Jessica Kojadinovic

Paco Bustamante
email: paco.bustamante@univ-lr.fr

Carine Churlaud

Richard P Cosson

Matthieu Le Corre

Mercury in seabird feathers: Insight on dietary habits and evidence for exposure levels in the western Indian Ocean

Keywords: Marine birds, Trophic ecology, Mercury, Bioaccumulation, Contamination, Tropical islands

Breast feathers were used to estimate mercury levels in six marine birds nesting in the tropical western Indian Ocean, i.e. Sooty Tern (Sterna fuscata), Brown Noddy (Anous stolidus), Lesser Noddy (Anous tenuirostris), Audubon Shearwater (Puffinus lherminieri bailloni), Barau's Petrel (Pterodroma baraui) and the White-tailed Tropicbird (Phaethon lepturus). Juveniles consistently showed lower plumage mercury than adults. The lowest mean level was noted in juvenile Sooty Terns from the Glorioso Archipelago (0.05 µg.g -1 ). The highest levels were obtained for adult Barau's Petrels from Reunion Island (0.96 µg.g -1 ). An inter-site analysis of Sooty Tern showed higher mercury levels in birds nesting on Juan de Nova Island. Levels were low in comparison with values reported in the plumage of seabirds worldwide. The potential impacts of the size, the type (fish/cephalopod) and the origin (epi-/meso-pelagic) of prey on mercury intake in birds are discussed. Although the diet composition of individuals within a species appeared to be quite variable, combining results on mercury levels with common knowledge of each species allowed additional information on their dietary and foraging habits to be unraveled.

Introduction

Seabirds are a major component of tropical oceanic environments. Yet, knowledge of tropical seabirds in general, and of their trophic ecology in particular, is still lacking. This is particularly obvious in the tropical western Indian Ocean, home to many avian species that breed on very dispersed islands, forming large colonies sometimes numbering hundreds of thousands of pairs [START_REF] Le Corre | Assessment of the seabird community of the Mozambique Channel and its potential use as an indicator of tuna abundance[END_REF]. This lack of knowledge may be attributed in part to the logistical difficulties involved in studying animals that scatter over huge areas. Consequently, most knowledge of feeding ecology obtained by conventional methods is restricted to the bird's breeding season because sightings usually occur close to islands and stomach contents (generally obtained at the colony) only gives information on the seabird's last meal. Global Positioning System (GPS) satellite tracking methods, however, have recently been used to investigate the foraging ecology of non-breeding birds (Weimerskirch et al., 2005a).

Because of its bioaccumulation specificities, the analysis of mercury (Hg) in the tissues of seabirds and their prey is a useful technique to complement conventional studies. Mercury levels in an organism depend on a certain number of factors such as its species, its size and the type (fish/cephalopod), the size and/or the origin (demersal vs. pelagic or epivs. meso-pelagic) of its prey [START_REF] Monteiro | Mercury concentrations in prey fish indicate enhanced bioaccumulation in mesopelagic environments[END_REF]Stewart et al., 1997b;[START_REF] Monteiro | Relationship between mercury levels and diet in Azores seabirds[END_REF][START_REF] Becker | Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age[END_REF][START_REF] Na | Mercury in feathers from Chilean birds: influence of location, feeding strategy, and taxonomic effiliation[END_REF][START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF]. Interpreting mercury levels in birds with respect to these factors may offer clues to the birds' principal dietary mercury sources and hence provide information on their dominant prey. Mercury is also the only metal known to be clearly biomagnified in the food chain [START_REF] Atwell | Biomagnification and bioaccumulation of mercury in an arctic marine food web: insights from stable nitrogen isotope analysis[END_REF]. Its determination in seabirds also serves as reference for environmental contamination monitoring as seabirds are considered as good bioindicators of mercury levels in the marine environment [START_REF] Furness | Birds as Monitors of Environmental Changes[END_REF].

Many studies of mercury in marine birds rely on the use of feathers (e.g. [START_REF] Doi | Mercury in feathers of wild birds from the mercury-polluted area along the shore of the Shiranui Sea, Japan[END_REF][START_REF] Burger | Lead, cadmium, selenium and mercury in seabird feathers from the tropical mid-Pacific[END_REF][START_REF] Thompson | Long-term changes in mercury concentrations in herring gulls Larus argentatus and common terns Sterna hirundo from the German North Sea coast[END_REF]. The non-invasive sampling of feathers allows them to be used for the study of threatened or endangered species, permits the sampling of large numbers of birds, and enables repeated sampling from the same individual over many years. During feather formation, mercury present in the blood and mercury mobilized from various tissues such as liver or kidneys (essentially methyl mercury) is sequestered in the growing feathers, where it binds to sulfhydryl groups of keratin [START_REF] Goede | The use of bird feather parts as a monitor for metal pollution[END_REF][START_REF] Burger | Metals in feathers of brown noddy (Anous stolidus): Evidence for bioaccumulation or exposure levels?[END_REF]. Once the feather is formed, the blood ir-rigation subsides implying that no further metal can be deposited. Therefore, feathers remain as a record of exposure during the months preceding feather growth. Body feathers and wing coverts are the major elimination routes for mercury in the plumage [START_REF] Lewis | Mercury accumulation and excretion in laboratory reared black-headed gull Larus ridibundus chicks[END_REF]. Breast feathers, that will be used in this study, are particularly interesting because they do not reflect molt sequence as do flight feathers. Furthermore, they represent the plumage as a whole and usually show low variability in mercury levels providing the best approximation of the bird's exposure [START_REF] Lewis | Mercury levels in eggs, tissues, and feathers of herring gulls Larus argentatus from the German Wadden Sea Coast[END_REF]Burger and Gochfeld, 2001a).

This investigation is based on the use of breast feathers to estimate mercury levels in six marine birds inhabiting the tropical western Indian Ocean, viz. the Sooty Tern (Sterna fuscata), the Brown Noddy (Anous stolidus), the Lesser Noddy (Anous tenuirostris), Audubon's Shearwater (Puffinus lherminieri bailloni ), Barau's Petrel (Pterodroma baraui ) and the White-tailed Tropicbird (Phaethon lepturus). Phylogenetic, biological (age), ecological (diet) and geographical (breeding location) factors influencing mercury levels in feathers were investigated. The results were discussed in the light of the bio-and ecological specificities of each species to reveal information on their dietary habits. The tropical zone of the Indian Ocean houses very little anthropogenic activity. It is suspected that this situation may result in lower mercury levels in the avian top predators inhabiting this oceanic area in comparison with those found in temperate oceans, and perhaps in other tropical waters under anthropogenic influences. Mercury levels determined in these six seabirds were thus compared with mercury in feathers of marine birds originating from other areas worldwide.

Materials and methods

Study sites

The seabirds studied during this investigation originated from colonies established across the western Indian Ocean. The following islands were chosen in the intention of giving a representative overview of the region (Figure 1

Studied seabirds

The main morphological and biological characteristics of the studied species are presented in Table 1 and in the following sections.

Table 1

Morphological and biological characteristics of seabirds breeding on islands of the south-western Indian Ocean (Schreiber & Burger 2001[START_REF] Schreiber | Sooty tern[END_REF], Ramos & Pacheco 2003, Surman & Wooler 2003, Le Corre pers. observ.). Data given for specific islands correspond to measures originating from this study.

Geographical distribution and breeding patterns

The Sooty Tern, a widely scattered pan-tropical seabird, is the most numerous of the six species with nearly 6 million pairs living in the western Indian Ocean where they breed on various islands: Cosmoledo, Europa, Glorioso, Juan de Nova, Madagascar, Mauritius, Rodrigues, Seychelles and Zanzibar [START_REF] Schreiber | Sooty tern[END_REF][START_REF] Le Corre | Assessment of the seabird community of the Mozambique Channel and its potential use as an indicator of tuna abundance[END_REF]Feare et al., in press). They breed synchronously in very large colonies although breeding seasons vary among islands (in austral winter at Europa and Seychelles, in austral summer at Juan de Nova), and among years (on Lys Island for example) [START_REF] Le Corre | Breeding seasons of seabirds at Europa Island (southern Mozambique channel) in relation to seasonal changes in the marine environment[END_REF]. The Brown Noddy and the White-tailed Tropicbird are also widely distributed throughout tropical oceans and breed on numerous islands of the Indian Ocean. Both are asynchronous breeders although Brown Noddies are fairly synchronous in Seychelles. The synchronous Lesser Noddy is present in Australia, and breeds extralimitally in the tropical Indian Ocean in Rodrigues, Seychelles, Saint Brandon (Mauritius), the Chagos Archipelago, and the Maldives [START_REF] Higgins | Handbook of Australian[END_REF]. Audubon's Shearwater is also asynchronous, and is found in the Indian Ocean (Comoros, Europa, Mayotte, Reunion and Seychelles), the western Pacific around the Galapagos Islands, as well as in the Antilles, the Bahamas and the Caribbean. However, the birds considered in this study belong to the subspecies P. l. bailloni which is known to breed only on Reunion and Europa islands [START_REF] Le Corre | Taxonomic affinities of Audubon's shearwater from Europa Island[END_REF]. The least widespread of these seabirds is Barau's Petrel which is endemic of Reunion, where its population is estimated at 4 000 to 6 500 pairs, with a breeding season extending from November to May (Le Corre et al., 2001).

Diet

Diet composition of the studied species is known only for the breeding period from stomach content of adults and chicks.

In Seychelles, Sooty Terns eat mostly goatfish and squid (Ramos 2000, pers. comm.). In Reunion, stomach content analysis indicates that the diet of Audubon Shearwaters is composed of 50% squid (mainly Sthenoteuthis oualaniensis) and 50% fish, that Barau's Petrel is mostly squid-eating, with 98% squid (S. oualaniensis and Taonius sp.) and 2% fish and that the White-tailed Tropicbird eats 80% squid (mainly S. oualaniensis) and 20% fish (Mollidae and Dactylopteridae) (Le Corre, unpubl. data). The local dietary prey composition of the two Noddies is not known. In the Mozambique Channel, Sooty Terns generally prey on fish of the following families: Carangidae, Exocoetidae, Hemiramphidae, Scombridae and Sphyraenidae (Jaquemet et al, in prep.). All squids consumed belong to the Ommastrephidae family with the dominant species being S. oualaniensis. More specifically, Sooty Terns from Europa mainly feed on flying squids and juveniles of epi-pelagic and meso-pelagic fishes. At Juan de Nova, Sooty Terns mostly consume small fish and especially larvae of coastal Mullidae and Engraulidae. On Lys Island, their diet is composed of flying squids, juveniles and larvae of epi-pelagic fish, and larvae of reef fish (Jaquemet et al, pers. com.). The diet of the two Noddies from Lys Island is unknown.

Bird sampling

Feather sampling was conducted in 2004 on each island except for Reunion Island where the sampling effort extended from 2001 to 2004. Sampling acts were conducted in colonies, during the species' breeding period, except for the Lesser Noddies which were sampled at night while non-breeders were roosting on Lys Island. The number of birds sampled on each island is given in Table 2. Breast feathers were collected on live birds caught on the ground with a net, or on birds which died from accidental causes, i.e. on healthy birds. Because of their spontaneous attraction to light, petrels and shearwaters often die from fatal collisions with urban light sources. This is particularly frequent in Reunion Island (Le Corre et al., 2001) where bird colonies are situated mostly inland forcing birds to fly over a considerably urbanized coastline. Simultaneously to the feather sampling, each individual was assigned to one of two age-classes, juvenile or adult, on the basis of beak and plumage features.

Sample preparation and mercury analysis

Pectoral feathers were washed vigorously in two series of triple baths of 0.25 N sodium hydroxide solution alternated with triple baths of deionized water, and dried for 24 h at 50 • C in order to remove adherent external deposits and airborne contamination [START_REF] Walsh | The use of seabirds as monitors of heavy metals in the marine environment[END_REF][START_REF] Burger | Diving depths of shearwaters[END_REF] which could alter the results of the analysis of mercury sequestrated in the feathers. Thereby, the existence of external mercury contamination was substantiated by the comparison of mercury levels in 10 feather samples of which one aliquot was washed according to the latter protocol and the second aliquot was simply agitated in deionized water. The results showed significantly higher mercury levels in the second aliquots (by 1.6 to 5.9 fold) in comparison with the aliquots washed in sodium hydroxide (ttest : p < 0.001).

Total mercury analyses were carried out with an Advanced Mercury Analyzer (ALTEC AMA 254) on aliquots ranging from 5 to 20 mg of dried sample weighed to the nearest 0.01 mg. The detection limit, accuracy and reproducibility of the preparation were tested by preparing replicates of lobster hepatopancreas (TORT-2) reference standards (National Research Council, Canada) and blanks along with each set of samples. The detection limit, determined as 3× the value of at least 8 blanks, was equal to 2.5 ng.g -1 . Accuracy (mean±SD) was estimated as the closeness of each measured value to the certified value of TORT-2 (0.27±0.06 µg.g -1 ); measured values were always within 6% of this reference value. Reproducibility was estimated as the closeness of replicate measurements: the coefficient of variation given by relative standard deviations was always lower than 20%. Mercury levels are expressed in µg.g -1 of dry weight (d.w.).

Interpretation of plumage mercury levels

Most adult seabirds undergo molt at sea during the non-breeding season [START_REF] Cramp | Handbook of the birds of Europe, the Middle East and North Africa , Vol. 1 Ostrich to ducks[END_REF]. As there is no documentation on the molting processes of the studied species in the western Indian Ocean and as no molting adult was found in the colonies, we have assumed here that they all molt during the non-breeding season. As mercury levels in feathers correspond to part of the dietary input since the last molt, their analysis in breast feathers of juvenile birds reflects the dietary mercury intake since hatching, and are thus indicators of the bioavailable mercury in prey present in the water masses in which adults forage to feed their young. On the contrary, the breast feathers collected on adults present on the island during the breeding period were grown while the bird was at sea. The mercury levels in these feathers testify of the mercury availability in the waters adults fed from during the non-breeding season.

Statistical analysis

Statistical analyses were performed using the GNU R statistical system (R Development Core Team, 2005). All statistical samples submitted to tests were first checked for normality by means of Shapiro-Wilk test. In all cases, nonparametric tests were used as mercury values did not follow Gaussian trends. The influence of species and location on mercury levels was tested by means of Kruskal-Wallis (KW ) test followed, in case of significant differences, by multiple pairwise comparison Wilcoxon (W ) test. Wilcoxon tests were also applied to test the influence of age classes on mercury levels. Levels of significance of these tests were divided into classes of p-values (NS ≥ 0.05 ; * < 0.05 ; * * < 0.01 ; * * * < 0.001), with standard deviation (SD), sample size (n) and coefficient of variation (CV) indicated.

Results

Feather mercury levels for each species x age x location subgroup are presented in Table 2. All samples were above the method detection limit. The highest mean mercury level was noted in adult Barau's Petrels from Reunion Island and the lowest in juvenile Sooty Terns from Lys Island in the Glorioso Archipelago (19 times lower). These large differences in mercury impregnation across subgroups testify of the influence of age, species and location on the plumage mercury levels of these seabirds.

Table 2 Mercury levels (Mean ± SD, µg.g -1 d.w.), inter-specific and inter-locational comparison results in feathers of juvenile and adult seabirds from islands of the southwestern Indian Ocean. 8

Age-class mercury levels

The influence of age on mercury level in the feathers was tested on 4 of the 6 species. Age-class comparisons were conducted for the islands of Lys and Reunion separately (Figure 2). Adult:juvenile mercury ratios were similar among species from the same study site, and slightly different between islands: 3.6, 3.2 and 2.9 for Audubon Shearwater, Barau's Petrel and White-tailed Tropicbird respectively in Reunion, and 4.6 and 4.0 for Sooty Tern and Brown Noddy respectively in Lys.

Species related differences within sites

On Lys, there were no significant differences in feather mercury levels among adult Brown Noddies, Lesser Noddies and Sooty Terns (Table 2). On Reunion Island, Barau's Petrels and White-tailed Tropicbirds had significantly (p < 0.001) higher mercury levels than Audubon Shearwaters in both, juveniles and adults. Adult Brown Noddies had significantly (p = 0.023) higher levels of mercury than adult Audubon Shearwaters and significantly (p = 0.014) lower levels than adult petrels.

Geographical mercury patterns

Plumage mercury levels were significantly higher in adult Sooty Terns from Juan de Nova than in those breeding on Europa (p = 0.001), Lys (p = 0.012) or Bird islands (p = 0.001) (Table 2). Differences among the later islands were not significant. From the few results obtained for Brown Noddies, seabirds breeding on Reunion Island seem to be subject to higher mercury exposure than those breeding on Lys Island (Table 2).

Discussion

Comparison with other species and areas

In Table 3, feather mercury levels in seabirds from this study are compared to the same, or phylogenically related species from other locations in the world. Elemental levels in juveniles and adults are presented separately.

Worldwide, mercury in tern feathers covers a large range of levels generally between 0.10 and 12 µg.g -1 d.w. [START_REF] Burger | Risk, mercury levels, and birds: relating adverse laboratory effects to field biomonitoring[END_REF]. Mean mercury levels in adult terns from the western Indian Ocean (this study) were close to the lower limit of this range of values. Moreover, they were lower than levels reported for the same tern species from the Pacific and Atlantic Oceans, and those measured in Bridled Tern, Roseate Tern, Common Tern, Arctic Tern and Black Noddy originating from tropical, temperate and arctic environments (Table 3).

Procellariiformes are known to concentrate much higher elemental levels than many other seabirds, with burdens leveling off at 24 µg.g -1 wet weight (w.w.) in the Southern Indian Ocean White-chinned Petrel for example (Kim et al., 1996b). Adult Barau's Petrels had the highest mean mercury levels among the studied species with 0.96 µg.g -1 d.w. It, nonetheless, fitted in the 0.6-2.7 µg.g -1 d.w. range previously reported for other Petrels and Shearwaters [START_REF] Burger | Risk, mercury levels, and birds: relating adverse laboratory effects to field biomonitoring[END_REF]. In fact, Barau's Petrels had much lower mercury levels than other petrel species of equivalent or smaller sizes originating from other oceans [START_REF] Gochfeld | Metals in feathers of Bonin Petrels, Christmas Shearwaters, Wedge-tailed Shearwaters and Red-tailed Tropicbird in the Hawaian islands, Northern Pacific[END_REF][START_REF] Na | Mercury in feathers from Chilean birds: influence of location, feeding strategy, and taxonomic effiliation[END_REF]. Mean mercury levels in adult Audubon Shearwaters were also below most reported values (Table 3).

The Phaethon genus has received very limited attention regarding elemental bioaccumulation. Feather mercury levels in White-tailed Tropicbirds from Reunion were more than seven times lower than those found in Red-Tailed Tropicbirds from Midway Island [START_REF] Burger | Metal levels in feathers of 12 species of seabirds from Midway Atoll in the northern Pacific Ocean[END_REF].

Since seabirds are considered as good bioindicators of mercury levels in the marine environment [START_REF] Furness | Birds as Monitors of Environmental Changes[END_REF], the above comparisons of mercury levels in seabird feathers may serve in comparing worldwide environmental mercury levels. The comparison of singles species (or closely related species) from various areas suggests that mercury availability in the western Indian Ocean is low in comparison to other marine environments, including other tropical ecosystems.

Although some of the comparison areas presented above can be considered as relatively enriched in mercury, such as the Mediterranean Sea and some regions of the Atlantic (e.g. the Azores), the recorded values are much lower than levels noted in coastal polluted areas. For example, levels equal to 4.1 µg.g -1 d.w.

were noted in Little Egrets sampled in Hong Kong [START_REF] Connell | Risk to breeding success of Ardeids by contaminants in Hong Kong: evidence from trace metals in feathers[END_REF]. Mercury levels in feathers of Herring Gulls from the German North Sea coast showed a peak of 12 µg.g -1 d.w. during the 1940s, presumably due to mercury discharges during the Second World War [START_REF] Thompson | Long-term changes in mercury concentrations in herring gulls Larus argentatus and common terns Sterna hirundo from the German North Sea coast[END_REF]. Also, piscivorious seabirds collected along the shores of the Shiranui Sea, where Minamata disease occurred in the 1960s had mean mercury levels of 7.1±3.7 µg.g -1 d.w. [START_REF] Doi | Mercury in feathers of wild birds from the mercury-polluted area along the shore of the Shiranui Sea, Japan[END_REF]. Although laboratory studies that examine mercury levels in feathers associated with behavior, growth, or reproductive abnormalities are scarce, a range of 5-40 µg.g -1 d.w. is considered associated with adverse effects [START_REF] Burger | Risk, mercury levels, and birds: relating adverse laboratory effects to field biomonitoring[END_REF]. In this study, the highest mercury level recorded among all birds was more than five times lower than this toxicity level. It can therefore be presumed that the marine birds considered during this work do not suffer from adverse effects of mercury.

Age-related differences in mercury levels

Most studies that examine the differences in mercury levels between adult and juvenile birds report higher levels in adults (e.g. [START_REF] Becker | Feather mercury levels in seabirds at South Georgia: influence of trophic position, sex and age[END_REF] which substantiates our results. Mercury levels in adults are higher than in young birds because they may eat larger, more contaminated prey, and/or because they have accumulated mercury in their tissues over a longer period of time. Indeed, although feathers act as a route of mercury excretion, a certain percentage of the body burden is not evacuated into the plumage [START_REF] Lewis | Mercury accumulation and excretion in laboratory reared black-headed gull Larus ridibundus chicks[END_REF]Kim et al., 1996a) which leads, to some extent, to an increase of mercury with age in some species.

Site and species differences in relation to food sources

Variations in mercury levels between bird species potentially reflect many factors including feeding and migratory habits, body size, life span, molt strategy and taxonomic influences on physiology [START_REF] Walsh | The use of seabirds as monitors of heavy metals in the marine environment[END_REF][START_REF] Stewart | Influence of phylogeny, diet, moult schedule and sex on heavy metal concentrations in New Zealand Procellariiformes[END_REF]. However, dietary differences have often been proposed as the best explanation of mercury variability [START_REF] Honda | Metal concentrations in pelagic seabirds from the north Pacific Ocean[END_REF][START_REF] Fiona | Influence of phylogeny, diet, moult schedule and sex on heavy metal concentrations in New Zealand Procellariiformes[END_REF] since food is the major intake path in marine vertebrates [START_REF] Walsh | The use of seabirds as monitors of heavy metals in the marine environment[END_REF][START_REF] Stewart | Influence of phylogeny, diet, moult schedule and sex on heavy metal concentrations in New Zealand Procellariiformes[END_REF]. Detailed information on the diet of pelagic seabirds is scarce as they may be studied only during their breeding season while they are on land. Moreover, feeding habits can change with habitat and season, diminishing the reliability of information obtained by stomach content analysis or foraging observations. Although it is difficult to link observed mercury levels with uptake from the diet without analyzing the potential prey, interspecies differences in plumage mercury levels may offer relative information on the dietary composition.

Tropical seabirds consume a wide variety of prey among which the two major taxonomic groups are fish and squid [START_REF] Ballance | Foraging ecology of tropical seabirds[END_REF]. Fish prey are known to be a source of mercury contamination for top predators because of the high levels they accumulate with time (e.g. [START_REF] Van Den Broek | Mercury levels in some New Zealand sea fishes[END_REF][START_REF] Jaffar | Selected trace metal concentrations in different tissues of fish from coastal waters of Pakistan (Arabian Sea)[END_REF]) and because mercury is chiefly under a bioavailable form (Me-Hg) in the muscle tissue [START_REF] Wagemann | Percent methylmercury and organic mercury in tissues of marine mammals and fish using different experimental and calculation methods[END_REF]. On the other hand, studies on the bioaccumulation of mercury in squid offer conflicting results [START_REF] Mcarthur | Mercury in the marine food chain in the Southern Ocean at Macquarie Island: an analysis of a top predator, Patagonian toothfish (Dissostichus eleginoides) and a mid-trophic species, the warty squid (Moroteuthis ingens)[END_REF][START_REF] Seixas | Accumulation of mercury in the tissues of the common octopus Octopus vulgaris (L.) in two localities on the Portuguese coast[END_REF]. Recent work has however shown that most (70-90%) of the total mercury in cephalopods is in an organic form [START_REF] Bustamante | Total and organic Hg concentrations in cephalopods from the North East Atlantic waters: influence of geographical origin and feeding ecology[END_REF]. Moreover, squids are considered more as vectors of Cd and Persistent Organic Pollutants (POPs) transfers [START_REF] Bustamante | Cephalopods as a vector of the transfer of cadmium to top marine predators in the North-East Atlantic Ocean[END_REF][START_REF] Weisbrod | Species, tissue and gender-related organochlorine bioaccumulation in white-sided dolphins, pilot whales and their common prey in the Northeast Atlantic[END_REF]. The difference in bioaccumulation potential of these two types of prey may explain part of the difference noted between the mercury-enriched Sooty Terns from Juan de Nova and Sooty Terns breeding on the other three islands. Indeed, according to current knowledge, Sooty Tern diets differ among study sites. Squid constitutes the major dietary resource in Europa and Lys (in reconstituted mass) while fish prey are predominant in Juan de Nova (Section 2.2.2, Jaquemet et al., pers. com.).

It has been demonstrated in previous studies that the inclusion of meso-pelagic prey in seabird diet has an influence on mercury accumulation over and above that of trophic status [START_REF] Monteiro | Relationship between mercury levels and diet in Azores seabirds[END_REF]. More specifically, it has been suggested that the comparatively high mercury levels in Barau's Petrels are due to them feeding on meso-pelagic squids of the Taonius genus [START_REF] Kojadinovic | Trace elements in three marine birds breeding on Reunion Island (Western Indian Ocean) Part 1: Factors influencing their bioaccumulation[END_REF]. This supposition relies on the hypothesis that these cephalopods are mercury enriched because they originate from deep, poorly oxygenated waters where mercury is mainly in its organic, more bioavailable form [START_REF] Monteiro | Relationship between mercury levels and diet in Azores seabirds[END_REF][START_REF] Thompson | Seabirds as biomonitors of mercury inputs to epipelagic and mesopelagic marine food chains[END_REF]). Barau's Petrel is most probably a scavenger (Ballance and Pitman, 1999, Le Corre unpubl. data). Although it has not been observed, Barau's Petrels could also feed on carcasses of larger animals such as marine mammals floating at the water surface. The access to such high trophic level and, consequently, potentially more mercury-enriched food due to mercury biomagnification, could be another source of mercury intake for Barau's Petrels.

Finally, considering that the major internal mercury source in birds is food and that plumage mercury levels differ with prey size, type and origin, coefficient of variation values (Table 2) may be interpreted as the variability of the diet compositions within species×age-class×location subgroups. Most CV values were fairly high, indicating that individuals of the same subgroup have diversified diets. These birds are indeed most likely opportunists as they have little choice of prey in these oligotrophic environments. Sooty Terns require a minimal mass of meal but that the nature of the prey is less important (Jaquemet et al., pers. com.).

Conclusion

The present work on mercury in the plumage of six seabird species, along with a recent investigation on mercury in pelagic fish [START_REF] Kojadinovic | Mercury content in commercial pelagic fish and its risk assessment in the Western Indian Ocean[END_REF], suggests that mercury availability in the western Indian Ocean is low in comparison to other marine environments.

The study of the impact of diet on mercury levels is far from simple in tropical species for which trophic relationships are often complex because of the large numbers of prey species available [START_REF] Ballance | Foraging ecology of tropical seabirds[END_REF]. The present investigation has nonetheless enabled us to point out some of the factors influencing mercury intake in the birds considered and obtain clues to certain aspects of their dietary and foraging habits. The investigation of mercury levels of the main prey species of these birds would be useful in pointing out the mercury sources for each seabird, which in turn would help in estimating the importance of each prey in its diet. It would also be of great interest to associate these mercury values to more complete stomach content analysis as well as stable isotope studies to further explore the trophic relationships existing in these seabird communities.

Fig. 1 .

 1 Fig. 1. Map of the study sites (western Indian Ocean).

Fig. 2 .

 2 Fig.2. Comparison of mercury levels (µg.g -1 d.w.) in feathers of juvenile (J) and adult (A) seabirds. AS: Anous stolidus; PLB: Puffinus lherminieri bailloni; PB: Pterodroma baraui; PL: Phaethon lepturus; SF: Sterna fuscata. The significances of the level differences between juveniles and adults are indicated below the boxplots. Significances were not given when at least one of the tested subgroups had too few values (-).

Table 3

 3 Mercury levels (Mean±SD, µg.g -1 ) in seabird feathers.

Acknowledgments

We acknowledge a grant to J.K. from the Conseil Régional de La Réunion and the European Social Fund. The authors would like to thank S. Jaquemet (University of La Réunion) and the SEOR (Société d'Etudes Ornithologiques de La Réunion) for providing the birds, as well as E. Robert (University of La Réunion) for her help in the field work and in the sample preparation. They also thank J. Ramos and P.C. Tavares for their helpful comments on the biology of seabirds breeding in Seychelles.


Bruno Dubois
email: bruno.dubois@aphp.fr

MD Nicolas Villain

MD Giovanni B Frisoni

MD Gil D Rabinovici

MD Marwan Sabbagh

MD Stefano Cappa

MD Alexandre Bejanin

PhD Stéphanie Bombois

MD Stéphane Epelbaum

Marc Teichmann

MD Marie-Odile Habert

Agneta Nordberg

MD Kaj Blennow

Douglas Galasko

MD Yaakov Stern

PhD Christopher C Rowe

MD Stephen Salloway

MD Lon S Schneider

MD Jeffrey L Cummings

Howard H Feldman

Prof Bruno Dubois

Clinical diagnosis of Alzheimer's disease: recommendations of the International Working Group

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

Introduction

In 2018, the National Institute of Aging (NIA) and the Alzheimer Association (AA) proposed the amyloid, tau, neurodegeneration (ATN) research framework for the definition and diagnosis of Alzheimer's disease (AD) 1 . This framework has enabled the move from a clinical-biological diagnosis to a purely biological definition of AD that can be applied in both the asymptomatic and symptomatic stages of the disease. The increased accessibility to biomarkers and the perspective to have blood-based information on the underlying pathophysiological process in the future make it necessary to consider the limitations of biomarkers in the diagnosis of AD and to propose recommendations for how these biomarkers might and might not be used for the diagnosis of AD in a clinical setting.

I -Evolution of the diagnostic frameworks for Alzheimer's disease

Over the last 15 years, there has been a remarkable evolution in the development and availability of in-vivo AD biomarkers, in the characterization of the natural history of the disease, and in turn in the application of this new knowledge to revised diagnostic research frameworks for AD (see Table 1). The first revision to the National Institute of Neurological and Communicative Disorders and Stroke and the Alzheimer's Disease and Related Disorders Association criteria 2 was provided by the International Working Group (IWG) in 2007 3 . It was the first research framework to propose AD as a clinical-biological entity based on in-vivo biomarkers with specific clinical phenotypes of AD and to extend the definition to the prodromal (predementia) dementia stages. In 2010, the IWG introduced a new supporting lexicon for AD with recommendations in the classification of the preclinical stages without symptoms 4 including a) "asymptomatic at-risk" for those with biomarker evidence of Alzheimer pathology; b) "presymptomatic" for those carrying monogenic AD mutations 4 . The next important development occurred in 2011 with the NIA-AA criteria that defined three different preclinical stages, according to the amyloid cascade hypothesis: amyloid lesions first, then tau pathology causing neurodegeneration, and finally occurrence of subtle cognitive changes 5,6 . In 2016, an IWG and NIA-AA consensus advanced the classification scheme to include AD for research purposes diagnosed at a preclinical stage of the disease, on the presence of both in-vivo amyloid and tau positivity "where the risk of a further progression to clinical AD is high" 7 , a proposal that was further developed by Jack and collaborators 1,8 . In the latest 2018 NIA-AA diagnostic framework, AD diagnosis is centred exclusively around a biomarker definition of disease according to ATN status 1 . Even in the absence of any cognitive symptoms, those subjects having both abnormal amyloid and tau biomarkers (A+T+) were defined as 'Alzheimer's disease'.

The development of 'positive' in-vivo biomarkers has moved the field from diagnosing AD at the dementia stage, towards earlier AD diagnosis at the prodromal stage, and have introduced the potential for preclinical diagnosis. These developments are particularly relevant in advancing testable therapeutic hypotheses towards secondary prevention of AD.

II -Limitations of a purely biological definition of AD

These 2016 consensus meeting and 2018 criteria 1,7 have engendered significant debate about applying a biomarker-based disease diagnosis, with clinical symptoms and phenotype being used only for staging 9,10 . Four years after their introduction, a reevaluation of a diagnostic approach based only on biological markers is warranted, both on conceptual and evidence-based practical levels.

Risk of confusion between presence of brain lesions and disease

Based on A/T/N definition, AD may be considered as a purely biological condition, dissociated from a clinical component or patient status. By dissociating AD from a clinical phenotype, the disease equates to AD neuropathologic changes, whereas in 2012, neuropathologists stated that "There is consensus to disentangle the clinicopathologic[al] term 'Alzheimer's disease' from [Alzheimer's disease] neuropathologic[al] change" 11 . As a consequence, it provides a classification, which gathers under the same label -AD -an extended continuum from cognitively normal individuals to severely demented patients.

Low predictive accuracy (Panel 1)

A major limitation of a purely biological definition of AD is the lack of predictive accuracy.

Several recent studies indicate that the presence of tau and amyloid positivity is insufficient to definitively predict the invariable occurrence of symptoms (mild cognitive impairment [MCI] or dementia) in those subjects without clinical impairment (evidence is summarized in Panel 1).

Another challenge to a biomarker-only diagnosis of AD is that the presence in-vivo of biomarkers of AD lesions may not certify the existence of AD as the primary diagnosis: such lesions are a frequent co-pathology in other neurodegenerative diseases, dementia with Lewy bodies (DLB) being most frequent 12 . These patients should not be considered as having a primary diagnosis of AD. These exemplars of co-pathologies will create potential confusion for those receiving a biomarker-based diagnosis in such circumstances.

Presence of other pathologies

Conversely, neuropathologically-defined AD is frequently associated with other pathologies.

Pure AD pathology is the exception rather than the rule in autopsy cases (~3-30% of cases according to age 12 ). AD, as described years ago in its "pure" form, is a model that may not apply especially in late-life dementia where multiple proteinopathies are more frequent. Co-pathologies, including synucleinopathy, vascular pathology, non-AD tauopathies -in particular argyrophilic grain disease and cortical aging-related tau astrogliopathy -and TAR DNA binding protein 43 (TDP-43) pathology -in particular limbic-predominant age-related TDP-43 encephalopathy [LATE] neuropathological changesinfluence significantly both clinical trajectory and phenotypes of AD (~50-60% of dementia of the Alzheimer type cases are estimated to be attributable to these co-pathologies) [12][13][14][15][16] . Unfortunately, pathological biomarkers are currently unavailable for the non-AD proteinopathies, and disentangling AD from non-AD neurodegenerative diseases remains phenotypic or cannot be achieved in life.

Uncertainty regarding the pathogenesis model of AD

The biological model that supports the A/T/N classification opens up the possibility for research into the biological changes prior to the onset of symptoms, which is necessary to develop drugs for the earliest stage. Advocates for a biological definition of AD often refer to the cancer model where a long asymptomatic phase of the disease can exist and where all affected patients, located anywhere along this disease continuum, will benefit from the same therapeutic approach even at a preclinical stage. However, the follow-up of cognitively unimpaired biomarker positive individuals suggests that they do not all progress overtime. At the present time, the determination of whether AD fits better with the long and asymptomatic 'prostate cancer' continuum or with an at-risk model of the disease (where the asymptomatic (A+T+) would be the at-risk state, i.e. the precancerous condition, and where clinical phenotype would be the disease state, i.e. the cancer state) is not yet determined.

Finally, defining the disease by its pathological lesions only -and not by a clinical phenotypeleads to the risk of creating confusion particularly within the healthy oldest old where memory complaints and low levels of AD pathology are the rule 17,18 . Publications in the field of dementia, which propose that the disease is a myth [START_REF] Whitehouse | The myth of Alzheimer's : what you aren't being told about today's most dreaded diagnosis[END_REF] or a decoy [START_REF] Jean | Alzheimer, le grand leurre[END_REF] , illustrate the potential for confusion between AD and old age.

Taken together, these data indicate that amyloid and tau biomarkers are not sufficient to confidently predict the trajectory towards prodromal AD or AD dementia to define the AD continuum without clinical input. The relationships between co-occurrence of tau and amyloid pathology on the one hand, and the development of cognitive decline and neurodegeneration on the other hand remain uncertain at an individual level. Besides the dominating amyloid cascade model, additional pathogenesis models have been advanced, including the role of endosomal recycling deficiency [START_REF] Small | Endosomal recycling reconciles the Alzheimer's disease paradox[END_REF] , of immunity, of lipid metabolism and endocytosis deficiency [START_REF] Van Der Kant | Amyloid-β-independent regulators of tau pathology in Alzheimer disease[END_REF] , of vascular dysfunction [START_REF] Reitz | Alzheimer disease: Epidemiology, diagnostic criteria, risk factors and biomarkers[END_REF] , etc.

Difficulty in classifying cognitively unimpaired biomarker positive subjects (Panel 2)

The challenge to a purely biological definition of AD is mostly centred on the preclinical stage of the disease, i.e. in conditions where, by definition, cognitive testing does not support the presence of an AD phenotype. In case of symptomatic patients, the identification of the specific clinical phenotype (interfering or not with independence in everyday activities) is a major step for the diagnosis as it expresses the brain dysfunction that the biomarkers are signalling. Cognitively unimpaired individuals with biomarker positivity are not invariably doomed to subsequent cognitive decline: the best current estimates of lifetime dementia risk range from 5% to 42% [START_REF] Brookmeyer | Estimation of lifetime risks of Alzheimer's disease dementia using biomarkers for preclinical disease[END_REF] .

Because of the uncertainty of their evolution over time (Panel 1), asymptomatic individuals who are biomarker positive should be classified as "at-risk for progression" -ARP -distinguishing two different subgroups for clinical and research purposes: A) Some individuals will remain stable over long periods of time: this subgroup includes people who may never develop symptoms. These persons may compensate and maintain normal functioning for many years even in the presence of an on-going neurodegenerative process, or some may have no accelerated neurodegeneration despite AD brain lesions. [START_REF] Arenaza-Urquijo | Resistance vs resilience to Alzheimer disease[END_REF] B) Other will progress: this subgroup includes individuals who demonstrate signs of accelerated neurodegeneration and whose compensatory mechanisms are overcome. They can be reliably predicted to progress to prodromal AD and AD dementia in the future.

For research purposes, both subgroups have to be separated in order to define factors of prevention and to develop algorithms to predict progression. This risk of progression depends on several modulating factors whose magnitude and interactions still remain to be determined (see Panel 2). These factors are either risk markers or risk factors and are related to brain resilience (e.g. cognitive reserve) and resistance [START_REF] Arenaza-Urquijo | Resistance vs resilience to Alzheimer disease[END_REF] , biological and genetic factors that are directly related to the amyloid, tau, and their neurodegeneration-induced pathways (genetic protection and risk factors regarding tau binding proteins and amyloid precursor protein metabolism; protection and risk factors related to immunity, endocytosis, and lipid metabolism [START_REF] Kunkle | Genetic meta-analysis of diagnosed Alzheimer's disease identifies new risk loci and implicates Aβ, tau, immunity and lipid processing[END_REF][START_REF] Jansen | Genome-wide meta-analysis identifies new loci and functional pathways influencing Alzheimer's disease risk[END_REF][START_REF] Hamelin | Distinct dynamic profiles of microglial activation are associated with progression of Alzheimer's disease[END_REF][START_REF] Monsell | Genetic Comparison of Symptomatic and Asymptomatic Persons With Alzheimer Disease Neuropathology[END_REF][START_REF] Medina | Toward common mechanisms for risk factors in Alzheimer's syndrome. Alzheimer's[END_REF] ; newly described brain cellular senescence mechanisms [START_REF] Swanson | DT-01-07: TREATMENT OF EARLY AD SUBJECTS WITH BAN2401, AN ANTI-Aβ PROTOFIBRIL MONOCLONAL ANTIBODY, SIGNIFICANTLY CLEARS AMYLOID PLAQUE AND REDUCES CLINICAL DECLINE[END_REF] ), and co-pathology. For instance, is a 60-year-old APOE 4 carrier with a negative tau PET and an amyloid PET scan just below the threshold at less risk than an 85-year-old who is A+T+ with cerebrovascular lesions on MRI? We anticipate the emergence of age-and risk factor-adjusted individualized predictions and the ability to rank the risk by analogy with the example of cardiovascular risk factors [START_REF] Perk | European Guidelines on cardiovascular disease prevention in clinical practice (version 2012). The Fifth Joint Task Force of the European Society of Cardiology and Other Societies on Cardiovascular Disease Prevention in Clinical Practice (constituted by re[END_REF] . Such modelling is in its early stages but exemplified through the example of age adjusted polygenic hazard scores [START_REF] Desikan | Genetic assessment of age-associated Alzheimer disease risk: Development and validation of a polygenic hazard score[END_REF] . However, disclosing individual patient-level risk with the current state of knowledge is premature and to be avoided as current individualized prediction models perform insufficiently well in cognitively unimpaired subjects compared to those in the prodromal stage of AD [START_REF] Van Maurik | Personalized risk for clinical progression in cognitively normal subjects -The ABIDE project[END_REF] . Besides conceptual issues, there are several practical aspects that may limit the use of biomarkers for the pure biologically-based diagnosis of AD. They concern the thresholds, generalization, metric performance, accessibility, amongst others.

Biomarker thresholds

A biological diagnosis of AD that is linked to positive biomarkers brings the invariable need to define and be certain about thresholds and cut-off points of positivity, as any modification of these thresholds would significantly impact both the diagnosis and the stages of the disease. The clear-cut separation between negative and positive subjects in relation to a given biomarker is somewhat artificial and different from one site or one study to another. Factors contributing to this uncertainty include the specifics of the biomarker used as well as the threshold determination. Most of all, this binary threshold does not reflect the reality of amyloid and tau pathology which is continuous and present at a minimal level in almost all the aging population 18 , with important discrepancies between pathology burden and clinical symptoms at intermediate levels (Panel 1). It is noteworthy that neuropathology criteria of AD provide no cut-off for establishing AD but only define 'low, intermediate or high' levels of AD neuropathologic change 11 .

For amyloid biomarkers, the use of binary thresholds of amyloid PET standard uptake value ratio (SUVR) has long restricted the measure of amyloid to intermediate and high levels of amyloid deposition [START_REF] Villeneuve | Existing Pittsburgh Compound-B positron emission tomography thresholds are too high: Statistical and pathological evaluation[END_REF] .The use of different amyloid PET tracers is also a known factor of variability in measurements, though the centiloid quantification approach helps at diminishing its impact [START_REF] Klunk | The Centiloid project: Standardizing quantitative amyloid plaque estimation by PET[END_REF] .

Recent works (using the centiloid scale or longitudinal amyloid PET) open the possibility to identify earlier stages of amyloid accumulation [START_REF] Villain | Regional dynamics of amyloid-β deposition in healthy elderly, mild cognitive impairment and Alzheimer's disease: a voxelwise PiB-PET longitudinal study[END_REF][START_REF] Joie | Multisite study of the relationships between antemortem [ 11 C]PIB-PET Centiloid values and postmortem measures of Alzheimer's disease neuropathology[END_REF] . In CSF, a positive amyloid measure despite a negative amyloid PET or the use of novel biomarkers (for instance, CSF A34/42 ratio 39 ) might also prove to be reliable biomarkers of early amyloid deposition stages [START_REF] Liebsch | Aβ34 is a BACE1-derived degradation intermediate associated with clearance and Alzheimer's disease progression[END_REF][START_REF] Palmqvist | Cerebrospinal fluid analysis detects cerebral amyloid-β accumulation earlier than positron emission tomography[END_REF] .

For tau biomarkers, there is also a strong discrepancy between neuropathological identification (in post-mortem studies, the presence of tau Braak stages I-II is almost universal after the age 70) 18,[START_REF] Duyckaerts | Prevalence, incidence and duration of Braak's stages in the general population: Can we know?[END_REF] and in-vivo measures of tau aggregates on PET (~36% of (T+) detected after the age of 70 [START_REF] Jack | Associations of Amyloid, Tau, and Neurodegeneration Biomarker Profiles with Rates of Memory Decline among Individuals Without Dementia[END_REF] ). The current in-vivo definition ability of tau PET to detect 'tau positivity' using flortaucipir PET seems to correspond only to widespread tau pathology in the brain (i.e. Braak stages ≥IV) [START_REF] Fleisher | Positron Emission Tomography Imaging with [18F]flortaucipir and Postmortem Assessment of Alzheimer Disease Neuropathologic Changes[END_REF] , while CSF phospho-tau (p-tau) elevation may reflect earlier stages of tauopathy [START_REF] Mattsson-Carlgren | Aβ deposition is associated with increases in soluble and phosphorylated tau that precede a positive Tau PET in Alzheimer's disease[END_REF] . In the near future it might become possible to have in-vivo tau measurements that detect very early tau deposits, i.e. tau Braak stages I-II with 2 d generation tau PET tracers or by detecting other phosphorylated species of tau such as p-tau 217 in the CSF or the plasma [START_REF] Palmqvist | Discriminative Accuracy of Plasma Phosphotau217 for Alzheimer Disease vs Other Neurodegenerative Disorders[END_REF] .

In clinical practice, such an extension to "earlier" thresholds in amyloid or tau biomarkers may lengthen the duration of the asymptomatic stages and decrease the lifetime clinical progression probability. Biomarkers thresholds also depend on the intended use and no consensus on the context of use is currently available 1 .

On the other hand, evaluating cognitive changes and determining the stages in patients with AD (i.e. identification of objective cognitive impairment, MCI or dementia) also raise threshold issues. The emergence of lower thresholds in cognitive testing to define the 'objective subtle cognitive changes' entity further extends the clinical stages of neurodegenerative diseases, before the occurrence of MCI 6 . However, this increased sensitivity in the detection of cognitive decline comes at the expense of a reduced specificity, and numerous other etiologies can be responsible for the observed changes, such as personality traits, psychiatric disorders, and sleep apnea amongst others [START_REF] Chipi | Biomarker-based signature of alzheimer's disease in pre-MCI individuals[END_REF] .

Generalization and accessibility in clinical practice

Considering general medical practice and standard of care, the six currently available Cochrane reviews regarding the use of CSF or amyloid PET biomarkers have consistently led to the same conclusion: "we cannot recommend the routine use of [these biomarkers] in clinical practice" 47- 52 . These reviews considered the ability of biomarkers to predict the future occurrence of clinical AD dementia in patients with MCI. Their recommendation answers the pragmatic question in clinical practice: "will my patient decline?". As underscored by the Cochrane reviews, the prognostic value of biomarker diagnosis remains limited by the fact that 1) there is a high variability of decline rate amongst biomarker-positive AD patients [START_REF] Jang | Prediction of fast decline in amyloid positive mild cognitive impairment patients using multimodal biomarkers[END_REF] and 2) non-AD neurodegenerative diseases contribute to cognitive decline. The Cochrane reviews further point out the "the heterogeneity in the conduct and interpretation of the biomarkers and the lack of defined thresholds for determination of test positivity". The choice of lower or higher biomarker thresholds correspond to earlier or later pathological burden and may change the duration of the asymptomatic stages and decrease the lifetime clinical progression probability. Finally, the Cochrane reviews underline the high financial cost of PET exams and the invasiveness of CSF measurements, which limit their interest and applicability in clinical practice, especially in lowincome countries. While these limitations were highlighted for patients with MCI, but data are even more sparce for cognitively unimpaired individuals as available currently models are lacking in the clinical setting, especially in cognitively unimpaired subjects. As seen in Panel 1, the magnitude effect of biomarker positivity on clinical progression remains weak to moderate and was established on selected volunteers from research cohorts. The generalization of these cohort findings to clinical practice also faces several obstacles: the risk of attrition bias from these data is strong, especially in the Alzheimer's Disease Neuroimaging Initiative (ADNI) cohort with many individuals lost to follow-up [START_REF] Insel | Determining clinically meaningful decline in preclinical disease[END_REF] , and the risk of selection bias is even stronger since individuals included in these longitudinal cohorts were usually selected by advertisement and do not represent the variety of individuals in clinical practice to whom the biomarker investigations will be applied.

An increasing number of persons now seek consultation in memory clinics complaining of subjective memory problems or cognitive decline (SCD) while scoring normally on formal cognitive testing. SCD may be a risk factor of clinical progression [START_REF] Ebenau | ATN-classification and clinical progression in subjective cognitive decline: the SCIENCe project[END_REF] but the magnitude of prediction ability of SCD regarding cognitive decline remains low (OR ~1.5-3 [START_REF] Slot | Subjective cognitive decline and rates of incident Alzheimer's disease and non-Alzheimer's disease dementia[END_REF] ). SCD may result from many factors besides aging: anxiety, depression, fatigue, sleep disorders, attention deficits, and drug side effects amongst others [START_REF] Balash | Subjective memory complaints in elders: depression, anxiety, or cognitive decline?[END_REF] . In this regard, it is noteworthy that the frequency of CSF AD profiles between those with normal cognition and SMC is similar [START_REF] Jansen | Prevalence of cerebral amyloid pathology in persons without dementia: A meta-analysis[END_REF] . The heterogeneity of this population is important according to the nature of complaint (self-report versus report by an informant) and to the cognitive status (normal cognition versus subtle cognitive changes) 17 .

Other individuals do not report cognitive problems and may be just worried about future cognitive decline due to family history, results from commercial direct-to-consumer genetic testing or APOE status, or simply concerned about preserving their memory and general cognitive abilities (sometimes referred to as "worried well" patients). Such individuals are seeking to understand their risk and their future. They represent up to 20-30% of new patients in some specialty memory clinics [START_REF] Verity | The Worried Well? Characteristics of Cognitively Normal Patients Presenting to a Rural and Remote Memory Clinic[END_REF][START_REF] Van Der Flier | Optimizing patient care and research: The Amsterdam dementia cohort[END_REF][START_REF] Hejl | Potentially reversible conditions in 1000 consecutive memory clinic patients[END_REF] and in some of these cases, biomarker investigations may have been performed. In these situations, reliance on a biomarker-only diagnosis would require a very secure and tightly elaborated natural history connecting the positivity of biomarkers with an invariable -or at least extremely high probability of -subsequent expression of clinical symptoms. This is not the case today. Recent experience with the Sokrates study underscores some of the uncertainties (e.g. inability to make an accurate short-or medium-term prediction about cognitive decline for an individual), which are inherent in revealing amyloid PET results alone to cognitively unimpaired individuals [START_REF] Mozersky | Comprehension of an elevated amyloid positron emission tomography biomarker result by cognitively normal older adults[END_REF] . This issue also applies for patients who may undergo a work-up for other medical conditions where AD biomarkers are included in the absence of any clinical context, and are positive [START_REF] Epelbaum | How many patients are eligible for diseasemodifying treatment in Alzheimer's disease? A French national observational study over 5 years[END_REF] .

As a whole, evidence for the use of biomarkers in clinical practice remains highly disputed and suffers from a lack of evidence-based data to recommend biomarker assessments for those with subjective memory complaints (SMC).

Ethical concerns

Diagnostic disclosure is ethically challenging when physicians inform cognitively unimpaired individuals that they have an irreversible disease based on biomarkers when the clinical trajectory towards prodromal AD or AD dementia is uncertain and where there is no way to significantly prevent the development of symptoms in the absence of modifiable risk factors or specific therapies [START_REF] Schermer | On the reconceptualization of Alzheimer's disease[END_REF] . Disclosing biomarker results and the related risk profile should be seen as different from disease diagnosis disclosure. Within the lay community, AD is amongst the most feared diseases given its outcomes including profound disability and loss of personal dignity [START_REF]What America Thinks MetLife Foundation Alzheimer's Survey[END_REF] . A discourse by physicians where AD equates AD neuropathological changes is profoundly discrepant with patients' narratives where AD equates dementia, dependency, and death, and this can adversely impact the therapeutic alliance 9 . In the future, being said to be 'at-risk for progression' instead of 'in the preclinical stage of AD' may help in the discussion of the 'Risk-Benefit balance' regarding a putative treatment and its side effects.

III-The IWG recommendations for the clinical setting

Based on this evidence, the IWG proposes the following recommendations:

1. The diagnosis of AD is clinical-biological. It requires the presence of both a specific clinical phenotype of AD (P+) and biomarker evidence of AD pathology (A+T+).

Specific clinical phenotype commonly associated with AD pathology (common AD

phenotypes) are: the amnestic syndrome of the hippocampal type [START_REF] Dubois | Amnestic MCI or prodromal Alzheimer's disease?[END_REF] (typical), the posterior cortical atrophy variant [START_REF] Crutch | Consensus classification of posterior cortical atrophy[END_REF] , and the logopenic variant primary progressive aphasia [START_REF] Gorno-Tempini | Classification of primary progressive aphasia and its variants[END_REF][START_REF] Bergeron | Prevalence of amyloid-β pathology in distinct variants of primary progressive aphasia[END_REF] (Supplementary Figure 1). Other phenotypes, including the behavioural/dysexecutive variant [START_REF] Ossenkoppele | The behavioural/dysexecutive variant of Alzheimer's disease: clinical, neuroimaging and pathological features[END_REF][START_REF] Perry | Clinicopathological correlations in behavioural variant frontotemporal dementia[END_REF] , the cortico-basal variant [START_REF] Armstrong | Criteria for the diagnosis of corticobasal degeneration[END_REF][START_REF] Lee | Clinicopathological correlations in corticobasal degeneration[END_REF] , and the other variants of primary progressive aphasia [START_REF] Bergeron | Prevalence of amyloid-β pathology in distinct variants of primary progressive aphasia[END_REF][START_REF] Mesulam | Asymmetry and heterogeneity of Alzheimer's and frontotemporal pathology in primary progressive aphasia[END_REF] (Supplementary Figure 1), are less commonly related to AD pathology (uncommon AD phenotypes). These phenotypes may or may not interfere with independence in everyday activities.

3. With these common phenotypes, amyloid, and tau biomarkers positivity establishes an AD diagnosis (Table 2). The positivity of both amyloid and tau biomarkers is required because an amnestic phenotype with only amyloid positivity is not specific to AD, being observed in other neurodegenerative diseases with amyloid co-pathology, including LATE, DLB, etc 1,12,[START_REF] Villain | Alzheimer's Disease Including Focal Presentations[END_REF] or in patients with cerebral amyloid angiopathy and amnestic vascular cognitive impairment [START_REF] Jang | Cerebral Amyloid Angiopathy: A report of two cases[END_REF] (Supplementary Figure 1). On the other hand, an isolated amnestic syndrome of the hippocampal type with only tau biomarker positivity can be encountered in primary age-related tauopathy (PART) [START_REF] Bell | Neuropathologic, genetic, and longitudinal cognitive profiles in primary age-related tauopathy (PART) and Alzheimer's disease[END_REF][START_REF] Toledo | Alzheimer's disease cerebrospinal fluid biomarker in cognitively normal subjects[END_REF] or in atypical presentations of mixed 3R/4R frontotemporal lobar degeneration (FTLD) tau [START_REF] Villain | Alzheimer's Disease Including Focal Presentations[END_REF][START_REF] Mattsson-Carlgren | The implications of different approaches to define AT(N) in Alzheimer disease[END_REF] (Supplementary Figure 1). Finally, uncommon phenotypes with positive AD biomarkers should not be a priori classified as an established AD (Table 2): in such cases the clinician may deem that AD is not the dominant pathology driving the clinical phenotype but only a copathology. 4. Recommended biomarker measures for amyloid pathology are low CSF A42, increased CSF A40/42 ratio (which is, if possible, preferred to low CSF Aβ42), or increased tracer retention in amyloid PET; for tau pathology, we recommend high CSF p-tau (not total tau [t-tau] due to its lack of specificity [START_REF] Antonell | Synaptic, axonal damage and inflammatory cerebrospinal fluid biomarkers in neurodegenerative dementias[END_REF] ) or increased ligand retention in tau PET. Amyloid and tau PET recommendation for use in clinical practice is conditional on regulatory approval and reimbursement by payers in different countries.

Diagnosis conclusion requires clinician expertise in both the critically assessment of

clinical and biomarker results. The different situations encountered in clinical practice are summarized in Table 2. In the case of cognitive testing and/or biomarkers in the 'grey zone', it may be useful to complete the workup with another investigation (repeated measure of pathophysiological biomarker, clinical follow-up, use of neurodegeneration biomarkers such as FDG-PET, etc).

6. CSF investigation is prioritized as it provides simultaneous information on the 2 types of biomarkers (amyloid and tau) and is less expensive than amyloid-PET and/or tau-PET.

Where lumbar puncture is contraindicated, PET investigations are an alternative consideration.

7. In clinical practice, plasma biomarkers for amyloid and tau pathology are not currently recommended. Although promising, they require further standardization and validation before they can be broadly regarded as secure biomarker evidence of AD pathology (A+T+) [START_REF] Palmqvist | Discriminative Accuracy of Plasma Phosphotau217 for Alzheimer Disease vs Other Neurodegenerative Disorders[END_REF][START_REF] Nakamura | High performance plasma amyloid-β biomarkers for Alzheimer's disease[END_REF] .

8. In clinical practice, the investigation of pathophysiological biomarkers in cognitively unimpaired individuals is not recommended, given their current inability to predict reliable clinical trajectories of those who are asymptomatic with biomarker positive status (A+T+). In the future, if therapies or prevention programs show significant efficacy in delaying onset of disease it will probably change the need for biomarker investigations in these individuals, although the problem of the prediction of clinical trajectories in cognitively unimpaired biomarker positive individuals will still remain. 9. In cases where a biomarker investigation is performed in cognitively unimpaired individuals (for any specific situations: will to know, brain health services, expert centres, research project, cohort study, diagnostic workup for other conditions…), a risk stratification of biomarkers is proposed (Panel 3) distinguishing an absolute risk group (carriers of autosomal dominant monogenic mutations for AD) [START_REF] Dai | The genes associated with early-onset Alzheimer's disease[END_REF] , a high-risk group [START_REF] Jack | Associations of Amyloid, Tau, and Neurodegeneration Biomarker Profiles with Rates of Memory Decline among Individuals Without Dementia[END_REF] , and an undefined risk group, to be further clarified in the future as additional evidence accrues [START_REF] Ebenau | ATN-classification and clinical progression in subjective cognitive decline: the SCIENCe project[END_REF][START_REF] Yu | Frequency and longitudinal clinical outcomes of Alzheimer's AT(N) biomarker profiles: A longitudinal study[END_REF][START_REF] Younes | Identifying changepoints in biomarkers during the preclinical phase of Alzheimer's disease[END_REF] . The proposed stratification is a starting point for research purposes.

Validation studies on large cohorts with long periods of follow-up are needed. The challenge for the future is to reliably and predictably define the level of risk for further progression.

In these situations, subjects should be counselled before AD biomarker investigation so that they can be informed of the ins and outs of the test results and can decide whether or not the result will be disclosed. If they decide to receive the results and they return positive, subjects should be counselled that they are at-risk for subsequent clinical progression (ARP) to prodromal AD or AD dementia and not clinically diagnosed as having AD.

10. SMC and SCD, when isolated and not supported by objective cognitive impairment, are not specific enough to be considered as part of the AD phenotype [START_REF] Jessen | Prediction of dementia of Alzheimer type by different types of subjective cognitive decline[END_REF] . In cognitively unimpaired individuals, self-reported complaints and complaints reported by an informant should be clearly distinguished as the latter are more at-risk of progression [START_REF] Caselli | Subjective cognitive decline: Self and informant comparisons[END_REF] and merit a closer follow-up with regular clinical and neuropsychological evaluations.

11. Alzheimer's disease can be associated with other brain pathologies, including synucleinopathy [START_REF] Hamelin | Distinct dynamic profiles of microglial activation are associated with progression of Alzheimer's disease[END_REF] , vascular pathology, non-AD tauopathies, and TDP-43 pathology [12][13][14][15] .

Alternatively, lesions of the Alzheimer type are frequently observed as co-pathology in post-mortem examination of other neurodegenerative diseases 12,[START_REF] Bergeron | Prevalence of amyloid-β pathology in distinct variants of primary progressive aphasia[END_REF][START_REF] Perry | Clinicopathological correlations in behavioural variant frontotemporal dementia[END_REF] . In both situations, pathophysiological AD biomarkers can be positive [START_REF] Niemantsverdriet | Added Diagnostic Value of Cerebrospinal Fluid Biomarkers for Differential Dementia Diagnosis in an Autopsy-Confirmed Cohort[END_REF] . This is particularly ambiguous in the case of behavioural variant frontotemporal dementia (bvFTD), cortico-basal syndrome (CBS), semantic or non-fluent variants of primary progressive aphasia where the presence of positive AD biomarkers can be considered either as AD co-pathology or atypical forms of AD [START_REF] Bergeron | Prevalence of amyloid-β pathology in distinct variants of primary progressive aphasia[END_REF][START_REF] Perry | Clinicopathological correlations in behavioural variant frontotemporal dementia[END_REF][START_REF] Lee | Clinicopathological correlations in corticobasal degeneration[END_REF][START_REF] Mesulam | Asymmetry and heterogeneity of Alzheimer's and frontotemporal pathology in primary progressive aphasia[END_REF] (Supplementary Figure 1). In all these situations, it is recommended that the physician relies more on the phenotype and follow-up to determine the final diagnosis (AD or co-pathology) (Table 2). In some complex cases, only post-mortem evaluation will provide definitive information.

12. It is recommended that the physician objectively evaluates the added-value of biomarker investigation for each symptomatic patient according to the clinical situation (age, risk of comorbidity, complexity of the phenotype), the life context, the wishes of the patient to know the most likely diagnosis, the possibility to participate in a disease-modifying trial, and more globally the appreciation of how exactly this information will change the management of the patient. Biomarker investigations may also be limited by the availability, cost, and health care payment coverage of biomarkers across countries and centres as well as clinical situations.

13. When pathophysiological biomarkers are not available, patients should have a clinical syndromic diagnosis: 'amnestic AD phenotype' or 'logopenic variant primary progressive aphasia' etc (P+ with A and T unknown), where staging (MCI or dementia) may still be applied. In these situations, more attention should be given to rule out non-degenerative causes [START_REF] Villain | Alzheimer's Disease Including Focal Presentations[END_REF] . If a positive neurodegeneration biomarker (FDG-PET hypometabolism, T1-MRI atrophy, CSF NFL, etc) is associated with a common AD phenotype, the term 'neurodegenerative disease of Alzheimer type' can be used (Table 2).

Conclusion

While the definition of AD based exclusively on biological markers has gained significant traction in research settings, emerging studies suggest that the biomarker definition is not ready for application in clinical settings and for diagnosis of individuals without cognitive impairment.

The evidence of the natural history of those asymptomatic at-risk with positive biological markers is insufficient to predict subsequent cognitive decline and dementia. In light of these findings, we provide recommendations for diagnosis and disclosure in the clinical setting which avoid labelling AD in individuals who are biomarker positive and cognitively unimpaired and who are at-risk for progression to prodromal AD or AD dementia. We recommend that the diagnosis of AD in the clinical setting remains tied to the clinical phenotypic presentation.

There are several critical requirements to achieve the utility of biomarkers to predict progression to clinical stages of AD. The first is the relationship between pathological burden, biomarker thresholds, and the respective impact and weight of modulating factors in relation to future risk of clinical progression. The second concerns the pathogenesis of AD itself. As detailed above, data suggest that AD may result not only from tau and amyloid pathologies but from synergy and interactions among these pathologies which lead to the highest stages of protein accumulation, the highest rates of cognitive decline. Investigating such synergies and understanding protective factors in those who are asymptomatic with biomarkers of AD pathology represent both a direction to better define the disease and to prevent it in the future.

Glossary of terms

Alzheimer's disease: a clinico-biological entity defined by a specific clinical phenotype (either typical or atypical) associated with in-vivo evidence of Alzheimer's pathology Alzheimer's pathology: can be approached today in-vivo by biomarkers of amyloid pathology (decreased A42 or elevation of A 1-40/A 1-42 ratio in CSF; increased tracer retention in amyloid PET) and of tau pathology (increased P-tau in CSF; increased tracer retention in tau PET).

Prodromal Alzheimer: refers to the early symptomatic and pre-dementia phase of AD.

Alzheimer dementia: refers to the phase of AD where cognitive symptoms are sufficiently severe to interfere with social functioning and instrumental activities of daily living.

Typical AD: refers to the most frequent clinical phenotype of AD that is characterized by an initial progressive and predominant episodic memory deficit (not normalized by retrieval facilitation) and is followed by or associated with other cognitive impairments (executive dysfunction, language, praxis and complex visual processing impairments) and neuropsychiatric changes.

Atypical AD: refers to the less frequent clinical phenotypes of AD. These clinical syndromes include logopenic variant primary progressive aphasia, posterior cortical atrophy, behavioural/dysexecutive variant, cortico-basal syndrome, and the other variants of primary progressive aphasia.

Common AD phenotype: refers to the phenotypes where AD pathology is the most common underlying primary pathology. These phenotypes include amnestic AD, logopenic variant primary progressive aphasia, and posterior cortical atrophy.

Uncommon AD phenotype: refers to the phenotypes where AD pathology can be uncommonly the underlying primary pathology (common underlying primary pathology include FTLD-Tau, FTLD-TDP, etc). These phenotypes include behavioural/dysexecutive variant, cortico-basal syndrome, and other variants of primary progressive aphasia.

Neurodegenerative disease of Alzheimer type: refers to cases where pathophysiological biomarkers are missing and common AD phenotypes are observed together with neurodegeneration biomarkers (FDG-PET hypometabolism, T1-MRI atrophy, CSF NFL, etc).

Asymptomatic at-risk (ARP): refers to cognitively unimpaired individuals who have in-vivo evidence of Alzheimer's pathology. Some can remain stable over long period of time. Other will progress.

Pathophysiological biomarkers: refer to in-vivo biological changes that reflect the underlying Alzheimer's pathology. They are markers of diagnosis, more targeted at identifying AD.

Markers of neurodegeneration: refer to topographical biomarkers or downstream markers. They can be structural (atrophy/MRI) or metabolic (hypometabolism/FDG-PET). They are more targeted at assessing changes over time and predicting outcomes.

Clinical-biological diagnosis

Panel 1 -Evidence limiting the predictive accuracy of amyloid and tau lesions for a further cognitive decline in cognitively unimpaired subjects

A -Cross-sectional data

There is an overlap between AD pathological changes in cognitively normal subjects and in patients with AD dementia. Post-mortem  Numerous cognitively normal and impaired individuals have comparable burden of AD brain lesions, confirmed with large post-mortem cohorts using quantification and digital neuropathological methods 88,89  All stages AD brain lesions (including both amyloid and tau lesions) are found in 2/3 of subjects aged of 70 years in systematic post-mortem examination regardless of clinical status 18 , well beyond the expected prevalence of having cognitive impairment (30%) [START_REF] Knopman | Mild cognitive impairment and dementia prevalence: The Atherosclerosis Risk in Communities Neurocognitive Study. Alzheimer's Dement Diagnosis[END_REF] .  Neurofibrillary tangles in the medial temporal regions are found in almost all cognitively unimpaired individual over 70 years old 18,[START_REF] Duyckaerts | Prevalence, incidence and duration of Braak's stages in the general population: Can we know?[END_REF]

B -Longitudinal molecular neuro-imaging data

They are insufficient to predict an invariable occurrence of symptoms:  Amyloid positivity is associated to an overall cognitive decline although a majority of (A+) subjects remain cognitively stable over time even after several years [START_REF] Jack | Associations of Amyloid, Tau, and Neurodegeneration Biomarker Profiles with Rates of Memory Decline among Individuals Without Dementia[END_REF][START_REF] Ebenau | ATN-classification and clinical progression in subjective cognitive decline: the SCIENCe project[END_REF][START_REF] Yu | Frequency and longitudinal clinical outcomes of Alzheimer's AT(N) biomarker profiles: A longitudinal study[END_REF][START_REF] Younes | Identifying changepoints in biomarkers during the preclinical phase of Alzheimer's disease[END_REF][START_REF] Burnham | Clinical and cognitive trajectories in cognitively healthy elderly individuals with suspected non-Alzheimer's disease pathophysiology (SNAP) or Alzheimer's disease pathology: a longitudinal study[END_REF][START_REF] Albert | Predicting progression from normal cognition to mild cognitive impairment for individuals at 5 years[END_REF][START_REF] Dubois | Cognitive and neuroimaging features and brain βamyloidosis in individuals at risk of Alzheimer's disease (INSIGHT-preAD): a longitudinal observational study[END_REF][START_REF] Sperling | The impact of amyloid-beta and tau on prospective cognitive decline in older individuals[END_REF][START_REF] Parnetti | Prevalence and risk of progression of preclinical Alzheimer's disease stages: A systematic review and metaanalysis[END_REF][START_REF] Vogel | Subjective cognitive decline and β-amyloid burden predict cognitive change in healthy elderly[END_REF] [START_REF] Yu | Frequency and longitudinal clinical outcomes of Alzheimer's AT(N) biomarker profiles: A longitudinal study[END_REF][START_REF] Younes | Identifying changepoints in biomarkers during the preclinical phase of Alzheimer's disease[END_REF] , even more in case of (SCD) [START_REF] Ebenau | ATN-classification and clinical progression in subjective cognitive decline: the SCIENCe project[END_REF] ; -Longitudinal tau PET studies showed no or only minimal acceleration of tau tracer uptake in the following 1 or 2 years in A(+) versus A(-) cognitive unimpaired elderly 101 *****. ___________________________________________________________________________ * Primary Age Related Tauopathy (PART) has emerged in the lexicon as an age-related normal occurrence of tauopathy in the absence or with a low extent of amyloid pathology (Thal ABeta Phase ≤ 2 102 ). The cognitive decline of these patients (who could be considered (T+A±) according to sensitive in vivo detection methods of biomarkers [START_REF] Toledo | Alzheimer's disease cerebrospinal fluid biomarker in cognitively normal subjects[END_REF] ) is significantly slower than that of patients with AD [START_REF] Bell | Neuropathologic, genetic, and longitudinal cognitive profiles in primary age-related tauopathy (PART) and Alzheimer's disease[END_REF]

:

  refers to a diagnosis based on both clinical and biomarker findings.

  Biological diagnosis: refers to a diagnosis only based on biomarker evidence Co-pathology: co-morbid pathological changes superimposed to a primary pathological diagnosis. Prevention of AD: becomes a major challenge. Discovery of pathophysiological biomarkers makes possible to distinguish: -Primary prevention based on interventions before the presence of positive AD biomarkers -Secondary prevention based on interventions performed when positive biomarkers are present.

*

  Molecular neuro-imaging cohorts  Numerous cognitively normal and impaired individuals have comparable amyloid and tau PET burden 91,92 .  Both amyloid and diffuse tau (i.e. outside the medial temporal lobe) pathologies are found in 19% of 576 cognitively unimpaired elderly subjects (mean age 71 years) 93 **.

Panel 2: Toward a personalized risk profile in Asymptomatic at-risk 1-Factors that may increase the risk of progression to AD

  , a noteworthy finding indicating that low (A+) (i.e. Thal ABeta Phase ≤ 2) associated with (T+) does not necessarily lead to an accelerated cognitive decline and dementia. **Probable intermediate or high level of AD pathology according to neuropathological criteria11

	 Increased Age
	 Frailty
	 Female sex
	 Low education level
	 Heterozygous APOE 4 status
	 Polygenic risk factors beyond APOE
	 Familial history of AD
	 Memory complaint/SCD
	 Magnitude of brain lesions, inferred from pathophysiological biomarker results
	especially if searched with PET
	 Presence of markers of neurodegeneration (isolated hippocampal atrophy on MRI,
	FDG-PET hypometabolism or elevated CSF NFL…)
	 Co-pathology
	2-

Factors that may decrease the risk

   Protective genes, such as the presence of the APOE 2 allele and homozygous, APOE 3 Christchurch, A673T APP Icelandic mutation  High cognitive reserve

3-Factors that need to be further confirmed

 Pattern of neuroinflammation  Functional brain marker of Cognitive Reserve (e.g. using fMRI connectivity)  Lifestyle factors: physical activity, sleep, social activities…  Psychiatric diseases (depression…)

Table 2 -AD diagnosis in clinical setting

 2

	CLINICAL	BIOMARKER	Likelihood	Further investigations
	PHENOTYPE		of AD as a
			primary
			diagnosis
	Common AD	A(+) T(+)	Highly	None required
	phenotypes (amnestic		probable -
	variant, lvPPA, and		Established
	PCA)	A(+) T(unknown)	Probable	Consider tau measure (PET, CSF)
		A(+) T(-)	Probable	Consider additional tau measure (PET, CSF)
		T(+) A(unknown)	Possible	Consider amyloid measure (PET, CSF)
		T(+) A(-)	Possible	Consider additional amyloid measure (PET, CSF)
		A(-) T(unknown)	Unlikely	Full etiology work-up* and consider tau measure (PET, CSF)
		A(unknown) T(-)	Unlikely	Full etiology work-up* and consider amyloid measure (PET, CSF)
		A(-) T(-)	Highly	Full etiology work-up*#
			unlikely -
			Excluded
		A(unknown)	Unassessab	Consider tau and amyloid measures (PET, CSF)
		T(unknown)	le
	Uncommon AD	A(+) T(+)	Probable	None required
	phenotypes			Careful follow-up needed: an incongruent phenotype and neurodegeneration
	(behavioural-			pattern should trigger a new etiology work-up*
	dysexecutive variant,	A(+) T(unknown)	Possible	Consider tau measure (PET, CSF)
	CBS, nfvPPA, and	A(+) T(-)	Possible	Consider additional tau measure (PET, CSF)
	svPPA)	T(+) A(unknown)	Unlikely	Full etiology work-up* and consider amyloid measure (PET, CSF)
		T(+) A(-)	Unlikely	Full etiology work-up*
		A(-) T(unknown)	Highly	Full etiology work-up*#
			unlikely -

Author's contribution

Supplementary Material