Jana Brunner

Phil Hoole

Pascal Perrier

Motor equivalent strategies in the production of /u/ in perturbed speech -published in Proceedings of Acoustics'08

Several articulatory strategies are available during the production of /u/, all resulting in a similar acoustic output. /u/ has two main constrictions, at the velum and at the lips. A perturbation of either constriction can be compensated at the other one, e.g wider constriction at the velum by more lip protrusion, wider lip opening by more tongue retraction. This study investigates whether speakers use this relation under perturbation. Six speakers were provided with palatal prostheses which were worn for two weeks. Speakers were instructed to make a serious attempt to produce normal speech. Their speech was recorded via EMA and acoustics several times over the adaptation period. Formant values of /u/-productions were measured. Velar constriction width and lip protrusion were estimated. For four speakers a correlation between constriction width and lip protrusion was found. A negative correlation between lip protrusion and F1 or F2 could sometimes be observed, but no correlation occurred between constriction size and either of the formants. The results show that under perturbation speakers use motor equivalent strategies in order to adapt. The correlation between constriction size and lip protrusion is stronger than in studies investigating unperturbed speech. This could be because under perturbation speakers are inclined to try out several strategies in order to reach the acoustic target and the co-variability might thus be greater.

Introduction

Some sounds such as rounded back vowels or the American English /r/ allow for several possibilities to produce them. In rounded back vowels, for example, speakers can vary lip protrusion, lip opening, larynx height and tongue height in dependence on each other. Several vocal tract configurations can thus lead to the same acoustic output. Lowering the larynx, for example, lowers the formants. This effect can also be reached by protruding the lips. Similarly, raising and retracting the tongue leads to a lower first and second formant, which can be compensated for by less lip protrusion. "Rounded" back vowels can even be produced with open lips and no rounding at all when the velar constriction is retracted.

To give another example, the American English /r/ can be produced with a bunched tongue shape or a retroflex tongue. Both ways to articulate the sound result in similar acoustic outputs (e.g. ( 2), [START_REF] Zhou | Acoustic cues of "retroflex" and "bunched" American English rhotic sound[END_REF], but see [START_REF] Espy-Wilson | Acoustic differences between "bunched" and "retroflex" variants of American English /r[END_REF] for slight acoustic differences).

Different articulatory strategies which lead to the same acoustic output, as the ones discussed for rounded back vowels and /r/ are called motor equivalent strategies. A number of studies show that speakers in fact use these motor equivalent strategies in their speech. The study presented in [START_REF] Perkell | Trading Relations Between Tongue-body Raising and Lip Rounding in Production of the Vowel /u/: a Pilot 'Motor Equivalence' Study[END_REF] shows that during several productions of /u/ speakers vary in their application of tongue position and lip rounding. Three of the four speakers presented in this study moved the tongue to a higher and more retracted position when they had little lip protrusion. When they had more lip protrusion they produced a wider constriction by lowering and fronting the tongue. The acoustic output of all these productions was similar.

The study presented in [START_REF] Perkell | Trading Relations Between Tongue-body Raising and Lip Rounding in Production of the Vowel /u/: a Pilot 'Motor Equivalence' Study[END_REF], however, shows a problem when investigating motor equivalent strategies: Even if there are clear interspeaker differences in the way speakers produce /u/, the effect is normally not as obvious within a speaker. Speakers seem to have a preference for a certain lip protrusion in /u/. Similarly, investigating /r/ one often finds speakers using a bunched OR a retroflex strategy exclusively, or one finds speakers alternating between the two according to context (cf. (2)). A reason for the fact that different strategies can often only be observed across speakers or at least across contexts, but hardly ever within a given speaker and context might be that speakers are used to a certain articulatory strategy involving little articulatory effort in a given context.

Perturbation experiments have shown that if one forces speakers to explore other articulatory possibilities by confronting them to a perturbation, the articulatory changes carried out in order to keep the acoustic output constant can be quite remarkable even if the context is not changed. For example, in the perturbation experiment presented in ( 6) and ( 7) speakers' lips were hold open and speakers were asked to produce the vowel /u/ which is usually rounded in unperturbed speech. Some of the speakers managed to do so by retracting the constriction.

To give another example, results presented in (8) for a short term experiment on North American English /r/, where speakers vocal tract shape was changed by a palatal prosthesis, show that some speakers who used to have bunched tongue shapes switched to retroflex shapes under perturbation.

Thus, speech perturbation seems to be a useful means to explore motor equivalent strategies. The study presented here shows results of a long term perturbation experiment where speakers' palate shape was changed in a similar way as it was done in [START_REF] Tiede | Perturbed palatal shape and North American English /r/ production[END_REF]. The articulation of /u/ was investigated. As in the two perturbation experiments discussed above speakers in our experiment were thus encouraged to use other strategies leading to the same acoustic result, either because the old strategy did no longer lead to the correct result or because it involved too much articulatory effort.

Our hypothesis was that being confronted with a palatal prosthesis lowering the palate speakers should at first lower their tongue in order to keep the same constriction size. The degree of lip protrusion should stay about the same. Later they might notice that the acoustic output does not match with the one they used to have in the unperturbed condition, or they might look for a strategy involving less articulatory effort. Thus, they should use different degrees of lip protrusion and vary the constriction size correspondingly. Over sessions one should therefore find a positive correlation between lip protrusion and constriction size. Furthermore, if speakers make efficient use of motor equivalence, no correlation between either of the two articulatory parameters and one of the formants should be found.

Methods

A two-week perturbation experiment was carried out with six German speakers. Their articulation was perturbed by a palatal prosthesis. There were two types of prostheses, the first one moved the alveolar ridge posteriorily ("alveolar palate"), the second one made the palate flatter and lower by filling out the palatal arc ("central palate"). The thickness of the palates differed from speaker to speaker and depended on the anatomical conditions of the speaker. However, they all had a maximal thickness of around 1 cm. Four speakers (A1 to A4) were recorded with an alveolar prosthesis and two (C1 and C2) with a central prosthesis. Speakers were asked to wear the prosthesis all day for two weeks and to make a serious effort to improve their speech.

Speakers' articulator movements and the resulting acoustic signal were recorded via electromagnetic articulography. Four speakers were recorded with the AG 100 at the ZAS Berlin and two speakers were recorded with the AG 500 at the IPS Munich. On the first day of the experiment three different sessions were recorded. First, speakers were recorded without the prosthesis (session nopert, meaning "no perturbation") in order to record their habitual articulation. In the second session, the artificial palate inserted and speakers' auditory feedback was masked with white noise (session wnpert, "white noise -perturbed"). In the third session speakers were recorded with auditory feedback available (session pert, "perturbed"). After one week adaptation time speakers returned to the laboratory and were recorded with the prosthesis in place (session 1wpert, "one week, perturbed"). A final perturbed session was recorded after two weeks (session 2wpert, "two weeks, perturbed"). Then speakers removed the prosthesis and were recorded with their normal vocal tract shape (session 2wnopert, "two weeks, no perturbation") in order to investigate possible after effects.

Since the experimental setup, especially for the first session, is extremely complex and difficult to coordinate the session with auditory feedback masking (wnpert) was left out for one speaker who was recorded towards the beginning of the study (speaker A2).

Eight sensor coils were attached to the subject. Three were glued to the tongue, one around a centimeter behind the tongue tip, one at the part opposite the border between hard and soft palate and the third one in the middle between these two. Another sensor was placed below the lower incisors in order to track jaw movements. In order to record lip movements, two further sensors were glued to the upper and the lower lip. Two sensors at the upper incisors and the bridge of the nose served as reference sensors to compensate for head movements.

Since the sensors had to be glued to the articulators anew on each recording day, the positions of the tongue sensors differed slightly for different recording days. Whereas it was easy to find approximately the same place for the sensor at for example the lower incisors (midsagittaly below the incisors), it was hard to find landmarks on the tongue which could serve as points of orientation for finding the same position in a further recording. Photos of the tongue with the sensors on it were taken and the distance between the sensors was measured, in order to find the positions again, however, this method turned out to be not completely satisfactory.

After the recording a number of preprocessing steps were carried out which included correction algorithms for head movement, filtering of the data, rotation and translation of the position data and synchronisation with the acoustic data. The acoustic data were downsampled to 24 kHz.

The target vowel /u/ was recorded in the nonsense word /'tu:ta/ which was embedded in the carrier phrase Ich sah ... an. (I looked at ...). The sentences were in general repeated 20 times per session in randomised order with other items. Thus, over sessions around 120 productions of /u/ were recorded.

As a first analysis step an acoustic segmentation of the material was carried out. /u/ was segmented from the onset of the second formant to the offset of the second formant. These segmentations were later used in order to carry out semiautomatic formant measurements of F1 and F2 in each /u/-production. In these measurements the formants were at first calculated fully automatically by just adapting the LPC order according to the sex of the speaker. Afterwards, the measurements were corrected manually by determining the frequency ranges for each formant in each production and calculating formants for these ranges.

Measuring the formants of one speaker (A4) turned out to be problematic since she had a rather breathy voice.

Lip protrusion was measured by calculating the difference in the horizontal dimension between the upper lip sensor and the upper incisor sensor at the middle of the interval for which the formants had been measured. By doing this it was assumed that the upper incisor sensor and the upper lip sensor were glued in about the same location on different recording days, which seemed to be basically true.

The constriction size was measured as the shortest Euclidean distance between the palate and the tongue. In order to carry out this measurement, the palatal contour for each session was estimated by plotting all positional data of the complete speech material recorded in the session. Then the palatal contour was estimated at the upper border of these tongue positions. In order to estimate the tongue contour, spline functions going through the three tongue sensors were calculated at the acoustic target position. A comparison of the position of just the tongue back sensor (as was done in (3)) was not possible since the sensor position on the tongue varied between different experimental sessions. Afterwards, the constriction size was estimated as the smallest Euclidean distance between tongue contour and palate contour.

Bivariate Pearson correlations for the four parame-ters (F1, F2, lip protrusion and constriction size) were calculated with SPSS 15.0.

Results

At first the results for the formant measurements will be discussed. Afterwards, results of the measurements of the articulatory parameters will be presented. In section 3.3 the results of the correlation measurements can be found. Section 3.4 deals with articulatory parameters measured within a single session. Section 3.5 discusses the development across sessions.

Acoustic parameters: F1 and F2

Figure 1 shows mean values of F1 (upper subplot) and F2 (lower subplot) for each session (abscissa). Different line styles and grey shades represent different speakers. The speakers are given either on the left or on the right of each graph in the same height and in the same colour as the graph. If one looks at sessions unpert, wnpert and pert first, one can see that both formants deviate somewhat in the session when no auditory feedback is there (wnpert) as compared to the unperturbed session. In the session thereafter (pert), however, the speakers regain the original values. 1 An exception is speaker A1 with more deviation in F1 in session pert than wnpert. In the later sessions the speakers do not stay with these values measured for session pert but the values vary in a rather inconsistent manner. The values reached in the last perturbed session (2wpert), which presents the end of the practice period and should therefore give the "best possible" adaptation, are not more similar to the original values than the ones already measured in session pert. Repeated measures ANOVAs for data split by speaker showed a significant influence of the session on both formants except for the second formant of speaker C2. However, even if the influence is significant, it might not be perceptually relevant. 2 In general, the variation over sessions within a speaker (about 100 Hz around the F1 of the initial session and about 250 Hz around the F2 of the initial session) is much lower than the variation across speakers. Furthermore, the variation in F1 is not much higher than the "compensation threshold" of 60 Hz found in a study by Purcell & Munhall (4) and 64 Hz in a later study [START_REF] Purcell | Psychophysical detection threshold of formant manipulations during speech production[END_REF]. An exception is speaker A4 with large deviations in both F1 and F2. However, an alternative explanation for the inconsistent results could be the measurement problems for this speaker discussed above.

Lip protrusion and constriction size

Before discussing the results of the articulatory parameters two problems which arose during the analysis will be described. First, for one speaker (A4) the data of the upper lip sensor were unreliable in the sessions recorded on the first day, so these data could 1 This cannot be seen for speaker A2 of course since there was no session wnpert.

2 This is being tested at the moment. not be used for further analysis and they are missing in the figure below as well as in the calculation of the correlation.

The second problem was that sometimes the constriction size could not be measured because it was in the velar region. For one speaker (C2) the constriction was in all the sessions so far in the velar region that the palatal contour above the tongue could not be estimated with certainty and the constriction size could consequently not be measured either. The data for this speaker are therefore missing in the figure below. A similar problem occurred for speaker A4, but only in one session, session 1wpert. In all the other sessions the constriction was in the palatal region. For this speaker constriction size was measured normally for all the sessions except 1wpert. For the problematic session the most retracted measureable point at the end of the hard palate was taken as constriction location. Looking at the upper subplot showing lip protrusion one can see that it varies over session. It is difficult to find a tendency for all speakers. Whereas speakers A1 (black solid line) and A2 (dark grey solid line) in general increase lip protrusion until the end of the adaptation time (session 2wpert), speaker C1 (light grey solid) rather has an oscillating pattern (increase -decrease -increase). The other speakers keep the degree of lip protrusion about equal.

Looking at the constriction size (lower subplot) one can see that there is indeed a measurement problem for speaker A4 (dark grey dotted) in session 1wpert where the constriction was assumed to be behind the end of the hard palate. Probably it was not the constriction which was measured here but a point in front of the constriction so that the distance measured is too large. Apart from this one problematic case, the constriction sizes go parallel with the results for lip protrusion: There is an increase in lip protrusion for speakers A1 (black solid) and A2 (dark grey solid) and an increase-decrease-increase pattern for speaker C1. Repeated measures ANOVAs showed that the influence of the session on both parameters is significant.

Correlations

Results were gained for five speakers (all except C2 because no constriction could be measured). For four speakers significant correlations for at least one of the parameter pairs could be found. For speaker A4 no significant correlations could be found. This can be assumed to be due to methodological problems (one sensor missing in the three first sessions, difficulties while measuring formants).

Table 1 gives the results of the correlations between lip protrusion, constriction size and the two acoustic parameters for the four remaining speakers. The first line gives the speaker, the second line shows the correlation coefficients for the correlation between lip protrusion and constriction size. If speakers use motor equivalent strategies this correlation should be positive: For more lip protrusion the constriction should be larger. The other lines give the correlations of the two articulatory parameters with F1 and F2. If speakers use motor equivalent strategies effectively in order to keep the acoustic output constant, there should be no correlations between the articulatory and the acoustic parameters.

A significant positive correlation between lip protrusion and constriction size could be found for four speakers. For three speakers (A1, A2 and A3) there are also significant correlations between lip protrusion and one or both of the formants. These could be seen as cases of ineffective compensation: Either there is too much lip protrusion or a too narrow constriction.

Relation within a session

In order to show more details the results for lip protrusion vs. constriction size for speaker A1 are shown in figure 3 has more lip protrusion it also has a wider constriction. Numbers represent different sessions in chronological order. The figure is meant to illustrate something which could be seen for all the four speakers: Within single sessions the correlation between the parameters either does not exist or it is weaker or it is even negative. For the speaker shown here a positive correlation exists for session 4 (1wpert) but for example not for session 1 (nopert). For session 5 (2wpert) there is a clear negative correlation. The reasons for this result are so far not entirely clear. It is possible that the measuring accuracy of our method which can, for constriction size be assumed to be around 1 mm is too low in order to see a relation within a session. The correlation between lip protrusion and constriction size might be blurred when both parameters change only little (as they do within a session) but it might become clear when they change more (as is the case across sessions).

The fact that the parameters change more across sessions than within sessions shows in any case that within each session the speaker has a preferred strategy: a preferred constriction size and a preferred degree of lip protrusion. In session unpert ('1'), for example the lips are only slightly protruded and the constriction is rather small. In the last perturbed session ('5') there is a lot of lip protrusion, and the constriction is larger.

Development across sessions

There is an initial increase in constriction size in session wnpert as compared to session unpert (cf. figure 2, lower subplot). This could be because the speakers try to avoid having too much linguo-palatal contact and to thus produce a fricative rather than a vowel. Some of the speakers compensate for this lower tongue position by more lip protrusion even in the initial perturbed session. Afterwards, with some practice, speakers try out different strategies by varying both lip protrusion and constriction size. The development over sessions is quite individual and might be due to speaker specific morphological differences which lead to preferences for certain strategies.

Conclusion

This study has dealt with motor equivalent strategies in /u/. The results show that for all speakers for whom there were no methodological problems motor equivalent strategies could be found. Speakers vary the tongue position and in order to compensate for that they produce more or less lip protrusion. However, the compensation is not complete: For three of the four speakers there is a negative correlation between one of the articulatory parameters and one or even both of the acoustic parameters. The constriction is thus too small, or there is too much lip protrusion in order to keep the acoustic output completely stable. An alternative explanation for the negative correlations between one of the articulatory parameters and the acoustic parameters could be that other articulatory parameters are changed in order to keep the acoustic output constant. As discussed above, the lip opening area, for example, can vary in dependence on the constriction location. Neither of these additional parameters has been investigated here.

The correlations found here are a little stronger (higher correlation coefficients) than the ones presented in [START_REF] Perkell | Trading Relations Between Tongue-body Raising and Lip Rounding in Production of the Vowel /u/: a Pilot 'Motor Equivalence' Study[END_REF]. A reason for that could be that, in contrast to this earlier study, in the present study the speakers are confronted with a perturbation. They are thus more inclined to try out several articulatory strategies. Judging from the sensor plots presented in (3), lip protrusion in all cases varies considerably less than a centimetre. The ranges for the speakers presented here are between 7 and 12 mm.

Figure 1 :

 1 Figure 1: Mean values of formants of /u/ over sessions. Abscissa: Sessions in chronolgical order. Upper subplot: results for F1, lower subplot: results of F2. Each graph refers to one speaker. Speakers are given on the left or the right of the graph in the same colour as the corresponding graph. Error bars show standard error.

Figure 2 :

 2 Figure 2: Mean values of lip protrusion (upper subplot) and constriction size (lower subplot) of /u/ over sessions. Abscissa gives sessions. Lip protrusion data of the first three sessions for speaker A4 and constriction data of speaker C2 are missing. Error bars show standard error.

Figure 3 :

 3 Figure 3: Lip protrusion and constriction size measurements for speaker A1. Different numbers represent different sessions in chronological order: unpert: 1, wnpert: 2, pert: 3, 1wpert: 4, 2wpert: 5, 2wunpert: 6.

  . The abscissa shows lip protrusion, the ordinate constriction size. A correlation between the two parameters can clearly be seen: If a production

	speaker	A1	A2	A3	C1
	lip-const 0.568*** 0.537*** 0.235*	0.660***
	lip-F1	-0.126	-0.330** -0.351*** 0.006
	lip-F2	-0.220*	0.065	-0.313**	0.118
	const-F1 -0.173	-0.197	-0.014	0.037
	const-F2 -0.033	-0.108	0.042	0.093
	Table 1: Correlation coefficients and significance
	levels for four speakers (first line) of the correlation
	between lip protrusion and constriction size (second
	line), lip protrusion and F1 (third line), lip
	protrusion and F2 (fourth line), constriction size and
	F1 (fifth line), constriction size and F2 (sixth line).
	Significance levels: *p < 0.05, **p < 0.01,
			***p < 0.001.

Acknowledgements

This study was supported by grants of the Deutsche Forschungsgemeinschaft (PO 334/4-1 and HO 3271/1-1) to Bernd Pompino-Marschall and Phil Hoole as a project in the framework of the German-French network POPAART, and a grant of the Ministère délegué à l'enseignement et à la recherche of France. We would like to thank Jörg Dreyer from the Zentrum für Allgemeine Sprachwissenschaft for technical support during the recordings, and Olessia Panzyga, Vivien Hein and Susanne Waltl from the Humboldt Universität zu Berlin and the Ludwig-Maximilians-Universität München for acoustic segmentation and formant measurements.


Isabelle Hoxha

Alvaro Feito Boirac

Gabriel Orellana

Lianne Klaver

Jonathan Coutinho

Fabio Solari

A Feito Boirac

Four grid cell modules can code for a precise location at a low error

Introduction

Grid cells in the rat entorhinal cortex fire according to the animal's spatial location. Grid cells belong to modules with different field sizes, combined to encode location precisely. Previous studies have addressed the importance of the architecture on location accuracy (Wei et al., 2015;Sorscher et al., 2019).

Four grid cell modules can code for a precise location at a low error Isabelle Hoxha 1 , Alvaro Feito Boirac 2 , Gabriel Orellana 3 , Lianne Klaver 4 , Jonathan Coutinho 5 , Fabio Solari 6 1-Université Paris-Saclay, CIAMS, Faculté des Sciences du Sport, Orsay Cedex, France, 2-Bermuda Automation, Warwick, Bermuda, 3-Protera Biosciences, Santiago, Chile, 4-Virginia Tech, School of Neuroscience, Blacksburg, VA, United States of America, 5-Queen's University, Centre for Neuroscience Studies, Kingston, ON, Canada, 6-University of Genova, Department of Informatics, Bioengineering, Robotics and Systems Engineering, Genova, Italy

Hypothesis

The more scales, the more precise the localization


Quentin Le Hingrat

Benoit Visseaux
email: benoitvisseaux@aphp.fr

Cédric Laouenan

Sarah Tubiana

Lila Bouadma

Yazdan Yazdanpanah

Xavier Duval

Charles Burdet

Houria Ichou

Florence Damond

Detection of SARS-CoV-2 N-antigen in blood during acute COVID-19 provides a sensitive new marker and new testing alternatives

Keywords: COVID-19, SARS-CoV-2, Antigen, Antigenemia, Diagnostic, Serum, Plasma, Blood

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

Detection of SARS-CoV-2 N-antigen in blood during acute provides a sensitive new marker and new testing alternatives

Quentin LE HINGRAT, 1,2 * Benoit VISSEAUX, 1,2 * Cédric LAOUENAN, 1,3 Sarah TUBIANA, 1,3 Lila BOUADMA, 1,4 Yazdan YAZDANPANAH, 1,5 Xavier DUVAL, 1,3 Charles BURDET, 1,3 Houria ICHOU, 2 Florence DAMOND, 1,2 Mélanie BERTINE, 1,2 Nabil BENMALEK, 2 French COVID cohort management committee, CoV-CONTACT study group, Christophe CHOQUET, 1,6 Jean-François TIMSIT, 1,4 Jade GHOSN, 1,5 Charlotte CHARPENTIER, 1,2 Diane DESCAMPS, 1,2 Nadhira HOUHOU-FIDOUH 2

INTRODUCTION

Molecular assays on nasopharyngeal swabs remain the cornerstone of COVID-19 diagnostic. Despite massive efforts, the high technicalities of nasopharyngeal sampling and molecular assays, as well as scarce resources of reagents, limit our testing capabilities. Several strategies failed, to date, to fully alleviate this testing process, e.g. saliva sampling [START_REF] Rao | Comparing nasopharyngeal swab and early morning saliva for the identification of SARS-CoV-2[END_REF][START_REF] Wyllie | Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2[END_REF] or antigen testing on nasopharyngeal samples [START_REF] Flower | Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 seroprevalence survey[END_REF][START_REF] Dinnes | Rapid, point-of-care antigen and molecular-based tests for diagnosis of SARS-CoV-2 infection[END_REF]. Nucleocapsid-antigen (N-antigen) has been detected in the serum of SARS-CoV-infected patients and, recently, it has been demonstrated in SARS-CoV-2 infected patients by a single study with a global sensitivity of 41/64 patients [START_REF] Ogata | Ultra-sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease[END_REF][START_REF] Che | A Patient with Asymptomatic Severe Acute Respiratory Syndrome (SARS) and Antigenemia from the 2003-2004 Community Outbreak of SARS in Guangzhou, China[END_REF].

In this work, we assessed the performances of N-antigen sera detection in a large patients' population using the first commercially available assay, the COVID-19 Quantigene® (AAZ France)

providing a low limit of detection at 2.98 pg/mL.

METHODS

Patients and ethics.

Negative samples are composed of 50 pre-pandemic samples (collected between December 2, 2019 and January 13, 2020) and 13 pandemic samples from SARS-CoV-2 noninfected patients that tested positive for other microbial antigens (i.e. NS1 antigen, HBs antigen, HIV-1 p24 antigen, HKU1 coronavirus or malaria antigens). Positive samples were collected between January 25, 2020 and September 2, 2020 from study participants included in the French COVID (clinicaltrials.gov NCT04262921) and CoV-CONTACT cohorts (clinicaltrials.gov NCT04259892). We selected the first serum samples available after COVID-19 diagnosis (cf. Supplementary Figure 2). The following serum samples of those patients, when collected at physician discretion, were also included. They have provided written informed consent for the use of their samples for research.

Ethics approval was given by the French Ethics Committee CPP-Ile-de-France 6 (ID RCB: 2020-A00256-33 and ID RCB: 2020-A00280-39) and the French National Data Protection Commission (approval #920102).

For COVID-19 patients, available sera samples were classified into different categories according to the delay since symptom onset: sera collected ≤14 days post-symptoms onset (142 sera from 114 patients), samples collected >14 days post-symptoms onset (81 samples from 72 patients), samples collected from asymptomatic patients (3 sera from 3 patients), and patient without date of symptom onset (1 sera from 1 patient). Distribution of sera samples according to date of sampling and hospitalization status is detailed in Supplementary Figure 2.

N-antigen levels assessment. Prior to analysis, sera samples were stored at -80°C. N antigenemia levels were determined with a being marketed CE-IVD ELISA microplate assay, COVID-Quantigene® (AAZ), according to manufacturer recommendations. Briefly, in each well of 96-wells microplates previously coated with anti-SARS-CoV-2 N-antibodies, 50 µL of a solution containing biotinylated anti-SARS-CoV-2 N antibodies and 50 µL of sera were added. After incubation at 37°C for 60 minutes, plates were washed 5 times with a phosphate buffer solution. Then, 100 µL of a solution containing HRP-conjugated streptavidin were added, followed by incubation for 30 minutes at 37°C. Plates were washed 5 times with the phosphate buffer solution, then 50 µL of a solution containing the substrate [START_REF] Flower | Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 seroprevalence survey[END_REF][START_REF] Flower | Clinical and laboratory evaluation of SARS-CoV-2 lateral flow assays for use in a national COVID-19 seroprevalence survey[END_REF][START_REF] Ogata | Ultra-sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease[END_REF]) and 50 µL of a second solution containing urea were added.

After 15 minutes at 37°C, the colorimetric reaction was stopped by adding 50 µL of H 2 SO 4 .

Absorbance values were measured at 450nm, with a reference set at 630nm. In each plate, standards made of recombinant N antigens were tested, to quantify the N antigenemia levels for each patient's sample. As the purpose of this study was to assess the sensitivity of this new assay, samples with titers above 180 pg/mL were not diluted for precise quantification.

RT

Data availability.

A file compiling all data used in this article is available on Mendeley Data public repository (https://data.mendeley.com/datasets/fjz6zbkxvm/1). 1A and Supplementary Appendix 1A).

RESULTS

Specificity

For 91 patients, 104 paired samples of sera and nasopharyngeal swabs collected in the same 24 hours were available, allowing us to compare N-antigen detection with E gene Ct values for those patients (Figure 1-D). Among patients with E gene Ct value below 30 and below 33 on their nasopharyngeal swab, only 1/50 and 4/67 tested negative for N-antigenemia, respectively (Supplementary Figure 1B). For patients with positive NP samples with Ct values ≥33, only 15/25 (60%) were positive for N-antigenemia. Interestingly, 8 out of 12 patients with a negative nasopharyngeal RT-PCR presented positive N-antigenemia. The lower respiratory tract was explored for 6 of these 8 patients either the same day or in the 5 following days. RT-PCR on the lower respiratory tract sample was positive in 5 of these 6 patients.

DISCUSSION

This is the first evaluation of a commercially available SARS-CoV-2 N-antigen serum or plasma detection. This assay presented a low detection limit at 2.98 pg/mL and a sensitivity above 90%

during the acute phase of the disease (i.e. <14 days after symptoms onset in PCR confirmed COVID-19 patients). In the first two weeks, N-antigen negativity was associated with anti-N IgG detection (6/10) and/or low nasopharyngeal viral load in the same 24 hours (7/7, Ct value >30). This sensitivity could allow its use for COVID-19 diagnostic and is in line with RT-PCR on NP samples whose reported sensitivity rates ranged between 71 and 98%, based on negative RT-PCR tests which were positive on repeat testing [START_REF] Arevalo-Rodriguez | False-negative results of initial RT-PCR assays for COVID-19: a systematic review[END_REF].

Detection of viral antigens in the blood of COVID-19 patients has been recently described by Ogata and collaborators, who detected N and S1 antigens in the blood of 41 out of 64 COVID-19 patients [START_REF] Ogata | Ultra-sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease[END_REF]. If antigens blood circulation is not uncommon in infectious diseases, antigenemia tests usually target blood-borne pathogens, notably Dengue, CMV, HBV or HIV. In respiratory diseases, antigen circulation into non-respiratory body fluids is usually not considered, even if likely, because of the focal nature of the infection or possible pre-existence of antibodies. Antigen detection in nonrespiratory fluids is still used for two respiratory bacteria: Streptococcus pneumonia and Legionella pneumophila. Interestingly, it has also been reported in SARS-CoV-1 infection [START_REF] Che | A Patient with Asymptomatic Severe Acute Respiratory Syndrome (SARS) and Antigenemia from the 2003-2004 Community Outbreak of SARS in Guangzhou, China[END_REF]. Whether the circulation of free viral antigens has an impact on disease physiopathology should be assessed in future studies.

N-antigenemia was also detectable in outpatients but the decrease seems to occur earlier in our This innovative marker may also be of help for prognostic evaluation of patients. An association between high N-antigen levels and higher ICU admission rates has been reported by Ogata et al. [5].

In our study, we observed higher N-antigen levels in serum of viremic patients, in line with the possible association of viremia, or RNAemia, with disease severity and/or immunosuppression [START_REF] Veyer | Highly sensitive quantification of plasma SARS-CoV-2 RNA shelds light on its potential clinical value[END_REF][START_REF] Buetti | Risk factors for SARS-CoV-2 detection in blood of critically ill patients[END_REF][START_REF] Chen | Detectable serum SARS-CoV-2 viral load (RNAaemia) is closely correlated with drastically elevated interleukin 6 (IL-6) level in critically ill COVID-19 patients[END_REF].

Our study presents several limitations. We included a very few number of outpatients and this population needs to be explored in larger cohorts, ideally with longitudinal samples of paucisymptomatic and asymptomatic patients. The case-control design is another limitation and the test performances could be slightly deteriorated in real life condition.

In conclusion, sensitive N-antigen detection in serum or plasma provides a valuable new marker for COVID-19 diagnosis, only requiring a blood draw, scalable in all clinical laboratories. It allows potential new developments to design rapid antigen blood test or combined ELISA assays, detecting both antigens and antibodies. It also raises new questions about the physiological mechanisms at play explaining blood circulation of this antigen and its potential correlation to disease severity.

FIGURE LEGEND Figure 1:

(A) Evolution of N-antigen sera levels in SARS-CoV-2-infected patients according to hospitalization status (N=227 serum samples from 165 patients); sequential samples are connected with a gray line, while the positivity threshold value for N-antigen (2.97 pg/mL) is indicated with a dashed red line. (B) N-antigenemia levels according to delay since symptoms onset. (C) N-antigen sera levels according to positive and negative RNAaemia status (n=146 sera, N=89 patients). (D) Nantigen sera levels according to E-gene cycle threshold value of 104 nasopharyngeal swabs collected within 24 hours (N=91 patients).

FUNDING

This study has been funded in part by the REACTing (REsearch & ACTion emergING infectious diseases) consortium, by a grant from the French Ministry of Health (PHRC n°20-0424) and by the ANRS (Agence Nationale de la Recherche sur le SIDA et les hépatites virales). The study was supported by AAZ (Boulogne-Billancourt, France) in the form of free consumables and they had no role in conceptualization, design, data collection and analyses, decision to publish and manuscript preparation.

ACKNOWLEDGEMENT

The French COVID cohort was sponsored by Inserm. We wish to thank the ANRS (Agence Nationale de la Recherche sur le SIDA et les hépatites virales).

  -PCR assays. For all patients included in this study, diagnosis of SARS-CoV-2 infection was performed in the virology department of Bichat-Claude Bernard University Hospital by RT-PCR on naso-pharyngeal swabs, as recommended. Different techniques were performed throughout the study period for nasopharyngeal samples, due to frequent shortages issues and requirements for fast turnaround time: RealStar® SARS-CoV-2 (Altona, Hamburg, Germany), Cobas® SARS-CoV-2 (Roche Diagnostics, Branchburg, NJ, USA), Simplexa® COVID-19 Direct (DiaSorin, Gerenzano, Italy), BioFire® SARS-CoV-2 (BioMerieux, Salt Lake City, UT, USA), QIAstat-Dx® Respiratory SARS-CoV-2 (Qiagen, Hilden, Germany) and NeumoDX® (QIAgen, Hilden, Germany) using the IP2 Institute Pasteur and the WHO E gene primers[7]. E gene cycle threshold (Ct) values, available for all techniques except Simplexa® COVID-19 Direct and BioFire® SARS-COV-2, were used as a proxy for viral load for 104 samples from 91 patients with paired naso-pharyngeal swabs and sera (i.e. collected in the same 24 hours). For a subset of 146 samples, corresponding to 89 patients included in the French COVID-19 cohort, paired sera and plasma samples were available, allowing to determine the presence of viral RNA in plasma. Briefly, viral nucleic acids were extracted from 200 µL of plasma with the MagNA Pure LC Total Nucleic Acid Isolation Kit -Large Volume (Roche Diagnostics, Branchburg, NJ, USA) and eluted in 50 μL. RT-PCR was performed on 10 μL of eluate using the RealStar® SARS-CoV-2 assay (Altona, Germany), according to the manufacturer recommendations. Samples with RT-PCR cycle threshold values above 40 were considered negative. Detection of anti-SARS-CoV-2 nucleocapsid IgG. For a subset of 85 sera, corresponding to 80 patients (ICU patients: n=21, ward patients: n=36 and outpatients: n=23), we performed a chemiluminescent microparticle immunoassay detecting anti-N immunoglobulins G (Architect SARS-CoV-2 IG Assay, Abbott). Results were reported as a signal to cut-off (S/Co) value. The positivity threshold was set to 1.4, as recommended by the manufacturer.

  study. Detection of viral antigens in this population was not evaluated in the study byOgata and collaborators. Detection of N-antigenemia was higher for patients with either high NP viral loads, i.e. Ct below 30, or active replication in the lung, i.e. high Ct values (>30) in NP samples but positive RT-PCR in lower respiratory tract samples.

  Figure 1

CONFLICT OF INTEREST

Benoit Visseaux reports grants, personal fees and non-financial support from Qiagen, personal fees from BioMérieux, personal fees from Hologic, personal fees from Gilead, all outside the submitted work. Xavier Duval reports grants from Sanofi Pasteur, grants from Pfizer, all outside the submitted work. Charles Burdet reports personal fees and non-financial support from Da Volterra, personal fees from Mylan, all outside the submitted work. Jean-François Timsit reports grants and personal fees from MSD, grants and personal fees from Pfizer, grants and personal fees from beckton dickinson, personal fees from biomerieux, personal fees from Medimune, personal fees from Gilead, all outside the submitted work. Jade Ghosn reports grants and personal fees from Gilead Sciences, grants and personal fees from ViiV Healthcare, personal fees from MSD, all outside the submitted work.

Charlotte Charpentier reports personal fees from ViiV Healthcare, personal fees from Gilead, personal fees from Theratechnologie, all outside the submitted work. Diane Descamps reports personal fees from ViiV Healthcare, personal fees from Gilead, all outside the submitted work.

Yazdan Yazdanpanah has been a board member receiving consultancy fees from Abbvie, BMS, Gilead, MSD, J&J, Pfizer, and ViiV Healthcare, all outside the submitted work and all these activities have been stopped in the 3 past years. The other authors have nothing to disclose. Supplementary Table 1.

AUTHORS CONTRIBUTION

QLH


Lucas Laplanche

Alexandre Arnoult

Quentin Gravelier

Stéphane Calvez

Christophe Viallon

Guilhem Almuneau

Monolithically integrated 850-nm VCSEL / Electro-Absorption Modulator structures grown by MBE

published or not. The documents may come   L'archive ouverte pluridisciplinaire

Optical results

Room temperature post-growth FTIR reflectivity measurement of the grown EAM-VCSEL structure (black) Numerical calculation of the reflectivity in the case of:

 Strong coupling (red)  Moderate coupling (blue, EAM cavity deviation of -3nm)

 Overcoupling (orange, deviation of +7.5nm)

The grown structure exhibits a case of strong coupling

Room temperature post-growth measurements of the cavities FP resonance by FTIR at different locations on the wafer



  Vertically integrated VCSEL / Electro-absorber modulator (EAM): Original and efficient approach for overpass the limits of directly-modulated VCSELs  Design/modelling of electro-optical behavior of the EAM-VCSEL  MBE growth with in-situ monitoring: reflectometry + wafer curvature  EAM-VCSEL growth requires high precision: Best performances (moderate coupling) requires an error of less than +/-0.5% on the EAM cavity thickness High speed VCSEL applications: • Short-reach optical links in Datacenters, supercomputers,.Marigo-Lombart et al. doi.org/10.1088/2515-7647/aaf5af  Splitting up Emission / Modulation: CW VCSEL emission + EA Modulation  Electro-absorption modulator: Very high 3dB bandwidth > 65Ghz (Grillanda et al. 2019)  High devices density, small footprint  Solution not based on direct-current modulation: Not (intrinsically) limited by carriers/laser dynamics Contact : almuneau@laas.fr



  EAM cavity FP resonance ranges from 848 to 852 nm: best configuration possible  The difference of the two FP resonance wavelengths is constant → coupling is constant across the wafer Wafer curvature by EZ-CURVE (LAAS patent)  Optical reflectometry 700-1100 nm range Real-time reflectometry full spectrum allows to: -Check the DBR central wavelength, -Track the VCSEL FP resonance, -Track and adjust the middle and top DBR wavelength -Track and adjust the EAM FP detuning → Fab yield increase Refractive index profile of the EAM-VCSEL Field distribution in the case of: -Strong coupling (red) -Moderate coupling (blue) Double microcavity resonant system:  Frequency modulation response, is correlated to the optical coupling between VCSEL and EAM cavities (K. Panajotov et al. doi.org/10.3390/app10176128)  Strong coupling disrupts the VCSEL CW emission  Moderate coupling gives maximized frequency bandwidth  Weak coupling results in small modulation contrast From Cheng et al. doi.org/10.3390/photonics9020107 Data rate improvement requires:  Increased intrinsic bandwidth (limited)  Advanced modulation formats  Wavelength or Frequency Demultiplexing λ VCSEL = 850 nm λ VCSEL = 850 nm L. Laplanche, A. Arnoult, Q. Gravelier, S. Calvez, C. Viallon, G. Almuneau LAAS-CNRS, Université de Toulouse, CNRS, F-31400, France