Patrick D Mathews
email: patrickmathews83@gmail.com

Omar Mertins

Luis L Espinoza

Tiago Milanin

Gema Alama-Bermejo

Fabienne Audebert

André C Morandini

Taxonomy and 18S rDNA-based phylogeny of Henneguya multiradiatus n. sp. (Cnidaria: Myxobolidae) a parasite of Brochis multiradiatus from Peruvian Amazon

Keywords: Cnidaria, Endocnidozoa, Hognosed catfish, Myxosporean, 18S rDNA sequencing, Peru

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

Introduction

Myxozoans are microscopic endoparasites with complex life cycles involving invertebrate and vertebrate hosts [START_REF] Atkinson | Myxozoans: Ancient metazoan parasites find a home in phylum Cnidaria[END_REF]. Recently, due to synapomorphies and cnidarianspecific genes, myxozoans were placed within the phylum Cnidaria and currently represent about 20% of the known cnidarian species diversity around the world [START_REF] Atkinson | Myxozoans: Ancient metazoan parasites find a home in phylum Cnidaria[END_REF].

While predominantly known to infect freshwater and marine fishes, myxozoans can also infect other groups of vertebrates such as amphibians, reptiles, birds, and terrestrial small mammals [START_REF] Lisnerová | Mechanisms and drivers for the establishment of life cycle complexity in myxozoan parasites[END_REF]. Within fish-infecting myxozoans, Henneguya Thélohan, 1892, is one of the largest genera, with more than 195 described species within a wide geographical range [START_REF] Eiras | Checklist of new species of Henneguya Thélohan, 1892 (Myxozoa: Myxosporea, Myxobolidae) described between 2002 and 2012[END_REF]. Henneguya spp. are histozoic parasites and they show high hostspecificity and organ-or tissue tropism, with some species inducing severe henneguyosis in wild and cultured fish [START_REF] Pote | Small subunit ribosomal RNA sequences link the cause of proliferative gill disease in channel catfish to Henneguya n. sp. (Myxozoa: myxosporea)[END_REF][START_REF] Gupta | 18S and 28S rDNA identity and phylogeny of two novel myxosporeans infecting gills of cyprinid carps inhabiting a cold-water wetland in northern India[END_REF][START_REF] Figueredo | Henneguyosis in gills of Metynnis hypsauchen: an Amazon freshwater fish[END_REF][START_REF] Katharios | Native parasite affecting an introduced host in aquaculture: cardiac henneguyosis in the red seabream Pagrus major Temminck & Schlegel (Perciformes: Sparidae) caused by Henneguya aegea n. sp. (Myxosporea: Myxobolidae)[END_REF].

It is well known that the growing global pet trade, including aquarium market, plays a role in the spread of pathogens [START_REF] Mohanty | The global pet trade in amphibians: species traits, taxonomic bias, and future directions[END_REF]. According to Hallett et al. [START_REF] Hallett | Myxozoans on the move: dispersal modes, exotic species and emerging diseases[END_REF], the aquatic pet trade would very likely stand as a primary mode for international transport of myxozoan parasites. In fact, there are some reports about the introduction of myxosporeans through commercial transfers of pets with some cases of ecological and economic impacts [START_REF] Camus | Molecular characterization and histopathology of Myxobolus koi infecting the gills of a koi, Cyprinus carpio, with an amended morphological description of the agent[END_REF][START_REF] Sitjà-Bobadilla | Can myxosporean parasites compromise fish and amphibian reproduction?[END_REF][START_REF] Hartigan | Myxosporean parasites in Australian frogs: Importance, implications and future directions[END_REF]. As such, there is a growing need for myxozoans screening during parasitological surveys in these aquatic pet animals.

The armored catfish corydoradines are members of the family Callichthyidae. With over 190 described species, it is one of the most diverse siluriform assemblages in the Neotropics [START_REF] Britto | Phylogeny of the subfamily Corydoradinae Hoedeman, 1952 (Siluriformes: Callichthyidae), with a definition of its genera[END_REF]. These neotropical freshwater catfishes are endemic to South America [START_REF] Britto | Phylogeny of the subfamily Corydoradinae Hoedeman, 1952 (Siluriformes: Callichthyidae), with a definition of its genera[END_REF]. Among these corydoradines, Brochis multiradiatus Orcés V., 1960 is found in the Napo River in Ecuador and Peru [START_REF] Froese | FishBase[END_REF]. It can reach up to 6.7 cm in length and is largely marketed in the aquarium trade, being one of the most popular in the Chinese ornamental fish market [START_REF] Xu | Complete mitochondrial genome of Brochis multiradiatus[END_REF]. Although corydoradines are highly appreciated in the international aquarium trade, there are few parasitological surveys about this fish, especially those concerning myxozoan parasites.

To our knowledge, nothing is known about myxozoan parasites infecting any member of the genus Brochis. Herein, based on morphological, biological/ecological traits and molecular data, we described a new Henneguya species infecting the ornamental fish B. multiradiatus from the Amazon region of Peru, a key supply region of wild ornamental fishes for the international aquarium trade. family and current status of the host fish (valid species name or synonym) were reviewed using FishBase [START_REF] Froese | FishBase[END_REF].

Material and methods

In

Morphometric analyses were performed on mature myxospores following the criteria outlined by Lom and Arthur [START_REF] Lom | A guideline for the preparation of species descriptions in Myxosporea[END_REF]. Measurements and photographs were taken from 30 myxospores using a computer equipped with Axiovision 4.1 image capture software coupled to an Axioplan 2 Zeiss microscope (Carl Zeiss AG, Oberkochen, Germany).

The dimensions of the myxospores were given in micrometers (μm) and expressed as the mean ± standard deviation, followed by the range in parentheses. Prevalence of infection [(number of infected fish/total number of fishes examined) ×100] was calculated according to Bush et al. [START_REF] Bush | Parasitology meets ecology on these terms: Margolis et al. revisited[END_REF]. Smears containing free myxospores were airdried, fixed with methanol and stained with Ziehl-Neelsen to mount on permanent slides that were deposited in the cnidarian collection of the zoology Museum of the University of São Paulo -USP, São Paulo, Brazil (MZUSP).

For molecular studies, a plasmodium was dissected from the host tissue and fixed in absolute ethanol. The genomic DNA was extracted from a single plasmodium using DNeasy® Blood & Tissue Kit (Qiagen, Valencia, USA), in accordance with the manufacturer's instructions. The DNA concentration was measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, USA). Polymerase chain reactions (PCRs) were performed in accordance with Milanin et al. [START_REF] Milanin | Molecular phylogeny of the gill parasite Henneguya (Myxosporea: Myxobolidae) infecting Astyanax lacustris (Teleostei: Characidae) from fish farm in Brazil[END_REF] and conducted in a final volume reaction of 25 μL, which comprised 10-50 ng of extracted DNA, 0.2 pmol for each primer, 12.5 μL of Dream Taq Green PCR Master Mix (Thermo Scientific) and nuclease-free water. Partial 18S rDNA sequence was amplified using the primer pairs ERIB1 [START_REF] Barta | Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences[END_REF] -ACT1r [START_REF] Hallett | Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia[END_REF] and Myxgen4F [START_REF] Diamant | A new species of Sphaeromyxa (Myxosporea: Sphaeromyxina: Sphaeromyxidae) in devil firefish, Pterois miles (Scorpaenidae), from the northern Red Sea: morphology, ultrastructure, and phylogeny[END_REF] -ERIB10 [START_REF] Barta | Phylogenetic relationships among eight Eimeria species infecting domestic fowl inferred using complete small subunit ribosomal DNA sequences[END_REF] (Table 1), which amplified two fragments containing approximately 1.000 and 1.200 bp respectively. PCR amplification was done by initial denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 1 min, 64 °C (ERIB1-ACT1r) or 58 °C (Myxgen4F-ERIB10) for 1 min, 72 °C for 2 min, and then final elongation at 72 °C for 5 min. PCRs were performed in an AG22331 Hamburg Thermocycler (Eppendorf, Hamburg, Germany). PCR products were subjected to electrophoresis in 2.0% agarose gel (BioAmerica, Irvine, USA) in a TAE buffer (Tris-Acetate EDTA: Tris 40 mM, acetic acid 20 mM, EDTA 1 mM), stained with Sybr Safe DNA gel stain (Invitrogen by Life Technologies, Carlsbad, USA), and then analyzed with a Stratagene 2020E trans illuminator (Stratagene California, San Diego, USA). The size of the amplicons was estimated by comparison with the 1 Kb Plus DNA Ladder (Invitrogen by Life Technologies). PCR products were purified using USB® ExoSap-IT® (Thermo Fisher Scientific) in accordance to the manufacturer's instructions and sequenced using the same PCR primers plus two additionally MC5 and MC3 primers used to overlap the obtained amplification fragments [START_REF] Molnár | Morphological and molecular biological studies on intramuscular Myxobolus spp. of cyprinid fish[END_REF]. Sequencing was performed at Paulista Medical School, Federal University of São Paulo, using a BigDye® Terminator v3.1 Cycle Sequencing kit (Applied Biosystems Inc., California, USA) in an ABI 3130 automatic DNA analyzer (Applied Biosystems Inc.™). The obtained sequence was visualized, assembled and edited using BioEdit 7.1.3.0 software [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. A standard nucleotide BLAST (blastn) search was conducted to verify the similarity of the sequence obtained in this study with other sequences available in GenBank [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. Phylogenetic analysis was conducted using the most closely related myxozoans sequences with similarity > 80%.

The sequences were aligned with ClustalW within BioEdit version 7.1.3.0 [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF].

Phylogenetic analysis was performed using Maximum Likelihood (ML) method with a Kimura 2-parameter (K2P) evolution sequence model in MEGA 6.0 [START_REF] Tamura | MEGA 6: molecular evolutionary genetics analysis version 6.0[END_REF]. Bootstrap analysis (1000 replicates) was employed to assess the relative robustness of the branches in ML tree. Ceratonova shasta sequence was used as outgroup. The pairwise method with the p-distance model in MEGA 6.0 [START_REF] Tamura | MEGA 6: molecular evolutionary genetics analysis version 6.0[END_REF] was performed to evaluate the genetic distance between the myxozoan species clustering together with the new sequence obtained.

Results

Whitish plasmodia macroscopically visible measuring 2.1 to 3 mm in diameter and containing large quantities of myxospores belonging to the genus Henneguya were found in the serous membrane of the visceral cavity of wild specimens of B. multiradiatus (Fig. 1A). The prevalence of the myxozoan parasites in these fish hosts was 13.3% (4/30) and no signs of diseases were observed in the infected individuals.

Morphological characterization

Mature myxospores were ellipsoidal in shape from the frontal view, measuring 44.5 ± 0.6 μm (43.9-45.1) in total length, 18.7 ± 0.9 μm (16.8-19.6) in spore body length, 7.1 ± 0.2 μm (6.6-7.4) in width and 5.5 ± 0.3 μm (4.9-5.6, n=5) in thickness (Figs. 1B and2). Two elongated polar capsules, equal in size, measuring 9.1 ± 0.1 μm (8.8-9.4) in length and 1.7 ± 0.1 μm (1.6-1.8) in width, occupying half of myxospore body (Figs.

1B-C and 2). Polar tubules had 10 to 11 coils, perpendicular to long axis of polar capsule (Figs. 1C and2). Non-bifurcate caudal appendage, measuring 25.8 ± 0.6 μm (24.7-26.5) in length (Figs. 1B and2).

Molecular characterization and phylogenetic analysis

Discussion

The Peruvian Amazon is the second largest portion of the Amazon rainforest concentrating a high aquatic biodiversity, with several commercially exploited fish species for human consumption and global aquarium trade [START_REF] Albert | Fishes of the Fitzcarrald[END_REF][START_REF] Rivadeneyra | Histopathology associated with infection by Procamallanus (Spirocamallanus) inopinatus (Nematoda) in farmed Brycon cephalus (Characiformes) from Peru: a potential fish health problem[END_REF]. However, little is known about the diversity of myxozoans in this region, with only three species described to date: Henneguya loretoensis, Henneguya peruviensis and Myxidium peruviensis [START_REF] Espinoza | A new Myxidium species (Myxozoa: myxosporea) infecting the gallbladder of the turtle Podocnemis unifilis (Testudines: podocnemididae) from Peruvian Amazon[END_REF][START_REF] Mathews | Morphology and small subunit rDNAbased phylogeny of a new Henneguya species, infecting the ornamental fish Corydoras leucomelas from the Peruvian Amazon[END_REF][START_REF] Mathews | Morphology and 18S rDNA sequencing of Henneguya peruviensis n. sp. (Cnidaria: Myxosporea), a parasite of the Amazonian ornamental fish Hyphessobrycon loretoensis from Peru: A myxosporean dispersal approach[END_REF]. In contrast, over 40 myxozoan species have been described in the Brazilian Amazon region [START_REF] Adriano | Myxosporea in catálogo taxonômico da fauna do Brasil[END_REF][START_REF] Zatti | Novel Henneguya spp. (Cnidaria: Myxozoa) from cichlid fish in the Amazon basin cluster by geographic origin[END_REF][START_REF] Mathews | A new Myxobolus (Cnidaria: Myxosporea) infecting the ornamental catfish Corydoras schwartzi from the Purus River in Brazil[END_REF], indicating that there are likely many more myxozoan parasites to be identified in Peruvian Amazon, considering the recognized high diversity. This study reports, for the first time, a myxozoan species infecting a fish from the genus Brochis in the Amazon basin.

For robust identification and description of new myxosporeans taxa, it has been recommended, that the studies integrate multiple characters, including morphology, biological traits, host ecology factors and molecular data [START_REF] Atkinson | Approaches for characterising myxozoan species[END_REF]. Following these delineated criteria for classifying myxozoans, we described a novel Henneguya species, Henneguya multiradiatus n. sp. from an important Amazonian ornamental fish. In the morphological comparison, considering the twenty-two Henneguya species previously described to infect fishes from Amazon basin, the most morphologically similar species to the new species were Henneguya torpedo Azevedo, Casal, Matos, Alves and Matos, 2011 and Henneguya testicularis Azevedo, Corral and Matos, 1997. Nevertheless, these differ from H. multiradiatus n. sp. in myxospore body length (28.5 ± 0.3 μm in length for H. torpedo, 14.0 μm for H. testicularis and 18.7 ± 0.9 μm to the new species), number of coils of the polar tubule (12 to 13 in H. torpedo, six to seven in H. testicularis and ten in the new species) and in the length of the caudal appendage, which is substantially larger in the new species (19.6 ± 0.4 μm in H. torpedo, 13.5 μm in H. testicularis, and 25.8 ± 0.6 μm in H. multiradiatus n. sp.). Plasmodia of H. multiradiatus n. sp. were located in the serous membrane of the visceral cavity, while H. torpedo and H. testicularis were described infecting brain, spinal cord, and testis respectively [START_REF] Azevedo | Light and ultrastructural data on Henneguya testicularis n. sp. (Myxozoa, Myxobolidae), a parasite from the testis of the Amazonian fish Moenkhausia oligolepis[END_REF][START_REF] Azevedo | Henneguya torpedo sp. nov. (Myxozoa), a parasite from the nervous system of the Amazonian teleost Brachyhypopomus pinnicaudatus (Hypopomidae)[END_REF]. On the same premise, host species and locality of collection are indispensable traits for accurately distinguishing new histozoic platysporines species, since host-specificity and geography may play an important role in speciation [START_REF] Wang | The impacts of geographic and host species isolation on population divergence of Myxobolus lentisuturalis[END_REF]. Accordingly, differences were observed in the infected host, e.g., H. multiradiatus n. sp. infecting B. multiradiatus, a Siluriform fish; H. torpedo infecting Brachyhypopomus pinnicaudatus (Hopkins 1991), a Gymnotiform fish; and H. testicularis infecting Moenkhausia oligolepis (Günther 1864), a Characiform fish. Besides, differences in the locality of these species were noted, with H. multiradiatus n. sp. found in the Napo River, a tributary of the upper Amazon River, near the village of Cabo Pantoja in Department of Loreto, Peru, and H. torpedo and H. testicularis found in the lower Amazon River, both near Belém city in Pará state, Brazil. In addition to the large geographic distance between these species (2, 965 km in straight line), the nonmigratory behavior of their hosts is an important ecological character for the separation of these species, taking in account that host behavior may drive both parasite endemism and the radiation within the Amazon basin [START_REF] Zatti | Novel Henneguya spp. (Cnidaria: Myxozoa) from cichlid fish in the Amazon basin cluster by geographic origin[END_REF]. Unfortunately, there is no molecular data available for H. torpedo and H. testicularis in GenBank, so it is not possible to compare them with H. multiradiatus n. sp. However, differences observed in the integrative comparison, including morphology, geography, biological traits in conjunction with the ecological characters of their hosts, are sufficient arguments to conclude that H. multiradiatus n. sp. is a separate species.

In our phylogenetic analysis, Henneguya spp. showed tendency to cluster, largely based on vertebrate host fish family, and this corroborates the results pointed out by other authors who evidenced that host group is a strong evolutionary signal within the Myxobolidae [START_REF] Zatti | Novel Henneguya spp. (Cnidaria: Myxozoa) from cichlid fish in the Amazon basin cluster by geographic origin[END_REF][START_REF] Carriero | Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species[END_REF][START_REF] Liu | Phylogeny of Myxobolidae (Myxozoa) and the evolution of myxospore appendages in the Myxobolus clade[END_REF]. Henneguya multiradiatus n. sp. was placed in a wellsupported subclade of parasites of callichthyids fishes, with H. loretoensis as the closet related species (Fig. 3). This close phylogenetic relationship was corroborated by the pairwise analysis, which showed a slight genetic divergence of 1.8% on their 18S rDNA sequences. Nevertheless, there is no exact value for determining the level of genetic variation in 18S rDNA that equates to species differentiation within this enigmatic group of parasites [START_REF] Carriero | Molecular phylogeny of the Myxobolus and Henneguya genera with several new South American species[END_REF][START_REF] Gunter | Seven new species of Ceratomyxa Thélohan, 1892 (Myxozoa) from the gall-bladders of serranid fishes from the Great Barrier Reef, Australia[END_REF]. In this context, species differentiation should be assessed for each individual case and always with the aid of biology and/or ecology traits of the organisms, such as their morphology, tissue and/or organ tropism, host species, geography, and host ecological aspects such as endemic character, migratory behavior and distribution [START_REF] Lom | A guideline for the preparation of species descriptions in Myxosporea[END_REF][START_REF] Gunter | Seven new species of Ceratomyxa Thélohan, 1892 (Myxozoa) from the gall-bladders of serranid fishes from the Great Barrier Reef, Australia[END_REF]. In our study, remarkable morphometrical differences can be observed between these two corydoradines Henneguya parasites, with myxospores substantially larger in total length and width in the newly identified species (44.5 ± 0.6 μm × 7.1 ± 0.2 μm vs 36.2 μm × 5.1 μm for H. loretoensis), larger body length (18.7 ± 0.9 μm vs 14.3 ± 0.1 μm for H. loretoensis), longer polar capsules (9.1 ± 0.1 μm vs 5.1 ± 0.1 μm in H. loretoensis), higher number of coils in the polar tubule (ten to eleven coils vs five coils in H. loretoensis) and the caudal appendage which is substantially longer (25.8 ± 0.6 μm vs 21.9 ± 0.1 in H. loretoensis). Furthermore, H. multiradiatus n. sp. the caudal appendage is not bifurcated, as occurs in H. loretoensis. Differences can also be outlined concerning the host-, tissue-and organ infected, with the new species proposed found in the serosa layer of the visceral cavity of B. multiradiatus n. sp. whereas H. loretoensis is found in gill filaments of Corydoras leucomelas Eigenmann and Allen, 1942, a host of a different genus. While these two corydoradines parasites inhabit the Amazon biome of Peru (H. multiradiatus n. sp. in Napo River and H. loretoensis Nanay River), they are separated by 381 km from each other. Another important point to consider in establishing H. multiradiatus n. sp. as a new species, is in regards to the endemic character and the absence of migratory reproduction, characteristics which are highly recognized to corydoradines fish [START_REF] Mathews | A new Myxobolus (Cnidaria: Myxosporea) infecting the ornamental catfish Corydoras schwartzi from the Purus River in Brazil[END_REF].

In the present study, an integrative taxonomy approach was employed in the description of an unknown myxosporean. Given that these criteria are compelling evidence for the characterization of new species [START_REF] Lom | A guideline for the preparation of species descriptions in Myxosporea[END_REF][START_REF] Gunter | Seven new species of Ceratomyxa Thélohan, 1892 (Myxozoa) from the gall-bladders of serranid fishes from the Great Barrier Reef, Australia[END_REF], we confidently considered that this isolate is a new species, H. multiradiatus n. sp. Hence, this new data contributes to increase the knowledge of the myxosporean diversity from the Amazon biome, as well to clarify the relationships of myxozoan parasites of corydoradines, an economic important assemblage of catfishes in the lucrative global aquarium industry.

Legends

  September 2019, thirty wild specimens of B. multiradiatus were collected by a local fisherman. The fishes were caught in the Napo River, in an adjacent area of the village Cabo Pantoja (0°57′25″S, 75°27′11″W), Department of Loreto, Peru. The fish were transported alive to the field laboratory where they were euthanized by neural pithing and examined. The procedures were approved by the Federal University of São Paulo-UNIFESP Ethics Committee (CEUA No. 9209080214), in accordance with Brazilian law for scientific use of animals (Federal Law No. 11794, dated 8 October 2008). The

  Henneguya multiradiatus n. sp. Taxonomic summary Phylum: Cnidaria Verrill, 1865 Class: Myxosporea Bütschli, 1881 Order: Bivalvulida Shulman, 1959 Family: Myxobolidae Thélohan, 1892 Genus: Henneguya Thélohan, 1892 Type host: Brochis multiradiatus (Orcés V., 1960) (Siluriformes: Callichthyidae) Site of infection: Serous membrane of the visceral cavity Type locality: Napo River, adjacent area of the village Cabo Pantoja (0°57′25″S, 75°27′11″W), Department of Loreto, Peru. Prevalence: 13.3 % (4/30). Type of material: Syntypes-air-dried slide stained with Ziehl-Neelsen deposited in the cnidarian collection of Zoology Museum of the University of São Paulo -USP, São Paulo, Brazil (MZUSP 8508). Partial 18S rDNA sequence (1400bp) was deposited in GenBank under accession number MT415832. Etymology: The specific name (H. multiradiatus) is based on host species name.

Fig. 1 .

 1 Fig. 1. Plasmodium and myxospores of Henneguya multiradiatus n. sp. from Brochis multiradiatus. A-Whitish plasmodium macroscopically visible (black arrow) in the serosa of the visceral cavity (S). Scale bar= 3 mm. B-Mature myxospore stained with

Fig. 2 .

 2 Fig. 2. Schematic representation of myxospore of Henneguya multiradiatus n. sp. from Brochis multiradiatus. Scale bar= 7 µm.

Fig. 3 .

 3 Fig. 3. Maximum likelihood phylogenetic tree based on partial 18S rDNA sequences containing Henneguya multiradiatus n. sp. and closely related myxozoans based on BLAST. GenBank accession numbers and host family are given in front of species. Bootstrap values above 50 are indicated at the nodes.

  Partial 18S rDNA sequence obtained from mature myxospores of H. multiradiatus n. sp. resulted in 1400 bp with a CG content of 49.4% and did not match any other myxozoans available in GenBank. The comparison of the new sequence obtained by BLAST revealed the highest sequence similarity to Henneguya loretoensis Mathews, Naldoni and Adriano 2017 (GenBank accession number MF434827.1, query coverage 99%, maximum identities 98.2%). The phylogenetic analysis placed H. multiradiatus n. sp. in a well-supported subclade of Henneguya parasites of callichthyids fish, including Henneguya loretoensis, and Henneguya guanduensis Abdallah, Azevedo, Luque and Bomfim 2007 (Fig. 3). This subclade, was close to a lineage formed by Henneguya species described from Amazonian cichlids. 18S rDNA pairwise comparisons between H. multiradiatus n. sp. described and the closest relatives resulted in 1.8% to H. loretoensis, 5.3% to H. guanduensis, 6.1% to Henneguya peruviensis Mathews, Mertins, Pereira, Maia and Adriano 2018, 7.6% to Hennguya jariensis, 9.5% to Henneguya tapajoensis, 7.2% to Henneguya tucunarei Zatti, Atkinson, Maia, Bartholomew and Adriano 2018 and 8.6% to Henneguya paraensis Velasco, Videira, Nascimento, Matos, Gonçalves and Matos 2016.

Table 1 .

 1 Primers used in the amplification and sequencing of the 18S rDNA gene of Henneguya multiradiatus n. sp.

Table 2 .

 2 Comparative data of Henneguya multiradiatus n. sp. with other Henneguya species parasites of Amazon fish. Spore dimensions, infection sites, and fish host are given. TL: total length; BL: body length; APCL: caudal appendage length; SW: spore width; ST: spore thickness; PCL: polar capsule length; PCW: polar capsule width; NCT: number of coils of polar tubules; -: no data. All measurements are range in μm and/or means ± SD.

Acknowledgments

The authors thank the São Paulo Research Foundation, FAPESP, for Post-Doc fellowship awarded to P.D. Mathews (grant no. 2018/20482-3 and 2019/13147-6). A.C. Morandini was funded by CNPq (309440/2019-0) and FAPESP (2015/21007-9). T. Milanin was supported by a Post-Doc fellowship from FAPESP (grant no. 2015/18807-3). L.L. Espinoza thanks Vice-rectorado de Investigación y Postgrado, Universidad Nacional Mayor de San Marcos for research financial support (grant no. A17080131).

The authors thank Dr K. Liittschwager (USA) for revision of the English idiom.

Primers

Sequences (5′-3′) References

ACT1r AATTTCACCTCTCGCTGCCA Hallett and Diamant [START_REF] Hallett | Ultrastructure and small-subunit ribosomal DNA sequence of Henneguya lesteri n. sp. (Myxosporea), a parasite of sand whiting Sillago analis (Sillaginidae) from the coast of Queensland, Australia[END_REF] ERIB1 ACCTGGTTGATCCTGCCAG Barta et Henneguya peruviensis [START_REF] Mathews | Morphology and 18S rDNA sequencing of Henneguya peruviensis n. sp. (Cnidaria: Myxosporea), a parasite of the Amazonian ornamental fish Hyphessobrycon loretoensis from Peru: A myxosporean dispersal approach[END_REF] Henneguya loretoensis [START_REF] Mathews | Morphology and small subunit rDNAbased phylogeny of a new Henneguya species, infecting the ornamental fish Corydoras leucomelas from the Peruvian Amazon[END_REF] Henneguya tucunarei Crenlcichla lepldota [START_REF] Rocha | Henneguya amazonica n. sp. (Myxozoa, Myxobolidae), parasitizing the gills of Crenicichla lepidota Heckel, 1840 (Teleostei, Cichlidae) from Amazon river[END_REF] (55-65.9) (11.5-14.9) (41.7-52.1) (5.2-6.3) (2.7-3.6) (1.1-1.9)


Augustin Lerebours
email: augustin.lerebours@utc.fr

Clémence Demangel
email: c.demangel@critt-mdts.com

Lucas Dembinski

Salima Bouvier
email: salima.bouvier@utc.fr

Alain Rassineux
email: alain.rassineux@utc.fr

Christophe Egles
email: christophe.egles@utc.fr.

Effect of the residual porosity of CoCrMo bearing parts produced by additive manufacturing on wear of polyethylene

Keywords: Additive manufacturing, Selective laser melting, Polyethylene wear, Cobalt-chromiummolybdenum alloy, Porosity, Total joint replacement

Additive manufacturing (AM) has proven to be a flexible technique to create complex designs layer-by-layer. Among others, this versatility has disabled the production of advanced total joint replacements. The high degree of customization offered by AM provides countless opportunities to empower the fabrication accuracy of complex biomaterial constructs to tailor patient-specific implants according to their morphology and pathology.

In addition, AM offers interesting microarchitecture control of surfaces, which influences biotribological performances. Porosity is an inherent defect of AM-based metallic material and has been shown to affect mechanical performances such as tensile stress. Thus, in the context of bearing surfaces of artificial joints, this article aims to understand the physical behavior of AM-created cavities on the wear performance of polyethylene in a synovial-like environment with a multidirectional pin-on-disc machine at 37°C.

The article provides quantitative ultra-high molecular weight polyethylene wear results against AM-based cobalt-based alloy parts with a gradient of cavities. The results demonstrate a strong correlation between mass loss of UHMWPE and the total surface porosity defects in the metallic counterparts. Reducing the surface porosity of AM parts is therefore beneficial for improving the wear resistance of UHMWPE. This work not only underlines the negative effect of metallic surface cavities in the wear resistance of UHMWPE but also gives some understanding of the physical behavior of metallic cavities produced by AM. The AM-created cavities modify adhesion wear and abrasion wear mechanisms. Interestingly the pores also limit the abrasive effect of a third body particles by acting as debris collector.

Introduction

Additive manufacturing (AM) allows the creation of parts with complex internal and external geometry by adding material layer by layer directly from computer-assisted-design (CAD) according to ASTM standard F2792-10 and ISO 17296. AM enabled customized manufacturing of implants matching patient's anatomy which may be further adapted to the stage attained by the pathology [START_REF] Lerebours | A voxelbased method for designing a numerical biomechanical model patient-specific with an anatomical functional approach adapted to additive manufacturing[END_REF][START_REF] Lethaus | Additive manufacturing for microvascular reconstruction of the mandible in 20 patients[END_REF]. Compared to traditionally manufactured implants with standard dimensions, AM creates unique and accurate implants for various orthopedic and traumatological purposes such as knee arthroplasty, spinal deformities and dental replacement [START_REF] Deshmukh | Rapid prototyping assisted fabrication of the customised temporomandibular joint implant: A case report[END_REF]. Accurately fitting total joint replacement implants along with specific surgical guides help the surgeon complete the surgical implantation precisely and successfully within a shorter operative time [START_REF] Javaid | Additive manufacturing applications in orthopaedics: A review[END_REF]. In fact, demand for patient-specific bone and joint implants has become inherent in order to palliate osteoporosis, arthritis and traumatic musculoskeletal injuries [START_REF] Hutchinson | The Burden of Musculoskeletal Diseases in the United States: Prevalance, Societal and Economic Cost, 1st Edition[END_REF].

Among AM technologies, production of metal parts through Selective Laser Melting (SLM) has been possible thanks to high-power laser which progressively and locally fuses metal powder in a controlled atmosphere (Ar, N2, H2).

The manufacturing process is complex as it possesses many parameters which all play a role in producing fully dense parts with minimum internal stress. These parameters include (i) the laser parameters (power, scanning speed, beam diameter, etc), (ii) the system parameters (thickness of the powder layer, the treated powder surface, hatch space, the scanning strategies, etc), (iii) the environmental parameters (preheating temperature, the atmosphere and gas pressures) and (iv) the powders characteristics (morphology, size, distribution) [START_REF] Joguet | Porosity content control of CoCrMo and titanium parts by Taguchi method applied to selective laser melting process parameter[END_REF]. Metal additive manufacturing offers many advantages: (i) freedom of design in relation to subtractive processes, (ii) lighter structures possible either by lattice structures or by the design of parts where the material is only where it is needed without further constraints, (iii) new functions such as complex internal channels, (iv) net shape process leading to less raw material consumption and reducing the number of assembly operations and tools needed, (v) short production cycle time for complex parts.

SLM creates unique material characteristics which thereby might modify the mechanical, biological and physico-chemical properties. The process consists of melting and solidifying a small surface of metallic powders with a laser beam of high energy. The thermal history due to the rapid melting and solidification of the material leads to fine microstructural properties. Most of the materials made by SLM are found to be anisotropic. An anisotropy in the Z direction is due to the superposition of layers while an anisotropy in X-Y directions is related to the scanning strategy (scan vector lengths and scan vector rotation) [START_REF] Frazier | Metal Additive Manufacturing: A Review[END_REF][START_REF] Ali | Effect of scanning strategies on residual stress and mechanical properties of Selective Laser Melted Ti6Al4V[END_REF]. Typical defects can be observed including partially melted particles, lack of fusion, pores, cracks and inclusions [START_REF] Frazier | Metal Additive Manufacturing: A Review[END_REF]. Such defects can be linked to poor process parameters optimization, construction strategy, part orientation or inadequate powder quality.

Pores in the parts created by SLM can significantly affect their mechanical behavior and therefore their performance [START_REF] Dadbakhsh | Effect of selective laser melting layout on the quality of stainless steel parts[END_REF][START_REF] Wilson-Heid | Characterization of the Effects of Internal Pores on Tensile Properties of Additively Manufactured Austenitic Stainless Steel 316L[END_REF]. Thus, in many applications, the objective is to obtain full dense parts without any porosity. Parameters optimization may be a challenge for some materials that poorly accommodate high internal stress during the fabrication process [START_REF] Gokuldoss | Additive manufacturing processes: Selective laser melting, electron beam melting and binder jetting-Selection guidelines[END_REF].

Finely tuned manufacturing parameters coupled with specific post-treatments (e.g. hot isostatic pressing) allows a reduction of the porosity but not its complete disappearance. The presence of open pores to the surface are particularly difficult to remove [START_REF] Tammas-Williams | The Effectiveness of Hot Isostatic Pressing for Closing Porosity in Titanium Parts Manufactured by Selective Electron Beam Melting[END_REF][START_REF]Energie-und kosteneffizienter Extremleichtbau mit Hybridwerkstoffen View project Twin-roll casting of aluminium-steel clad strips View project[END_REF]. Thus, in the context of bearing surfaces of artificial joints, this article aims to understand the physical behavior of AM-based cavities on the wear performance of polyethylene in a synovial-like environment.

Metal additive manufacturing especially SLM has enabled the production of cobaltchromium-molybdenum (CoCrMo) parts with complex geometries [START_REF] Hedberg | In vitro biocompatibility of CoCrMo dental alloys fabricated by selective laser melting[END_REF][START_REF] Lerebours | Additive manufacturing process creates local surface roughness modifications leading to variation in cell adhesion on multifaceted TiAl6V4 samples[END_REF]. CoCrMo alloy is a biocompatible material which has been widely used for orthopedic implant [START_REF] Milošev | CoCrMo Alloy for Biomedical Applications[END_REF][START_REF] Disegi | Cobalt-Base Alloys for Biomedical Applications[END_REF] along with ultra high molecular weight polyethylene (UHMWPE). This material combination is common for articular implants such as knee and hip replacement implants [START_REF] Mantripragada | An overview of recent advances in designing orthopedic and craniofacial implants[END_REF] and therefore was selected for this work. The cobalt based alloy was produced by selective laser melting and UHMWPE was molded (ASTM F72, ISO 5834-2).

Our study focuses on the polyethylene mass loss under metallic surfaces that have a gradient of porosity defect under lubricated conditions in order to control the performance of the parts for further artificial joint implant applications. Joint fluid was simulated by diluted bovine serum.

This work is particularly important regarding the development of complex geometric AMbased implant.

Firstly, we tackled the production feasibility of selective laser melted CoCrMo parts with a surface texture in accordance with the specifications of ISO 21534:2007 standard. Secondly, in order to meet application requirements, we used a superfinishing process which can also be applied to complex geometric parts. This is the first report to study the biotribological physical behavior of the cavities produced by metal-AM on the UHMWPE wear.

2 Material and methods

Specimens

Cylindrical discs of a 10mm radius and 5mm thickness were manufactured by SLM in CoCrMo. The control material was a cast-CoCrMo. UHMWPE pins were machined from an extruded UHMWPE bar GUR 1020 (chirulen®, Quadrant TM ) [START_REF]Implants for surgery -Ultra-high-molecular-weight polyethylene -Part 2: Moulded forms[END_REF]. The polyethylene bar was thermally stabilized under inert atmosphere and was not irradiated. Pins comprise respectively a radius and a length of 3.5mm and 30mm.

The manufacturing process of the CoCrMo discs includes (i) SLM of the disc from CoCrMo powder, (ii) removal of the SLM supports, (iii) cleaning of the specimens by a sandblasting process (iv) superfinishing of the specimens by micromachining process and (v) removal of the micromachining supports.

Three different types of CoCrMo-discs were produced with gradual porosity defects in order to understand their impact on the wear behavior of UHMWPE. Surface texture and cavity characteristics are subsequently described in the results and discussion section.

Selective laser melting

Selective laser melting (SLM) principle and the parameters used for this study are described in Fig. 2. During the overall SLM process, discs are digitalised and sliced in a stack of layers (width 24µm). Each slice is developed with a scan path and adequate supports are added by the SLM software. A stripe pattern was chosen for this study, each stripe is separated by a distance of 105µm also known as the hatch vector.

Through lenses and scanning mirrors, a laser beam of 125W selectively scans and melts the powders with a specified scan path for each slice. Once a layer is finished, the building platform is lowered by an amount equal to the layer thickness, and a new layer of powders is provided from metal powder supply platform with a recoater. The process repeats until the completion of the whole part. To prevent oxidation, the process was performed under Argon gas.

Superfinishing by micromachining process

Once the parts have been manufactured, surfaces usually have to be polished to achieve mirror or extremely smooth finishes to meet articular implant specifications [START_REF]Non-active surgical implants -Joint replacement implants -Specific requirements for knee-joint implants[END_REF]. Compared to standard-sized implants, complex and unique AM-based implant are no fitted to traditional superfinishing techniques. In an attempt to be as close as possible to the application, we used a superfinishing process that can also be applied for complex geometric parts. Specimens were superfinished by a micromachining method. It is a physico-catalyst surface treatment applied to specimens inside a liquid environment. The media used is unique and developed by the company BESTinCLASS TM (BinC TM ). The parameters of the superfinishing processes have been chosen by the industrial provider of the process directly.

The advantage of the process is that it removes a uniform quantity of material and produce smooth surface mirror-like finish below 0.1µm (Ra). Our approach was to mirror-like polish our specimen using a more reproducible and automatic method [START_REF] Bordonado | Traitement sélectif de la rugosité des surfaces[END_REF] which might be used for complex additive manufactured implants. The test configuration is more representative of the AM-based implant reality. This is an additional element compared to literature.

Before testing, specimens were cleaned in an ultrasonic bath with, consecutively, sodium dodecyl sulfate at the critical micelle concentration 8.2 mM, acetone, ethanol, and pure water for 15min each followed by a drying of the specimens.

Multidirectional Pin-On-Disk Testing

Multidirectional pin-on-disc machine was used to perform wear tests representative of those endured by articular implant devices as seen in Fig. 3. It allows to understand the general wear behavior of these materials, under the described conditions. The operating variables were chosen to mimic in vivo physiological and biomechanical conditions. The machine is a custom built equipment, co-designed and assembled by RSI concept© (Lux). It allows the conduction of 3 tests simultaneously with an identicale triangular kinematics (variable point coordinates, variable speed, variable number of cycle) and the application of independent loads (variable up to 500 N). Resistances allow the heating (variable temperature) of each pin-on-disc system individualy and silicon gaiters ensure the sealing and avoids evaporation or cooling of any lubricant medium (variable volume). Therefore, this pin-on-disc machine is able to apply cross shear through several changes of direction during each cycle. In the literature, it has been demonstrated that wear on polyethylene is more relevant (and debris size and shape also) when shear forces are added to motion. More specifically, the wear rate is directly correlated to the shear rate [START_REF] Wang | A unified theory of wear for ultra-high molecular weight polyethylene in multi-directional sliding[END_REF][START_REF] Patten | Quantifying cross-shear under translation, rolling, and rotation, and its effect on UHMWPE wear[END_REF] when nearly no mass loss is detected with unidirectional motions [START_REF] Bragdon | A new pin-on-disk wear testing method for simulating wear of polyethylene on cobaltchrome alloy in total hip arthroplasty[END_REF]. Indeed, the alignment of polymeric molecular chains at material surface follows the principal direction of sliding. As a consequence, multidirectional motion is necessary to generate realistic wear rates [START_REF] Bragdon | A new pin-on-disk wear testing method for simulating wear of polyethylene on cobaltchrome alloy in total hip arthroplasty[END_REF][START_REF] Saikko | A Hip Wear Simulator with 100 Test Stations[END_REF]. The standard also suggests running the test at 37 ± 2 °C to reflect body temperature. At higher test temperatures, a degradation of the protein content in the lubricant might occur creating artefacts in the results.

Physiological contact pressure was set up at 3.9MPa and applied on 7mm diameter pins taking also into account the surface states. It is representative of the contact stresses of human joints in general (between 0,5 to 5 Mpa) [START_REF] Brand | Joint contact stress: a reasonable surrogate for biological processes?[END_REF]. The lubricant was bovine serum from

Gravimetric measurements

Wear rate were analyzed by gravimetric measures. Before weighting, UHMWPE samples are rinsed with water and sodium hypochlorite (bleach), cleaned in ultrasonic bath during 10min, rinsed with water and dried with compressed air.

Gravimetric weight loss per pin was determined every 0.12, 0.24, 0.34, 0.48 and 0.84 million cycles (SARTORIUS ME 235S® with ±0.01mg of resolution and ±0.2 mg of accuracy). The weight measurements were carried out in a temperature and humidity controlled room.

Surface texture measurements

Prior to the tests, it is necessary to describe the surface texture induced by the superfinishing process, and to verify the possibility to attain Sa values in accordance with the specification of metallic bearing part of articular implant described in the standard ISO 21534: 2007.

Surface texture measurements were carried out using a confocal microscope laser (Sensofar®) on the specimens before wear testing. Roughness parameters were analyzed (SensoMAP premium® software) in order to characterize the surface of the discs at 2 different scales:

-the entire discs (assembling of zones), reflecting the geometrical deviation induce by the SLM process, -zone of 132, 10µm x 177,44µm with an applied form removal filter (polynomial order 6). It reflects the superfinishing results of the physico-catalytic process.

Surface texture measurements were also done after the tests on the wear grooves of the CoCrMo surface and on the worn-out pins. These results were completed by SEM images (Zeiss Σigma instrument, Carl Zeiss SMT Ltd TM ) and optical microscope images. The SEM accelerating voltage used was 20kV.

Microstructure and chemical composition analysis

The

Hardness measurements

Microstructural changes are often associated with hardness modification of the material.

The hardness was measured prior the tests using a microdurometer (ZWICK ZHV-µs(H115))

in accordance to ISO-6507 and ASTM E384. The test consists of making an impact with a pyramid-shaped diamond indenter. The normal load is 300g and the residence time is 10s.

Porosity defects analysis in the sliding path

Finally, the porosity defects of the AM-CoCrMo parts were finely analyzed. Images of the cavities were taken using a digital optical microscope (Keyence®, VHX-6000) and the cavities present in the sliding path of the UHMWPE were extracted and analyzed using the integrated software (Keyence®, VHX-6000, §measure of grains). Morphology parameters including area, diameter and circularity were measured for each cavity. The circularity is described by the following equation:

= √2
where C is the circularity, S the area and P the perimeter.

3 Results and discussion

Specimens Characteristics

This work focused on the tribological performance of polyethylene against superfinished SLM samples. The characteristics of the sliding surface of SLM-CoCrMo discs as well as the UHMWPE pins are detailed here. which is explained by the thermal history of the process. During SLM, materials experience fast local melting along the tracks of a high energy laser followed by rapid solidification. Due to severe temperature gradients, this material's microstructure tends to have metastable structure [START_REF] Zhou | Textures formed in a CoCrMo alloy by selective laser melting[END_REF]. The matrix is face centered cubic (fcc) (Fig. 5.(i)) with precipitates at the joints of dendritic-shaped grains. No carbide was found by EDX analysis. Microstructural features changes are often impacting material properties. In our case, the resulted hardness was found to be at 400 HV0,3 with a slight increase of 20 HV0,3 in the direction of the building direction but not significant (Fig. 5.(ii)). Comparatively, the microstructure of the cast alloy features dendrite-like structural pattern with a larger grain size [START_REF] Hedberg | In vitro biocompatibility of CoCrMo dental alloys fabricated by selective laser melting[END_REF][START_REF] Xin | Surface Properties and Corrosion Behavior of Co-Cr Alloy Fabricated with Selective Laser Melting Technique[END_REF]. Cast alloys also contain blocks of carbides of about 50 μm homogeneously spread into the fcc matrix. They are known to reduce the wear rate because of their very high hardness [START_REF] Liao | CoCrMo metal-onmetal hip replacements[END_REF].

The cavities of the metallic discs located on the sliding path were fully described. Pore shape was extracted and analyzed by a set of parameters shown in fig. 6.(i) and (ii)-(vii) respectively.

The 3 specimens feature a gradient of porosity, 0.14%, 9,0% and 17,4% respectively for specimen #1, #2 and #3, in the sliding area as seen in fig. 6.(i) and (iv). Most of the pores are small, as 75% of the pores have an area inferior to 3500µm 2 (fig. 6.(ii)). These pores are due to gas entrapment during the process as seen in fig. 4 (i). The analysis of the perimeter of each pore features the same tendency because of its spherical characteristics (fig. 6.(iii)). The small number of large and irregular pores is the discriminating factor which leads to a difference in the ratio (%) of the overall porosity between the specimens (fig. 6.(ii-iv-vi).

Specimens #2 and #3 have a similar number of pores (≈1500) and a similar number of pores presenting similar size distribution. The main difference results in the number of pores that are superior to 10 5 µm 2 in term of area. Specimen #1 has significantly less pores (<500). All its pores are smaller than 10 4 µm 2 . Analysis of the overall porosity perimeter shows a gradient between specimens which is equivalent to that of the area (fig. 6.(v)). The UHMWPE mass loss results of the pins against SLM-CoCrMo discs featuring a gradient of cavities are shown in Fig. 7. Throughout the test, the cumulative mass loss of the UHMWPE is higher when sliding against specimen #3 than specimen #2 followed by specimen #1. The control (cast-CoCrMo) induces the lowest wear rate. The non-cumulative mass loss per million cycle shown in Fig 7 .(ii) demonstrates an initial high wear which decreases in the first 240 000 cycles. At 1 million cycle, specimen #1 (i.e. 0.4% vol porosity) has a similar wear rate than the cast version although the microstructural properties are different. Our results are within the range of wear rates reported in the litterature, which are about 7-10mg/million cycles for a standard UHMWPE [START_REF] Bragdon | A new pin-on-disk wear testing method for simulating wear of polyethylene on cobaltchrome alloy in total hip arthroplasty[END_REF][START_REF] Atwood | Tradeoffs amongst fatigue, wear, and oxidation resistance of cross-linked ultra-high molecular weight polyethylene[END_REF]. For a cross-linked polyethylene, the wear rate is about 2-4 mg/million cycles [START_REF] Saikko | A Hip Wear Simulator with 100 Test Stations[END_REF][START_REF] Atwood | Tradeoffs amongst fatigue, wear, and oxidation resistance of cross-linked ultra-high molecular weight polyethylene[END_REF] where the wear rate decreases with the rise of PE crosslinking rate [START_REF] Wang | A unified theory of wear for ultra-high molecular weight polyethylene in multi-directional sliding[END_REF]. The mass loss values of the UHMWPE per million cycles are within the specifications recommended by the ASTM F732 standard after 240 000 cycles for all the specimens. The mass loss values of the UHMWPE per million cycles seemed linearly and positively correlated to the total area of porosity (Fig. 7 (iii)) and the total perimeter of the pores (Fig. 7 (iv)). Therefore, an increase of 0,213mg of polyethylene mass loss per mm 2 of pores' area is estimated. Similarly, an increase of 0,007mg of polyethylene mass loss per mm of pores' perimeter is estimated. According to Fig. Some UHMWPE wear debris were embedded in the SLM-CoCrMo discs after the test as seen in Fig. 8. In the case of specimens #1 and #2, the polyethylene debris filled the pores which are mostly small and circular. In the case of specimen #3, the polyethylene debris have also filled the small circular pores but are additionally seen at the edge of the irregular pores (Fig. 8.#3). In this last configuration, the debris are much larger (Fig. 8.#3) and some have a flake shape. Specimen #2 has the same polyethylene debris type than specimen #3 but in a lower content and smaller debris size (Fig. 8.#2).

Polyethylene Wear behavior against SLM-CoCrMo

All the polyethylene pins' surfaces featured triangular scratching (Fig. 9 (i)) as can be explained by the kinematics of the test. The increase in pin roughness (Sa, Sq) coincides with the increase in porosity (Fig. 9 (ii) and (iii)). This confirms the abrasive behavior of the pore edge. Similarly, there were scratches visible on all the CoCrMo discs (Fig. 10). Traces of burnt polyethylene were also seen at the surface of all the discs (Fig. 8). Many traces are visible on the specimen #1 compared to specimens #2 and #3. These traces are typical of polymer adhesion mechanisms.

The wear tracks visible onto the surface of the disc suggest that metallic third-body particles were created during the tests. Specimen #1 had larger and deeper wear tracks compared to specimen #3. Specimen #2 had wear tracks as wide as specimen #1, but less deep. The width of the wear tracks can be explained by the morphology of the 3rd body that was formed during the test. The wear tracks of specimens #1 and #2 might reflects flat and wide debris while the wear tracks of specimen #3 might reflects small debris. The fact that specimen #3 had more irregular pores which might release metallic particles with time tends to confirm this hypothesis. The depth of the wear tracks might be explained by the potential effect of the large pores to collect debris. Indeed, these 3 rd bodies tend to slide over a shorter period of time as in the presence of large pores, leading to reduced superficial wear tracks. A total absence of debris will not ever be possible. There will always be wear and tear. However, by reducing the abrasiveness of pore edges, it would be possible to exploit their debris trapping characteristic.

Abrasion wear was therefore found on all configuration of tests resulting from two different mechanisms: (i) abrasion from the sharp pore edges on the surface and (ii) abrasion from the metallic third-body particles trapped between the surfaces.

Our work is not without limitations. Firstly, the UHMWPE samples could undergo irradiation in order to improve the wear rate [START_REF] Saikko | Effect of counterface roughness on the wear of conventional and crosslinked ultrahigh molecular weight polyethylene studied with a multi-directional motion pin-on-disk device[END_REF]. Indeed, cross-linked polyethylene is mostly used for hip implants, however for other articular devices, as knee implants, it is unclear whether its superiority depends on the type of wear [START_REF] Boyer | Is Cross-Linked Polyethylene an Improvement Over Conventional Ultra-High Molecular Weight Polyethylene in Total Knee Arthroplasty?[END_REF]. Because of its higher cost, production struggle and tough reticulation treatment, the standard existing polyethylene (GUR 1020) was chosen for our project. UHMWPE GUR 1050 is also used in medical joint implants; the main difference remains in its higher molecular weight (3-6 million g/mol). No statistical difference is expected between GUR 1050 and GUR 1020 in our test conditions [START_REF] Atwood | Tradeoffs amongst fatigue, wear, and oxidation resistance of cross-linked ultra-high molecular weight polyethylene[END_REF].

Secondly, the results of our work show that mass loss of polyethylene is greater when the number of cavities in the AM-CoCrMo counterpart is high. However, by increasing the surface area of cavities onto the surface, the contact area decreases. It has been reported

that wear factor drops with increasing contact stress [START_REF] Vassiliou | Is the wear factor in total joint replacements dependent on the nominal contact stress in ultra-high molecular weight polyethylene contacts?[END_REF]. Under constant load, larger contact area led to a larger wear volume.

Finally, this study was performed without soak control specimens during wear testing.

Literature indicates a negligible effect since presoaking parts for 3 weeks could only induce an increase in weight between 0,2 to 0,5mg [START_REF] Yao | Effect of fluid absorption on the wear resistance of UHMWPE orthopedic bearing surfaces[END_REF]. Considering that less than 30% of the UHMWPE pins are in contact with lubricant and that the test duration is 5 days in this work, the lack of soaking would likely not change general results of the performed comparative tests.

Conclusion

This study investigated the physical behavior of porosity defects in additive manufactured CoCrMo, by selective laser melting. A particular focus was put on the wear performance of polyethylene. It was evaluated with multidirectional pin-on-disc experiments in synovial fluid-like lubricant at 37°C. The testing conditions were set to simulate the wear of a total joint arthroplasty implant. Samples were produced by selective laser melting with percentage of surface porosity ranging from 0.4 to 19.7%. Under the present testing conditions, all polyethylene wear rates were in accordance with the ASTM F732 standard acceptance criteria

The paper allows the following conclusions to be drawn:

-There is a strong correlation between wear rate of UHMWPE and the total area of the porosity in AM-CoCrMo discs. Reducing porosity is beneficial for improving the wear resistance of UHMWPE.

-The edges of irregular pores in SLM samples are abrasive, which leads to increased numbers if polyethylene debris and an accelerated UHMWPE mass loss during the lubricated sliding process.

-Adhesion wear was identified particularly in the nearly full-dense disc configuration.

It was generated by the transfer of polyethylene material on the metallic surface.

-Abrasion wear was identified on all configuration of tests because of (i) sharp edges asperities on the surface and (ii) third -body particles trapped between the surfaces.

-The pores also limit the abrasive effect of the third body particles by acting as debris collectors.

2. 1 . 1

 11 CoCrMo powderCoCrMo parts are produced through selective laser meting which is based on the melting and rapid solidification of powder particles. The CoCrMo powder was produced by gas atomization under Ar and produced by LPW Technology Ltd TM . SEM imaging (Zeiss Σigma instrument, Carl Zeiss SMT Ltd TM ) at 15kV shows that particles have spherical shape (Fig.1.(i)); The average particle diameter ( ̅ ) was measured at 33.53 μm by a granulometer (Fig.1.(ii)). The values d(0.1) and d(0.9) are the diameters where 10% / 90% of the data is below, respectively. The chemical composition was measured through energy-dispersive Xray (EDX) detector (EDAX TM ), the signal was processed and recorded by a NumeriX-DXP X10P unit, and the spectrum analysis was carried out by the software: IDFix©. The EDX system was coupled to the SEM. The values are similar to the ASTM F75 alloy as seen in Fig.1.(iii).

Figure 1 :

 1 Figure 1 : CoCrMo Powder analysis: (i) morphology of the powder observed by SEM imaging, (ii) dry granulometry of the powder and (iii) chemical composition of the powder by EDX and comparison to the ASTM F75 standard

Figure 2 :

 2 Figure 2 : Selective laser melting (SLM) process: (i) SLM principle, (ii) Manufacturing parameters strategy used

  Biowest (Cat. S0400 -Sterile and Filtered) diluted in demineralized water with an antibiotic (Proclin 300). The total protein concentration in the mixture is 30g/L which is a clinically relevant concentration. It is filtered before use and heated at 37°C. An individual silicon gaiter ensures the sealing and avoids evaporation or cooling of the lubricant. The tests conditions were inspired by the ASTM F 732-17 Annex 2: « Wear testing of polymeric materials used in total joint prostheses », as seen in Fig.3. During the test, less than 30% of the UHMWPE pin is in contact with the diluted bovine serum and the test duration is 5 days.

Figure 3 :

 3 Figure 3 : Pin-on-disc wear test: (i) photograph of the test running, (ii) test conditions used in this work.

  microstructure and chemical composition of SLM-CoCrMo parts were analyzed to have a better understanding of the material changes because of the process. To investigate the microstructure of SLM-CoCrMo, the specimens were mounted in epoxy pucks and polished up to 4000 grit paper, and finally chemical-mechanical polished with an OP-S colloidal silica solution (Struers). The microstructural-crystallographic characterization was carried out on a SEM (Zeiss Σigma instrument, Carl Zeiss SMT Ltd©) at 15kV coupled with an Electron backscatter diffraction (EBSD) system (camera: TSL-EDAX, acquisition software: Nordif, Indexation software: OIM Data collection, processing, analysis software: OIM Analysis) at 25kV. The chemical composition was measured by EDX (detector: Brucker TM , signal processing: NumeriX-DXP X10P unit, spectrum analysis software: IDFix©. 5 measurements were realized on different zones on the samples to have an average value and standard deviation.

Figure 4 .

 4 Figure 4. (i) shows SEM micrograph taken from the superfinished sliding surface of an SLM-CoCrMo discs. The superfinished surfaces appear to be highly polished with brilliant mirrorlike finished surface. The superfinishing process has also revealed pores on the surface specific to SLM materials. It indicates that the SLM samples were not fully dense. The pores have various sizes from a few microns to 1500µm. Two type of pores were revealed: (i) small and round shaped pores which are due to gas entrapment and (ii) irregular shaped pores which are the result of incomplete fusion, and/or incomplete adhesion between two successive layers, and/or a collapse of parts structure due to either a lack of support material or poor orientation. The characteristics of the pores are further described for each CoCrMo samples.

Figure 4 Figure 5 :

 45 Figure 4 Superfinished surface: (i) SEM images showing the surface of the discs after superfinishing and the type of pores small and round pores (1) due to gas entrapment and irregular pores (2) due to sintering process, (ii) Chemical composition of the surface by EDX analysis, (iii) Surface texture measurements The chemical composition of the superfinished surface revealed an increase in the Iron, Copper and Aluminum content compared to ASTM F75 alloy standard and the chemical composition of the CoCrMo powder (Fig.4. (ii)). It might be a contamination of the superfinishing process that was not removed by the cleaning step.

Figure 6 :

 6 Figure 6 : Pores analysis: (i) Optical microscope images showing the path of the UHMWPE pin on the CoCrMo discs and the porosity distribution on the sliding area, (ii) area distribution of the pores, (iii) perimeter distribution of the pores, (iv) Total ratio (%) of the pores areas compared to the sliding area, (v) total perimeter and (vi) number of pores regarding the pores size.

Figure 7 :

 7 Figure 7 : Polyethylene wear against SLM-CoCrMo discs characterized by a gradient of pores: (i) cumulative mass loss of the UHMWPE pins, (ii) evolution of the non-cumulative mass loss per million cycle during the test, (iii) evolution non-cumulative mass loss results per million cycle regarding the total area of the pores, (iv) evolution of the non-cumulative mass loss results per million cycle regarding the total perimeter of the pores.

  7 (iii), a polyethylene mass loss of 5,5mg/million cycles is expected against a full dense SLM-CoCrMo disc. The data of Fig.7.(iv) gives an estimate of 5,1mg/million cycles. Sun et al.[START_REF] Sun | Sliding Wear Characteristics and Corrosion Behaviour of Selective Laser Melted 316L Stainless Steel[END_REF] performed pin-on-dics tribological test on SLM-316L against spherical stainless steel pin under dry conditions. They found that the wear rate of SLM 316L is positively correlated to the porosity percentage in terms of volume. Although the tests were performed with a different couple of materials and different tribological parameters (e.g. unlubricated condition, rotating sliding motion), the results confirm the deteriorating effect of the porosity on the wear performance of SLM parts. It is therefore essential to minimize porosity defects during the manufacturing of SLM components.

Figure 8 :

 8 Figure 8 : UHMWPE material on the SLM-CoCrMo discs after the test; transfer of polyethylene on the metallic surface. 1 : entirelly filled pore with polyethylene; 2: trace of adhesive polyethylene; 3 : large flake-like polyethylene debris on a pore edge; 4 : round and small (10-15µm) debris of polyethylene inside a large pore; 5.

Figure 9 :Figure 10 :

 910 Figure 9 : Surface texturation of the UHMWPE pins after the wear test : (i) optical microscope, (ii) Non-contact 3D optical profiler of the entire surface, (iii) Non-contact 3D optical profiler of the entire surface of a zone of 500x700µm

Acknowledgements

We thank CRITT MDTS for their excellent expertise in biotribology and for the use of their cutting-edge equipment and machines.

Funding source

The work was supported by the "Agence National de la Recherche" (ANR-11-IDEX-0004-02) under Idex "Sorbonne Universités" by an IUIS project for the promotion of healthcare innovation. Augustin Lerebours was recipient of a Ph.D. fellowship from the French Ministry of Science and Technology.

Abbreviations

Conflicts of interests

The authors have no conflicts to declare.


Rik Schrijvers

Bárbara Kong Cardoso

Jean Luc Bourrain

M.D. Ph.D Pascal Demoly

M.D. Ph.D Anca Mirela Chiriac
email: chiriac@chu-montpellier.fr

M D Phd

Bárbara Kong

Dr Anca Mirela

Delayed positive skin tests in patients with immediate hypersensitivity reactions to beta-lactams

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

Clinical implications

Skin tests for presumed IgE-mediated reactions to beta-lactams may be negative on immediate reading, but rarely demonstrate positivity on delayed reading with wheal-and-flare. Clinicians should be aware of this possible presentation.

The allergy work-up of a presumed immediate drug hypersensitivity reaction (DHR) is well established for beta-lactams (BL) [START_REF] Brockow | Skin test concentrations for systemically administered drugs -an ENDA/EAACI Drug Allergy Interest Group position paper[END_REF] . Patients undergo skin tests (ST) (prick (SPT) and intradermal (IDT) tests), followed by an immediate reading. In case of negative results and in the absence of contraindications, a drug provocation test (DPT) is performed, often on the same day.

We describe three cases of immediate DHR to BL in which ST were negative on immediate reading, yet all developed a late wheal-and-flare reaction, that contra-indicated DPT (Table 1).

Case 1, 64-year-old female, presented with palm and sole pruritus, malaise and dyspnea 30 minutes after taking amoxicillin/clavulanic acid (AxC), 500/62.5mg for a dental infection. She returned to her dentist where she was treated with prednisolone 80mg. The treatment was ineffective and the reaction worsened (vomiting, loss of consciousness, systolic pressure 60mmHg). Adrenaline was administered, and she was transferred to the emergency department. Tryptase measurements at the time of the event and baseline were 9.8 and 6.2 µg/mL, respectively. She had no atopy but suffered from SAPHO (Synovitis, Acne, Pustulosis, Hyperostosis and Osteitis) syndrome and hypothyroidism treated with infliximab and L-thyroxine, respectively. She took 1g of paracetamol 9h before the event.

Intake of amoxicillin 4 months earlier was uneventful. ST to penicillin G (Pen G), AxC, ampicillin (Amp) 4

weeks later were all negative on immediate reading. When the patient returned for the DPT, she mentioned a late reaction at the prior ST had occurred. IDTs were repeated and revealed again negative immediate reading. However, 4h and 6h later a dose-dependent wheal for IDT to AxC and Amp was observed, fading over the following days (Figure 1-Case1). The patient was considered allergic to AxC and Amp. A paracetamol DPT was negative.

Case 2, 26-year-old male was on noradrenaline because of circulatory instability after a trauma. He received suxamethonium, ethomidate, midazolam, sufentanyl to induce general anaesthesia. After achieving circulatory stability, he was rushed to emergency orthopedic surgery (where propofol, sufentanyl, paracetamol were administered). Two hours after the beginning of the procedure and 30 minutes after the (presumably) second injection of AxC (2g; the patient seemed to have received AxC and gentamycin while in the emergency unit, but this information could not be undoubtedly confirmed) he experienced rapidly increasing circulatory instability, demanding increasing doses of noradrenalin and then adrenalin syringe-pump. After cardio-circulatory recovery, the patient developed urticaria. Tryptase and histamine levels 30 minutes after the onset were 4.8 µg/mL and 36.9 nmol/L (normal <10 nmol/L), respectively. Baseline tryptase was <1 µg/L. The patient had a history of allergic rhinoconjunctivitis, took no maintenance treatment and previously underwent general anesthesia uneventfully. He didn't recall prior penicillin use. ST with PenG, AxC, Amp 6 weeks later were negative on immediate reading. When the patient returned for the DPT, he described a late reaction on the previous ST that lasted for several days. IDTs were repeated demonstrating negative immediate reading, yet wheal-and-flare at PenG, AxC, and Amp 24h later, lasting for several days, with an accompanying lymphangitis on day 3 (Figure 1-Case2). Further allergy work-up was negative. The patient was considered allergic to penicillins. He tolerated cefuroxime and ceftriaxone.

Case 3, 49-year-old male, presented 1 hour and 30 minutes after the start of renal stent placement with isolated generalized erythema while in the post-operative care unit. The patient had received propofol, sufentanil, mivacurium, betamethasone, cefazolin, ketoralac, paracetamol, and desflurane. Baseline tryptase level was 10.6 µg/L, no acute measurement was performed. The patient suffered from Crohn's disease but received no maintenance treatment. ST performed 10 weeks later were negative on immediate reading. A few hours later, a reaction was observed at the cefazolin ST. The IDT for cefazolin was repeated, together with other BL ST, and negative on immediate reading yet an itchy wheal-and-flare appeared at the cefazolin ST appeared 3h30 and again waned 8h30 later (Figure 1-Case3). The patient was considered allergic to cefazoline, a DPT for cefuroxime was negative.

An immediate IgE-mediated reaction on ST is expressed by a wheal-and-flare reaction caused by mast cell degranulation. The release of histamine, the major mediator in this reaction, begins within 5 minutes after allergen exposure and peaks at 30 minutes. Histamine and other mediators can trigger the release of substance P by axonal reflexes and additionally expand histamine release from the cutaneous mast cells 2 . Any IgE-mediated reaction consists of an immediate and a late-phase reaction (LPR). It is the former that is used in clinical practice as a reference for ST reading.

The IgE-mediated LPR in ST is a reaction that begins several hours after the exposure of the allergen via SPT or IDT (peaking at 6-12h and resolving in approximately 24-48h). Generally, this reaction occurs when the immediate reading of the IDT reveals a large wheal-and-flare reaction, as shown for respiratory allergens [START_REF] Yates | Delayed Hypersensitivity Skin Testing[END_REF][START_REF] Barata | IL-4-and IL-5-positive T lymphocytes, eosinophils, and mast cells in allergen-induced late-phase cutaneous reactions in atopic subjects[END_REF] .

Several mechanisms have been put forward to explain the LPR, involving other mediators than histamine (such as TNF-alpha, IL-6 [START_REF] Nagai | Role of mast cells in the onset of IgEmediated late-phase cutaneous response in mice[END_REF] ) that orchestrate the recruitment of a mixed cellular infiltrate [START_REF] Galli | IgE and mast cells in allergic disease[END_REF] , a prolonged stimulation of mast cells, and altered expression of adhesion molecules on vascular endothelium at the reaction site [START_REF] Barata | IL-4-and IL-5-positive T lymphocytes, eosinophils, and mast cells in allergen-induced late-phase cutaneous reactions in atopic subjects[END_REF] . Late-onset presumed IgE-mediated reactions (urticaria, anaphylaxis) have been described 7 mostly after bee-stings. The authors suggested a stimulation of IgE antibodies with persisting venom antigen. In asthmatics, intradermal administration of allergens was able to elicit a late phase asthmatic reaction without an early, IgE-mediated response, and was attributed as T-cellmediated [START_REF] Galli | IgE and mast cells in allergic disease[END_REF][START_REF] Kay | Allergy and allergic diseases. First of two parts[END_REF][START_REF] Haselden | Immunoglobulin E-independent major histocompatibility complexrestricted T cell peptide epitope-induced late asthmatic reactions[END_REF] . Here, skin reaction was absent both on immediate and LPR [START_REF] Haselden | Immunoglobulin E-independent major histocompatibility complexrestricted T cell peptide epitope-induced late asthmatic reactions[END_REF] .

In our work, case 1 and 2 suffered from an immediate DHR with ST positivity only on delayed reading suggesting a mitigated early phase ST response. In case 3, concordant late-onset index reaction and ST were observed, possibly alluding to a reaction to a metabolite. Of note, none of the index reactions were reminiscent of a type IV reaction. The peculiarity of the cases is that no immediate phase reaction preceded the alleged LPR and, to our knowledge, there is no previous report of this in beta-lactam allergy.

We did not perform confirmatory DPTs for ethical reasons and our observations do not provide a definite mechanistic explanation. Delayed ST positivity in the evaluation of a presumed IgE-mediated DHR is rare. We do not envision that this approach changes the recommendation of the use of ST, immediately followed by DPT in case of negative ST on immediate reading. However, if ST performed for a possible immediate DHR results in a delayed reaction, this should be taken into consideration despite the atypical presentation, before exposing patients to a potentially high risk DPT.

Table 1 -Characterization of Clinical Cases (on separate file)

Figure 1 -Skin Test positive results in the 3 cases Legend: Skin test results in the 3 cases, on different time points, using the indicated drugs (penicillin G, Pen G; ampicilline, Amp; amoxicilline, Ax; amoxicillin clavulanic acid, AxC; cefuroxim, Cefu; cefazoline, Cefa) demonstrate late skin test positivity with a wheal-and-flare-like reaction for Amp and AxC in case 1; Pen G, AxC, Amp in case 2; and Cefa in case 3. The outer right picture in case 2 demonstrates an accompanying lymphangitis on day 3. ST were performed according to ENDA/DAIG recommendations 1 (maximal concentration (1/1) for Cefu 2 mg/mL; Amp 25 mg/mL; Ax 25 mg/mL; AxC 25 mg/mL of Ax; Pen G 10,000 IU/mL), except for benzyl penicilloyl-polylysine (PPL, e.g. PRE-PEN, ALK Abello, USA) and minor determinant mixture (MDM) associated with increased ST sensitivity for diagnosing penicillin allergy, that are not available in France and Cefa IDT at 30 mg/ml in case 3, being negative in 20 controls. Co-sensitization to clavulanic acid was deemed unlikely but testing for clavulanic acid alone is not available in France.

Table 1 -

 1 Characterization of Clinical CasesAbbreviations: ST, skin tests; AxC, amoxicillin clavulanic acid; Cefa, cefazoline; CS, corticosteroids; IV, intravenous; ND, not determined; po, per os; we, weeks; * Skin test results were independently confirmed. £ It remains uncertain whether a 'first' dose was actually given (discordant recordings in emergency mobile unit, emergency department, and surgery unit concerning a first dose early before the event). Alternatively, a reaction after a first dose was missed due to co-administration of noradrenaline, or a delayed presentation of anaphylaxis with concordant late skin test positivity occurred, with unknown mechanism.

	Case Age,	Index	Culprit	Reaction	Reaction	Reaction	Test	ST	Duration	Tryptase	Tryptase	Maintenance
		sex	reaction	(admi-	interval	duration	treatment	interval	positivity*	of ST	acute	baseline	treatment(s)
				nistration						positivity	(µg/L)	(µg/L)
				route)						(start-
										end)
	#1	64,	Anaphylaxis	AxC (po)	30 min	Short	CS,	4 we	Amp, AxC	4h -4d	9.8	6.2	Infliximab, L-
		F				(Minutes	adrenaline						thyroxine
						-hours)
	#2	26,	Anaphylaxis	AxC (IV)	30 min	Short	Nor-	6 we	Amp, AxC,	24h -5d	4.8	<1	None
		M			after 2 nd	(Minutes	adrenaline		Pen G
					dose £	-hours)
	#3	49,	Generalized	Cefa (IV)	1h30	Short (30	CS, anti-H2	10 we	Cefa	3h30-	ND	10.6	None
		M	erythema			min)				8h30
			Legend:

Rik Schrijvers is recipient of a FWO (Fonds Wetenschappelijk Onderzoek) senior clinical investigator fellowship; Bárbara Kong Cardoso is an EAACI clinical fellowship recipient (2018).


Prof Hubert Blain
email: h-blain@chu-montpellier.fr

Yves Rolland

Edouard Tuaillon

Nadia Giacosa

Mylène Albrand

Audrey Jaussent

Athanase Benetos

Stéphanie Miot

Jean Bousquet

Antonin Balmes

Efficacy of a Test-Retest Strategy in Residents and Health Care Personnel of a Nursing Home facing a COVID-19 Outbreak

Keywords: 5. Graphics count: 1 table, 1 figure COVID-19, nursing home, rRT-PCR, antibodies against SARS-CoV-2

A wide testing strategy is effective in detecting asymptomatic COVID-19 residents and HCP in a NH facing COVID-19 outbreak. Symptomatic residents and HCP as well as asymptomatic HCP with negative testing may also play a role in the virus spread within the NH.

Introduction

After identification of a COVID-19 case in a Nursing Home (NH), residents are at high risk of serious illness and death from COVID-19, with a rapid and widespread transmission of SARS-CoV-2 . [START_REF] Mcmichael | Epidemiology of Covid-19 in a Long-Term Care Facility in King County, Washington[END_REF][START_REF] Arons | Presymptomatic SARS-CoV-2 Infections and Transmission in a Skilled Nursing Facility[END_REF] The standard COVID-19 diagnosis is based on SARS-CoV-2 nucleic acid testing by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). [START_REF] Nandini | Interpreting Diagnostic Tests for SARS-CoV-2[END_REF] Residents and health care personnel (HCP) with positive RT-PCR can be asymptomatic at the time of testing and may contribute to transmission. [START_REF] Arons | Presymptomatic SARS-CoV-2 Infections and Transmission in a Skilled Nursing Facility[END_REF] Control strategies focusing only on symptomatic residents are therefore insufficient. This explains why the recent American Testing Guidance for Nursing Homes recommends (i) testing of all residents and HCP in the NH if there is a confirmed case of COVID-19 and (ii) repeated weekly testing of all previously negative residents until no new cases of COVID-19 are detected for at least 14 days since the most recent positive result. [START_REF]Interim Testing Guidance in Response to Suspected or Confirmed COVID-19 in Nursing Home Residents and Healthcare Personnel[END_REF] Seroconversion with SARS-CoV-2 antibodies generally occurs rapidly in adult subjects. [START_REF] Tuaillon | Detection of SARS-CoV-2 antibodies using commercial assays and seroconversion patterns in hospitalized patients[END_REF] The immune response to viruses may be influenced by aging, and seroconversion in frail older subjects is uncertain. It is unclear whether residents and HCP with repeated negative testing may develop antibodies against SARS-CoV-2.

A study was carried out on all residents and HCP of a NH facing a COVID-19 outbreak. The aim was to assess clinical and serological parameters for the efficacy of infection prevention and control (IPC) measures adapted to (i) symptoms and (ii) results of repeated testing.

Methods

Setting: Single NH Participants: From March 3 rd to 6 th , 2020, three NH residents were hospitalized for severe non-respiratory COVID-19 symptoms. All three developed respiratory symptoms (cough with fever and dyspnea) 7 to 10 days after admission, and RT-PCR following nasopharyngeal swab test confirmed COVID-19. Seven days after the first diagnosis, all residents or HCP were enrolled in the study.

No ethics committee was required as this is an observational study.

Outcomes: COVID-19 symptoms were examined for 14 days before the first test and then followed daily for 6 weeks.

Nasopharyngeal testing for SARS-CoV-2 using rRT-PCR was performed in all residents and HCP. It was repeated weekly in all previously negative subjects until no new cases were identified and in all positive subjects until testing was negative. COVID-19 IPC measures were applied in all residents and HCP with positive testing or with new COVID-19 symptoms, including diarrhoea, delirium, or falls.

Six weeks after initial testing, all residents and HCP underwent blood testing for IgG antibodies directed against the SARS-CoV-2 nucleocapsid protein using an ELISA CE-IVD marked kit (ID screen ® SARS-CoV-2-N IgG indirect ID. Vet, Montpellier, France). 5

Results

Residents

Among the 79 residents, 38 (48%) had a positive RT-PCR (Table 1). 36 were diagnosed at baseline and two at day 7. The residents who tested positive were distributed throughout the 4 floors of the NH (10, 9, 10, 9).

The mean age of residents was similar in positive and negative RT-PCR groups. Diabetes and renal disease were more common in RT-PCR positive residents.

Thirteen residents died two to seven days after testing due to respiratory symptoms. Twelve (7 men) had a positive RT-PCR. Six RT-PCR-positive residents (16%) were asymptomatic before testing.

Six weeks after initial testing, seven residents still had at least one typical COVID-19 symptom (particularly fever or cough) or a significant functional impairment. Among them, 5 (83%) were RT-PCR-positive.

The RT-PCR test became negative 14, 21, or 28 days after initial positive testing in 2 (14%), 7 (27%), and 12 (46%) residents. In the 5 (19%) who still had positive RT-PCR 28 days after initial testing, one recovered completely and 4 had long-lasting symptoms (fever and hypothermia; shortness of breath; dry cough; impaired health status).

Health Care Personnel

Discussion

The present study shows the clinical efficacy of a symptom-and repeated testing-based strategy in a NH facing a COVID-19 outbreak. This experience validates the American Testing Guidance for Nursing Homes updated in May 2020. [START_REF]Interim Testing Guidance in Response to Suspected or Confirmed COVID-19 in Nursing Home Residents and Healthcare Personnel[END_REF] All residents and HCP were tested and there was no selection bias. This study was conducted before any other COVID-19 cases had been detected in the county. The presence of antibodies in residents and HCP is therefore almost certainly linked with the COVID-19 outbreak in that NH.

In the present study, 16% of residents and one third of HCP with positive RT-PCR were asymptomatic in the 14 days before testing. This confirms that all residents and HCP should be tested if there is a confirmed case of COVID-19, whatever the symptoms. [START_REF]Interim Testing Guidance in Response to Suspected or Confirmed COVID-19 in Nursing Home Residents and Healthcare Personnel[END_REF] Two residents and two HCP who tested negative at baseline were tested positive for COVID-19 7 days after baseline. This suggests that a repeated weekly testing of all previously negative residents and HCP until no new COVID-19 cases are identified is also essential in preventing the SARS-CoV-2 spread. [START_REF]Interim Testing Guidance in Response to Suspected or Confirmed COVID-19 in Nursing Home Residents and Healthcare Personnel[END_REF] Positive RT-PCR was associated with a severe prognosis (death in 32%), especially in men (death in 58%), confirming previous studies. [START_REF] Mcmichael | Epidemiology of Covid-19 in a Long-Term Care Facility in King County, Washington[END_REF][START_REF] Arons | Presymptomatic SARS-CoV-2 Infections and Transmission in a Skilled Nursing Facility[END_REF] Among the 22 negative RT-PCR residents presenting COVID-19 symptoms, one died and the others recovered completely, suggesting that severe COVID-19 outcomes could be generally, but not always, predicted by positive testing.

Testing remained positive for 3 weeks or more in two thirds of the RT-PCR positive residents.

One remained positive for 8 weeks, indicating that NHs facing a COVID-19 outbreak should be prepared to maintain prolonged protective measures in residents tested positive for SARS-CoV-2. In accordance with our regional guidelines [START_REF]COVID-19 Strategy for Prevention in Older Subjects[END_REF] , this NH was considered to be COVID-19-free when none of the residents and HCP were diagnosed within the 14 days after the last positive result. COVID-19 free NHs apply regional recommended measures to prevent any further COVID entrance and spread. In our Occitanie region, these measures include 6 : 1.

Checking that RT-PCR testing in HCP and visitors with COVID-19 symptoms or in those having had contact with COVID-19 suspected or confirmed cases (daily screening) is negative before entering the premises. 2. Checking that RT-PCR testing in all new residents and in all residents having spent more than 24 h outside the NH (especially after hospitalization) is negative before entering. 3. Checking that residents, HCP, and visitors previously tested positive for COVID-19 meet all 3 follow-up NH entrance criteria: (i) resolution of fever (without use of fever-reducing medications) and of other COVID-19 symptoms within the past 48 hours; (ii) two consecutive negative RT-PCR results collected ≥24 hours apart, (iii) the first control test collected at least 7 days after the positive testing or 7 days after the first COVID-19 symptoms. 4. Obliging visitors allowed to enter the NH to sign a charter in which they agree to adhere to standard and transmission-based precautions to prevent COVID-19 spread in the NH (systematic face mask wearing, hand hygiene, and especially social distancing) as well as a registry with contact details to facilitate testing and contact tracing should a new case be diagnosed in the NH. 5. Admission to a private room and 14 days of isolation for every new resident and every resident having left the NH for at least 24h (especially after hospital stay). 6. Testing of every resident having left the NH for less than 24 hours (especially for medical consultation) 5 to 7 days after a possible contact with COVID-19. 7. Daily screening of all residents for COVID-19 symptoms (including atypical symptoms) and testing if there is any doubt. 8. If regular testing of HCP, visitors, and residents at high risk of encountering COVID-19 subjects outside the NH (health care workers who have a care activity outside the NH, visitors of several NHs, hemodialysed patients, etc…) can be justified in regions with moderate or substantial community transmission, this measure is not recommended in our region in which community transmission is now considered as low.

Residents and HCP with positive RT-PCR developed IgG antibodies against the SARS-CoV-2 in 96% and 100%, respectively, suggesting that most frail older adults living in a NH, as well as the HCP, can produce an antibody response against SARS-CoV-2.

Two residents (5%) with negative RT-PCR developed antibodies and all had fever or respiratory symptoms consistent with COVID-19 in the 14 days before. This suggests that residents with COVID-19 symptoms should benefit from the same IPC strategy as residents with positive RT-PCR, even if tested negative. Five HCP with negative testing developed antibodies against SARS-CoV-2 (11%) and some of them had no COVID-19 symptoms. This suggests that if specific COVID-19 IPC measures must be implemented in HCP with confirmed or suspected COVID-19, all HCP should wear a facemask, even if asymptomatic and with negative testing.

Conclusions and Implications

The present study supports the recent American testing guidance for NHs. It demonstrates that

  Among the 34 HCP, 6 had positive RT-PCR at baseline and 2 at day 7 (23.5%). No new COVID-19 diagnosis was made later. Two thirds of the positive RT-PCR HCP had COVID-19 symptoms, often mild. Seroconversion Six weeks after nasopharyngeal testing, 25 residents (92.3%) and all 8 HCP (100%) with positive RT-PCR developed SARS-CoV-2 IgG antibodies. Two (5%) RT-PCR negative residents and 5 (11.5%) RT-PCR negative HCP developed antibodies. All 2 residents and 3/5 HCPs had typical COVID-19 symptoms.

  (i) testing all NH residents and HCP as soon as a new case of COVID-19 is diagnosed and (ii) repeating tests in all previously negative subjects once a week until the testing identifies no new COVID-19 cases is effective in detecting asymptomatic COVID-19 residents and HCP. It also shows that (iii) proposing COVID-19 IPC measures in residents and HCP tested positive or with COVID-19 symptoms and (iv) taking precautions in all other HCP should be effective in blocking the dissemination of the virus in NHs facing a COVID-19 outbreak. Conflicts of Interest: The authors declare no conflicts of interest/Competing interests

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