Shadi Damough

Masoumeh Sabzalinezhad

Yeganeh Talebkhan
email: talebkhan@pasteur.ac.ir

Leila Nematollahi

Elham Bayat

Fatemeh Torkashvand

Ahmad Adeli

Hoda Jahandar

Farzaneh Barkhordari

Fereidoun Mahboudi

Optimization of culture conditions for high-level expression of soluble and active tumor necrosis factor-α in E. coli

Keywords: Affinity purification, recombinant, tumor necrosis factor-α

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

Introduction

Tumor necrosis factor alpha (TNF-α) is a pluripotent inflammatory cytokine produced from macrophages, CD4 lymphocytes, neutrophils, mast cells, eosinophils, and natural killer (NK) cells in response to the inflammatory agents, including bacterial toxins [START_REF] Takahiko | Transmembrane TNF-a: structure, function and interaction with anti-TNF agents[END_REF]. Increased expression of this cytokine has been involved in the development of rheumatoid arthritis (RA), psoriatic arthritis, and Crohn's disease (CD) [START_REF] Kollias | On the role of tumor necrosis factor and receptors in models of multi organ failure, rheumatoid arthritis, multiple sclerosis and inflammatory bowel disease[END_REF][START_REF] Lipsky | Infliximab and Methotrexate in the treatment of rheumatoid arthritis, anti-tumor factor trial in rheumatoid arthritis with concomitant therapy study group[END_REF]. Its cytotoxic activity is mediated by binding to specific receptors and induction of proto-oncogenes, such as c-fos [START_REF] Emch | TNF-α-induced c-Fos generation in the nucleus of the solitary tract is blocked by NBQX and MK-801[END_REF] and c-myc [START_REF] Jänicke | Nuclear c-Myc plays an important role in the cytotoxicity of tumor necrosis factor alpha in tumor cells[END_REF], within fibroblasts. This circulating anti-tumorigenic cytokine is made up of 233 amino acids as a 26kDa transmembrane protein type 2, forming stable active homotrimers [reviewed in 6]. TNF-α immature protein loses a part of the molecule during metalloprotease activity and can be released as a 17kDa mature TNF-α [START_REF] Jonese | The structure of tumour necrosis factor -implications for biological function[END_REF] compromising 157 amino acids and a single disulfide-bond (Cys69-Cys101) [START_REF] Surovtseva | A new Escherichia coli strain producing human tumor necrosis factor[END_REF]. The monomeric 17.35kDa protein is active, and a soluble protein without any glycosylation and its isoelectric pH is about 5.8. This fundamental molecule has attracted a great deal of attention during the last decades for its immunological, diagnostic, and medicinal applications. For instance, for detecting TNF-α in blood samples through immunochemical approaches, recombinant TNF-α has been expressed for rabbit immunization to obtain specific monoclonal antibodies [START_REF] Surovtseva | A new Escherichia coli strain producing human tumor necrosis factor[END_REF][START_REF] Kuznetsova | A new system for TNF-α quantification in human blood samples[END_REF]. On the other hand, due to its suggested anti-tumor activities in combination with chemotherapy agents [START_REF] Benderska | Apoptosis signalling activated by TNF in the lower gastrointestinal tract-review[END_REF], several studies have been conducted to express the recombinant TNF-α protein [START_REF] Zhang | Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant[END_REF][START_REF] Alizadeh | A simple and rapid method for expression and purification of functional TNF-α using GST fusion system[END_REF]. For neutralizing the excess amount of endogenous TNF-α in patients suffering from RA and CD, several TNF-α inhibitors have been developed and approved by FDA for clinical usage, raising due to the TNF-α double-edged signaling pathway.

Biological activity assessment of these anti-TNF-α molecules needs the heterologous recombinant expression of TNF-α as well.

Escherichia coli (E. coli) expression system is one of the most widely used systems for heterologous expression of simple human proteins without glycosylation or other posttranslational modifications. Preference of E. coli host is due to its fast growth, easy genetic manipulation, and simple scale-up for industrial production and purification [START_REF] Rosano | Recombinant protein expression in Escherichia coli: advances and challenges[END_REF]. This expression system, however, has its specific disadvantages, particularly in posttranslational modifications, codon bias, and, importantly, the formation of inclusion bodies, making protein purification more expensive, complicated, and time-consuming, especially in industrial scale.

Besides genetic manipulations, critical strategies, including incubation temperature, postinduction duration, type of medium, and IPTG concentration, are the main parameters studied frequently to explore the best conditions for heterologous protein expression. A good balance between biomass growth conditions and criteria involved in protein production is, therefore, essential to achieve high productivity. In this regard, not only the choice of appropriate vector and promoter system but also the composition of the medium, cultivation temperature, and induction condition is important in the final destination of the desired protein in being correctly folded or aggregated as insoluble cytoplasmic or periplasmic inclusion bodies [START_REF] Rosano | Recombinant protein expression in Escherichia coli: advances and challenges[END_REF][START_REF] Berrow | Recombinant protein expression and solubility screening in Escherichia coli: a comparative study[END_REF][START_REF] Morowvat | Optimization of fermentation conditions for recombinant human interferon beta production by Escherichia coli using the response surface methodology[END_REF][START_REF] Diederichs | Phenotyping the quality of complex medium components by simple online-monitored shake fask experiments[END_REF][START_REF] Marini | Experimental design approach in recombinant protein expression: determining medium composition and induction conditions for expression of pneumolysin from Streptococcus pneumoniae in Escherichia coli and preliminary purifcation process[END_REF][START_REF] Villaverde | Protein aggregation in recombinant bacteria: biological role of inclusion bodies[END_REF][START_REF] Fahnert | Inclusion bodies: formation and utilization[END_REF].

Here, we report the cloning, optimization of E. coli cytoplasmic expression, and native affinity purification of TNF-α, and evaluation of its biological activity using Altebrel as a biosimilar of Etanercept.

Methods

Materials

E. coli BL21 (DE3) and pET28a expression vector (Novagen, USA) were used for expression experiments. Restriction enzymes were purchased from Fermentas (Lithuania). Nickel Nitrilotriacetic acid (Ni-NTA) agarose resin was purchased from ABT (Spain). CM sensor and other required materials for Surface Plasmon Resonance (SPR) were purchased from GE Healthcare Life Sciences (USA). All media, chemicals, reagents and antibodies required for experiments and buffers were purchased from Sigma Chemicals (USA) unless otherwise stated.

TNF-α gene cloning

The amino acid sequences encoding the active human TNF-α molecules were obtained from available data banks, including drug and NCBI protein data bank. The consensus sequence was selected, and the corresponding nucleotide sequence was codon-optimized for protein expression in E. coli expression host cells. Using NcoI and HindIII restriction enzymes under the T7 promoter, the synthesized gene fragment harboring N-terminal Histidine tag was subcloned in the pET28a expression vector.

Expression of TNF-α protein

Transformed E. coli BL21 (DE3) strain was inoculated into 5 ml LB broth medium supplemented with 15µg/ml Kanamycin on a shaker incubator at 37°C until the optical density at 600nm reached 0.4. 0.5mM IPTG was used to induce protein expression. The tubes were incubated at 37°C for a further 4 hours. The bacterial pellet was collected by centrifugation at 9,000rpm (3min), resuspended in 2X loading buffer, and ran on 12% SDS-PAGE. The identity of the expressed protein was confirmed using western blotting by anti-His antibody or Altebrel, a TNF-α blocking biosimilar product (Aryogen, Iran). Briefly, protein bands were transferred on nitrocellulose membrane (GE Healthcare, USA) using a semi-dry blotting system (Bio-RAD, USA) under 18 volts for 35min. The membrane was blocked by 3% (w/v) skim milk in phosphate buffer saline (PBS) overnight at 4°C. Horseradish peroxidase (HRP)-conjugated anti-His antibody was added to the membrane in a 1:1000 dilution and incubated for 2h at 30ºC. After the washing step (5 times with 0.05% Tween 20 in PBS, one time with PBS alone), DAB (3, 3'-Diamonobenzidine) solution, HRP substrate, was added to the membrane until the desired bands appeared. In a further experiment, Altebrel was used as the first antibody (2mg/ml), and after 1 h incubation at 30ºC and the washing step (5 times), 1:10,000 dilution of HRP-conjugated rabbit anti-human IgG (Invitrogen, USA) was added to the transferred membrane and incubated for 1h at 30ºC. After the washing step (5 times with 0.05% Tween 20 in PBS, one time with PBS alone), DAB was added to the membrane until the bands appeared.

Optimization of protein expression

Two broth media (Luria Broth; LB and Terrific Broth; TB), different Isopropyl ß-D-1thiogalactopyranoside (IPTG) concentrations (0.25, 0.5, 0.75 and 1mM) under various temperatures (25, 30 and 37°C), and different post-induction incubation times (2, 4, 6 and 24h) were examined in the optimization of TNF-α expression. Recombinant E. coli strain was inoculated into 50ml culture medium. After reaching to the appropriate optical density (OD) at 600nm, the culture medium was aliquoted in sterile tubes and subjected to different conditions mentioned above. At the end of each experiment, bacterial pellet was harvested, and the expression of recombinant TNF-α protein (~17kDa) was determined on 12% SDS-PAGE after staining with Coomassie Brilliant Blue G-250 dye. The ImageJ analyzer (National Institute of Health, USA) was used to measure the intensity of the expressed protein.

Solubility of the recombinant protein

Large scale protein expression (500ml) was performed under the optimized conditions. In the lysis buffer (50mM NaH2PO4, 300mM NaCl, 10mM imidazole; pH8.0), the bacterial pellet was resuspended and sonicated by 9 pulses (30sec sonication with the same intervals at 25% amplitude). The suspension was centrifuged at 10,000 rpm (10min) at 4°C. The presence of the recombinant protein and its intensity was investigated separately in the pellet and supernatant of the sonicated bacteria through running on 12% SDS-PAGE.

Purification of His-tagged TNF-α under native conditions

The supernatant of sonicated bacteria was loaded on the Ni-NTA column after equilibration with the binding buffer (50mM NaH2PO4, 300mM NaCl, 10mM Imidazole; pH8.0) in the FPLC purification system (Bio-RAD, USA) (under 1ml/min flow rate). The column was washed with the buffer I (50mM NaH2PO4, 300mM NaCl, 20mM Imidazole, 0.1% Triton-X114; pH8.0) (20 volumes of the column) to remove the residual endotoxin content. The next wash was done with the same washing buffer for removing Triton-X114 from the column (50 volumes of the column). His-tagged TNF-α molecules were eluted from the column using the elution buffer (50mM NaH2PO4, 300mM NaCl, 250mM Imidazole; pH8.0) under 1ml/min flow rate. The eluted soluble recombinant protein sample was dialyzed to remove imidazole. The final protein was equilibrated in 20mM phosphate buffer containing 100mM NaCl (pH7.2). Nanodrop was used to measure protein concentration at 280nm. Protein samples were stored at -70°C for further studies.

Binding activity of r-TNF-α through indirect ELISA

To investigate the affinity of anti-TNF-α molecules against the purified recombinant protein, the purified protein was coated in 96-well ELISA plates in a final 200 ng/ml concentration at 4°C overnight. After 3 times washing with PBS (pH7.2), the wells were blocked using the blocking buffer (PBS with 1% bovine serum albumin) for 2h at 30ºC. Altebrel was used as the first antibody (anti-TNF-α molecule) in a serial dilution for 1h at 30ºC. After washing with PBS buffer (supplemented with 0.05% Tween 20), HRP-conjugated Rabbit anti-human IgG (1:10,000 dilution) was added to each well and incubated for 1h at 30ºC. After the second washing, the TMB substrate was added to each well, and the reaction was stopped using 2M H2SO4. The optical densities were measured at 450nm using an ELISA reader (BIOHIT, Finland).

Assessment of r-TNF-α binding activity by SPR

To evaluate the binding activity of the home-made recombinant TNF-α to Cimzia (anti-TNF-α inhibitor, Certolizumabpegol) (UCB, Belgium), surface plasmon resonance (SPR) experiments were accomplished by Biacore X100 (GE Healthcare) at 25°C using HBS-EP running buffer (10mM HEPES; pH7.4, 150mM NaCl, 3mM EDTA, 0.005% v/v p20 detergent; pH7.4) on CM5 sensor chip coated with Fab binder (Biacore Fab capture kit, GE Healthcare) and its corresponding amine coupling kit. Briefly, Cimzia was injected in an appropriate concentration at a flow rate of 30µl/min for 180sec with a surface density of about 300 resonance units (RU).

The home-made recombinant TNF-α was injected at different concentrations (0.5, 16.6, 50.166, 150 and 450nM) in HBS-EP buffer at a flow rate of 30µl/min for 180sec. In order to remove all bound proteins, chip regeneration was performed with the recommended regeneration buffer (10mM glycine-HCl at pH2.1).

Biological activity of r-TNF-α (IC50 determination of r-TNF-α)

The biological activity of r-TNF-α was assessed using the MTT assay. The L929 cell line (murine fibroblast cells obtained from Department of Cell Bank, Pasteur Institute of Iran) was seeded at a density of 20,000 cells/well in 96-well plate and incubated for 24h at 37°C under 5% CO2. The medium was removed, and different concentrations of r-TNF-α and actinomycin-D (1µg/ml) were added. Before adding MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), the plates were incubated at 37°C for further 24h. After 4h incubation, 1% SDS (Sodium dodecyl sulfate) was added to the wells, and the plates were incubated at 37°C overnight. The optical density was measured at 600nm, and the IC50 value was calculated.

Further MTT test was performed to study the inhibitory effect of anti-TNF-α molecules, such as Altebrel, on the expressed recombinant TNF-α. Briefly, the initial 50ng/ml concentration of Altebrel was mixed with serially diluted TNF-α and incubated for 2h at 37°C. The mixture was added to the adherent L929 cells (20,000 cells/well) and incubated at 37°C (24h). The solution was removed, and the MTT substrate was added to the wells. After adding 1% SDS and incubating overnight at 37°C, the optical density was read at 600nm. Then, the anti-TNFα inhibitory effect of Altebrel was calculated.

Statistical analysis

SPSS software (SPSS Inc., v. 17.0) was used to perform statistical analyses. Protein expression levels were expressed as mean ± SE, and differences were determined by nonparametric Mann Whitney U and two-sided Student's t-tests. P-values below 0.05 were considered statistically significant.

Results

Design of the contrast

The designed construct was synthesized by Biomatik Company (Canada) and subcloned into pET28a expression vector (Fig. 1). The designed vector was confirmed via restriction digestion analysis using NcoI and HindIII, representing 506 and 5246bp fragments on 1% agarose gel (data not shown).

Expression of the recombinant protein

An approximately 17kDa protein was observed when the expression of the protein was induced in the LB medium using 0.5mM IPTG at 37°C (Fig. 2). The expressed recombinant protein (~17kDa) was detected by immunoblotting using an anti-Histidine monoclonal antibody and anti-TNF-α Altebrel (Fig. 3A andB).

Optimization of TNF-α expression

Optimization of protein expression was performed under different conditions. Initial screening showed the highest expression of r-protein in LB medium at 37°C in 0.5mM IPTG 6h postinduction (approximately 56.6% of E. coli total protein), which was significantly different from other tested inducer concentrations (Fig. 4A). In contrast, within TB medium at the same incubation temperature, the highest expression was observed in 0.75mM IPTG 4h post-induction (58.2% of total host protein) (Fig. 4B). Further optimization was performed at different temperatures in several post-induction time points. It was observed that the highest expression level of recombinant protein in the LB broth medium was achieved at 30 or 37°C 6h postinduction (Fig. 4C). In TB medium, although the highest expression was achieved under 37°C and 4 to 6h post-induction (Fig. 4D), its overall protein expression level was lower than the LB medium.

Protein purification procedure

Assessment of protein solubility showed that recombinant TNF-α had been expressed in both soluble and inclusion body (IB) formats (data not shown), where the soluble form was used in the protein purification process. Protein expression was performed under optimum conditions. A soluble fraction of bacterial lysate was injected into Ni-NTA affinity chromatography. The eluted samples were dialyzed against phosphate buffer and analyzed on 12% SDS-PAGE, showing a purity of more than 95% (Fig. 5).

Measurement of r-TNF-α binding affinity through indirect ELISA

A home-made ELISA test using a constant concentration of purified recombinant TNF-α represented the affinity of Altebrel, an anti-TNF-α molecule, with an EC50 value of 1.31 µg/ml (Fig. 6).

Absorbance at 450 nm Fig. 6. TNF-α ELISA using altebrel. Serially diluted altebrel was used as the primary antibody.

Results are the mean values ± SEM of triplicates.

Measurement of r-TNF binding affinity through SPR

Biacore sensorgram was obtained using surface plasmon resonance and binding affinity of Cimzia as a TNF-α inhibitor to the developed r-TNF-α was shown (Fig. 7). The result revealed that home-made recombinant TNF-α was bound to Cimzia like commercial TNF-α proteins previously used for binding assessment of Cimzia antibody. In the present study, the calculated KD value of this antibody molecule (Cimzia) towards the recombinant TNF-α was 7.281 E -13 M.

This result confirmed that Histidine tag does not interfere with the binding kinetic of the TNF-α.

In vitro neutralization of r-TNF-α cytotoxicity

The cytotoxic effect of TNF-α on the mouse fibroblast cell line L929 is due to the apoptosis induction following its attachment to the surface receptors. This apoptotic effect can be suppressed by anti-TNF-α molecules as Altebrel inhibiting TNF-α attachment to the corresponding receptors. In the present study, TNF-α cytotoxicity and its neutralization by Altebrel were investigated. Although actinomycin-D alone decreased the viability of the treated cells by 58%, recombinant TNF-α represented significantly increased cytotoxicity (up to 75%).

Altebrel significantly decreased the TNF-α induced cytotoxicity in higher concentrations (in the range of 5 to 0.3125ng/ml) (Fig. 8).

Discussion

Although the expression of TNF-α as inclusion bodies has been repeatedly reported in E. coli under different inducible promoters (T7 or tryptophan), its refolding strategy needed large volumes of buffers in which yield of the final protein could be dramatically reduced [START_REF] Surovtseva | A new Escherichia coli strain producing human tumor necrosis factor[END_REF][START_REF] Kuznetsova | A new system for TNF-α quantification in human blood samples[END_REF]. Due to the difficulties industrial biotechnologists face in the commercialization of the proteins in the form of insoluble inclusion bodies, monitoring the effective extrinsic factors, including incubation temperature, IPTG concentration, and post-induction time, have been studied in details to achieve the most soluble format of recombinant proteins from E. coli host cells.

Several studies have reported increased solubility of the examined proteins by decreasing the temperature [START_REF] Sørensen | Soluble expression of recombinant proteins in the cytoplasm of Escherichia coli[END_REF][START_REF] Volontè | Optimization of glutaryl-7-aminocephalosporanic acid acylase expression in E. coli[END_REF][START_REF] Piserchio | Optimized bacterial expression and purification of the c-Src catalytic domain for solution NMR studies[END_REF][START_REF] Farshdari | The Improvement of Recombinant PirA Antigen Expression in Escherichia coli M15[END_REF]. On the other hand, the IPTG potential toxicity as the main inducer, besides its negative effect on host metabolism, growth, and viability, has been repeatedly reported [START_REF] Xu | Modular optimization of multi-gene pathways for fatty acids production in E. coli[END_REF][START_REF] Kosinski | Isopropyl-β-Dthiogalactopyranoside influences the metabolism of Escherichia coli[END_REF][START_REF] Andrews | Selective disadvantage of non-functional protein synthesis in Escherichia coli[END_REF][START_REF] Malakar | Effect of substrate and IPTG concentrations on the burden to growth of Escherichia coli on glycerol due to the expression of Lac proteins[END_REF]. This chemical inducer is often consumed at 0.5-1.0mM final concentrations [START_REF] Xu | Modular optimization of multi-gene pathways for fatty acids production in E. coli[END_REF][START_REF] Leonard | Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli[END_REF][START_REF] Anthony | Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4, 11-diene[END_REF] that should be optimized in the case of each experiment. Thus, finding the best and the least IPTG concentration has been assumed as a key factor in preventing cytotoxicity within E. coli cells. In order to minimize this effect, E. coli host cells such as BL21 (DE3) or similar strains with the DE3 lysogen have commercially been developed and attracted too much attention in making a balance between the bacterial metabolic pathways [START_REF] Wu | Metabolic engineering of Escherichia coli for (2S)-pinocembrin production from glucose by a modular metabolic strategy[END_REF][START_REF] Fang | High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway Microb[END_REF]. Zhang et al., developed a tag-free form of TNF-α purified in a two-step purification protocol, representing a recovery of over 75% in 24h [START_REF] Zhang | Facile purification of Escherichia coli expressed tag-free recombinant human tumor necrosis factor alpha from supernatant[END_REF]. In an additional study, Alizadeh et al., expressed a GST-tagged TNF-α fusion protein in E. coli host cells for achieving the higher yield of correctly folded recombinant protein [START_REF] Alizadeh | A simple and rapid method for expression and purification of functional TNF-α using GST fusion system[END_REF]. Although the authors suggested the methodology as an effective and cost-benefit approach, using the GST tag requires a tag removal step that makes the procedure time-consuming and more expensive for large scale industrial production. Alternatively, affinity purification of the recombinant proteins using the fused poly-histidine tag can be performed under either native or denaturing conditions. The use of mild buffer conditions and imidazole as the eluent for the soluble cytoplasmic proteins, besides secreted proteins, often yields biologically active proteins.

However, native purification conditions are not applicable in the case of insoluble inclusion bodies, and multi-subunit proteins possess a complicated tertiary structure in which polyhistidine affinity tag may be skinned. In such cases, proteins can be purified using the denaturing reagents such as 6M guanidine hydrochloride or 8M urea in sample preparation and purification buffers. Proteins purified under denaturing conditions should then be refolded into their active form by removing the denaturants [START_REF] Wingfield | Current Protocols in Protein Science[END_REF].

The final goal of the present study was to produce TNF-α in its soluble format. Optimization of TNF-α expression, therefore, was performed, and high yield of the soluble recombinant protein achieved in TB medium led to successful native affinity chromatography through its N-terminal histidine tag. A higher rate of protein expression in TB medium compared with LB can be explained by the additional source of carbon and energy within the TB medium due to the presence of glycerol. In contrast, LB does not have any additional carbohydrates, relying almost entirely on amino acids as the source of carbon and energy. On the other hand, the potassium phosphate components of the TB medium act as mild buffers in comparison to the NaCl content of LB [START_REF] Kram | Rich medium composition affects Escherichia coli survival, glycation, and mutation frequency during long-term batch culture[END_REF].

The total yield of the r-TNF-α protein was measured as 7.2 mg/lit of bacterial medium, being significantly cost-effective in large scale production of this cytokine in assessing biological activity and affinity of TNF-α inhibitor molecules in in vitro and in vivo studies. Production of biologically active TNF-α was analyzed by cytotoxicity assessment via MTT assay on TNF-α sensitive L929 cell line. The activity of the produced recombinant TNF-α was neutralized by serial dilutions of the applied TNF-α inhibitors (Certolizumab pegol and Etanercept). Etanercept, a TNF-α inhibitor molecule, is a fusion protein that compromises the extracellular region of human TNFR2 and C-terminal human IgG1 Fc fragment. This protein has been approved for human use by blocking TNF-α induced inflammatory and cytotoxicity [START_REF] Molta | Biologics in General Medicine[END_REF]. Certolizumab pegol (Cimzia) is another FDA approved anti-TNF-α inhibitor, which is structurally a Fab' molecule conjugated chemically to a PEG moiety [START_REF] Goel | Certolizumab pegol[END_REF]. SPR evaluated binding kinetics of the His-tagged TNF-α towards Cimzia re-confirmed that Histidine affinity tag does not have any effect on protein-protein interactions.

Conclusion

Although higher expression systems, such as yeast, Chinese hamster ovary (CHO), and Human embryonic kidney 293 (HEK293) cells, have been used in commercial production of TNF-α, the use of E. coli expression system takes less time and energy in the production of large protein amounts in which only one disulfide bond and no glycosylation modification have been reported.

That is how the optimization of parameters involved in the soluble protein expression could significantly increase the yield of correctly folded TNF-α, which has been approved in biological assessments of the present study.

Fig. 1 .

 1 Fig. 1. Schematic design of the expression vector.

Fig. 2 .

 2 Fig. 2. Protein expression (12% SDS-PAGE).Lanes #1: Recombinant E. coli before induction

Fig. 3 .

 3 Fig. 3. Immunoblotting against r-TNF-α: A) anti-histidine antibody; B) Altebrel. Lanes 1, 7:

Fig. 4 .

 4 Fig. 4. Optimization of protein expression. A, B: LB and TB media at 37°C; C, D: LB and TB

Fig. 5 .

 5 Fig. 5. Native Ni-NTA affinity purification. Lanes 1: Initial sample (IS); 2: Flow through (FT);

Fig. 7 .

 7 Fig. 7. Binding sensogram of Cimzia and r-TNF-α

Fig. 8 .

 8 Fig. 8. In vitro neutralization of TNF-α in L929 cell-based assay. Neutralization of serially

Acknowledgments

We acknowledge Dr. Amir Farshchi, CEO, AryoGen Pharmed, Iran, for the kind gift of anti-TNF-α Altebrel. The authors wish to express their deep gratitude to all who provided technical supports.

Conflict of interest

The authors declare no conflict of interest.

Author contributions

Y. T. and F. M. were the chief investigators. M.S. was involved in MTT assays. F. T. assisted in binding studies. E.B., F.B. and A.A. assisted in data collection. Sh. D. was responsible for the major experimental work and contributed in writing the main manuscript. Y. T., L. N. and H. J. contributed revising the main manuscript. All authors approved the final version of the manuscript.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Jean Dupouy-Camet
email: jean.dupouy-camet@orange.fr

Mélanie Gay

René Houin

De nouvelles habitudes alimentaires, de nouveaux risques parasitaires : l'exemple du poisson* New eating habits, new parasitic risks: the example of fish

Étant donné le contexte sanitaire épidémique lié à la Covid-19 en 2020, la présentation de cette communication en séance à l'Académie a été reportée. Déclaration d'intérêt : Les auteurs ne déclarent aucun conflit d'intérêt concernant ce travail.. RESUME Introduction Il y a une vingtaine d'années l'explosion massive de restaurants « japonais » servant du poisson cru a popularisé de nouvelles habitudes culinaires en France. Parallèlement, les consommateurs ont pris pour habitude de préparer eux-mêmes, à domicile, des plats à base de poissons crus ou marinés. En conséquence, la mise en évidence de larves de vers parasites vivants dans la chair de poisson crue est fréquente et une source d'inquiétude pour les professionnels ou les cuisiniers amateurs. Parfois, ces vers sont recrachés ou extirpés après fibroscopie chez des patients ayant présenté de violentes douleurs épigastriques rapidement après consommation de poissons crus. L'objectif de cet article est de faire le point sur les principales parasitoses transmises à l'Homme par la consommation de poisson cru en France.

Introduction

En France, l'explosion massive du nombre de restaurants dit « Japonais » et servant du poisson cru a popularisé il y a une vingtaine d'années de nouvelles habitudes culinaires. La plupart des grandes surfaces proposent des « sushis » et autres plats à base de poisson cru préparés sur place et plusieurs enseignes assurent la livraison à domicile de tel produits.

L'achat de poissons crus préparés par les ménages, a augmenté de 390 % entre 2005 et 2018 [START_REF] Franceagrimer | Consommation des produits de la pêche et de l'aquaculture[END_REF]. Parallèlement, les consommateurs ont pris pour habitude de préparer eux-mêmes, à domicile, des plats à base de poissons crus ou marinés... En conséquence, la mise en évidence de larves de vers parasites vivants dans la chair de poisson crue est fréquente et une source d'inquiétude pour les professionnels ou les cuisiniers amateurs. À l'échelon mondiale la consommation de poisson et d'autres produits de la mer vient au premier rang des consommations de chair animale [START_REF] Lewandowski | Bioeconomy: Shaping the Transition to a Sustainable Biobased Economy[END_REF]. Entre 2012 et 2014, la Food and Agriculture Organization (FAO) et l'Organisation Mondiale pour la Santé (OMS/WHO) ont mis en place un groupe de travail visant à fournir au Codex Committee on Food Hygiene un avis et des recommandations sur les parasites transmis par les aliments, leur impact sur la santé publique et sur les échanges commerciaux [START_REF] Fao | Multicriteria-based ranking for risk management of food-borne parasites[END_REF]. Ce groupe de travail a d'abord sollicité des experts du monde entier pour lister les parasites transmis par l'alimentation et les classer selon leur impact socio-économique et sur la santé humaine. Cette première étape a résulté en un classement multicritères mondial des parasites transmis par les aliments. Ce classement a été décliné en 2018 pour l'Europe, réalisé par la Cost Action Euro-FBP [START_REF] Bouwknegt | The Euro-Fbp Workshop Participants. Prioritisation of food-borne parasites in Europe[END_REF]. Dans les deux classements, sur 24 parasites, huit étaient soit spécifiquement soit partiellement associés à l'eau et aux produits aquatiques : Cryptosporidium spp., Giardia lamblia, les Anisakidae, les Opisthorchidae, Diphyllobothrium sp., Paragonimus spp., les Heterophyidae et Spirometra sp. Pour l'Europe, seuls les Anisakidés font parties des 10 parasites transmis par l'alimentation les plus préoccupants. Les parasitoses transmises par la consommation de poissons crus concernaient principalement jusqu'à la fin du vingtième siècle, les populations du Sud Est Asiatiques confrontées aux graves Clornorchis et Opistorchis. Ces petites douves hépatiques sont considérées comme des cofacteurs carcinogéniques dans la survenue de cancers hépatiques. Quinze millions de sujets sont toujours infectés par ces redoutables parasites et 200 millions sont exposés [START_REF] Tang | Current status and perspectives of Clonorchis sinensis and clonorchiasis: epidemiology, pathogenesis, omics, prevention and control[END_REF]. Il y a une décennie, plusieurs épidémies d'opistorchiase à Opistorchis felineus ont été rapportées en Italie chez des convives de restaurants ayant consommé des préparations à base de tanche crue [START_REF] Pozio | Opisthorchis felineus, an emerging infection in Italy and its implication for the European Union[END_REF]. Manger du poisson cru en France expose à des maladies parasitaires provoquées par des vers ronds (les Anisakidae qui sont les « ascaris » des mammifères marins) ou des vers plats du genre Dibothriocephalus (autrefois appelé Diphyllobothrium, encore avant bothriocéphale ou « taenia » du poisson). La responsabilité des poissons dans la transmission de protozoaires tels que Cryptosporidium n'est pas clairement établie et ne sera pas discutée ici même si des cryptosporidies zoonotiques ont été retrouvées aussi bien chez des poissons d'eau douce que chez des poissons d'eau de mer [START_REF] Certad | Identification of Cryptosporidium Species in fish from Lake Geneva (Lac Léman) in France[END_REF][START_REF] Certad | Prevalence, molecular identification, and risk factors for Cryptosporidium infection in edible marine fish: a survey across sea areas surrounding France[END_REF][START_REF] Gantois | First Report on the prevalence and subtype distribution of Blastocystis sp. in edible marine fish and marine mammals: a large scale-study conducted in Atlantic Northeast and on the coasts of Northern France[END_REF].

Les anisakidoses

Les anisakidoses sont une infestation parasitaire digestive provoquée par l'ingestion de chair crue de poisson de mer contenant des larves du ver nématode parasite Anisakidae. Ces vers parasites, appartenant à plusieurs genres (Anisakis, Pseudoterranova, Contracaecum…) se voient à l'oeil nu. Les infections par des parasites du genre Anisakis donnent des anisakioses (ou anisakiases). Les adultes parasitent le tractus digestif des mammifères marins (cétacés pour Anisakis, pinnipèdes pour Pseudoterranova) ou d'oiseaux aquatiques (Contracaecum).

Les oeufs sont émis dans le milieu marin et libèrent des larves qui sont consommées par les microcrustacés du plancton et qui se logent dans la cavité générale de ce zooplancton source de l'alimentation de nombreuses espèces de poissons carnivores. Une fois ingérées par ces poissons, les larves sont libérées dans l'estomac et migrent ensuite vers les organes de la cavité viscérale (foie, gonades, mésentères) et plus rarement vers les filets. Ce n'est que lors du passage dans le tractus digestif des mammifères marins que ces larves deviennent adultes.

De nombreuses espèces sont cause d'anisakidose

Les Anisakidae sont présents dans les océans et mers du monde entier. Ils ont été décrits dans plus de 200 espèces de poissons [START_REF] Mattiucci | Molecular systematics, phylogeny and ecology of anisakid nematodes of the genus Anisakis Dujardin, 1845: an update[END_REF][START_REF] Mattiucci | Molecular epidemiology of Anisakis and anisakiasis: an ecological and evolutionary road map[END_REF]

Des pathologies digestives et allergiques

Ces parasites peuvent induire des pathologies digestives et/ou allergiques pour l'Homme suite à la consommation de produits de la pêche infestés. Ces parasitoses sont connues en France depuis longtemps chez l'Homme et les poissons mais ont pris un regain d'actualité ces dernières années [START_REF] Angot | Épidémiologie et prophylaxie de l'anisakiase. Enquête sur l'infestation de 3 espèces de consommation courante en France[END_REF][START_REF] Bourée | Anisakidosis: report of 25 cases and review of the literature[END_REF][START_REF] Catsaras | Anisakiase et poisson cru[END_REF][START_REF] Chord-Auger | L'anisakiase dans la région nantaise. De l'étal du poissonnier au cabinet du médecin[END_REF][START_REF] Huang | Anisakidés et anisakidoses humaines -Première partie : Données bibliographiques[END_REF][START_REF] Huang | Anisakidés et anisakidoses humaines -Deuxième partie : Enquête sur les Anisakidés de poissons commerciaux du marché parisien[END_REF][START_REF] Hubert | L'anisakiase en France[END_REF][START_REF] Hubert | Epidemiology of human anisakiasis: incidence and sources in France[END_REF][START_REF] Mudry | Anisakiase humaine, cinq cas dans le Nord de la France[END_REF][START_REF] Petithory | Diagnostic sérologique de l'anisakiase (granulome éosinophile digestif) par précipitation en milieu gélifié (Ouchterlony, électrosynérèse, immunoélectrophorèse)[END_REF][START_REF] Petithory | Etude biologique d'un cas d'anisakiase colique[END_REF][START_REF] Petithory | Données nouvelles sur l'anisakidose[END_REF][START_REF] Petithory | Actualités sur l'anisakidose[END_REF][START_REF] Petter | Enquête sur les Nématodes des sardines pêchées dans la région nantaise. Rapport possible avec les granulomes éosinophiles observés chez l'homme dans la région[END_REF]. Les anisakidoses se présentent cliniquement sous plusieurs formes aiguës ou chroniques résumées dans le tableau I [START_REF] Petithory | Actualités sur l'anisakidose[END_REF][START_REF] Fréalle | L'anisakidose, une helminthose humaine aux manifestations allergiques émergentes[END_REF]. Le plus souvent ces vers Anisakidae sont naturellement recrachés ou extirpés après fibroscopie chez des patients ayant présenté de violentes douleurs épigastriques rapidement après consommation de poissons crus. Des localisations oesophagiennes ont aussi été décrites [START_REF] Dupouy-Camet | L'atteinte oesophagienne : une complication rare de l'anisakidose à Pseudoterranova[END_REF][START_REF] Brieau | Acute dysphagia and odynophagia revealing an unusual case of oesophageal anisakiasis[END_REF]. Les formes allergiques sont également fréquentes avec des manifestations classiques mais aussi des formes intestinales graves oedémateuses provoquant des occlusions. Exceptionnellement des formes pseudotumorales ont été observées : c'est le classique granulome éosinophilique centrée sur une larve [22 ; 29]. Le diagnostic repose sur l'interrogatoire, sur la fibroscopie et la coloscopie, sur l'identification parasitologique et moléculaire des larves, sur l'anatomopathologie et sur les sérologies avec essentiellement le dosage des IgE et IgG4 spécifiques [START_REF] Petithory | Diagnostic sérologique de l'anisakiase (granulome éosinophile digestif) par précipitation en milieu gélifié (Ouchterlony, électrosynérèse, immunoélectrophorèse)[END_REF][START_REF] Fréalle | L'anisakidose, une helminthose humaine aux manifestations allergiques émergentes[END_REF][START_REF] Yera | Molecular confirmation of Anisakis pegreffii as a causative agent of anisakidosis in France[END_REF][START_REF] Brunet | Molecular diagnosis of Pseudoterranova decipiens s.s in human, France[END_REF]. Pour ces tests, des réactions croisées sont connues avec les crustacés et les acariens.

Incubation

Dibothriocéphalose

Alors que le parasite semblait absent de France, des cas de dibothriocéphalose (« ténia du poisson ») étaient à nouveau signalés, au début des années 2000, sur les bords du lac Léman [START_REF] Desvois | Enquête sur l'incidence de la bothriocéphalose en Haute-Savoie (1993-2000)[END_REF] ainsi qu'autour des lacs alpins [START_REF] Dupouy-Camet | Current situation of human diphyllobothriasis in Europe[END_REF][START_REF] Houin | Fasciolose, trichinellose et autres zoonoses parasitaires[END_REF]

  . La plupart des espèces de poissons couramment consommées en France ont été décrites comme hôte pour les Anisakidae. Neuf espèces d'Anisakis ont été décrites, six d'entre elles (A. simplex sensu stricto (s.s.), A. pegreffii, A. berlandi (anciennement A. simplex C), A. typica, A. ziphidarum et A. nascettii) appartiennent au groupe A. simplex sensu lato (s.l.), qui regroupe toutes les espèces dont les larves sont morphologiquement proches d'A. simplex et qui forment le clade I en phylogénie moléculaire. La même situation existe pour le genre Pseudoterranova. La dénomination Pseudoterranova decipiens s.l. est un complexe d'espèces regroupant P.decipiens s.s., P. krabbei, P. bulbosa, P. azarasi, P. cattani. L'Homme est une impasse parasitaire pour les Anisakidae. Les larves de stade L3 présentes dans les poissons et les céphalopodes et ingérées par l'Homme meurent au bout de quelques heures à quelques jours.

2. 4 .

 4 Une diminution de l'incidence des cas humains, une prédominance féminine et un potentiel allergique émergent En complément du programme Fish-Parasites, une enquête rétrospective sur l'anisakidose humaine a été réalisée sur les années 2010 à 2014 et a consisté en un recueil des cas auprès de tous les laboratoires hospitalo-universitaires de parasitologie-mycologie de France et a été complétée par une analyse des données du réseau national d'allergovigilance. Trente-sept cas d'anisakidose ont pu être répertoriés par les laboratoires de parasitologie : 6 cas certains avec mise en évidence du ver dans un prélèvement digestif, 13 cas possibles définis par des douleurs abdominales après consommation de poisson cru et une recherche positive de précipitines anti-Anisakis, et 18 cas d'anisakidose allergique définis par des manifestations allergiques aiguës après consommation de poisson et associées à la présence d'IgE anti-Anisakis. Dans cette enquête, une prédominance féminine significative (67 %) a été observée. Six cas supplémentaires d'allergie sévère aux Anisakidés ont été rapportés au réseau national d'allergovigilance sur cette même période [40, 41]. Par rapport à des études antérieures [18, 19], cette enquête a objectivé une diminution des cas d'anisakidose, mais a montré que le potentiel allergisant des Anisakidés était en émergence. En France également, Van Cauterem et al [42] estimait le nombre de cas annuels d'anisakidose entre 4 et 15 sur la période 2008-2013. Ce potentiel allergisant des Anisakidés avait déjà été rapporté par Petithory dès 2007 devant l'Académie nationale de médecine [23]. La faible incidence des cas humains d'anisakidose parasitologique contraste donc avec la forte prévalence du parasite chez les poissons. Cela est certainement dû à l'exigence réglementaire de congeler le poisson destiné à être consommé cru en restauration collective, à une augmentation de l'approvisionnement par la filière aquaculture et par l'élimination des parasites par les professionnels lors du parage des filets.

4 .

 4 Conclusions et perspectivesDepuis quelques années, suite à la modification des habitudes alimentaires et à l'augmentation de la consommation de produits crus ou insuffisamment transformés, les autorités sanitaires ont exprimé une préoccupation grandissante vis-à-vis du risque lié aux parasites dans les produits de la pêche. Ces inquiétudes se sont traduites par des saisines auprès de l'Anses (ou AFSSA) et de l'EFSA[START_REF] Efsa | Scientific opinion on risk assessment of parasites in fishery products[END_REF][START_REF] Afssa | Demande d'évaluation du risque relatif à la présence d'Anisakidés dans les produits de la pêche et extension de la dérogation à l'obligation de congélation assainissante pour les produits de la pêche dont l'alimentation est maîtrisée ainsi que pour certaines espèces de poissons sauvages saisine[END_REF]. Ces avis scientifiques faisaient état d'un manque important de connaissances sur les parasites de poissons. Du point de vue de la biologie de ces parasites, ces avis indiquaient la nécessité d'acquérir des données sur le cycle biologique complet, la distribution géographique et saisonnière, la prévalence, l'intensité et la localisation anatomique précise des parasites d'importance en santé publique dans les poissons sauvages capturés. D'un point de vue plus appliqué, l'EFSA recommandait d'acquérir de meilleures connaissances sur les moyens de maîtrise de ces parasites : évaluation de l'efficacité de traitements physiques et/ou chimiques et procédés de transformation pour détruire ces parasites ; impact de différentes pratiques d'aquaculture sur la présence de ces parasites dans les poissons issus de l'élevage. Enfin, l'EFSA mentionnait la nécessité de disposer d'informations claires et pratiques pour le corps médical, les professionnels des produits de la pêche et le grand public. Beaucoup de ces objectifs ont été atteints au cours de l'étude Fish-Parasites et un pôle de compétence en parasitologie des parasites de poisson existe maintenant à l'ANSES de Boulogne sur Mer. En France, les professionnels de la filière des produits de la pêche ont longtemps occulté les risques liés aux parasites. Leur attitude a fortement évolué au cours des dernières années avec une réelle prise de conscience de ce risque et la volonté de mettre en place des outils et des démarches pour mieux le maîtriser. Par ailleurs, pour donner suite aux résultats du plan de surveillance de 2017 et à la constatation d'un manque de maîtrise du risque par les acteurs de la filière, la DGAL a mis à jour l'instruction technique sur la maîtrise du risque parasitaire dans les produits de la mer et d'eau douce[START_REF] Dgal | Ministère de l'Agriculture, Instruction technique DGAL[END_REF].

  mer étaient parasités par des Anisakidae. Des prévalences élevées ont été observées chez la lingue bleue, la cardine franche, le lieu noir, la lotte et le merlu. Aucun parasite n'a été retrouvé chez le saumon d'élevage ni le bar d'élevage[START_REF] Seesao | Monitoring of four DNA extraction methods upstream of high-throughput sequencing of Anisakidae nematodes[END_REF][START_REF] Seesao | Caractérisation des Anisakidae dans les poissons marins : développement d'une méthode d'identification par séquençage à haut-débit et étude de prévalence[END_REF]. Lors de ce

	programme de recherches, plus de 54 000 larves d'Anisakidae ont été identifiées par des
	outils moléculaires. Ces données ont permis de décrire de nouveaux hôtes et de nouvelles
	Principaux symptômes aires de répartition géographiques pour certaines espèces d'Anisakidae. Entre 2013 et 2016, Cause Fréquence Complications
	De 1 à 12 heures un programme européen, PARASITE, a permis entre autres, de mettre en place la plus Anisakidose gastrique douleur pseudo-ulcéreuses Anisakidose oesophage +++ -larves +/-importante étude épidémiologique sur les parasites de poissons, avec plus de 17 000 poissons
	brûlures rétro-sternales, analysés appartenant à 16 espèces de poissons téléostéens. Cette étude a permis de quantifier pyrosis De 12 heures à Anisakidose intestinale les niveaux d'infestation ainsi que la distribution spatiale des Anisakidae dans les espèces de
	plusieurs jours De quelques heures à 24 heures Plusieurs mois Granulome éosinophile syndromes appendiculaires et péritonéaux, formes coliques ou iléales basses Anisakidose gastro-allergique douleurs abdominales Anisakidose allergique urticaire, allergies respiratoires intestinal symptomatologie pseudo tumorale Tableau I : Les différentes formes d'anisakidoses chez l'Homme. larves +/-larves + allergènes + allergènes +++ larves +/-poissons cibles. Une méta-analyse récente de 123 publications parues de 1967 à 2017 examinant la présence Occlusion intestinale par oedème localisé OEdème de Quincke, choc anaphylactique Occlusion intestinale
	de ce ver parasite chez 56 778 poissons appartenant à 215 espèces différentes a montré que la
	2.3. Des prévalences très importantes chez les poissons sauvages quantité d'Anisakis par poisson avait été multipliée par 283 au cours de ces 50 dernières
	De 2011 à 2014, l'action Fish-Parasites, financée par l'ANR (Agence Nationale de la années [39].
	Recherche) a évalué la prévalence du parasitisme chez le poisson. Le programme a comporté
	un échantillonnage de poissons de mer (campagnes sur des navires océanographiques de

À noter que les allergènes d'Anisakis sont très divers

[START_REF] Aibinu | Anisakis nematodes in fish and shellfishfrom infection to allergies[END_REF]

, thermostables et gardent leur caractère allergisant même après cuisson ou congélation

[START_REF] Ventura | Immediate and cell-mediated reactions in parasitic infections by Anisakis simplex[END_REF]

.

Les

traitements sont chirurgicaux (prélèvement de la larve ou d'un granulome) et médicamenteux associant ou non antiparasitaires (albendazole 400 mg matin et soir 7 jours sans que l'efficacité de ce traitement soit prouvée) et antiallergiques (antihistaminiques et corticoïdes). l'Ifremer, achats à des professionnels de Boulogne-sur-Mer) et d'eau douce (achats auprès de pêcheurs professionnels du lac Léman et pêches scientifiques de l'INRA pour les lacs d'Annecy et du Bourget). C'est ainsi que 1768 poissons de mer appartenant à 18 espèces et poissons de L'influence de facteurs tels que la taille des poissons et la zone de pêche sur les niveaux d'infestation a été évaluée et des données de génétique des populations parasitaires ont été acquises [36]. En 2017, un plan de surveillance, défini par la Direction Générale de l'Alimentation (DGA) et réalisé par les Directions Départementales de Protection des Populations avait pour objectif d'estimer les niveaux d'infestation par les Anisakidae, des poissons mis sur le marché, quelle que soit leur présentation au consommateur final. Sept espèces d'importance commerciale ont été prélevées : la cardine franche, le lieu noir, la lingue bleue, la lingue franche, la lotte, le merlan et le merlu. Les niveaux d'infestation observés selon des méthodes de détection non destructives (utilisées par les professionnels) et par une méthode de détection destructive et exhaustive ont été comparés. Globalement, une observation à l'oeil nu a permis d'identifier 42,93 % des échantillons comme étant parasités et la méthode exhaustive a permis de détecter 62,93 % des échantillons comme étant parasités.

Les prévalences d'infestation étaient significativement différentes entre les espèces de poisson. Les flancs des poissons étaient significativement plus infestés que les filets

[START_REF] Gay | Niveaux d'infestation par les Anisakidae chez sept espèces de poisson prélevées au stade de la distribution en France[END_REF][START_REF] Gay | Infection levels and species diversity of ascaridoid nematodes in Atlantic cod, Gadus morhua, are correlated with geographic area and fish size[END_REF]

.

  . Les Dibothriocephalidae sont des cestodes dont le stade larvaire (plérocercoïde) est présent dans les filets de certains poissons d'eau douce (perche) ou marins (saumon du Pacifique). Plusieurs espèces sont connues : Dibothriocephalus latus européen et transmis par les poissons d'eau douce, D. dendriticus dans les zones arctiques, D. nihonkaiensis transmis par les saumons du Pacifique et Les pathologies (diphyllobothriose) sont généralement peu graves, avec principalement des troubles digestifs. Entre 1987 et 2015, plus de 200 cas humains ont été recensés autour des lacs alpins (France, Italie, Suisse) majoritairement suite à la consommation de filets de perche crus [49]. L'incidence du nombre de cas humains est donc faible. Un des volets du programme Fish-Parasites a comporté un échantillonnage de poissons d'eau douce (achats auprès de pêcheurs professionnels du lac Léman et pêches scientifiques de l'INRA pour les lacs d'Annecy et du Bourget). 138 poissons d'eau douce appartenant à 11 espèces ont été examinés. Des larves de Dibothriocephalus latus ont été retrouvées dans les brochets, perches et lottes du lac Léman mais chez aucun des poissons examinés des lacs d'Annecy ou du Bourget [49, 50]. Mais, entre 2016 et 2018, sept cas d'infestation par Dibothriocephalus nihonkaiense, une espèce inféodée au saumon sauvage du Pacifique et absente d'Europe, ont été diagnostiqués en l'espace de deux ans au CHU de Rennes [51, 52]. Un cas similaire d'infestation par Dibothriocephalus nihonkaiense a aussi été signalé à Strasbourg [53]. Des cas similaires avaient été signalés en France dès 2006 et 2009 [54, 55] après consommation de saumon sauvage frais importé du Canada.

	Adenocephalus pacificus transmis par les poissons de mer du Pacifique [46]. Ces parasites
	sont indiscernables morphologiquement et seule la biologie moléculaire permet de les
	différencier [47, 48]. L'Homme est un hôte définitif tout comme d'autres mammifères
	piscivores [49].


Antoine Piau
email: piau.a@chu-toulouse.fr.

PhD Claude Vautier

PhD Adelaïde De Mauleon

Achille Tchalla

PhD Pierre Rumeau

Fati Nourhashemi

Maria Soto-Martin

PhD Maria Soto

Health workers perception on telemedicine in management of neuropsychiatric symptoms in long-term care facilities: Two years follow-up

Keywords: assisted living facilities, behavioral symptoms, dementia, telemedicine

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

Introduction

Neuropsychiatric symptoms (NPS) of dementia affect 80% of patients during the disease course. [START_REF] Seitz | Prevalence of psychiatric disorders among older adults in long-term care homes: a systematic review[END_REF][START_REF] Selbaek | The prevalence and course of neuropsychiatric symptoms in nursing home patients with dementia: a systematic review[END_REF] Hallucinations, agitation and other disturbing NPS are associated with a poorer disease prognosis, inappropriate treatments, an increase in professional caregiver burden and health care costs. [START_REF] Schmidt | Distress experienced by nurses in response to the challenging behaviour of residents -evidence from German nursing homes[END_REF][START_REF] Zuidema | Psychotropic drug prescription in nursing home patients with dementia: influence of environmental correlates and staff distress on physicians' prescription behavior[END_REF][START_REF] Pitfield | A systematic review of stress in staff caring for people with dementia living in 24-hour care settings[END_REF] Management of NPS is a challenge for the staff of long-term care facilities (LTCF) [START_REF] Normann | Attitudes of registered nurses towards patients with severe dementia[END_REF][START_REF] De Rooij | Professional caregivers' mental health problems and burnout in small-scale and traditional long term care settings for elderly 19 people with dementia in the Netherlands and Belgium[END_REF] where the prevalence of dementia can be as high as 70%. [START_REF] Rolland | Descriptive study of nursing home residents from the REHPA network[END_REF][START_REF] Matthews | UK Medical Research Council Cognitive Function and Ageing Study. Prevalence of dementia in institutional care[END_REF][START_REF] Helmer | Dementia in subjects aged 75 years or over within the PAQUID cohort: prevalence and burden by severity[END_REF] NPS also frequently result in inappropriate use of emergency facility networks [START_REF] Soto | Improving care of older adults with dementia: description of 6299 hospitalizations over 11 years in a special acute care unit[END_REF] despite the negative impact on patients' functional autonomy and quality of life. [START_REF] Wang | Emergency department visits by nursing home residents in the United States[END_REF][START_REF] Becker | Predictors of emergency commitment for nursing home residents: the role of resident and facility characteristics[END_REF][START_REF] Hurd | Monetary costs of dementia in the United States[END_REF] The older population living in LTCFs is particularly exposed to the risk of decompensation in the event of stressful situations such as transportation to emergencies. While the situation is not new, the recent viral pandemic is a dramatic reminder of this fact. Social distancing measures also pose significant challenges to the health and wellbeing of older people with cognitive impairment. Reduced cognitive stimulation that accompanies socialization can aggravate cognitive symptoms and NPS. [START_REF] Steinman | Meeting the Care Needs of Older Adults Isolated at Home During the COVID-19 Pandemic[END_REF] Older patients are paying the heaviest price in terms of COVID-19 mortality rates. [START_REF] Wu | Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72 314 Cases From the Chinese Center for Disease Control and Prevention[END_REF] However, they also have high rates of morbidity and mortality from other acute and chronic conditions. Thus, containment measures have potentially deprived them of necessary specialist care. This has been a key factor in the rapid development of telemedicine tools for teleconsultation or tele-expertise to provide minimal access to care for these populations.

Non-pharmacological therapeutic approaches of NPS show the best evidence-based results and are the first line of treatment considering that environmental causes are among the most common NPS triggers. [START_REF] Gitlin | Nonpharmacologic management of behavioral symptoms in dementia[END_REF][START_REF] Livingston | A systematic review of the clinical effectiveness and cost-effectiveness of sensory, psychological and behavioural interventions for managing agitation in older adults with dementia[END_REF] These approaches are based on a comprehensive multidisciplinary assessment and a subsequently tailored person-centered care plan. This explains why evaluation outside a real life context (e.g. hospital setting) is biased and partial. Furthermore, this evaluation requires a change of environment that might aggravate the patient's behavior. Telemedicine (TM) could be a useful support to connect LTCF staff to specialized units and thus provide a comprehensive and ecological evaluation without aggravating NPS.

Several studies have evaluated TM for older patients with encouraging results [START_REF] Van Den Berg | Telemedicine and telecare for older patients--a systematic review[END_REF] and data suggests its validity for dementia diagnosis. [START_REF] Ramos-Ríos | Telepsychogeriatrics: a new horizon in the care of mental health problems in the elderly[END_REF][START_REF] Martin-Khan | The diagnostic accuracy of telegeriatrics for the diagnosis of dementia via video conferencing[END_REF][START_REF] Harrell | Telemedicine and the evaluation of cognitive impairment: The additive value of neuropsychological assessment[END_REF] However, health professionals are facing a low level of diffusion of TM in real clinical care settings, including long-term care facilities. [START_REF] Greenhalgh | SCALS: A fourth-generation study of assisted living technologies in their organisational, social, political and policy context[END_REF] Decades after the first TM initiatives, we should be focusing on how to overcome this relative failure. Besides technical, economical and regulatory issues, all innovations have a social component. Sociology reminds us that individuals confronted with an innovation often begin by counteracting it. [START_REF] Crozier | L'acteur et le système. Les contraintes de l'action collective[END_REF] Such reactions do not only reflect fits of temper but sometimes reveal plausible fears (e.g. quality of care for the patients). [START_REF] Proulx | La sociologie des usages, et après ?[END_REF] This multicenter study evaluates health workers perception on telemedicine in management of neuropsychiatric symptoms in long-term care facilities.

Materials and Methods

Study design and procedure

This study is a multicenter, open-label trial. We evaluated LTCF staff perceptions of TM for NPS management before and after its implementation. Ten LTCF were recruited for a 24month follow-up period. The study received ethical approval from the local institutional review board (September 09, 2014). Written informed consent was required from all participants. The trial was registered in ClinicalTrials.gov, June 1, 2015. Full details of the research and assessment procedure have previously been described. [START_REF] Piau | Telemedicine for the management of neuropsychiatric symptoms in long-term care facilities: the DETECT study, methods of a cluster randomised controlled trial to assess feasibility[END_REF] When a patient presented a disruptive NPS (defined as NPS that can be stressful and/or difficult to manage, based on family or professional assessment and which requires a specialist consultation or an unscheduled hospitalization according to LTCF staff) a TM consultation was scheduled within 72 hours. The LTCF physicians were acting as medical investigators and requested the TM consultation from memory expert centers. The study involved two expert memory centers (University Hospitals) in two French regions (named 'Region 1' and 'Region 2'). The TM session was led by a geriatrician trained in NPS management along with specialized NPS nurses. The LTCF participants were LTCF physicians, nurses, psychologists, the patient's GP when possible and the patient if required. A tailored personal plan with a therapeutic strategy was established after the TM session. A second follow-up TM consultation took place after 1 month.

Study outcomes and analysis

We used a qualitative research design using semi-structured interviews. Our primary outcome was a qualitative assessment of TM perceptions after implementation among the LTCF staff. Our hypothesis was that perception of TM by LTCF staff would be improved after a 2 year implementation.

A senior social science researcher (University Sociology Laboratory) conducted face to face group interviews in both sites ('Region 1' and 'Region 2'): before and after experiencing TM. The first staff meeting took place before the inclusion of patients, and the second staff meeting took place after TM implementation, two years later. Each interview lasted half a day and was designed to explore key questions relating to TM perception by the LTCFs' staff. . The interviews were conducted as following :

-For the first interviews (before the implementation of TM), non-directive exchanges were conducted in order to identify recurring themes. These themes were then used as a guide to finalize the interviews in a semi-directive mode (reformulation and theme development).

-For the second session (Two years later), semi-structured interviews were conducted based on the same themes previously identified, and an additional questionnaire was distributed individually (a combination of open and closed questions addressing these themes, see supplementary file 1).

All interviews were transcribed. The transcripts were analyzed using a conventional content analysis along with a summative qualitative content analysis. [START_REF] Hsieh | Three Approaches to Qualitative Content Analysis[END_REF] The Strengths, Weaknesses, Opportunities and Threats (SWOT) method [START_REF] Dyson | Strategic development and SWOT analysis at the University of Warwick[END_REF] was applied to illustrate future determinants of larger implementation. For the description of LTCF and the analysis of the questionnaires, variables were described in percentages, means and standard deviations. Statistical analyses were performed using STATA software.

Results

Descriptive analysis of the LTCF

A total of 10 LTCF were included for 24 months and were equipped with TM for NPS management in addition to the standard health care services available in their area (for full details of the procedure see Piau et al. [START_REF] Piau | Telemedicine for the management of neuropsychiatric symptoms in long-term care facilities: the DETECT study, methods of a cluster randomised controlled trial to assess feasibility[END_REF] ) The LTCF had a mean number of 85 beds (min 60, max 133). The distribution of the number of beds, "specialized" professions in the LTCF (in addition to nurses, auxiliary nurses and night shift workers), full-time equivalent jobs for each profession, and whether or not non-pharmacological approaches were used in the LTCF are presented in Table 1.

Perception of TM among the LTCF staff (primary outcome)

During the first interviews (before the implementation of TM), we identified recurring keywords/codes from the first non-directive exchanges. Then, we identified several key themes to conduct the rest of the interview in a semi-structured mode (example codes are given in brackets):

-Organizational issues (LTCF organization, healthcare system organization); -Financial impact (the financing of the LTCF, healthcare system financing); -LCTF staff issues (staff involvement, staff cohesion, valuation of staff work, valuation of staff expertise, staff relationship with families and GPs, knowledge transfer, continuing medical education); -Quality of care (NPS diagnosis quality, administered treatments appropriateness, transfers and hospitalizations relevance, specialized healthcare access).

During the 24-month follow-up period, 90 patients were included and each one benefited from two TM sessions; a total of 180 were conducted. Perception among LTCF staff was evaluated beforehand and two years after TM implementation.

There was a clear change in staff members' perceptions between the first meeting (before TM implantation) and the second one (two years later, after TM implantation). At the first meeting, 49% of the LTCF staff expressed positive perceptions concerning TM in 'Region 1' and 40% in 'Region 2'. Two years later these figures rose to 78% and 76% respectively. On the other hand, negative perceptions classified as weaknesses expressed by participants increased twofold (15 to 22% in "Region 1" and 8 to 15% in "Region 2"), while those identified as potential threats clearly decreased (52 to 9% in "Region 2") or even disappeared (36 to 0% in "Region 1").. No one expressed a clearly negative or even hostile perception during either meeting. The labelling of the statements by social science researchers did not raise any problems since they were all clearly positioned. Table 2 summarizes the main issues raised during the two meetings. Table 3 present the aggregated results of the questionnaire analysis (2 nd meeting) for both regions 1 and 2. ‡ Note. Empirical scoring and interpretation was done by the sociology team after summarization of the open and closed questions. A "positive" interpretation means a positive perception of the potential influence of TM, e.g. for the item "TM could cause GPs to lose interest in the care of LCTF patients", the global perception that emerges is that "no", TM would not cause GPs to lose interest.

We observed that the impact of TM on health care organization was positively perceived by participants. They saw TM as an opportunity to tackle the lack of specialized health care in remote areas. However, despite this generally positive perception, there were also concerns that an unequal healthcare system could develop. With respect to the participation of general practitioners (GPs), the discussion highlighted the difficulties in motivating several of them. Some refused to implement the proposed pharmacological interventions, which was very frustrating for the staff. When staff members met with the GPs, the results were more satisfactory. There were no major difficulties in the TM process implementation in 'Region 1'.

The situation was different in 'Region 2' where two concerns were highlighted the lack of time and the difficulties of several staff members to cope with change. LTCF staff issues are addressed in table 3. Concerning staff cohesion, the valuation of staff work, and experience sharing, the perceptions of TM impact are almost all very positive, there were few negative or mixed comments.

Concerning patients' family involvement and relations, in the 'Region 1' there were no difficulty obtaining family consent (100%). In comparison, the 'Region 2' staff had some difficulties with family adherence in more than one out of three TM sessions (36%). When relatives participated in the TM sessions (11 occasions) this involvement was considered as positive three out of 4 times in 'Region 1' (75%) and 6 out of 7 in 'Region 2' (86%). With regard to the feared risk of a possible dehumanized medicine offering a lower quality of care, table 3 clearly shows that this concern has disappeared after two years. It is also clear that the first meeting identified negative perceptions (Table 2) that no longer exist two years later. LTCF staff considers that the estimated number hospitalizations avoided is 13 for 'Region 1' and 20 for 'Region 2'. The two emergency TM sessions held during the study were similarly considered to have had a positive impact for the patient.

A final summary of staff perceptions after TM implementation (second meeting) using a SWOT template is presented in table 4. We observed that staff members felt fully acknowledged for their specific skills, and TM was perceived as positive for patient care.

Discussion

Main results

In this study, we focused on assessing perceptions of telemedicine in the real world, which is a key issue for future deployment. This is not an observational study to assess the willingness of end-users to adopt TM, but rather an interventional prospective study to assess the perception of TM before and after its actual use. This is a global and relevant primary criterion that meets the overall objectives of this type of study because large implementation of health care technologies largely depends on end-users' perception.

The health care professional positive perceptions at the very beginning are confirmed at the end of the study: the theoretical advantages prior to implementation became a reality according to end-users after 2 years of field experience. On the other hand, the negative aspects of TM perceived by staff members during the first meeting were much more varied and numerous compared to the second meeting two years later. We could say that some issues which seemed to be very important before the TM experience were less significant retrospectively.

After two years, some negative aspects whose potential consequences could be overestimated a priori became mere inconveniences to be taken into account but not disqualifying. It could be said that potential threats became weaknesses. Real-life experience with TM use concretized some doubts into certainties and there was no more questioning. Participants initially raised questions about the possibility of LTCF disorganization, dehumanized medicine, and a focus on cost minimization rather than quality of care. These fears did not disappear but were rationalized.

They are now possibilities that require attention but are not inevitable. None of the concerns about a possible decrease in the quality of care were reiterated. Not only do staff members think that TM is not a downgraded version of medicine but they also believe it could improve the quality of care and make patients the focus of care again.

The auxiliary nurses expressed a very positive feelings of recognition of their professional qualifications and that they had not been listened to enough in the past (although they are very close to the patients and can provide useful information and solutions). The results of our study reveal positive LTCF staff perceptions about the potential influence of TM on staff work, staff consideration, and the quality of care. They also indicate the necessity to involve staff members in the change processes and to address local organizational issues to ensure successful implementation and better adherence to telemedicine as a service.

Our results in the context of previous research

Despite the fact that TM could be an emerging method of providing care for LTCF residents presenting NPS, to our knowledge, our study is the first trial specifically designed to explore this specific topic. Among the numerous studies that assessed remote cognitive assessment of patients living in LTCF, [START_REF] Harrell | Telemedicine and the evaluation of cognitive impairment: The additive value of neuropsychological assessment[END_REF][START_REF] Greenhalgh | SCALS: A fourth-generation study of assisted living technologies in their organisational, social, political and policy context[END_REF][START_REF] Crozier | L'acteur et le système. Les contraintes de l'action collective[END_REF][START_REF] Grob | Psychiatric assessment of a nursing home population using audiovisual telecommunication[END_REF][START_REF] Lee | A telemedicine system as a care modality for dementia patients in Korea[END_REF][START_REF] Lyketsos | Telemedicine use and the reduction of psychiatric admissions from a long-term care facility[END_REF][START_REF] Johnston | Telepsychiatry consultations to a rural nursing facility: a 2year experience[END_REF] few addressed TM implementation for NPS management. None of them used a social evaluation approach. Several preliminary noncomparative pilot studies described TM solutions that linked a single LTCF [START_REF] Lee | A telemedicine system as a care modality for dementia patients in Korea[END_REF][START_REF] Lyketsos | Telemedicine use and the reduction of psychiatric admissions from a long-term care facility[END_REF] or nursing care unit of a rural hospital [START_REF] Johnston | Telepsychiatry consultations to a rural nursing facility: a 2year experience[END_REF] to a specialized center with encouraging results. In a more recent paper Georgeton et al. [START_REF] Georgeton | General practitioners adherence to recommendations from geriatric assessments made during teleconsultations for the elderly living in nursing homes[END_REF]

Limitations of the study

We included LTCF staff in the early stages of project specification, including the definition of the tele-expertise process. Their participation from the very beginning of the project has probably influenced our results, which was a desired bias of our approach. Measuring the perception of a procedure has the disadvantage of confronting people with a preconceived project without any opportunity to modify it. To avoid this, we choose a method more in line with participatory design approaches that emphasize the involvement of users in the innovation process. [START_REF] Merkel | Participatory Design in Gerontechnology: A Systematic Literature Review[END_REF] Our overall positive results tend to validate such an approach. We should accept the fact that all new organization could be scrutinized and criticized by end-users. Collective consultation prior to the project enables involvement of those with ideas to enrich the project and at the same time those who, without this critical examination, could became unconditional objectors.

The questionnaire used during the second session was developed for this study based on the themes identified during the first interviews session and we are not in a position to provide elements of reliability and validity. Moreover, a single researcher carried out the semi-structured interviews (scoring and interpretation of the staff feedback relied on its subjective interpretation).

This therefore limits the generalization of our results.

This protocol main limitation was the duration of follow-up per patient (2 months), and the total duration of the study (2 years). It could be insufficient to assess organizational aspects and perceptions over a longer period once the innovation becomes routine. We have also deliberately set aside technical issues (robustness of service and service failures) in this paper (that are fully explored elsewhere).

Perspectives

Although older adults living in LTCF are highly susceptible to death from COVID-19, their non-COVID-19 care should not be forgotten. In this very particular context, it is necessary to bear in mind both the risk of presenting an atypical form of COVID infection (such as delirium or NPS) and at the same time not to neglect the other pathologies in this population. [START_REF] Steinman | Meeting the Care Needs of Older Adults Isolated at Home During the COVID-19 Pandemic[END_REF] Similarly, while telehealth is a very promising and relevant tool in this population, it complements rather than fully substitutes for other care options. We must be careful not to offer the same care in all situations, which are very heterogeneous, and not to delay hospitalization if it is necessary. The COVID pandemic has been a major accelerator in the large-scale deployment of telemedicine. The challenge now is to make it an additional option for better quality of care rather than a backup solution.

Conclusion

Our results reveal positive LTCF staff perceptions regarding TM influence on the health care system, staff work and staff consideration, and the quality of care. These results have been confirmed and even reinforced after the actual use of telemedicine. We also highlighted staff members' willingness to be involved in change. The results of our study could be useful both to extend existing TM networks in other regions and to design a national multicenter randomized trial to assess the efficacy of TM for NPS management.

Table 1 .

 1 Description of the LTCF

	Ergotherapist	6 (60)
	Psychologist	10 (100)
	Psycho-motor therapist	4 (40)
	Number of LTCF that benefited from non-pharmacological therapy (%)
	Any non-pharmacological therapy (yes)	8 (80)
	Balneotherapy (yes)	5 (50)
	Luminotherapy (yes)	0 (0)
	Aromatherapy (yes)	0 (0)
	Music therapy (yes)	5 (50)
	Snoezelen (yes)	1 (10)
	Pet therapy (yes)	6 (60)
	Full-time equivalent jobs, mean (SD)
	Nurse	4.0 (2.4)
	Auxiliary nurse	3.9 (1.8)
	Night shift workers	3.6 (2.4)
	Ergotherapist	0.5 (0.7)
	Psychologist	0.2 (0.4)
	Psycho-motor therapist	0.3 (0.5)
	† Abbreviations: SD, Standard deviation; LTCF, long term care facilities.

Characteristics of the 10 LTCF that were included Admission capacity, Mean (SD) Number of beds 84.9 (26.8) Number of beds in the special dementia unit 10.2 (11.4) Number of LTCF that benefited from "special" professionals (%)

Table 2 .

 2 Summary of Staff perceptions concerning TM Several regions of France are impacted by a decrease in the number of doctors, particularly GPs. In some regions this can have an impact on the quality of care, it is now referred to as the medical desertification.

	First meeting (2015)		Second meeting (2017)
	'Region 1' LTCF	'Region 2' LTCF	'Region 1' LTCF	'Region 2' LTCF
	Positive perceptions
	Tackles the lack of health care	Tackles the lack of
	in remote areas	specialized care in remote
		areas
	Lowest health care costs
	Stronger team spirit and	Improved expression by all	Involvement of all	Better team mobilization
	interdisciplinary collaboration	staff participants and	participants, families	and interdisciplinary
		interdisciplinary teams	and auxiliary nurses	collaboration
			Increased knowledge of	Positive effect of
			the LTCF staff	exchange of views
			Better valuation of	LTCF staff work
			LTCF staff work	valuation
		Better evaluation of patients		Better evaluation in
		in their own environment		patients' own
				environment
	Easiest and faster access to	Improved access to health		Easiest access to
	specialized health care	care		specialized health care
	Minimization of patient	Minimization of transfers		Fewer transfers and less
	transfers and hospitalizations	and stress for patients		stress for patients
		Emergency solutions for		Positive impact on NPS
		crisis situations
				Promotion of non-
				pharmacological
				treatments
	Negative perceptions
	TM is a "spare wheel" that does
	not solve the lack of specialized
	care in remote areas
	Concerns about a possible loss	Concerns about possible	Concerns about CPs
	of interest by GPs	opposition from GPs	opposition
	Possible disorganization of the	Possible disorganization of	Lack of time and
	current health care network	LTCF functioning	workforce for TM
		TM is not a financial priority
	Concerns about the adoption of	Concerns about TM adoption		Difficulty changing, a
	TM by LTCF staff	by LTCF staff		sense of intrusion
	Concerns about a possible loss
	of interest by LTCF staff and
	systematic referral to TM
	TM introduces two-tiered	TM introduces two-tiered
	dehumanized medicine	medicine
		Concerns about the ethics of
		remote health care and health
		data security
	† Abbreviations: LTCF, long term care facilities; TM, telemedicine
	‡ Note.

Table 3 .

 3 Results of the questionnaire analysis

	Issues addressed by open and closed questions	LTCF staff response rating (%)
		Positive Split	Negative
	Organizational issues, TM does…
	… tackle the lack of healthcare in remote areas	55.0	20.0	25.0
	… provide real solutions for the lack of healthcare in remote areas 45.4	9.1	45.5
	… not introduce two-tiered medicine	66.7	9.5	23.8
	… lower costs for the health care system	63.7	9.0	27.3
	… lower costs for the LTCF	55.5	11.2	33.3
	… benefit the organization of health care	75.0	12.4	12.6
	… have a beneficial impact on LTCF organization	63.2	15.8	21.0
	… not cause GPs to lose interest in the care of LCTF patients	63.6	18.2	18.2
	LTCF staff issues, TM does…
	… contribute to interdisciplinary collaboration	77.8	11.0	11.2
	… contribute to team cohesion	90.9	0.0	9.1
	… contribute to knowledge transfer and continuing training	84.2	5.3	10.5
	… promote valuation of LTCF staff	81.8	18.2	0.0
	… promote valuation of auxiliary nurse expertise	72.6	18.3	9.1
	… enable greater involvement of staff in NPS management	62.6	10.3	27.1
	… provide external support for burdensome situations	80.8	19.2	0.0
	Patient-related issues, TM does…
	… improve the quality of NPS diagnosis	77.6	11.5	10.9
	… improve the speed of NPS diagnosis	58.3	25.0	16.7
	… promote the re-evaluation of psychotropic drugs	83.3	16.7	0.0
	… promote faster re-evaluation of psychotropic drugs	72.8	18.0	9.2
	… improve access to specialized care	66.7	25.1	8.2
	… reduce patient transfers	75.0	13.1	11.9
	… reduce hospitalizations	76.9	15.4	7.7
	… reduce patient stress	54.4	9.3	36.3
	… make the patient the focus of care again	82.1	0.0	17.9
	† Abbreviations: LTCF, long term care facilities; TM, telemedicine.

Table 4 .

 4 Summary of staff perception of TM after implementation (2 nd meeting)

	Strengths	Weaknesses
	Organizational aspects • Easiest access to specialized health care	• Difficulties involving GPs
	LTCF staff issues • Greater involvement of Staff in NPS management	• Difficulty coping with change • Feeling of intrusion
	Increased knowledge transfer • LTCF staff work valuation
	Family issues • Greater involvement of families	• Difficulties obtaining family consent in several
		cases
	Patient issues • Better evaluation of patients in their own	• None
	environment
	• Positive impact on NPS • Promotion of non-pharmacological treatments
	Opportunities	Threats
	Organizational aspects • Tackling the lack of specialized care in remote	• Economic issues at the LTCF level
	areas
	• Lowering costs for the health care system
	LTCF staff issues • Improved continuing training • Better team cohesion and interdisciplinary • Full recognition of staff members' specific skills	• Lack of time and workforce for TM development
	collaboration
	Family issues • More trusting relationship with staff	• None
	Patient issues • Fewer transfer and hospitalizations and less	• Introduction of a two-tiered medicine
	stress

  reported TM activity in three nursing homes. The main aim of the study was not TM evaluation for NPS but most of the teleconsultations concerned neuropsychological issues (83%). Catic et al. evaluated a TM solution specifically dedicated to NPS management of long-term care residents with dementia, and the results were encouraging.[START_REF] Catic | ECHO-AGE: an innovative model of geriatric care for long-term care residents with dementia and behavioral issues[END_REF] This one-year follow-up non-comparative pilot study proposed bimonthly TM sessions to 11 long-term care sites to discuss cases with a specialized team. However, this study differed from our protocol because the intervention focused more on staff training than on the deployment of a TM care service. In fact, staff training is a valuable potential collateral benefit of our procedure (table2) that helps LTCF staff to acquire up-to-date skills for NPS management. Staff training interventions have also been shown to reduce work-related stress and staff turnover and to decrease antipsychotic prescription for patients with dementia living in LTCF.[START_REF] Spector | A systematic review of staff training interventions to reduce the behavioural and psychological symptoms of dementia[END_REF][START_REF] Burgio | Teaching and maintaining behavior management skills in the nursing home[END_REF][START_REF] Elliott | Building capacity and resilience in the dementia care workforce: a systematic review of interventions targeting worker and organizational outcomes[END_REF] Moreover, the best evidence-based NPS treatment is demonstrated by non-pharmacological approaches that provide education and training for caregivers.[START_REF] Livingston | A systematic review of the clinical effectiveness and cost-effectiveness of sensory, psychological and behavioural interventions for managing agitation in older adults with dementia[END_REF]

Acknowledgments

None.

Acknowledgments

None.

Funding Statement

This research is conducted with the support of an interregional French grant (French Ministry of Public Health, PHRC N° 13 7031 08).

Funding statement

This research is conducted with the support of an interregional PHRC French grant (French Ministry of Public Health, PHRC N° 13 7031 08).

Conflict of interest

None.

Conflict of interest

None

Ethical approval information

The study received ethical approval from the local institutional review board (September 09, 2014). Written informed consent was required from all participants (or legal representatives).

The trial was registered in ClinicalTrials.gov, June 1, 2015.