J S Louis

F Odille

D Mandry
email: mandry@chru-nancy.fr

C De Chillou

O Huttin
email: huttin@chru-nancy.fr

J Felblinger
email: j.felblinger@chru-nancy.fr

C Venner
email: clemvenner@gmail.com

M Beaumont

DESIGN AND EVALUATION OF AN ABBREVIATED PIXELWISE DYNAMIC CONTRAST ENHANCEMENT ANALYSIS PROTOCOL FOR EARLY EXTRACELLULAR VOLUME FRACTION ESTIMATION

ABSTRACT:

Introduction T1-based method is considered as the gold standard for extracellular volume fraction (ECV) mapping. This technique requires at least a 10 minutes delay after injection to acquire the post injection T1 map. Quantitative analysis of Dynamic Contrast Enhancement (DCE) images could lead to an earlier estimation of an ECV like parameter (2 minutes). The purpose of this study was to design a quantitative pixelwise DCE analysis workflow to assess the feasibility of an early estimation of ECV.

Methods

Fourteen patients with mitral valve prolapse were included in this study. The MR protocol, performed on a 3T MR scanner, included MOLLI sequences for T1 maps acquisition and a standard SR-turboFlash sequence for dynamic acquisition. DCE data were acquired for at least 120s. We implemented a full DCE analysis pipeline with a pre-processing step using an innovative motion correction algorithm (RC-REG algorithm) and a post-processing step using the extended Tofts Model (ECVETM).

Estimated ECVETM maps were compared to standard T1-based ECV maps (ECVT1) with both a Pearson correlation analysis and a group-wise analysis.

Results

Image and map quality assessment showed systematic improvements using the proposed workflow. Strong correlation was found between ECVETM, and ECVT1 values (r-square=0.87).

Conclusion

A DCE analysis workflow based on RC-REG algorithm and ETM analysis can provide good quality parametric maps. Therefore, it is possible to extract ECV values from a 2 minute-long DCE acquisition that are strongly correlated with ECV values from the T1 based method.

KEYWORDS: Cardiac MR; DCE-MRI; REGISTRATION; PERMEABILITY; ECV

Manuscript: 1 INTRODUCTION :

Myocardial extracellular volume (ECV) is considered as a key biomarker for the quantification of myocardial fibrosis using magnetic resonance imaging (MRI) [START_REF] Andrew | Equilibrium Contrast Cardiovascular Magnetic Resonance for the Measurement of Diffuse Myocardial Fibrosis[END_REF], [START_REF] Fontana | Comparison of T1 mapping techniques for ECV quantification. Histological validation and reproducibility of ShMOLLI versus multibreath-hold T1 quantification equilibrium contrast CMR[END_REF].

It was first qualitatively studied on local infarcted area with the so-called late gadolinium enhancement or delayed enhancement technique [START_REF] Kim | How we perform delayed enhancement imaging[END_REF], which is based on the signal enhancement observed in the myocardium few minutes (15-20 minutes i.e. at the equilibrium of concentrations between compartments) after the injection of a paramagnetic contrast agent (CA) distributing within the extracellular extravascular space (EES) [START_REF] Dulce | MR imaging of the myocardium using nonionic contrast medium: signal-intensity changes in patients with subacute myocardial infarction[END_REF]. The accumulation of contrast agent within large EES, such as in infarcted tissues, leads to a brighter signal on T1-weighted (T1w) images than on the healthy myocardium [START_REF] Thomson | Magnetic resonance imaging for the assessment of myocardial viability[END_REF]. Quantitative assessment of ECV has been lately developed using T1 measurements before injection and once the equilibrium state of CA concentrations between vascular space and EES has been reached [START_REF] Arheden | Measurement of the Distribution Volume of Gadopentetate Dimeglumine at Echo-planar MR Imaging to Quantify Myocardial Infarction: Comparison with 99mTc-DTPA Autoradiography in Rats[END_REF]. This technique is well-known and denoted as ECV mapping. The very straightforward formula and its ease of use (as long as specific cardiac T1 mapping sequences are available) made it remarkably popular in the last few years and yield very promising results for diffuse fibrosis diagnosis [START_REF] Andrew | Equilibrium Contrast Cardiovascular Magnetic Resonance for the Measurement of Diffuse Myocardial Fibrosis[END_REF], [START_REF] Ugander | Extracellular volume imaging by magnetic resonance imaging provides insights into overt and sub-clinical myocardial pathology[END_REF]. However, the major disadvantage of this technique lies in its duration, with a required long waiting time before reaching the steady-state concentration between injection and acquisition of the post-injection T1 map (>10 minutes according to the latest statement of the SCMR community [START_REF] Messroghli | Clinical recommendations for cardiovascular magnetic resonance mapping of T1, T2, T2* and extracellular volume: A consensus statement by the Society for Cardiovascular Magnetic Resonance (SCMR) endorsed by the European Association for Cardiovascular Imaging (EACVI)[END_REF]).

Cardiac MRI (CMR) exams are among the longest MRI exams and there is a crucial need to shorten the total duration of CMR exam. Several options have been investigated in order to assess myocardial tissue pathophysiological state in a shorter time. Non-contrast enhanced imaging with diffusion weighted imaging (DWI) has been investigated for more than a decade [START_REF] Croisille | Imagerie de diffusion cardiaque : vers une nouvelle approche de l'ischémie myocardique ?[END_REF]. However, despite its huge potential, cardiac DWI is very complex to set up and improvements are still required before its use in clinical routine with a high reproducibility [START_REF] Nielles-Vallespin | Cardiac Diffusion: Technique and Practical Applications[END_REF]. Another avenue of research consists in taking advantage of T1w Dynamic Contrast Enhanced (DCE) acquisition, which could bring early and new insights on the myocardium viability. From the early works of Wilke et al. [START_REF] Wilke | Contrast-enhanced first pass myocardial perfusion imaging: Correlation between myocardial blood flow in dogs at rest and during hyperemia[END_REF] and Jerosch-Herold et al. [START_REF] Jerosch-Herold | Magnetic resonance quantification of the myocardial perfusion reserve with a Fermi function model for constrained deconvolution[END_REF] to the latest works of Knot et al. [START_REF] Knott | Quantitative myocardial perfusion in coronary artery disease: A perfusion mapping study[END_REF] using fully automatic in-line perfusion analysis [START_REF] Kellman | Myocardial perfusion cardiovascular magnetic resonance: optimized dual sequence and reconstruction for quantification[END_REF], myocardial perfusion quantification has been mostly focusing on myocardial blood flow estimation from the first-pass analysis of the CA injection. Several models have been explored to quantify the myocardial blood flow; but constraint Fermi deconvolution [START_REF] Jerosch-Herold | Magnetic resonance quantification of the myocardial perfusion reserve with a Fermi function model for constrained deconvolution[END_REF] or Blood Tissue Exchange Model [START_REF] Bassingthwaighte | Blood-Tissue Exchange via Transport and Transformation by Capillary Endothelial Cells[END_REF] remain the most studied to this day. Bi-compartmental models like the Extended Tofts Model (ETM) based on the Kety model have also been investigated with various conclusions regarding the impact on myocardial viability assessment [START_REF] Pack | Comparison of myocardial perfusion estimates from dynamic contrast-enhanced magnetic resonance imaging with four quantitative analysis methods[END_REF]. Larsson et al. [START_REF] Larsson | Myocardial perfusion modeling using MRI[END_REF] showed that the extracellular extravascular volume fraction Ve extracted from pharmacokinetic models could be a good predictive factor of the interstitial volume, which is also quantified with the ECV method. A little bit more than a decade ago, Pack et al. [START_REF] Pack | Quantitative myocardial distribution volume from dynamic contrast-enhanced MRI[END_REF] investigated the relationship between the compartmental parameter Ve and the so called "steadystate" tissue enhancement method. They demonstrated a non-significant difference between these two parameters. However, they could not compare their results to ECV extracted from T1 maps largely validated against histology. More recently, Kunze and colleagues compared measurements of ECV using the T1 maps method (equilibrium ECV) with the ECV extracted from perfusion quantification (perfusion ECV) [START_REF] Kunze | Measurement of extracellular volume and transit time heterogeneity using contrast-enhanced myocardial perfusion MRI in patients after acute myocardial infarction[END_REF]. They demonstrated a good correlation between ECV values extracted from both methods. They used a hierarchical modeling approach to estimate ECV from perfusion data using up to 5 parameter-models. This approach did not include standard permeability model such the 3 parameter-ETM.

The present work aims to pursue these investigations by comparing ECV values obtained from T1 maps (ECVT1) with those derived from parameters of the ETM analysis of perfusion imaging (ECVETM).

The first step in the development of a full DCE analysis pipeline was to implement a motion correction algorithm for images acquired in free breathing. This step presented a critical challenge due to the joint presence of contrast variation and respiratory and cardiac motion . Our vendor-provided in-line motion correction algorithm, MOCO [START_REF] Xue | Unsupervised Inline Analysis of Cardiac Perfusion MRI[END_REF], aligns each image of the series to the previous one. It is known to be prone to registration error propagation and to eventually introduce distortion in registered images, especially when acquisition time exceeds first-pass (about 1min) [START_REF] Xue | Unsupervised Inline Analysis of Cardiac Perfusion MRI[END_REF]. Thus, myocardial first-pass DCE registration has been and is still extensively investigated using various strategies such model-based registration [START_REF] Adluru | Model-based registration for dynamic cardiac perfusion MRI[END_REF], independent component analysis [START_REF] Milles | Fully Automated Motion Correction in First-Pass Myocardial Perfusion MR Image Sequences[END_REF] or using robust large displacement optical flow [START_REF] Benovoy | A Robust Universal Nonrigid Motion Correction Framework for First-Pass Cardiac Magnetic Resonance Perfusion Imaging[END_REF]. This problem was already assessed in DCE studies on other organs. For instance, Hamy et al. used a robust Principal Component Analysis to register DCE data from bowel [START_REF] Hamy | Respiratory motion correction in dynamic MRI using robust data decomposition registration -Application to DCE-MRI[END_REF] employing a similarity indicator robust to intensity variation called residual complexity (RC) [START_REF] Myronenko | Intensity-Based Image Registration by Minimizing Residual Complexity[END_REF]. We hypothesized that such similarity indicator could also be a suitable solution to myocardial DCE registration.

Thereby, the goal of this study was two-fold. First, it aimed to assess our proposed registration algorithm compared to the vendor's in-line registration algorithm MOCO.

Second, it aimed to investigate the feasibility of an early ECV estimation from 2min-long DCE-MRI analyzed with the Extended Tofts Model compared to the regular 1 10min ECV estimation from the T1 mapping method. 2

MATERIALS AND METHODS:

Acquisition of DCE data and T1 maps

MR data of 14 patients, with suspected or diagnosed mitral valve prolapse were extracted from the STAMP (STretch and Myocardial Characterization in Arrhythmogenic Mitral Valve Prolapse) study (NCT02879825), and were analyzed for the purpose of this study. STAMP was approved by the local committee of ethics, wherein all participants provided written informed consent. Every patient had a single measure of hematocrit (hct) that was used for ECV calculation. The CMR protocol was performed on a 3Tesla (T) MR scanner (Prisma, Siemens Healthineers, Erlangen, Germany).

Injection consisted in a 0.15mmol/kg bolus Gd-DOTA (Dotarem ® , Guerbet, France) at least 6 seconds after the beginning of the dynamic acquisition at a rate of 4mL/s. The pre-bolus T1 map (T10 map) was acquired with a single shot MOLLI set of 8 TI samples (5(3)3) on 8 slices covering the heart from the left atrium to the mid ventricular area (TR/TE: 283/1.12ms, matrix: 256x220, in plane resolution:

1.40x1.40mm², Slice Thickness: 8mm). DCE series lasted for at least 116 seconds in free breathing or partial free-breathing using a SR-TurboFLASH sequence (TI: 95ms, TR/TE: 116/0.97ms, flip angle:10°, matrix: 160x116, in plane resolution: 2.25x2.25mm², slice thickness: 8mm). The acquisition was cardiac gated meaning temporal resolution varied (mean: 0.95±0.13s) depending on the patient's heart rate.

To ensure a temporal resolution equal to one RR-interval, the maximum number of slices was defined by the acquisition time available within one RR interval. This resulted in the acquisition of one to two slices maximum par RR interval. Post-T1 map was acquired with a single shot MOLLI sequence set of 9 TI samples for 4(1)3(1)2 at least 15 minutes after the injection T10 map, DCE series and post-T1map slices were set to be automatically acquired at the same location with the same orientation and extra care was taken to ensure the acquisitions were performed during the same cardiac phases.

ECV mapping using extended Tofts modelling

The processing pipeline of this study (Figure 1) was implemented in Matlab® (The Mathworks, Natick,MA, USA).

The first step consisted in the registration of the magnitude images of the DCE series using our homemade registration algorithm (RC-REG). DCE acquisitions were normalized using a pre-scan calibration in order to attenuate signal inhomogeneity from the coil profile across the slice. The specific difficulty met with DCE images registration is the contrast change from one image to another, especially during the bolus arrival. The originality of this registration algorithm was the use of two reference images for the successive registration of pre-bolus and post-bolus acquisitions.

Registration of DCE images was based on a 2-dimensional non-rigid algorithm using b-spline deformation model with gradient descend optimization using the residual complexity (RC) as similarity measure included in the Medical Image Registration Toolbox developed by Myronenko et al. [START_REF] Myronenko | Intensity-Based Image Registration by Minimizing Residual Complexity[END_REF]. RC-REG algorithm is further described in Figure 2.

The algorithm was set to iterate until both the mean and standard deviation (SD) of the Structure Similarity (SSIM) [START_REF] Wang | Image Quality Assessment: From Error Visibility to Structural Similarity[END_REF] scores of the registered DCE series reached empirically determined cutoff values (mean > 0.81 and std < 0.028). Thus, the algorithm kept iterating using the newly registered DCE images as a starting point. This allowed the algorithm to manage DCE series with large respiratory motion, which leads to blurry averaged DCE series after the first iteration.

The T10 map was resized and downsampled to fit the DCE series resolution and FoV. Then, T10 map was aligned to the registered DCE series averaged image using the Matlab® multimodal 2-dimensional rigid registration algorithm (function imregister). A pixel-wise signal to CA concentration conversion on the DCE series was then performed.

To shorten post-processing, automatic cropping was applied to all DCE images. First, the region of interest was selected based on both the maximum intensity of the DCE series and shape segmentation of the Left Ventricle (LV) chamber. Then, the coordinates of a rectangle were calculated to widen the region of interest area and to prevent over cropping. The resulting cropping was applied to the corresponding T10 map. Registration quality measurements were evaluated on this area of interest. DCE signals were interpolated using a piecewise cubic Hermite interpolation algorithm to a constant temporal resolution of 0.5s. AIF was measured for each exam. Since it has been shown that RC algorithm does not alter signal of the corrected image [START_REF] Hamy | Respiratory motion correction in dynamic MRI using robust data decomposition registration -Application to DCE-MRI[END_REF], [START_REF] Myronenko | Intensity-Based Image Registration by Minimizing Residual Complexity[END_REF], we considered that sampling the AIF from the RC-REG series was the best solution to extract reliable signal intensity dynamics of the vascular space without being polluted by motion. A manual region of interest (ROI) was placed in the center of LV cavity to extract blood signal intensity dynamics.

Pixel-wise dynamic T1 values were processed from (pixel-wise) normalized signal intensity curves associated with baseline signal mean value, i.e. before injection (acquired for at least 6 seconds), and corresponding T10 voxel value using the signal equation of the SR-turboFLASH sequence. Ultimately, CA concentration was calculated for each pixel using the linear relationship between T1 and CA concentration:

, , = • , , + , , (2)
with i,j the coordinates of the pixel of interest, k the time, T1i,j,k the T1 value; r the relaxivity of the CA at 3T, 3.6 mM -1 .s -1 , [CA]i,j,k the CA concentration and T10 i,j the T1 extracted from T10map.

Extended Tofts modeling was achieved using a trust region least square algorithm generating Ktrans, Kep the flux rate parameters and Vb the blood volume fraction maps using the following formula:

( ) = ( ) * • ! "# • + ( ) • $ , (3)
with Ct, the tissue concentration and Cb the blood concentration. Ve' maps were calculated using the relation Ve' = Ktrans/Kep. Before fitting, AIF was temporally shifted to adjust the bolus arrival time to the one of the dynamic tissue concentration curve Ct(t) by computing cross-correlation between the two curves. Extracellular extravascular volume fraction Ve was calculated by correcting Ve' with each subject's hematocrit (hct) so that: Ve = Ve' • (1-hct). Likewise, plasma volume fraction was computed such as Vp = Vb • (1-hct). ECV corresponds to the non-cellular volume fraction of a tissue [START_REF] Andrew | Equilibrium Contrast Cardiovascular Magnetic Resonance for the Measurement of Diffuse Myocardial Fibrosis[END_REF], [START_REF] Moon | Myocardial T1 mapping and extracellular volume quantification: a Society for Cardiovascular Magnetic Resonance (SCMR) and CMR Working Group of the European Society of Cardiology consensus statement[END_REF], and therefore to the EES plus the plasma volume fraction.

We calculated the ECV values from ETM analysis so that ECVETM = Ve+Vp. ECVT1

were computed using the classical T1 maps technique and individual hct values [START_REF] Ugander | Extracellular volume imaging by magnetic resonance imaging provides insights into overt and sub-clinical myocardial pathology[END_REF].

Native and post blood T1 were measured within a ROI manually drawn in the center of the LV cavity. Full size native and post T1 maps were registered with the same multimodal 2D rigid registration algorithm used to register the T10 map and the mean DCE series. ECV maps were computed with the following formula: , where ;1 <=>/@A>>B C> / DE correspond to post/native 1/T1 of the myocardial/blood.

% $ ( , ) = (1 -ℎ) ) • +, -./ #/

Evaluation of the motion correction and permeability maps

The registration algorithm was qualitatively assessed by extracting dynamic line profiles of DCE series. Two line-profiles were extracted for each DCE series. Line profiles from unregistered (UNREG), in-line MOCO algorithm (MOCO) and RC-REG series were compared. Registration quality of the proposed algorithm was also assessed on permeability maps, looking at the absence of abnormal deformation of the LV. Also, homogeneous and/or a less "noisy" map was considered of a global higher quality.

Permeability parameter measurement was achieved within ROI manually drawn around the endocardium and epicardium of the LV and divided into the regular AHA (American Heart Association) segments [START_REF]Standardized Myocardial Segmentation and Nomenclature for Tomographic Imaging of the Heart[END_REF] on one to two slices per subject (from basal to medial level). Because slices were basal to medial, we concatenated segments from these slices into a single segment value to finally extract 6 segment values per subject. Permeability measurements were performed only when the thickness of the whole myocardium wall was sufficient to avoid any partial volume effect.

Comparison between ECVETM and ECVT1

Diffuse fibrosis is a process at the root of several cardiomyopathies and is not restricted to a specific location but that affects the whole myocardium. Therefore, we considered the global myocardial mean value of ECVETM and ECVT1 as representative of the general state of fibrosis infiltration. For each patient, we therefore calculated the ECV median± median absolute deviation (mad) values from the measured segments.

Pearson correlation with a significance level of 0.05 was used to compare ECVETM from the three series and ECV T1 myocardial values for the three series: UNREG, MOCO and RC-REG.

We chose to evaluate the discrimination power of ECVETM versus ECVT1 as follows:

an ECVT1value higher than 0.30 was considered as abnormal, splitting the patients into two groups. We then evaluated the difference between the two groups of ECVT1

and associated ECVETM values with a permutation test.

RESULTS:

Evaluation of the motion correction and permeability maps

Line profiles inspection of the fourteen exams showed, except for a few exceptions mentioned later, that respiratory motion correction was possible with MOCO and RC-REG algorithms (i.e. respiratory patterns were smoothened). Figure 3 Two exams had to be discarded from the analysis because the myocardium thickness (<2 pxls) did not allow an ROI to be placed without including voxels affected by partial volume effect.

Comparison between ECVETM and ECVT1

Figure 5 highlights the excellent correlation between ECVETM and ECVT1values (r-square=0.87) with a unity slope but a slight offset (= -0.05).

As shown in Figure 6, the 12 exams were split into two small groups of unequal size (n=8 for ECV≤0. [START_REF] Menys | Dual registration of abdominal motion for motility assessment in free-breathing data sets acquired using dynamic MRI[END_REF]

Evaluation of the motion correction and permeability maps

In order to proceed to ECVETM evaluation, we first proposed a novel DCE imaging registration algorithm. This algorithm used RC as a similarity indicator between two images. Well known in the post-processing community [START_REF] Sotiras | Deformable Medical Image Registration: A Survey[END_REF], residual complexity has been used in few MRI studies and mostly for abdominal exams [START_REF] Hamy | Respiratory motion correction in dynamic MRI using robust data decomposition registration -Application to DCE-MRI[END_REF], [START_REF] Menys | Dual registration of abdominal motion for motility assessment in free-breathing data sets acquired using dynamic MRI[END_REF], [START_REF] Feng | Liver DCE-MRI Registration in Manifold Space Based on Robust Principal Component Analysis[END_REF]. Results of our study confirmed that RC-based algorithms offer better registration for free-breathing DCE cardiac MRI images than the vendor's in-line MOCO in terms of line profiles (figure 3), and permeability maps quality (figure 4). MOCO is well known to incrementally introduce distortions on the corrected images as the processed image is further away from the bolus arrival in the LV. This phenomenon is well described in literature [START_REF] Xue | Unsupervised Inline Analysis of Cardiac Perfusion MRI[END_REF], [START_REF] Ledesma-Carbayo | Motion corrected free-breathing delayed-enhancement imaging of myocardial infarction using nonrigid registration[END_REF], [START_REF] Pontré | An Open Benchmark Challenge for Motion Correction of Myocardial Perfusion MRI[END_REF] and can be observed in figure 3 and supplementary videos.

RC-REG is a 2D registration algorithm (like MOCO), which implies that it is intrinsically unable to register through-plane motion leading to lower similarity metric scores from one image to another. It is also a post-processing algorithm and therefore dependent on the quality of the ECG gating during the prospective image acquisition. This acquisition technique is known to be sensitive to irregular heart rates [START_REF] Salerno | Advances in Parametric Mapping With CMR Imaging[END_REF], [START_REF] Salerno | Recent Advances in Cardiovascular Magnetic Resonance Techniques and Applications[END_REF] and even if DCE images were correctly aligned, the apparent thickness of the left ventricle may vary if such mistriggered data are present. This issue was not address in this work but it is visible on supplementary material 1. Such isolated mistriggered acquisitions could be potentially discarded from the DCE dataset and replaced using temporal interpolation with very low impact on the final analysis.

However, several consecutive acquisitions at wrong cardiac phases during the bolus arrival might lead to strongly biased parameter estimation, which was not encountered during this study. Despite these common limitations (shared between MOCO and other registration algorithms) our solution demonstrated better results both qualitatively and quantitatively compared to MOCO. It also showed comparable results compared to other more complex algorithms proposed in the literature [START_REF] Benovoy | A Robust Universal Nonrigid Motion Correction Framework for First-Pass Cardiac Magnetic Resonance Perfusion Imaging[END_REF],

[32], [START_REF] Zhou | Simple motion correction strategy reduces respiratory-induced motion artifacts for k-t accelerated and compressed-sensing cardiovascular magnetic resonance perfusion imaging[END_REF].

Two exams were acquired in a partial free-breathing paradigm, i.e. the patient was asked to hold his/her breath as long as possible [START_REF] Salerno | Advances in Parametric Mapping With CMR Imaging[END_REF]. The reference image for postinjection data was found to be in a very different location from the reference image for pre-injection data for these patients. Our registration technique did not fully overcome this limitation resulting in a slight shift of the data particularly visible in the right ventricle (figure 3.B). Nonetheless, it had no visible impact on the processed permeability maps. It has to be noted that the registration between reference 1 (from baseline series) and reference 2 (from entire series), resulting images would also be inadequate in the case of poor cardiac triggering between the baseline acquisition and the acquisition after the bolus arrival. The non-rigid nature of this deformation would lead to incomplete registration of those two images and would end as being discarded in the study. The use of a non-rigid registration for the alignment of these two reference images would be an interesting avenue of research, although it would not guarantee that the aligned pixels would coincide with a real volume alignment because of the complex nature of cardiac fibers motion between cardiac phases.

Permeability maps from RC-REG series appeared to be less noisy and provided more homogeneous ETM parameter values than MOCO and UNREG maps.

Distorted registration with MOCO yielded distorted ETM parameters maps as shown in figure 4.

It is noteworthy that two exams had to be discarded from the segment analysis because of a too thin left ventricle myocardial wall, which did not allow for the extraction of a sufficient number of voxels not affected by partial volume effect. This underlines the drawback of low-resolution acquisitions in first-pass protocols. This is a common limitation to all quantitative perfusion methods and developments still have to be made especially for application to patients with dilated cardiomyopathy.

In one case, T10 map and DCE slices were not acquired in the exact same locations.

This exam represents one limitation of our workflow, which requires the T10 information (DCE baseline) from a separate breath-held acquisition of T1 map.

Cernicanu and Axel method [START_REF] Cernicanu | Theory-Based Signal Calibration with Single-Point T1 Measurements for First-Pass Quantitative Perfusion MRI Studies[END_REF] for the signal to [CA] conversion could bring a good solution to avoid the use of T1 mapping and its drawbacks. Another point of improvement of our workflow would be to include a better way to measure or correct the AIF which would guarantee a better signal to CA concentration conversion and ultimately a more accurate measure of ETM parameters. There is still no consensus on which method to use but dual bolus [START_REF] Christian | Absolute Myocardial Perfusion in Canines Measured by Using Dual-Bolus First-Pass MR Imaging[END_REF], [START_REF] Hsu | Quantitative myocardial perfusion analysis with a dual-bolus contrast-enhanced first-pass MRI technique in humans[END_REF] or dual sequence [START_REF] Kellman | Myocardial perfusion cardiovascular magnetic resonance: optimized dual sequence and reconstruction for quantification[END_REF], [START_REF] Gatehouse | Accurate assessment of the arterial input function during high-dose myocardial perfusion cardiovascular magnetic resonance[END_REF] methods are valuable, although imperfect candidates to ensure a more reliable AIF measurement.

Comparison between ECVETM and ECVT1

We found a very strong correlation between ETM and T1-based ECV average myocardial values. ECVETM values exhibited lower absolute values compared to ECVT1. In their study, Kunze et al. [START_REF] Kunze | Measurement of extracellular volume and transit time heterogeneity using contrast-enhanced myocardial perfusion MRI in patients after acute myocardial infarction[END_REF] also reported both strong correlation between perfusion and ECVT1 and lower values for ECV estimated from DCE data. This underestimation was slightly more pronounced in our study. The use of ETM, which is a suitable model only under certain conditions of perfusion and flow in the tissue [START_REF] Sourbron | On the scope and interpretation of the Tofts models for DCE-MRI[END_REF], could explain a sub-optimal estimation of ECV. Although, the presented results, in line with the literature, illustrate the feasibility of ECV measurement using DCE-MRI.

ECVETM values were able to separate the two groups as distinctly as ECVT1 values.

One might add that the cutoff ECV value was chosen from the reported values in the literature to discriminate between healthy and pathologic subjects, but this arbitrary separation requires a larger study to confirm its clinical utility..

Between the two ECV measurement methods, the ETM technique is more error prone as more steps are required for the creation of an ECV map. However, the quality of T1-based ECV map is also dependent on the successful registration of the T1 weighted images necessary to reconstruct each T1 map and the registration of both native and post injection T1 maps. Low resolution acquisition needs to be improved to enable a more accurate permeability and ECV quantification using the ETM method, and to avoid partial volume effects in myocardium edge voxels.

However, in the context of the characterization of a diffuse pathology like fibrosis (therefore not located to a specific spot) such low resolution could be sufficient to assess the general state of the myocardial tissue. Furthermore, the low-resolution analysis of the first 2 minutes after injection could help the radiologist to decide whether it worth waiting for a 10 to 15 minutes high-resolution post injection T1 map or even an LGE acquisition. This could therefore be a useful tool to shorten CMR exams and to personalize them to each patient's requirements.

The acquisition duration of at least 2 minutes is enough to guarantee an estimation error of Ve inferior to 5% in healthy myocardium, but could lead to slightly more imprecision in the infarcted area, as demonstrated by Pack et al. [START_REF] Pack | Quantitative myocardial distribution volume from dynamic contrast-enhanced MRI[END_REF]. In the same study, they also shown an increase of such error if the acquisition duration is below this limit. According to different studies, the optimal DCE duration ranged from 2 to 5 minutes [START_REF] Pack | Quantitative myocardial distribution volume from dynamic contrast-enhanced MRI[END_REF], [START_REF] Sourbron | On the scope and interpretation of the Tofts models for DCE-MRI[END_REF]- [START_REF] Tofts | T1-weighted DCE Imaging Concepts: Modelling, Acquisition and Analysis[END_REF]. A longer acquisition duration, e.g. 3 minutes, could improve the precision of Ve estimation (and other ETM parameters) and therefore, reduce the wider distribution of ECVETM values compared to ECVT1 ones as reported in figure 6.

This wider distribution could also result from the fact that the ECVETM parameter is obtained from dynamic data. It is therefore more prone to influences of the local perfusion/permeability regime unlike T1-based ECV, estimated at equilibrium.

Despite these limitations, this study demonstrated a clear correlation between compartment parameter estimation from a dynamic perfusion analysis and those measured with the longer static T1-based method. All these results are in agreement with previous related works, but further investigations on a larger population is still needed to fully validate these findings.

FIGURES AND LEGENDS:

  It is only very recently that Scannell et al. used RC as a similarity indicator integrated in aRobust principal component analysis (RPCA) based registration algorithm for first pass perfusion imaging[START_REF] Scannell | Robust Non-Rigid Motion Compensation of Free-Breathing Myocardial Perfusion MRI Data[END_REF]. Compared to the latter work, our algorithm is based on an iterative optimization process of 2 similarity indicators (SSIM and RC), which could improve the quality of the registration compared to a two-step registration process. In this study, we also demonstrated the feasibility to register perfusion data from longer acquisition than what was assessed previously. Furthermore, the impact of the proposed registration algorithm was evaluated on both native DCE data and permeability maps.

Figure 1 :

 1 Figure 1: Framework diagram. The framework is split in 3 major parts: 1) pre-

Figure 2 :

 2 Figure 2: RC-REG algorithm diagram. The algorithm is based on the determination

Figure 3 :

 3 Figure 3: Example of two slice profiles from two exams with different acquisition

Figure 4 :

 4 Figure 4: Example of two sets of permeability parameters. A and B sets were

Figure 5 :

 5 Figure 5: ECVETM and ECVT1 myocardial mean values correlation. The RC-REG

CONCLUSION:

We presented a complete workflow for the processing of DCE-MRI for compartmental analysis of the myocardial tissue. We were able to demonstrate better registration quality of the presented RC-REG algorithm over the in-line MOCO algorithm. RC-REG algorithm allowed us to compute good quality parametric maps. Resulting ECVETM parameters exhibited strong correlation with ECV values extracted from MOLLI T1 maps. These results give an additional asset to quantitative cardiac perfusion and permeability MRI in order to assess ECV with a low resolution 2 minutes DCE-MRI acquisition against the current 10-15 minutes T1-based method.

Acknowledgements:

Authors want to acknowledge the work of the radiology technician's team of the Hospital and the help of the vendor's clinical scientist Khalid Ambarki, Ph.D., for its technical help. We also acknowledge J. Oster, Ph.D., for his valuable comments and corrections on the manuscript.

Funding sources:

Results incorporated in this article received funding from the Investments for the Future program under grant agreement: No ANR-15-RHU-0004.


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Hadj Salah N'

Demodex and ocular surface disease

Keywords: Demodex, blépharite, dysfonction meibomienne, rosacée, huile d'arbre à thé, ivermectine Demodex, blepharitis, meibomian gland dysfunction, roacea, tea tree oil, ivermectine. Conflits Demodex, blépharite, dysfonction meibomienne, rosacée, huile d'arbre à thé, ivermectine Demodex, blepharitis, Meibomian gland dysfunction, rosacea, tea tree oil, ivermectin

Résumé :

Les Demodex sont des acariens saprophytes des phanères, qui peuvent dans certaines circonstances proliférer au niveau de la peau du visage et sur le bord palpébral. Ils sont impliqués dans la rosacée du visage (en particulier papulo-pustuleuse) et participent au développement ou à l'aggravation de blépharites antérieures et/ou postérieures voire de kératoconjonctivites, volontiers associées à des atteintes cutanées, et dont la physiopathologie est souvent multifactorielle. Les symptômes sont aspécifiques, mais la présence de manchons cylindriques péri-ciliaires est très évocatrice d'une infestation, et certaines techniques d'examen biomicroscopique ou d'imagerie, telle que la microcopie confocale in vivo, permettent de visualiser le parasite. La confirmation diagnostique repose néanmoins sur l'examen parasitologique des cils, dont la rentabilité peut être améliorée par une bonne technique de prélèvement. L'hygiène des paupières et les pommades grasses constituent la base du traitement. De nouveaux traitements spécifiques, notamment les topiques contenant de l'huile d'arbre à thé, l'ivermectine, mais aussi la thérapie par lumière pulsée et la micro-exfoliation du bord libre palpébral permettent de diminuer la charge parasitaire et d'améliorer les symptômes.

Introduction

Les Demodex sont des acariens saprophytes de la peau et des phanères de tous les mammifères. Parmi la centaine d'espèces connues, deux colonisent l'homme : D. folliculorum et D. brevis. Si leur pathogénicité a longuement été controversée, certaines conditions pourraient favoriser leur prolifération qui serait en cause dans certaines dermatoses ainsi que dans certaines formes de blépharites [START_REF] Elston | Demodex mites[END_REF]. Les démodécidoses sont ainsi définies comme des ectoparasitoses causées par la prolifération de Demodex dans l'appareil pilosébacé, à l'origine de signes inflammatoires palpébraux et/ou cutanés [START_REF] Chang | Role of Demodex mite infestation in rosacea: A systematic review and meta-analysis[END_REF][START_REF] Cheng | Recent advances on ocular Demodex infestation[END_REF][START_REF] Fromstein | Demodex blepharitis: clinical perspectives[END_REF]. Dans cette revue générale, nous aborderons d'abord les aspects parasitologiques et physiopathologiques de l'infection, puis les différentes formes cliniques des démodécidoses pour enfin se focaliser sur les atteintes oculaires.

Parasitologie

Histoire naturelle et physiopathologie générale des infections à Demodex

La colonisation par les Demodex semble se faire au fur et à mesure de la vie, au gré des contacts entre personnes. Le portage est rare chez l'enfant, atteint 3 à 55% chez les personnes de plus de 18 ans lorsqu'il est évalué par recherche de parasite mais peut aller jusqu'à 100% lorsqu'il est évalué par détection d'ADN parasitaire [START_REF] Thoemmes | Ubiquity and diversity of human-associated Demodex mites[END_REF]. Chaque comédon ou follicule pileux peut contenir un à plusieurs Demodex… Dans la grande majorité des cas, la présence de ces acariens est sans conséquence : ils font partie du microbiote cutané et vivent en symbiose avec leur hôte, contribuant à nettoyer la peau, notamment du sébum.

Dans certaines conditions, les Demodex prolifèrent et sont à l'origine de démodécidoses. Les facteurs de risques de prolifération des Demodex, retrouvés dans la littérature sont multiples : immunodépression (y compris iatrogène par traitements systémiques ou topiques), diabète, grossesse, cancers, malnutrition, stress, insuffisance rénale, mauvaise hygiène, exposition à la lumière, port de lunettes etc.... [START_REF] Kurt | Increased density of Demodex folliculorum mites in pregnancies with gestational diabetes[END_REF][START_REF] Kosik-Bogacka | Demodex folliculorum and Demodex brevis in healthy and immunocompromised patients[END_REF][START_REF] Karincaoglu | Pseudozoster clinical presentation of Demodex infestation after prolonged topical steroid use[END_REF][START_REF] Antille | Induction of rosaceiform dermatitis during treatment of facial inflammatory dermatoses with tacrolimus ointment[END_REF].

Pathogénicité des Demodex :

Certains auteurs pensent que la présence de Demodex pourrait être bénéfique, jouant un rôle dans [START_REF] Nicholls | Demodex species in human ocular disease: new clinicopathological aspects[END_REF] :

-Le contrôle de la prolifération locale des bactéries en les ingérant -La défense contre d'autres parasites vermiformes -La protection contre certains allergènes environnementaux.

Le pouvoir pathogène des Demodex ne fait pas l'unanimité en raison de leur prévalence au niveau des follicules pilo-sébacés et des comédons des sujets sains. Néanmoins, certaines observations cliniques suggèrent que leur prolifération et/ou leur présence sur des terrains particuliers, puisse faire passer leur statut de saprophyte bénin à parasite pathogène.

Histologiquement, le Demodex peut effectuer des brèches dans les parois folliculaires. En outre, certaines bactéries vivant en symbiose dans l'organisme des Demodex (en particulier Bacillus oleronius), seraient responsables d'une activation du système immunitaire de l'hôte et impliquées dans la physiopathologie des atteintes cutanées et oculaires [START_REF] Clanner-Engelshofen | Corynebacterium kroppenstedtii subsp. demodicis is the endobacterium of Demodex folliculorum[END_REF][START_REF] Mcmahon | Exposure of a corneal epithelial cell line (hTCEpi) to Demodexassociated Bacillus proteins results in an inflammatory response[END_REF].

Le rôle des Demodex est désormais reconnu dans la physiopathologie de certaines dermatoses au premier rang desquels l'acné rosacée, et certaines formes de blépharites que nous détaillerons ci-après.

Démodécidoses cutanées :

Dans une méta-analyse récente, Chang et Huang rapportaient que les prélèvements cutanés retrouvent une fréquence et une densité plus importante de D. folliculorum chez les patients atteints de rosacée (quelles que soient les formes cliniques) que chez les contrôles [START_REF] Chang | Role of Demodex mite infestation in rosacea: A systematic review and meta-analysis[END_REF]. Toutefois, la variabilité inter-étude ne permettait pas d'établir un lien de causalité, qui reste fortement suspectée par d'autres spécialistes de la rosacée [START_REF] Cribier | Physiopathology of rosacea[END_REF][START_REF] Moran | Demodex and rosacea revisited[END_REF]. Les atteintes cutanées peuvent d'ailleurs être associées aux atteintes oculaires [START_REF] Brown | Severe demodexfolliculorum-associated oculocutaneous rosacea in a girl successfully treated with ivermectin[END_REF][START_REF] Forton | Rosacea and Demodicosis: Littleknown Diagnostic Signs and Symptoms[END_REF][START_REF] Liang | In vivo confocal microscopy evaluation of ocular and cutaneous alterations in patients with rosacea[END_REF] (Figure 2). Sur le plan histologique, les lésions associées aux infestations cutanées comportent un infiltrat périfolliculaire et périvasculaire lympho-histiocytaire riche en neutrophiles, avec cellules multinucléées, associé à la présence de nombreux Demodex dans l'infundibulum folliculaire, les pustules infundibulaires et les infiltrats périfolliculaires [START_REF] Hsu | Demodicosis: a clinicopathological study[END_REF]. Un autre argument très en faveur de ce lien est l'efficacité de l'ivermectine topique (voire systémique) sur les formes papulopastuleuses de rosacée, rapportée initialement dans des cas cliniques spectaculaires [START_REF] Brown | Severe demodexfolliculorum-associated oculocutaneous rosacea in a girl successfully treated with ivermectin[END_REF], puis confirmée par de nombreuses études contrôlées [START_REF] Husein-Elahmed | Efficacy of topical ivermectin and impact on quality of life in patients with papulopustular rosacea: A systematic review and meta-analysis[END_REF][START_REF] Ebbelaar | Topical Ivermectin in the Treatment of Papulopustular Rosacea: A Systematic Review of Evidence and Clinical Guideline Recommendations[END_REF]. Ce traitement fait d'ailleurs désormais partie des traitements de première intention de cette pathologie.

L'implication du Demodex dans les pathologies dermatologiques a également été retenue dans des tableaux cliniques évocateurs de dermites péri-orales, ou d'autres folliculites du visage [START_REF] Hsu | Demodicosis: a clinicopathological study[END_REF][START_REF] Alniemi | Perioral Demodex folliculitis masquerading as perioral dermatitis in the peripartum period[END_REF].

Demodex et blépharites :

Le rôle des Demodex dans les blépharites est à ce jour moins consensuel que celui qu'ils jouent dans l'acné rosacée. La controverse sur le lien de causalité entre leur présence et les symptômes repose d'une part sur le fait que les Demodex sont retrouvés sur de nombreux sujets asymptomatiques [START_REF] Wesolowska | Prevalence of Demodex spp. in eyelash follicles in different populations[END_REF], et d'autre part que la corrélation entre le niveau d'infestation et les symptômes n'est pas toujours solidement établie dans la littérature [START_REF] Wesolowska | Prevalence of Demodex spp. in eyelash follicles in different populations[END_REF][START_REF] Biernat | Occurrence of Demodex species in patients with blepharitis and in healthy individuals: a 10-year observational study[END_REF].

Toutefois, de nombreuses études ont montré une association entre la présence de ces parasites et la survenue de pathologies de la surface oculaire, au premier rang desquelles les blépharites [START_REF] Biernat | Occurrence of Demodex species in patients with blepharitis and in healthy individuals: a 10-year observational study[END_REF][START_REF] Luo | Ocular Demodicosis as a Potential Cause of Ocular Surface Inflammation[END_REF][START_REF] Liang | Significant correlation between meibomian gland dysfunction and keratitis in young patients with Demodex brevis infestation[END_REF]. En outre, des hypothèses physiopathologiques solides sont étayées par des études morphologiques, microbiologiques et biochimiques (voir ci-dessous).

Demodex et blépharites antérieures :

Elles sont secondaires à la colonisation des cils et de leurs follicules par D. folliculorum [START_REF] Bhandari | Blepharitis: always remember demodex[END_REF][START_REF] Liu | Pathogenic role of Demodex mites in blepharitis[END_REF] :

-Les régurgitations du Demodex se mélangent avec la kératine, les débris de cellules épithéliales pour former des dépôts cylindriques autours de la base des cils (manchons cylindriques). Ces dépôts et les Demodex contiennent des lipases immunoréactives l'origine des symptômes irritatifs [START_REF] Jimenez-Acosta | Demodex mites contain immunoreactive lipase[END_REF].

-Les streptocoques et staphylocoques présents à la surface du Demodex sont impliqués dans les blépharites antérieures et postérieures microbiennes.

-Les débris des Demodex peuvent par ailleurs engendrer une réaction d'hypersensibilité retardée [START_REF] Liu | Pathogenic role of Demodex mites in blepharitis[END_REF].

L'ensemble de ces processus physiopathologiques entraine une inflammation du bord libre.

Demodex et blépharites postérieures :

D. brevis, de plus petite taille, peut se loger dans les glandes sébacées des paupières mais surtout dans les glandes de Meibomius, dont il peut bloquer mécaniquement les sécrétions [START_REF] Liang | In vivo confocal microscopy evaluation of ocular and cutaneous alterations in patients with rosacea[END_REF][START_REF] Sabeti | Management of meibomian gland dysfunction: a review[END_REF][START_REF] Cheng | The correlation between the microstructure of meibomian glands and ocular Demodex infestation: A retrospective case-control study in a Chinese population[END_REF][START_REF] Gutgesell | Histopathology of meibomian gland dysfunction[END_REF]. En outre, l'exosquelette du parasite peut être à l'origine de réactions granulomateuses à corps étranger et les excrétions du parasite peuvent provoquer une réaction immunitaire / inflammatoire. On comprend alors mieux comment les Demodex peuvent contribuer au développement de dysfonctions des glandes de Meibomius et aux récurrences de chalazions [START_REF] Yam | Ocular demodicidosis as a risk factor of adult recurrent chalazion[END_REF].

Demodex et inflammation de la surface :

Les blépharites antérieures et postérieures peuvent s'accompagner d'atteintes conjonctivales et cornéennes (voir diagnostic clinique) [START_REF] Kheirkhah | Corneal manifestations of ocular demodex infestation[END_REF].

Diagnostic :

Diagnostic clinique :

Les symptômes de blépharite à Demodex (quand il y en a) ne sont pas spécifiques (symptômes de sécheresse, irritation, sensation de corps étranger, larmoiements, cils collés…). En revanche, une anamnèse de blépharite résistante au traitement bien conduit doit faire évoquer le diagnostic.

Les signes liés à l'infestation par D. folliculorum sont essentiellement la présence de manchons cylindriques périciliaires (Figure 3) : ces derniers sont constitués de débris cireux et/ou secs à la base des cils formant un cylindre très évocateur de la présence d'un grand nombre de Demodex [START_REF] Gao | High prevalence of Demodex in eyelashes with cylindrical dandruff[END_REF].

Certains auteurs suggèrent d'observer la base de cils à fort grossissement en lampe à fente, après avoir réalisé des mouvements de rotation et traction des cils, permettant de faire sortir le corps des Demodex au niveau du follicule pileux, prenant un aspect typique en forme de cigare (Figure 4) [START_REF] Muntz | Improved Demodex diagnosis in the clinical setting using a novel in situ technique[END_REF][START_REF] Mastrota | Method to identify Demodex in the eyelash follicle without epilation[END_REF].

Muntz et al. ont même comparé plusieurs techniques d'examen de la base

des cils à fort grossissement, dont ils évaluaient la rentabilité en comparaison avec l'examen en microscopie confocale in vivo (IVCM) (considéré dans cette étude comme le gold standard) : 1) rotation du cil pour en exposer la base, 2) ablation du manchon cylindrique à la pince, puis exposition de la base du cil, ou enfin 3) ablation du manchon cylindrique puis traction latérale du cil à la pince à monofilament pour mieux exposer la base du cil. C'est la 3 ème technique qui était la plus rentable [START_REF] Muntz | Improved Demodex diagnosis in the clinical setting using a novel in situ technique[END_REF]. Toutefois, l'examen considéré comme référence (IVCM) dans cette étude est discutable, même s'il est considéré comme très sensible et spécifique (voir ci-dessous) [START_REF] Wang | Prospective Study of the Diagnostic Accuracy of the In Vivo Laser Scanning Confocal Microscopy for Ocular Demodicosis[END_REF]

Traitements :

Traitements non spécifiques :

L'hygiène quotidienne des paupières, prescrite pour toute blépharite a une efficacité modérée pour limiter la prolifération des Demodex et améliorer les symptômes [START_REF] Navel | Efficacy of treatments for Demodex blepharitis: A systematic review and meta-analysis[END_REF]. Les produits émollients facilitent dans ce cas l'ablation des croûtes présentes à la base des cils. Les autres traitements médicamenteux des blépharites et dysfonctions meibomiennes (collyre et pommade antibiotiques, doxycycline per os), sont à adapter au tableau clinique, et à une éventuelle rosacée associée. Les corticoïdes et la ciclosporine topiques peuvent être nécessaires en cas d'atteinte cornéenne [START_REF] Kheirkhah | Corneal manifestations of ocular demodex infestation[END_REF].

Traitements spécifiques :

Le but du traitement n'est pas d'éradiquer, mais de diminuer la prolifération du Demodex, qui recolonisera tôt ou tard les téguments [START_REF] Navel | Efficacy of treatments for Demodex blepharitis: A systematic review and meta-analysis[END_REF].

Autres topiques et traitements physiques :

La crème à l'ivermectine à 1% (Soolantra  ) est bien tolérée en application palpébrale quotidienne. Son efficacité pour le traitement des atteintes oculaires et cutanées des rosacées est désormais bien démontrée [START_REF] Moran | Demodex and rosacea revisited[END_REF][START_REF] Sobolewska | Efficacy of Topical Ivermectin for the Treatment of Cutaneous and Ocular Rosacea[END_REF][START_REF] Schaller | A randomized phase 3b/4 study to evaluate concomitant use of topical ivermectin 1% cream and doxycycline 40-mg modified-release capsules, versus topical ivermectin 1% cream and placebo in the treatment of severe rosacea[END_REF]. En revanche, bien que l'effet démodécide de l'ivermectine soit bien établi, aucune étude n'a pour l'instant relié l'effet bénéfique sur les atteintes oculaires à une diminution de la charge en Demodex, ni sa tolérance.

Légendes des figures :

Les

  Demodex spp. sont un genre d'acariens de la famille de Demodicidae. Ils sont retrouvés chez pratiquement tous les mammifères, domestiques ou non mais ont une spécificité d'hôte : il est exceptionnel d'avoir une infestation humaine par un Demodex animal. Les Demodex spp. sont très difficilement cultivables in vitro, par conséquent, les connaissances sur leur biologie et leur physiologie sont relativement limitées. Le cycle parasitaire du Demodex dure entre 14 et 16 jours. Les parasites pondent dans les follicules pilosébacés ou dans les glandes sébacées (ou meibomiennes) ou sur les cils. Après 7 jours, l'oeuf donne naissance à une larve qui migre à la surface de la peau saine, grossit et subit plusieurs mues. L'accouplement a lieu vers 28 jours. La femelle s'enfonce alors dans le follicule ou la glande sébacée pour pondre. La durée de vie de ces parasites est d'environ de 2 à 3 mois. Ils apprécient une température de 16 °C à 20 °C et sont détruits en-dessous de 0 °C et au-dessus de 54 °C [5]. Deux espèces sont présentes chez l'homme : D. folliculorum plus grand (0,3 à 0,4mm) et colonisant les cils, et D. brevis (0,2 à 0,3mm) qui colonise plus volontiers les glandes sébacées et meibomiennes. Ces petits arthropodes sont translucides et dotés de huit pattes (Figure 1 C). Le corps de D. folliculorum est couvert d'écailles qui lui permettent de s'ancrer dans le follicule pileux. Ils peuvent quitter le follicule pileux et évoluer sur la peau, généralement la nuit. Ils se nourrissent de sébum et de déchets de cellules épithéliales. Bien que dotés d'une bouche, les Demodex n'ont pas d'appareil digestif excréteur : le matériel non digéré est donc régurgité. La synchronicité du cycle décrite pour ces espèces, pourrait expliquer d'une part l'intermittence des symptômes et d'autre part la présence d'une seule forme parasitaire (le plus fréquemment adultes et plus rarement larves ou oeufs) au moment du prélèvement.

  La plupart des traitements proposés dans la littérature sont efficaces à des degrés variables mais n'ont pas d'AMM dans cette indication. Quels que soient les traitements, deux cures ou plus sont le plus souvent nécessaires vu la chronicité de l'infestation et la fréquence des récidives. De nombreux topiques ont une efficacité sur les Demodex, mais finalement assez peu sont disponibles en France. A noter que les Demodex sont résistants aux shampoings et à un grand nombre d'antiseptiques, y compris la povidone iodée à 10% [35]. L'existence d'une pathologie inflammatoire sous-jacente explique parfois l'inefficacité de ces traitements. Les traitements topiques à base d'huile d'arbre à thé (TTO pour Tea Tree Oil) en application locale ont fait leur preuve [3, 42]. Cette huile essentielle dérivée de Melaleuca alternifolia a des propriétés démodécides liées à sa forte teneur en Terpinène-4-ol, un principe actif fongicide et parasiticide. Les produits à base de TTO doivent être appliqués sur le bord des cils. Ils provoquent ainsi une migration des follicules pileux des cils vers la peau et ainsi éviter l'accouplement, la ponte et l'éclosion. La méta-analyse de Navel et al. les place parmi les traitements topiques les plus efficaces en termes de réduction du nombre de parasites [42]. Pour être efficaces, les traitements doivent être effectuées quotidiennement pendant plusieurs semaines. A côté des huiles en flacon, nous avons désormais à notre disposition des lingettes spécifiquement destinées à l'hygiène des paupières, imprégnées d'une lotion concentrée à 2,5% de Terpinen-4-ol (Blephademodex  ). L'efficacité parasitologique et clinique de ces dernières semble très intéressante [43]. Un autre topique pour application palpébrale (mousse micellaire Steriblef  ) contient également (entre autres) de l'huile d'arbre à thé.

Figure 1 :

 1 Figure 1 : Demodex en microscopie optique. A) Demodex folliculorum sur un

Figure 2 :

 2 Figure 2 : Patiente présentant une atteinte cutanée et palpébrale causée par

Figure 3 :

 3 Figure 3 : Manchons péri-ciliaires typiques d'une infestation à Demodex

Figure 4 :

 4 Figure 4 : A) Aspect typique « en cigare » du corps du Demodex sortant de

Figure 5 :

 5 Figure 5 : Examen en microscopie confocale du bord libre palpébral d'une

  . D'autres signes non spécifiques peuvent être associés aux blépharites à Demodex prélevés en raison de la difficulté de l'absence de quantification des Demodex restés au fond du follicule pileux (ou même dans les glandes de Meibomius). Murphy et al. ont même montré que l'examen

	Demodex : trichiasis, distichiasis, madarose, érythème du bord libre
	palpébral. Il est plus difficile d'identifier D. brevis in situ. La présence d'une biomicroscopique à fort grossissement de la base du cil, après rotation, était
	blépharite postérieure avec dysfonction meibomienne et éventuellement plus rentable que l'examen parasitologique pour compter le nombre de
	chalazions à répétition [33], est évocatrice lorsqu'elle résiste au traitement Demodex [40].
	bien conduit. La microscopie confocale in vivo permet la visualisation directe des
	Les complications conjonctivo-cornéennes des blépharites à Demodex ne Demodex au niveau des cils et des follicules pileux (Figure 5), et parfois des
	sont pas spécifiques, et s'apparentent aux complications des blépharites
	d'autres étiologies : kératite ponctuée superficielle inférieure (Figure 2C)
	infiltrats	marginaux,	néovascularisation	périphérique	superficielle,
	opacification cornéenne, lésions phlycténulaires conjonctivales ou
	Examens complémentaires :
	La présence de Demodex sur des cils n'est pas suffisante pour l'incriminer
	dans la symptomatologie clinique, c'est surtout le fait d'objectiver un nombre
	important (un à 3 par cil sur un prélèvement, selon les laboratoires) qui
	aurait une signification pathologique.

cornéennes, cicatrices cornéennes voire nodules de Salzmann

[START_REF] Kheirkhah | Corneal manifestations of ocular demodex infestation[END_REF]

. Le lien de cause à effet entre les atteintes cornéennes et la présence de Demodex est difficile à établir. Toutefois, Liang et al., sur une série de 60 patients atteints de démodécidose palpébrale, rapportaient une corrélation entre la sévérité des atteintes cornéennes, le nombre de D. Brevis sur les cils et l'atrophie meibomienne évaluée en meibographie infrarouge

[START_REF] Liang | Significant correlation between meibomian gland dysfunction and keratitis in young patients with Demodex brevis infestation[END_REF]

.

Le prélèvement consiste à épiler 5 cils non adjacents par paupière en préférant ceux gainés d'un manchon puis de les adresser rapidement en parasitologie pour identifier et quantifier le nombre de parasites observés sur chaque cil

[START_REF]Pratiques diagnostiques : ectoparasites et parasitoses associées : Demodex[END_REF]

. Cette technique semblerait sous-estimer le nombre de glandes de Meibomius

[START_REF] Randon | In vivo confocal microscopy as a novel and reliable tool for the diagnosis of Demodex eyelid infestation[END_REF]

. Cet outil d'imagerie aurait une sensibilité et une spécificité supérieures à l'examen parasitologique (qui reste malgré tout la référence), mais il nécessite un opérateur entrainé et la bonne coopération du patient (d'autant plus pour l'examen des glandes de Meibomius). En outre, la projection des images des cils et des manchons pose parfois des difficultés pour différencier les Demodex des structures adjacentes.

  En outre, l'ivermectine peut provoquer une somnolence et des vertiges pouvant affecter la conduite automobile ou le maniement de machines, et est contre-indiquée chez la femme enceinte et pendant l'allaitement. La toxicité potentielle de ce traitement et l'absence d'AMM dans cette indication doivent inciter à bien évaluer l'indication avant prescription. Pour conclure, l'infestation par les Demodex doit être évoquée devant des blépharites résistantes aux traitements bien conduits, ou en présence de signes cliniques évocateurs. De nouvelles techniques permettant d'améliorer la rentabilité de l'examen clinique, l'imagerie par microscopie confocale in vivo permettent de confirmer le diagnostic, et peuvent, dans certains cas remplacer l'examen parasitologique. Ce dernier reste la référence, mais nécessite un prélèvement de bonne qualité. De nouveaux traitements topiques, bien tolérés, efficaces et disposant d'une AMM, sont désormais disponibles.

	parasitologique mais expose aux effets indésirables en cas de passage sur la Le traitement systémique par ivermectine per os (Stromectol  , 200µg par kg,
	surface oculaire. Les topiques au métronidazole ont un effet sur les soit 3 à 5 comprimés à 3mg en une prise, à renouveler au bout de 2-3
	symptômes, mais leur efficacité pour réduire le nombre de parasites n'a pas semaines) peut être proposé dans les formes résistantes, ou en cas
	été évaluée [42]. d'atteinte cutanée associée étendue [51]. Si aucun effet indésirable sévère
	La micro-exfoliation du bord libre palpébral, par application d'une brosse n'a été rapporté dans le contexte des démodécidoses, d'exceptionnelles
	rotative sur le bord libre et la base des cils (Blephex  ) serait également être réactions sévères ont été décrites dans le traitement d'autres parasitoses
	Selon efficace dans le traitement des démodécidoses oculaires en éliminant (syndrome de Lyell, Stevens Johnson, troubles hématologiques sévères) [52,
	la sévérité, la durée du traitement peut varier de 4 semaines à 4 mois. Ce mécaniquement les Demodex et leurs oeufs. Les séances doivent être 53].
	traitement n'est pas recommandé pendant la grossesse et l'allaitement. répétées à des intervalles de 3 à 6 mois. Toutefois, une étude récente ne
	Les topiques à base de miel de Manuka (Lepstotermum scoparium, ou myrte
	de Nouvelle-Zélande) constituent une autre option prometteuse. Ce miel a la
	particularité d'être particulièrement riche en méthylglyoxal, connu pour ces
	propriétés bactéricides et parasiticides [46]. Dans une étude publiée
	récemment, Craig et al. retrouvaient une amélioration significative de la charge
	parasitaire et des symptômes après 1 mois d'application nocturne d'une crème
	contenant 100 mg/kg de miel de Manuka, sur les paupières d'un oeil, par
	rapport à l'autre oeil non traité [46]. Bien que l'efficacité in vitro du miel de testaient cette option in vitro en soumettant des parasites aux paramètres
	Manuka semble comparable à celle de l'huile de TTO. Des études cliniques utilisés habituellement en ophtalmologie. La température des parasites était
	comparant ces topiques restent toutefois nécessaires pour définir leurs places portée à 49°C, et aucun ne survivait au traitement. En clinique, plusieurs
	respectives [47]. études semblent en faveur d'une efficacité sur le nombre de parasites et les
	Les autres traitements locaux sont le plus souvent prescrits sous forme de symptômes, mais les critères de jugement varient sensiblement d'une étude
	pommades à appliquer sur les cils pour piéger les Demodex dans la à l'autre [49, 50], et des études complémentaires sont nécessaires dans ce
	pommade lors de leur sortie nocturne et ainsi bloquer le cycle parasitaire, en domaine.
	plus de leur action démodécide propre. Traitements systémiques :
	L'oxyde de mercure 1% en pommade ophtalmique est actif sur les Demodex
	mais n'est plus disponible en France. De même, le gel à la pilocarpine à 4%
	(non disponible en France), présente une bonne efficacité clinique et

mettait pas en évidence de différence significative quant à la densité de Demodex et à l'amélioration des symptômes entre hygiène des paupières avec lingettes imbibées de terpinène-4-ol, précédée, ou non, de microexfoliation du bord libre palpébral

[START_REF] Murphy | The efficacy of tea tree face wash, 1, 2-Octanediol and microblepharoexfoliation in treating Demodex folliculorum blepharitis[END_REF]

.

La photothérapie par lumière pulsée intense (IPL pour Intense Pulsed Llight), utilisée pour le traitement des dysfonctions meibomiennes, pourrait également être efficace pour lutter contre les Demodex. Fishmann et al.

La méta-analyse de Navel et al. permet de conclure qu'aucun traitement systémique n'a de supériorité par rapport aux traitements tropiques les plus efficaces

[START_REF] Navel | Efficacy of treatments for Demodex blepharitis: A systematic review and meta-analysis[END_REF]

.


François Jussiaux

Cécile Miot-Sertier

Darrène Nguyen-Lopez

ORCID Cécile Badet

Johan Samot
email: johan.samot@u-bordeaux.fr

Reliability of MALDI-TOF Mass Spectrometry to identify oral isolates of Streptococcus salivarius and Lactobacillus spp

Keywords: Bacteria, Probiotics, Mass spectrometry, MALDI-TOF, Oral cavity

Objective:

The aim of this study is to evaluate the performance of MALDI-TOF mass spectrometry in identifying bacteria isolated in the oral cavity known to be of probiotic interest.

Design

: We evaluated Bruker MALDI Biotyper for the identification of 92 clinical oral isolates of probiotic interest (31 Streptococcus salivarius and 61 Lactobacillus spp.) by comparing direct colony method with on-plate formic acid extraction. Isolates were previously identified by use of biochemical methods and molecular biology.

Results: Using the manufacturer's suggested genus and species level cutoff scores, the direct colony method identified 42 (45.7%) isolates at the genus level and 35 (38%) at the species level while the on-plate extraction method correctly identified 90 (97.8%) isolates at the genus level and 82 (89.1%) at the species level. The difference between the two methods was statistically significant at the genus and species levels (P ≤ 0.0001). After dividing the isolates into two subgroups, the analysis was repeated. The direct colony method identified correctly all isolates of Streptococcus salivarius at the species level. In contrast, the direct colony method allowed the identification of only 11 (18%) lactobacilli at the genus level and 4 (6.6%) at the species level. The on-plate extraction method was statistically (P ≤ 0.0001) more efficient since 59 (96.7%) lactobacilli were identified at the genus level and 51 (83.6%) at the species level.

Conclusions: MALDI Biotyper can efficiently identify Streptococcus salivarius regardless of the preparative method but on-plate extraction is superior to direct colony method for the identification of lactobacilli.

Introduction

The identification of clinical isolates from polymicrobial flora is a lengthy procedure that usually involves several techniques such as GRAM staining, biochemical methods and molecular biology. In recent decades, identification by mass spectrometry has provided new solutions. Indeed, since the first descriptions of bacterial identification by mass spectrometry in the 1970s, the performance of these methods have constantly evolved to allow, through the constitution of increasingly precise spectral databases of proteins, a more sensitive identification of microorganisms [START_REF] Anhalt | Identification of bacteria using mass spectrometry[END_REF][START_REF] Lay | MALDI-TOF mass spectrometry of bacteria[END_REF][START_REF] Meuzelaar | A technique for fast and reproducible fingerprinting of bacteria by pyrolysis mass spectrometry[END_REF][START_REF] Simmonds | Whole microorganisms studied by pyrolysis-gas chromatographymass spectrometry: significance for extraterrestrial life detection experiments[END_REF]. Currently, using a soft ionization principle, Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry (MS) has become a gold standard for microbial identification in clinical microbiology laboratories [START_REF] Sauer | Mass spectrometry tools for the classification and identification of bacteria[END_REF][START_REF] Schubert | MALDI-TOF MS in the Microbiology Laboratory: Current Trends[END_REF][START_REF] Van Belkum | Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry in Clinical Microbiology: What Are the Current Issues?[END_REF].

The four present commercial MALDI-TOF systems (MALDI Biotyper, Saramis, Andromas and Vitek MS) can identify a wide range of microorganisms through the analysis of unique "fingerprints" of abundant proteins from whole cells or cell extracts [START_REF] Angeletti | Matrix assisted laser desorption time of flight mass spectrometry (MALDI-TOF MS) in clinical microbiology[END_REF][START_REF] Khot | Optimization of matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis for bacterial identification[END_REF]. MALDI-TOF mass spectrometry has been described as a fast, cost-effective and accurate method for the identification of bacteria [START_REF] Schulthess | Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine[END_REF] and can therefore be particularly useful for the study of oral ones. The human oral cavity is a complex ecosystem in which more than 700 species of bacteria live together in a fragile balance. Modern lifestyles, age, nutritional status and general state of health are all factors that can cause disturbances of the oral microbiome leading to dysbiosis, which can have harmful consequences on oral health (candidiasis, caries, periodontitis...). Because of the interplay between oral pathologies and numerous systemic disorders, it is essential to avoid the development and progression of oral diseases, especially infectious ones [START_REF] Dorfer | The relationship of oral health with general health and NCDs: a brief review[END_REF].

With the limited progress in the discovery of new antibiotics and the increase in emerging resistant pathogenic bacteria, it has become imperative to try new approaches such as biotherapy. The cornerstone of this biotherapy strategy is currently the use of beneficial microbes, commonly referred to as probiotics. According to little but increasing number of randomized double-blind clinical studies, beneficial bacteria may play a role in maintaining oral health through their interaction with the oral microbiome, thereby contributing to a healthy microbial equilibrium and preventing oral infectious conditions [START_REF] Bustamante | Probiotics as an Adjunct Therapy for the Treatment of Halitosis, Dental Caries and Periodontitis[END_REF][START_REF] Gruner | Probiotics for managing caries and periodontitis: Systematic review and meta-analysis[END_REF][START_REF] Nadelman | Are dairy products containing probiotics beneficial for oral health? A systematic review and meta-analysis[END_REF]. The persistence in the oral cavity of these beneficial bacteria is therefore essential and like other authors, we believe that an adequate strategy is to use microorganisms directly isolated from their natural oral habitat as oral probiotics [START_REF] Samot | Probiotic properties of lactobacilli that could be used against periodontitis[END_REF][START_REF] Samot | Adherence capacities of oral lactobacilli for potential probiotic purposes[END_REF][START_REF] Strahinic | Molecular and biochemical characterizations of human oral lactobacilli as putative probiotic candidates[END_REF][START_REF] Wescombe | Developing oral probiotics from Streptococcus salivarius[END_REF]. However, prior to any clinical use, the selection and evaluation of potential probiotic candidates requires a multi-step approach starting with precise strain identification (FAO/ WHO, 2002). Our aim was to evaluate the performance of MALDI-TOF mass spectrometry in identifying bacteria isolated in the oral cavity known to be of probiotic interest. In this study, we evaluated the Bruker Maldi Biotyper for identification of clinically isolates Streptococcus salivarius and lactobacilli using or not on-plate formic acid preparation.

Materiels and Methods

Bacterial isolates

Thirty-one oral strains of Streptococcus salivarius were isolated among children before and after dental care under general anesthesia. The strains were identified using both biochemical (API20Strep, BioMerieux, Marcy l'Etoile, France) and PCR method. The primers used were

Ssa442F

(5'-AACGTTGACCTTACGCTAGC-3') and Ssa2712R

(5'-GATTCTGTCAAAGAAGCC-3') targeting the dextranase gene of Streptococcus salivarius [START_REF] Igarashi | Identification of Streptococcus salivarius by PCR and DNA probe[END_REF]. Sixty-one lactobacilli strains, isolated from human salivary samples and previously identified, were used in this study [START_REF] Román-Méndez | Identification of oral strains of Lactobacillus species isolated from Mexican and French children[END_REF][START_REF] Samot | Probiotic properties of lactobacilli that could be used against periodontitis[END_REF]. Sequencing of the 16S-rDNA fragment was performed for all strains using the primers FD1 et RD1 according to [START_REF] Weisburg | 16S ribosomal DNA amplification for phylogenetic study[END_REF]. Streptococci were cultured in Trypticase Soy broth (Oxoid, Dardilly, France), and Lactobacilli were cultured in Man, Rogosa and Sharpe Medium (Fischer Scientific, Illkirch, France) at 37°C. All strains were cryo-preserved at -80°C.

DNA extraction

DNA was isolated from each bacterial strain by using GenElute Bacterial Genomic DNA kit (Sigma Aldrich, St Quentin Fallavier, France) according to the manufacturer's supplied protocol.

Mass spectrometry identification

To obtain fresh bacteria, isolates were cultured overnight or until visible growth was observed on Trypticase Soy agar (Oxoid, Dardilly, France) for Streptococci or on Man, Rogosa and Sharpe Agar (Fischer Scientific, Illkirch, France) for Lactobacilli. A colony of each isolate, taken directly from the agar plate, was deposited on a polished steel target (Bruker Daltonics, Bremen, Germany) in a single spot and allowed to dry at room temperature. One microliter of matrix solution (saturated solution of a cyano-4-hydroxycinnamic acid (HCCA) in 50% acetonitrile) was added to the sample and was then crystallized by air-drying at room temperature for 5 min. For the direct transfer-formic acid method, 1µl of 70% formic acid (Sigma-Aldrich) was added to the bacterial spot and allowed to air dry, before the matrix solution was added. The samples prepared by each method were applied to a MicroFlex LT mass spectrometer (Bruker Daltonik), and the results were analyzed using the database (containing 6,903 reference spectra of 2,641 microbial species at the time of the study) associated with MALDI Biotyper 3.1 software (Bruker Daltonik). Each measurement was performed only once for each culture and each preparation. Escherichia coli DH5α was used as a quality control as recommended by the manufacturer on each experiment.

Data analysis

According to the manufacturer's recommended score identification: a score of ≥2.0 indicated a species-level identification; ≥1.7 but <2.0, a genus-level identification and an isolate with a score <1.7 is considered to be unidentified. Discordant results between MALDI-TOF MS and molecular identification were categorized as misidentifications. The rates for different criteria (genus-level, species-level, unreliable identification, misidentifications) were calculated by the number of different criteria divided by the total number of isolates.

Statistical analysis

Comparisons of genus-or species-level identification using on-plate extraction versus direct colony methods were made by using McNemar's test of paired proportions. P values of less than 0.05 were considered statistically significant. All analyses were performed by using R 3.6.3 (R Core Team, 2020).

Results

Ninety-two isolates were included in this study. Molecular identifications were considered as the gold standard. Both the direct colony method and the on-plate extraction method were performed from a colony growing on one of the isolate preferred solid culture media. The "score" (ranging from 0.00-to-3.00) provided by the Biotyper database determined the level of identification for each isolate. For the direct colony method, results for each strain are

shown in Table 1. The direct colony method identified 42 (45.7%) isolates at the genus level and 35 (38%) isolates at the species level. Using the on-plate extraction method, the Bruker Biotyper correctly identified 90 (97.8%) isolates at the genus level and 82 (89.1%) isolates at the species level (Table 2). The difference between the two methods was statistically significant at the genus and species levels (P ≤ 0.0001). No isolate returned without identification after treatment with 70% formic acid (on-plate extraction) while with the direct colony method, 53.4% of the isolates remained unidentified. This positive evolution of the rate of unidentified isolates with the on-plate extraction method is to be modulated with the number of misidentified strains. Indeed, the number of misidentified strains increased from 7.6% (for the direct colony method) to 10.9% (for the on-plate extraction method).

Surprisingly, some misidentifications were at the genus level (2 enterococci detected instead of 2 lactobacilli) with the on-plate extraction method while all misidentifications with the direct colony method were at the species level. The direct colony method was sufficiently conclusive to identify correctly all isolates of Streptococcus salivarius at the species level (Figure 1). The direct colony method allowed the identification of only 11 (18%) lactobacilli at the genus level and 4 (6.6%) at the species level. The on-plate extraction method was statistically (P ≤ 0.0001) more efficient since 59 (96.7%) lactobacilli were identified at the genus level and 51 (83.6%) at the species level. Overall, MALDI-TOF mass spectrometry was less accurate for the identification of lactobacilli up to the precision of the species level.

Discussion

Lactobacilli as Streptococcus salivarius belong to the phylum Firmicutes, class Bacilli and order Lactobacillales. Bacteria belonging to this order are more commonly known as lactic acid bacteria (LAB). Lactic acid bacteria have a prominent place among bacteria of probiotic interest but because of their genetic closeness, it is sometimes complicated to identify them quickly and accurately [START_REF] Argyri | Selection of potential probiotic lactic acid bacteria from fermented olives by in vitro tests[END_REF][START_REF] Garcia | Identification of Lactic Acid Bacteria in Fruit Pulp Processing Byproducts and Potential Probiotic Properties of Selected Lactobacillus Strains[END_REF]. The purpose of our study was to estimate the value of MALDI-TOF mass spectrometry for the rapid identification of certain oral lactic acid bacteria. Our findings show that the method of sample preparation has a significant impact on the accuracy of strain identification. These results are consistent with other previous studies that have shown that a simple formic acid layer on the colony would be sufficient to increase the identification rate [START_REF] Hsu | MALDI-TOF MS identification of anaerobic bacteria: assessment of pre-analytical variables and specimen preparation techniques[END_REF][START_REF] Schulthess | Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine[END_REF].

Formic acid, by removing salt residues and some additives from the media, would allow obtaining clearer mass spectra, free of certain adducts [START_REF] Lay | MALDI-TOF mass spectrometry and bacterial taxonomy[END_REF][START_REF] Walker | Off-line coupling of capillary electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry[END_REF]. This aspect of the formic acid treatment is very interesting because it would make it possible to counterbalance the variations in the spectral fingerprint that a given strain may present depending on the agar culture medium on which it is grown. According to some previous studies, the complete extraction of proteins (a more laborious process) seems to offer only minimal gains and is above all not compatible with rapid routine identification methods [START_REF] Barcelos | Comparison of standard and on-plate extraction protocols for identification of mastitis-causing bacteria by MALDI-TOF MS[END_REF][START_REF] Schulthess | Identification of Gram-positive cocci by use of matrix-assisted laser desorption ionization-time of flight mass spectrometry: comparison of different preparation methods and implementation of a practical algorithm for routine[END_REF].

Our results also show that the performance of Bruker MALDI Biotyper also depends on the species we are trying to identify. The strains of Streptococccus salivarius were all correctly identified even with the direct colony method. This excellent identification rate of S. salivarius was also reported by the review by Fan et al [START_REF] Fan | Performance of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry system for rapid identification of streptococci: a review[END_REF]. In contrast, even with the on-plate formic acid extraction method, the MALDI biotyper had more difficulty in identifying lactobacilli species accurately, even leading to misidentification at the genus level (Enterococcus) for two poorly described species belonging to the L. buchneri phylogroup (L.

farraginis and L. parafarriginis). Another strain (L. kefiri) belonging to this phylogroup was also misidentified (L. fermentum). In view of the small number of representatives of certain lactobacilli species in this study, no general statistical data on the identification of lactobacilli by Bruker MALDI biotyper can be derived. Nevertheless, it can be observed that the MALDI biotyper tends to slightly generate a poor identification of lactobacilli belonging to the same phylogenetic group. This is particularly the case here for lactobacilli of the L. casei group (L.

casei, L. paracasei, L. rhamnosus, L. zeae) which accounts for more than a third (4/10) of misidentifications. This discordance in the identification of casei group lactobacilli by MALDI-TOF MS has already been documented, and has been explained as probably related to the fact that the four bacteria mentioned above cannot be distinguished by classical phenotypic tests [START_REF] Garcia | Identification of Lactic Acid Bacteria in Fruit Pulp Processing Byproducts and Potential Probiotic Properties of Selected Lactobacillus Strains[END_REF]. This tendency to discriminate less well between species of the same phylogenetic group seems to be confirmed by the misidentification of L. vaginalis as L. fermentum (L. reuteri group). In this study, however, two results nuance this hypothesis;

the above-mentioned misidentification of L. kefiri in L. fermentum and one of the four Lactobacillus gasseri (L. delbrueckii group) was misidentified as L. rhamnosus (L. casei group). The lower performance in the identification of lactobacilli can be explained by the complexity of this genus, which is regularly subject to taxonomic updates [START_REF] Huang | Genome-based reclassification of Lactobacillus casei: emended classification and description of the species Lactobacillus zeae[END_REF][START_REF] Salvetti | Comparative Genomics of the Genus Lactobacillus Reveals Robust Phylogroups That Provide the Basis for Reclassification[END_REF][START_REF] Zheng | A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae[END_REF]. To allow better identification of lactobacilli, an improvement in the quality and richness of information of the protein spectra in Bruker Biotyper database seems essential. An alternative solution for microbiology laboratories remains the possibility of developing in-house databases, which can have the advantage of taking into account variations in the bacterial proteome depending on the culture medium used, and can even be adapted to identify antibiotic resistances [START_REF] Bove | Metabolic and proteomic adaptation of Lactobacillus rhamnosus strains during growth under cheese-like environmental conditions compared to de Man, Rogosa, and Sharpe medium[END_REF][START_REF] Huang | Identification and Classification for the Lactobacillus casei Group[END_REF][START_REF] Oviano | Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for the Rapid Detection of Antimicrobial Resistance Mechanisms and Beyond[END_REF].

Conclusion

In summary, our data illustrate that the Bruker MALDI Biotyper can efficiently identify

Streptococcus salivarius regardless of the preparative method but that on-plate formic acid extraction is superior to direct colony testing for the identification of lactobacilli. The addition of protein spectra to the database to reflect the diversity of closely related species may further improve identification.

Figure legend Figure 1 .

 legend1 Figure legend

Table 1 .

 1 MALDI Biotyper identification of isolates of Streptococcus salivarius and Lactobacillus spp. to the genus and species levels by use of

	direct colony method

Table 2 .

 2 MALDI Biotyper identification of isolates of Streptococcus salivarius and Lactobacillus spp. to the genus and species levels by use of on-plate formic acid extraction

					Number of isolates
			correctly identified to each level Not identified	Misidentified (misidentification)
	Oral isolates tested	Total number of isolates	genus	species
	Streptococcus salivarius	31	31	31	0	0
	Lactobacillus rhamnosus	47	47	46	0	1 (Lactobacillus zeae)
	Lactobacillus casei					4 (2 Lactobacillus rhamnosus,
		4	4	0	0	2 Lactobacillus paracasei)
	Lactobacillus gasseri	4	4	3	0	1 (Lactobacillus rhamnosus)
	Lactobacillus farraginis	1	0	0	0	1 (Enterococcus faecium)
	Lactobacillus fermentum	1	1	1	0	0
	Lactobacillus kefiri	1	1	0	0	1 (Lactobacillus fermentum)
	Lactobacillus parafarraginis	1	0	0	0	1 (Enterococcus faecium)
	Lactobacillus plantarum	1	1	1	0	0
	Lactobacillus vaginalis	1	1	0	0	1 (Lactobacillus fermentum)
	Total				10 (8 at the species level, 2 at the genus
		92	90	82	0	level)
	% identification
			97,8	89,1	0	10,9

Acknowledgments

Biomedical research technicians Astrid Ducournau and Martine Saint-Marc are kindly acknowledged for the technical assistance in the laboratory. This research was funded by the Institut français pour la Recherche Odontologique (IFRO).

Declaration of competing interest

The authors declare no conflict of interest.