Keywords: entheses, enthesopathy, Musculoskeletal Stress Markers (MSM), senescence, activity, hormones, animal models, clinical studies entheses, enthesopathy, Musculoskeletal Stress Markers (MSM), senescence, activity, hormones, animal models, clinical studies

Over the past two decades, many articles have been published on entheseal changes (usually called "Musculoskeletal Stress Markers") as activity markers in past societies. Oversimplified methods and over-interpretation of past activities have generated robust critiques of research results in this area of enquiry. While some significant improvements regarding the recording systems for entheseal changes have been applied more recently, many bioarchaeologists appear not yet to be fully aware of the multi-factorial aetiology of these alterations. In this article, we review the anatomical and clinical literature to discuss some of the difficulties associated with the recording of entheseal changes and the multiple factors leading to their appearance in the human skeleton. Thus far fibrocartilagenous entheses appear to hold more promise for activity-related reconstruction than do fibrous ones, but these relationships remain an area of active research interest.

Introduction

La [START_REF] Cava | Enthesitis-traumatic disease of insertions[END_REF] appears to have been first to use the term "enthesis" for creating the word "enthesitis" to designate the inflammation of tendinous attachments. Subsequently, [START_REF] Ball | Enthesopathy of rheumatoid and ankylosing spondylitis[END_REF] and [START_REF] Niepel | Enthesopathy[END_REF] suggested using the words "enthesis" to designate the area where a tendon, a capsule or a ligament attaches to bone and "enthesopathy" to indicate pathological changes of this structure. "Enthesis" and "enthesopathy" are commonly used today in biomedical sciences, but other terms can also be found in the literature (table 1).

TABLE 1

An enthesopathy can be a radiological, clinical, histological or osteological finding, and the notion of pain is not necessarily associated with its occurrence (e.g. [START_REF] Ball | Enthesopathy of rheumatoid and ankylosing spondylitis[END_REF][START_REF] Resnick | Entheses and enthesopathy[END_REF][START_REF] François | Entheses and enthesitis: a histopathologic review and relevance to spondyloarthritides[END_REF]. In biological anthropology, several terms are used to designate the osteological changes seen in entheses: enthesopathies (e.g. [START_REF] Dutour | Enthesopathies (lesions of muscular insertions) as indicators of the activities of Neolithic Saharan populations[END_REF][START_REF] Hawkey | Use of upper extremety enthesopathies to indicate habitual activity patterns[END_REF], muscle crests [START_REF] Angel | Life stresses of the free Black community as represented by the First African Baptist Church, Philadelphia, 1823-1841[END_REF], musculoskeletal stress markers [START_REF] Hawkey | Activity-induced musculoskeletal stress markers (MSM) and subsistence strategy changes among ancient Hudson Bay Eskimos[END_REF], or muscle markings [START_REF] Robb | The interpretation of skeletal muscle sites: a statistical approach[END_REF]. Recently, [START_REF] Jurmain | Terminology. Entheses in medical literature and physical anthropology: a brief review[END_REF] F o r P e e r R e v i e w 3 proposed the generic and more neutral term "entheseal changes" to designate all "particular" aspects of entheses seen in skeletal material.

The interest of biological anthropologists in entheseal changes of the human skeleton is older than a century (e.g. [START_REF] Lane | A remarkable example of the manner in which pressure-changes in the skeleton may reveal the labour history of the individual[END_REF][START_REF] Lane | Anatomy and physiology of the shoemaker[END_REF][START_REF] Testut | Recherches anthropologiques sur le squelette quaternaire de Chancelade (Dordogne)[END_REF]. Three main factors account for this interest. First, the areas of tendon or ligament attachment are usually easily visible on dry bones. Second, entheses exhibit a number of morphological variations, with more or less pronounced changes, such as irregularity and porosity, so changes can be scored. Finally, as entheses are regularly under heavy strain during physical activity, changes can, hypothetically, be used to reconstruct past physical activities. This type of study also has a long and contentious disciplinary history (for reviews, see [START_REF] Kennedy | Skeletal markers of occupational stress[END_REF][START_REF] Dutour | Chasse et activités physiques dans la Préhistoire : les marqueurs osseux d'activités chez l'homme fossile[END_REF]Jurmain et al., 2012), with some notable detractors (e.g. [START_REF] Jurmain | Story from the skeleton[END_REF][START_REF] Zumwalt | The effect of endurance exercise on the morphology of muscle attachment sites[END_REF][START_REF] Jurmain | Juggling the evidence: the purported 'acrobat' from Tell Brak[END_REF][START_REF] Cardoso | Enthesopathy formation in the humerus: Data from known age-at-death and known occupation skeletal collections[END_REF] as well as supporters (e.g. [START_REF] Dutour | Enthesopathies (lesions of muscular insertions) as indicators of the activities of Neolithic Saharan populations[END_REF][START_REF] Hawkey | Activity-induced musculoskeletal stress markers (MSM) and subsistence strategy changes among ancient Hudson Bay Eskimos[END_REF][START_REF] Molnar | Tracing prehistoric activities: musculoskeletal stress marker analysis of a stone-age population on the Island of Gotland in the Baltic sea[END_REF]Villotte et al., 2010aVillotte et al., , 2010b;;[START_REF] Havelková | Enthesopathies and activity patterns in the Early Medieval Great Moravian population: Evidence of division of labour[END_REF].

Methodological research recently carried out by several scholars [START_REF] Mariotti | Enthesopathies -Proposal of a standardized scoring method and applications[END_REF][START_REF] Mariotti | The study of entheses: proposal of a standardised scoring method for twenty-three entheses of the postcranial skeleton[END_REF][START_REF] Zumwalt | A new method for quantifying the complexity of muscle attachment sites[END_REF][START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF][START_REF] Henderson | Quantitative recording of entheses[END_REF][START_REF] Henderson | Scoring entheseal changes: proposal of a new standardised method for fibrocartilaginous entheses[END_REF]Villotte et al., 2010a) provides a renewed interest for the study of entheseal changes as potential markers of activity, as it can be seen, for instance, by the success of the Workshop in Musculoskeletal Stress Markers (MSM): limitations and achievements in the reconstruction of past activity patterns, held at the University of Coimbra, in Portugal (2 nd -3 rd July 2009) [START_REF] Santos | The Coimbra Workshop in Musculoskeletal Stress Markers (MSM): an annotated review[END_REF]. However, this relative success could turn against itself, and one cannot entirely dismiss previous remarks on the limitations and pitfalls of this kind of study.

The purpose of this article is not to discuss the reliability of entheseal changes for the reconstruction of past activities, nor the strictly methodological aspects of this kind of study because those considerations have been largely discussed elsewhere [START_REF] Dutour | Activités physiques et squelette humain : le difficile passage de l'actuel au fossile[END_REF][START_REF] Robb | The interpretation of skeletal muscle sites: a statistical approach[END_REF][START_REF] Jurmain | Story from the skeleton[END_REF][START_REF] Mariotti | Enthesopathies -Proposal of a standardized scoring method and applications[END_REF][START_REF] Mariotti | The study of entheses: proposal of a standardised scoring method for twenty-three entheses of the postcranial skeleton[END_REF][START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF]Villotte, , 2008a[START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF][START_REF] Henderson | Quantitative recording of entheses[END_REF][START_REF] Cardoso | Enthesopathy formation in the humerus: Data from known age-at-death and known occupation skeletal collections[END_REF]Villotte et al., 2010a;Jurmain et al., 2012;Millela et al., n.d.). The goal here is rather to attempt to clarify several points to anticipate continued use of entheseal changes as potential markers of activity in past populations. This perspective is based on the following premises: the most reliable data on entheses and entheseal changes are provided by their use in the biomedical sciences (even if some studies, as case reports, have apparently little broader application), and data from Anatomical background 1) Types of enthesis: a brief presentation Two groups of entheses can be distinguished according to the tissue type present at the skeletal attachment site: fibrocartilaginous and fibrous [START_REF] Benjamin | The anatomical basis for disease localisation in seronegative spondyloarthropathy at entheses and related sites[END_REF][START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF]. Recently, the importance of this anatomical distinction for recording entheseal changes in skeletal material has been highlighted, independently, by Henderson [START_REF] Henderson | Quantitative recording of entheses[END_REF][START_REF] Cardoso | Enthesopathy formation in the humerus: Data from known age-at-death and known occupation skeletal collections[END_REF] and by [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF]Villotte ( , 2008a[START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF]Villotte et al., 2010a).

Briefly, fibrocartilaginous entheses occur close to joints of the long bones, but also on short bones and some parts of vertebrae; fibrous entheses occur on the diaphysis of long bones and also on the vertebral column (see tables A1 and A2 in Villotte et al., 2010a for a list of the main post-cranial fibrous and fibrocartilaginous entheses). Four histological zones are distinguished in fibrocartilaginous entheses [START_REF] Cooper | Tendon and ligament insertion. A light and electron microscopic study[END_REF][START_REF] Benjamin | The histology of tendon attachments to bone in man[END_REF]:

1) tendon or ligament, 2) uncalcified fibrocartilage, 3) calcified fibrocartilage, and 4) subchondral bone. Zones 2 and 3 are separated by a regular calcification front called the ''tidemark". The tidemark, which is relatively rectilinear and not crossed by blood vessels, is the point at which soft tissues are removed during maceration [START_REF] Benjamin | The histology of tendon attachments to bone in man[END_REF].

Contrary to fibrocartilaginous attachments, anatomical and anatomo-pathological descriptions for fibrous entheses are extremely rare. They attach soft tissues to bone directly or via a mediating layer of periosteum [START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF]. The anchorage is achieved through collagen fibers from periosteum, tendon, or ligament, which are embedded into bone [START_REF] François | Entheses and enthesitis: a histopathologic review and relevance to spondyloarthritides[END_REF][START_REF] De Pinieu | Qu'est-ce qu'une enthèse ? In Tendons et enthèses[END_REF]. At fibrous entheses, blood vessels from the tendon or the ligament may anastomose with those of the bone [START_REF] Dörfl | Vessels in the region of tendinous insertions. II. Diaphysoperiosteal insertion[END_REF].

2) Defining a "normal" and a "changed" enthesis, is it simple? The characteristic aspect of a healthy fibrocartilaginous enthesis is described by Benjamin and colleagues (2002: 939) in the following manner: "as the tidemark is relatively straight and the fibrocartilage zones avascular, the site of attachment in a healthy enthesis is smooth, well circumscribed and devoid of vascular foramina." This description fits well with the appearance of several attachment sites seen on the skeleton (Fig. 1). Moreover, the accurate and numerous descriptions of changes that occur in enthesopathies in living people -erosion of the calcified fibrocartilage and subchondral bone, tidemark irregularity, vascularization of the fibrocartilage, calcification and ossification of soft tissues, cysts, and avulsions can be observed in skeletal material [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF]Villotte et al., 2010a and clinical references therein). Thus, it seems conceivable to define a "healthy" (or "normal") fibrocartilaginous enthesis in skeletal material as a smooth, well-defined imprint on the bone, without vascular foramina, and with a regular margin, and an enthesopathy in the other cases [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF], Villotte et al., 2010a). However, this definition can be applied for only some fibrocartilaginous attachments; it does not work as well, for instance, for the entheses of ligamenta flava on the vertebral column [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF] or for small attachment sites like the insertion of the M. brachialis (pers. obs., and Mariotti, pers. comm.). In the case of the M. brachialis, this may be related to the relatively thin layer of uncalcified fibrocartilage at the insertion, and the fact that this muscle is already attached to the bone at birth (cf. [START_REF] Benjamin | The structure of the insertions of the tendons of biceps brachii, triceps and brachialis in elderly dissecting room cadavers[END_REF].

FIGURE 1

Regarding the paucity of clinical and anatomical data for fibrous entheses, the definition of a "normal" type is far more complex [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF][START_REF] Cardoso | Enthesopathy formation in the humerus: Data from known age-at-death and known occupation skeletal collections[END_REF]. Osseous irregularity in the area of fibrous attachments is common in human skeletal remains, even in the first decades of adulthood [START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF]. Since the term "enthesopathy" implies a pathological condition, it is not appropriate to designate all of these very common and probably asymptomatic changes as pathological [START_REF] Jurmain | Terminology. Entheses in medical literature and physical anthropology: a brief review[END_REF].

3) Possible pitfalls of anatomical over-simplification

Changes in the two types of enthesis do not indicate the same phenomena, and it is to be expected that biological anthropologists will no longer combine them in a single study. Although the distinction between fibrous and fibrocartilaginous entheses seems clear enough, one should not forget that some amendments to their apparent distinctiveness have been made. First, some attachments are "mixed." [START_REF] Hems | Tendon entheses of the human masticatory muscles[END_REF] showed that the majority of the entheses of the masticatory muscles are of this type. Thus, the insertion of M. masseter is partly periosteal, partly osseous and partly fibrocartilaginous. Second, in a fibrocartilaginous enthesis, the periphery has little or no fibrocartilage [START_REF] Benjamin | The histology of tendon attachments to bone in man[END_REF][START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF]. Third, fibrocartilage may exist in a small quantity at a fibrous enthesis, particularly on the metaphysis, an example of which is the M. pectoralis major insertion on the humerus [START_REF] Benjamin | The histology of tendon attachments to bone in man[END_REF][START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF]. Finally, one should not forget the morphological diversity in our species. For example, the most distal part of the insertion of the M. iliopsoas, at the junction between the lesser trochanter and the femoral shaft, may correspond to the inconstant fibrous insertion of the M. iliacus [START_REF] Polster | MRI and gross anatomy of the iliopsoas tendon complex[END_REF] and this region is highly variable as a consequence (Fig. 2).

FIGURE 2

Several authors have discussed the boundaries of the concept of enthesis (for fibrocartilaginous attachments), and all agree that it cannot be reduced to the attachment of a tendon or ligament. 1 In addition to the attachment zone, [START_REF] Niepel | Enthesopathy[END_REF] include under the term "enthesis", all of the following: peritenon, associated bursae, fibrous tissues, fat-pads and sesamoid bones. Benjamin and colleagues (2001Benjamin and colleagues ( , 2002Benjamin and colleagues ( , 2004) ) formalized this concept and suggest using "enthesis organ" for the complex consisting of these anatomical structures around the fibrocartilaginous entheses sensu stricto because fibrocartilage is almost systematically observed in these structures. It is important that biological anthropologists consider this point before scoring entheseal changes. For instance, recent methodological papers [START_REF] Mariotti | Enthesopathies -Proposal of a standardized scoring method and applications[END_REF][START_REF] Mariotti | The study of entheses: proposal of a standardised scoring method for twenty-three entheses of the postcranial skeleton[END_REF][START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF][START_REF] Henderson | Quantitative recording of entheses[END_REF][START_REF] Henderson | Scoring entheseal changes: proposal of a new standardised method for fibrocartilaginous entheses[END_REF]Villotte et al., 2010a) do not mention bursae and whether or not bursopathies are scored. Admittedly, this is a thorny problem. [START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF], perhaps wrongly, did not score bone changes that can occur at the location of bursae (Fig. 3) for several reasons: at present, data on bursopathies are still too scarce to accurately analyze the consequences on the 1 Opinions differ on the structures to be associated with the enthesis sensu stricto. For instance, Fournié proposed the extension of the concept of enthesis to include amphiarthroses and diarthro-amphiarthroses [START_REF] Fournié | Elargissement du concept d'enthèse[END_REF][START_REF] Fournié | Pathology and clinico-pathologic correlations in spondyloarthropathies[END_REF]. First, in histological and functional terms, these joints are closer to fibrocartilaginous entheses than to true diarthroses. Second, these joints and fibrocartilaginous entheses are predilected in seronegative spondyloarthropathies. skeleton, the presence of bursae is inconstant, and their exact location varies. However, these data may be of interest in the discussion of past activities -bursae, as a part of the "enthesis organ", participate actively in the dissipation of mechanical stresses. Moreover, it should be noted that sometimes the distinction between a bony involvement of the tendon attachment site or of the bursa is difficult to establish.

FIGURE 3

Entheseal changes through life: the effects of age, hormones and activity Trauma and micro-trauma can produce entheseal changes, among numerous others factors [START_REF] Resnick | Entheses and enthesopathy[END_REF]. Clinical literature also reports the presence of entheseal changes in many diseases. The goal is not to present these conditions in detail here (listed in [START_REF] Henderson | When hard work is disease: the interpretation of enthesopathies[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF], but rather to focus on lesser-known aspects of entheseal changes related to age, hormones and physical activities. However, it seems necessary to present briefly the two main causes of non-degenerative enthesopathies: spondyloarthropathies and diffuse idiopathic skeletal hyperostosis (DISH or hyperostotic disease). DISH is characterized by para-articular bridging osteophytes in the anterolateral aspect of the spine [START_REF] Forestier | Senile Ankylosing Hyperostosis of the Spine[END_REF][START_REF] Resnick | Diffuse idiopathic skeletal hyperostosis (DISH): Forestier's disease with extraspinal manifestations[END_REF]. Exuberant bone production is seen at extra-spinal fibrocartilaginous and fibrous entheses [START_REF] Resnick | Diffuse idiopathic skeletal hyperostosis (DISH): Forestier's disease with extraspinal manifestations[END_REF]. Very early on, researchers recognized the enthesis as a primary target in ankylosing spondylitis and other spondyloarthropathies [START_REF] Ball | Enthesopathy of rheumatoid and ankylosing spondylitis[END_REF]Paolaggi et al., 1984aPaolaggi et al., , 1984b)). The inflammation occurs at the level of fibrocartilaginous entheses, leading to erosion of the fibrocartilage [START_REF] Benjamin | The anatomical basis for disease localisation in seronegative spondyloarthropathy at entheses and related sites[END_REF][START_REF] Fournié | Pathology and clinico-pathologic correlations in spondyloarthropathies[END_REF]. This erosive process is followed by deposition of reactive bone and the formation of an enthesophyte [START_REF] Ball | Enthesopathy of rheumatoid and ankylosing spondylitis[END_REF][START_REF] Resnick | Entheses and enthesopathy[END_REF]. It is noteworthy that entheseal changes seen in cases of spondyloarthropathy are characterized, at least for some fibrocartilaginous entheses, by erosive lesions uncommonly seen in other individuals [START_REF] Villotte | Remodelages des enthèses fibrocartilagineuses dans les spondylarthropathies inflammatoires Bulletins et[END_REF].

In studies of entheseal changes in skeletal samples with known age-at-death, age is the main etiological factor identified [START_REF] Shaibani | The significance of enthesopathy as a skeletal phenomenon[END_REF][START_REF] Cunha | What can bones tell about labour and occupation: the analysis of skeletal markers of occupational stress in the Identified Skeletal Collection of the Anthropological Museum of the University of Coimbra (preliminary results)[END_REF]Mariotti et [START_REF] Cardoso | Enthesopathy formation in the humerus: Data from known age-at-death and known occupation skeletal collections[END_REF]Villotte et al., 2010a;[START_REF] Niinimäki | What do muscle marker ruggedness scores actually tell us?[END_REF][START_REF] Milella | The effect of age, sex, and physical activity on entheseal morphology in a contemporary Italian skeletal collection[END_REF]. However, the precise relation between age and entheseal changes remains poorly described (and in some aspects poorly understood).

Properties of the entheses during skeletal immaturity are mainly described in studies of animal models and, to a lesser extent, in studies of human cadavers. For adulthood, data derive mainly from sports medicine or that associated with the aged, though the study of identified skeletal collections provides informative results.

1) Secondary ossification centres

In early development of humans and other mammals, the tendon or ligament attaches to the perichondrium [START_REF] Hurov | Soft-tissue bone interface: How do attachments of muscles, tendons, and ligaments change during growth? A light microscopic study[END_REF][START_REF] Gao | An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint[END_REF][START_REF] Wei | The postnatal development of the insertions of the medial collateral ligament in the rat knee[END_REF][START_REF] Shaw | Development of the human Achilles tendon enthesis organ[END_REF].

During growth, entheses seem to act as growth plates; the cartilage is resorbed at the inner side and produced at the outer side, possibly by metaplasia [START_REF] Gao | An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint[END_REF][START_REF] Nawata | Development of the attachment zones in the rat anterior cruciate ligament: changes in the distributions of proliferating cells and fibrillar collagens during postnatal growth[END_REF]. The classic appearance of a fibrocartilaginous enthesis (i.e. the four histological zones, see supra) appears in non-human mammals when growth slows or stops [START_REF] Wei | The postnatal development of the insertions of the medial collateral ligament in the rat knee[END_REF][START_REF] Nawata | Development of the attachment zones in the rat anterior cruciate ligament: changes in the distributions of proliferating cells and fibrillar collagens during postnatal growth[END_REF][START_REF] Wang | Age-dependent changes in matrix composition and organization at the ligament-to-bone insertion[END_REF]. For instance, in the attachment zones in the rat anterior cruciate ligament, the boundary between uncalcified and calcified fibrocartilage is not clearly distinguishable before growth slows [START_REF] Nawata | Development of the attachment zones in the rat anterior cruciate ligament: changes in the distributions of proliferating cells and fibrillar collagens during postnatal growth[END_REF]. The process is not described for humans, but this progressive organization of the enthesis during growth and development could explain the lack of a clearly distinguishable area of attachment in juvenile human skeletons (Fig 4, compare with Fig. 1). Indeed, in skeletal remains, the classic appearance of a fibrocartilaginous enthesis is seen when epiphyses of short and flat bones (also called apophyses) and long bones are partially or fully fused. The most common activity-related change occurring before the complete fusion of the epiphysis is a total or a partial bony avulsion (e.g. [START_REF] Resnick | Entheses and enthesopathy[END_REF][START_REF] Nakanishi | MR arthrography of elbow: evaluation of the ulnar collateral ligament of elbow[END_REF][START_REF] Stevens | Imaging features of avulsion injuries[END_REF][START_REF] Adirim | Overview of injuries in the young athlete[END_REF]. Consequences of these avulsions (total or partial) can be observed on the adult skeleton (Villotte et al., 2010b;[START_REF] Knüsel | Men Take Up Arms for War: Sex and Status Distinctions of Humeral Medial Epicondylar Avulsion Fractures in the Archaeological Record[END_REF].

FIGURE 4

During adulthood, the degenerative process related to age and mechanical demands affects both the tendon and the fibrocartilaginous enthesis. Within the aging tendons, the amount of denatured collagen and proteolytic cleavage of matrix components increase [START_REF] Riley | The pathogenesis of tendinopathy. A molecular perspective[END_REF]. These changes lead to deterioration in the physical properties of the tendon [START_REF] Riley | The pathogenesis of tendinopathy. A molecular perspective[END_REF], which, in turn, may favour the occurrence of mechanically induced alterations in the enthesis [START_REF] Rodineau | Pathogénie des enthésopathies du membre supérieur[END_REF][START_REF] Bard | Physiopathologie, réparation, classification des tendinopathies mécaniques[END_REF]. From the sixth decade onwards, the fibrocartilaginous enthesis itself is the target of the degenerative process [START_REF] Durigon | Enthèse au cours de la vie[END_REF][START_REF] Rodineau | Pathogénie des enthésopathies du membre supérieur[END_REF][START_REF] Bard | Physiopathologie, réparation, classification des tendinopathies mécaniques[END_REF]. Those degenerative changes are well described [START_REF] Durigon | Enthèse au cours de la vie[END_REF][START_REF] Lagier | Enthèses normales et enthésopathies[END_REF][START_REF] Kumagai | The collagen types in the attachment zone of rotator cuff tendons in the elderly: an immunohistochemical study[END_REF][START_REF] Jiang | Trabecular microstructure and surface changes in the greater tuberosity in rotator cuff tears[END_REF][START_REF] Milz | Molecular composition and pathology of entheses on the medial and lateral epicondyles of the humerus: a structural basis for epicondylitis[END_REF][START_REF] Benjamin | Microdamage and altered vascularity at the enthesis-bone interface provides an anatomic explanation for bone involvement in the HLA-B27-associated spondylarthritides and allied disorders[END_REF][START_REF] Benjamin | Evidence for a distinctive pattern of bone formation in enthesophytes[END_REF]. They are:

-Microtears or microdamage of one of the four histological zones of the enthesis (tendon, Disturbance of enthesis organization and subsequent healing processes are visible on skeletal remains: vascularization, enthesophytes, calcific deposits, cysts, and irregularity of the surface are common in the skeletons of old individuals [START_REF] Shaibani | The significance of enthesopathy as a skeletal phenomenon[END_REF][START_REF] Cunha | What can bones tell about labour and occupation: the analysis of skeletal markers of occupational stress in the Identified Skeletal Collection of the Anthropological Museum of the University of Coimbra (preliminary results)[END_REF][START_REF] Mariotti | Enthesopathies -Proposal of a standardized scoring method and applications[END_REF][START_REF] Mariotti | The study of entheses: proposal of a standardised scoring method for twenty-three entheses of the postcranial skeleton[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF][START_REF] Cardoso | Enthesopathy formation in the humerus: Data from known age-at-death and known occupation skeletal collections[END_REF]Villotte et al., 2010a;[START_REF] Milella | The effect of age, sex, and physical activity on entheseal morphology in a contemporary Italian skeletal collection[END_REF] Bony spur formation typically occurs in the most fibrous part of an enthesis [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF][START_REF] Benjamin | Evidence for a distinctive pattern of bone formation in enthesophytes[END_REF]. It should be noted that, contrary to other fibrocartilaginous entheses, there is no correlation between frequency and size of enthesophytes at the ligamenta flava attachment sites and age-at-death [START_REF] Cunha | What can bones tell about labour and occupation: the analysis of skeletal markers of occupational stress in the Identified Skeletal Collection of the Anthropological Museum of the University of Coimbra (preliminary results)[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF].

Excessive mechanical stress in terms of frequency, speed and/or intensity can cause a series of micro-traumatic insults that tend to disturb the tissue structure of the fibrocartilaginous enthesis [START_REF] Husson | Physiopathologie et anatomopathologie des enthésopathies d'insertion par sollicitations mécaniques du système tendinomusculaire[END_REF][START_REF] Khan | Histopathology of common tendinopathies. Update and implications for clinical management[END_REF][START_REF] Benjamin | Where tendons and ligaments meet bone: attachment sites ("entheses") in relation to exercise and/or mechanical load[END_REF]. In young adults, these mechanical stresses are the main factor in the occurrence of an activity-related enthesopathy [START_REF] Rodineau | Pathogénie des enthésopathies du membre supérieur[END_REF]. In the older individual, on the contrary, it is the gradual depletion of tendon vascularity close to the insertion that favours the occurrence of lesions [START_REF] Rodineau | Pathogénie des enthésopathies du membre supérieur[END_REF].

Biomechanical parameters are, in this case, a secondary factor. Other factors may increase the (Commandré, 1977;[START_REF] Rodineau | Pathogénie des enthésopathies du membre supérieur[END_REF][START_REF] Bard | Physiopathologie, réparation, classification des tendinopathies mécaniques[END_REF]. The work of several authors, including Khan andcollaborators (1999) andMilz andcollaborators (2004), clearly indicate that overuse enthesopathies are similar to degenerative ones described in older individuals. Thus, micro-and macro-bony avulsions, tidemark irregularity (disorganization of the layer of calcified fibrocartilage), vascularisation of the fibrocartilage, calcification, and ossification of soft tissues can be observed in cases of overuse enthesopathy [START_REF] Dupont | Tendinites corporéales et ostéotendinites, essai de définition des lésions et de leur traitement chirurgical[END_REF][START_REF] Husson | Physiopathologie et anatomopathologie des enthésopathies d'insertion par sollicitations mécaniques du système tendinomusculaire[END_REF][START_REF] Saillant | Aspect macroscopique des enthésites. Rapport avec le socle osseux. Bursites associés[END_REF][START_REF] Potter | Lateral epicondylitis: correlation of MR imaging, surgical and histopathologic findings[END_REF][START_REF] Selvanetti | Overuse tendon injuries: basic science and classification[END_REF]. It is noteworthy that in these sports medicine reports, skeletal alterations are, in most cases, inconspicuous.

Sports and occupational injuries at tendons and entheses seem, at present, more common in women [START_REF] Punnett | Work-related musculoskeletal disorders: is there a gender differential, and if so, what does it mean?[END_REF][START_REF] Bard | Physiopathologie, réparation, classification des tendinopathies mécaniques[END_REF]. However, these differences are not observed for all anatomical sites and many sex-related parameters (e.g. muscle mass, fat mass, size, morphology), may interact (e.g. [START_REF] Punnett | Work-related musculoskeletal disorders: is there a gender differential, and if so, what does it mean?[END_REF][START_REF] Bard | Physiopathologie, réparation, classification des tendinopathies mécaniques[END_REF]. Among the factors involved, ovarian hormones, including estradiol and relaxin, could play an important role. The contribution of these hormones in reducing the amount of glycosaminoglycans and collagen has been demonstrated for fibrocartilaginous joints [START_REF] Naqvi | Relaxin's induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants[END_REF][START_REF] Hashem | Relaxin and betaestradiol modulate targeted matrix degradation in specific synovial joint fibrocartilages: progesterone prevents matrix loss[END_REF].

Moreover, these hormones promote hyper-laxity and increase the risk of intrinsic mechanical lesions [START_REF] Punnett | Work-related musculoskeletal disorders: is there a gender differential, and if so, what does it mean?[END_REF][START_REF] Bard | Physiopathologie, réparation, classification des tendinopathies mécaniques[END_REF]. At the menopause, blood levels of estrogen drop significantly [START_REF] Sowers | Menopause: its epidemiology[END_REF]. This decrease causes a change in the composition of collagen connective tissue, including ligaments, associated with loss of elasticity [START_REF] Falconer | Changes in paraurethral connective tissue at menopause are counteracted by estrogen[END_REF][START_REF] Ewies | Changes in extracellular matrix proteins in the cardinal ligaments of post-menopausal women with or without prolapse: a computerized immunohistomorphometric analysis[END_REF].

2) Diaphyses

As for fibrocartilaginous entheses, the following discussion on properties of fibrous entheses during skeletal immaturity is based on animal models. These models are of two kinds: those focusing on gross anatomy and those studying the histological properties of these entheses.

The first type indicates that during growth, there is a relationship between muscle activity/properties and the morphology of attachment sites. For instance, Dyzart and colleagues (1989) demonstrated that denervation of the rat forelimb is followed by an abnormally formed humerus, notably a smaller and less curved deltoid tuberosity. Based on these findings they postulated "that muscle pull affects periosteal tension and consequently et al., 1989: 158). In a study of mutant strains of mice, Montgomery and colleagues (2005: 819) reached a slightly different conclusion: "These findings suggest that muscle attachment sites expand during growth in order to accommodate increases in muscle size and mass, but that expansion of these bony regions is not necessarily dependent on increases in muscle contractile strength […]".

If these experiments provide interesting insights into the effects of muscle properties and activity on bone morphology, the study of histological properties of attachment sites appears more informative for a better understanding of the "normal" appearance of fibrous entheses in the immature human skeleton. In dog, rabbit and rat studies on diaphyseal entheses, tendons and ligaments attach via periosteum during growth [START_REF] Laros | Influence of physical activity on ligament insertions in the knees of dogs[END_REF]Dörfl, 1980aDörfl, , 1980b;;[START_REF] Hurov | Soft-tissue bone interface: How do attachments of muscles, tendons, and ligaments change during growth? A light microscopic study[END_REF][START_REF] Matyas | The development morphology of a "periosteal" ligament insertion: Growth and maturation of the tibial insertion of the rabbit medial collateral ligament[END_REF][START_REF] Gao | An immunohistochemical study of enthesis development in the medial collateral ligament of the rat knee joint[END_REF][START_REF] Wei | The postnatal development of the insertions of the medial collateral ligament in the rat knee[END_REF]. Both osteoclastic and osteoblastic activity are seen at fibrous attachment sites during this period and appear to be mainly related to the migration of the attachments of tendons and ligaments during the growth in length of long bones [START_REF] Hoyte | Wolff's law and the problem of muscle attachment on resorptive surface of bone[END_REF]Dörfl, 1980aDörfl, , 1980b;;[START_REF] Hurov | Soft-tissue bone interface: How do attachments of muscles, tendons, and ligaments change during growth? A light microscopic study[END_REF]. It is noteworthy that muscular traction plays no role in this migration (Dörfl, 1980a(Dörfl, , 1980b;;[START_REF] Grant | The effect of changes in muscle function and bone growth on muscle migration[END_REF]. All these animal model studies included the tibial insertion of the medial collateral ligament. This ligament attaches to the tibia of growing individuals in an area called the "metaphyseal depression", where growth-related osteoclastic resorption is more predominant than osteoblastic activity (Dörfl, 1980a;[START_REF] Matyas | The development morphology of a "periosteal" ligament insertion: Growth and maturation of the tibial insertion of the rabbit medial collateral ligament[END_REF][START_REF] Wei | The postnatal development of the insertions of the medial collateral ligament in the rat knee[END_REF]. Osteoclasts are most obvious at the periosteal side of the bone but they are also seen at the endosteal or marrow side [START_REF] Wei | The postnatal development of the insertions of the medial collateral ligament in the rat knee[END_REF]. It is noteworthy that the "metaphyseal depression" disappears in mature rabbits [START_REF] Matyas | The development morphology of a "periosteal" ligament insertion: Growth and maturation of the tibial insertion of the rabbit medial collateral ligament[END_REF], but a shallow depression persists in rats up to 120 days of age, which was interpreted by [START_REF] Wei | The postnatal development of the insertions of the medial collateral ligament in the rat knee[END_REF] as a sign of continuing growth. Many biological anthropologists (e.g. [START_REF] Saunders | The development and distribution of discontinuous morphological variation of the human infracranial skeleton[END_REF][START_REF] Castex | Insertion du grand pectoral et du grand rond sous forme de fosse[END_REF][START_REF] Stirland | Femoral non-metric traits reconsidered[END_REF][START_REF] Mariotti | Enthesopathies -Proposal of a standardized scoring method and applications[END_REF] reported high frequencies of a "fossa" in juveniles and young adults for several metaphyseal attachment sites (e.g. humeral insertions of the Mm. pectoralis major and teres major and the femoral insertion of the M. gluteus maximus). Actually, these grooves or "fossae" are very common in immature human skeletons, in frequency but also in their distribution in the body (Fig. 5), and it may be tentatively suggested that they are related to a process similar to that described for the tibial "metaphyseal depression" in rats and rabbits. These changes, especially for the humeral insertion of the M. pectoralis major, seem to be more dramatic in males during late adolescence and early adulthood, before the complete fusion of the epiphysis [START_REF] Mariotti | Enthesopathies -Proposal of a standardized scoring method and applications[END_REF]. Consequently, linking these erosions in young adult males solely to mechanical [START_REF] Hawkey | Activity-induced musculoskeletal stress markers (MSM) and subsistence strategy changes among ancient Hudson Bay Eskimos[END_REF] appears, at least, highly hazardous (see Villotte, 2008b for a review of the possible causes of a "fossa" at the humeral insertion of the M. pectoralis major in adults). Moreover, the bottom of these grooves is usually not smooth in juvenile human skeletons: marked porosity, short striae and small asperities are often present [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF]. One could speculate that those changes are related to the irregularity of the mineralization front (i.e. the superficial cortex) before skeletal maturity in other mammals [START_REF] Matyas | The development morphology of a "periosteal" ligament insertion: Growth and maturation of the tibial insertion of the rabbit medial collateral ligament[END_REF][START_REF] Wei | The postnatal development of the insertions of the medial collateral ligament in the rat knee[END_REF].

FIGURE 5

In mature animals the periosteal layer may or may not disappear, depending on the species and the enthesis. In adult humans, periosteal fibrous entheses are seen where muscles attach to a large area by short fibrous ends [START_REF] Kenesi | Anatomie et biomécanique des enthèses[END_REF], and for some masticatory muscles [START_REF] Hems | Tendon entheses of the human masticatory muscles[END_REF]. As the mediating layer of periosteum often disappears with age and leaves the soft tissue attaching directly to bone [START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF], it has been hypothesized that the physiological transition from a periosteal to a bony attachment in early adulthood may explain the high frequency of skeletal changes (i.e. irregularity) seen in young/middle-aged adults [START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF]. Benjamin and collaborators (2002: 934) note that "relatively little attention has been paid to fibrous entheses, even though they are associated with some of the largest and most powerful muscles in the body [...]. This partly reflects a clinical bias toward fibrocartilaginous entheses -which are more vulnerable to overuse injuries, but also the attraction of working with a richer variety of tissues that such entheses can offer." In a study that focuses mainly on fibrocartilaginous entheses, Benjamin and collaborators (2007) briefly describe two modifications observed in elderly subjects at the fibrous insertion of M. pronator teres: a bony production and a vascular invasion of the fibrous tissue. Micro-trauma at fibrous entheses are described mainly for periosteal attachment sites (e.g. [START_REF] Condouret | Périostites tibiales[END_REF]; they lead to a periostitis. Only a few cases were reported for bony ones: enthesopathy at the M. deltoideus insertion on the humerus in golfers and "pala" players (Commandré, 1977: 67) and small resorptive areas at the humeral insertion of the M. pectoralis major in gymnasts [START_REF] Fulton | Cortical desmoid-like lesion of the proximal humerus and its occurrence in gymnasts (ringman's shoulder lesion)[END_REF]. To conclude this section, it seems important to report an interesting study on the effect of inactivity for several entheses in dogs [START_REF] Laros | Influence of physical activity on ligament insertions in the knees of dogs[END_REF]. In active immature dogs, normal metaphyseal remodelling was seen with a marked bone resorption at the tibial insertion of the medial collateral ligament (i.e. a normal appearance, cf. supra). In inactive adolescent dogs, the reaction was more generalized. Contrary to the other entheses under study, simple caging for six weeks produced resorption at the tibial insertion of the medial collateral ligament in adult dogs. Moreover, after several weeks of immobilization in a plaster cast, resorptive changes at this enthesis were seen for the immobilized limb of adult dogs, but also in a lesser extent for the non-immobilized limb, free for activity and weight-bearing! With continued caging (over a period of six months or more) and in dogs sacrificed twelve weeks after removal of plaster immobilization, bone resorption healed as fibrous tissue replaced resorbed bone and then became mineralized.

Conclusion

In the last twenty years, researchers have published important works on the limits and pitfalls of interpretations of entheseal changes as activity markers (e.g. [START_REF] Dutour | Activités physiques et squelette humain : le difficile passage de l'actuel au fossile[END_REF][START_REF] Jurmain | Story from the skeleton[END_REF]Jurmain et al., 2012), mainly related to the problem of false positives (in our case, an entheseal change not related to physical activity). Based on our experience, exuberant bone production is mainly seen in older individuals, individuals with systemic disease or, locally, in cases of trauma. In many cases major entheseal changes are probably not directly related to physical activity, and certainly not only to micro-trauma at the enthesis.

While some biological anthropologists seem to have been completely unaware of these problems, others, notably [START_REF] Weiss | Understanding muscle markers: aggregation and construct validity[END_REF][START_REF] Weiss | Understanding muscle markers: lower kimbs[END_REF][START_REF] Weiss | Sex differences in musculoskeletal stress markers: Problems with activity pattern reconstructions[END_REF], have attempted to identify the "confounding" factors, but without adequately documented material. The archaeological record does not represent the best samples from which to identify the processes that produce alterations of an attachment site, for at least one good reason: the age-at-death assessment. This contribution illustrates the usefulness of clinical studies and studies based on identified skeletal collections for the understanding of entheseal changes. Based on the data obtained thus far, it seems that most of the changes seen for fibrous entheses cannot be directly associated with activity. In fact, some of the most common changes considered in biological anthropology -cortical defects at metaphyseal sites -may be related to growth and development, or even to inactivity. This hypothesis was formulated previously by [START_REF] Mafart | Quelques particularités anthropologiques et paléopathologiques d'une population de moniales médiévales de l'abbaye de Saint-Pierre de l'Almanarre (XIIIe-XIVe siècles, Hyères, Var). In L'identité des populations archéologiques[END_REF], though on archaeological criteria. If the study of enthesopathies for fibrocartilaginous [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies : nouvelle méthode[END_REF]. Black arrow: salient margin at the junction between the lesser trochanter and the femoral shaft, which can occur independently and was not taken into account in the same scoring system. Scale: 1 cm. Figure 5. Proximal tibial shaft of an immature individual (6-9 years old). The area of attachment of the M. soleus displays the classic appearance of immature metaphyseal entheses, a "fossa" with porosity, short striae and small asperities/irregularities at its bottom. Scale: 1 cm.

  are far better than those provided by archaeological samples because of numerous inherent biases (e.g. the effect of age and the difficulty to assess the ageat-death in archaeological samples, their unknown genetic background).

  uncalcified and calcified fibrocartilage, bone); -Formation of enthesophytes (bony spurs at the enthesis), induced by the healing process, after microtears; -Disturbance of collagen fibers and of the organization of cell columns; -Calcific deposits; -Increase of the thickness of the calcified fibrocartilage layer; -Vascularization of the calcified and uncalcified fibrocartilage layers; -Erosion of the surface and bone resorption beneath the enthesis.

  enthesopathy, for instance cold temperatures, the use of unsuitable equipment, very heavy muscular stresses endured without training or appropriate warm-up, and abnormal structures disrupting joint biomechanics

  growth in length" (Dyzart

  promising to attempt to reconstruct past activities, one cannot deny the numerous difficulties associated with their recording and the multiple factors leading to their appearance in the human skeleton.

Figure 3 .

 3 Figure3. Radial tuberosity. Changes occur at the medial part of the tuberosity -i.e. the attachment of the distal tendon of the M. biceps brachii (white arrow), and the lateral part that is the location of the bursa associated with this tendon (black arrow). Scale: 1 cm.

Figure 4 .

 4 Figure 4. Proximal humeral epiphysis of an immature individual (6-9 years old). The area of attachment of the M. supraspinatus and M.infraspinatus on the greater tubercle (white arrow) is not clearly distinguishable. Scale: 1 cm.

  al.,

	2004, 2007; Villotte, 2009; Alves
	F o
	r
	P
	e e r
	R e v i e w
	8

Table 1 .

 1 Common terminology used in biomedical sciences. There is no consensus in favour of either "entheseal" or "enthesial" and both are in standard use. Figure 1. Greater trochanter, insertion of the M. gluteus medius. Smooth imprint with regular margins. Scale: 1 cm.Figure 2. Lesser trochanter, insertion of the M. iliopsoas. White arrow: salient margin at the trochanter, taken into account in

	Tendon-to-bone insertion / Ligament-to-bone
	insertion
	Zone of insertion / Zone of attachment

*:

http://mc.manuscriptcentral.com/oa

http://mc.manuscriptcentral.com/oa International Journal of Osteoarchaeology

Acknowledgements

The authors thank Charlotte Henderson and Francisca Alves-Cardoso for the invitation to participate in the Symposium and the two reviewers for their useful comments.


MD, DrPH Arnaud Fontanet
email: fontanet@pasteur.fr

MSc, MPH Rebecca Grant

MSc Laura Tondeur

PhD Yoann Madec

Ludivine Grzelak

MSc Isabelle Cailleau

MD, PhD Marie-Noëlle Ungeheuer

MD Charlotte Renaudat

PhD Sandrine Fernandes Pellerin

MD Lucie Kuhmel

Isabelle Staropoli

François Anna

Pierre Charneau

Caroline Demeret

PhD Timothée Bruel

PhD Olivier Schwartz

MD, PhD Bruno Hoen

Affiliations

SARS-CoV-2 infection in primary schools in northern France: A retrospective cohort study in an area of high transmission

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

Introduction

As the coronavirus (COVID-19) pandemic continues to evolve, the extent of SARS-CoV-2 infection in children has not been well documented and the role children may play in virus transmission remains unclear. During the first epidemic wave, many countries included school closures among the measures implemented to limit viral transmission, largely based on the evidence of the impact of school closures on influenza transmission [START_REF] Cauchemez | Estimating the impact of school closure on influenza transmission from Sentinel data[END_REF][START_REF] Litvinova | Reactive school closure weakens the network of social interactions and reduces the spread of influenza[END_REF] . As many schools are now reopening, it is critical to evaluate the risk of viral circulation among pupils and their teachers in schools [START_REF] Esposito | School Closure During the Coronavirus Disease 2019 (COVID-19) Pandemic: An Effective Intervention at the Global Level?[END_REF] . Initial epidemiological data from China indicated that children were significantly less affected than adults, whether considering the total number of clinical cases, disease severity or fatal outcomes [START_REF] China | The Epidemiological Characteristics of an Outbreak of 2019 Novel Coronavirus Diseases (COVID-19) -China[END_REF] .

Similar findings were also reported in other countries [START_REF] Docherty | Features of 16,749 hospitalised UK patients with COVID-19 using the ISARIC WHO Clinical Characterisation Protocol[END_REF][START_REF]Coronavirus Disease 2019 in Children -United States[END_REF][START_REF] Riccardo | Epidemiological characteristics of COVID-19 cases in Italy and estimates of the reproductive numbers one month into the epidemic[END_REF] . It is now understood that children, when infected, present with mild and asymptomatic forms of the disease more often than adults [START_REF] Cao | SARS-CoV-2 infection in children: Transmission dynamics and clinical characteristics[END_REF][START_REF] Dong | Epidemiology of Covid-19 among children in China[END_REF][START_REF] Choi | Epidemiology and clinical features of coronavirus disease 2019 in children[END_REF] , with severe and fatal outcomes remaining rare in children [START_REF] Shekerdemian | Characteristics and Outcomes of Children With Coronavirus Disease 2019 (COVID-19) Infection Admitted to US and Canadian Pediatric Intensive Care Units[END_REF][START_REF] Verdoni | An Outbreak of Severe Kawasaki-like Disease at the Italian Epicentre of the SARS-CoV-2 Epidemic: An Observational Cohort Study[END_REF] .

Younger children are generally thought to be less susceptible to infection as compared to adults [START_REF] Gudbjartsson | Spread of SARS-CoV-2 in the Icelandic Population[END_REF][START_REF] Viner | Susceptibility to and transmission of COVID-19 amongst children and adolescents compared with adults: a systematic review and meta-analysis[END_REF][START_REF] Lavezzo | Suppression of COVID-19 outbreak in the municipality of Vo[END_REF][START_REF] Stringhini | Seroprevalence of anti-SARS-CoV-2 IgG antibodies in Geneva, Switzerland (SEROCoV-POP): a population-based study[END_REF][START_REF] Cohen | Assessment of spread of SARS-CoV-2 by RT-PCR and concomitant serology in children in a region heavily affected by COVID-19 pandemic[END_REF] , and, when infected, are usually contaminated by a household member [START_REF] Qiu | Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19) in Zhejiang, China: an observational cohort study[END_REF] . Some studies have nevertheless documented secondary attack rates in families as high in children as in adults [START_REF] Bi | Epidemiology and transmission of COVID-19 in 391 cases and 1286 of their close contacts in Shenzhen, China: a retrospective cohort study[END_REF] .

Children, when infected, may carry the virus in their throats for 9-11 days [START_REF] Qiu | Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19) in Zhejiang, China: an observational cohort study[END_REF] and for up to one month in stools [START_REF] Jiehao | A Case series of children with 2019 novel coronavirus infection: clinical and epidemiological features[END_REF] . Viral loads have been found to be similar between infected children and adults [START_REF] Jones | An analysis of SARS-CoV-2 viral load by patient age[END_REF][START_REF] Huillier | Shedding of infectious SARS-CoV-2 in symptomatic neonates, children and adolescents[END_REF] , which would suggest that children may be as infectious as adults. It is therefore unclear why children would be less susceptible, and less infectious, as compared to adults [START_REF] Patel | Nasal ACE2 Levels and COVID-19 in Children[END_REF] . Seroepidemiological studies are thus needed to determine the extent of infection in children and to decipher the role they may play in transmission To our knowledge, the number of SARS-CoV-2 secondary transmissions in school setting documented in scientific literature is limited, with very few or no secondary cases in investigations in Australia [START_REF] Ncfiras | COVID-19 in schools -the experience in NSW[END_REF] , Ireland [START_REF] Heavey | No evidence of secondary transmission of COVID-19 from children attending school in Ireland[END_REF] , and France 26 , with the exception of one important cluster in a high school north of Paris in February 2020 [START_REF] Fontanet | Cluster of COVID-19 in northern France: A retrospective closed cohort study[END_REF] .

It is all the more important to understand the extent of infection in children and the role they may play in transmission given the likely negative effects of school closures on educational achievement and economic outcomes 3 . To investigate the extent of infection in younger children, a follow-up seroepidemiologic investigation to that in the high school in a city north of Paris was conducted across primary schools in the same city. Here, we present the results of the follow up investigation in primary schools.

Methods

Study setting

An initial retrospective epidemiological investigation was conducted in the Crépy-en-Valois city (15,000 inhabitants) north of Paris, France after the diagnosis of the first case of COVID-19 on 24 February 2020. This investigation identified an epidemic around a local high school with two teachers having symptoms consistent with COVID-19 as early as on 2 February 2020. Since there was no known circulation of SARS-CoV-2 at that time in the region, no public health or social measures intended to limit the viral transmission had been implemented and no active SARS-CoV-2 testing had been conducted. A preliminary rapid investigation among symptomatic adults and pupils at the high school on 5-6 March 2020 revealed that 11/66 (16.7%) adults and 2/24 (8.3%) pupils had acute infection, as determined by a positive RT-PCR test result. As a follow-up to this rapid investigation, the decision was made to further examine by serological testing the extent of infection among pupils, their parents and relatives, teaching staff and non-teaching staff of 1) the high school where the two teachers worked and 2) the primary schools in the same city. The high school investigation has previously been reported [START_REF] Danis | Cluster of coronavirus disease 2019 (COVID-19) in the French Alps[END_REF] . Here, we describe the follow-up seroepidemiologic investigation across six primary schools from the same city, among children aged 6 to 11 years.

Study design

A retrospective cohort study was conducted by inviting all pupils, teachers and non-teaching staff (administrative, cleaners, catering) from each of the six primary schools who were registered at the school from the beginning of the epidemic (estimated around 13 January 2020) up to the time of the investigation (28-30 April 2020). Since pupils were minor, at least one parent was invited to provide informed consent for their child. The parents were also invited to participate in the study, as well as any of the other children or relatives over the age of 5 years living in the same household.

Following informed consent, participants (with the help of their parents in the case of pupils) completed a questionnaire that covered sociodemographic information, underlying medical conditions, history of recent symptoms, and history of COVID-19 diagnosis prior to this investigation.

A 5-mL blood sample was taken from all participants.

Laboratory analyses

Samples were transferred to and stored in the Clinical Investigation and Access to BioResources (ICAReB) biobank platform at the Institut Pasteur (Paris, France), which collects and manages human bioresources for scientific purposes, following ISO 9001 and NF S 96-900 quality standards (BRIF code n°BB-0033-00062). Serological testing was conducted using the S-Flow assay, a flowcytometry-based serological test developed by the Institut Pasteur. The assay is based on the recognition of the SARS-CoV-2 Spike protein expressed at the surface of 293T cells. In previous studies, the sensitivity of the assay was estimated at 99.4% (95% confidence interval (CI) = 96.6% -100%) on a panel of 160 RT-PCR confirmed mild forms of COVID-19 [START_REF] Fafi-Kremer | Serologic responses to SARS-CoV-2 infection among hospital staff with mild disease in eastern France[END_REF] , while its specificity was found to be 100% (one-sided 97.5% CI = 97.4% -100%) on a panel of 140 pre-epidemic sera [START_REF] Grzelak | SARS-CoV-2 serological analysis of COVID-19 hospitalized patients, pauci-symptomatic individuals and blood donors[END_REF] .

Case definitions

Any participant with a positive serology at the time of blood sampling was considered as having had SARS-CoV-2 infection. Each infection was categorized as symptomatic if any recent symptoms were reported by the participant up until 7 days prior to the date of sample collection to allow sufficient time for seroconversion [START_REF] Wölfel | Virological assessment of hospitalized patients with COVID-2019[END_REF][START_REF] Zhao | Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019[END_REF] , or, alternatively, as asymptomatic. Symptoms were further categorized as major (fever, dry cough, dyspnea, anosmia and ageusia) or minor (sore throat, rhinitis, muscle pain, diarrhea, headache, asthenia, vomiting, nausea, chest pain, abdominal pain).

Statistical analyses

The IAR was defined as the proportion of all participants with SARS-CoV-2 infection based on antibody detection in the collected blood sample by the end of the first COVID-19 epidemic wave.

Participants were further categorized as children if under 18 years of age (pupils, relatives of the pupil living in the same household) and adults if 18 years or older. The analysis was also broken down by school, and by time period (before and after February 14, date of the school closure for two-week holidays immediately followed by a local lockdown on 1 March 2020). The IAR was compared according to participants' characteristics using chi-square test and Fisher exact test, where appropriate. All statistical analyses were performed using Stata 15.0 (StataCorp, College Station, TX, USA).

Ethical considerations

This study is included as part of the registration on ClinicalTrials.gov under the identifier NCT04325646, cohort 'CORSER-2c,' and received ethical approval by the Comité de Protection des Personnes Ile de France III. Informed consent was obtained from all participants.

Results

From 28 to 30 April 2020, 1047 pupils and 51teachers, from six primary schools, with children aged 6 to 11 years, were invited by email to participate in the investigation. Of these, 541 (51.5%) pupils and 46 (90.2%) teachers accepted to participate in the study. Thirty-one pupils were excluded as they refused phlebotomy, as were four teachers not directly affiliated with any of the six schools. This resulted in 510 pupils and 42 teachers with blood sample to be analyzed. In addition, 641 parents of pupils, 119 relatives of pupils sharing the same household, and 28 non-teaching staff completed the study population (Figure 1). Table 1 indicates the characteristics of the 1340 participants. Pupils and their parents constituted the majority of the study population (38.1% and 47.8%, respectively).

Most participants were female (57.4%), particularly among teaching (90.5%) and non-teaching (89.3%) staff. The pupils were aged 6-11 years, while the median (IQR) age was 40 (37-44) years for parents, 47.5 (40-51) years for teachers, and 47.5 (32-54) years for non-teaching staff (Table 1).

The overall IAR across study participants was 139/1340 (10.4%). It did not differ by gender, age categories, or type of participants (Table 2). The epidemic curve, based on symptoms experienced by participants with SARS-CoV-2 antibodies, had no specific pattern, and transmission does not appear to have been impacted by the closure of schools for holidays on February 14 (end of week 7) (Figure 2A). There were three instances in three separate schools of high suspicion of SARS-CoV-2 infection in pupils before the closure of the school for holidays (end of week 7), one in week 6, and two in week 7. There were no secondary cases in pupils, teachers and non-teaching staff of the corresponding schools in the 14 days following these initial cases, except for one teacher who had onset of symptoms nine days later, but also had a close contact with a confirmed case outside of the school five days prior to becoming sick. Parents of infected pupils had higher IAR compared to parents of non-infected pupils (61.0% versus 6.9%; P <0.0001), and relatives of infected pupils had higher IAR compared to relatives of non-infected pupils (44.4% versus 9.1%; P = 0.002) (Table 2 & Figure S1). Among adults, fever, cough, dyspnea, ageusia, anosmia, muscle pain, sore throat, headache, asthenia, and diarrhea were all associated with the detection of SARS-CoV-2 antibodies (Table 3). Ageusia and anosmia, reported among 48% of adult participants, had a high positive predictive value for infection: 75.0% and 90.7%, respectively. In children, only asthenia and diarrhea were associated with SARS-CoV-2 antibodies. Only two children with SARS-CoV-2 antibodies experienced anosmia and ageusia, and both were 15 years of age. Among the 139 participants with SARS-CoV-2 antibodies, only two (1.4%, 95% CI = 0.2% -5.1%), both parents, were hospitalized. There were no deaths.

Across the study period, 9.9% of seropositive adults, and 41.4% of seropositive children, reported no symptoms (P <0.001). Symptoms of respiratory infections -fever, cough, rhinitis -were common among the participants without SARS-CoV-2 antibodies during the study period, with a marked decrease after lockdown was introduced on 1 March 2020 (Figure 2B).

Discussion

This study in one of the first seroepidemiologic investigations on SARS-CoV-2 in the primary school setting. Despite three introductions of the virus into three primary schools, there appears to have been no further transmission of the virus to other pupils or teaching and non-teaching staff of the schools.

In families of infected pupils, the prevalence of antibodies was very high, suggesting intrafamilial clustering of infections. Finally, children experienced mild forms of disease, with many of them being asymptomatic.

We can infer from the reported date of symptom onset among seropositive individuals that viral circulation in the study population presumably began around week 5 (27-31 January 2020).

Transmission continued to increase up to week 10 (2-6 March), with no effect of school closure for holidays (14 February). Transmission stabilized and then declined after week 13 (23-27 March). Since there was no reported circulation of SARS-CoV-2 during the month of February in the region until the diagnosis of the first local case on 24 February 2020, adherence to any public health or social measures intended to limit the transmission of the virus was likely low, allowing us to document the natural circulation of the virus in the community.

We could identify three symptomatic SARS-CoV-2 infected pupils in three separate schools during the three weeks preceding closure of the school for holidays and then lockdown. There were no secondary cases in pupils, teachers and non-teaching staff of the corresponding schools in the 14 days following these initial cases. These findings are in line with previous studies from Australia [START_REF] Ncfiras | COVID-19 in schools -the experience in NSW[END_REF] , Ireland [START_REF] Heavey | No evidence of secondary transmission of COVID-19 from children attending school in Ireland[END_REF] , and France [START_REF] Danis | Cluster of coronavirus disease 2019 (COVID-19) in the French Alps[END_REF] . They differ however from the results of the study performed in the high school of the same city in France, in which 38% of pupils, 43% of teaching staff and 59% of nonteaching staff who participated in the investigation had anti-SARS-CoV-2 antibodies [START_REF] Fontanet | Cluster of COVID-19 in northern France: A retrospective closed cohort study[END_REF] . This study would suggest that high school aged children have similar susceptibility to SARS-CoV-2 infection as adults, and can transmit SARS-CoV-2 efficiently. The reasons why the observed onwards transmission from primary school aged children was lower than in adolescents warrant further investigation. Given that viral load among infected children and adults have been found to be similar [START_REF] Jones | An analysis of SARS-CoV-2 viral load by patient age[END_REF][START_REF] Huillier | Shedding of infectious SARS-CoV-2 in symptomatic neonates, children and adolescents[END_REF] , milder symptoms among children may explain the reduced onward transmission. In households, we found a high prevalence of antibodies among parents and relatives of infected pupils (61% and 44%, respectively). Considering the low onward transmission from pupils in schools, the high prevalence figures observed in families more likely resulted from transmission from the parents to the child rather than the opposite, which has already been suggested by others [START_REF] Qiu | Clinical and epidemiological features of 36 children with coronavirus disease 2019 (COVID-19) in Zhejiang, China: an observational cohort study[END_REF] .

In adults, symptoms associated with SARS-CoV-2 infection were fever, cough, shortness of breath, ageusia, anosmia, headache, asthenia, muscle pain, sore throat, and diarrhea, all known features of the disease. Symptoms with highest predictive values for SARS-CoV-2 infection were anosmia and ageusia, as previously reported [START_REF] Bénézit | Utility of hyposmia and hypogeusia for the diagnosis of COVID-19[END_REF] . Symptoms were less specific in children, with only fatigue and diarrhea being associated with SARS-CoV-2 infection. Anosmia and ageusia were rarely seen (1% of children), and only in those aged 15 years or older. This study also gave an opportunity to estimate the proportion of asymptomatic forms among infected. Here, we found that the asymptomatic fraction was higher in children than in adults (41.4% vs 9.9%, respectively; P < 0.001).

Our results are limited by the short timeframe for studying the impact of the presence of infected pupils in schools before closure, which happened only two weeks after the first cases of COVID-19 developed in pupils. Still, the absence of well characterized viral spread in the primary school as opposed to what had been observed in the nearby high school at the same time suggests that children aged 6-11 years may be less contagious than high school aged pupils. Another limitation was the incomplete sampling of classes and families, preventing from a full exploration of viral circulation in the schools and households. The clinical findings of our investigation were also limited by the fact that information on symptoms was collected retrospectively, and that other respiratory viruses were circulating concurrently in the study population.

Conclusion

The findings of our investigation are in line with other reports which suggest limited transmission of SARS-CoV-2 in primary schools. These findings suggest that reopening of primary schools can be considered carefully, with continuous monitoring of possible resurgence in infections and strategies to limit transmission such as hand hygiene, physical distancing, respiratory etiquette and masks for older children.

Tables and Figures

Figure 1 .Figure 2 .

 12 Figure 1. Flowchart of enrolment of participants Figure 2. Timeline of symptom onset among (A) 107 symptomatic individuals who were seropositive for anti-SARS-CoV-2 antibodies; (B) 631 symptomatic individuals who were seronegative for anti-SARS-CoV-2 antibodies.

Table 1 .

 1 Sociodemographic characteristics of the 1340 participants of the SARS-CoV-2, France from 28-30 April 2020

Table 2 .

 2 Infection attack rate (IAR) according to sociodemographic characteristics

Table 3 .

 3 IAR (%) by symptoms and type of participant

Table 3 .

 3 IAR (%) by symptoms and type of participant

		61 (12.0)	67 (10.4)	20 (16.8)	163 (12.2)
		90 (17.6)	108 (16.8)	18 (15.1)	228 (17.0)
		68 (13.3)	78 (12.2)	15 (12.6)	171 (12.8)
		87 (17.1)	116 (18.1)	24 (20.2)	239 (17.8)
		117 (22.9)	151 (23.6)	25 (21.0)	304 (22.7)
	3 (10.7)	87 (17.1)	121 (18.9)	17 (14.3)	235 (17.5)

* Only among participants who declared symptoms .

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

Acknowledgements

We would like to thank the Directorate General for Health for facilitating the initiation of the study, Santé publique France for linking with their investigation team, the Agence Régionale de Santé des Hauts de France, and the Academia of Amiens for their continuous support throughout the realization of the study, especially Ms. Delphine Maskara. We would like to thank the mayor of Crépy-en-Valois, Bruno Fortier and his team, Mrs Sandra Riveros, the local police, and local paramedical staff, Nathalie Bourgeois, Mélissa Mannevy, and Michaël Mannevy, for their help in the implementation of the study. We would like also to thank Nathalie de Parseval, Claire Dugast, Valentine Garaud, Soazic Gardais, Caroline Jannet, Fanny Momboisse, Isabelle Porteret, Hantaniaina Rafanoson, Sandrine Ropars who participated in the organization and the realization of the field investigation. Julian Buchrieser and Rémy Robinot who assisted in the development of serological tests. And finally Nathalie Jolly and Cassandre van Platen, who took care of the regulatory aspects of the study, and Christine Fanaud who informed the participants of their serological status.

Role of the funding source

The study was funded by Institut Pasteur, and several laboratories participating in the study receive funding from the Labex IBEID (ANR-10-LABX-62-IBEID), REACTing and the INCEPTION project (PIA/ANR-16-CONV-0005) for studies focusing on emerging viruses. OS lab is funded by Institut Pasteur, ANRS, Sidaction, the Vaccine Research Institute (ANR-10-LABX-77), "TIMTAMDEN" ANR-14-CE14-0029, "CHIKV-Viro-Immuno" ANR-14-CE14-0015-01 and the Gilead HIV cure program.

LG is supported by the French Ministry of Higher Education, Research and Innovation.