Lucile Garrigues

Louna Maignien

Eric Lombard

Jasbir Singh

Stéphane E Guillouet
email: stephane.guillouet@insa-toulouse.fr

Isopropanol production from carbon dioxide in Cupriavidus necator in a pressurized bioreactor

Keywords: Ralstonia eutropha, Cupriavidus necator, isopropanol, carbon dioxide, hydrogen, bioreactor design, gas fermentation, autotrophy, pressure

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

INTRODUCTION

Developing bioconversion processes based on CO2 has gained increasing interest in the microbial production of biofuel and chemical synthons. To develop future bioprocesses for bio-based chemical synthesis using CO2, the bacterium Cupriavidus necator is particularly attractive due to its efficient utilization of this sustainable carbon source. This facultative chemolithoautotroph, formerly also classified as Ralstonia eutropha, is the best-reported microorganism based on CO2 as the sole carbon source. It has the ability to grow at high rates (in the range of 0.3-0.4 h -1 ) to high cell densities using gas mixtures of CO2/H2/O2 [START_REF] Tanaka | Production of poly(D-3hydroxybutyrate) from CO2, H2, and O2 by high cell density autotrophic cultivation of Alcaligenes eutrophus[END_REF]. It has also demonstrated higher energy efficiencies in producing biomass and bioproduct, poly(3hydroxybutyrate) (P(3HB)) compared to plants or microalgae [START_REF] Lu | Comparison analysis on the energy efficiencies and biomass yields in microbial CO2 fixation[END_REF]. C. necator has also been extensively studied for P(3HB) production in heterotrophy [START_REF] Reinecke | Ralstonia eutropha strain H16 as model organism for PHA metabolism and for biotechnological production of technically interesting biopolymers[END_REF][START_REF] Budde | Production of poly(3hydroxybutyrate-co-3-hydroxyhexanoate) from plant oil by engineered Ralstonia eutropha strains[END_REF][START_REF] Koller | Production of Polyhydroxyalkanoates from Agricultural Waste and Surplus Materials[END_REF][START_REF] Schlegel | Alcaligenes Eutrophus and Its Scientific and Industrial Career[END_REF] and autotrophy [START_REF] Tanaka | Production of poly(D-3hydroxybutyrate) from CO2, H2, and O2 by high cell density autotrophic cultivation of Alcaligenes eutrophus[END_REF]. This natural ability to store excess carbon in the form of P(3HB) is of great interest as numerous interesting chemicals share the same production pathway precursor acetyl-coA, which implies that fewer genetic modifications would be required to divert P(3HB) precursors into the final product [START_REF] Crépin | Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production[END_REF][START_REF] Marc | Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production[END_REF].

Recently, C. necator was successfully engineered for the production of various compounds under autotrophic conditions from a CO2/H2/O2 mixture. Up to 250 mg L -1 isopropanol [START_REF] Marc | Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production[END_REF], 180 mg L -1 methyl ketones [START_REF] Muller | Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones[END_REF], 4.4 mg L -1 alka(e)nes [START_REF] Crépin | Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production[END_REF] and 6 mg L -1 α-humulene [START_REF] Krieg | CO2 to Terpenes: Autotrophic and Electroautotrophic alpha-Humulene Production with Cupriavidus necator[END_REF] were obtained under autotrophic conditions. The limited amount of product reported here is due to a combination of strain efficiency and unsuitability of the laboratory-scale bioreactor for gas fermentation. The previous cultivation systems using classical bioreactor flushed with commercial gas mixtures have demonstrated production levels suitable only for proof of concept studies. In order to further develop the engineered strains for future CO2-based bioprocesses, laboratory-scale bioreactor systems have to be designed specifically for gas fermentation, allowing higher titres of biomass to be achieved and to characterize the strains under more realistic conditions in terms of product titres, productivity and yields.

Bioreactor designs for gas fermentation have previously been reported for cultivation of H2 oxidising bacteria such as C. necator. They generally consist of gas mixtures containing CO2, H2 and O2 added through a single sparger. The optimal gas composition employed in the literature for autotrophic growth of C. necator is typically H2/O2/CO2=7:1:1 [START_REF] Ishizaki | Batch culture of Alcaligenes eutrophus ATCC 17697 using recycled gas closed ciruit culture system[END_REF][START_REF] Takeshita | Influence of hydrogen limitation on gaseous substrate utilization in autotrophic culture of Alcaligenes eutrophus ATCC 17697[END_REF][START_REF] Volova | Cell growth and P(3HB) accumulation from CO2 of a carbon monoxide-tolerant hydrogen-oxidizing bacterium, Ideonella sp. O-1[END_REF],

H2/O2/CO2=7:2:1 [START_REF] Takeshita | Influence of hydrogen limitation on gaseous substrate utilization in autotrophic culture of Alcaligenes eutrophus ATCC 17697[END_REF], or H2/O2/CO2=6:2:1 [START_REF] Ammann | Gas consumption and growth rate of Hydrogenomonas eutropha in continuous culture[END_REF][START_REF] Volova | Kinetic parameters of a culture of the hydrogen-oxidizing bacterium Ralstonia eutropha grown under conditions favoring polyhydroxybutyrate biosynthesis[END_REF]. However, these gas mixtures lie within the explosive composition range (which is normally regarded as being above 6% O2 in the presence of H2) and limits the cell densities reached. In order to avoid this O2 limitation during autotrophic growth of C. necator, pure oxygen has been fed to the bioreactor [START_REF] Tanaka | Production of poly(D-3hydroxybutyrate) from CO2, H2, and O2 by high cell density autotrophic cultivation of Alcaligenes eutrophus[END_REF]. This was combined with a basket-type agitator which generated huge increases in the kLa (up to 2970 h -1 ), allowing a high cell density of 91g L -1 and a residual biomass containing 68% of PHB (29 g L -1 of catalytic biomass concentration) to be reached.

The design of a small laboratory-scale bioreactor dedicated to CO2 fermentation of H2oxidizing bacteria is described here. The challenges tackled were (i) the danger of gas explosion above the limit of 6% O2 in the presence of excess H2 and (ii) the gas transfer efficiency at small scale. Reducing the size of a bioreactor is always more challenging for parameters such as gas transfer and necessitates the application of process engineering and biochemical engineering principles. The engineering solution to poor gas transfer is usually limited to the volumetric gas transfer coefficient, kLa, increase through changes in sparger and stirring arrangement as mentioned above. However, this offers relatively limited scope for improvement. An effective alternative is to operate the bioreactor at elevated pressure as this can, in principle, increase the gas transfer rate several fold without changes to sparging or agitation. This concept has already been successfully applied in bioreactors with various microorganisms, such as Arxula adeninivorans, Corynebacterium glutamicum, C. necator, Escherichia coli, and Saccharomyces cerevisiae [START_REF] Belo | Fed-batch cultivation of Saccharomyces cerevisiae in a hyperbaric bioreactor[END_REF][START_REF] Knoll | High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors[END_REF][START_REF] Knabben | High celldensity processes in batch mode of a genetically engineered Escherichia coli strain with minimized overflow metabolism using a pressurized bioreactor[END_REF][START_REF] Matsui | High cell density cultivation of recombinant E. coli for hirudin variant 1 production by temperature shift controlled by pUC18-based replicative origin[END_REF][START_REF] Yu | Gas fermentation enhancement for chemolithotrophic growth of Cupriavidus necator on carbon dioxide[END_REF]. There is, however, a lack of information on its impact on cell viability and productivity with engineered microorganisms during long fermentations runs. Moreover, in order to have more flexibility in terms of gas composition and therefore higher gas solubility, the bioreactor gas-supply system was designed with three independent gas lines supplying three spargers (CO2, H2 and air). A safety system was designed to control O2 level in the headspace in order to avoid an explosive mixture within the bioreactor. Experimental validation was performed with autotrophic growth and production of isopropanol with an engineered C. necator strain.

MATERIALS AND METHODS

Strains

In this study, the reference strain used was C. necator Re2133, deficient for P(3-HB) biosynthesis (H16 ∆phaCAB) obtained from [START_REF] Lu | Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha[END_REF]. The isopropanol strain tested in this study was C. necator Re2133 bearing the plasmid pEG7b carrying a synthetic isopropanol operon under the constitutive pTac promoter [START_REF] Grousseau | Isopropanol production with engineered Cupriavidus necator as bioproduction platform[END_REF]. The strains were stored at -80°C in liquid TBS medium (dextrose-free tryptic soy broth, Becton Dickinson, France) with 20 % glycerol (v/v).

Media

The rich medium consisted of 2.75% (w/v) dextrose-free tryptic soy broth (TSB, Becton Dickinson, France). The minimal medium A used for preculture in flask was previously described by [START_REF] Lu | Studies on the production of branched-chain alcohols in engineered Ralstonia eutropha[END_REF] and [START_REF] Crépin | Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production[END_REF]. Fructose (20 g L -1 ) was used as the only carbon source. NH4Cl (0.5 g L -1 ) was used as the nitrogen source to reach a biomass concentration of about 1 g L -1 . The minimal medium B used for the cultures in the bioreactor consisted of 0.19 g L -1 nitrilotriacetic acid, 0.06 g L -1 ferrous ammonium citrate, 0.5 g L -1 of MgSO4.7H2O, 0.01 g L - 1 of CaCl2.2H2O, and 1 ml of trace element solution. The composition of the latter was 0.3 g

L -1 H3BO3, 0.2 g L -1 CoCl2.6H2O, 0.1 g L -1 ZnSO4.7H2O, 0.03 g L -1 MnCl2.4H2O, 0.03 g L -1
Na2MoO4.2H2O, 0.02 g L -1 NiCl2.6H2O, and 0.01 g L -1 CuSO4.5H2O. (NH4)2SO4 (2 g L -1 ) was used as nitrogen source to reach a biomass concentration of about 4 g L -1 . After autoclaving the above medium, 40 ml of a sterile phosphate solution containing 224 g L -1 Na2HPO4.12H2O and 37.5 g L -1 KH2PO4 was aseptically added to the fermenter.

Seed cultivations

After thawing at room temperature, one glycerol stock was streaked on a TSB agar Petri dish. and incubated for 36 h at 30°C. One isolated colony was used to inoculate the first seed culture grown for 24 h with 5 mL of TSB supplemented with 10 mg L -1 gentamycin and 200 mg L -1 kanamycin, in a 50 mL baffled Erlenmeyer flask. This culture was used to inoculate a 250 mL Erlenmeyer baffled flask containing 50 mL of mineral medium A. The seed was cultivated at 30°C, 100 rpm for 18h. The culture was then centrifuged (5 min, 10000g), the supernatant was discarded and the cell pellet was resuspended in 30 mL sterile fresh medium B to eliminate the residual fructose. The suspension was then used to inoculate the bioreactor containing 300 mL of sterile mineral medium B.

Culture conditions and bioreactor system

The cultures were performed in a 500 ml total volume Xplorer® (HEL Ltd, UK) bioreactor that was modified specifically for this study. The working liquid volume was 330 mL. The bioreactor was first used with a commercial gas mixture containing H2/O2/CO2/N2 (molar %: 60:2:10:28) (Air Liquide, France) provided into the medium through a sparger. For the experiments carried out with individual gases, the bioreactor was then set up with three spargers in order to deliver air, pure CO2 and pure H2 independently. A fourth inlet gas was added in the headspace for pure N2 supply as a safety system. Each gas inlet was controlled by a gas mass flow meter. The bioreactor was equipped with pH, dissolved oxygen (DO), temperature and pressure controllers. The Xplorer® software handled the on-line monitoring and control systems of the reactor. Both CO2 and O2 in the outlet gases were analysed using a Tandem Pro gas analyzer (Magellan BioTech HEL Ltd , UK). The bioreactor was safely controlled by the O2 concentration in the headspace in order to avoid any explosive risk. The headspace was flushed with pure N2 if the O2 surpassed 5% in the headspace. The DO level in the reactor was controlled below 5% of air saturation at atmospheric pressure by varying stirring speed and/or inlet air flow rate in order to restrict the known inhibition of C. necator hydrogenases by the dissolved O2 concentration [START_REF] Siegel | Kinetics of Growth of the Hydrogen-Oxidizing Bacterium Alcaligenes eutrophus (ATCC 17707) in Chemostat Culture[END_REF]. Temperature was maintained at 30°C.

The pH was maintained at 7.0 by addition of a 14% (v/v) NH4OH solution.

Characterization of the biomass

Cell growth was followed spectrophotometrically at 600 nm with a DR3900 spectrophotometer (Hach, Loveland, Colorado, USA) after calibration against cell dry weight measurements. For cell dry weight determination, culture medium was harvested and filtered through 0.2 μm pore-size polyamide membranes (Sartorius AG, Göttingen, Germany), which were then dried to a constant weight at 60°C under partial vacuum (200 mmHg, i.e.

approximately 26.7 kPa).

Cell viability was measured with a BD Accuri C6® flow cytometer (BD Biosciences, US) after cell staining with propidium iodide (PI) dye (Molecular Probes, Invitrogen, USA) commonly used to monitor membrane integrity as indicator of viability [START_REF] Marc | Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production[END_REF] Metabolites and statistical analyses

Metabolites (isopropanol, acetone, pyruvic and acetic acids) were quantified by chromatography as described in [START_REF] Grousseau | Isopropanol production with engineered Cupriavidus necator as bioproduction platform[END_REF].

Statistical analyses

Concentrations of metabolites and biomass are given as the mean value of 2 to 3 independent analyses. The error on the specific growth rate (determined as being the slope of ln[Optical Density] = f(t)) was calculated as the standard deviation (SD) of the slope. The instantaneous specific growth rates were calculated upon fitting process of the experimental data and the derivative calculation. For the yield determination, the state variables were plotted pairwise in a scatter plot within the considered period of the culture. A linear regression was applied to determine the yield (as the slope) and the error (as the SD of the slope). For the calculation of the CO2 to isopropanol conversion yield, the isopropanol amount was plotted versus the amount of consumed CO2, calculated from a classical mass balance from the analyses of inlet and outlet gas composition.

RESULTS & DISCUSSION

Benefits of elevated pressure on autotrophic growth of C. necator Re2133 on a commercial gas mixture

Autotrophic production of chemicals by C. necator was previously reported in a classical CSTR bioreactor where gases were supplied by flushing the reactor with a commercial gas mixture containing H2/O2/CO2/N2 mix (molar %: 60:2:10:28) [START_REF] Crépin | Metabolic engineering of Cupriavidus necator for heterotrophic and autotrophic alka(e)ne production[END_REF][START_REF] Marc | Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production[END_REF]. Here, this mixture was used first to characterize the growth of the reference strain C. necator Re2133 in the newly designed bioreactor (Figure 1). After 6 h cultivation, the maximum values of agitation and ventilation rates were reached. The biomass concentration was low at 0.2 g L -1 . An alternative way to provide higher gas transfer is to operate the bioreactor at elevated pressure. Then the dissolved O2 concentration was kept at 2% saturation, at various pressures in the reactor up to 3 bar overpressure. Growth was then extended to 15 h leading to a final biomass concentration of 0.5 g L -1 . Growth followed an exponential profile throughout the cultivation with a maximum specific rate of 0.13 ± 0.01h -1 . Cell viability was analysed by flow cytometry after staining with propidium iodide. The non-PI stained cells were considered viable, and the percentage was >95% over the duration of the culture, even when overpressure reached 3 bar, indicating their robustness towards high pressure.

Benefits of independent gas sparging on autotrophic growth of C. necator Re2133

The relatively low final biomass concentration obtained even under high pressure was likely due to the low gas transfer capacity when using the mixture containing H2/O2/CO2/N2 (molar %: 60:2:10:28). This composition was far from optimal for autotrophic growth of C. necator.

However, they are all within the explosive range for H2 and O2. The cultivation of the Re2133 strain was initiated by sparging the 3 independent gases in order to reach 88% H2, 2% O2 and 2.5%CO2 gas mixture in the reactor (62, 6.7 and 1.75 mL/min, for H2, air and CO2). It allowed ensuring an H2-rich (H2 being the less soluble gas) and safe environment controlling the O2 % in the headspace below the explosive range (6% of air saturation at atmospheric pressure). The level of dissolved O2 was controlled to meet the increasing biological demand while maintaining the level at 2% saturation in order to limit the reported inhibition of the hydrogenases [START_REF] Siegel | Kinetics of Growth of the Hydrogen-Oxidizing Bacterium Alcaligenes eutrophus (ATCC 17707) in Chemostat Culture[END_REF][START_REF] Cracknell | A kinetic and thermodynamic understanding of O2 tolerance in [NiFe]-hydrogenases[END_REF] by increasing the ratio air/H2 flow rate within the range of 6.7-25 mL/min for air and 62-86 mL/min for H2 (Figure 2). The duration of growth at atmospheric pressure was 15 h compared to 6 h with the reactor operating with the gas mix, illustrating the benefit of three independent spargers. Growth followed an exponential profile throughout the cultivation at atmospheric pressure with a maximum specific rate of 0.11 ± 0.01 h -1 . At 15 h, inlet gas composition was 70% H2, 5% O2 and 2.8% CO2 (71, 26 and 2.8 mL/min for H2, air and CO2). When the inlet gas composition for O2 reached the safety level (5%) defined to ensure a safe environment, the dissolved O2 level was maintained by switching the strategy to a step-wise increase in total reactor pressure up to >3 bars. The growth profile was still exponential without any observed drop in specific rate (0.11 ± 0.01 h -1 ). A final pressure up to 4.2 bars was applied with a concomitant increase in H2 (117 mL/min) and a decrease in O2 level in the inlet gas (3.7%) in order to maintain the dissolved oxygen at a low level. The amount of CO2 in the outlet gas was always found to be lower than in the inlet and never exceeded 1.6%. During this period, the specific growth was lower but constant at 0.07 ± 0.005 h -1 . Cell viability was assessed by flow cytometry and did not show any fall in the percentage of PI unstained cells.

Isopropanol production under autotrophic condition

Isopropanol autotrophic production was tested in the designed bioreactor using an engineered strain Re2133/pEG7b [START_REF] Grousseau | Isopropanol production with engineered Cupriavidus necator as bioproduction platform[END_REF]. Cultivation was initiated by sparging the three independent gases in order to reach 87% H2, 2% O2 and 3%CO2 mixture in the reactor (59, 6.7 and 2 mL/min respectively for H2, air and CO2) allowing an H2-rich, O2-low environment to be reached. The level of dissolved O2 was monitored throughout by increasing the ratio air/H2 flow rate and the overpressure in the bioreactor in order to fulfill the increasing microbial O2 demand (Figure 3). The gas flow rates ranged between 59-200 mL/min for H2, 6.7 -75 mL/min for air and 2-6 mL/min for CO2. C. necator Re2133/pEG7b grew for 20 h at an average specific rate of 0.051±0.006 h -1 . Growth slowly decreased to 0.023±0.001 h -1 for the remaining period of the cultivation. The final biomass concentration reached at 85 h was 2.9 g L -1 . Compared to the reference strain Re2133, the lower growth rate was likely due to inhibition by isopropanol and acetone as previously observed with the same strain with fructose (0.05 vs 0.17 h -1 , for Re2133 and Re2133/pEG7b respectively) [START_REF] Grousseau | Isopropanol production with engineered Cupriavidus necator as bioproduction platform[END_REF]. Isopropanol and acetone were produced concomitantly to reach maximum concentrations in the liquid phase of 3 and 0.65 g L -1 respectively. Any isopropanol and acetone evaporated with the gas stream was trapped in water and quantified during the cultivation. Taking this evaporation into account, the maximal concentrations were equivalent to 3.5 and 0.9 g L -1 of isopropanol and acetone, respectively.

Reproducibility was assessed with a second independent cultivation and led to the production of 1.96 g L -1 biomass, 2.1 g L -1 isopropanol and 1 g L -1 acetone. These performances were remarkable when compared to that obtained with fructose: Re2133/pEG7b was able to produce up to 2.2 g L -1 isopropanol and 1 g L -1 acetone in 88 h with fructose [START_REF] Grousseau | Isopropanol production with engineered Cupriavidus necator as bioproduction platform[END_REF]. The overall specific production rate of isopropanol in C. necator Re2133/pEG7b from CO2 was 0.013 ggDCW -1 h -1 (0.030 ggDCW -1 h -1 with fructose) and peaked at 0.053 ggDCW -1 h -1 with CO2 (0.085 ggDCW -1 h -1 with fructose). Cell viability was assessed by flow cytometry after staining with PI during the cultivation. The percentage of PI unstained cells ranged from 82-98%, which was slightly lower cultivation of Re2133 in the pressurized bioreactor. Concomitantly with the reduction in the µmax, this could be attributed to an inhibitory effect of isopropanol on Re2133/pEG7b.

The proof of concept of producing isopropanol from CO2 with an engineered C. necator strain was previously demonstrated: 250 mg L -1 isopropanol was obtained from 1 g L -1 biomass using a classical bioreactor and commercialized gas mix containing H2, O2 and CO2 (60:2:10) [START_REF] Marc | Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production[END_REF]. It is clearly demonstrated here that designing a pressurized bioreactor with independent gas feeds significantly improved gas transfer to reach higher biomass and consequently higher isopropanol concentrations of up to 3.5 g L -1 . The calculation of the carbon balance in this fermentation showed that 70-80% of the CO2 was recovered into biomass, isopropanol and acetone. The yield of CO2 converted into isopropanol was 0.13 Cmol Cmol -1 in this study, slightly lower than 0.2 Cmol Cmol -1 reached previously. [START_REF] Marc | Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production[END_REF]. However, strain Re2133/pEG7b used here constitutively expressed the isopropanol synthetic operon whereas Re2133/pEG23 previously tested had an inducible isopropanol operon controlled by the arabinose promoter.

Thus uncoupling of growth and production allowed the carbon flow to be better directed towards the isopropanol pathway, as reported with fructose [START_REF] Grousseau | Isopropanol production with engineered Cupriavidus necator as bioproduction platform[END_REF].

CONCLUSIONS

The design of a dedicated gas bioreactor incorporating the independent management of the three gases, CO2, H2 and Air, coupled with the use of high pressure has enabled a dramatic improvement in gas transfer in a small scale bioreactor. C. necator showed an ability to sustain a pressure of over 4 bars, leading to a higher biomass concentration and an isopropanol production of up to 3.5 g L -1 . This is the first reported demonstration successfully producing compounds at the g L -1 scale from engineered autotrophic bacteria and raises the prospect of the near-future development of CO₂-based commercial bioprocesses.

Figure 1 :Fig. 2 :

 12 Figure 1: (A) Evolution of the dissolved O2 level (dashed line), the total gas flow rate (thin black line) and the overpressure (thick black line) in the bioreactor. (B) Evolution of the biomass concentration (black circles) and cell viability (open triangles)

Fig. 3 :

 3 Fig.3: Autotrophic isopropanol production of C. necator Re2133/pEG7b in the gas bioreactor modified with 3 independent gas spargers. (A) Evolution of the relative pressure (thick line) and the partial pressure of dissolved O2 (dashed line). (B) Evolution of the inlet gas composition in H2 (solid thick line), O2 (dashed line) and CO2 (solid thin line). (C) Evolution of biomass concentrations (•), % of PI unstained cells (∆) and isopropanol ( ), acetone (∇) concentrations in the bioreactor liquid phase. Total isopropanol ( ) and acetone (▼) production were calculated taking amounts evaporated into account.

ACKNOWLEDGMENT

We thank M. Appleton (H.E.L., UK) for all the efforts and discussions provided on the design of the dedicated bioreactor. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


MD Julien Ternacle
email: julien.ternacle.hmn@gmail.com

MD, PhD; Marie-Annick Clavel

Assessment of Aortic Stenosis Severity: a multimodality approach

Keywords: aortic stenosis, aortic valve calcification, calcium scoring, echocardiography, computed tomography, multimodality imaging

Key points

1. Transthoracic echocardiography is the cornerstone to confirm the severity of aortic stenosis.

2. In patients with high-gradient severe aortic stenosis, the stroke volume should be evaluated to assess the prognosis.

3. In patients with a low-gradient and tight aortic valve area pattern, a multimodality imaging approach should be used to confirm the true severity of aortic stenosis before decision making.

SYNOPSIS:

Severe aortic stenosis (AS) is associated with a progressive cardiac remodeling which ultimately leads to heart failure and death if the valve is not replaced. The confirmation of AS severity is therefore crucial to adequately manage patients with AS. Transthoracic echocardiography is the first line exam to confirm AS severity, but it may be inconclusive in discordant cases. In this setting, AS severity should be confirmed using a multimodality imaging approach. This review gives an overview of how to assess and/or confirm AS severity, especially in case of discordance.

Introduction:

Assessing aortic stenosis (AS) severity is essential in the management of patients. Indeed, class I indications for aortic valve replacement (AVR) are only recommended in patients with severe AS. Doppler-echocardiography is the cornerstone for AS evaluation because of its high performance, low-cost and large availability. The first line evaluation after auscultation of a heart sound or in patients at risk, such as patients with known congenitally bicuspid aortic valve is the transthoracic echocardiography (TTE). In the majority of cases TTE allows to confirm the diagnosis, determine the severity, and evaluate the myocardial repercussion of AS. Moreover, indication for aortic valve intervention in asymptomatic patients are predominantly based on echocardiography. However, a multimodality imaging approach could be mandatory, especially in patients with discordance in echocardiographic parameters used to grade AS severity.

Transthoracic Doppler-echocardiography: the first line of AS evaluation

Assessment of AS severity is mostly based on three parameters (Figure 1): transaortic valvular peak jet velocity (Vmax), mean pressure gradient (∆Pm) between the left ventricle (LV) and the aorta, and aortic valve area (AVA) calculation. Severe AS is defined by (Table 1) a Vmax ≥4 m/s, a ∆Pm ≥40 mmHg, and an AVA <1.0 cm 2 or <0.6cm 2 /m 2 after indexation to body surface area (BSA) [START_REF] Baumgartner | Recommendations on the echocardiographic assessment of aortic valve stenosis: A focused update from the European Association of Cardiovascular Imaging and the American Society of Echocardiography[END_REF] . The evolution of these parameters over time is also crucial to identify patients with a rapid AS progression (increase in Vmax ≥0.3m/s/year).

-Vmax and ∆Pm of the antegrade systolic flow across the narrowed aortic valve are measured using continuous-wave Doppler (CWD) ultrasound. The maximum velocity is measured at the outer edge of the dark signal, and the outer edge of the spectral Doppler envelope is traced to provide both the velocity-time integral (VTI) for the continuity equation and ∆Pm (using the simplified Bernoulli equation) (Figure 1A). In order to obtain the highest Vmax, the CWD must be aligned with the ejection flow and Doppler angle correction should be avoided. Hence, a multiple acoustic window exploration including the right parasternal way 2 , if possible with the use of a small dual-crystal CWD transducer (PEDOF), after a careful patient positioning is strongly recommended (Figure 1B) [START_REF] Baumgartner | Recommendations on the echocardiographic assessment of aortic valve stenosis: A focused update from the European Association of Cardiovascular Imaging and the American Society of Echocardiography[END_REF] . The window where the highest Vmax is recorded should be mentioned in the TTE report for further evaluations.

-AVA is calculated using the continuity equation which is based on the concept of flow preservation: the stroke volume (SV) ejected through the left ventricular outflow tract from the valve should be avoided because of the risk of underestimation (i.e. septal bulging and non-tubular LVOT). In addition, positioning the LVOT d within 0.3-1.0cm of the valve may impact the evaluation reproducibility. LVOT d assessment may be difficult in case of extensive calcifications, and might require the use of transesophageal echocardiography (TEE) or cardiac computed tomography (CT) to accurately define it [START_REF] Ng | Comparison of aortic root dimensions and geometries before and after transcatheter aortic valve implantation by 2-and 3dimensional transesophageal echocardiography and multislice computed tomography[END_REF] . LVOT VTI is measured using pulsed-wave Doppler (PWD) modality from an apical approach. The PWD sample is positioned just proximal to the aortic valve, ideally at the same level as the LVOT d is measured. The recording should show a smooth velocity curve with a well-defined peak, and a narrow band of velocities throughout systole (Figure 1 C). In case of important spectral dispersion, the sample volume should be slowly moved from the valve towards the apex.

Thirty to 40% of patients with severe AS may have a discordance between AVA (<1.0 cm 2 , i.e. in the severe range) and hemodynamic parameters (Vmax <4 m/s and ∆Pm <40 mmHg, i.e. in the moderate range). A careful evaluation of LVOT d measures should be done in these cases, and second line parameters (Table 1, Figure 1) should be assessed if the discordance is confirmed: velocity or VTI ratio, acceleration time and its ratio over ejection time, and AVA planimetry [START_REF] Baumgartner | Recommendations on the echocardiographic assessment of aortic valve stenosis: A focused update from the European Association of Cardiovascular Imaging and the American Society of Echocardiography[END_REF] . These parameters reduce the risk of error related to the LVOT d measurement.

-Velocity or VTI ratio (Figure 1C) is the ratio Vmax or VTI between LVOT and AV levels. The normal value is 1, while in a severe AS the ratio is <0.25 (or 25%). This parameter is particularly interesting when LVOT d measurement is unclear.

Velocity ratio = LVOT Vmax

AV Vmax

VTI ratio =

LVOT VTI AV VTI -The analysis of the ejection dynamics transvalvular velocity using CWD may provide additional semiquantitative parameters to better characterize AS severity. Acceleration time (AT) is the delay between the beginning and the maximal peak of the aortic ejection velocity and ejection time is the overall ejection duration (ET) (Figure 1D). It was demonstrated that an AT >110ms or an AT/ET >0.36 was associated with AS severity and outcomes [START_REF] Griguer | Clinical significance of ejection dynamics parameters in patients with aortic stenosis: an outcome study[END_REF] . However, in low flow patients, these parameters could be increase in non-severe stenosis due to the lack of strength of the flow to open a stenosis that is only moderate.

-AVA planimetry (Figure 3A) is an interesting additional parameter which offers a direct anatomic visualization of the narrowed AV orifice (cutoff <1.0 cm 2 ). However, several limits exist: 1) the level of the orifice area assessment must be well positioned at the tips of the leaflets in order to avoid overestimation and thus positioning should be performed with 3D imaging modality, 2) planimetry can be very difficult in case of extensive calcifications (shadows and reverberations), and 3) because of flow contraction, the hemodynamic calculated AVA is could be up to 0.6-fold smaller than the anatomic AVA by planimetry. Thus, severe AS diagnosis should not be challenged on this parameter alone, but it may provide an additional information in difficult cases. TEE using two-(2D) or three-dimensional (3D) modalities, and cardiac CT with contrast injection are more accurate than TTE for AVA planimetry.

All hemodynamic parameter measured by echocardiography are flow dependent: a decrease in flow will decrease gradient and velocity but also velocity/VTI ratio and AVA by continuity equation and planimetry.

Finally, AS severity should also be assessed using a semi-quantitative approach based on valvular calcification extension (Figure 4A) because of its strong correlation with the stenosis severity and outcomes [START_REF] Rosenhek | Predictors of outcome in severe, asymptomatic aortic stenosis[END_REF] . This additional parameter may also help in case of discordant severe AS.

Transesophageal echocardiography: an anatomic evaluation

AS severity assessment can require the use of TEE in specific conditions: 1) patient with poor TTE echogenicity, 2) inaccurate LVOT d measurement, and 3) associated HVD.

In patients with extensive aortic valve calcification, the risk of LVOT d underestimation by TTE can require the use of TEE [START_REF] Ng | Comparison of aortic root dimensions and geometries before and after transcatheter aortic valve implantation by 2-and 3dimensional transesophageal echocardiography and multislice computed tomography[END_REF] . Moreover, the LVOT area calculation is based on the 2D LVOT d measurement, while the aortic annulus has an elliptic shape [START_REF] Piazza | Anatomy of the aortic valvar complex and its implications for transcatheter implantation of the aortic valve[END_REF] . This geometric assumption may lead to underestimate AVA in case of misalignment with the larger LVOT diameter (Figure 2B). Thus, a 3D TEE (Figure 2C) may be performed to more accurately assess LVOT dimensions 3 in case of discordance between a small AVA (<1.0 cm 2 ) and a low transvalvular flow (Vmax <4 m/s and ∆Pm <40 mmHg). 3D TEE automated software can provide a rapid and reliable assessment of aortic annulus and root [START_REF] Prihadi | Feasibility, accuracy, and reproducibility of aortic annular and root sizing for transcatheter aortic valve replacement using novel automated three dimensional echocardiographic software: comparison with multi-detector row computed tomography[END_REF] . However, threshold of AVA used to assess aortic stenosis severity should probably be modified (cf. 3. Fusion imaging).

As previously explained in the TTE section, TEE can be used to directly measure the AVA at the tip of leaflets using 2D and 3D planimetry (Figure 3B). In addition, TEE allows a more accurate assessment of the aortic root dimensions and the aortic valve anatomy (i.e. bicuspid valve) than TTE.

Cardiac computed tomography: calcium scoring and fusion imaging

-Non-contrast CT: Calcium scoring These last years, the quantification of aortic valve calcific burden (i.e. calcium scoring) by cardiac CT revolutionized AS evaluation. Calcium scoring (Figure 4B) requires a low-dose (<1mSv) cardiac CT with ECG gating but without contrast injection. The quantification of calcific deposition is performed according to the Agatston method using a semi-automated software. Several studies demonstrated the excellent reproducibility and performance of calcium score for the AS severity and prognosis evaluations. Moreover, calcium score may provide a more reliable prognostic evaluation than usual hemodynamic parameters [START_REF] Cueff | Measurement of aortic valve calcification using multislice computed tomography: correlation with haemodynamic severity of aortic stenosis and clinical implication for patients with low ejection fraction[END_REF][START_REF] Clavel | Impact of aortic valve calcification, as measured by MDCT, on survival in patients with aortic stenosis: results of an international registry study[END_REF][START_REF] Pawade | Computed tomography aortic valve calcium scoring in patients with aortic stenosis[END_REF] .

According to current guidelines, calcium scoring should be used in patients with discordant parameters of severe AS and both preserved or reduced LV ejection fraction (LVEF <50%), in order to confirm the severity [START_REF] Baumgartner | ESC/EACTS guidelines for the management of valvular heart disease: The Task Force for the management of valvular heart disease of the European Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery (EACTS)[END_REF] and avoid unnecessary or delayed intervention.

Interestingly, the calcium score cutoff to define severe AS (Table 1) differs according to sex with a lower value in women (≥1,200 AU) than in men (≥2,000 AU) [START_REF] Clavel | Impact of aortic valve calcification, as measured by MDCT, on survival in patients with aortic stenosis: results of an international registry study[END_REF][START_REF] Pawade | Computed tomography aortic valve calcium scoring in patients with aortic stenosis[END_REF] . This difference may be explained by the presence of a smaller aortic annulus, in part, and especially a more fibrotic pattern of valve disease in women [START_REF] Simard | Sex-related discordance between aortic valve calcification and hemodynamic severity of aortic stenosis: is valvular fibrosis the explanation?[END_REF] . In addition, ethnicity may also influence the calcium deposition [START_REF] Patel | Racial differences in the prevalence of severe aortic stenosis[END_REF] , but further investigations are required to define additional cutoffs.

-Contrast CT: AVA and LVOT planimetry -fusion imaging The high 3D spatial resolution of contrast cardiac CT allows a very accurate evaluation of the anatomy and the dimensions of cardiac structures. Hence, contrast CT may be used in AS patients to better define LVOT, AV morphology and AVA planimetry and aortic root dimensions.

As previously explained for TEE, AVA planimetry may be accurately measured by contrast CT (Figure 3C) in case of discordance between AVA and transvalvular flow in order to better characterize AS severity (AVA <1.0 cm 2 , Table 1).

The elliptic shape of the LVOT may lead to AVA underestimation when evaluated by echocardiography, while a 3D evaluation by CT using both mean LVOT d and LVOT area may be more accurate [START_REF] Ng | Comparison of aortic root dimensions and geometries before and after transcatheter aortic valve implantation by 2-and 3dimensional transesophageal echocardiography and multislice computed tomography[END_REF] . Hence, some authors evaluated if an hybrid approach to calculate AVA using TTE-derived Doppler parameters and contrast cardiac CT-derived LVOT dimensions (Figure 2D) could reconcile AVA and flow in discordant cases [START_REF] Kamperidis | Low gradient severe aortic stenosis with preserved ejection fraction: reclassification of severity by fusion of Doppler and computed tomographic data[END_REF][START_REF] Clavel | Aortic valve area calculation in aortic stenosis by CT and doppler echocardiography[END_REF][START_REF] Arangalage | Anatomic characterization of the aortic root in patients with bicuspid and tricuspid aortic valve stenosis does fusion of Dopplerechocardiography and computed tomography resolve discordant severity grading?[END_REF] . Interestingly, this approach lead to find larger LVOT dimensions but the result was not unequivocal because of a similar or even a higher overall percentage of misclassified patients compared to TTE findings [START_REF] Arangalage | Anatomic characterization of the aortic root in patients with bicuspid and tricuspid aortic valve stenosis does fusion of Dopplerechocardiography and computed tomography resolve discordant severity grading?[END_REF] . Indeed, some patients with a classical severe AS (AVA <1.0 cm 2 and ∆Pm ≥40 mmHg) became discordant (∆Pm ≥40 mmHg but AVA ≥1.0 cm 2 ), while patients with a severe discordant AS (AVA <1.0 cm 2 and ∆Pm <40 mmHg) became moderate (AVA ≥1.0 cm 2 and ∆Pm <40 mmHg). This could be explain by the fact that the optimal AVA cutoff to predict outcomes was larger than using LVOT measured by TTE (AVA ≤1.2cm 2 vs. 1.0cm 2 ) [START_REF] Clavel | Aortic valve area calculation in aortic stenosis by CT and doppler echocardiography[END_REF] . Thus, an hybrid approach may be proposed but only in case of discordant severe AS and using a larger cutoff of AVA (Table 1) [START_REF] Delgado | How do we reconcile echocardiography, computed tomography, and hybrid imaging in assessing discordant grading of aortic stenosis severity?[END_REF] .

Finally, contrast cardiac CT is essential before transcatheter aortic valve replacement (TAVR) to define the prosthesis size according to the aortic annulus dimensions, the AV anatomy (bicuspid or tricuspid), and the aortic root characteristics (aortic dimensions and coronary artery position), in addition to the vascular access assessment (cf. Article 10).

Cardiac magnetic resonance imaging

Cardiac magnetic resonance (CMR) is the gold standard method for the assessment of ventricular volumes and function, and for the characterization of myocardial abnormality, while its use in HVD is relatively recent. Intracardiac flows can be evaluated by 2D phasecontrast or more recently by four-dimensional (4D) flow imaging acquisitions. The latter has the advantage of analyzing all the components of the flow and not only those perpendicular to the acquisition plane, which is the main limitation of phase-contrast data. The most validated indication is aortic regurgitation in both native and prosthetic valves, especially after TAVR [START_REF] Ribeiro | Cardiovascular magnetic resonance to evaluate aortic regurgitation after transcatheter aortic valve replacement[END_REF] . Several publications also reported a good accuracy compared to TTE for assessing AS severity [START_REF] Mantini | Grading of aortic stenosis severity: a head-to-head comparison between cardiac magnetic resonance imaging and echocardiography[END_REF] in both bicuspid and tricuspid valves. CMR parameters to quantify AS severity are similar to those of TTE: AVA and antegrade systolic flow across the aortic valve.

Transvalvular aortic flow parameters (Vmax, ∆Pm, and aortic antegrade volume) are obtained from phase-contrast imaging between the leaflet tips and the proximal ascending aorta portion, and AVA is calculated using the continuity equation. LVOT area measurement is obtained from cine-images using the same method as cardiac CT and antegrade LVOT volume is derived from a phase-contrast acquisition at the LVOT level. This method is time consuming and requires a previous TTE analysis (Vmax) to accurately encode CMR velocity. Thus, CMR is not a first-line exam for the evaluation of AS severity but may be used in dedicated conditions as in patients without any correct acoustic window by TTE. In addition, LVOT area by CMR could be used to calculate AVA using a hybrid approach as previously described for cardiac CT. Finally, a phase-contrast acquisition at the tip of aortic leaflets may be used for direct AVA planimetry.

Stress echocardiography for aortic stenosis severity evaluation: an obsolete exam?

Two distinct situations should be considered regarding the use of stress echocardiography in patients with AS: 1) the prognostic evaluation of asymptomatic patients with severe AS using exercise-stress echocardiography, and 2) the confirmation of AS severity when TTE at rest is inconclusive.

The first aspect is very controversial. Some studies found that exercise-stress echocardiography parameters (LVEF variation, ∆Pm increase) might be useful for the prognostic stratification of asymptomatic severe AS patients in order to identify patients who would benefit from early surgery [START_REF] Masri | Predictors of long-term outcomes in asymptomatic patients with severe aortic stenosis and preserved left ventricular systolic function undergoing exercise echocardiography[END_REF][START_REF] Maréchaux | Usefulness of exercise stress echocardiography for risk stratification of true asymptomatic patients with aortic valve stenosis[END_REF] . However, recent reports did not find any additional value of echocardiography over the stress-test alone [START_REF] Goublaire | Prognostic value of exercise-stress echocardiography in asymptomatic patients with aortic valve stenosis[END_REF] . In view of the lack of reliable data, the use of exercise-stress echocardiography in asymptomatic severe AS was removed from the latest guidelines [START_REF] Baumgartner | ESC/EACTS guidelines for the management of valvular heart disease: The Task Force for the management of valvular heart disease of the European Society of Cardiology (ESC) and the European Association for Cardio-Thoracic Surgery (EACTS)[END_REF] .

Conversely, the use of stress echocardiography with low dose (up to 20 µg/kg/min) dobutamine infusion is recommended by current guidelines to distinguish severe (true-severe) form moderate (pseudo-severe) AS (Table 1) in patients with discordant parameters of severe AS (AVA <1.0 cm 2 and ∆Pm <40 mmHg) and reduced LVEF (<50 %, i.e. classical lowgradient severe AS). The first step is to determine if the patient has a flow reserve (increase in SV ≥20%). Indeed, AS severity evaluation is very complex when the low-flow state related to LV systolic dysfunction is not corrected by dobutamine infusion. In patients with a flow reserve, the second step is to quantify AVA and transvalvular aortic flow variations at peak stress. Severe AS is confirmed by an increase in Vmax ≥4 m/s or ∆Pm ≥40 mmHg but with an AVA that remains <1.0 cm 2 (Figure 5), while moderate AS is defined by an increase in AVA ≥1.0 cm 2 . However, many patients, even with contractile reserve will not reach a normal flow rate and will remain discordant at peak stress evaluation. To overcome this limitation, the projected AVA calculation may help to identify true-severe AS [START_REF] Clavel | Validation of conventional and simplified methods to calculate projected valve area at normal flow rate in patients with low flow, low gradient aortic stenosis: the multicenter TOPAS (True or Pseudo Severe Aortic Stenosis) study[END_REF][START_REF] Annabi | Dobutamine stress echocardiography for management of low-flow, low-gradient aortic stenosis[END_REF] . Finally, in patient without contractile reserve, the projected AVA could be calculated if the increase in flow is higher than 15%. If not, the severity of AS remained in determined by stress echocardiography. In all of these patients, AS severity could be confirmed/assessed by CT calcium scoring.

These guidelines are mainly based on publications that predate the era of TAVR [START_REF] Monin | Low-gradient aortic stenosis: operative risk stratification and predictors for long-term outcome: a multicenter study using dobutamine stress hemodynamics[END_REF][START_REF] Monin | Aortic stenosis with severe left ventricular dysfunction and low transvalvular pressure gradients[END_REF][START_REF] Quere | Influence of preoperative left ventricular contractile reserve on postoperative ejection fraction in low-gradient aortic stenosis[END_REF] . It was demonstrated that patients with a pseudo-severe AS had the same prognosis than matched patients with heart failure, whereas patients with a true-severe AS had worse outcomes [START_REF] Fougères | Outcomes of pseudo-severe aortic stenosis under conservative treatment[END_REF] . In addition, the absence of flow reserve was a major risk marker for postoperative mortality. However, surgical aortic valve replacement (SAVR) was beneficial in true-severe AS patients (confirmed by the surgeon) regardless of the presence or absence of flow reserve compared to medical therapy alone [START_REF] Tribouilloy | Outcome after aortic valve replacement for low-flow/low-gradient aortic stenosis without contractile reserve on dobutamine stress echocardiography[END_REF] . The prognosis impact of flow reserve was not confirmed in SAVR [START_REF] Annabi | Dobutamine stress echocardiography in low-flow, low-gradient aortic stenosis: flow reserve does not matter anymore[END_REF] patients as well as after TAVR [START_REF] Ribeiro | Transcatheter aortic valve replacement in patients with low-flow, low-gradient aortic stenosis: The TOPAS-TAVI registry[END_REF] . To summarize, all patients with a classical low-gradient true-severe AS will benefit from AVR, especially using a percutaneous approach, whether or not having a flow reserve. Hence, the current use of dobutamine stress echocardiography is probably less relevant, since the AS severity can be directly confirmed by calcium scoring (Proposal 1, Figure 6).

Confirmation and classification of severe aortic stenosis: a multimodality approach

Aortic stenosis is classified in different categories according to transvalvular mean pressure gradient (∆Pm cutoff: 40 mmHg), LVEF (cutoff 50%) and LVOT flow (SVi cutoff: 35ml/m 2 ).

High-gradient (HG) severe AS corresponds to classical severe AS and requires no further exam to confirm its severity. However, AS patients with a HG low-flow (LF) pattern may have a worse prognosis in absence of early treatment compared to HG normal-flow (NF) patients [START_REF] Rusinaru | Impact of low stroke volume on mortality in patients with severe aortic stenosis and preserved left ventricular ejection fraction[END_REF] . Conversely, LG (or discordant) severe AS requires a multimodality approach to confirm its true severity before considering AV intervention (Figure 6). The first step consists in confirming the measurements (especially LVOT d ) and in using additional parameters (i.e. VTI ratio, AVA planimetry, AT or AT/ET, hybrid AVA) to exclude moderate AS. Next step requires the assessment of LVEF to distinguish LG AS patients with preserved (≥50%) from reduced LVEF (<50%). In patient with LG AS and reduced LVEF (i.e. classical LG-LF AS), the true severity of AS should be confirmed by dobutamine stress echocardiography or by calcium scoring when inconclusive. In patients with LG AS but preserved LVEF, the SV assessment should be used to discriminate NF from paradoxical LF LG AS. LG-NF AS is classified as severe AS unlikely by the current guidelines, but recent studies suggest that 50% patients may have a true-severe stenosis and may benefit from AVR [START_REF] Zusman | Intervention versus observation in symptomatic patients with normal flow-low gradient severe aortic stenosis[END_REF] . Hence, the use of calcium scoring may help to identify these patients (proposal 2, Figure 6). Patients with a paradoxical LG-LF AS also require calcium scoring by CT to confirm AS severity, moreover there prognosis is darker and interventional risk higher due to the low flow state. The Figure 6 is an adapted version of the different steps reported in the current guidelines to classify and confirm severe AS. In addition, we provide a simplify proposal (proposal 1) to confirm AS severity with calcium scoring in patients with a LG pattern, regardless of the LVEF and SV levels.

Conclusion

Aortic stenosis is a serious cardiac disease with a significant morbi-mortality. AS severity confirmation is essential to adequately manage such patients. TTE is the key exam to confirm the diagnosis in a large majority of patients. However, parameter discordance is not uncommon and requires a multimodality approach to confirm AS severity.

TABLES

Low-Gradient AS

(

  LVOT) is equal to SV ejected through the aortic valve orifice: Where LVOT d is the diameter of LVOT. This equation requires the measurement of two additional echocardiographic parameters: the LVOT VTI (LVOT VTI ) and the LVOT diameter (LVOT d ) to calculate LVOT area. LVOT d is a key measure to define AS severity. It should be measured at the aortic annulus level (inner edges of leaflet insertion) from a parasternal long axis view zoomed on the aortic valve during the systolic phase, when the diameter is x LVOT VTI = AVA x AV VTI maximal (Figure 2A). Although recommended for years, LVOT d measurement away

Figure 1 :

 1 Figure 1: Multiparametric evaluation of aortic stenosis severity: peak jet velocity (Vmax) and

Figure 2 :

 2 Figure 2: Left ventricular outflow tract diameter (LVOT d ) assessment in a same patient

Figure 3 :

 3 Figure 3: Aortic valve orifice planimetry in (A) a same patient using transthoracic and (B)

Figure 4 :

 4 Figure 4: Calcium scoring evaluation in a patient with severe aortic stenosis using (A) a

Figure 5 :

 5 Figure 5: Evaluation of aortic stenosis severity using dobutamine stress echocardiography in

Figure 6 :

 6 Figure 6: Algorithm to confirm and classify severe aortic stenosis. Velocity or VTI ratio, AT

Table 1 :

 1 Parameters defining severe aortic stenosis using a multimodality approach

	Imaging modality	Parameters
	Echocardiography
	First line (also available for CMR)
	-Peak jet velocity	≥4 m/s
	-Mean pressure gradient	≥40 mmHg
	-Aortic valve area	≤1.0 cm 2
	-Aortic valve area index	≤0.6 cm 2 /m 2
	Second line
	-Velocity or VTI ratio	<0.25
	-Acceleration time	>110 ms
	-Acceleration time/ejection time	>0.36
	-Aortic valve area planimetry	≤1.0 cm 2
	Stress echocardiography
	-Peak jet velocity at peak	≥4 m/s
	-Mean pressure gradient at peak	≥40 mmHg
	-Aortic valve area at peak	≤1.0 cm 2
	Cardiac computed tomography
	-Calcium scoring	Men ≥2,000 AU
		Women ≥1,200 AU
	-Aortic valve area planimetry	≤1.0 cm 2
	Hybrid method (LVOT by CT or CMR)
	-Aortic valve area	≤1.2 cm 2

Aortic Valve Area < 1 cm 2 ? Stroke volume index

	Transvalvular Gradient/Velocity ∆ ∆ ∆ ∆Pm ≥ ≥ ≥ ≥ 40 mmHg and Vmax ≥ ≥ ≥ ≥ 4 m/s ?
	Moderate AS
	≤ ≤ ≤ ≤ 35 ml/m 2

High-Gradient Severe AS Yes No Normal-Flow Low-Gradient AS Severe AS Unlikely No Aortic Stenosis SV and SVi evaluation for prognosis SV < 55 ml or SVi < 30 ml/m 2 ?

		Low-Flow	Normal-Flow
		High-Gradient	High-Gradient
		Severe AS	Severe AS
	LVEF ≥ ≥ ≥ ≥ 50%?
		Low-Gradient AS with
	(Low-	preserved LVEF

Flow) Low-Gradient AS with reduced LVEF Paradoxical Low-Flow, Low-Gradient AS Dobutamine stress echocardiography Flow reserve and conclusive? No Yes Yes Confirm measures and 2 nd line evaluation* Alternative 1 Alternative 2 Yes No Calcium scoring by CT

		No
	Yes
		Yes	No
		Yes	No
		No
	Yes
	AVA ≤ ≤ ≤ ≤ 1cm 2 and ∆ ∆ ∆ ∆Pm ≥ ≥ ≥ ≥ 40 mmHg
	Or Projected AVA ≤ ≤ ≤ ≤ 1cm 2	-≥2000 in men -≥1200 in women
	Moderate AS	Severe AS

CMR for cardiac magnetic resonance, VTI for velocity time integral, LVOT for left ventricular outflow tract, CT for computed tomography, mmHg for millimeter of mercury.

Dr. Clavel has Core Lab contracts with Edwards Lifesciences, for which she receives no direct compensation; and research grant with Medtronic. Dr Ternacle has no disclosure. Julien Ternacle has received a fellowship grant from AREMCAR. Marie-Annick Clavel is scholar fellow from Fond de recherche du Québec en santé.


MD Xavier Jouven
email: xavier.jouven@inserm.fr

Bara I Diop

Kumar Narayanan

MD, DM Anicet Adoubi

Serigne Abdou Ba

MD Dahdi Balde

Jean-Marie Damorou

Mamadou Bacary Diarra

Anastase Dzudie

PhD Beatriz Ferreira

PhD, Bara I Diop Md

MD Jean-Marc Damorou

MD, Mamadou Bacary Diarra

PhD, Sedonougbo Martin Houenassi

Meo-Stephane Ikama

Abdoul Kane

Adama Kane

Samuel Kingue

Jean-Bruno Mipinda

Ana-Olga Mocumbi

PhD Ali Niakara

Merimee Ouankou

PhD Abdallahi Aly Sidi

PhD Jean- Laurent Takombe

Ibrahim Ali Toure

Patrice Zabsonré

MD David S Celermajer

PhD Antoine Frcp

Lafont

PhD Bernard Dodinot

Pascal Sagnol

Eloi Marijon

Cardiac Pacing in Sub-Saharan Africa JACC International

Keywords: Developing Countries, Pacing, Heart, Sudden Death, Technology, Education, Training

Introduction

Since it was first performed in 1958, cardiac pacing has expanded exponentially, with now nearly 1,000 implantations annually per million population, in developed countries. [START_REF] Bonny | Cardiac Arrhythmias in Africa: Epidemiology, Management Challenges, and Perspectives[END_REF][START_REF] Kusumoto | ACC/AHA/HRS Guideline on the Evaluation and Management of Patients With Bradycardia and Cardiac Conduction Delay: A Report of the American College of Cardiology/American Heart Association Task Force on Clinical Practice Guidelines and the Heart Rhythm Society[END_REF]While the need for cardiac pacing is similar worldwide, developing regions of the world lag significantly behind their developed counterparts in having reasonable (or in some cases, any) access to this potentially life-saving procedure.

Sub-Saharan Africa, one of the poorest parts of the world, is undergoing an epidemiologic transition, with the incidence of deaths related to cardiovascular causes increasing rapidly over time.(3) However, except for South Africa, there has been very little implementation of cardiac pacing [START_REF] Bonny | Cardiac Arrhythmias in Africa: Epidemiology, Management Challenges, and Perspectives[END_REF][START_REF] Thiam | Cardiac pacing in West Africa: feasibility, problems, and perspectives[END_REF][START_REF] Jouven | Cardiac pacing in Western Africa[END_REF][START_REF] Bonny | Statistics on the use of cardiac electronic devices and interventional electrophysiological procedures in Africa from 2011 to 2016: report of the Pan African Society of Cardiology (PASCAR) Cardiac Arrhythmias and Pacing Task Forces[END_REF]. Lack of adequate expertise in the form of skilled physicians is a major limitation to provide cardiac pacing to the population overall, along with scarcity of resources, funding and technology.

Through a dedicated effort over the past two decades (1996-2018), we have carried out a project to develop cardiac pacing in Sub-Saharan Africa, based on training local healthcare teams and adapting to regional constraints. This unique project aimed to establish and build a sustainable local capacity for cardiac pacing.

Methods

Scope of the Program

The main objective of Africa-Pace was to initiate and build sustainable local and regional capacity for cardiac pacing in countries of sub-Saharan Africa, by partnership with local cardiologists and healthcare teams providing clinical services mainly within the framework of public institutions, such as teaching hospitals. The pacing program, initiated in 1996, is part of a broader initiative called Cardiology and Development, launched by the European Georges Pompidou Hospital and the French Institute of Health and Medical Research in Paris.

Cardiology and Development is a specific Non-Governmental Organizations aiming to link

Care-Research-Teaching in emerging countries, especially in Africa and Asia [START_REF] Antignac | Quality Assessment of 7 Cardiovascular Drugs in 10 Sub-Saharan Countries: The SEVEN Study[END_REF][START_REF] Marijon | Prevalence of rheumatic heart disease detected by echocardiographic screening[END_REF][START_REF] Ranque | Early renal damage in patients with sickle cell disease in sub-Saharan Africa: a multinational, prospective, cross-sectional study[END_REF][START_REF] Dubert | Degree of anemia, indirect markers of hemolysis, and vascular complications of sickle cell disease in Africa[END_REF][START_REF] Celermajer | Cardiovascular disease in the developing world: prevalences, patterns, and the potential of early disease detection[END_REF][START_REF] Marijon | Early detection of rheumatic heart disease and prevention of heart failure in sub-Saharan Africa[END_REF].

The pacing program was based on a concept of doorstep delivery of technology and onsite training. This comprised both theoretical teaching as well as carrying out planned missions to perform procedures in local hospitals, with the involvement of the local cardiologists/support staff, so that center-specific challenges could be identified and overcome by collaborative effort.

Through this approach, we aimed to: (i) provide services to the patients most in need of cardiac pacing during the initial collaborative missions; (ii) achieve proficiency among participating local cardiologists after missions, and (iii) enhance collaborations between African countries to help make the program sustainable. Furthermore, we additionally hoped to raise awareness among policy makers regarding the urgent need to develop this lifesaving therapy, and to promote education of the healthcare community.

Preparation for the Missions

Each mission was carried out by agreement and invitation from the Health Ministry of each particular country. Africa-Pace staff, in conjunction with each local team sought necessary approvals from hospital and national authorities to undertake the mission, including customs for devices, visas for visiting staff, and obtaining authorization for visiting physicians to work in the country.

We aimed to develop a local pacemaker team consisting of an engaged and committed cardiologist (who would perform the implantation) and two nurses (who would be trained onsite). If available, a local surgeon was trained as well. Commitment to the program and (likely) permanence of staff at the hospital were key requirements.

Local cardiologists screened patients who might require cardiac pacing and selected those deemed suitable for the planned intervention. A minimum of 15 patients was necessary to plan a mission. The list of patients and indications for implantation were discussed with the local team prior to the mission by conference call. Pre-procedure work-up (chest X-ray, full blood count, electrolytes and creatinine) was performed by the local team, aimed at identifying any contraindications to implantation. Preparation of basic infrastructure, such as x-ray equipment and sterile instruments, was also arranged by the local team.

The Africa-Pace team had two experienced pacemaker implanters, initially from European and subsequently from African countries (to ensure that the program could be sustained long-term), who were also able to adapt and innovate as necessary to overcome challenges in resource-constrained settings. Africa-Pace was responsible for arranging the pacemaker equipment (pulse generators and leads). As funding was very limited, new devices with expiry dates close to the date of the mission were provided by industry, free of charge. At least three missions were planned to one site, aiming for about 10 to 15 implantations during each mission; hence in most cases the local implanter performed about 30 to 40 procedures under supervision before becoming truly independent and doing implantations in the absence of the proctor.

Carrying out the Missions

The first day on site was dedicated to verifying that all requirements were in place for the procedures. The visiting cardiologist met with the local cardiologist at the hospital with the rest of the team. The procedure room as well as all consumables and equipment brought by the visiting team were double-checked. The cardiologist leading the mission reviewed the plan and the expected duties of each member of the team in detail. All medical records were then discussed and the patients admitted thereafter. Rules of good practices, including sterility and hygiene, were provided to the nurses and other local staff.

The visiting cardiologist performed the first procedure (closely observed and assisted by the local team) and then guided the local cardiologist (trainee) for the remaining procedures.

Pacemaker implantation was done under local anesthesia. Intravenous antibiotic coverage was administered at the time of skin incision. During the following second and third missions to each site, the visiting cardiologist was available in the lab and supervised the trainee without scrubbing in, with the aim of enabling the local team to operate independently.

Patients were usually discharged on the 4 th post-procedure day if there were no complications. Inspection of the incision site and ruling out pocket complications was emphasized. Only basic training on checking the pacemaker by means of a magnet was provided.

Remote support continued, via regular conference calls, once the local team was proficient to do procedures independently, to encourage continued maintenance of good clinical standards. All patients were systematically invited for a routine visit 4 weeks after implant, either at the implant center or a closer medical center for patients living in very remote areas.

Outcomes of the mission were summarized and made public through national and regional media, with a special focus on the new local team. Meetings with policy makers were scheduled in order to gain support for future missions and the expansion of the local cardiology clinic. Traditionally, the visiting physician also gave at least one lecture at the local University on a variety of topics in order to optimize the educational component of each mission.

Data Collection and Statistical Analysis

Data on implanted patients were prospectively obtained during missions according to a pre-specified Case Report Form, and collected by the Paris Cardiovascular Research Center (French Institute of Health and Medical Research, Paris, France), in collaboration with the implantation center. The data comprised recording of device functionality and any related events/complications. In parallel with the systematic collection of data on first implantations during the 64 missions, further efforts were directed towards following the deployment of pacing nationally in each of the 14 countries involved in the program. This focused especially on the number of implantations in each country in order to provide incidence of implantation per million inhabitants and ascertaining peri-procedure events, if any. All data were collected by the French Institute of Health and Medical Research, at Unit 970, Cardiovascular Epidemiology.

Data were analyzed using SAS version 9.3 (SAS Institute, Cary, North Carolina). All tests were 2-tailed, and P values of less than 0.05 were considered to indicate statistical significance.

Results

Setting and Participants

Three missions were undertaken every year, eventually targeting 14 countries between 1996 and 2018, for a total of 64 missions. In 11 of these countries, cardiac pacing programs were initiated for the first time (first pacemaker implant in the country was through the mission) (Figure 1, dark yellow). Median duration per mission was 7 days (IQR: 3-10). Out of the 1,077 patients initially listed, 542 (50.3%) were eventually implanted. The majority of the remaining patients had died suddenly while waiting for pacemaker implantation, prior to mission arrival.

The median waiting time between diagnosis and intervention was 18.4 (IQR: 3.2-26.4) months.

The mean age was 63±9 years (from 14 to 92 years) and 336 (62%) were male (Table 1).

The mean heart rate on the day of implantation was 37±8 bpm. The majority of patients were diagnosed with paroxysmal or permanent complete AV block (94%), with the others having sinus node dysfunction. All, except 15 patients (3%), were symptomatic, mainly with dizziness/syncope (44%) and shortness of breath (53%), with 108 (20%) having experienced more than one symptom. Hypertension (304, 64%), rheumatic heart disease (67, 14%), and coronary artery disease (78, 16%) accounted for the majority of the associated cardiac pathologies.

Pacemaker Implantations

A single chamber (ventricular) pacemaker was most often used (482 patients, 89%). In 2017-2018, four cardiac resynchronization therapy pacemakers were also implanted during the missions by visiting experts. Only one implantable cardioverter defibrillator has been implanted during the missions.

Successful pacing was eventually achieved in all cases. There was no peri-procedural mortality. During the procedure, however, cardiac arrest occurred in approximately 20%, requiring temporary chest compression. This was usually due to third degree AV block without escape rhythm, particularly in patients with end-stage conduction disease. Threshold testing was not commonly performed due to the lack of testing cables or the perceived risk of infection from improperly sterilized cables.

No deaths occurred during implantation or during the in-hospital stay. Peri-operative complications occurred in 33 patients (6%), including lead dislodgment and local hematoma, requiring reintervention in 19 patients (3%). Other complications included pocket infection in 2% and pericardial effusion in 0.5%.

Capacity Building

Overall, this program resulted in a significant increase in local implantation rates over time from 0.04 (95% CI 0.02-0.06) to 0.65 (95% CI 0.54-0.75) per million inhabitants per year for the 1996-2000 and the 2011-2018 periods respectively (P<0.0001). Figure 2 depicts the progressive increase in the number of implantations performed over the years, across the 11 of these countries where cardiac pacing programs were initiated for the first time.

The most advanced center in 1996 was the Heart Institute in Abidjan (Ivory Coast), but civil war considerably delayed its progression, whereas the best progression was achieved in Dakar (Senegal). Importantly, as seen in the figure, the number of implantations performed by the visiting team remained the same over the years, while there was a rapid increase in independently performed implantations by local staff. The proportion of the implantations that were completely handled by local teams moved up from 3% in 1996 to 98% in 2018. Senegal, Ivory Coast, Mauritania, Burkina Faso, Gabon and Cameroon have successfully established their own completely autonomous pacing programs. Other countries including Niger, Mozambique, Mali, Benin and Togo have achieved partial proficiency and are expected to establish their own programs within the coming years. Congo Brazzaville joined this group recently, with the first mission performed in 2012. 13 Finally, a specific program was recently started in Democratic Republic of Congo (Kinshasa), as well as a first mission performed in February 2014 in Guinea just before the outbreak of Ebola which stopped the program; this is to be resumed shortly. 14 The most recent mission took place in Burundi in May 2019.

Discussion

We have described the results of a novel and scalable initiative to make cardiac pacing achievable and sustainable in Sub-Saharan Africa. Over the past two decades, this project has successfully performed pacemaker implants in 14 African countries, making cardiac pacing available for the first time in most of these countries. Despite working in resource-poor settings and having to adapt to unique local circumstances in each country, the outcomes and complication rates observed are acceptable, demonstrating the feasibility and practicality of this approach. More importantly, the key achievement of this program has been the fact that implantations performed independently by local cardiologists have increased considerably over the duration of the program, with half the countries now having successfully established their own independent pacing programs. This has been possible not only through the training of African cardiologists by proctor missions, but also by the subsequent initiatives of the initially trained African physicians to reach out to neighboring countries, resulting in transfer of knowledge between different developing countries. [START_REF] Ikama | Permanent cardiac pacing: first Congolese experiment[END_REF][START_REF] Balde | Permanent cardiac pacing: first experience in Guinea[END_REF][START_REF] Diop | Permanent cardiac stimulation in Senegal: preliminary experience at the Cardiology Clinic of Dakar[END_REF][START_REF] Tchoumi | Follow up in a developing country of patients with complete atrio-ventricular block[END_REF][START_REF] Yayehd | Management of high-grade atrioventricular block in Lomé, Togo[END_REF] This experience serves as a potential roadmap to help disperse necessary medical technology to all corners of the developing world.

While some may question the allocation of resources to cardiac pacing as compared to other health needs in sub-Saharan Africa, two issues merit consideration. Firstly, pacing is usually a lifesaving procedure. This is amply demonstrated by the fact that approximately 50% of patients initially shortlisted did not survive the waiting period, prior to mission arrival. If one assumes a death rate without pacing of 50% of selected patients (as we observed) and a population of adults in Sub-Saharan Africa of 973 million,(18) then widespread adoptions of this program, with the eventual goal of reaching one tenth of the implantation rate observed in Western countries, could save approximately 50,000 lives each year. Secondly, cardiac pacing is a key requirement to the advancement of cardiology in any region, without which a reasonable standard of cardiac care is simply not possible. While there has been a major focus on infective diseases in the third world, changing demographic trends are resulting in an increasing burden of morbidity secondary to non-communicable disease, mainly cardiovascular disease. Though there are continued increases in life expectancies in developing nations with corresponding population expansions, years lost to disability have proportionately increased as well, thus contributing to an expanding morbidity pool.(3) Thus it is important to address this discrepancy. For instance, in the Democratic Republic of Congo, a country with 80 million inhabitants, not a single pacemaker implantation had been performed prior our first mission in 2014 (13) (Figure 3). Important hurdles to the development of pacing in developing countries include cost, availability of devices, equipment (sterilization facilities, x-ray) as well as lack of skilled personnel. [START_REF] Gakenheimer | Societal views of pacemaker reutilization for those with untreated symptomatic bradycardia in underserved nations[END_REF][START_REF] Wunderly | Using reconditioned pacemakers to treat bradycardia in Africa[END_REF] The traditional approach to address the gap in technology and skill between developed versus developing countries has been to either sponsor procedures for patients in modern centers in Europe/North America through charitable missions or to help physicians from developing nations to travel abroad for training in advanced centers with the expectation that they would in turn return and benefit their home country. Both these approaches have shortcomings. Indeed, experience suggests that, for a multitude of reasons, doctors from developing countries may be likely to remain in these developed countries, rather than to return home. Furthermore, training in western countries often occurs in the setting of access to the best equipment, facilities and support personnel, which is a problem when translating this experience, to the country of origin. 21,22 Hence indigenous training is the need of the hour; for instance a regional university teaching program was set up in 2017 by Professor Adama Kane at St louis University, Senegal to deliver specific courses in cardiac arrhythmias, pacing and electrophysiology to students from West-Africa. 23 France is in a vantage position to help countries, especially in Francophone Africa, to address health as a global security issue. Beyond supporting transfer of some patients into expert university hospitals in France, the more sustainable solution may be to actually take technology to the "doorstep" of the country in need and develop the program "in the trenches" adapting to local constraints and challenges. While this may seem initially daunting, we have demonstrated that this is feasible and can result in successful growth of local programs, which is crucial for long-term results and sustainable development. In developing such programs, it is important to have meticulous planning and a careful start, to ensure early success in the program to establish trust and facilitate local acceptance. In our mission, the first few implantations in any country were especially crucial and good results were important to ensure that the program took root and continued to grow. Although it is possible that cardiologists trained elsewhere, outside of our missions, may have contributed partly to the increase in implantations, due to our strong collaboration network with the cardiologists of these countries, we are aware that the subsequent growth of implantations has been for the major part driven by the cardiologists initially trained by us and by those whom they trained subsequently (South-to-South collaboration).

Our work reflects a real-world experience and results should be interpreted thoughtfully; rigorous and complete follow-up for all cases could not be ensured. The reported complication rates should be interpreted with caution as there could have been some underreporting and incomplete follow up. Further studies with more rigorous long term follow up are required. Also, there may be concerns regarding the adequacy of training for the local cardiologist, as compared to international standards, however our experience suggests that outcomes are reasonable and further long-term data would undoubtedly be useful. Healthcare needs and economic/political conditions are varied and diverse and therefore our results may not be generalizable. While no formal cost effectiveness analysis was performed, the main long term sustenance of this program is based on implantation by local cardiologists with only a first few on site missions by the traveling team, hence we feel that it is very likely to be cost effective per life saved as compared to many other contemporary therapies in cardiology.

Although implantation rates increased over time, they still remain many-fold lower than in developed regions. An important limitation to the success of such programs is the availability of devices at affordable costs and for this all relevant parties including local government, NGOs and industry need to be actively engaged to find ways to make the therapy affordable. Finally, our work serves to demonstrate that "doorstep technology delivery" can be potentially applied to other areas in cardiology (especially interventional cardiology), but also more generally in medicine to reduce healthcare gaps in developing countries. As part of the Cardiology and Development initiative, we aim to address not only cardiac pacing but also other common disease conditions, such as rheumatic heart disease and sickle cell disease in Africa. [START_REF] Antignac | Quality Assessment of 7 Cardiovascular Drugs in 10 Sub-Saharan Countries: The SEVEN Study[END_REF][START_REF] Marijon | Prevalence of rheumatic heart disease detected by echocardiographic screening[END_REF][START_REF] Ranque | Early renal damage in patients with sickle cell disease in sub-Saharan Africa: a multinational, prospective, cross-sectional study[END_REF][START_REF] Dubert | Degree of anemia, indirect markers of hemolysis, and vascular complications of sickle cell disease in Africa[END_REF][START_REF] Celermajer | Cardiovascular disease in the developing world: prevalences, patterns, and the potential of early disease detection[END_REF] The network built could also be potentially useful to enhance collaboration in other needed areas, especially relevant to the third world.

Conclusions

Our findings demonstrate the feasibility and effectiveness of a specific on-site proctorship program to develop cardiac pacing in Sub-Saharan Africa based on "doorstep" delivery of technology and training to develop local expertise. However, persisting unmet needs and major gaps in implantation rates (still 500-fold lower, compared to industrialized nations) require further diligent efforts.

Main messages

• Many countries in Sub-Saharan Africa, lack access to the lifesaving technology of cardiac pacing.

• Through a multinational program, we demonstrated the feasibility and effectiveness of a proctorshipbased approach to the development of local cardiac pacing capabilities in Sub-Saharan African nations.

• Further studies should focus on the long-term outcomes of such programs as well as the issue of making such technologies available and affordable through engagement of all relevant stakeholders.

FIGURES LEGENDSFigure 1 .

 1 FIGURES LEGENDS

Figure 2 .

 2 Figure 2. Number of pacemaker implanted in countries where cardiac pacing programs

Figure 3 .

 3 Figure 3. First mission in Congo Brazzaville, January 2012