Florence Gaunet
email: florence.gaunet@univ-amu.fr

Carine Savalli

Thierry Legou

An exploratory study on dogs' vocalizations towards their owner and food in an unsolvable task

Keywords: dog, unsolvable task, vocalization, whine, bark 50 51

An exploratory study on dogs' vocalizations towards their owner and food in an unsolvable task

Florence Gaunet, Carine Savalli, Thierry

Introduction

Dogs communicate to their human partners about an inaccessible target mainly by using gaze alternation between the human and the target and by positioning themselves close to the target. Extensive research using the unsolvable task paradigm has determined that these gaze and positioning modalities fit the criteria of functional referential and intentional communication [START_REF] Gaunet | How do guide dogs of blind owners and pet dogs of sighted owners (Canis familiaris) ask their owners for food?[END_REF][START_REF] Gaunet | Functionally referential and intentional communication in the domestic dog: effects of spatial and social contexts[END_REF][START_REF] Gaunet | Marked referential communicative behaviours, but no differentiation of the "knowledge state" of humans in untrained pet dogs versus 1-yearold infants[END_REF][START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF][START_REF] Miklósi | Intentional behavior in dog-human communication: Experimental analysis of "showing" behaviour in the dog[END_REF]Townsend et al., 2016). In addition, these signals convey attentiongetting and deictic information. The abovementioned authors have also shown that dogs communicate using sonorous mouths licks, touch the owner, whine and bark. To date, however, no attention has been focused on the potential differences in vocal coding of the target according to the direction of dogs' gaze nor on the acoustic compositions of these vocalizations.

Initially discovered in vervet monkeys which produce three distinct alarm call variants according to the predator encountered (Seyfarth et al., 1980ab), vocal signals meeting the criteria of functional reference (see [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF] for the criteria) have since been described for social, food, and alarm calls for a range of primate taxa [START_REF] Clay | Food-associated vocalizations in mammals and birds: what do these calls really mean?[END_REF];[START_REF] Fischer | Meaning, intention, and inference in primate vocal communication[END_REF]Seyfarth et al., 1980ab;[START_REF] Seyfarth | Vocal development in vervet monkeys[END_REF][START_REF] Slocombe | Food-associated calls in chimpanzees: responses to food types or relative food value?[END_REF]Wheeler, 2010;Zuberbühler, 2003). Functionally referential vocalizations contain contextual features defining the ''referent'' and elicit specific responses in perceivers even in the absence of the supposed referent (Wheeler and Fischer, 2012). Studies on chimpanzees and bonobos have also revealed that calls vary according to the food presented and trigger different reactions: information related to appetence/motivation, if not the exact nature of the food (referentiality) is included in the signal [START_REF] Clay | Bonobos extract meaning from call sequences[END_REF]Slocombe andZuberbühler 2005, 2006).

In canids, domestication has had an effect on communicative mechanisms involving people in interactions. As a point of fact, [START_REF] Gogoleva | Explosive vocal activity for attracting human attention is related to domestication in silver fox[END_REF] showed that aggressive and unselected foxes selectively produced vocal activity for the duration of the presence of an unfamiliar human at their cage, while foxes of tame strain displayed bursts of vocal activity for the first minute after the appearance of an unfamiliar human, which faded quickly. Affiliative behaviors were also displayed, similar to domestic dogs, indicating a positive emotional state arising from interactions with people, acting to attract human attention. Dog vocalizations have also been found to vary according to context, especially social context. For instance, barks differ according to whether the dog is playing, in isolation or in the presence of a stranger [START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF][START_REF] Pongrácz | Human listeners are able to classify dog barks recorded in different situations[END_REF]Yin and McCowan, 2004). Dogs reliably produce different growl variants depending on the external social event, particularly during threatening situations compared to when playing with a human (Faragó et al., 2010a). Using playbacks, variations in growls have been shown to be meaningful to receivers. Dogs spent less time in contact with a food item after being played back food-guarding growl variants than either play or 'threatening stranger' growls. Because dogs were just as likely to approach the food source while hearing 'threatening stranger' growls as play growls, the authors argue that the dogs were not responding purely to the emotional information in the call; instead, the calls provided specific information to receivers regarding the ongoing social context (Faragó et al., 2010a). Dog barks also have a considerable effect on humans' inner state, following [START_REF] Morton | On the occurrence and significance of motivation-structural rules in some bird and mammal sounds[END_REF] motivation-structural (MS) rules: low-pitched barks with short inter-bark intervals were rated as aggressive, while high-pitched barks with long intervals were considered playful and happy [START_REF] Pongrácz | Acoustic parameters of dog barks carry emotional information for humans[END_REF]. Meaning attribution to vocalizations has been shown to some extent [START_REF] Pongrácz | Human listeners are able to classify dog barks recorded in different situations[END_REF][START_REF] Pongrácz | Acoustic parameters of dog barks carry emotional information for humans[END_REF]: humans can determine if a situation involves aggression, fear or isolation from dog vocalizations, whether or not they are dog owners.

There also appear to be breed-typical differences in the production of barks. In a study on nine different breeds of dogs, [START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF] found a high level of variability in their vocalizations: each breed produced between two and twelve subtypes of barking based on spectrographic features and behavioral correlates. Categories of function or emotion included ongoing social play, play soliciting, exploration, caregiving, social contact and "greeting,", loneliness, and agonistic behaviors. Furthermore, [START_REF] Molnár | Classification of dog barks: a machine learning approach[END_REF][START_REF] Molnár | Dogs discriminate between barks: The effect of context and identity of the caller[END_REF] categorized individual dogs based on their barks, but no study has demonstrated individual recognition based on auditory information [START_REF] Taylor | Size communication in domestic dog, Canis familiaris, growls[END_REF]. Additionally, these authors and Faragó et al. (2010b) showed that dogs perceive size-related information in growls.

The vocal repertoire of dogs thus conveys a range of socially relevant information about the context and the signaler, including its breed and body size, motivational state and almost certainly some measure of individual identity [START_REF] Taylor | Size communication in domestic dog, Canis familiaris, growls[END_REF].

Still, there is not yet a full and exhaustive repertoire of dogs' vocalizations according to context or intra-or inter-species interactions [START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF][START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF]Tembrock, 1976). On the basis of spectrographic evidence, [START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF] identified twelve basic vocal sound types of the canid species (mews, grunts, whines/whimpers, yelps, yips, howls, coos, growls, coughs, barks, clicks, tooth snaps). [START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF] described ten classes of vocalizations in dogs and wolves, grouped into three categories of sounds: harmonic/tonal (whines, yelps, screams, whimpers), intermediate (moans, growls, barks) and noisy (woofs, snarls, yaps); see also [START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF]). Finally, Tembrock (1976) described the temporal structure and the frequency composition of ten classes of vocalizations in various canids. The major factors of variability are breed and context. There is a full consensus that, in a social context, dogs pant [START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF], mouth-lick [START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF][START_REF] Gaunet | Marked referential communicative behaviours, but no differentiation of the "knowledge state" of humans in untrained pet dogs versus 1-yearold infants[END_REF][START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF], bark, growl, whine and sometimes howl (see [START_REF] Taylor | Auditory communication in[END_REF][START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF].

As is the case in human communication, fundamental frequency (F0) and duration are two of the most meaningful characteristics in bioacoustic investigations of vocal communication in dogs, among others including harmonic-to-noise ratio, between vocalization intervals, intensity or tonality (e.g. Pongrácz et al., 2006, Riede and[START_REF] Riede | Vocal tract length and acoustics of vocalization in the domestic dog (Canis familiaris)[END_REF]. Since only F0 and duration are robust in regarding recording conditions, we will focus on these characteristics in the present study in the case of barks and whines. Barks have been characterized as short, plosive, and possibly noisy, produced in sequence or isolated, and with variable pitch across breeds, individuals and contexts [START_REF] Taylor | Auditory communication in[END_REF]. Their spectral shape looks like a 'Christmas tree' formation due to a progressive increasing followed by a lowering of energy in higher frequencies [START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF][START_REF] Scott | Genetic variation and the evolution of communication[END_REF]. A bark unit can be described as follows:

Feddersen-Petersen (2000): F0: 120-1640 Hz, duration: 69-1000 ms, for 6 breeds; [START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF]: F0: maximum of 2000 Hz, duration: < 500 ms; duration varies between 100 and 500 ms as compiled by [START_REF] Lord | Barking and mobbing[END_REF][START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF] reported bark durations between 0.2 and 0.6 sec. Whines have been characterized as tonal, high pitched, noiseless vocalizations.

The nuances of whines were described by [START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF] for six breeds: 400-8800 Hz, duration: 38-2025 ms. Yelps, screams and whimpers have also been described (Cohen and Fox, 1976: F0: 400-3500 Hz, no description for duration).

Yeon (2007) demonstrated that barks are produced during contexts involving alerts/warnings, territorial defense/rivalry/defense, individual identity, social facilitation, play solicitation, greetings, or calls for attention; whines are produced during greetings, active submission, or frustration or attention-seeking contexts. The present study addressed the following question: is there a difference between dogs' vocalizations toward the target and their vocalization toward the owner when the target is a desirable food inaccessible to the dog?

In the absence of existing research, the present study uses existing material produced by [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF]2016). One of the present aims is to quantify vocalizations produced in a food request context in terms of percentages of dogs and numbers of vocalizations per type of vocalizations found. We also examine differences in the acoustic characteristics of the vocalizations a dog produces when gazing at the target compared to vocalizations when gazing at their owner, as a mean to study if vocalizations at the target and at the owner differ. In food (or toy) requesting tasks, dogs have been shown to display different behaviors towards the owner as compared to the target. Using videos and sounds collected with a unidirectional microphone placed in the center of a room [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF], the present study is the first to use an unsolvable task to explore qualitative and quantitative descriptions of the two main canid vocalizations: barks and whines and their related vocalizations (but see [START_REF] Pongrácz | Should I whine or should I bark? Qualitative and quantitative differences between the vocalizations of dogs with and without separation-related symptoms[END_REF] in the case of a separation context). [START_REF] Slocombe | Food-associated calls in chimpanzees: responses to food types or relative food value?[END_REF] showed that chimpanzees may include the exact nature of the food may be included in the signal (referentiality) in chimpanzees, and [START_REF] King | Come dine with me: food-associated social signalling in wild bottlenose dolphins (Tursiops truncatus)[END_REF] demonstrated that in the case of dolphins, specific social signals were significantly correlated with food call production, and that these calls rarely occurred in the absence of food. Given the analogies and convergence on referential communication behaviors of monkeys, apes and dogs, we expected dogs to discriminate the target and the social agent when vocalizing toward them, both in terms of types of vocalizations (and their number) and acoustic characteristics. Although [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF]2016) found that gaze at the food and at the owner fitted all the operational criteria for functionally referential and intentional communication (previously found in a study with chimpanzees, [START_REF] Leavens | Intentionality as measures in the persistence and elaboration of communication by chimpanzees (Pan troglodytes)[END_REF], this was not the case for the number and duration of vocalizations, likely because there were too few occurrences. Accordingly, due to the lack of an exhaustive and dedicated study testing referentiality and intentionality of vocalizations directed at the target and receiver in the pet dogs, these vocalizations are thought of as reflecting the inner state of the dogs, as an attention-getting signal.

Methods

Data collection and participants

Vocalizations were collected by [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF] in an unsolvable task: after habituation to the room, a helper took a pet dog to an experimental room with food, put the food on one of two shelves (randomly chosen) that were inaccessible to the dog, and left the room. The owner entered the room and remained quietly standing, equidistant from the two shelves, for 30 seconds during which the owner visually followed the dog with his/her gaze; see [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF] for all details on the procedure. This condition, called "Food+Owner", was used three times in the 2014 study; see the video in [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF].

The identical condition in [START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF] was called "Visual following." For the aim of this study, just these four identical conditions were taken into account. In total, 51 pet dogs living in households were tested by [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF]. Behaviors analysed in these two previous studies were the visual communicative behaviors (e.g. gaze alternation, gaze direction). No dog had been training to vocalize on command. Their multimodal behaviors (including vocalizations but without dissociating the types of vocalizations, i.e. barks, whines, etc.) were also recorded by two video cameras and a unidirectional microphone placed in the center of the room and attached to a crossbar connecting the two sides of the room (Rode NT55A); see Figure 1. Their durations and numbers were extracted using Actogram Kronos software (Octarés Edition, http://www.actogram.com/index.htm) by [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF]. As a byproduct of the [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF] studies, the present study kept and analyzed vocalizations produced when the dog's head/nose was oriented toward the food and towards the owner's head and face. These additional data were recorded to address the present aim to compare vocalizations towards the food and towards the owner.

A total of 189 vocalizations produced by eleven dogs were collected and categorized. A small number of vocalizations were removed from the data set (total = 5, among these 3 barks and 2 whines) because of background noise or ambiguity over the direction of the dog's head; see Table 1 for characteristics of the dogs included in the analysis.

INSERT FIGURE 1 ABOUT HERE INSERT TABLE 1 ABOUT HERE

Ethics Statement

This study was approved by the Committee for Ethical Research in Animals of the Institute of Psychology of the University of São Paulo (N° 004.2012). Owners consented to their dogs' participation.

Extraction of acoustical boots and categorization

For each dog, the soundtrack (16 bits -44.1kHz) was extracted from the video, and then analyzed with Praat software, version 4.4.32 (Boersma and Weenik, 1996).

The videos were re-used to determine the periods of interest for acoustic analysis with Praat: vocalizations produced while gazing at the target and at the owner. Next, vocalization boundaries were precisely defined using both the oscillogram and the spectrogram. An auditory perceptual evaluation of the vocalization coupled with a visual inspection of oscillograms and spectrograms was used to further subdivide vocalizations into units when necessary. The envelope was taken into account for the visual inspection of the oscillogram, whereas frequency composition and voiced/unvoiced mode were used for the spectrogram.

The name and category of each vocalization as well as its segmentation (overall boundaries and subunits) were agreed upon by three judges (two of the authors and Stéphanie Mana).

Several acoustic parameters were extracted using Praat. Duration and F0 (fundamental frequency) were calculated for voiced segments.

Qualitative and quantitative analysis

We first provide the percentage of dogs who vocalized out of the 51 dogs recorded, and we indicate the mean and standard deviation of the number of vocalizations. We then provide the extracted list of vocalizations and their description from the corpus collected, i.e. the vocal repertoire in terms of typical whines/barks and whine/bark-related vocalizations. We collected the occurrences (189) of these two types of vocalizations when they were directed at the target or at the owner. The comparison between the number of vocalizations according to the type (i.e. typical whines or barks, and whine-or bark-related vocalizations) and their direction (target or owner), as well as the interaction between these two factors were carried out using a GEE (Generalized Estimating Equation) with the number of vocalizations as a counting variable and the logarithm as a link function. The Wald statistic was used for tests. The within-dogs' dependence was incorporated by using an exchangeable structure, assuming that correlations among measures taken from the same dog were the same. Additionally, we analyzed the distribution of the vocalizations for the four types of whines and the five types of barks also identified using GEE models with the same specification for the two directions of gazes separately.

In the next section, we provide an Overall description of the acoustic characteristics of each type of whine. We then provide information about the Distribution of dogs according to the four types of whines (GEE). The acoustic parameters (F0 and Duration) according to the direction are then presented. The effect of the direction of the dog's gaze (Owner or Target) on the frequency F0 and duration of whines was analyzed with linear mixed models (LMM) considering the dog's identity as a random effect (within-subject effect). The mixed model was compared to the independent model (excluding random effect) based on the evaluation of Akaike Information Criteria. For all models, except for the duration of the bark sub-unit a, the lowest Akaike Information Criterion (AIC) was obtained when the random effect of the dog was incorporated. Residuals were visually analyzed and considered acceptable for all models.

Finally, we describe the number of bouts in a row (from 1 to 7) of whines towards the target and the owner.

In the last section, we first provide an Overall description of the acoustic characteristics of barks. Next, we focus on the typical bark, first with a description of its acoustic characteristics (subdivided into units).

We then describe the Distribution of the composition of a typical bark (i.e. which dogs used the various units and their number of vocalizations), with a GEE model. The acoustic parameters (F0 and Duration) of the sub-units according to the direction of the typical bark (owner vs. target) are then presented. The effect of the direction of gaze (Owner or Target) on F0s and durations of the bark sub-units were analyzed with linear mixed models (LMM) considering the dog's identity as a random factor (within-subject factor), as mentioned above.

Additionally, the number of bouts in a row (from 1 to 4) of the typical bark towards the target and the owner are presented, which we fed into a GEE model. Finally, we describe the other types of barks in terms of distribution of occurrences across dogs and across types of barks, as well as their acoustic forms.

A second naïve observer independently coded 46% of the sample, i.e. frequency of typical barks when gazing at the owner and at the target. The exact same occurrences were found. Additionally, we calculated the Kendall's concordance coefficient for durations of these barks that were respectively 0.83 and 0.81, indicating a good agreement between raters.

Results

Vocalization description and categorization

The proportion of dogs that vocalized was 21.6% (i.e. 11 out of 51 dogs). Additionally, the number of vocalizations varied considerably between dogs, from one to 51 occurrences (mean = 17.2, sd = 16.2), with a total of being 189.

Using spectrogram and auditory analyses, we identified two types of vocalizations directed at the target and at the owner: whines composed of typical whines and whine-related vocalizations (3 types), and barks composed of typical barks and bark-related vocalizations (4 types). Table 2 presents the distribution of vocalizations according to the types of vocalizations found and the direction of the dog's gaze (target or owner) for 189 occurrences produced by 11 dogs.

INSERT TABLE 2 ABOUT HERE

Table 2 shows that most vocalization occurrences consisted in barks, then in whines (67.72% and 32.27%, respectively). They were mostly directed at the owner (67.7%), then at the target (32.3%). Table 2 also reveals that the main vocalizations (see the four cells in italics, i.e. all whines and typical barks) were not produced by all dogs, but only by four to six different dogs out of the eleven dogs, in each of the four cases.

The GEE model (estimated parameters presented in Figure 2) for the number of vocalizations according to the type (whines or barks) and their direction (target or owner) indicated a possible interaction between types of vocalizations (2) and gaze directions (2) (Wald = 3.50, df = 1, p = 0.0615). As a matter of fact, there was no difference between numbers of barks and of whines when directed at the owner (Wald = 0.64, df = 1, p = 0.4237); however, there were significantly more barks than whines directed at the target (Wald = 4.65, df = 1, p = 0.0310). No difference was found between the number of barks directed at the owner compared to the target (Wald = 0.39, df = 1, p = 0.5314). However, the number of whines directed at the owner was significantly greater than those directed at the target (Wald = 6.27, df = 1, p = 0.0123).

INSERT FIGURE 2 ABOUT HERE

In the case where the dogs were gazing at the target, it was not possible to compare the distribution of the four types of whines and the number of vocalizations using the GEE model due to the small number of occurrences recorded. In the case where the dogs were gazing at the owner, yelps could not be included in the analysis, but the GEE model used to compare the three other distributions indicated that there was no difference between them (Wald = 1.28, df = 2, p = 0.5266). In contrast, there were considerably more typical barks directed in each direction than the four other types of barks (at the target: Wald = 604.31, df = 4, p < 0.0001; at the owner: Wald = 14.17, df = 4, p = 0.0068, respectively).

Typical whine and whine-related vocalizations

Overall description

Our recordings of whine vocalizations matched all the types of whines described by Tembrock (1976) and Feddersen-Pettersen (2000). The vocalizations that we recorded were high frequency, tonal, narrow-banded and noiseless, along with the whines in Figures 3,4, 5 and 6 below. Their intensity was largely stable and their harmonic composition sometimes visible (cf. the four types over time).

Typical Whines. These whines were high frequency / high pitched, with the energy of harmonics that varied over time; cf. Fig. 3.

INSERT FIGURE 3 ABOUT HERE

Screams. This vocalization was high frequency, tonal, narrow-banded, and noiseless, along with a clear harmonic composition; cf. Fig. 4.

INSERT FIGURE 4 ABOUT HERE

Whimpers. This vocalization was high frequency, tonal, nearly monofrequency and noiseless (i.e. strong and stable pitch) with no harmonic composition visible; cf. Fig. 5.

INSERT FIGURE 5 ABOUT HERE

Yelps. This vocalization was high frequency and tonal, with a clear dominant frequency, and was noiseless with little harmonic composition; cf. Fig. 6.

INSERT FIGURE 6 ABOUT HERE

Distribution of dogs according to the four types of whines

Eight of the eleven dogs who vocalized produced whine vocalizations. Each type of whine was produced by a similar number of dogs (i.e. three or four). Additionally, only four dogs produced two or three types of whines and whines-related vocalizations (Cachoro: yelps and whimpers; Phoebe: whines, screams and yelps; Mel: whines and whimpers; Mancha: whimpers and screams). The four other dogs produced only one type of whine-related vocalization. Finally, the GEE model indicated that there was no difference in the number of dogs that produced the four types of whines (Wald = 1.75, df = 3, p = 0.6269). See Table 1 in the supplementary material.

Direction of whines and concomitant acoustic parameters (F0 and duration)

The results obtained from the linear mixed models revealed that the F0 and durations of whines addressed to the owner and the target did not differ; see The number of bouts for all whines when the vocalization was produced toward the owner (see Table 3 in the supplementary material.) tended to be produced alone or in a sequence of two bouts rather than in a sequence of three or more; however, it was not possible to run the GEE model to validate or invalidate this observation.

Typical barks and bark-related vocalizations

Overall description

Barks, or what we call 'typical barks' are usually described in the literature as presenting a Christmas-tree shape, i.e. a rising part with an increasing pitch (and harmonics) over time, then a more or less symmetric decreasing pitch (and harmonics) of a more or less similar duration, including a stable part, cf. Faragó et al., 2014 and Fig. 1a in the supplementary material, see the second bark for a stable part; Feddersen-Petersen, 2000 and Fig. 1b in the supplementary material, mostly described in [START_REF] Scott | Genetic variation and the evolution of communication[END_REF].

Typical barks

In our case, out of 87 barks, we recorded only five barks that did not include the stable part of the Christmas tree (see Fig. 7 for an item representative of a Christmas-tree-like bark); all five were produced by a single dog. [START_REF] Scott | Genetic variation and the evolution of communication[END_REF] noted that "barks differ in quality and quantity"; our recordings regarding quality are in agreement with his observation, and see Table 2 for variations in quantity.

INSERT FIGURE 7 ABOUT HERE

Based on the observation of typical spectrograms of barks', a clear stable central and tonal segment could be preceded by a noisy segment (the onset) and sometimes followed by a short segment (the offset) of the bark. In order to analyze and compare all the barks and take into account potential onsets or offsets, we considered three sub-units in typical barks; cf. Fig.

8.

• Sub-unit "a": the start of the vocalization. This sub-unit is a transition to the sub-unit "b" and corresponds to the left part of the Christmas tree. In general, no pitch was detected at the beginning of sub-unit "a"; it appeared and rose quickly. This segment was quite noisy.

• Sub-unit "b": this was the stable part of the vocalization. The harmonic structure was clearly visible and stable, and no modulation (intensity, frequency) was observed.

• Sub-unit "c": this sub-unit, when it existed in our corpus, signaled the end of the call; it corresponded to the right-hand part of the Christmas tree.

INSERT FIGURE 8 ABOUT HERE

Distribution of the composition of the typical bark

Only four of the eleven dogs that vocalized produced typical barks that could be composed differently with their a, b or c parts, presenting different configurations; see the supplementary material. Each type of bark configuration was produced by a similar number of dogs (one or four); all four dogs produced the configuration a, ab and abc, while ac, bc or b configurations were each produced by one dog (Nana or Mila). Finally, a very small percentage of barks (4.65%) present the Christmas-tree pattern per se (ac) and most of the barks (45.36 %) present ab sub-units; however, it was not possible to run a GEE model to validate or invalidate the observation due to instability of estimation.

Direction of the typical bark and concomitant acoustic parameters (F0 and duration)

Table 3 shows the adjusted mean and standard errors for F0 and durations obtained from linear mixed models for the sub-units a, b, and c of barks toward the owner and the target. The results revealed that barks addressed toward the owner and the target did not differ. We found a tendency for sub-unit b to have a higher F0 and lower duration when dogs gazed at the owner compared to the target; however, it was not significant at a level of 5%.

INSERT TABLE 3 ABOUT HERE

Number of bouts in a row for typical barks towards the target and the owner

To extract the number of bouts within a production for barks composed of the 3 subunits, we considered all barks regardless of the number of sub-units (ac, a, ab, abc, bc, or b).

We found mainly single bouts when the vocalization was produced toward the owner or the target (see Table 5 in supplementary material); however, it was not possible to run a GEE model to validate or invalidate the observation.

Other barks

Four novel types of barks were found in our data, representing a small percentage of the total vocalizations (22.64%, cf. Table 2). These four types of barks had not been found in the previously quoted literature. Given the small number of occurrences, we will describe them briefly and only qualitatively. Table 6 in the supplementary material shows that the distribution of these barks varies considerably between dogs.

'Dull Bark' (Fig. 9). This vocalization had a muffled sound. The characteristics of dull barks were stable in time and could not be decomposed into sub-units. The durations ranged from 76 to 280ms. For this weakly tonal and strongly noisy (small harmonic to noise ratio) vocalization, the detection of the first two formants was easy and reliable. F0, F1 and F2 stability over time showed very little variation, in congruence with a monosegmentation.

INSERT FIGURE 9 ABOUT HERE

'Push Bark' (Fig. 10). This vocalization resembled a breathy cough; it was partially tonal and always very noisy. Durations lasted from 99 to 222ms. To our knowledge, this type of vocalization has not been previously described in the literature.

Of the eight vocalizations of this type, most (five) were composed of two segments:

• Sub-unit "a": this part of the vocalization was noisy with no pitch detected, and was the sub-unit more closely resembling a cough.

• Sub-unit "b": this segment was voiced, and still contained a lot of noise. The pitch ranged from 90Hz to 583Hz. This segment presented a higher degree of breathiness than a bark.

INSERT FIGURE 10 ABOUT HERE

'Cooing Flat Bark' (Fig. 11). This bark was an atonal vocalization lasting from 101 to 439 ms and sounded like an exhaled breath. The frequency composition was stable along the vocalization with no perceptible modulation. This vocalization was the noisiest of the repertoire.

The spectrogram clearly shows two formants. The first formant (F1), around 1100Hz, signed the jaw aperture; the second formant (F2), around 2200Hz, indicated the tongue position, i.e. antero-posterior.

INSERT FIGURE 11 ABOUT HERE

'Cooing Bark' (Fig. 12). This cooed vocalization showed a low frequency (270Hz to 470 Hz) band energy concentration slightly modulated around 10%. This sound lasted from 230ms to 715ms, and the five items found were emitted alone (i.e. not in a row). The cooed effect resembles a perceptual vibrato. This vocalization was similar to ones found by Feddersen-Petersen (2000, called 'vibrato growling'), and by Tembrock (1976, see Figure 6, 'Snarl'), in a smooth/soft version. Both authors characterized this vocalization as wide-banded, composed of extended sounds, a long ascension time, changes in amplitude with a tendency to form cycles, the appearance of more bands, and a primary frequency with an increase in intensity, with vibrato involved in both pitch and intensity.

INSERT FIGURE 12 ABOUT HERE

Discussion

The aim of the present exploratory study was to quantify vocalizations produced in an unsolvable task where food was inaccessible in terms of percentage of dogs and of vocalizations per type of vocalizations found. We also sought to determine whether dogs vocalize differently toward their owner and food in terms of repartition and the acoustic composition of canine vocalizations. The results indicate that this is indeed the case.

Firstly, and interestingly, only 21.6 % of the fifty-one dogs recorded in [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF]2016) vocalized. Breed was investigated [START_REF] Scott | Genetic variation and the evolution of communication[END_REF][START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF] but not revealed to be a factor. A plausible explanation then is that many of the non-vocalizing dogs had previously learned from their owners not to vocalize in situations involving food requests [START_REF] Duranton | Effects of shelter housing on dogs' sensitivity to human social cues[END_REF][START_REF] Henschel | Effect of shared information and owner behavior on showing in dogs (Canis familiaris)[END_REF]. We also found that the number of vocalizations varied considerably between the 11 dogs (1-50 for the total of 189 found).

The vocalizing dogs in contrast, may have produced a vocalization at one point when in the presence of their owner and food. And this vocalization may have been rewarded by food.

Accordingly, vocalizations can be of various forms, in accordance with the various types we found. Only four to six different dogs produced all the whines and the typical barks, and the various types of whines and barks and units of the barks were not produced by all dogs; see Tables 2, 3 and 6. This variability in vocal productions between dogs may reasonably be based on an effect of the dogs' previous experience with their owners [START_REF] Duranton | Effects of shelter housing on dogs' sensitivity to human social cues[END_REF][START_REF] Henschel | Effect of shared information and owner behavior on showing in dogs (Canis familiaris)[END_REF], in accordance with [START_REF] Scott | Genetic variation and the evolution of communication[END_REF] observation that "barks differ in quality and quantity,".

What did canine vocalizations look like in the present study? Only whine and whinerelated and bark and bark-related vocalizations were found [START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF]Farrago et al., 2014;[START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF][START_REF] Taylor | Auditory communication in[END_REF]Tembrock, 1976). In the first category, we identified four types of vocalizations already described in the literature. We described five types in the second category, including four new types of barks; we postulate an effect of context.

Most of the 189 vocalizations (67.72%) consisted in typical and related barks, and the other third in whines and whine-related vocalizations. Interestingly, the majority of vocalizations (67.7%) were directed at the owner. The tendency of an interaction between the direction of the vocalization and types of vocalizations, with significantly fewer whines directed at the target, suggest firstly that vocalizations tended to be mostly used to attract the owner's attention to the dog itself rather than to designate the target per se, and thus were essentially a social [START_REF] King | Come dine with me: food-associated social signalling in wild bottlenose dolphins (Tursiops truncatus)[END_REF], attention-seeking signal [START_REF] Gaunet | Marked referential communicative behaviours, but no differentiation of the "knowledge state" of humans in untrained pet dogs versus 1-yearold infants[END_REF][START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF][START_REF] Henschel | Effect of shared information and owner behavior on showing in dogs (Canis familiaris)[END_REF]. Secondly, while the present protocol does not make it to assume a referential property of barks (see the end of the Introduction), these results suggest a different inner state for attention-getting when they bark, comparatively to when they whine. Importantly, the interaction clearly indicates that dogs treat their owner and the food differently by way of their vocal productions. Furthermore, the distribution of the four types of whines appeared to be similar when dogs were gazing at their owner or at the target, indicating flexible use of the different types of whines. In contrast, there were more typical barks than related barks when dogs were gazing at their owner or at the target, indicating that typical barks are the main bark pattern.

Statistical analyses did not show that the means of F0 and of durations statistically differed for each type of vocalization according to the direction of the dogs' gaze toward the target and the owner. There was however a tendency for bark b to have higher F0 and lower duration when gaze was directed at the owner compared to the target. In addition, the number of items was too small to be able to compare the distribution of the different lengths of bouts when whine-related vocalizations were produced toward the target or the owner; however, barks appeared to be produced mostly alone.

The whine per se and whine-related vocalizations we found matched those reported in the literature based on acoustic characteristics [START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF]Tembrock, 1976).

They were high frequency, tonal, narrow-banded and noiseless vocalizations, with harmonic composition sometimes visible, corresponding to calls used for short range communication [START_REF] Taylor | Auditory communication in[END_REF]. The present ranges of F0 for gazes at the target and at the owner corresponded to those reported in the literature. F0 has been reported to vary from 400 Hz [START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF]Tembrock, 1976) to 8800 Hz [START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF][START_REF] Schassburger | Vocal communication in the timber wolf, Canis lupus, Linnaeus: structure, motivation, and ontogeny[END_REF], and F0 is known to depend on dog size and breed [START_REF] Riede | Vocal tract length and acoustics of vocalization in the domestic dog (Canis familiaris)[END_REF], elements that are variable in our experiment. Possible differences with the literature could also be due to context. Whining is reported in contexts involving greetings, frustration, active submission, and attention-seeking (Yeon, 2007), whereas our recordings likely involved attention-seeking to get food [START_REF] Gaunet | Marked referential communicative behaviours, but no differentiation of the "knowledge state" of humans in untrained pet dogs versus 1-yearold infants[END_REF][START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF]. We thus propose that the range of F0 we found may be linked to the socio-physical recording contexts that often differ between studies. Our range is likely to be specific to an attention-getting communicative activity, though variability may occur according to sizes and breeds of dogs. In contrast, the durations of whine-related vocalizations are not systematically reported in the literature. Still, the durations we found were in the lower range of the durations previously reported (Federsen-Petersen, 2000;[START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF][START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF].

Regarding the production of barks, we found the typical Christmas-tree production [START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF][START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF] to be the most common compared to the four other related barks we found. While whine and whine-related vocalizations were produced by eight dogs, only four dogs barked; we believe this is likely due to the request from owners for their dogs not to bark at food in general. Again, in contrast with whining, typical barks were directed equally towards the owner and the target. The acoustic characteristics of the typical barks found matched those recorded in the literature, especially in non-aggressive contexts (i.e. higher peak, mean, and range of F0, [START_REF] Feddersen-Petersen | Vocalization of European wolves (Canis lupus lupus L.) and various dog breeds (Canis lupus f. fam.)[END_REF]Yin, 2002). The presence of three segments in the typical bark may reveal a specific vocal coding. The in-depth analysis of the acoustic characteristics we performed revealed a statistical tendency for the b segment of the typical bark to have higher F0 and lower duration during gazes at the target than during gazes at the owner. Adding more dogs and more occurrences of barks to the study could reveal more marked differences.

None of the four related barks (dull, push, cooing flat and cooing barks) is described in the literature. Interestingly, the latter resembled a variant of the typical bark with a poor harmonic structure and a high noise level. We hypothesize that these barks were generated and maintained by positive reinforcement within each specific human-dog pair in earlier interactions, as has been shown for other communicative behaviors [START_REF] Gaunet | How do guide dogs of blind owners and pet dogs of sighted owners (Canis familiaris) ask their owners for food?[END_REF][START_REF] Gaunet | Functionally referential and intentional communication in the domestic dog: effects of spatial and social contexts[END_REF][START_REF] Gaunet | Marked referential communicative behaviours, but no differentiation of the "knowledge state" of humans in untrained pet dogs versus 1-yearold infants[END_REF][START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF][START_REF] Savalli | Eye contact is crucial for referential communication in pet dogs[END_REF][START_REF] Miklósi | Intentional behavior in dog-human communication: Experimental analysis of "showing" behaviour in the dog[END_REF]Townsend et al., 2016).

Barks have been found to be produced in various contexts and thus seem a priori to be non-specific: they are used for long distances and proximal interactions, in various contexts such as aggressive signaling, the defense of self and territory, drawing attention, separation and submissive behaviors, and playful and positive situations [START_REF] Cohen | Vocalizations in wild canids and possible effects of domestication[END_REF][START_REF] Faragó | The information content of wolf (and dog) social communication[END_REF][START_REF] Taylor | Auditory communication in[END_REF]. Barks and whines thus convey different attention-getting inner-state content, with effects of breed, size and individual experience potentially at play. Given the convergence of communicative behaviors between dogs, monkeys and apes, and that vocalization in dogs is said to have developed to enable dogs to communicate with humans [START_REF] Gogoleva | Explosive vocal activity for attracting human attention is related to domestication in silver fox[END_REF][START_REF] Larranaga | Comparing supervised learning methods for classifying sex, age, context and individual Mudi dogs from barking[END_REF][START_REF] Pongrácz | Acoustic parameters of dog barks carry emotional information for humans[END_REF], the present results indicate specific vocal coding in dogs' vocalizations worth examining in a more systematic manner. Note that only 11 vocalizations occurred while the dogs were not gazing either at the target or at the owner, confirming the importance of vocalizations associated with gaze direction at a social agent or a desirable target.

To conclude, does the present study suggest that dogs' vocalized signals should be conceived as reflecting different attention-getting signals? In other words, was there a difference between dogs' vocalizations toward the target or toward the owner? The differential distribution of whines and barks according to gaze direction and the tendency for the stable segment of the typical bark to differ according to the owner vs. the food suggest that this is the case. So, although we found a limited differentiation of vocal characteristics per se according to the direction of gaze (owner vs. food) in an unsolvable task, this is likely due to a limited control of the position of articulators that introduce variations in formant positions, and to the anatomy of dogs and in particular the length of the vocal tract (see [START_REF] Taylor | Auditory communication in[END_REF]. In this exploratory study, we provide a path to expand research on the content of vocalizations, since some argue that the vocal communication of the canid species developed with humans during domestication [START_REF] Gogoleva | Explosive vocal activity for attracting human attention is related to domestication in silver fox[END_REF][START_REF] Larranaga | Comparing supervised learning methods for classifying sex, age, context and individual Mudi dogs from barking[END_REF][START_REF] Pongrácz | Acoustic parameters of dog barks carry emotional information for humans[END_REF]. Though we started with a large number of dogs (51), only 11 vocalized, resulting in a total of 189 vocalization occurrences, a fact which should be taken into account in future studies which should include an even larger number of dogs to form a larger corpus of vocalizations. Future studies should also focus on only one breed, and take place outdoors and with a toy; these two last experimental characteristics would enable a greater proportion of dogs to vocalize. Adding these characteristics to the paradigm by [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF] will make it possible to study referentiality and intentionality in the acoustical content of dogs' vocalization, and possibly show evidence of more than an affective state within the vocalizations. In other words, if acoustical characteristics of vocalizations differ when directed at an owner or at food, and the criteria for referential and intentional communication are fulfilled, we may hypothesize proto-semanticity in dog vocalizations. Finally, temporal characteristics (cf. Yin, 2002 for barks and [START_REF] Taylor | Context-related variation in the vocal growling behaviour of the domestic dog (Canis familiaris)[END_REF] for growls) might provide a guide to motivational state because these characteristics are influenced by changes in the rate of respiration and/or muscular tension in the vocal folds [START_REF] Hauser | The sound and the fury: primate vocalizations as reflections of emotion and thought[END_REF][START_REF] Bachorowski | Vocal expressions of emotion[END_REF]. A study of intercalls should thus be another avenue to pursue to find evidence of specific vocal coding in dogs' vocalizations, since the variability of a signal may reflect the function of the signal [START_REF] Scott | Genetic variation and the evolution of communication[END_REF].

The present study opens perspectives in terms of understanding needs expressed by dogs. domestic dogs: vocal signalling in the extended social environment of a companion animal, in Kaminski, J., Marshall-Pescini, S. (Eds), The Social Dog, Behavior and Cognition. Academic Press, Amsterdam, pp. 131-163. Tembrock, G., 1976. Canid vocalizations. Behav. Proc., (dog as random effect) for F0 and durations of the three types of units (a, b and c), for barks produced when gazing at the owner and at the target.

Figure captions

Fig. 1. Experimental layout (schema from [START_REF] Savalli | Are dogs able to communicate with their owners about a desirable food in a referential and intentional way[END_REF], with the position of the unidirectional microphone that registered vocalizations of dogs.

Fig. 2 .

 2 Fig. 2. Adjusted means and standard error of the logarithm of the number of vocalizations estimated by the GEE model.

Fig. 3 .

 3 Fig. 3. Whine-related vocalizations (typical whines) from Mila. Duration = 57.5ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 4 .

 4 Fig. 4. Whine-related vocalizations (screams) from Netuno. Duration =146ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 5 .

 5 Fig. 5. Whine-related vocalizations (whimpers) from Cachorro. Duration =205ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 6 .

 6 Fig. 6. Whine-related vocalizations (yelps) from Cachorro. Duration =284ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 7 .

 7 Fig. 7. The Christmas-tree-like bark of Nana. Duration = 353ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 8 .

 8 Fig. 8. Detailed description of the 3 sub-units of the barks. Duration=236ms -Top: oscillogram (arbitrary unit), Middle: spectrogram (Hz), Bottom: annotation line that shows « a » (left part), « b » (middle part) and « c » (right part) sub-unit boundaries.

Fig. 9 .

 9 Fig. 9. Dull Bark from Eros. Duration = 164ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 10 .

 10 Fig. 10. Push Bark from Barto. Duration = 186ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 11 .

 11 Fig. 11. Cooing Flat Bark from Netuno. Duration = 285ms-Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 12 .

 12 Fig. 12. Cooing Bark from Netuno. Duration = 286ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 1 .

 1 Fig. 1. Experimental layout (schema from Savalli et al., 2014), with the position of the

Fig. 3 .

 3 Fig. 3. Whine-related vocalizations (typical whines) from Mila. Duration = 57.5ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 4 .

 4 Fig. 4. Whine-related vocalizations (screams) from Netuno. Duration =146ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 5 .

 5 Fig. 5. Whine-related vocalizations (whimpers) from Cachorro. Duration =205ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 6 .

 6 Fig. 6. Whine-related vocalizations (yelps) from Cachorro. Duration =284ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 7 .

 7 Fig. 7. The Christmas-tree-like bark of Nana. Duration = 353ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 8 .

 8 Fig. 8. Detailed description of the 3 sub-units of the barks. Duration=236ms -Top: oscillogram (arbitrary unit), Middle: spectrogram (Hz), Bottom: annotation line that shows « a » (left part), « b » (middle part) and « c » (right part) sub-unit boundaries.

Fig. 9 .

 9 Fig. 9. Dull Bark from Eros. Duration = 164ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 10 .

 10 Fig. 10. Push Bark from Barto. Duration = 186ms -Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

Fig. 11 .

 11 Fig. 11. Cooing Flat Bark from Netuno. Duration = 285ms-Top: oscillogram (arbitrary unit), Bottom: spectrogram (Hz).

  Table 2 in the supplementary material. 4.2.4. Number of bouts in a row of whines towards the target and the owner

Table 2 .

 2 1(1), 57-75.Townsend, S.W., Koski, S.E., Byrne, R.W.,Slocombe, K.E., Bickel, B., Boeckle, M., ... Glock, H.J., 2017. Exorcising G rice's ghost: an empirical approach to studying intentional communication in animals. Biol. Rev., 92(3), 1427-1433. Wheeler, B.C., 2010. Production and perception of situationally variable alarm calls in wild tufted capuchin monkeys (Cebus apella nigritus). Behav. Ecol. Sociobiol., 64, 989-1000. Wheeler, B.C., Fischer, J., 2012. Functionally referential signals: a promising paradigm whose time has passed. Evol. Anthrop.: Iss., News Rev., 21(5), 195-205. Yeon, S.C., 2007. The vocal communication of canines. J.Vet. Behav.: Clin. App. Res., 2(4), Referential signalling in non-human primates: cognitive precursors and limitations for the evolution of language. Adv.Stud. Behav., 33, Numbers of vocalizations by category and direction of gaze. In italics, the number of different dogs that presented whines, whine-related and typical bark 732 vocalizations for both directions of gaze (with the number of occurrences per dog).

	141-144.
	Yin, S., 2002. A new perspective on barking in dogs (Canis familaris). J Comp. Psych.,
	116(2), 189.
	Yin, S., McCowan, B., 2004. Barking in domestic dogs: context specificity and individual
	identification. Anim. Behav., 68(2), 343-355.
	Zuberbühler, K., 2003. Tables
	Dogs	Breed	Sex	Age (in	Weight	Number of
				years)	(Kg)	Vocalizations
	Mancha	Mongrel+Border	F	8.8	33	16
	Cacchoro	Mongrel	M	3	29	13
	Netuno	American	M	5.5	40	43
		Staffordshire terrier

Table 3 .

 3 Adjusted Means, Standard Error and inferential statistics from Linear Mixed Models

Acknowledgments

We thank the Psychology Institute of USP -University of Sao Paulo, for having provided with the dog laboratory for this research. We also thank Fernanda Torello for helping with the experiments. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.


Valérie Potard
email: valerie.potard@iplesp.upmc.fr

Ana Canestri

Sebastien Gallien

Dominique Costagliola

Use of darunavir in HIV-1 infected individuals in routine clinical practice from 2012 to 2016 in France

Objectives:

We assessed virological outcomes of darunavir use in France from 2012 to 2016, in three groups of people living with HIV (PLHIV):(1) antiretroviral (ARV)-naïve PLHIV;(2) ARVexperienced PLHIV switching to darunavir while failing therapy;(3) ARV-experienced PLHIV switching to darunavir while virologically controlled.

Methods:

Virological success (VS) was defined as a plasma HIV-1 viral load (VL)<50 copies/mL and virological failure (VF) as two consecutive VL>50 copies/mL or one VL>50 copies/mL followed by a treatment switch prior to the next VL measurement. The cumulative incidence of VS was assessed considering darunavir discontinuation, loss to follow-up, and death as competing risks, while estimates of cumulative incidence of VF accounted for loss to followup and death.

Results: Among the 3235 ARV-naïve PLHIV initiating darunavir, the four-year cumulative incidence of VS was 80.9% and was associated with lower VL and higher CD4. Among the 3485 ARV-experienced PLHIV switching to darunavir while failing therapy, the four-year cumulative incidence of VS was 82.2% and was associated with lower VL. Among the 3005 ARV-experienced PLHIV switching to darunavir while virologically controlled, the four-year cumulative incidence of VF was 12.6%. The risk of VF was higher with darunavir monotherapy (subdistribution hazard ratio (sHR)=1.67, 95%CI, 1.15-2.42) while no difference was observed with dual therapy (sHR=1.00, 95%CI, 0.71-1.42) relative to triple therapy or more.

Conclusion:

Darunavir-containing regimens yielded similarly high rates of viral suppression in PLHIV whether they were ARV-naïve or ARV-experienced switching to darunavir while failing therapy, or of maintaining VS in ARV-experienced PLHIV switching to darunavir while virologically controlled.

Introduction

The primary objective of antiretroviral (ARV) therapy is to achieve and maintain an HIV viral load below the detection limit of current assays, in order to promote immune reconstitution, to reduce HIV-related morbidity and mortality due to HIV infection, and to avoid the accumulation of resistance mutations. [START_REF] Gazzard | British HIV Association Guidelines for the treatment of HIV-1 infected adults with antiretroviral therapy[END_REF][START_REF] Hf | Antiretroviral treatment of adult HIV infection. 2014 recommendations of the international AIDS society-USA panel[END_REF] In parallel, viral load suppression prevents the transmission of the virus. [START_REF] Montaner | Association of highly active antiretroviral therapy coverage, population viral load, and yearly new HIV diagnoses in British Columbia, Canada: a population-based[END_REF] WHO ambitions an implementation of the 90-90-90 target to accelerate progress towards ending the AIDS epidemic in the post-2015 era: 90% of all people living with HIV infection (PLHIV) diagnosed, 90% of people with an HIV diagnosis treated, and 90% of all people receiving antiretroviral therapy with viral suppression by 2020. [START_REF] Who | Consolidated Guidelines on the use of Antiretroviral Drugs for Treating and Preventing HIV infection: Recommendations for a public health approach second edition[END_REF] Darunavir is a potent PI, with a high genetic barrier, used in treatment-naïve and experienced PLHIV. [START_REF] Meyer | Characterisation of virologic failure patients on darunavir/ritonavir in treatment-experienced patients[END_REF][START_REF] Clotet | Efficacy and safety of darunavir-ritonavir at week 48 in treatment-experienced patients with HIV-1 infection in POWER 1 and 2: a pooled subgroup analysis of data from two randomised trials[END_REF] The French expert group recommends using darunavir, if a PI is chosen as the third drug in ARV-naïve PLHIV or as a new third drug in ARV-experienced PLHIV. [START_REF] Pulido | Dual therapy with darunavir and ritonavir plus lamivudine vs triple therapy with darunavir and ritonavir plus tenofovir disoproxil fumarate and emtricitabine or abacavir and lamivudine for maintenance of human immunodeficiency virus type 1 viral suppression: randomized, open-label, noninferiority DUAL-GESIDA 8014-RIS-EST45 Trial[END_REF] French guidelines also propose a simplified darunavir-containing regimen, such as dual therapy or monotherapy to optimize treatment in ARV-experienced virologically controlled PLHIV. [8][START_REF] Paton | Protease inhibitor monotherapy for long-term management of HIV infection: a randomised, controlled, open-label, non-inferiority trial[END_REF][START_REF] Mathis | Effectiveness of Protease Inhibitor Monotherapy versus Combination Antiretroviral Maintenance Therapy: A Meta-Analysis[END_REF] Several studies have shown an increasing risk of virological rebound with a shorter duration of viral load suppression prior to switching to monotherapy. [START_REF] Stohr | Factors associated with virological rebound in HIV-infected patients receiving protease inhibitor monotherapy[END_REF][START_REF] Guiguet | Boosted protease inhibitor monotherapy as a maintenance strategy: an observational study[END_REF][START_REF] Curran | Effectiveness of ritonavir-boosted protease inhibitor monotherapy in the clinical setting: same results as in clinical trials? The PIMOCS Study Group[END_REF] The expert group thus recommends waiting for at least two years of sustained virological suppression before starting monotherapy. [START_REF] Pulido | Dual therapy with darunavir and ritonavir plus lamivudine vs triple therapy with darunavir and ritonavir plus tenofovir disoproxil fumarate and emtricitabine or abacavir and lamivudine for maintenance of human immunodeficiency virus type 1 viral suppression: randomized, open-label, noninferiority DUAL-GESIDA 8014-RIS-EST45 Trial[END_REF] it is thus important to generate data from real-life settings to assess how darunavir is used in routine care in France and to evaluate its virological efficacy, in the context of WHO targets and simplification strategies, as a complement to clinical trials. The purposes of this observational study were to describe the routine use of darunavir in ARV-naïve PLHIV, ARV-experienced PLHIV failing therapy, and ARV-experienced virologically-controlled PLHIV, to assess its effectiveness in terms of virological outcomes and whether it differs depending on its being given in the context of monotherapy, dual therapy or triple therapy or more.

Individuals and methods

Individuals and data sources

The French Hospital Database on HIV (FHDH) is a hospital-based multicentre open cohort in which inclusions have been ongoing since 1989. [START_REF] Mary-Krause | Cohort Profile: French hospital database on HIV (FHDH-ANRS CO4)[END_REF] Individuals are eligible if they have documented HIV-1 or HIV-2 infection and give their written informed consent to participate. Data are collected prospectively by trained research assistants using standardized forms which include demographic characteristics, biological markers such as the CD4 cell count and plasma HIV RNA level, and antiretroviral treatments. The FHDH project was approved by the French data protection authority (Commission National de l'Informatique et des Libertés on 27 November 1991, Journal Officiel, 17 January 1992).

Study population

This study was restricted to HIV-1 infected individuals of at least 18 years of age who started darunavir between January 1 st , 2012 and December 31 th , 2016, at least one year before the last recorded FHDH visit in the centre, with available viral load (VL) and CD4 cell count values within six months before initiating darunavir. If they satisfied the inclusion criteria, individuals were included in one of the following groups: (1) ARV-naïve PLHIV; (2) ARV-experienced PLHIV switching to darunavir while failing therapy; (3) ARV-experienced PLHIV switching to darunavir while virologically controlled.

Statistical analysis

The baseline for all analyses was the date of darunavir initiation. Continuous variables were expressed as the median and IQR and categorical variables as counts and percentages.

Virological success (VS) was defined as a VL < 50 copies/mL and virological failure (VF) as two consecutive VL values > 50 copies/mL, or one VL value > 50 copies/mL followed by a treatment switch prior to the next VL measurement. For ARV-naïve PLHIV and ARVexperienced PLHIV switching to darunavir while failing therapy, we assessed the cumulative incidence of VS considering the discontinuation of darunavir as a competing risk (individuals who discontinue darunavir are likely to be those experiencing a slower reduction in VL). This approach avoids a situation in which most individuals switch from darunavir and achieve a reduction in VL while on an alternative treatment. The events of loss to follow-up and death were also considered as competing risks for VS. For ARV-experienced PLHIV switching to darunavir while virologically controlled, the cumulative incidence of VF was estimated using an intention-to-continue-treatment approach, ignoring treatment change in order to also adopt a conservative approach. Only the events loss to follow-up and death were considered as competing risks. Individuals were considered to be "lost to follow-up" when there was an interval of more than 18 months between the last follow-up visit and the last database update for the centre in which they were followed. Individuals who experienced neither the outcome of interest nor the competing events were censored at the last follow-up or 48 months, whichever occurred first.

Univariable and multivariable competing-risk regression models, yielding subdistribution hazard ratios (sHR) were used to assess the influence of the type of combination (monotherapy, dual therapy, triple therapy or more) on VS or VF. [START_REF] Andersen | Competing risks in epidemiology: possibilities and pitfalls[END_REF] The following potential confounding factors were accounted for: age, gender and transmission group (MSM, injecting drug users, other men, other women), sub-Saharan origin, prior AIDS event, baseline CD4 T cell count (<200/mm 3 , 200-350/mm 3 , 350-500/mm 3 , ≥ 500/mm 3 ) and HCV antibody status (negative, positive) for the three groups, and VL at baseline in ARV-naïve PLHIV, VL at baseline and number of prior ARVs in ARV-experienced PLHIV switching to darunavir while failing therapy and duration of viral suppression prior to baseline in ARVexperienced PLHIV switching to darunavir while virologically controlled. SAS software (v9.4; SAS Institute Inc, Cary, NC, USA) was used for all statistical analyses.

Results

Baseline characteristics

The baseline characteristics according to group are shown in Table 1. A total of 3235 ARVnaïve PLHIV initiated darunavir (group 1), 3485 ARV-experienced PLHIV switched to darunavir while failing therapy (group 2) and 3005 ARV-experienced PLHIV switched to darunavir while virologically controlled (group 3). Almost all ARV-naïve PLHIV started a triple therapy or more (96.3%). Among ARV-experienced PLHIV who switched to darunavir while failing therapy, 85.9% were prescribed triple therapy or more and among those who switched to darunavir while virologically controlled, 76.4% received triple therapy or more, 14.5% dual therapy and 9.1% monotherapy. Triple therapy with two NRTIs, particularly tenofovir and emtricitabine, was the most prescribed for all groups. The median follow-up was 2.3 years (IQR: 1.5-3.2) for group 1, 2.5 years (IQR: 1.4-3.4) for group 2, and 2.5 years (IQR: 1.6-3.4) for group 3.

ARV-naïve PLHIV

Of the 3235 ARV-naïve PLHIV who initiated darunavir, 2546 achieved a VL<50 copies/mL, whereas 461 discontinued darunavir, 107 were lost to follow-up and 10 died before controlled VL. Cumulative incidence function estimates showed the probability of VS, discontinuation, loss to follow-up, and death at one year to be 72.3% (95% CI, 70.8-73.9), 12.2% (95% CI, 11.1-13.4), 2.7% (95% CI, 2.2-3.4) and 0.2% (95% CI, 0.1-0.4) respectively, and at four years to be 80.9% (95% CI, 79.8-82.1), 14.9% (95% CI, 13.6-16.3), 3.4% (95% CI, 2.8-4.1) and 0.4% (95% CI, 0.2-0.6) respectively. The probability of VS was associated with a lower VL at baseline, whereas individuals with baseline CD4 T cell counts < 200/mm 3 were less likely to achieve a VL<50 copies/mL (sHR=0.85, 95% CI, 0.75-0.97) (Figure 1a and Table 2). The type of combination was not associated with VS.

The main reason for discontinuing darunavir before VS was adverse events (42.2%).

Among the 2546 individuals with VS, 1435 discontinued darunavir after reaching a VL < 50 copies/mL, the probability of discontinuing darunavir at four years was 72.3 [69.7-75.0]. The main reason for discontinuing darunavir after VS was simplification (63.6%).

ARV-experienced PLHIV switching to darunavir while failing therapy

Among the 3485 ARV-experienced PLHIV switching to darunavir while failing therapy, 2762 reached a VL< 50 copies/mL, whereas 370 discontinued darunavir, 105 were lost to followup and 25 died before reaching this endpoint. Cumulative incidence function estimates showed the probability of VS, discontinuation, loss to follow-up, and death at one year to be 70.0% (95% CI, 68.5-71.6), 8.0% (95% CI, 7.1-8.9), 2.1% (95% CI, 1.7-2.6), and 0.4% (95% CI, 0.2-0.6) respectively, and at four years to be 82.2% (95% CI, 80.7-83.8), 11.6% (95% CI, 10.6-12.7), 3.4% (95% CI, 2.8-4.1), and 0.9% (95% CI, 0.5-1.4) respectively. The probability of VS was associated with lower VL at baseline, whereas the type of combination was not associated with VS and individuals who were exposed to more than three prior ARVs were less likely to reach a VL<50 copies/mL (Figure 1b and Table 3).

The main reason for the discontinuation of darunavir before VS was adverse events (41.2%). Among the 2762 individuals with VS, 783 discontinued darunavir after reaching a VL<50 copies/mL. The probability of discontinuing darunavir at four years was 41.7 [39.0-44.5]. The main reason for discontinuing darunavir after VS was simplification (48.5%).

ARV-experienced PLHIV switching to darunavir while virologically controlled

Among the 3005 ARV-experienced PLHIV switching to darunavir while virologically controlled, 308 experienced VF (294 with two consecutive VL > 50 copies/ml and 14 with one VL > 50 copies/ml followed by a treatment switch prior to the next VL measurement), whereas 207 were lost to follow-up and 17 died. Cumulative incidence function estimates showed the probability of VF, loss to follow-up, and death at one year to be 7.1% (95% CI, 6.3-8.1), 3.4% (95% CI, 2.8-4.1), and 0.1% (95% CI, 0.05-0.4) respectively, and at four years to be 12.6% (95% CI, 11.1-14.2), 8.9% (95% CI, 8.0-10.0), and 1.0% (95% CI, 0.6-1.6) respectively. The probability of VF was higher when the duration of viral suppression prior to baseline was shorter (Table 4). The probability of VF at four years after switching to darunavir ranged from 6.5% (95% CI, 4.8-8.8) in participants for which the duration of viral suppression was > 5 years, to 17.5% (95% CI, 15.4-19.8) among individuals with a duration of viral suppression < 2 years (figure 2a). The one-year and four-year probabilities of VF, were respectively 8.9% (95% CI, 6.6-11.9) and 15.6% (95% CI, 11.1-22.0) among individuals undergoing darunavir monotherapy, 6.1% (95% CI, 4.5-8.9) and 10.9% (95% CI, 7.8-15.1) among those undergoing dual therapy, and 7.1% (95% CI, 6.2-8.1) and 12.6% (95% CI, 11.0-14.3) among those undergoing triple therapy or more (figure 2b). In the multivariable model (Table 4), monotherapy was associated with a higher risk of VF than triple therapy or more (sHR=1.67 (95% CI, 1.15-2.42)) while no difference was evidenced between dual therapy and triple therapy or more (sHR=1.00 (95% CI, 0.71-1.42)). The effect of duration of viral suppression prior to baseline on the risk of VF was similar for participants undergoing monotherapy, dual therapy or triple therapy or more (sHR=0.73 (95% CI, 0.61-0.86) for monotherapy, 0.89 (95% CI, 0.78-1.02) for dual therapy, and 0.86 (95% CI, 0.81-0.91) for triple therapy or more).

A total of 1149 individuals discontinued darunavir, of whom 1014 individuals (88.3%) had not experienced VF. Among individuals who had not experienced VF, the probability of discontinuing darunavir at four years was 53.8 [51.2-56.5]. The main reason for discontinuation was "simplification" (45.0%).

Discussion

In this observational study, we showed that darunavir-based regimens were associated with a VS rate > 80% at four years, in three similar sized groups of ARV-naïve (group 1), or ARV-experienced PLHIV switching to darunavir while failing therapy (group 2) or while virologically controlled (group 3).

The darunavir one-year VS rate of 72.2% in ARV-naïve individuals was lower than those reported in randomized controlled trials, such as the Artemis trial (84% at week 48), [START_REF] Ortiza | Efficacy and safety of once-daily darunavir/ritonavir versus lopinavir/ritonavir in treatment-naïve HIV-1-infected patients at week 48[END_REF] or the Flamingo trial (83% at week 48), [START_REF] Clotet | Once-daily dolutegravir versus darunavir plus ritonavir in antiretroviral-naive adults with HIV-1 infection (FLAMINGO): 48 week results from the randomised open-label phase 3b study[END_REF] or the Kidar observational study (82% at week 48). [START_REF] Podzamczer | Abacavir/lamivudine plus darunavir/ritonavir in routine clinical practice: a multicentre experience in antiretroviral therapy-naive andexperienced patients[END_REF] This lower VS probability could be partly explained by the real life with less control over observations as compared to randomized controlled trials. We think the discrepancy is more likely explained by a difference in the level of baseline VL at darunavir initiation. Indeed, 51% of ARV-naïve individuals had a baseline VL>100 000 copies/mL whereas the proportion of such individuals was 34% in the Artemis trial, 25% in the Flamingo trial and 28% in the Kidar study. The rates of patients with a baseline VL<100 000 copies/mL reaching a VL <50 copies/mL at week 48 of our study (81%) were similar to those of the Artemis (86%) and Flamingo (87%) trials. Moreover, an observational Canadian study, which included 45% ARV-naïve individuals with a baseline VL>100 000 copies/mL, showed the cumulative incidence of VL<50 copies/mL at month 12 in those receiving darunavir to be 73% similar to our findings. [START_REF] Antoniou | Comparison of atazanavir/ritonavir and darunavir/ritonavir based antiretroviral therapy for antiretroviral naïve patients[END_REF] In our study, the most frequent reason for discontinuing darunavir before VS was adverse events and after VS treatment simplification. An observational study in Belgium that included HIV-infected individuals between 2010 and 2014 also found the main reasons for darunavir discontinuation to be treatment simplification and adverse events. [START_REF] Wit | Long-term use of darunavir/ritonavircontaining regimens in daily practice in Belgium: retrospective observational cohort data of 1701 HIV-patients[END_REF] The increasing popularity of available novel and simpler therapies among practitioners between 2011 and 2016 may explain the high rate of darunavir discontinuation for simplification. For example, the Swedish InfCare study showed that starting ARV in 2011 or later increased the risk of early discontinuation of treatment relative to treatment started between 2009 and 2010. [START_REF] Häggblom | HIV drug therapy duration; a Swedish real world nationwide cohort study on InfCareHIV 2009-2014[END_REF] Among ARV-experienced individuals switching to darunavir while failing therapy, the estimated rate of achieving a VL < 50 copies/mL at one year (70%) was similar to that of randomized trials such as Titan (71%) and Odin (72.1% for once-daily darunavir, 70.9% for twice-daily darunavir). [START_REF] Madruga | Efficacy and safety of darunavir-ritonavir compared with that of lopinavir-ritonavir at 48 weeks in treatment-experienced, HIVinfected patients in TITAN: a randomised controlled phase III trial[END_REF][START_REF] Cahna | Week 48 analysis of once-daily vs. twice-daily darunavir/ritonavir in treatment-experienced HIV-1-infected patients[END_REF] Lower rates of VS were obtained in participants with a baseline VL>100 000 copies/mL (55% in our study and the Titan trial).

During the period of this study, only a small number of ARV-experienced PLHIV with controlled VL switched to darunavir monotherapy or as part of a dual therapy, probably because the French guidelines that propose a simplified darunavir-containing regimen, consisting of dual therapy or monotherapy are recent. [START_REF] Pulido | Dual therapy with darunavir and ritonavir plus lamivudine vs triple therapy with darunavir and ritonavir plus tenofovir disoproxil fumarate and emtricitabine or abacavir and lamivudine for maintenance of human immunodeficiency virus type 1 viral suppression: randomized, open-label, noninferiority DUAL-GESIDA 8014-RIS-EST45 Trial[END_REF] In this observational study, the rates of VF at one year among ARV-experienced PLHIV switching to darunavir while virologically controlled was similar to that of the Monet trial among patients switching to darunavir monotherapy (8.7%) and those switching to darunavir-containing triple therapy (5.4%). [START_REF] Arribas | The MONET trial: darunavir/ritonavir with or without nucleoside analogues, for patients with HIV RNA below 50 copies/mL[END_REF] The rate of VF was slightly higher in the Monoi trial among patients switching to darunavir monotherapy (11.0%) at week 48. [START_REF] Katlama | Efficacy of darunavir/ritonavir maintenance monotherapy in patients with HIV-1 viral suppression: a randomized open-label, noninferiority trial, MONOI-ANRS 136[END_REF] As in other studies, the duration of viral suppression was associated with the risk of VF. [START_REF] Stohr | Factors associated with virological rebound in HIV-infected patients receiving protease inhibitor monotherapy[END_REF][START_REF] Guiguet | Boosted protease inhibitor monotherapy as a maintenance strategy: an observational study[END_REF][START_REF] Curran | Effectiveness of ritonavir-boosted protease inhibitor monotherapy in the clinical setting: same results as in clinical trials? The PIMOCS Study Group[END_REF] Interestingly the effect was similar regardless of the type of combination (monotherapy, dual therapy or triple therapy or more). Prolonged viral suppression was a strong predictor because it may be a marker of several factors associated with VF: good adherence to antiretroviral therapy, favourable genetic factors, low viral reservoir size, and high CD4 cell counts. Another independent factor associated with VF was the strategy of treatment. There was a higher risk of VF with monotherapy than triple therapy or more. A meta-analysis of four clinical trials reporting 8 VS at week 48, [START_REF] Arribas | The MONET trial: darunavir/ritonavir with or without nucleoside analogues, for patients with HIV RNA below 50 copies/mL[END_REF][START_REF] Katlama | Efficacy of darunavir/ritonavir maintenance monotherapy in patients with HIV-1 viral suppression: a randomized open-label, noninferiority trial, MONOI-ANRS 136[END_REF][START_REF] Guaraldi | Randomized trial to evaluate cardiometabolic and endothelial function in patients with plasma HIV-1 RNA suppression switching to darunavir/ritonavir with or without nucleoside analogues[END_REF][START_REF] Antinori | Week 48 efficacy and central nervous system analysis of darunavir/ritonavir monotherapy versus darunavir/ritonavir with two nucleoside analogues[END_REF] also showed the efficacy of triple therapy to be superior to that of monotherapy. [START_REF] Antinori | Efficacy and safety of boosted darunavir-based antiretroviral therapy in HIV-1-positive patients: results from a meta-analysis of clinical trials[END_REF] Finally, the risk of VF was similar for dual therapy and triple therapy, as shown in the Dual-Gesida trial. 8 The main strength of our study was its large size and routine clinical setting, providing additional evaluation of the use of darunavir in monotherapy or in combination with other antiretrovirals in all groups of patients. In this observational setting, we were unable to adjust the results for the genotypic susceptibility score or adherence which are not recorded in the FHDH. However, as we adjusted for number of prior ARVs in ARV-experienced PLHIV switching to darunavir while failing therapy and for prolonged viral suppression in ARV-experienced PLHIV switching to darunavir while virologically controlled, we feel our results are nevertheless robust.

In conclusion, this real-world nationwide cohort shows that darunavir was widely used in France from 2012 -2016 in ARV-naïve and ARV-experienced PLHIV switching to darunavir either while failing therapy or while virologically controlled with a high level of efficacy, similar to that seen in clinical trials. Ours results do not support the use of darunavir monotherapy in ARV-experienced PLHIV switching to darunavir while virologically controlled, while dual therapy was associated with high level efficacy. The cumulative incidence of VL<50 copies/ml was estimated considering DRV discontinuation, loss to follow-up and death as competing risks The cumulative incidence of virological failure was estimated considering loss to follow-up and death as competing risks

Fig. 1 .

 1 Fig. 1. Cumulative incidence of virological success (VL<50 copies/ml) according to VL at baseline in (a) ARV-naïve individuals and (b) ARV-experienced individuals switching to DRV while failing therapy

Fig. 2 .

 2 Fig. 2. Cumulative incidence of virological failure (two consecutive VL>50 copies/ml or one VL>50 copies/ml followed by a treatment switch prior to another VL measurement) according to (a) duration of viral suppression prior to baseline and (b) type of combination for ARV-experienced individuals switching to DRV while virologically controlled a. Duration of viral suppression prior to baseline

Table 1 . Individual characteristics at DRV initiation according to the clinical situation ARV naive (n=3235)

 1

		Switch to DRV while	Switch to DRV while
		failing therapy	virologically controlled
		(n=3485)	(n=3005)
	n, median	%, [IQR] n, median	%, [IQR] n, median %, [IQR]

CD4 (cells/mm 3 ) at baseline

  Abbreviations: ARV, antiretrovirals; DRV, darunavir; NNRTI, non-nucleoside reverse transcriptase inhibitor; VL, viral load; PCP, pneumocystis pneumonia

				Switch to DRV while	Switch to DRV while
				failing therapy	virologically controlled
		ARV naive (n=3235)	(n=3485)	(n=3005)
		n, median	%, [IQR] n, median	%, [IQR] n, median %, [IQR]
	HCV antibody status
	Negative	3105	96.0	3078	88.3	2581	85.1
	Positive	130	4.0	407	11.7	424	14.1
	PCP prophylaxis
	Not eligible (CD4>200)	2171	67.1	2352	67.5	2854	95.0
	No	674	20.8	911	26.1	136	4.5
	Yes	390	12.1	222	6.4	15	0.5
		303	[142-475]	330	[148-529]	587 [416-793]
	<200	1064	32.9	1133	32.5	151	5.0
	200-350	773	23.9	706	20.3	336	11.2
	350-500	673	20.8	669	19.2	614	20.4
	≥500	725	22.4	977	28.0	1904	63.4
	CD4/CD8 at baseline	0.3	[0.2-0.5]	0.4	[0.2-0.6]	0.8 [0.5-1.1]
	Missing	320		285		291
	<0.5	2062	70.7	2076	64.9	649	23.9
	≥0.5	853	29.3	1124	35.1	2065	76.1
	Viral load (copies/mL) at baseline	5.0	[4.4-5.5]	3.7	[2.5-4.9]	-	-
	<50	-	-	-	-	3005	100
	<5000	323	10.0	1721	49.4	-	-
	5000-100000	1275	39.4	1072	30.8	-	-
	>100000	1637	50.6	692	19.9	-	-
	Duration of viral suppression prior to baseline
	(year)
	<2					1340	44.6
	2-5					798	26.6
	≥ 5					867	28.9
	Prior AIDS event
	No	2787	86.2	2348	67.4	2198	73.1
	Yes	448	13.8	1137	32.6	807	26.9

Acknowledgments

Clinical Epidemiology Group of the FHDH-ANRS CO4

Funding FHDH is supported by the Agence Nationale de Recherches sur le Sida (ANRS), Institut National de la Santé et de la Recherche Médicale (INSERM) and the French Ministry of Health. This study was funded by Janssen. Transparency declaration DC reports HIV grants from Janssen (2017-2018, 2019-2020) and MSD France (2015-2017), personal fees from Janssen (2016, 2018), MSD France 2017) and Gilead (2018) for lectures, personal fees from Innavirvax (2016) and Merck Switzerland (2017) for consultancy, outside the submitted work. AC has received lecturer or travel fees from Gilead, Janssen, MSD and ViiV during the last three years. SG has received lecturer or travel fees from Gilead, Janssen, MSD, ViiV and Pfizer during the last three years. VP has conducted post-marketing studies for Janssen and MSD during the last three years.

Author's contribution

DC and VP designed the study, analyzed the data, drafted the manuscript, had full access to the data and had final responsibility for the decision to submit the study for publication. AC and SG revised the analysis plan. All authors were involved in the interpretation of the data and critical revision of the manuscript and approved the final version.