PhD Muriel Abbaci
email: muriel.abbaci@gustaveroussy.fr

MD Angelica Conversano

PhD Frederic De Leeuw

PhD Corinne Laplace-Builhé

MD PhD Mazouni Chafika

Near-infrared fluorescence imaging for the prevention and management of breast cancer-related lymphedema: a systematic review

Keywords: breast cancer related lymphedema, indocyanine green, ARM procedure, lymphography, supermicrosurgery

Sentinel lymph node identification by near infrared (NIR) fluorescence with indocyanine green (ICG) is recognized in the literature as a useful technique. NIR fluorescence technology could become key in the prevention and management of lymphedema after axillary dissection for breast cancer. Here, we conducted a systematic review focusing on ICG imaging to improve lymphedema prevention and treatment after axillary surgery.

A systematic literature review was performed using MEDLINE and Embase to identify articles focused on ICG imaging for breast-cancer-related lymphedema (BCRL). Qualitative analysis was performed to summarize the characteristics of reported ICG procedures. In situ tissue identification and functionality assessment based on fluorescence signal were evaluated. Clinical outcomes were appraised when reported.

Studies relating to axillary reverse mapping, BCRL and upper limb supermicrosurgery combined with ICG imaging were identified using MEDLINE and Embase. We included a total of 33 relevant articles with a total of 2016 patients enrolled. ICG imaging for axillary reverse mapping was safe for all 951 included patients, with identification of arm nodes in 80% to 88% of patients with axillary lymph nodes dissection. However, the papers discuss the oncologic safety of the approach and how -regardless of the contrast agent -concerns limit its adoption. ICG lymphography is openly supported in BCRL management, with 1065 patients undergoing this procedure in 26 articles. The technique is reported for lymphedema diagnosis, with high sensitivity and specificity, staging, intraoperative mapping and patency control in lymphaticovenous anastomosis. The substantial advantages/disadvantages of ICG imaging procedures are finally described.

Introduction

The most prevalent cancer in the world is breast cancer, with 266 000 new cases in the US each year (SEER database). In fact, 12.4% of women in the world will have breast cancer.

The outcomes for patients with breast cancer are improved if they have access to a full range of treatment options in a multidisciplinary setting [START_REF] Roohan | Hospital volume differences and five-year survival from breast cancer[END_REF][START_REF] Guller | High hospital volume is associated with better outcomes for breast cancer surgery: analysis of 233,247 patients[END_REF]. Mastectomy and axillary dissection can damage the upper-limb lymphatic system, with undesirable consequences for its ability to remove excess water, proteins and other cell products, leading to their accumulation in the extracellular spaces of the arm and hand [START_REF] Muhammed | Lymphatic mapping and preoperative imaging in the management of post-mastectomy lymphoedema[END_REF]. It is a chronic and progressive disorder with internal and external signs of anomalous lymph transport and lymphatic system deficiency.

After axillary lymph node dissection (ALND), incidence of lymphedema is between 7% and 49%, and even after sentinel lymph node biopsy there is still a risk of lymphedema in up to 13% of cases [START_REF] Petrek | Lymphedema in a cohort of breast carcinoma survivors 20 years after diagnosis[END_REF][START_REF] Vieira Ra Da | Risk Factors for Arm Lymphedema in a Cohort of Breast Cancer Patients Followed up for 10 Years[END_REF][START_REF] Mclaughlin | Prevalence of lymphedema in women with breast cancer 5 years after sentinel lymph node biopsy or axillary dissection: patient perceptions and precautionary behaviors[END_REF][START_REF] Leidenius | The consequences of long-time arm morbidity in node-negative breast cancer patients with sentinel node biopsy or axillary clearance[END_REF]. Furthermore, when radiotherapy is added to ALND, the risk of lymphedema increases with an odds ratio of 2.74 [START_REF] Shaitelman | Radiation therapy targets and the risk of breast cancer-related lymphedema: a systematic review and network meta-analysis[END_REF]. Although the majority of lymphedema cases are reported to develop within one year following breast cancer surgery, it can develop at any time after surgery. It may be mild to severe and, if left untreated, arm lymphedema has a significant tendency to increase with time, both in terms of the volume of the edema and the stage of tissue fibrosis [START_REF] Gillespie | Breast cancer-related lymphedema: risk factors, precautionary measures, and treatments[END_REF]. For this reasons, efforts have been made to prevent the risk of severe damage to the lymphatic system at the time of surgery, by reducing indication for extensive surgery with axillary sentinel lymph node (SLN) biopsy in routine procedure.

Different agents have been used, based on various kinetics, such as blue dye, isotope and more recently indocyanine green (ICG). Thus, near-infrared (NIR) fluorescence seems to be a useful diagnosis tool for SLN identification in breast cancer surgery and promising results are reported in a hundred articles [START_REF] Niebling | A systematic review and metaanalyses of sentinel lymph node identification in breast cancer and melanoma, a plea for tracer mapping[END_REF]. In particular, real-time imaging to guide surgical procedures has attracted interest with the development of a new generation of NIR fluorescence devices used in combination with ICG [START_REF] Low | Fluorescence-guided surgery of cancer: applications, tools and perspectives[END_REF]. ICG is a heptamethine cyanine fluorophore, and is the only NIR fluorophore clinically approved by the FDA for ophthalmic angiography, and for determining cardiac output, hepatic function and liver blood flow. These approvals have been in place for several decades but ICG has not yet been granted approval in oncology. Nevertheless, ICG is the most reported fluorescent contrast agent for fluorescence-guided cancer surgery, primarily for liver surgery and SLN identification. ICG is safe to use, with very few side effects and low toxicity [START_REF] Obana | Survey of complications of indocyanine green angiography in Japan[END_REF]. The maximum spectral absorption of ICG is 800 nm (between 780 nm to 805 nm) and its emission peak is at 820 nm, allowing fluorescence imaging without autofluorescence background noise. The fluorescent dye is commonly injected by systemic administration but subcutaneous injection is also successfully reported for fluorescence imaging of lymphatic pathways and lymph nodes [START_REF] Ogata | Novel Lymphography Using Indocyanine Green Dye for Near-Infrared Fluorescence Labeling[END_REF][START_REF] Li | Precise intraoperative sentinel lymph node biopsies guided by lymphatic drainage in breast cancer[END_REF].

With real-time lymphatic pathways highlighting, ICG imaging could answer many expectations to reduce incidence and treat Breast Cancer Related Lymphedema (BCRL) [START_REF] Yamamoto | Versatility of indocyanine green lymphography navigation in lymphatic surgeries[END_REF]. The purpose of this systematic review is to give a complete overview of the major developments in ICG fluorescence imaging for BCRL to analyse the scope of clinical results and to provide directions for future research.

Methods and materials

Literature search strategy

The preferred reporting items for systematic reviews and meta-analyses (PRISMA) statement was followed [START_REF] Moher | Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement[END_REF]. A literature review was conducted until December 2018 with no restrictions on publication date, study design, population, type of breast cancer surgery or type of lymphatic surgery by MA and FDL. All titles and abstracts in English were retrieved from the search and screened and assessed for eligibility. Furthermore, the reference lists of the selected papers were reviewed for additional articles. Any discrepancy in the search was discussed between the two authors and consensus was reached following discussion. The search strategy is illustrated in Figure 1.

Data extraction

The extraction of data was based on study design, type of surgery, number of patients, ICG posology, type of NIR fluorescence camera, imaging protocol and optical signal analysis.

Information sources

The database search included the MEDLINE and Embase databases. clinicaltrial.gov was also used to find relevant sources. Although every attempt was made to eliminate redundancy in the data, we cannot rule out the possibility that one or more individuals may have participated in more than one study.

Inclusion criteria

To be included, studies had to meet the following criteria: (1) use of fluorescence ICG imaging, (2) treatment of breast cancer, (3) breast cancer related lymphedema, (4) in vivo data acquisition on human tissues, (5) article in English.

Exclusion criteria

The following studies were excluded: (1) ICG-SLN studies, (2) reviews, letters and comments on articles, (3) abstracts and proceedings.

Assessment of study quality

For the systematic review, all clinical studies were assigned a level of evidence (LOE) adapted from the Oxford Centre for Evidence-Based Medicine, on methodology and study design, and from the Centre for Reviews and Dissemination of the University of York. The assigned levels were LOE1, randomized control trial (1a) and quasi-experimental study (that is, non-randomized control trial, before and after study) (1b); LOE2, cohort study; LOE3, case-control study; LOE4, case series; and LOE5, case report.

Statistical analysis

The paucity of randomized control trials and prevalence of observational studies led to the decision to perform a qualitative synthesis using only descriptive statistics. No assessment of heterogeneity, publication bias or any other statistical assessment was planned.

Results

Study identification and characteristics

We identified 33 relevant articles with a total of 2016 patients enrolled. Most of the series were observational. Based on the definition of cohort studies and case series by Mathes et al. [START_REF] Mathes | Clarifying the distinction between case series and cohort studies in systematic reviews of comparative studies: potential impact on body of evidence and workload[END_REF] and Dekkers et al. [START_REF] Dekkers | Distinguishing Case Series From Cohort Studies[END_REF], 21% of clinical studies were cohort studies (LOE2) and 66% were case series (LOE3). Designs were: cohort study [START_REF] Leidenius | The consequences of long-time arm morbidity in node-negative breast cancer patients with sentinel node biopsy or axillary clearance[END_REF], case series [START_REF] Thompson | Axillary reverse mapping (ARM): a new concept to identify and enhance lymphatic preservation[END_REF] and case report (4).

Study design and level of evidence

Based on the literature, ICG imaging for BCRL prevention and management was evaluated for three applications after ICG SLN exclusion. The primary outcome in breast cancer surgery was the identification of arm lymph nodes and lymphatic ducts using NIR fluorescence when axillary reverse mapping was achieved during SLN procedures or axillary lymph node dissection. Two cohort studies and five case series reported data on the subject. The second clinical application evaluated was ICG lymphography for diagnosis, mapping and assessment of evolution of BCRL. Three cohort studies and five case series with n≥15 patients, and one case report patients were reviewed. The last approach described in two cohort studies, twelve case series and three case reports was NIR fluorescence-guided supermicrosurgery to support surgery for patients who were refractory to conservative treatment.

Arm lymph node identification by NIR fluorescence imaging

Since the introduction of SLN biopsy, indication for extensive ALND has been reduced, thereby reducing the incidence of arm lymphedema. However, ALND cannot be avoided in all patients with invasive breast cancer [START_REF] Woods | A Review of Options for Localization of Axillary Lymph Nodes in the Treatment of Invasive Breast Cancer[END_REF] and a meta-analysis showed that patients with ALND have incidence of lymphedema that is four times higher than that of patients with SLN [START_REF] Disipio | Incidence of unilateral arm lymphoedema after breast cancer: a systematic review and meta-analysis[END_REF]. Several studies have shown promising results of sparing arm nodes on reducing the incidence of lymphedema after ALND [START_REF] Ahmed | Systematic review of axillary reverse mapping in breast cancer[END_REF]. In 2007, axillary reverse mapping (ARM) was proposed by Thomson et al. [START_REF] Thompson | Axillary reverse mapping (ARM): a new concept to identify and enhance lymphatic preservation[END_REF] and Nos et al. [START_REF] Nos | Blue dye injection in the arm in order to conserve the lymphatic drainage of the arm in breast cancer patients requiring an axillary dissection[END_REF] to identify and preserve arm lymphatic drainage during ALND. Based on the theory that the arm and the breast each have specific paths of lymphatic drainage, the ARM nodes are identified by subcutaneous or intradermal injection of tracer into the arm prior to surgery, followed by a brief massage and elevation of the arm. The tracer then diffuses through the lymphatic ducts to the axillary lymph nodes draining the arm [START_REF] Shao | Axillary reverse mapping (ARM): where to go[END_REF]. Initial investigators used blue dye to identify the ARM nodes but showed low detection rates (61%-71%) of ARM nodes. Moreover, it was difficult to identify lymphatic networks [START_REF] Thompson | Axillary reverse mapping (ARM): a new concept to identify and enhance lymphatic preservation[END_REF][START_REF] Nos | Blue dye injection in the arm in order to conserve the lymphatic drainage of the arm in breast cancer patients requiring an axillary dissection[END_REF]. In addition, an inconvenience of blue dye injection was that staining at the injection site could persist over months. Furthermore, some surgeons are not keen to use blue dye given that hypersensitivity reactions are observed in up to 2% of cases [START_REF] Nos | Blue dye injection in the arm in order to conserve the lymphatic drainage of the arm in breast cancer patients requiring an axillary dissection[END_REF]. To improve the identification rate of ARM nodes and to prevent persistent blue staining at the injection site, ARM node detection using radioisotope injection and gamma probe has been described [START_REF] Britton | Lymphatic drainage pathways of the breast and the upper limb[END_REF]. However, the use of radioisotope alone does not enable the visualization of the lymphatic ducts, yet the purpose of ARM procedure is to preserve the lymphatic pathway.

First described by Noguchi et al. [START_REF] Noguchi | Axillary reverse mapping with indocyanine fluorescence imaging in patients with breast cancer[END_REF], NIR fluorescence imaging combined with ICG injection has since been reported in the breast oncological setting for ARM node identification and lymphatic visualization (Figure 2) in two cohort studies and five case series (Table 1).

Among the studies, we noticed variation in the volume of subcutaneously injected ICG and in injection sites [START_REF] Noguchi | Feasibility study of axillary reverse mapping for patients with clinically node-negative breast cancer[END_REF][START_REF] Sakurai | Axillary reverse mapping using fluorescence imaging is useful for identifying the risk group of postoperative lymphedema in breast cancer patients undergoing sentinel node biopsies[END_REF][START_REF] Foster | Axillary reverse mapping with indocyanine green or isosulfan blue demonstrate similar crossover rates to radiotracer identified sentinel nodes[END_REF][START_REF] Ikeda | The influence of axillary reverse mapping related factors on lymphedema in breast cancer patients[END_REF]. The fluorescence technique was always safe with ICG injection to be given at anaesthesia time to more than 2 hours before surgery [START_REF] Sakurai | Axillary reverse mapping using fluorescence imaging is useful for identifying the risk group of postoperative lymphedema in breast cancer patients undergoing sentinel node biopsies[END_REF]. Given that a lack of standardization of the protocol and the optimal dose of ICG to be injected, an objective comparison of the clinical results is not possible. In practice, no side effects were reported, and only a transitory green stain at the injection site (which disappeared within 10 days) was described by Noguchi et al. [START_REF] Noguchi | Axillary reverse mapping using a fluorescence imaging system in breast cancer[END_REF]. For a total of 951 patients, authors showed that ARM nodes can be identified in 80% to 88% of patients using NIR fluorescence during ALND, and to a lesser extent during SLN incision (21% to 50%) (Table 1). Tausch et al. reported failure to detect ARM nodes outside of the axilla or when there was a high tumour burden in the axilla [START_REF] Tausch | Can axillary reverse mapping avoid lymphedema in node positive breast cancer patients?[END_REF]. Moreover metastatic lymph nodes that limit ICG dye diffusion could also partly explain ARM node identification rate. The difference between ALND and SLN for ARM node identification may be explained by a reported localization of fluorescent ARM nodes mainly between the axillary vein and the second intercostobrachial nerve and close to the anterior edge of the latissimus dorsi muscle regardless of the contrast agent injected, as described by Ikeda et al. [START_REF] Ikeda | Evaluation of the metastatic status of lymph nodes identified using axillary reverse mapping in breast cancer patients[END_REF]. In terms of number of ARM nodes identified by fluorescence, we noted variation in the studies that we examined from 1.2 to 7.2 (Table 1). Higher numbers of identified lymph nodes might be explained by a long delay between ICG injection and axillary dissection, with diffusion of fluorescent dye along the subsequent lymph nodes. Not only the fluorescence technique was useful for detecting the ARM lymph nodes but also the lymphatic ducts (in 63% to 86% of cases in ALND) [START_REF] Noguchi | Axillary reverse mapping using a fluorescence imaging system in breast cancer[END_REF]. Difference in the visualization of the lymphatic ducts in the axilla may be related to the delay between the ICG injection and the lymphatic ducts imaging. In practice, when the drug-light interval increases, this delay may result in one lower rate of lymphatic vessels visualization. Besides, Foster et al. compared injection of blue dye and ICG for ARM node mapping and they reported better visualization of lymphatic pathways with ICG (39.1% vs 60.9%) [START_REF] Foster | Axillary reverse mapping with indocyanine green or isosulfan blue demonstrate similar crossover rates to radiotracer identified sentinel nodes[END_REF].

The authors CM and AC are breast specialists with a long experience in breast oncological surgery. They first used ICG for sentinel lymph node biopsy: the technique is considered easy to perform, intuitive, with a short learning curve [START_REF] Mazouni | Prospective evaluation of the limitations of near-infrared imaging in detecting axillary sentinel lymph nodes in primary breast cancer[END_REF]. No more operative time is necessary.

Transposition of the same procedure to perform axillary reverse mapping was the natural progression of surgical skills. They conducted a recent clinical trial (NCT02994225) in which ARM node detection and excision were fast and in the surgery time procedure, simple and available for almost 99% of patients. Training procedure is short (two patients) and surgeons get experienced after only few procedures for selecting fluorescent lymph node from lymphatic leak and vessels around axillary and thoracic veins. Most of the clinical studies used the handheld PDE (photodynamic eye, Hamamatsu Photonics) fluorescence imaging system (Table 1). Given that a new generation of NIR cameras with more sensitive detectors is now available, the quality of lymphatic mapping using ICG may increase in a near future.

Technically, the routine use of radioisotope Tc 99m to identify SLNs could be combined with ICG ARM procedure to preserve the lymphatic system of the arm when ALND is required.

Both methods could be complementary and not competitive when radioisotope and blue dye are also injected for SLN procedures. Despite the promising results, there are some aspects that need to be considered before fluorescence ARM procedure can be used in routine clinical practice in the surgical oncology setting. Recent reviews have discussed the oncologic safety of ARM procedures, independent of the injected tracer (blue dye, radioisotope and ICG), as the major issue for technique adoption [START_REF] Ngui | Axillary reverse mapping in patients with breast cancer: Is it oncologically safe?[END_REF][START_REF] Han | The Feasibility and Oncological Safety of Axillary Reverse Mapping in Patients with Breast Cancer: A Systematic Review and Meta-Analysis of Prospective Studies[END_REF]. To date, predictors for metastatic ARM node are poorly studied but a recent meta-analysis by Han et al. of 24 prospective studies reported a metastatic rate of ARM nodes of 16.9% (95% CI, 14.2%-20.1%) [START_REF] Han | The Feasibility and Oncological Safety of Axillary Reverse Mapping in Patients with Breast Cancer: A Systematic Review and Meta-Analysis of Prospective Studies[END_REF]. Ikeda et al. showed (in 98 patients followed for up to 3 years after ALND and ICG ARM procedure) that patients with metastatic ARM nodes identified using fluorescence were significantly more affected by lymphedema than patients with ARM nodes without involvement (42% vs 13%) [START_REF] Ikeda | The influence of axillary reverse mapping related factors on lymphedema in breast cancer patients[END_REF]. At last, Noguchi et al. [START_REF] Noguchi | Is axillary reverse mapping feasible in breast cancer patients?[END_REF] and Schunemann et al. [START_REF] Doria | Prospective study evaluating oncological safety of axillary reverse mapping[END_REF] studied the link between the clinical status of lymph nodes, pNx and ARM node involvement. The reviewing of published clinical results argues for the contraindication of ARM procedure for patients with clinically node positive due to oncological concern. They proposed the ARM procedure for patients with cN0 and SLN-positive, when the arm node is not the SLN, as arm node involvement is reported in most cases from cN1.Only randomized controlled studies can determine whether the ARM technique is secure and may reduce lymphedema when ARM nodes are preserved in ALND with a selected population, as proposed by Parks et al. [START_REF] Parks | Axillary reverse mapping in N0 patients requiring sentinel lymph node biopsy -A systematic review of the literature and necessity of a randomised study[END_REF]

ICG imaging in lymphedema mapping and staging

Diagnosis and staging of lymphedema are usually based on patients' reported experiences (for example, pain and heaviness), characteristic physical findings (for example, mobility, arm volume and arm circumference) and standard lymphoscintigraphy with the injection of radioisotope-labelled colloids into the interdigital space to monitor the lymphatic flow along the limb using a gamma camera. Lymphoscintigraphy is the gold standard but it is timeconsuming and expensive, requiring licensing and a nuclear medicine department. In addition, its poor spatial resolution makes detailed analysis of the lymphatic ducts impossible [START_REF] Visconti | Innovative surgical treatment of peripheral lymphedema after breast cancer surgery[END_REF]. ICG fluorescence imaging of the lymphatic pathway of the upper limb was first described by Yamamoto et al. for the diagnosis of lymphedema without exposure to radiation [START_REF] Yamamoto | Indocyanine green-enhanced lymphography for upper extremity lymphedema: a novel severity staging system using dermal backflow patterns[END_REF].

ICG lymphography for lymphedema mapping and staging has been described in BCRL, in three cohort studies, five case series and one case report [START_REF] Hara | Blocking of the Lymphatic Vessel in Lymphedema[END_REF], corresponding to data from 449 patients (Table 2). The protocols used in the studies that we evaluated show slight differences to each other but ICG was typically injected subcutaneously into the web spaces of the hand and then the fluorescent dye diffusion was measured in the lymphatic ducts from the hand to the axilla (Table 2). Fluorescence imaging of collectors, precollectors, and capillary channels can be conducted from multiple angles, in real-time and in a user friendly way. A temporary green stain at the injection sites is reported after two weeks [START_REF] Narushima | Indocyanine Green Lymphography Findings in Limb Lymphedema[END_REF]. Mihara et al. reported sensitivity and specificity of 100% for ICG lymphography for secondary lymphedema [START_REF] Mihara | Indocyanine green (ICG) lymphography is superior to lymphoscintigraphy for diagnostic imaging of early lymphedema of the upper limbs[END_REF].

Moreover, the results of a study by Akita et al. suggested that ICG lymphography could be useful for detecting lymphatic disorder before clinical symptoms, such as arm volume change, thereby increasing the chance of disease regression by compression therapy at an early stage [START_REF] Akita | Early Detection of Lymphatic Disorder and Treatment for Lymphedema following Breast Cancer[END_REF]. In practice, for lymphatic vessels of the healthy limb, a fluorescent linear pattern is usually observed a few minutes after dye injection. In the case of lymphedema, Yamamoto et al. established a severity staging system, the arm dermal backflow (ADB) stage, to classify ICG data into five stages (I to V) [START_REF] Yamamoto | Indocyanine green-enhanced lymphography for upper extremity lymphedema: a novel severity staging system using dermal backflow patterns[END_REF]. The ADB stage is qualitatively subdivided into three categories, ordered splash, stardust and finally diffuse. When many patterns are visible on the arm, the most severe pattern is selected for the final diagnosis. However, ICG lymphography cannot be used to detect lymph vessels at a depth of more than 1 cm below the skin surface [START_REF] Mihara | Indocyanine green (ICG) lymphography is superior to lymphoscintigraphy for diagnostic imaging of early lymphedema of the upper limbs[END_REF]. In addition, Zaleska et al. reported that ICG diffuses rapidly through the lymphatic wall and then may combine with tissue proteins, resulting in inaccurate pictures of lymphatics [START_REF] Zaleska | Imaging lymphatics in human normal and lymphedema limbs-Usefulness of various modalities for evaluation of lymph and edema fluid flow pathways and dynamics[END_REF].

In terms of quantitative measurement, Yamamoto et al. proposed two parameters to assess lymphatic function with the determination of ICG velocity (i.e dye diffusion distance from the injection point divided by time) and the transit time after subcutaneously ICG injection in 15 patients with BCRL [START_REF] Yamamoto | Dynamic indocyanine green (ICG) lymphography for breast cancer-related arm lymphedema[END_REF]. In the future, real-time ICG lymphatic flow imaging during massage for decongestive lymphatic therapy could indicate to the therapist the compression force to apply to the tissues to move the lymphatic fluid [START_REF] Tan | Assessment of lymphatic contractile function after manual lymphatic drainage using near-infrared fluorescence imaging[END_REF][START_REF] Zaleska | Indocyanine green near-infrared lymphangiography for evaluation of effectiveness of edema fluid flow under therapeutic compression[END_REF][START_REF] Lopera | Investigating the short-term effects of manual lymphatic drainage and compression garment therapies on lymphatic function using near-infrared imaging[END_REF].

NIR fluorescence-guided lymphatic surgery

The quality of fluorescence images, and the suitability and safety of ICG lymphography have persuaded surgeons to support lymphatic surgery. Lymphaticovenular anastomosis (LVA) and lymph node transfer (LNT) are now proposed to patients who are refractory to conservative treatment. Intraoperative ICG imaging for physiological reconstructive surgery has been reported for a total of 616 patients: two cohort studies, twelve case series (of which four included less than 15 patients [START_REF] Mihara | Upper-Limb Lymphedema Treated Aesthetically with Lymphaticovenous Anastomosis using Indocyanine Green Lymphography and Noncontact Vein Visualization[END_REF][START_REF] Liu | The use of a microscope with near-infrared imaging function in indocyanine green lymphography and lymphaticovenous anastomosis[END_REF][START_REF] Yang | Intraoperative identification and definition of "functional" lymphatic collecting vessels for supermicrosurgical lymphatico-venous anastomosis in treating lymphedema patients[END_REF][START_REF] Yamamoto | Navigation Lymphatic Supermicrosurgery for the Treatment of Cancer-Related Peripheral Lymphedema[END_REF] ) and three were case reports [START_REF] Yamamoto | Minimally Invasive Lymphatic Supermicrosurgery (MILS) Indocyanine Green Lymphography-Guided Simultaneous Multisite Lymphaticovenular Anastomoses via Millimeter Skin Incisions[END_REF][START_REF] Yamamoto | Supermicrosurgical deep lymphatic vessel-to-venous anastomosis for a breast cancer-related arm lymphedema with severe sclerosis of superficial lymphatic vessels[END_REF][START_REF] Shih | Use of Indocyanine Green-SPY Angiography for Tracking Lymphatic Recovery After Lymphaticovenous Anastomosis[END_REF] (Table 3).

LVA corresponds to an anastomosis created between the lymphatic and venous system to improve lymphatic flow [START_REF] Garza | A comprehensive overview on the surgical management of secondary lymphedema of the upper and lower extremities related to prior oncologic therapies[END_REF][START_REF] Scaglioni | Systematic review of lymphovenous anastomosis (LVA) for the treatment of lymphedema[END_REF]. In practice, ICG can be injected for preoperative staging and again for intraoperative mapping of superficial lymphatic ducts with good spatial resolution of images. First, individual lymphatic ducts can be detailed quickly by the NIR fluorescence signal and guide surgeons in real-time to mark (with a pen) appropriate lymphatic pathways and incision sites [START_REF] Chang | A Prospective Analysis of 100 Consecutive Lymphovenous Bypass Cases for Treatment of Extremity Lymphedema[END_REF]. As a consequence, operative time are usually reduced with fluorescence-guided supermicrosurgery [START_REF] Yamamoto | Minimally Invasive Lymphatic Supermicrosurgery (MILS) Indocyanine Green Lymphography-Guided Simultaneous Multisite Lymphaticovenular Anastomoses via Millimeter Skin Incisions[END_REF]. In ICG-guided LVA, regions in which linear patterns are visible are generally advised for microsurgery, as incisions in these areas are likely to reveal appropriate, typically nonsclerotic, lymphatic vessels, particularly in patients with early-stage lymphedema [START_REF] Seki | The dynamic-lymphaticovenular anastomosis method for breast cancer treatment-related lymphedema: Creation of functional lymphaticovenular anastomoses with use of preoperative dynamic ultrasonography[END_REF]. However, Yang et al. pointed out that an incorrect ICG injection could fail to detect some functional lymphatic ducts [START_REF] Yang | Intraoperative identification and definition of "functional" lymphatic collecting vessels for supermicrosurgical lymphatico-venous anastomosis in treating lymphedema patients[END_REF]. Then, imaging with handheld NIR fluorescence camera can be completed using intraoperative NIR fluorescence microscopy for dissection guidance thanks to high-resolution fluorescence signal from lymphatic vessels [START_REF] Liu | The use of a microscope with near-infrared imaging function in indocyanine green lymphography and lymphaticovenous anastomosis[END_REF][START_REF] Yamamoto | Near-infrared illumination systemintegrated microscope for supermicrosurgical lymphaticovenular anastomosis[END_REF]. Finally, ICG diffusion inside the vein can be used as an aid to control intraoperative anastomosis patency. After the surgery, regression of lymphedema severity can be monitored by reproducible ICG lymphography without side effects over months [START_REF] Chen | Indocyanine Green Lymphographic Evidence of Surgical Efficacy Following Microsurgical and Supermicrosurgical Lymphedema Reconstructions[END_REF]. For repeatable ICG lymphography, Chen et al. mentioned the importance of a standardized protocol (posology and camera) and the need to use the same distance between the camera and the limb at each imaging session [START_REF] Chen | Indocyanine Green Lymphographic Evidence of Surgical Efficacy Following Microsurgical and Supermicrosurgical Lymphedema Reconstructions[END_REF]. Studies showed a reduction in arm volume of between 33% and 61% after 12 months and 38% after 3 years in patients treated by ICG-guided LVA [START_REF] Chang | A Prospective Analysis of 100 Consecutive Lymphovenous Bypass Cases for Treatment of Extremity Lymphedema[END_REF][START_REF] Winters | The efficacy of lymphaticovenular anastomosis in breast cancer-related lymphedema[END_REF]. In the literature, the long-term impact of ICG administration on the skin layers and on the components of lymphatic vessels and lymph nodes has not yet been studied but such data would be valuable for patients with lymphedema. Then, the repeated intradermal or subcutaneous ICG injection should not be prescribed, today, to patients with advanced lymphedema. The follow up of patients with early stage of lymphedema by ICG lymphography may be proposed with precautions.

However, when patients had only critical patterns of the ICG lymphography staging (that is, stardust and diffuse ADB), LVA is less satisfactory [START_REF] Liu | The use of a microscope with near-infrared imaging function in indocyanine green lymphography and lymphaticovenous anastomosis[END_REF]. In cases of severe lymphedema, surgeons may not find suitable lymphatic ducts for LVA. Thus, LNT can be proposed to reduce symptoms of lymphedema [START_REF] Tourani | Vascularized Lymph Node Transfer[END_REF]. Namely, LNT is indicated to reconstruct the damaged or missing lymphatic tissue [START_REF] Becker | Microlymphatic Surgery for the Treatment of Iatrogenic Lymphedema[END_REF]. Lymph nodes are harvested from a donor site and transferred as a free flap to the lymphedematous limb [START_REF] Tourani | Vascularized Lymph Node Transfer[END_REF][START_REF] Becker | Microlymphatic Surgery for the Treatment of Iatrogenic Lymphedema[END_REF]. For instance, few studies have described intraoperative ICG imaging to demonstrate lymph collecting in recipient sites by subdermal injection of fluorescent dye (0.3ml-0.6ml) proximal to the flap [START_REF] Patel | From Theory to Evidence: Long-Term Evaluation of the Mechanism of Action and Flap Integration of Distal Vascularized Lymph Node Transfers[END_REF] or by injection of 0.05ml ICG directly into the lymph nodes [START_REF] Cheng | The Mechanism of Vascularized Lymph Node Transfer for Lymphedema[END_REF].

Future perspectives in breast cancer related lymphedema management with optical tools

With the prolongation of patients' survival thanks to early diagnosis and modern tools, lymphedema management is becoming a major health problem following cancer treatment. Despite the value of ICG as a contrast agent for lymph node identification and management of BCRL, the dye is used 'off-label' for these applications (it is not FDA approved for these applications), limiting its adoption by medical centers. Intradermal or subcutaneous injection of ICG is not associated with an increase in adverse events or intra-or postoperative complications (for example, wound healing or tissue retraction). ICG imaging is however limited to superficial layers and cannot provide data for high body mass index patients. Then, the lymphatic vasculature is a complex network and some preclinical studies highlight a possible physiological state perturbation of lymphatic function when an exogenous agent such as ICG is introduced [START_REF] Weiler | Differential transport function of lymphatic vessels in the rat tail model and the long-term effects of Indocyanine Green as assessed with near-infrared imaging[END_REF]. Intraluminal pressure might also be modified by injection of such exogenous agents [START_REF] Blatter | Simultaneous measurements of lymphatic vessel contraction, flow and valve dynamics in multiple lymphangions using optical coherence tomography[END_REF]. Table 4 presents final pros and cons for ICG imaging in BCRL prevention and management.

Although there is reasonable data to suggest that ICG can guide the surgeons in ARM procedure to preserve lymphatic network and in supermicrosurgery to identify lymphatic ducts, the postoperative function of lymphatic vessels must be correlated to any improvements in hard clinical end-points through RCTs. The 2016 consensus document of the international society of lymphology reaffirms the importance of lymphoscintigraphy as current standard imaging. The document reports also the increasing use of ICG lymphography in medical centers and the superficial lymphatic imaging by ICG which is critical for proper assessment of arm lymphatic flow (table 4). The authors argue on the interest in combining imaging methods to add scientific rigor to the analysis of patients outcomes and encourages research in near-infrared fluorescence imaging field based on valuable randomized trials. For instance, identification of functioning lymphatic vessels was the most challenging step in the LVA procedure until the introduction of ICG imaging with handheld cameras for lymphatic mapping and selection of incision sites.

ICG administration routes described in the 33 clinical studies listed were heterogeneous in both ARM procedure, ICG lymphography and ICG guided supermicrosurgery. Intradermal (ID) (ie. into the dermis, just below the epidermis), subcutaneous (SC) (i.e into the adipose tissue layer just below the epidermis and dermis) and intracutaneous injection (IC) (i.e into the skin, indeed, it is unclear as to whether it was ID or SC). We must point out that ID and SC will lead to different results if the lymphatic vessels function has to be evaluated.

Comparison between two and one injection to view the lymphatic vascular network has not been yet evaluated in the literature. A procedure including two injections (using the same administration route) is more likely adapted for the mapping of the lymphatic vascular network (and more precise than a single injection). Guidelines about fluorescence imaging in BCRL are needed to setup reference protocol for ICG administration according to the application to be considered. Then the time interval between the administration of ICG and the imaging may be short if the physician only needs to look at the lymphatic network but may become longer for lymphatic vessels functional assessment with a significant impact on the operative time.

There is a need for substantial evidence regarding the effect of the fluorescence procedures on the incidence of lymphedema when ICG ARM node is preserved, the benefit of ICG lymphography to conservative treatment with a long-term follow-up on lymphedema index scores, and the impact of ICG imaging for LVA on surgery times (skin incisions and dissections) and anastomosis patency. Fluorescence imagers are relatively expensive but one device can be used across a general surgery department for other applications such as SLN mapping [START_REF] Mazouni | Prospective evaluation of the limitations of near-infrared imaging in detecting axillary sentinel lymph nodes in primary breast cancer[END_REF], flap perfusion assessment and endocrine surgery [START_REF] De Leeuw | Intraoperative Near-infrared Imaging for Parathyroid Gland Identification by Auto-fluorescence: A Feasibility Study[END_REF][START_REF] Abbaci | Parathyroid gland management using optical technologies during thyroidectomy or parathyroidectomy: A systematic review[END_REF]. Another point is that ICG imaging may be efficient but an optical method with deeper imaging, a higher spatial resolution and real-time information would be optimal. In consequence, new optical tools are under development for lymph node and lymphatic vessel imaging, and when possible by label-free techniques. Among them, NIR II fluorescence, optical coherence tomography (OCT) [START_REF] Blatter | Simultaneous measurements of lymphatic vessel contraction, flow and valve dynamics in multiple lymphangions using optical coherence tomography[END_REF] and photoacoustic imaging [START_REF] Martel | Photoacoustic lymphatic imaging with high spatialtemporal resolution[END_REF][START_REF] Forbrich | Photoacoustic imaging of lymphatic pumping[END_REF] have shown promising results in preclinical studies. First, recent animal studies report imaging at greater depths than previous studies and with comparable spatial resolution when specific fluorescent dyes are illuminated in the NIR II spectrum (from 1000nm to 1700nm). Vascular fluorescence imaging in the NIR II window is significantly improved due to less light scattering in tissues and a higher signal-to-noise ratio at these longer wavelengths [START_REF] Starosolski | Indocyanine green fluorescence in second near-infrared (NIR-II) window[END_REF][START_REF] Alam | Emerging Intraoperative Imaging Modalities to Improve Surgical Precision[END_REF]. Interestingly, ICG also exhibits a fluorescence signal in NIR II [START_REF] Starosolski | Indocyanine green fluorescence in second near-infrared (NIR-II) window[END_REF] and this novel approach could be evaluated for deep lymphatic imaging [START_REF] Carr | Shortwave infrared fluorescence imaging with the clinically approved near-infrared dye indocyanine green[END_REF]. Then, identification of the ICG ARM node could be combined with in situ OCT imaging to determine the lymph node status and to decide its surgical preservation [START_REF] Nguyen | Optical Coherence Tomography: The Intraoperative Assessment of Lymph Nodes in Breast Cancer[END_REF]. OCT based on low-coherence interferometry works in a similar manner to conventional ultrasound but uses light instead of sound waves [START_REF] Huang | Optical coherence tomography[END_REF]. Ex vivo OCT imaging of lymph nodes has already shown promise [START_REF] Nolan | Intraoperative optical coherence tomography for assessing human lymph nodes for metastatic cancer[END_REF][START_REF] Grieve | Assessment of Sentinel Node Biopsies With Full-Field Optical Coherence Tomography[END_REF] and, combined with Doppler capabilities, lymph flow, lymphatic vessel contraction and lymph valve dynamics, can also be followed at high resolution, enabling the refinement of quantitative imaging to better understand lymphatic function [START_REF] Blatter | Simultaneous measurements of lymphatic vessel contraction, flow and valve dynamics in multiple lymphangions using optical coherence tomography[END_REF]. Finally, Photoacoustic imaging (PAI) uses ultrasound for image acquisition, while photons are used for signal generation [START_REF] Grootendorst | Optical techniques for the intraoperative assessment of nodal status[END_REF]. Few preclinical studies have described the use of this technology to image lymphatic systems but PAI imaging has been demonstrated to a depth of 5mm and at a resolution of 110µm [START_REF] Martel | Photoacoustic lymphatic imaging with high spatialtemporal resolution[END_REF][START_REF] Forbrich | Photoacoustic imaging of lymphatic pumping[END_REF].

Conclusion

NIR fluorescence imaging is gaining interest in breast surgery, mainly for SLN identification but also for ARM node identification in axillary lymph node dissection and supermicrosurgery following BCRL. At present, it is not possible to conclude on its advantages over standard clinical techniques but the contribution of multidimensional realtime imaging that does not use a radiolabelled probe could undoubtedly provide new possibilities for BCRL prevention and management. Other non-invasive optical techniques to enable surgeons to accurately visualize the lymphatic system and flow (such as OCT, Doppler OCT and photoacoustic imaging) are also evolving rapidly, with intraoperative applications expected in the future.

Figure 1 :

 1 Figure 1: Systematic record selection.

Figure 2 :

 2 Figure 2: A. Fluorescence signal in a lymphatic pathway of the arm after intracutaneous ICG

Table 1 :

 1 Clinical studies reporting ICG fluorescence imaging during the axillary reverse mapping procedure..

	Authors	Number of	ICG	Imaging	Number of cases	Number	Cases with	Study	LOE
		patients	administration	system	with ARM node	of ARM	fluorescence imaging	design
					identification	nodes	of lymphatic
						identified	pathways (%)
	Noguchi [26]	20	0.1ml (0.25mg) inner side of the wrist	PDE	In SLNf: 6/12 (50%) In ALND: 7/8 (88%)	1.2 2.7	42% 63%	Case series	4
	Noguchi [31]	131	0.1ml (0.25mg) inner side of the wrist	PDE	In SLNf: 42/97 (43%) In ALND: 29/34 (85%)	1.7 5.3	19% 76%	Case series	4
	Ikeda [33]	60	0.5-1ml (5mg) at the upper inner ipsilateral arm	PDE	In ALND: 48/60 (80%)	1.6	76% without prior 86% with prior SNLB SLNB	Case series	4
			0.5-1ml (5mg)
	Ikeda [30]	98	at the upper inner ipsilateral	PDE	In ALND: 80/98 (82%)	1.6 LE-1.9 LE+	83%	Case series	4
			arm
	Sakurai [28]	327	0.15 ml subcutaneously into the interdigital area	PDE	In SLNf: 77/372 (21%) (SLN=ARM node)	NA	32%	Cohort study	2
	Noguchi [27]	292	0.1ml (0.25mg) inner side of the wrist	PDE	In SLNf: 90/292 (31%)	7.2	22%	Cohort study	2
			0.5-3mg/ml
			medial aspect		In SLNf: 8/23 (35%)
	Foster [29]	23	of the proximal arm at the	SPY	and 7/23 (31% after SLN	NA	61%	Case series	4
			intermuscular		resection)
			groove

Table 2 :

 2 Clinical studies (with n≥15 patients) on ICG lymphography for diagnosis and staging of breast cancer-related lymphedema. extremities at the second web space of the hand and the ulnar border of the palmaris longus tendon at the level of the wrist

	Authors	Patients selection	ICG administration	Imaging time	Image information	Study design	LOE
	Yamamoto [41]	Secondary unilateral UE-LED after breast resection and ALND ±	0.1 ml ICG 0.25% injected subcutaneously into the bilateral upper Immediately after injection	First UE-LED classification by ADB stage	Case series
		radiotherapy
					100 % specificity for ICG,
	Mihara [44]	Secondary unilateral UE-LED, ISL stage 1, without dropout	0.2 ml ICG 0.5% injected intracutaneously into the bilateral second interdigits	Few minutes after injection	MRI, CT and LSc and 100 % with unaffected limb as sensitivity for ICG and MRI	Case series
					control.
	Yamamoto [47]	Secondary unilateral UE-LED after mastectomy and ALND	0.1 ml 0.25% ICG injected subcutaneously into the second web space of the hand	Immediately after injection	Description of based on ICG signal quantification parameters	Case series
	Akita [45]	Breast cancer surgery±ALND or SLN	0.3 ml ICG injected subcutaneously into the first web space of the dorsal aspect of the hand	1 hour after injection	Observation of lymphatic before arm volume changes dysfunction with ICG,	Prospective cohort study
			0.2 ml ICG 0.5% intracutaneously injected into		Imaging of accessory
	Tashiro [83]	UE-LED after tumor resection	the second interdigits of the hand and the ulnar border of the palmaris longus tendon at the	15 to 24 hours after injection	lymphatic pathways in axillary region and UE with	Case series
			level of the wrist		ICG
	Zaleska [46]	UE-LED stage II to IV, post inflammatory, post traumatic, post-surgery (mastectomy)	0.2 ml 0.5% ICG injected subcutaneously into the second and fourth interdigital spaces of the hand	Few minutes after injection	Description of clinical data lymphography provided by ICG	Retrospective cohort study
	Zaleska [49]	UE-LED after mastectomy	0.2 ml 0.5% ICG injected subcutaneously into the second and fourth interdigital spaces of the hand	Few minutes after injection	Imaging of fluid flow by ICG manual drainage lymphography to support	Case series
					Diagnostic accuracy of
	Qin [84]	Suspicion of unilateral UE-LED	0.1 ml ICG 0.25% injected into the distal arm at and one injection in the volar wrist three separate sites: two interdigital injections	After injection after and again 6 hours	bioimpedance spectroscopy lymphography as reference in comparison with ICG	Retrospective cohort study
					procedure

Table 3 :

 3 Clinical studies (with n>15 patients) on ICG lymphography-guided supermicrosurgery for breast cancer-related lymphedema

Acknowledgements:

The authors thank the Canceropôle Ile de France for funding the ARMoNIC study (NCT02994225).

Authors


Delphine Girlich

Victor Grosperrin

Thierry Naas

Dr Laurent Dortet
email: laurent.dortet@aphp.fr

CHROMagar™ ESBL / mSuperCARBA bi-plate medium for detection of ESBL-and carbapenemase-producing Enterobacteriaceae from spiked stools

Keywords: carbapenemases, NDM, sensitivity, specificity, selective medium, chromogenic

The recently released CHROMagar™ ESBL/CHROMagar™ mSuperCARBA biplate medium was evaluated for the detection of ESBL-and carbapenemaseproducing Enterobacteriaceae. Spiked stools were used to mimic in vivo stool colonization. Two-hundred enterobacterial isolates were tested. Respective sensitivities of 93.9% and 97.8% were obtained for the detection of ESBL and carbapenemase producers.

Resistance to broad-spectrum cephalosporins and subsequently to carbapenems spreads rapidly among Enterobacteriaceae at an alarming rate, resulting in both nosocomial and community-acquired infections primarily due to the production of extended-spectrum β-lactamases (ESBLs) and/or carbapenemases [START_REF] Pitout | Extended-spectrum beta-lactamaseproducing Enterobacteriaceae: an emerging public-health concern[END_REF][START_REF] Rood | Review: Molecular detection of extended spectrum-beta-lactamase-and carbapenemase-producing Enterobacteriaceae in a clinical setting[END_REF]. Targeted surveillance of high-risk patients and screening are essential to prevent outbreaks of nosocomial infections by these organisms. In Enterobacteriaceae, clinically relevant carbapenemases belong to the Ambler class A (KPC-type), Ambler class B metallo-β-lactamases (NDM, VIM and IMP), and Ambler class D enzymes (OXA-48-like) [START_REF] Nordmann | Carbapenem resistance in Enterobacteriaceae: here is the storm![END_REF]. The level of resistance to carbapenems conferred by these carbapenemase producers may vary widely, making their detection difficult when just based on their in vitro susceptibility profile [START_REF] Landman | Susceptibility profiles, molecular epidemiology, and detection of KPC-producing Escherichia coli isolates from the New York City vicinity[END_REF]. ESBL are enzymes that mediate resistance to penicillins, extended-spectrum third generation cephalosporins (3GC) and monobactams. ESBL-producing Enterobacteriaceae appeared in the 1980s, and have since become highly prevalent in nosocomial infections, mainly due to Escherichia coli and Klebsiella spp. ChromID ESBL (bioMérieux) and CHROMagar™ ESBL (CHROMagar, Paris, France) are very useful for the detection of ESBL enterobacterial producers, nevertheless their ability to detect isolates producing OXA-48-like carbapenemase alone is limited [START_REF] Carrer | Use of ChromID extendedspectrum beta-lactamase medium for detecting carbapenemaseproducing Enterobacteriaceae[END_REF][START_REF] Hornsey | Evaluation of three selective chromogenic media, CHROMagar ESBL, CHROMagar CTX-M and CHROMagar KPC, for the detection of Klebsiella pneumoniae producing OXA-48 carbapenemase[END_REF].

Consequently, the detection of all carbapenemase-producing Enterobacteriaceae (CPE), including OXA-48-like producers, is based on the inoculation of at least two media able to efficiently identify (i) all types of carbapenemases with the exception of OXA-48-like enzymes (e.g. ChromID ®

CARBA medium, bioMérieux) and (ii) specifically OXA-48-like producers (e.g.

ChromID ® OXA-48 medium, bioMérieux). These two media are now marketed in a single bi-plate, the ChromID ® CARBA SMART medium that has been reported to efficiently detect all CPEs. However, this medium is designed for the screening of CPEs and not for ESBL-producers.

Another medium had already been developed for the detection of all CPEs, including OXA-48-like carbapenemase-producers, the SUPERCARBA medium [START_REF] Nordmann | Detection of carbapenemase producers in Enterobacteriaceae by use of a novel screening medium[END_REF]. The initial SUPERCARBA medium was further modified to improve its stability and facilitate the presumptive identification of the enterobacterial species by the addition of chromogenic molecules. This improved medium, named mSuperCARBA (CHROMagar Ltd., France), was demonstrated to be reliable for the screening of CPE [START_REF] Garcia-Fernandez | CHROMagar mSuperCARBA performance in carbapenem-resistant Enterobacteriaceae isolates characterized at molecular level and routine surveillance rectal swab specimens[END_REF][START_REF] Garcia-Quintanilla | CHROMagar mSuperCARBA and RAPIDEC(R) Carba NP test for detection of carbapenemase-producing Enterobacteriaceae[END_REF][START_REF] Genc | Chromogenic culture media or rapid immunochromatographic test: Which is better for detecting Klebsiella pneumoniae that produce OXA-48 and can they be used in blood and urine specimens[END_REF][START_REF] Soria Segarra | Utility of CHROMagar mSuperCARBA for surveillance cultures of carbapenemase-producing Enterobacteriaceae[END_REF]. However, as observed with the ChromID ® CARBA SMART medium, this single plate medium is not suitable for screening of ESBL producers.

Recently, the selective bi-plate medium CHROMagar™ ESBL/CHROMagar™ mSuperCARBA (CHROMagar Ltd., France) was designed for the rapid identification of ESBL-producing and carbapenem-resistant Enterobacteriaceae.

The aim of the present study was to evaluate the performance of this bi-plate medium using spiked stool samples with a collection of well-characterized CPE and ESBL-producers at the molecular level.

Two hundred and three enterobacterial isolates were tested (Table 1) was characterized by-end-point PCR followed by Sanger sequencing or by whole genome sequencing. In addition the Carba NP test was also performed on all isolates as previously described [START_REF] Dortet | Evaluation of the RAPIDEC(R) CARBA NP, the Rapid CARB Screen(R) and the Carba NP test for biochemical detection of carbapenemaseproducing Enterobacteriaceae[END_REF]. Fully-susceptible strains of E. coli ATCC 25922 and K. pneumoniae CIP53153, and ESBL-producing strains of E. coli CIP103983 (TEM-4) and K. pneumoniae ATCC 700603 (SHV-18) were used as controls (not included in the Table of results).

CHROMagar™ ESBL/mSuperCARBA is a ready-to-use bi-plate with a shelf life of 60 days at +4°C (+2°C to +12°C). Bacterial suspensions with a 0.5

McFarland (inoculum of ~5 x 10 7 to 10 8 CFU/mL) of the different isolates were serially diluted (10-fold dilutions) in water. Spiked fecal samples were prepared by adding 100 µL of each dilution to 900 µL of fecal suspension obtained by resuspending 4 g of freshly pooled feces from four healthy volunteers in 40 mL of distilled water, as previously described [START_REF] Naas | Real-time PCR for detection of NDM-1 carbapenemase genes from spiked stool samples[END_REF]. A fecal suspension without spiking was used as negative control. The lowest detection limits were determined by plating 10 µl of each spiked fecal samples on CHROMagar™ ESBL/mSuperCARBA medium. Viable bacteria were counted after 24 h of culture at 37°C. The lowest detection limit (LOD) corresponds the minimum number of bacteria that must be present in the sample to obtain a growth on the selective medium. In order to compare both selective media, the cut-off value was arbitrarily set at > 1 x 10 4 CFU/mL. A minimal concentration greater than 1

x 10 4 CFU/mL was considered as non-detection and sensitivity and specificity values were determined accordingly (Table 1).

Considering the 62 non-carbapenemase producers, all ESBL-producers with (n=13) or without an altered outer-membrane (n=13) grew on the CHROMagar™ ESBL medium, while 75% of the enterobacterial isolates with an acquired cephalosporinase did not grow with the exception of one DHA-2producing K. pneumoniae isolate (considered as a false positive). However, for the screening of GRE. In the field of laboratory automation in clinical microbiology [START_REF] Croxatto | Project management: importance for diagnostic laboratories[END_REF][START_REF] Lina | Automation in bacteriology: a changing way to perform clinical diagnosis in infectious diseases[END_REF], the panel of media that can be loaded onto the sorter of the inoculation system is limited [START_REF] Croxatto | Laboratory automation in clinical bacteriology: what system to choose?[END_REF]. Accordingly, the implementation of CHROMAGAR™ ESBL/mSuperCARBA bi-plate might be an advantage, since this medium can replace two or three of the currently used media for the screening of ESBL and CPE producers. The cost of these bi-plates will be approximately 3.25€/plate. E. coli 0.12 0.12 0.12 CTX-M-1 1 x 10 2 > 1 x 2 E7 E. coli 0.12 0.12 0.12 CTX-M-3 1 x 10 2 > 1 x 2 E8 K. pneumoniae 0.12 0.12 0.12 CTX-M-3 1 x 10 2 > 1 x 2 E9 E. coli 0.12 0.12 0.12 CTX-M-14 1 x 10 2 > 1 x 2 E10 E. coli 0.12 0.12 0.12 CTX-M-14 1 x 10 2 > 1 x 2 F1 K. pneumoniae 0.12 0.12 0.12 CTX-M-14 1 x 10 2 > 1 x 2 F2 E. coli 0.12 0.12 0.12 CTX-M-15 1 x 10 2 > 1 x 2 F3 E. coli 0.12 0.12 0.12 CTX-M-15 1 x 10 2 > 1 x 2 F4 K. pneumoniae 0.12 0.12 0.12 CTX-M-15 1 x 10 2 > 1 x 2 F5 K. pneumoniae 0.12 0.12 0.12 CTX-M-15 1 x 10 2 > 1 x 2 F6 K. pneumoniae 0.12 0.12 0.12 CTX-M-15 1 x 10 2 > 1 x 2 F7 E. cloacae 0.12 0.12 0.12 CTX-M-15 1 x 10 2 > 1 x 2 F8 E. cloacae 0.12 0.12 0.12 VEB-1 1 x 10

  , including 137 carbapenemase-producers [30 Ambler class A (KPC, Sme, IMI, GES-5, FRI-1), 50 Ambler class B (IMP, VIM, GIM-1, NDM-type), 43 Ambler class D (OXA-48-type and OXA-372), 14 multiple carbapenemase producers] and 62 non carbapenemase-producers [ESBL with or without impaired outermembrane (n=26), cephalosporinase-producers with or without an altered outer-membrane (n=27), concomitant ESBL and over-expressed cephalosporinase-producers (n=4), K1 penicillinase or extended-spectrum oxacillinases-producers (n=5)]. The β-lactamase content of all these isolates

Table 1 )

 1 19/23 isolates with overexpression of the cephalosporinase associated with decreased altered outer membrane grew on the CHROMagar™ ESBL medium.They were falsely considered as potential ESBL-producers. Only one of the three extended-spectrum oxacillinase-producers grew on the CHROMagar™ ESBL medium (thus giving two false negatives). Finally, the two penicillinase K1 overexpressing K. oxytoca were also falsely detected as ESBL-producers.CHROMagar™ mSuperCARBA medium for the detection carbapenemase producers was 97.8% [CI95 = 93.2% -99.4%] in this study. The specificity was 66.1% [CI95 = 52.9% -77.4%], which corresponds mainly to the growth of isolates with reduced susceptibility to carbapenems due to ESBL production and/or AmpC overexpression plus impermeability of the outer membrane (. Due to the low specificity of both sides of the bi-plate, confirmatory tests for ESBLs or carbapenemases detection should be undertaken on any growth. This is a major drawback that will increase the turn around time and costs if a rapid test is implemented as a confirmation test. However, this low specifity is mostly due to the design of the strains collection and might be better from clinical samples.

	Overall, the sensitivity of the CHROMagar™ ESBL medium for the detection of
	true ESBL-producers was 93.9% [95% confidence interval (CI95) = 78.4% -
	98.9%] in this study. Due to the high number of isolates with overexpressed-
	cephalosporinase associated with decreased outer-membrane permeability,

and the absence of isolates with wild-type phenotypes, the specificity of the CHROMagar™ ESBL medium was low (24.1%). Despite this limitation in the number of susceptible isolates tested, several studies have previously demonstrated that this CHROMagar™ ESBL medium is useful for the efficient detection of ESBL producers

[START_REF] Grohs | Comparison of five media for detection of extended-spectrum Beta-lactamase by use of the wasp instrument for automated specimen processing[END_REF][START_REF] Saito | Evaluation of a chromogenic agar medium for the detection of extendedspectrum ss-lactamase-producing Enterobacteriaceae[END_REF]

. Among the 137 CPEs, all but three cultured on the CHROMagar™ mSuperCARBA medium. One Sme-1-producing Serratia marcescens, one NDM-1 producing Providencia stuartii, and one VIM-1 producing E. coli (Table

1

) did not grow on the 'carba side of the plate', however, NDM-1 producing P. most (15/23) non carbapenemase-producers with overexpression of the cephalosporinase associated with decreased outer membrane permeability did not culture on mSuperCARBA medium (Table

1

). Overall, the sensitivity of the Our results suggest that the CHROMagar™ ESBL/mSuperCARBA biplate might be a reliable tool for the concomitant screening of patients colonized with ESBL and/or CPE. Currently, in France, hospitalized patients who have travelled abroad are screened for the presence of CPE and ESBL-producing Enterobacteriaceae. Different infection-control measures are implemented and maintained according to the screening results (

16

): (i) standard precautions [no ESBL producer, no CPE and no glycopeptide resistant Enterococcus faecium

Table 1 .

 1 Sensitivity of detection of the CHROMagar ™ ESBL /mSuperCARBA medium from spiked fecal samples.

	β-lactam resistance mechanism	Name of the strain	Species	MIC (µg/ml) IPM a ETP MEM	β-lactamase content	Limit of detection (UFC/ml) b,c ESBL mSuperCARBA
					NON CARBAPENEMASE PRODUCERS
		2	E2	E. coli	0.12 0.02 0.02 DHA-1		> 1 x 10 4	> 1 x
	Acquired Case	2 2	E3 E4	E. coli K. pneumoniae	0.12 0.12 0.12 ACC-1 0.12 0.5 0.12 DHA-2		> 1 x 10 4 1 x 10 2	> 1 x > 1 x
		2	E5	P. mirabilis	0.25 0.12 0.12 ACC-1		> 1 x 10 4	> 1 x
		2	E6
	ESBL

ACKNOWLEDGMENTS

We thank Saoussen Oueslati and Sandrine Bernabeu for helping constituting the panel of isolates.

FUNDING

This work was partially supported by the University Paris-Sud, France and Assistance Publique -Hôpitaux de Paris, Paris, France. LD, DG and TN are members of the Laboratory of Excellence in Research on Medication and Innovative Therapeutics (LERMIT) supported by a grant from the French National Research Agency (ANR-10-LABX-33).

TRANSPARENCY DECLARATION

None to declare

c Underlined CFU counts are considered as negative results (cut-off values set at ≥ 1 x 10 4 CFU/ ml).