de prédiction de l'hydratation des DIV. Comme prévu, ils ont montré une plus grande variation en raison de l'incertitude supplémentaire due à la prédiction d'une valeur individuelle.

IV -Discussion

L'hypothèse centrale, assumant la dépendance de l'hydratation au genre, à l'âge et au niveau segmentaire a été partiellement validée. L'influence du genre n'était pas clairement significative bien que le groupe des filles montrât des valeurs inferieures. Les deux populations étaient comparables en âge : moyenne d'âge des filles 13.37 ans (7-18,6 ans), moyenne d'âge des garçons 13.31 ans (7-19,2 ans). Cette tendance à la différence selon le genre pourrait être un argument d'implication du DIV dans la genèse de la SIA, plus fréquente chez les filles. Cet argument nécessite plus d'exploration, notamment des DIV au niveau thoracique, car cette étude portait sur le rachis lombaire alors que la SIA est thoracique à convexité droite.

L'influence du niveau segmentaire a été étudiée. Les segments thoraco-lombaires présentaient une hydratation supérieure, mais ce résultat reste une tendance non significative. L'hydratation augmentait avec l'âge et semblait se stabiliser chez les patients les plus âgés de cette cohorte. Une augmentation de 0.005 du ratio NP/AF chaque année d'âge supplémentaire a été observée sur l'intégralité de la cohorte. L'histologie montre que les DIV sont une structure particulière, dont le rôle est étroitement lié aux cartilages adjacents. Les cellules chondrocytes-like du NP produisent un collagène de type 2, alors que les cellules chondrocytes-like du cartilage et de l'AF produisent des collagènes de type 1 et 2 [START_REF] Roberts | Histology and Pathology of the human intervertebral disc[END_REF]. Contrairement à l'âge adulte, durant la première décennie de vie, on remarque la présence de vaisseaux sanguins dans l'AF et le cartilage adjacent. Leur nombre décroît progressivement [START_REF] Roberts | Histology and Pathology of the human intervertebral disc[END_REF]. Le contenu en eau dépend de la présence de protéoglycannes et des fibres de collagène formant un maillage rétentif [START_REF] Antoniou | The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration[END_REF][START_REF] Mwale | Quantitative MRI as a diagnostic tool of intervertebral disc matrix composition and integrity[END_REF]. Les protéoglycannes et l'eau d'un côté, et les fibres de collagène de l'autre, sont distribuées en opposition dans le DIV. Les premières augmentent alors que le collagène diminue de la périphérie vers le NP. On retrouve dans la littérature une différenciation entre la maturation et la dégénérescence, séparant des processus d'évolution avant 10 ans et après 20 ans [START_REF] Rajasekaran | ISSLS prize winner: a study of diffusion in human lumbar discs: a serial magnetic resonance imaging study documenting the influence of the endplate on diffusion in normal and degenerate discs[END_REF]. L'hydratation des DIV lombaires augmente avec la maturation pendant les deux premières décennies [START_REF] Krueger | Changes in T2 relaxation times associated with maturation of the human intervertebral disk[END_REF] puis diminue après 25 ans. L'hydratation d'un DIV est plus importante entre 2 et 5 ans, puis diminue après 25 ans [START_REF] Antoniou | The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration[END_REF], mais les données concernant les âges intermédiaires sont manquantes. Dans cette population pédiatrique symptomatique, une augmentation de l'hydratation entre 7 et 20 ans a été observée. Ces résultats devraient être applicables à la population d'enfants de 5 à 11 ans, cependant aucune IRM de rachis normal chez de jeunes enfants n'a pu être analysée. L'homéostasie du segment est due aux transports de masse entre le disque et les corps vertébraux à travers les plaques terminales vertébrales. La vascularisation du disque évolue progressivement depuis l'enfance jusqu'à disparaître à l'âge pré-adulte. Le disque présente de bonnes propriétés cliniques et mécaniques lorsque ces échanges sont bien établis. Le NP est très hydraté par rapport à l'AF. Par conséquent, l'exploration du rapport entre le volume de NP et le volume de DIV peut être considérée comme un indice de l'hydratation du disque et finalement elle peut être représentative de la santé du segment. En milieu clinique, l'hydratation est accessible par des séquences d'IRM. Dans le suivi des scolioses, l'association de l'IRM à l'hydratation discale permet d'explorer le comportement poro-mécanique des segments et peut guider la décision clinique (localisation et extension de l'instrumentation) et sa restauration post-opératoire pourrait fournir un indice permettant de prédire les complications cliniques ultérieures.

Cette étude est originale, mais présente certaines limites. La plupart des patients avaient une IRM pour un diagnostic urologique ou neurologique, mais 14% des patients de la cohorte ont eu une IRM pour exploration de lombalgies. Une pathologie du rachis a été infirmée par la lecture de l'imagerie par un radiologue spécialisé.

Comme il s'agit d'une étude rétrospective, l'heure de réalisation de l'IRM n'était pas contrôlée. Les acquisitions étaient généralement faites le matin pour éviter l'influence des activités quotidiennes. Cependant ce paramètre non contrôlé peut être considéré comme une randomisation favorable à la fiabilité des résultats. Toutes les données concernant nos patients n'ont pas pu être récupérées, notamment les données morphologiques telles que la cyphose, la lordose ou l'incidence pelvienne propre à chaque patient [START_REF] Rajasekaran | ISSLS prize winner: a study of diffusion in human lumbar discs: a serial magnetic resonance imaging study documenting the influence of the endplate on diffusion in normal and degenerate discs[END_REF][START_REF] Janssen | Sagittal spinal profile and spinopelvic balance in parents of scoliotic children[END_REF]. La maturité ainsi que le statut pubertaire restaient inconnus. Malgré ces limites, la taille de cette cohorte (98 examens), est acceptable, compte tenu des difficultés à la création d'une base de données pédiatriques.

V -Conclusion

La description des propriétés d'hydratation des DIV dans une population pédiatrique sans pathologie du rachis durant le pic de croissance est originale. Un groupe contrôle est proposé pour les études cliniques pour la première fois à notre connaissance. Le modèle générique établi en fonction du sexe, de l'âge et du niveau de segment était au coeur de l'étude. Il pourrait représenter une base de données contrôle pour l'étude du rôle des DIV dans l'évolutivité des courbures et la stabilité du rachis chez des adolescents atteints de SIA.

VII-Tableaux et figures

Tableau 1 -Distribution des variables quantitatives de la cohorte pédiatrique (n = 98). Les propriétés de volume et les hydratations sont classées par niveau de segment avec p25 : 25e percentile, p50 : 50e percentile, p75 : 75e percentile, DS : écart-type.

Tableau 2 -Description du modèle linéaire mixte multivarié de la cohorte pédiatrique. L'hydratation est explorée en fonction du niveau de segment, du groupe d'âge et du genre. Le témoin était l'hydratation du segment T12-L1.

Figure 1 -Figure 2 -

 12 Figure 1 -Segmentation semi-automatique et reconstruction automatique du volume (Biomechlab®) des DIV à partir de coupes IRM (L5-S1 à L1-L2)

Figure 3 -

 3 Figure 3-Répartition des schémas d'hydratation du DIV pour la cohorte pédiatrique (n = 49), par genre, âge et segment. La ligne continue représente le modèle de régression linéaire. L'aire continue représente l'intervalle de confiance à 95 % et la ligne pointillée représente les limites de l'intervalle de prévision à 95 %.

Remerciements

Nous remercions la Sofcot (France) pour la bourse attribuée à Manon Bolzinger et la Fondation Cotrel (Institut de France) pour son support constant à la recherche sur la scoliose idiopathique. Nous remercions également Jérôme Briot PhD, développeur du logiciel de traitement d'image Biomechlab®, pour son soutien efficace.


Philippe Gautret

Philippe Colson

Jean Christophe Lagier

Laurence Camoin-Jau

Audrey Giraud-Gatineau

Sophia Boudjema

Julie Finance

Hervé Chaudet

Didier Raoult
email: didier.raoult@gmail.com

Different pattern of the second outbreak of COVID-19 in Marseille, France

Keywords: COVID-19, seasonality, severity markers, lethality

Since its emergence in China at the end of 2019, SARS-CoV-2 spread worldwide with more than 29 Million cases of COVID-19 reported, as of 15 September, 2020. Currently, most cases have been reported from the US, India, Brazil and Russia (https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423 467b48e9ecf6). In Europe, since the beginning of the pandemic, the EU/EEA and the UK have reported 1,733,550 COVID-19 cases and 182,639 deaths, as of 2 August, 2020 (10% of all cases reported worldwide). In early April, the EU/EEA and the UK reached a peak in reported cases. Then the trend declined until June to reach a plateau. however in recent weeks there has been a resurgence, although it is currently lower than the first peak which occurred in April 1 . In France, the incidence of PCR-confirmed cases peaked on early April with 50.1/100,000 and declined to less than 10/100,000 in early May, however, on late July the incidence rose again over this threshold and reached 75/100,0000 in early September 2 .

In Marseille, the largest city in south of France and the second one in France, our institute adopted a strategy consisting in early and massive screening of SARS-CoV-2 infections and COVID-19 treatment using hydroxychloroquine and azithromycin, since the first case was documented. We recently described the characteristics of 3737 adult COVID-19 patients seen at our institute between 3 March and 27 April [START_REF] Lagier | Outcomes of 3,737 COVID-19 patients treated with hydroxychloroquine/azithromycin and other regimens in Marseille, France: A retrospective analysis[END_REF] . In this paper, we describe these characteristics in 743 new adult patients seen between 15 June and 15 August with the aim to investigate possible changes in the disease between these two time periods.

Starting from early March, our institute proposed massive testing of any person presenting at our facility regardless they had or not COVID-19 symptoms. On 16 March, the WHO Director-General called for testing every suspected COVID-19 case [START_REF]WHO Director-General's opening remarks at the media briefing on COVID-19 -16[END_REF] . In France, the generalization of SARS-CoV-2 PCR testing was officially authorized by the Ministry of Health on 4 May 5 . The number of individuals newly-tested at out institute for SARS-CoV-2 infection by PCR sharply increased from 765 per week in late February (week 9) up to 8105 per week at the peak at the end of March (week 13), and then progressively decreased to 1481 patients per week in early June (week 24) (Figure 1). It then increased again to reach 5007 per week on mid August (week 33). Interestingly, the proportion of individuals testing positive paralleled this curve with a slight shift of one week later. By contrast, numbers of deaths peaked to 33 per week during week 14 and progressively decreased to reach 0 week 24.

Compared to patients seen during late winter and spring, those seen in summer were significantly younger with a lower proportion of patients aged ≥65 years, and were more likely to be male, although differences were not marked (Table 1). No significant differences were seen with regards to anosmia and agueusia prevalence, according to period of study.

Hospitalization rate, proportion of patients transferred to ICU, and lethality rates were significantly lower in patients seen in summer than in those seen earlier. The mean Ct value of positive PCR results was significantly lower in patients seen in summer than in those seen earler, with a proportion of patients with high viral load (Ct≤ 16) tending to be higher in summer. Lymphocyte and platelet counts and fibrinogen and D-dimer levels were significantly lower in patients seen in summer as compared to those seen earlier.

The current resurgence of COVID-19 in Marseille that started in June shows a marked dissociation between numbers of cases and numbers of death. The number of deaths among COVID-19 patients seen at our institute did not show a significant rebound, so far, while the number of cases clearly re-increased. Since the kinetic of the proportion of individuals testing positive paralleled the total number of tested individuals, the current increase in the number of new patients is not an artifact due to an increase in the number of tested individuals. (Wilcoxon) [START_REF]Coronavirus disease 2019[END_REF] Fisher exact test was used to compare differences between proportions (unilateral test used when indicated). Quantitative data means were compared using Student's t-test or Wilcoxon's rank test.

Legend to figures

Figure 1 .

 1 Figure 1. Number of persons tested for SARS-CoV-2 (black curve), proportion testing

Table 1 . Characteristics of COVID-19 patients seen at Méditerannée Infection Institute according to seasons. Late winter and spring 3 March-27 April Summer 15 June-15 August P value 1

 1

	N patients	3737	768
	Mean age	45.3 years	39.2 years	<2.2 10 -16
		Sd=16.8	Sd=17.3
	<65 years	3284 (87.9%)	696 (90.6%)	0.0358
	≥65 years	453 (12.1%)	72 (9.4%)
	Male sex	1704 (45.6%)	406 (52.9%)	0.00028
	Hospitalization	673 (18.0%)	82 (10.7%)	9.499 10 -7
	Transfert to ICU	67 (1.8%)	7 (0.9%)	0.048 (unilat.)
	Death (all patients)	41 (1.1%)	1 (0.1%)	0.0098 (unilat.)
	Death (under 60 years)	0 (0%)	0 (0%)	-
	Anosmia	1442/3676 (39.2%)	249/644 (38.7%)	0.7931
	Agueusia	1389/3676 (37.8%)	255/644 (39.6%)	0.4032
	CT 2 value ≤16 at admission	113/3056 (3.7%)	36/700 (5.1%)	0.108
	Mean	25.0	23.6	4.051 10 -10
	Sd	5.3	5.1
	Mean lymphocyte counts (G/L)	1.8	1.6	1.15 10 -6
		Sd=0.9	Sd=0.8
	Mean platelet count (G/L)	233.7	225.9	0.005
		Sd=74.8	Sd=62.4

CT = cycle threshold

Funding source

None

different than that of patients seen earlier. Patients in the two periods do not present marked age and sex differences, but markers of severity are undoubtedly less prevalent in the summer period, associating with a 10 times decrease in the lethality rate from 1.1 to 0.1%. At the time of writing, it seems, as described in other diseases [START_REF] Nicolle | Destin des maladies infectieuses[END_REF] , that the start of the epidemic was characterized by higher lethality rates, in comparison with the current phase of the epidemic.

This could be possibly due to mutations of the virus. The current situation of the COVID-19 epidemic seems to be similar to that of other endemic coronavirus infections [START_REF] Su | Epidemiology, Genetic Recombination, and Pathogenesis of Coronaviruses[END_REF][START_REF] Yin | MERS, SARS and other coronaviruses as causes of pneumonia[END_REF] . Continuous analysis of epidemiological data is needed in the following months to confirm that this trend is continuous.

Author's note

Since this analysis was completed, and as of the 30 September 2020, 3557 additional patients were diagnosed at the Institut Méditerranée Infection with 33 deaths, resulting in an overall lethality rate of 0.8% in the 4325 patients seen between 15 June and 30 September 2020.

Competing interest

The authors declare that they have no competing interests.

Ethical approval

Approval was not required