Gagandeep Chadha

MD a ; Yohann Bohbot

MD a ; Pierre Lachambre

MD Dan Rusinaru

MD a ; Saousan Serbout

MD c ; Alexandre Altes

MD Agnès Pasquet

MD, PhD Sylvestre Maréchaux

MD Jean-Louis Vanoverschelde

Professor Christophe Tribouilloy
email: tribouilloy.christophe@chu-amiens.fr

Progression of Normal Flow Low Gradient "Severe" Aortic Stenosis With Preserved Left Ventricular Ejection Fraction

Keywords: normal-flow low-gradient aortic stenosis, progression, aortic stenosis severity

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

INTRODUCTION

Normal-flow, low-gradient "severe" aortic stenosis (NF-LG-SAS) is the most prevalent form of low-gradient aortic stenosis (AS) [START_REF] Clavel | Low-gradient aortic stenosis[END_REF], with preserved left ventricular ejection fraction (LVEF) [START_REF] Minners | Inconsistent grading of aortic valve stenosis by current guidelines: haemodynamic studies in patients with apparently normal left ventricular function[END_REF][START_REF] Lancellotti | Clinical outcome in asymptomatic severe aortic stenosis: insights from the new proposed aortic stenosis grading classification[END_REF]. Its management is a matter of debate, as there is uncertainty about the "true" severity of AS. Patients with NF-LG-SAS are classically elderly women, with a small body surface area (BSA), less concentric left ventricular (LV) hypertrophy and concentric remodeling, less left atrial dilatation, less impaired systolic longitudinal function, and larger aortic valve area (AVA) than other forms of severe AS [START_REF] Lancellotti | Clinical outcome in asymptomatic severe aortic stenosis: insights from the new proposed aortic stenosis grading classification[END_REF][START_REF] Mehrotra | Differential left ventricular remodelling and longitudinal function distinguishes low flow from normal-flow preserved ejection fraction low-gradient severe aortic stenosis[END_REF][START_REF] Barone-Rochette | Aortic valve area, stroke volume, left ventricular hypertrophy, remodeling, and fibrosis in aortic stenosis assessed by cardiac magnetic resonance imaging: comparison between high and low gradient and normal and low flow aortic stenosis[END_REF]. The outcome of this entity is a subject of debate. Indeed, several studies suggest that these patients have "true" severe AS and that aortic valve replacement (AVR) should be performed when symptoms appear [START_REF] Dayan | Outcome and impact of aortic valve replacement in patients with preserved LVEF and low-gradient aortic stenosis[END_REF][START_REF] Ozkan | Impact of aortic valve replacement on outcome of symptomatic patients with severe aortic stenosis with low gradient and preserved left ventricular ejection fraction[END_REF][START_REF] Melis | Systolic volume index by Doppler echocardiography is an useful marker for stratification and prognostic evaluation in patients with severe aortic stenosis and preserved ejection fraction[END_REF][START_REF] Tarantini | Valve replacement for severe aortic stenosis with low transvalvular gradient and left ventricular ejection fraction exceeding 0.50[END_REF][START_REF] Mohty | Outcome and impact of surgery in paradoxical low-flow, low-gradient severe aortic stenosis and preserved left ventricular ejection fraction: a cardiac catheterization study[END_REF][START_REF] Zusman | Intervention versus observation in symptomatic patients with normal flow-low gradient severe aortic stenosis[END_REF][START_REF] Clavel | The complex nature of discordant severe calcified aortic valve disease grading: new insights from combined Doppler echocardiographic and computed tomographic study[END_REF][START_REF] Saeed | The impact of aortic valve replacement on survival in patients with normal flow low gradient severe aortic stenosis: a propensity-matched comparison[END_REF], whereas others have considered this entity to be in the majority of cases a moderate form of AS [START_REF] Mehrotra | Differential left ventricular remodelling and longitudinal function distinguishes low flow from normal-flow preserved ejection fraction low-gradient severe aortic stenosis[END_REF][START_REF] Tribouilloy | Low-gradient, low-flow severe aortic stenosis with preserved left ventricular ejection fraction: characteristics, outcome, and implications for surgery[END_REF][START_REF] Jander | Outcome of patients with lowgradient "severe" aortic stenosis and preserved ejection fraction[END_REF][START_REF] Kang | Watchful observation versus early aortic valve replacement for symptomatic patients with normal flow, low-gradient severe aortic stenosis[END_REF][START_REF] Eleid | Flow-gradient patterns in severe aortic stenosis with preserved ejection fraction: clinical characteristics and predictors of survival[END_REF][START_REF] Chadha | Outcome of Normal-Flow Low-Gradient Severe Aortic Stenosis With Preserved Left Ventricular Ejection Fraction: A Propensity-Matched Study[END_REF]. Given the paucity of data on the evolution of hemodynamic parameters over time in NF-LG-SAS with preserved LVEF, we studied the evolution of echocardiography Doppler parameters over time in these patients. We hypothesized that this entity represents an "intermediate" stage between moderate AS and high gradient severe AS (HG-SAS) with preserved LVEF, tending to evolve towards the classic pattern of HG-SAS with preserved LVEF.

METHODS

We retrospectively identified all cases of NF-LG-SAS with preserved LVEF diagnosed from 2005 to 2015 at the echocardiography laboratories of three academic centers (Amiens, Lille, and Brussels). Inclusion criteria were (i) the presence of NF-LG "severe" AS with preserved LVEF diagnosed by transthoracic echocardiography based on the following criteria: AVA < 1 cm², mean pressure gradient (MPG) < 40 mmHg, indexed stroke volume (SVi) ≥ 35 mL/m², and LVEF ≥ 50%; and (ii) availability of a second follow-up Doppler echocardiographic examination at least 6 months after baseline echocardiography (Figure 1).

We excluded: (i) patients with more than mild aortic and/or mitral regurgitation; (ii) patients with prosthetic valves, congenital heart disease (with the exception of bicuspid aortic valves), supravalvular or subvalvular AS, or dynamic LV outflow tract obstruction; (iii) patients experiencing AVR between the two examinations; and (iv) patients who did not provide authorization to be included in the study. The second echocardiography was performed when scheduled during the patient's follow-up or when requested by the patient's referring cardiologist. When several follow-up echocardiograms were performed, we used the last one available for analysis. Rapid progression of AS was defined as an increase of peak aortic jet velocity ≥ 0.3 m/s/year [START_REF] Nistri | Hemodynamic progression and outcome of asymptomatic aortic stenosis in primary care[END_REF] Baseline clinical and demographic characteristics, including cardiovascular risk factors, the presence of symptoms according to New York Heart Association (NYHA) class, comorbidity status, and the presence of coronary artery disease (defined by the presence of a documented history of acute coronary syndrome, coronary artery disease previously confirmed by coronary angiography, or history of coronary revascularization), were retrospectively recorded from medical charts. The Charlson comorbidity index was calculated for each patient. We obtained institutional review board authorizations before conducting the study. The study was conducted in accordance with institutional policies, national legal requirements, and the revised Helsinki declaration. Given the retrospective nature of the study inform consent was waived.

Blood pressure and heart rate were measured for all patients at the time of echocardiography. LV outflow tract (LVOT) diameter was measured in a zoomed cine loop of the parasternal long-axis view in early systole at the level of aortic cusp insertion. Aortic flow was systematically recorded using continuous wave Doppler from several views (apical 5chamber, right parasternal, suprasternal, and epigastric) [START_REF] Benfari | Feasibility and relevance of right parasternal view for assessing severity and rate of progression of aortic valve stenosis in primary care[END_REF]. The view identifying the highest velocities was used to determine peak aortic jet velocity and calculate the aortic velocity-time integral (VTI) and MPG (using the simplified Bernoulli equation). Three consecutive measurements for patients in sinus rhythm or five consecutives measurements for patients in atrial fibrillation (AF) in this view were systematically averaged. The LVOT VTI was recorded using pulsed-wave Doppler from the apical 5-chamber view, with the sample volume positioned approximately 5 mm proximal to the aortic valve [START_REF] Chadha | Outcome of Normal-Flow Low-Gradient Severe Aortic Stenosis With Preserved Left Ventricular Ejection Fraction: A Propensity-Matched Study[END_REF]. The alignment of both pulsed and continuous wave Doppler was optimized to be parallel with aortic flow.

Effective AVA was calculated using the continuity equation: π x (LVOT area / 2)² x (LVOT VTI) / (Aortic VTI). Stroke volume was calculated by multiplying the LV outflow tract area with the LV outflow tract VTI and was indexed to BSA. The degree of calcification of the aortic valve was assessed during echocardiography [START_REF] Rosenhek | Predictors of outcome in severe, asymptomatic aortic stenosis[END_REF]. LVEF and ventricular volumes were calculated using the Simpson biplane method . LV wall thickness and dimensions were measured, when possible, from M-mode imaging or by default from two-dimensional images obtained in the parasternal long-axis view using the leading-edge methodology. LV mass was estimated by the formula on the basis of linear measurements and indexed to BSA. LV hypertrophy was defined as a LV mass index > 115 g.m -2 in men and > 95 g.m -2 in women and patients were classified according to four patterns of LV geometry: normal; LV concentric remodelling; eccentric LV hypertrophy; and concentric LV hypertrophy [START_REF] Lang | Recommendations for cardiac chamber quantification by echocardiography in adults: an update from the American Society of Echocardiography and the European Association of Cardiovascular Imaging[END_REF]. Systolic pulmonary artery pressure was recorded from the maximum peak tricuspid regurgitation velocity in any view using the simplified Bernoulli equation.

Statistical analysis was performed using SPSS version 25 (IBM, Armonk, New York).

Categorical variables were summarized as counts and frequency percentages. Because of skewed distribution, continuous variables were expressed as medians and interquartile ranges.

For comparison between baseline and follow up, categorical variables were compared using the Pearson chi-square test and continuous variables by matched-pair analysis using the paired t-test (for normally distributed variables) or paired samples Wilcoxon test (for non-normally distributed variables). Annualized progression was calculated as the difference between the last measurement and the first measurement divided by the duration of follow-up. Univariate and multivariate linear regression were performed to identify factors associated with annualized progression of Vmax. All covariates associated with Vmax increase in univariate analysis (p < 0.10) were entered in the model for multivariable analysis. The limit of statistical significance was p < 0.05.

RESULTS

The baseline demographic and clinical characteristics of the 96 patients are presented in Table 1. The median age was 79 years, with a predominance of women (62%). More than 75% of the study population had hypertension, one-quarter had diabetes mellitus, and almost 40% had coronary artery disease. The Charlson comorbidity index and Euroscore II were low at echocardiographic diagnosis, with a median of 3 and 2.01, respectively. Patients were mostly asymptomatic or pauci-symptomatic (83%) at diagnosis.

The baseline and follow-up echocardiographic parameters are displayed in Table 2.

The median time between the two echocardiographic examinations was 25 (15-52) months.

At follow-up, the peak aortic jet velocity increased by 0.65 ± 0.60 m/s over baseline (Figure 2), corresponding to a median annual rate of progression of 0.25 [0.08-0.44] m/s/year (mean 0.34 m/s/year). Overall, 80 patients (84%) showed an increase in peak aortic jet velocity at follow-up. In this group, the median annual progression rate was 0.32 [0.14-0.47] m/s/year (mean 0.42 m/s/year). The increase in Vmax between baseline and follow-up was ≥ 0.3m/s in 43 patients (44.8%).

At follow-up, MPG showed a 13 ± 11 mm Hg mean increase compared to baseline (Figure 2), corresponding to a median annual rate of progression of 4.3 [1.7-8.1] mm Hg/year (mean 6 mm Hg/year). Overall, we observed an increase in MPG in 84 patients (88%), a decrease in six (6%), and it remained unchanged in six (6%). The increase in MPG between baseline and follow-up was ≥ 4 mmHg/year in 55 patients (57.3%).

At follow-up, the reduction in AVA was -0.16 ± 0.16 cm² (Figure 2), reflecting a median annual progression rate of -0.05 [-0.10 to -0.02] cm²/year (mean -0.06 cm²/year). A decrease in AVA was observed in 80 patients (83%) at follow-up. In this group, the median annual reduction in AVA was -0.07 [-0.11 to -0.03] cm²/year (mean -0.09 cm²/year). The indexed AVA also showed a significant decrease at follow-up relative to baseline (p < 0.001).

At follow-up, 46 patients (48%) had acquired the features of classical HG-SAS (i.e. MPG ≥ 40 mm Hg, AVA < 1 cm²). In these patients, the median progression of peak aortic jet velocity, MPG, and AVA was 0.32 [0.18-0.45] m/s/year (mean 0.39 m/s/year), 6.6 [3.0-9.6] mm Hg/year (mean 8 mm Hg/year), and -0.06 [-0.11 to -0.04] cm²/year (mean -0.08 cm²/year), respectively. Factors associated with annualized progression of Vmax are displayed in Table 3. On multivariable linear regression analysis, age<70 years (r -0.284; p=0.014), Vmax at baseline ≤ 3m/s (r -0.220; p=0.030), and concentric LV hypertrophy (r 0.214; p=0.026) were associated with annualized progression of Vmax. At follow-up, 11 patients (11%) had acquired the features of low-flow low-gradient severe AS with preserved LVEF (i.e. SVi < 35 mL/m², MPG < 40 mm Hg, AVA < 1 cm² and LVEF ≥ 50%), whereas four (4%) had acquired those of low-flow low-gradient severe AS with reduced LVEF (LVEF between 32% and 49%).

No significant change over time were observed in terms of LV end-diastolic diameter, LV end-systolic diameter, indexed LV mass or LVEF (all p>0.14) (Table 2, Figure 3). However, over time, we observed an increase in the number of patients with concentric LV hypertrophy (from 55% to 70%, p = 0.025), at the expense of less eccentric LV hypertrophy (from 27% to 10%, p = 0.004), and a significant increase in left atrial area (p = 0.02), systolic pulmonary artery pressure (p = 0.04), and valvulo-arterial impedance (p < 0.001) (Table 2, Figure 4). Stroke volume and stroke volume indexed to BSA both showed a significant decrease at follow-up relative to baseline (p = 0.006 and p = 0.002, respectively) (Table 2,

Figure 4).

After the second echocardiography, 26 patients (27%) underwent AVR (mean time: 6±13 months) and 31 patients (32%) died (mean time: 14±27 months).

DISCUSSION

We demonstrate significant hemodynamic progression over time in NF-LG-SAS with preserved LVEF. Indeed, at follow-up, we observed worsening of at least one parameter of AS severity quantification (MPG, peak aortic jet velocity, or AVA) in > 80% of patients.

Mean MPG and peak aortic jet velocity were significantly higher at follow-up than at baseline (p < 0.001 both), with 48% of patients developing the hemodynamic profile of HG-SAS.

Median annual rates of progression of MPG and peak aortic jet velocity were 4.3 mm Hg per year and 0.25 m/s per year, respectively, with a simultaneous annual reduction of AVA of 0.05 cm² per year. Such hemodynamic progression was not associated with significant LVEF impairment. However, we observed more LV concentric hypertrophy over time and an increase in left atrial area.

Patients with NF-LG-SAS and preserved LVEF are classically elderly, with a small BSA and less concentric LV hypertrophy, less severe left atrial dilation, and less severely impaired systolic longitudinal function than other forms of severe AS [START_REF] Lancellotti | Clinical outcome in asymptomatic severe aortic stenosis: insights from the new proposed aortic stenosis grading classification[END_REF][START_REF] Mehrotra | Differential left ventricular remodelling and longitudinal function distinguishes low flow from normal-flow preserved ejection fraction low-gradient severe aortic stenosis[END_REF]. However, its outcome and its management are a matter of debate. Several studies have suggested a poor outcome for this form of AS [START_REF] Dayan | Outcome and impact of aortic valve replacement in patients with preserved LVEF and low-gradient aortic stenosis[END_REF][START_REF] Ozkan | Impact of aortic valve replacement on outcome of symptomatic patients with severe aortic stenosis with low gradient and preserved left ventricular ejection fraction[END_REF][START_REF] Zusman | Intervention versus observation in symptomatic patients with normal flow-low gradient severe aortic stenosis[END_REF][START_REF] Clavel | The complex nature of discordant severe calcified aortic valve disease grading: new insights from combined Doppler echocardiographic and computed tomographic study[END_REF][START_REF] Saeed | The impact of aortic valve replacement on survival in patients with normal flow low gradient severe aortic stenosis: a propensity-matched comparison[END_REF]. A meta-analysis even suggested that the prognosis of this entity is comparable to that of HG-SAS, reporting a 52% reduction in the relative risk of mortality when AVR was performed in this population [START_REF] Dayan | Outcome and impact of aortic valve replacement in patients with preserved LVEF and low-gradient aortic stenosis[END_REF]. On the other hand, we and others reported that patients with NF-LG-SAS show comparable survival to that of moderate AS patients when AVR was performed during follow-up, according to guidelines and suggest that AVR performed at the stage of NF-LG AS does not improve survival [START_REF] Mehrotra | Differential left ventricular remodelling and longitudinal function distinguishes low flow from normal-flow preserved ejection fraction low-gradient severe aortic stenosis[END_REF][START_REF] Tribouilloy | Low-gradient, low-flow severe aortic stenosis with preserved left ventricular ejection fraction: characteristics, outcome, and implications for surgery[END_REF][START_REF] Kang | Watchful observation versus early aortic valve replacement for symptomatic patients with normal flow, low-gradient severe aortic stenosis[END_REF][START_REF] Chadha | Outcome of Normal-Flow Low-Gradient Severe Aortic Stenosis With Preserved Left Ventricular Ejection Fraction: A Propensity-Matched Study[END_REF]. Moreover, we recently reported that patients with HG-SAS showed significant excess mortality compared to patients with NF-LG-SAS with preserved LVEF [START_REF] Chadha | Outcome of Normal-Flow Low-Gradient Severe Aortic Stenosis With Preserved Left Ventricular Ejection Fraction: A Propensity-Matched Study[END_REF]. These data suggest that most of these patients should be considered to have a moderate form of AS and should be initially treated conservatively [START_REF] Chadha | Outcome of Normal-Flow Low-Gradient Severe Aortic Stenosis With Preserved Left Ventricular Ejection Fraction: A Propensity-Matched Study[END_REF]. Accordingly, Kang et al. [START_REF] Kang | Watchful observation versus early aortic valve replacement for symptomatic patients with normal flow, low-gradient severe aortic stenosis[END_REF] reported that overall and cardiovascular mortality were not significantly different for symptomatic patients with NF-LG-SAS when a watchful observation strategy was applied compared to an early AVR strategy. Current European guidelines consider that patients with NF-LG-SAS usually only have moderate AS and should not be referred at this stage for AVR [START_REF] Baumgartner | ESC/EACTS Guidelines for the management of valvular heart disease[END_REF][START_REF] Nishimura | ACC/AHA Task Force Members. 2014 AHA/ ACC guideline for the management of patients with valvular heart disease: a report of the American College of Cardiology/American Heart Association Task Force on Practice Guidelines[END_REF].

Although changes in Doppler echocardiographic parameters over time in patients with low-flow low-gradient severe AS and preserved LVEF have been specifically investigated [START_REF] Tribouilloy | Progression of Low-Gradient, Low-Flow, Severe Aortic Stenosis With Preserved Left Ventricular Ejection Fraction[END_REF][START_REF] Dahl | Development of paradoxical low-flow, low-gradient severe aortic stenosis[END_REF], there is no specific data concerning the evolution over time of hemodynamic severity parameters in the NF-LG-SAS setting. These data are however essential for understanding the "true" severity of this form. Prospective studies on the rate of hemodynamic progression in AS have reported an average rate of increase in MPG of 7 mmHg/year, with an increase in aortic jet velocity of 0.3 m/s/year, and a decrease in AVA of 0.1 cm²/year [START_REF] Freeman | Spectrum of calcific aortic valve disease: pathogenesis, disease progression, and treatment strategies[END_REF]. Here, we report a comparable mean progression in patients with NF-LG-SAS and preserved LVEF (averages of 0.34 m/s/year, 6 mm Hg/year, and 0.06 cm²/year respectively), but with individual variations. Accordingly, Kang et al. [START_REF] Kang | Watchful observation versus early aortic valve replacement for symptomatic patients with normal flow, low-gradient severe aortic stenosis[END_REF] reported that MPG increased significantly in patients with NF-LG-SAS at a median echocardiographic follow-up of 1.9 years when a strategy of watchful observation was applied. Here, we observed that nearly half the patients (48%) acquired the features of classical HG-SAS, with a median follow-up of 25 months. Such a high rate of progression of NF-LG-AS with preserved LVEF towards classical HG-SAS supports careful and close echocardiographic monitoring for these patients, especially when MPG is close to 40 mm Hg or Vmax to 4 m/s.

During follow-up, we also observed changes in LV geometry, with more concentric LV hypertrophy associated with an increase of LA area, suggesting evolution of the aortic valve disease. The observed significant decrease in stroke volume index may suggest an alteration of LV systolic function. However, we did not observe a significant reduction in LVEF or left ventricular volume variation.

Our study had several limitations. First, it was subject to the limitations inherent in the analysis of retrospective data. Second, there was a certain degree of selection bias, as we selected patients who had at least one available follow-up echocardiography and no aortic valve intervention between the two examinations. Third, although cardiologists with expertise in valvular heart disease performed the diagnosis and follow-up, inherent measurement errors may have led to a certain degree of misclassification of NF-LG-SAS as another form of AS.

Fourth, valvular calcium scoring assessed by computed tomography, which has been identified as a predictor of AS progression was not available in our database. Fifth left ventricular global longitudinal strain data were not available. Finally, we identified some factors associated with the hemodynamic progression of AS. Unfortunately, the aortic valve calcium scoring using computer tomography, a classic factor of progression, is not available in our database.

In conclusion, these findings strongly suggest that NF-LG-SAS with preserved LVEF generally represents an "intermediate stage" of AS between classical moderate AS and classical HG-SAS with preserved LVEF. Over time, these patients present a high risk of rapid progression to high gradient AS, with LA and LV remodeling, without significant LVEF impairment. They should therefore be subject to close and rigorous clinical and echocardiographic monitoring and be referred for AVR when indicated, in accordance with current European guidelines.

Figure 1 .

 1 Figure 1. Flow-chart of the study.

Figure 2 .

 2 Figure 2. Changes in hemodynamic parameters of aortic stenosis severity over time: (A)

Figure 3 .

 3 Figure 3. Changes in left ventricular (LV) end-diastolic diameter (A), LV end-systolic

Figure 4 .

 4 Figure 4. Changes in indexed stroke volume (A), valvulo-arterial impedance (B), left atrial

Acknowledgments: none

Source of funding: none

Disclosure: none

Solid lines in the boxplots represent the median value and cross mark in the boxplots represent the mean value.


Pharm D Laura Foucault-Fruchard

Jean Baptiste Delaye

MD) c Virginie Morange

MD Emilie Beaufils

MD) e Coline Duwicquet

MD Isabelle Quadrio

Anna Chloé Balageas

Pharm D Diane Dufour-Rainfray

Jean Baptiste

Anna Chloé

An automated alert system based on the p-Tau/Tau ratio to quickly inform health professionals upon a suspected case of sporadic Creutzfeldt-Jakob disease

Keywords: Creutzfeldt-Jakob disease, iatrogenic transmission, alert system, hygiene measures

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

Introduction

Knowing the risk of potential sCJD instrument-contamination is essential in hospitals. We examined the relevance of the p-Tau/Tau ratio to exclude a probable case of sCJD in clinical practice, and we established an alert system to quickly inform health professionals in case of positivity.

Methods

This retrospective study was conducted on 143 CSF samples from patients suspected for sCJD. The distinction between probable cases of sCJD and other patients was based on clinical, paraclinical and biological (14-3-3, Tau, p-Tau, Aβ 1-42 ) data. From this experience, the health professionals developed an alert system to be implemented upon a suspected case of sCJD.

Results

A significant decrease in p-Tau/Tau ratio between sCJD and the other diseases was observed (p<0.001). The combined Tau test presented a sensitivity higher than 14-3-3 (100% versus 92.3%, p=0.006) and an equivalent specificity (90% versus 96.1%). The time required for obtaining results was higher for 14-3-3 due to the centralization of investigations in some laboratories (3 weeks versus 2 hours). In the presence of these elements, the triggering of the alert system was based on the p-Tau/Tau ratio. This system involves sending an automatic mail to the hospital department involved in the patient's care and the hospital hygiene team, which oversees the application of the procedures.

Conclusion

The p-Tau/Tau concentrations present the desired criteria for use in current medical practice to fight against iatrogenic transmission. The alert system confirms a probable case of sCJD instantly to health professionals. Hygiene and sterilization measures can be applied immediately.

Introduction

Creutzfeldt-Jakob disease (CJD) is characterized by rapid neurodegeneration leading to death in a few months to a few years. CJD belongs to the category of prion diseases, also known as transmissible subacute spongiform encephalopathies. These rare neurodegenerative diseases are characterized by the accumulation in the central nervous system (CNS) of the prion protein, at the origin of the transmission of the disease [START_REF] Johnson | Prion diseases[END_REF]. CJD can be of genetic, infectious or sporadic origin. The latter case represents 85% of all CJD diagnosed each year [START_REF] Manix | Creutzfeldt-Jakob disease: updated diagnostic criteria, treatment algorithm, and the utility of brain biopsy[END_REF] and its median survival is only 6 months [3].

The diagnosis of sCJD is based on clinical signs suggestive of recent onset and a rapid and progressive evolution (fast worsening dementia, myoclonus, vision or cerebellar disorders, pyramidal/extrapyramidal disorders, akinetic mutism). However, this diagnosis may not be possible early in the illness based on diagnostic surveillance criteria. Indeed, heterogeneity of symptoms, particularly in the early in the course of the disease, can be associated with another progressive, rapidly irreversible form of dementia. Diagnosis should be supported by electroencephalogram (EEG), magnetic resonance imaging (MRI) or biomarker testing in cerebrospinal fluid (CSF) [START_REF] Steinhoff | Accuracy and reliability of periodic sharp wave complexes in Creutzfeldt-Jakob disease[END_REF][START_REF] Newey | Variability in diagnosing Creutzfeldt-Jakob disease using standard and proposed diagnostic criteria[END_REF][START_REF]World Health Organization, Manual for Strengthening Diagnosis and Surveillance of Creutzfeldt-Jakob Disease[END_REF]. The definitive diagnosis of CJD is based on post-mortem neuropathological examination of the brain with positive identification of pathological prion protein [START_REF] Parchi | Incidence and spectrum of sporadic Creutzfeldt-Jakob disease variants with mixed phenotype and cooccurrence of PrPSc types: an updated classification[END_REF]. The 14-3-3 protein is widely used as a surrogate marker for the premortal diagnosis of sCJD [START_REF] Hsich | The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies[END_REF][START_REF] Sanchez-Juan | CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease[END_REF][START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. This biomarker has been included in the diagnostic criteria of sCJD by the World Health Organization (WHO) [START_REF]World Health Organization, Manual for Strengthening Diagnosis and Surveillance of Creutzfeldt-Jakob Disease[END_REF]. Neuronal lysis observed during sCJD is associated with the leakage of this protein into the CSF [START_REF] Hsich | The 14-3-3 brain protein in cerebrospinal fluid as a marker for transmissible spongiform encephalopathies[END_REF]. The investigation of the 14-3-3 protein being carried out in a few specialized laboratories spread over the territory, the time required for the obtaining of results is several weeks. Updated criteria for prion diseases suggest RT-QuIC (Real-Time Quaking-Induced Conversion) [START_REF] Manix | Creutzfeldt-Jakob disease: updated diagnostic criteria, treatment algorithm, and the utility of brain biopsy[END_REF]. The performance of this technique was compared to the performance of the 14-3-3 protein detection. RT-QuIC is as sensitive as 14-3-3 test but its undeniable advantage is its excellent specificity which can be considered at 100%, while 14-3-3 protein is only a lysis marker [START_REF] Hermann | Validation and utilization of amended diagnostic criteria in Creutzfeldt-Jakob disease surveillance[END_REF]. However, in addition to the requirement of specialised equipment and a significant cost, this reference technique presents three major disadvantages in general hospitals. First, this technique is based on amplification of the supposed pathogen. Therefore, RT-QuIC must be performed in a P3 laboratory in France [START_REF] Brandel | Les marqueurs du liquide céphalo-rachidien pour le diagnostic des maladies à prions[END_REF]. Secondly, this experimentation used recombinant prion protein. Its quality is essential and is a condition to the success of the experimentation. Thirdly, RT-QuIC analysis takes time (36h in the optimized versions) and is in plate (serial analysis).

Another alternative to the 14-3-3 protein for a diagnosis of probable sCJD is the quantification in the CSF of Tau protein and its phosphorylated form (p-Tau) [START_REF] Bahl | The diagnostic efficiency of biomarkers in sporadic Creutzfeldt-Jakob disease compared to Alzheimer's disease[END_REF]. Unlike 14-3-3 protein, Tau protein is increased at the onset of clinical signs [START_REF] Sanchez-Juan | CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease[END_REF][START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. Its concentration is correlated with the intensity of neuronal degeneration [START_REF] Blennow | Cerebrospinal fluid and plasma biomarkers in Alzheimer disease[END_REF]. A threshold concentration to guide the diagnosis to sCJD was defined as between 1200 and 1300 ng/L [START_REF] Sanchez-Juan | CSF tests in the differential diagnosis of Creutzfeldt-Jakob disease[END_REF][START_REF] Otto | Tau protein and 14-3-3 protein in the differential diagnosis of Creutzfeldt-Jakob disease[END_REF][START_REF] Skillbäck | Diagnostic performance of cerebrospinal fluid total tau and phosphorylated tau in Creutzfeldt-Jakob disease: results from the Swedish Mortality Registry[END_REF]. An increase of Tau protein, at a lower concentration, is also observed in Alzheimer's disease [START_REF] Blennow | CSF total tau, Abeta42 and phosphorylated tau protein as biomarkers for Alzheimer's disease[END_REF]. In contrast, a significant increase in p-Tau concentration is characteristic of Alzheimer's disease (> 60 pg/ml) due to an imbalance in cell kinases and phosphatases [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF]. Nevertheless, moderate increases in p-Tau concentrations have also been detected in the CSF of sCJD cases compared to other types of dementia [START_REF] Hyeon | Alternative application of Tau protein in Creutzfeldt-Jakob disease diagnosis: Improvement for weakly positive 14-3-3 protein in the laboratory[END_REF][START_REF] Llorens | Cerebrospinal Fluid Biomarker-Based Diagnosis of Sporadic Creutzfeldt-Jakob Disease: A Validation Study for Previously Established Cutoffs[END_REF]. Overall, a p-Tau/Tau ratio lower than 0.0713 allows the distinguishing of sCJD from other neurological disorders [START_REF] Llorens | Comparative analysis of cerebrospinal fluid biomarkers in the differential diagnosis of neurodegenerative dementia[END_REF]. The combination of increased Tau protein concentrations and decreased p-Tau/Tau ratio in patients with sCJD has high specificity and is a useful diagnostic test for sCJD [START_REF] Skillbäck | Diagnostic performance of cerebrospinal fluid total tau and phosphorylated tau in Creutzfeldt-Jakob disease: results from the Swedish Mortality Registry[END_REF]. The combined Tau test, i.e Tau and p-Tau protein assays, offers the advantage of being able to be performed by automated methods in many laboratories [START_REF] Otto | Tau protein and 14-3-3 protein in the differential diagnosis of Creutzfeldt-Jakob disease[END_REF]. Every day in healthcare facilities, patients with suggestive symptomatology of sCJD are hospitalized. It is essential to be able to provide a probable diagnosis of sCJDs in a very short time in suspected patients in order to put in place safety procedures to avoid the risk of iatrogenic transmission of the prion, especially during invasive procedures [START_REF]du 1er décembre 2011 relative à l'actualisation des recommandations visant à réduire les risques de transmission d'agents transmissibles non conventionnels lors des actes invasifs[END_REF].

In this context, the objective of our study was to assess the relevance of the p-Tau/Tau ratio for the diagnosis of sCJD in routine hospital practice and to establish an alert system to quickly inform health professionals in case of positivity in order to immediately apply hygiene and sterilization measures to avoid transmission.

Materials and Methods

Study population

This retrospective study was conducted on 143 CSF samples collected between 2011 and 2019 as part of routine care in patients suspected for sCJD. The samples were analysed by our institution's hormonology Laboratory. For each patient, the presence of protein 14-3-3 was investigated and the proteins Tau, p-Tau as well as the amyloid peptide Aβ1-42 were measured in the CSF. The study population included 2 confirmed cases by post-mortem examination and 11 probable cases of sCJD. According to the European MRI-CJD Consortium criteria [START_REF] Zerr | Updated clinical diagnostic criteria for sporadic Creutzfeldt-Jakob disease[END_REF], a patient is considered likely to have sCJD if at least 2 clinical criteria and 1 paraclinical criterion on the following list are present:

-Clinical signs: rapidly progressive dementia, myoclonus, cerebellar or visual disturbance, pyramidal or extrapyramidal dysfunction, akinetic mutism

Biomarker analysis in CSF

Lumbar punctures were performed according to the standardized procedure for routine diagnosis. The CSF samples were collected into polypropylene test tubes (Sarstedt) and centrifuged (4000g, 10min, 4 • C). The supernatants were aliquoted into polypropylene test tubes (Sorenson Bioscience) and frozen (-80 • C) within 2 hours of puncture. Western blot immunoassay for protein 14-3-3 was performed in the Neurochemistry Unit, Hospices Civils de Lyon according to the protocol previously described [START_REF] Fourier | Detection of CSF 14-3-3 Protein in Sporadic Creutzfeldt-Jakob Disease Patients Using a New Automated Capillary Western Assay[END_REF]. Positive and negative CSF control samples with trace test results were used in all experiments as internal quality control.

Biomarker analyses (Tau, p-Tau, Aβ1-42) were performed in our hospital with commercially available ELISA kits (Innotest hTau Ag, Innotest phospho-Tau181, Innotest β-amyloid1-42, Fujirebio®, Belgium) according to the manufacturer's instructions. Optical densities were measured by a photometer (Appliskan®, ThermoFisher Scientific, Germany). Each analysis was performed in duplicate. Longitudinal stability in the biomarker measurements was monitored, including controls at the beginning and end of each measurement series. Interassay coefficients of variation were 7.6% for Tau, 5.4% for p-Tau and 8.7% for Aβ1-42. All samples were obtained and analysed as part of routine care and in accordance with French legislation.

Development and implementation of an alert system to inform health professionals

Multidisciplinary meetings involving the biologists, neurologists, hygienists and the information technology department of our institution took place in order to determine the computer programs to be used and the data transmission circuit depending on the result of the ratio p-Tau/Tau (i.e, higher or lower than 0.0713). The role of each health professional of the circuit was clearly identified. Trials with fictitious patients (hospitalized in geriatrics or neurology, with ratio p-Tau/Tau > or ≤ to 0.0713) were carried out several times to verify this computer alert system. It was an essential preliminary step before its application in medical practice.

Data analysis

The results were analyzed using the GraphPad Prism v.7 software, California, USA.

Comparisons were adjusted for age and sex with a logistic regression. The Benjamini-Hochberg post-test was applied following the Kruskal-Wallis test in the event of significant differences between the groups. The sensitivity, specificity, positive and negative predictive values of the tests were compared with the Fisher's exact test.

Results

Diagnosis of sCJD in hospital practice

The demographic characteristics of the study population (median age 71 years old, sex 50/50) and CSF biomarker data are described in Table 1.

Table 1: Demographic characteristics of the study population and corresponding

cerebral spinal fluid biomarker data. For each disease represented in the population, the total number of cases, the demographic characteristics (age, sex) and the corresponding cerebral spinal fluid biomarker data (Tau, p-Tau, Aβ1-42 concentrations, p-Tau/Tau ratio, 14-3-3 protein) are reported. # median (25% -75% percentile). F: female, M: male, N: negative, P: positive, T: trace. Tau protein concentration was higher in sCJD than in AD, LBD, FTD and 'other disorders' groups but not significantly different after adjusting for age and sex. A significant increase in Tau protein concentration was observed in AD group versus LBD, FTD and 'other disorders' groups (Fig. 2a). Concerning p-Tau protein concentration, a significant increase was observed in AD group versus FTD and other neurological disorders groups. p-Tau protein concentration was also higher in sCJD group compared to all the other groups (Fig. 2b). There was a significant decrease (p<0.001) in p-Tau/Tau ratio values between sCJD and all the other groups (Fig. 2c). Concerning Aβ1-42 concentration, a significant decrease was observed in AD group versus sCJD, FTD and other neurological disorders (Fig. 2d). The 14-3-3 protein was positive in 13 patients (12 sCJD and 1 other neurological pathology) and doubtful in 4 cases

(1 AD, 1 FTD, 2 other neurological pathologies). The clinical characteristics of patients with sCJD and the main results of the studies are shown in Table 2. to lumbar puncture and sCJD patient's death were respectively 8 (± 3) and 13 (± 6) weeks.

F
Tau concentrations were dramatically increased for all probable cases of sCJD with an average at 10 467 ng/L (normal cut-off is 500 ng/L) since the first symptoms. Similarly, p-Tau/Tau ratio was decreased in these patients with an average at 0.0110 (cut-off is 0.0713).

The average time from lumbar puncture to results of the 14-3-3 immunoassay on CSF was about 3 weeks (19 days). In the context of our study, when the 14-3-3 test was doubtful, we considered that a case of sCJD could not be excluded. Hence, a doubtful test result was considered as a positive result. The sensitivity of the p-Tau/Tau test was significantly higher (p=0.006) than the 14-3-3 test (100% versus 92.3%). However, the positive predictive value of the combined Tau test was significantly lower (p<0.0001) than the 14-3-3 test (50% versus 70.6%) No statistical difference was observed for specificity (90% versus 96.1%), and negative predictive value (100% versus 99.2%) between these two tests (Table 3, Table 4).

An alert system to save time in setting up safety procedures (Fig. 3)

Work collaboration led to the hospital of a warning system aimed at alerting the hospital hygiene unit as soon as possible about the confirmation of a probable sCJD case. Hence, in order to save time in setting up security procedures, an alert email is automatically sent to the hospital hygiene team and the care service if a suspected patient of sCJD has a p-Tau/Tau ratio lower than the minimum threshold for the diagnosis of CJD. These alert emails mention the patient identification number and the ratio p-Tau/Tau value. This information is directly Other neurological pathology 5 125

14 extracted from the computerized patient record that includes the biological data entered by the biochemistry laboratory and is sent by automatic email from the laboratory computer system (Millenium® Cerner) to the hospital hygiene unit. On receipt of the alert, the hospital hygiene unit contacts the care service where the patient is hospitalized, then the teams of clinicians proceed with the declaration of the disease to the competent authorities. Any suspicion requires an approach to ascertain that previously the patient has not undergone any examination or intervention likely to involve a risk for another patient, including any invasive acts during the 6 months preceding the onset of symptoms. Other actions are carried out in the event of a probable case of sCJD: (i) Manage the patient with Standard Hygiene Precautions.

(ii) Remind healthcare teams that the use of single-use medical devices is to be preferred. (iii)

At the same time, the hospital hygiene unit, in collaboration with the pharmacy (sterilization department), ensures the management of any medical devices used and the adaptation of the treatment procedures involving equipment used if further investigations are to be carried out.

When prescribing, the prescriber reports the suspicion of sCJD so as to adapt the cleaning of medical devices to a prionicidal protocol and to limit acts according to benefit/risk assessment. (iiii) Schedule an autopsy to confirm the diagnosis. Description of an early warning system based on p-Tau/Tau ratio values to save time in setting up CJD security procedures in the event of probable sCJD.

Conclusion

In this study, we aimed to implement an alert system to quickly and automatically inform health professionals in cases of positive probability diagnosis for sCJD in order to avoid iatrogenic transmission. With this objective in mind, we provide an investigation into the relevant of CSF biomarkers in hospital practice to quickly diagnose a probable case of sCJD.

One of the study's strengths is the heterogeneity of the neurological diseases represented in our cohort study (especially, acute neurological disorders). Ethically, lumbar punctures are only performed if clinical signs suggest a neurological disorder. Hence, unlike a comparison "sCJD versus no neurological pathology", our study cohort is representative of clinical practice, thus limiting bias.

Knowing the risk of sCJD before proceeding with invasive investigations or surgical interventions, and thus being able to implement infection control measures with single use equipment, or using prionicidal protocol medical device disinfection, is essential in healthcare facilities. The probability diagnosis of sCJD remains challenging because symptoms and paraclinical examinations only become suggestive after a certain time in the course of the disease [START_REF] Eisenmenger | Evolution of Diffusion-Weighted Magnetic Resonance Imaging Signal Abnormality in Sporadic Creutzfeldt-Jakob Disease, With Histopathological Correlation[END_REF][START_REF] Connor | Clinical Laboratory Tests Used To Aid in Diagnosis of Human Prion Disease[END_REF]. Reliable and early biomarkers for diagnosis of sCJD would be very useful in medical practice.

The 14-3-3 protein is the reference biological marker for the probability diagnosis of sCJD according to the WHO [START_REF]World Health Organization, Manual for Strengthening Diagnosis and Surveillance of Creutzfeldt-Jakob Disease[END_REF]. The duration of the disease influences its sensitivity [START_REF] Collins | Determinants of diagnostic investigation sensitivities across the clinical spectrum of sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Baldeiras | Diagnostic value of CSF protein profile in a Portuguese population of sCJD patients[END_REF].

Neuronal lesions in other conditions, such as encephalitis, AD and other dementia disorders can produce false positive results [START_REF] Chapman | Misleading results with the 14-3-3 assay for the diagnosis of Creutzfeldt-Jakob disease[END_REF][START_REF] Burkhard | CSF detection of the 14-3-3 protein in unselected patients with dementia[END_REF]. Traumatic lumbar puncture can also lead to false positive since the 14-3-3 protein is present in large amounts in red blood cells [START_REF] Schmitz | Validation of 14-3-3 Protein as a Marker in Sporadic Creutzfeldt-Jakob Disease Diagnostic[END_REF]. A combination of elevated Tau protein concentration with the presence of 14-3-3 protein in CSF gives considerable specificity [START_REF] Bahl | The diagnostic efficiency of biomarkers in sporadic Creutzfeldt-Jakob disease compared to Alzheimer's disease[END_REF]. Hence, several research teams were interested in the value of the p-Tau/Tau ratio to discriminate sCJD from other neurological disorders. They highlighted a p-Tau/Tau sensitivity higher than 14-3-3 test (94% versus 44-95%) [START_REF] Bahl | The diagnostic efficiency of biomarkers in sporadic Creutzfeldt-Jakob disease compared to Alzheimer's disease[END_REF][START_REF] Connor | Clinical Laboratory Tests Used To Aid in Diagnosis of Human Prion Disease[END_REF][START_REF] Hamlin | A comparison of tau and 14-3-3 protein in the diagnosis of Creutzfeldt-Jakob disease[END_REF] and confirmed the robustness of this surrogate biomarker in sCJD reflecting the intensity of neuronal damage [START_REF] Llorens | Comparative analysis of cerebrospinal fluid biomarkers in the differential diagnosis of neurodegenerative dementia[END_REF][START_REF] Llorens | Progress in CSF biomarker discovery in sCJD[END_REF][START_REF] Riemenschneider | Phospho-tau/total tau ratio in cerebrospinal fluid discriminates Creutzfeldt-Jakob disease from other dementias[END_REF].

In our study, the p-Tau/Tau ratio test presented a higher sensitivity than 14-3-3. This result confirms the data published in previous studies [START_REF] Bahl | The diagnostic efficiency of biomarkers in sporadic Creutzfeldt-Jakob disease compared to Alzheimer's disease[END_REF][START_REF] Connor | Clinical Laboratory Tests Used To Aid in Diagnosis of Human Prion Disease[END_REF][START_REF] Hamlin | A comparison of tau and 14-3-3 protein in the diagnosis of Creutzfeldt-Jakob disease[END_REF]. Remarkably, although not significantly, the negative predictive value of the combined Tau test was higher than that of the 14-3-3 test, which is a considerable asset in limiting the risk of wrongly excluding a probable case of sCJD. Hence, this data attests that the use of the p-Tau/Tau ratio is clinically very relevant in confirming a probable case of sCJD. However, it should be noted that these encouraging values can be influenced in part by the small size of our cohort. In addition, the p-Tau/Tau ratio tends to be lower in the early stages of the disease, when the risk of doubtful paraclinical examinations is highest. Interestingly, one sCJD patient without typical EEG/IRM and a negative 14-3-3 protein 10 weeks after the first symptoms, but a ratio p-Tau/Tau positive, died after 17 weeks of disease progression. In this case, the patient's clinical evolution probably confirms the diagnosis of sCJD but a post-mortem diagnosis would have been helpful to confirm or deny with certainty the presence of this disease. Unfortunately, autopsy is often refused by the family. Another advantage of the p-Tau/Tau test is the possibility of carrying it out using automated chemiluminescent rapid methods and using commercial immunoassays, which are standardized and used worldwide in many laboratories in clinical routine because of the use of these biomarkers for the diagnosis of Alzheimer's disease [START_REF] Llorens | Progress in CSF biomarker discovery in sCJD[END_REF]. The duration of the analysis is a couple of minutes guaranteeing the possibility to inform the clinicians in less than 2 hours. In our study, the time from lumbar puncture to the return of the 14-3-3 test results was approximately 3 weeks. This delay is incompatible with clinical practice. The clinical evolution of the patient, or even his death, make the diagnosis highly probable before obtaining the result. It is worth noting the total time necessary to obtain the 14-3-3 test result, which includes: first, the time necessary to send the CSF sample to the reference laboratory, then, the time necessary to perform the analysis. In

France, as elsewhere in Europe, the detection of the 14-3-3 protein for diagnostic purposes is centralized in reference laboratories. These prion disease surveillance centers belong to the national CJD surveillance network which is integrated into the European surveillance network. Detection of 14-3-3 protein is usually performed by using western blot. The use of ELISA kits would probably save time compared to the western blot. However, in France, the ELISA kits for the detection of the 14-3-3 protein are reserved for research only. These kits do not have the CE marking which is mandatory for in vitro diagnostic devices used in medical practice. Moreover, a sufficiently large number of CSF samples is necessary to perform a series of analyses. CJD being a rare disease, the risk linked to ELISA is the freezing of the CSF for a long time and ELISA analysis on a small series is not financially attractive.

For the determination of p-Tau/Tau, automatic analysers are present in hospitals (e.g.

Lumipulse analysers from Fujirebio® or Cobas from Roche®). Thus, the measurement of these proteins takes place simultaneously which saves time. The cost of the p-Tau/Tau ratio test and the 14-3-3 test is roughly equivalent in France (108 euros versus 94.5 euros, respectively).

Altogether, our data confirms the idea that CSF p-Tau/Tau tests are robust and probably early sCJD biomarkers, which opens new perspectives on diagnosis and rapid patient management.

However, these results remain to be confirmed. Due to the focus on a specific hospital, the sample size is limited and lacks strength.

Taking into account the advanced elements, it would seem interesting that the WHO recommendations include in the future the dosage of the Tau ratio along with the detection of the 14-3-3 protein. Overall, we confirmed the relevant use of the p-Tau/Tau ratio in clinical practice to exclude probable cases of sCJD. This sporadic form is associated with a rapid evolution and an extraordinarily high neural lysis, which generates very high Tau protein concentrations in the CSF and consequently a low p-Tau/Tau ratio (ie, ≤ 0.0713) [START_REF] Lattanzio | Prion-specific and surrogate CSF biomarkers in Creutzfeldt-Jakob disease: diagnostic accuracy in relation to molecular subtypes and analysis of neuropathological correlates of p-tau and Aβ42 levels[END_REF]. This threshold allows us to obtain excellent performance in our study. However, depending on the disease subtype whose progression is slower or different, it is possible that this ratio may be lower, potentially leading to false negatives. It is therefore still important to have several markers to validate the diagnosis [3,[START_REF] Lattanzio | Prion-specific and surrogate CSF biomarkers in Creutzfeldt-Jakob disease: diagnostic accuracy in relation to molecular subtypes and analysis of neuropathological correlates of p-tau and Aβ42 levels[END_REF]. One of the main objectives of our study was to examine the relevance of the p-Tau/Tau ratio to provide the first hint of a sCJD disease suspicion. The use of this ratio is not intended to establish with certainty the diagnosis of sCJD but it aims to warn health professionals that a case of sCJD cannot be excluded.

Whatever the value of the Tau ratio, all paraclinical and biological examinations were performed (protein 14-3-3, MRI, EEG ...) to limit a false diagnosis. The interpretation of MRI in most European hospitals is not a coded or an encrypted data in the computerized patient record and therefore it cannot be taken into account by artificial intelligence, and more specifically, by our alert system. In addition, given the waiting times in hospitals, the MRI examination generally takes place after the dosage of the Tau and p-Tau proteins.

Given the rapid evolution of the disease from the onset of the first symptoms, it's essential that the coordination of health professionals and social care be put in place as soon as probable diagnosis of sCJD is made. Hygiene and sterilization measures must be applied immediately to prevent transmission by contaminated human tissues (e.g., corneal graft, dura mater). Thus, we wanted to develop an alert system based on the combined Tau test to aid early diagnosis.

The risk of iatrogenic CJD according to recent direct evidence appears to be low. However, the long asymptomatic incubation periods (20 years and more in some cases), the difficulties of eradicating prions from surgical instruments and the high levels of infectivity of CJD in the brain mean that there is a margin of uncertainty around quantifying the risk of iatrogenic transmission of CJD [START_REF] Stevenson | Interventions to reduce the risk of surgically transmitted Creutzfeldt-Jakob disease: a cost-effective modelling review[END_REF]. Although no studies have identified a new case over the past few years, many case reports reported a relationship between CJD and a surgical intervention [START_REF] Stevenson | Interventions to reduce the risk of surgically transmitted Creutzfeldt-Jakob disease: a cost-effective modelling review[END_REF][START_REF] Gnanajothy | A case of Creutzfeldt-Jakob disease following cataract surgery: sporadic versus iatrogenic cause[END_REF][START_REF] Moreno | Creutzfeldt-Jakob disease cluster in the health area of Meixoeiro Hospital[END_REF][START_REF] Puopolo | Transmission of sporadic Creutzfeldt-Jakob disease by blood transfusion: risk factor or possible biases[END_REF]. Hence, potential CJD instrument-contamination events may continue to occur, causing widespread hospital and patient concern. The current decision problem focuses on the risk of transmission of CJD via surgical instruments. Iatrogenic transmission can occur when surgical instruments, endoscopes or laryngoscopes are used during high-risk surgical procedures.

Conventional cleaning and sterilization techniques applied in hospitals to surgical instruments are not sufficient to completely deactivate prions. The early and automatic alert described in this article allows these complementary techniques to be implemented without delay and only in cases where the risk is proven. Complementary techniques and sequestration of this equipment is necessary before re-use.

Any clinical suspicion of sCJD should be reported to the hospital hygiene team and the completion of any invasive procedure should be preceded by a benefit/risk assessment.

According to French guidelines, in the event of positivity, single-use medical devices (or medical devices with single-use protection) should be favoured if they are compatible with safe and effective procedures for the patient. Failing this, reusable medical devices must be double-cleaned manually, inactivated and sequestered [START_REF]du 1er décembre 2011 relative à l'actualisation des recommandations visant à réduire les risques de transmission d'agents transmissibles non conventionnels lors des actes invasifs[END_REF] (Canada, France, Germany, Switzerland) or incinerated (Australia, New-Zealand, UK, USA) [START_REF] Barcelos | Atypical clinical presentation of variant Creutzfeldt-Jakob disease[END_REF]. Decontamination procedures may differ between European countries [START_REF]Review of guidelines for prevention of Creutzfeldt Jakob disease transmission in medical settings[END_REF]. The prevention of prion transmission also involves the protection of medical staff in some specific circumstances (e.g., an accident involving exposure to tissues or biological fluids with a medical device that has been used and

has not yet undergone the predisinfection and inactivation procedure). In addition, any probably sCJD case requires an approach whose purpose is to ensure that, previously, the patient has not undergone examination or intervention that may cause a risk for other patients.

Finally, when dealing with a pathology of such gravity (poor prognosis in the short term after the first clinical symptoms), medico-social coordination is essential. It is necessary to take special care, including optimal organization of nursing and paramedical care, assistance with daily life, psychological support including that of relatives, pain management and palliative care [START_REF] Circulaire | DGAS/DSS n° 2001-139 du 14 mars 2001 relative à la prise en charge des personnes atteintes d'encéphalopathies subaiguës spongiformes transmissibles[END_REF]. To date, no similar alert system has been described in the literature. We have not been able to assess the benefits associated with it, especially because of the low incidence of sCJD.

Pending validation and availability of an easy-to-use etiologic biomarker for sCJD, the alert system developed can strongly and rapidly indicate to health professionals, with a high level of specificity and sensitivity, a probable diagnosis of sCJD, thus allowing them to set up infection control measures with adequate reprocessing of medical devices and thereby limit iatrogenic transmission. The Tau and p-Tau protein concentrations in CSF present the desired criteria for use in current medical practice. The simplicity and speed of these dosages are valuable assets in fighting against healthcare-related infections.

-

  Tests: o EEG: periodic sharp wave complexes o 14.3.3 detection in CSF (in patients with a disease duration of less than 2 years) o MRI: high signal abnormalities in caudate nucleus and putamen or at least two cortical regions (temporal-parietal-occipital) either on DWI or FLAIR -Disease duration <2 years The distinction between probable cases of sCJD (n=13) and other suspected patients (control group) was the subject of multidisciplinary expertise (neurologists, psychiatrists, geriatricians and biologists) (Fig. 1). Of the 130 patients in the control group, four subpopulations were distinguished based on clinical, paraclinical and biological data: Alzheimer's disease group (AD) (n=23), frontotemporal degeneration group (FTD) (n=22), Lewy body dementia group (LBD) (n=8), other neurological pathologies group (n=77, including in particular stroke, Parkinson's disease and toxic-metabolic disorders).

Figure 1 :

 1 Figure 1: Patient distribution included in the study. The figure represents the stratification

Figure 2 :

 2 Figure 2: Cerebrospinal fluid biomarkers in subpopulations of patients. Comparative

Figure 3 :

 3 Figure 3: An alert system for probable sporadic Creutzfeldt-Jakob disease case (sCJD).

Table 2 : Clinical features of sporadic Creutzfeldt-Jakob disease patients and principal investigation results

 2 Among these patients, the diagnosis of sCJD was confirmed by post mortem anatomopathological examination in two cases. These did not present typical paraclinical examination (EEG/MRI atypical for one case, EEG typical but MRI doubtful in the other case). A typical EEG and MRI were both observed in half of the cases. The average time from first symptoms

											Time from
	/M	Age (years)	EEG	MRI	Time from first symptoms to lumbar puncture (weeks)	Time from first symptoms to death (weeks)	p-Tau (ng/L)	Tau (ng/L)	ratio p-Tau/ Tau	14-3-3	lumbar puncture to results of the protein 14-3-3 test
											(days)
	M		T	T	9	16	59	7175	0.0082	P	12
	M		NT	CI	17	20	70	4648	0.015	P	12
	M		T	T	3	10	43	3399	0.0127	P	9
	M		NT	NT	3	7	71	11131	0.0064	P	11
	M		T	T	6	10	192	7617	0.0252	P	10
	M		T	T	4	10	85	10689	0.008	P	20
	F		T	T	22	37	63	7072	0.0089	P	13
	M		NT	NT	10	17	54	7574	0.0071	N	12
	F		NT	T	2	5	80	18050	0.0044	P	28
	F		D	NR	5	10	68	6430	0.0106	P	31
	F		T	T	5	10	46	5109	0.009	P	17
	M		T	D	6	8	73	45021	0.0016	P	15
	F		T	T	7	8	55	2150	0.0253	P	54

. * CI, contra indication, D: doubtful, F: female, M: male, N: negative, NT: not typical, NR: no realized, P: positive, T: typical

Table 3 : Contingency tables with the values of true positives, true negatives, false positives, false negatives for combined Tau test

 3

	a		Combined TAU test
			Positive	Negative
			(≤ 0.0713)	(> 0.0713)
	Probable cases of sCJD		13		0
	Other neurological pathology	13		117
			(a) and 14-3-3 test (b). The 14-3-3 protein
	test was doubtful in 4 cases.
	Combined Tau test (%) p-Tau/Tau ratio ≤ 0.0713	14-3-3 test (%)	p-value
	Sensitivity	100		92.3	**
	Specificity	90		96.1	NS
	Positive predictive value	50		70.6	***
	Negative predictive value	100		99.2	NS
	NS: not significant, ** p<0.01, *** p<0.001

Table 4 : Sensitivity, specificity and predictive values for the 14-3-3 and combined Tau tests.

 4

Acknowledgements

We would like to thank the technicians of the Nuclear Medicine Laboratory for their valuable technical assistance.

Funding source

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of interest

None