He ´lène Guegan

Judith Fillaux

Ele ´na Charpentier

Florence Robert-Gangneux

Pamela Chauvin

Emilie Guemas

Je ´ro ˆme Boissier

Alexis Valentin

Sophie Cassaing

Jean-Pierre Gangneux

Antoine Berry

Xavier Iriart
email: iriart.x@chu-toulouse.fr

Real-time PCR for diagnosis of imported schistosomiasis

Background

The diagnosis of schistosomiasis currently relies on microscopic detection of schistosome eggs in stool or urine samples and serological assays. The poor sensitivity of standard microscopic procedures performed in routine laboratories, makes molecular detection methods of increasing interest. The aim of the study was to evaluate two in-house real-time Schistosoma PCRs, targeting respectively S. mansoni [Sm] and S. haematobium [Sh] in excreta, biopsies and sera as potential tools to diagnose active infections and to monitor treatment efficacy.

Methods

Schistosoma PCRs were performed on 412 samples (124 urine, 86 stools, 8 biopsies, 194 sera) from patients with suspected schistosomiasis, before anti-parasitic treatment. Results were compared to microscopic examination and serological assays (enzyme-linked immunosorbent assay (ELISA), indirect haemagglutination (HA) and Western Blot (WB) assay).

Results

Compared to microscopy, PCRs significantly increased the sensitivity of diagnosis, from 4% to 10.5% and from 33.7% to 48.8%, for Sh in urine and Sm in stools, respectively. The overall sensitivity of PCR on serum samples was 72.7% and reached 94.1% in patients with positive excreta (microscopy). The specificity of serum PCR was 98.9%. After treatment, serum PCR positivity rates slowly declined from 93.8% at day 30 to 8% at day 360, whereas antibody detection remained positive after 1 year.

Conclusion

Schistosoma PCRs clearly outperform standard microscopy on stools and urine and could be part of reference methods combined with WB-based serology, which remains a gold

Introduction

Schistosomiasis is a snail-borne parasitic disease of great concern worldwide, occurring mainly in Africa, Asia, and to a lesser degree in South America and the Middle East [START_REF] Colley | Human schistosomiasis[END_REF][START_REF] Ross | A new global strategy for the elimination of schistosomiasis[END_REF]. In nonendemic areas, there is a growing number of people potentially infected, given the increasing number of immigrants, foreign workers, and travelers. Recently, the emergence of urogenital schistosomiasis, due to S. haematobium and S. bovis hybrids, has been reported in a previously uninfected area in Corsica (France), with more than 120 confirmed cases among local people and tourists [START_REF] Berry | Schistosomiasis haematobium, Corsica, France[END_REF][START_REF] Boissier | Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study[END_REF].

The diagnosis of urogenital and intestinal schistosomiasis currently relies on microscopy procedures which are time-consuming, require trained operators and offer poor sensitivity, particularly when the parasite burden is low. Additionally, egg release and migration from tissues to the lumen (gut, bladder) is a long process (>6 weeks) [START_REF] Ross | A new global strategy for the elimination of schistosomiasis[END_REF], leading to diagnosis delay. The detection of specific antibodies is the most commonly applied alternative diagnostic approach in non-endemic routine laboratories [START_REF] Hinz | Serological approaches for the diagnosis of schistosomiasis-A review[END_REF][START_REF] Agbata | Effectiveness of Screening and Treatment Approaches for Schistosomiasis and Strongyloidiasis in Newly-Arrived Migrants from Endemic Countries in the EU/EEA: A Systematic Review[END_REF]. Among the current panel of serological techniques which are highly sensitive, the development of an immunoblotting assay using S. mansoni and S. haematobium adult worm extracts improved the performances of serological screening and proved to be relevant in both urogenital and intestinal schistosomiasis [START_REF] Berry | Evidence for a permanent presence of schistosomiasis in Corsica, France[END_REF][START_REF] Beltrame | Accuracy of parasitological and immunological tests for the screening of human schistosomiasis in immigrants and refugees from African countries: An approach with Latent Class Analysis[END_REF]. Although the usefulness of serological tools has been demonstrated, particularly for symptomatic travelers, their contribution to diagnosing patients living in endemic regions is limited due to their inability to differentiate between ongoing and previous infections. Moreover, in endemic populations, circulating adult worm-derived antigens have been reported to be good indicators of active infection, but still appear to lack sensitivity, especially in case of low parasite burden [START_REF] Fuss | Comparison of sensitivity and specificity of three diagnostic tests to detect Schistosoma mansoni infections in school children in Mwanza region, Tanzania[END_REF][START_REF] Utzinger | New diagnostic tools in schistosomiasis[END_REF][START_REF] Ogongo | Diagnosis of schistosomiasis mansoni: an evaluation of existing methods and research towards single worm pair detection[END_REF]. Excellent results were described in diagnosing S. mansoni infections by the detection of circulating cathodic antigens in urine and serum samples [START_REF] Fuss | Comparison of sensitivity and specificity of three diagnostic tests to detect Schistosoma mansoni infections in school children in Mwanza region, Tanzania[END_REF], but the performance was disappointing in S. haematobium detection [START_REF] Rubaba | Validation of a urine circulating cathodic antigen cassette test for detection of Schistosoma haematobium in uMkhanyakude district of South Africa[END_REF].

The development of molecular methods to improve the diagnosis of parasitic diseases has been encouraging active research in recent years [START_REF] He | Nucleic acid detection in the diagnosis and prevention of schistosomiasis[END_REF][START_REF] Cavalcanti | The Advances in Molecular and New Point-of-Care (POC) Diagnosis of Schistosomiasis Pre-and Post-praziquantel Use: In the Pursuit of More Reliable Approaches for Low Endemic and Non-endemic Areas[END_REF]. The detection of Schistosoma DNA by PCR in stools and urine appears to be a promising, highly sensitive, and specific tool that could improve and facilitate the diagnosis of schistosomiasis, particularly in non-endemic countries where the parasite burden can be lower [START_REF] Pontes | Comparison of a polymerase chain reaction and the Kato-Katz technique for diagnosing infection with Schistosoma mansoni[END_REF][START_REF] Oliveira | Evaluation of polymerase chain reaction as an additional tool for the diagnosis of low-intensity Schistosoma mansoni infection[END_REF][START_REF] De Carvalho | Polymerase chain reaction for the evaluation of Schistosoma mansoni infection in two low endemicity areas of Minas Gerais, Brazil[END_REF][START_REF] Obeng | Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana[END_REF]. A few previous studies demonstrated the potential interest of blood-based PCR for the diagnosis of acute schistosomiasis. As evidenced by Wichmann et al., the high potential of both Schistosoma haematobium (Sh) and Schistosoma mansoni (Sm) PCR assays is linked to the high repetition of the multicopy targeted genes, accounting for 12-15% of the whole Schistosoma genome [START_REF] Hamburger | Highly repeated short DNA sequences in the genome of Schistosoma mansoni recognized by a species-specific probe[END_REF][START_REF] Hamburger | Polymerase chain reaction assay based on a highly repeated sequence of Schistosoma haematobium: a potential tool for monitoring schistosome-infested water[END_REF].

This work aimed i) to assess the performance of Schistosoma PCR assays for the diagnosis of imported schistosomiasis in comparison to conventional microscopy in stool, urine and biopsy samples, ii) to evaluate the clinical relevance of Schistosoma DNA detection in the serum for the diagnosis of active infection and for the monitoring of treatment efficacy.

Materials and methods

Ethics statement

The study was approved by the ethics committee of the Rennes University Hospital (No. 18.111). Biological material was obtained only for standard diagnosis on the basis of physicians' prescriptions. Clinical data were made anonymous for analysis. According to French Public Health Law [START_REF]De ´cret n˚2017-884 du 9 mai 2017 modifiant certaines dispositions re ´glementaires relatives aux recherches impliquant la personne humaine[END_REF], protocols of this type are exempt from the requirement for formal informed consent.

Study design

Data from patients with suspected schistosomiasis examined at Toulouse and Rennes University Hospitals between January 2016 and March 2018 were collected. Only patients with at least a positive Schistosoma PCR and a serology analysis (including Western Blot), alone or combined with excreta examination, and without any previous anti-Schistosoma treatment were retained in the study. The diagnostic procedures were part of the standard diagnostic work-up for schistosomiasis investigation as described below.

The design of this study aimed to evaluate the performances of Schistosoma detection by PCR in two contexts: i) in stool, urine and biopsy samples in comparison to conventional microscopy diagnosis, and ii) in the serum for the diagnosis of active infection and monitoring of treatment efficacy.

For the evaluation of PCRs assays in sera, patients were classified according to results of microscopy, serology and blood eosinophil count. "Proven active Schistosoma infection" was defined by the microscopic detection of Schistosoma eggs in excreta or biopsy samples. "Probable active infection" was defined by a positive Schistosoma WB serology associated with a blood eosinophil count �0.5G/L, while cases were graded as "strictly serological schistosomiasis" when the WB was positive without blood eosinophil increase. Patients with "excluded schistosomiasis" were defined by a negative Schistosoma WB serology or both negative ELISA and HA, and negative microscopic egg detection in excreta, when performed.

In addition, an evaluation of PCR specificity was conducted in serum, stool and urine samples recovered from volunteers who had never visited schistosomiasis-endemic areas.

All the diagnostic tests (microscopy, detection of anti-Schistosoma antibodies including WB and Schistosoma PCR assays) were performed independently and blindly from one another on each anonymised sample. The same samples were used (excreta, biopsies, sera) for Schistosoma PCR assays or reference methods (microscopy, detection of anti-Schistosoma antibodies including WB).

Microscopy (ME)

Microscopic examination for the presence of Schistosoma eggs in urine samples was performed by examination of the pellet from micturition obtained after centrifugation. Fresh stool specimens were examined after 2 concentration techniques (flotation and diphasic methods) [START_REF] Anofel | Parasitologie et mycologie me ´dicales-Guide des analyses et des pratiques diagnostiques[END_REF]. Direct examination of bladder, gut and other biopsies was performed after mechanical grinding. Results of histological examination after biopsy staining were also collected, when performed.

Detection of anti-Schistosoma antibodies

Sera were screened for anti-Schistosoma antibodies using the Schistosoma IgG ELISA (Bordier Affinity Products, Crissier, Switzerland or Schistosoma Antibody Detection Test Kit, Scimedx, Dover, New Jersey) and the indirect hemagglutination (HA) test (Schistosomiasis Fumouze, Fumouze Diagnostics, Levallois-Perret, France) according to manufacturers' instructions. Sera with a positive or a doubtful result by at least one technique were then confirmed using a Western Blot (WB) assay (Schisto II Western Blot IgG kit (LD-BIO Diagnostics, Lyon France)), according to the manufacturer's instructions. In total, the serology was considered positive if the WB was positive. A serum was considered negative if the two screening techniques or the WB were negative.

DNA extraction

Two hundred fifty to 500 mg of stool sample were suspended in 1 mL of PBS and frozen at -20˚C before DNA extraction. Extraction was performed on 200 μL of stool suspension after bead-beating, using High Pure PCR Template Preparation Kit (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions.

For blood and urine, DNA extraction was performed using 1 mL of serum or 1 mL of urine pellet and eluted in a volume of 50 μL, using the MagNA Pure Compact Nucleic Acid Isolation Kit (Roche Diagnostics) on a MagNA Pure Compact Instrument (Roche Diagnostics).

Extraction/Inhibition control

DNA extracts were first screened for PCR inhibitors using the beta-globin gene amplification, as previously described [START_REF] Fabre | Comparative assessment of conventional PCR with multiplex real-time PCR using SYBR Green I detection for the molecular diagnosis of imported malaria[END_REF]. Briefly, the run was performed in a 10 μL reaction mix containing 2.5 μL DNA, 10X Taq SyBr, 0.1 μM of BgO7 and BgO8 primers and 4 mM of MgCl 2 . Amplification was conducted on a LightCycler 2 instrument (Roche Diagnostics) during 40 cycles of 15 s at 95˚C, 10 s at 70˚C and 20 s at 72˚C, followed by a fusion step.

As long as the beta-globin gene was detected, DNA extracts were considered suitable for Schistosoma PCR testing. In case of PCR inhibition (lack of amplification of beta-globin gene), samples were retested diluted to 1:4, and the result was considered uninterpretable if beta-globin remained undetected.

Schistosoma real-time PCR assays

Two species-specific in-house real-time PCRs were evaluated. S. mansoni DNA detection (SmPCR) was performed using primers (SRA1 and SRS2) and probe (SRP) from Wichmann et al., targeting the Sm1-7 tandem repeat sequence [START_REF] Wichmann | Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma[END_REF]. The repetitive Dra1 sequence of S. haematobium (ShPCR) was amplified using primers (Sh-FW and Sh-RV) from Hamburger et al. [START_REF] Hamburger | Polymerase chain reaction assay based on a highly repeated sequence of Schistosoma haematobium: a potential tool for monitoring schistosome-infested water[END_REF], and probe from Cnops et al. [START_REF] Cnops | A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants[END_REF].

PCR assays were performed in a 20 μL reaction mix containing 5 μL DNA, 10X LightCycler FastStart DNA Master HybProbe master mix, 5 mM of MgCl 2 , 0.5 μM of each primer and 0.25 μM of probe. Amplification was conducted on a LightCycler 2 instrument (Roche Diagnostics) and consisted of 10 min at 95˚C and 45 cycles of 15 s at 95˚C and 1 min at 60˚C. A test was positive when the threshold was attained within 45 PCR cycles (Ct-value<45).

Statistical analysis

Data were analyzed using GraphPad Prism software. Data were expressed as median with interquartile range [25 th ;75 th percentile]. Nonparametric Mann-Whitney and Kruskal-Wallis tests were performed for distribution comparison. For proportion comparisons, the chi-square test or Fisher's exact test was used, as appropriate. Kappa coefficient was used to measure the agreement between each test. Correlation between blood eosinophil count and PCR Ct values was assessed with the Spearman rank correlation test. Differences were considered statistically significant if the p-value was < 0.05. DNA burden during post-treatment follow-up was calculated as the ratio of (45-Ct value after treatment)/(45-Ct value before treatment) � 100.

Results

Patients

A total of 412 clinical samples (urine, stool, biopsy and serum samples) from 255 patients were analyzed for schistosomiasis diagnosis.

The study population for PCR evaluation in excreta consisted of 180 patients (Table 1). The performance of serum PCR was assessed on a series of 194 sera from 194 patients, graded as "proven active schistosomiasis" (n = 34), "probable active schistosomiasis" (n = 30), "strictly serological schistosomiasis" (n = 35) or "excluded schistosomiasis" (n = 95), according to serology, blood eosinophil count and Schistosoma detection in excreta by microscopy (Fig 1). Of all 99 suspected cases, 82 were immigrants, mainly young men (Fig 1), 11 patients were travelers for a period of �3 months, 3 were French expatriates for more than 7 years, and the length of

Evaluation of PCR specificity

The specificity of both Sm and ShPCR assays was evaluated on 3 biological matrices, i.e. serum, urine and stool samples, from subjects unlikely to have had previous contact with the parasite. The specificity was 100%, as neither of the two PCR assays yielded a positive result in the 20 serum, 20 urine, and 20 stool samples. Additionally, PCR specificity was also evaluated regarding other parasites, in 3 blood samples and 34 stools samples in which parasites had been detected by other diagnostic methods. No cross reaction was observed with Toxoplasma (n = 1), Plasmodium (n = 1), Leishmania

(n = 1), Enterocytozoon bieneusi (n = 1), Encephalitozoon sp (n = 1), Cryptosporidium sp (n = 1), Endolimax nana (n = 2), Blastocystis sp (n = 4)., Entamoeba hartmanni (n = 1), Entamoeba dispar (n = 1), Entamoeba histolytica (n = 1), Entamoeba coli (n = 1), Dientamoeba fragilis (n = 4), Giardia intestinalis (n = 5), Enterobius vermicularis (n = 1), Ascaris lumbricoides (n = 2), Trichuris trichiura (n = 1), Strongyloides stercoralis (n = 2), Ancylostomidae (n = 1), Hymenolepis nana (n = 3) and Taenia sp.(n = 2).

Performances of PCR assays compared to microscopy in urine, stool and biopsy samples

A total of 124 urine, 86 stool and 8 biopsy samples were collected from 108, 65, and 7 patients, respectively, with suspected infection with Schistosoma, before any anti-parasite treatment. A comparison of egg detection performance using microscopic methods (microscopic examination or anatomic pathology procedures) and Schistosoma DNA detection is presented in Table 1. All PCR results were interpretable.

Of the 124 urine samples, 13 (10.5%) yielded a positive ShPCR result, whereas parasite eggs were microscopically detected in only 5 samples (4%) (p<0.001). Two samples were positive by both PCRs. The 8 samples with discordant results were collected from 7 distinct patients, all having a positive WB-based serology, suggesting that these were 8 true-positive samples detected by PCR but not by microscopy. All positive-microscopy samples were positive by PCR.

In stools, 42 out of 86 stool samples (48.8%) yielded a positive SmPCR but only 29 with microscopy (33.7%) (p<0.001) (Table 1). For the 13 samples with discordant results, WBbased serology supported positive PCR results. All samples containing S. mansoni eggs yielded a positive SmPCR result. No positive ShPCR was found in stool samples.

The concordance of SmPCR and microscopy for biopsies was 100%, as the 2 colon and rectal specimens, sampled from the same patient, were positive both by direct examination and SmPCR.

The agreement between the results of microscopy and PCR assays varied between 84.9 and 100% according to the type of sample. Discordances concerned only probable true-positive samples detected by PCR but not by microscopy (Table 1). Therefore the specificity of PCR can be considered as 100%.

Moreover, 8 patients gave several stool or urine specimens (2 or 3) a few days apart (Table 2). Interestingly, the PCR consistently yielded a positive signal (except patient 5), whereas eggs were inconsistently detected by microscopy in 7/8 patients.

PCR Ct values appeared to be correlated with microscopic results. Indeed, median Ct values of both PCR assays were significantly lower in samples with microscopic egg detection, compared to those with negative microscopic results: 19. Overall, these results suggested that the molecular detection of Schistosoma by PCR is more efficient and reliable than microscopic examination of excreta, particularly in the case of low burden infections.

Performances of serum PCR compared to serology and excreta examination for schistosomiasis diagnosis

The overall rate of positive serum PCRs among the 99 patients with a microscopic and/or serological criterion in favor of schistosomiasis was 72.7% (72/99) (Table 3). All PCR results were interpretable. The specificity was excellent, reaching 98.9%. The only serum with a positive SmPCR among cases with excluded schistosomiasis was unlikely to be a false-positive result, as the ELISA serology was simultaneously positive, and the patient had been living for 28 years in Sudan and had abdominal pain. This patient was classified as "excluded schistosomiasis" because of a negative WB (probably a false-negative result). Interestingly, serum PCRs were positive in 85.9% (55/64) of active schistosomiasis (proven and probable groups), exceeding the microscopy yield which was only 53.1% (34/64) (Table 3). In the group of proven schistosomiasis, the positivity rate of serum PCRs reached 94.1% (32/ 34). Among the 2 missed PCR detections (2/34), the two occurred in patients with proven urogenital schistosomiasis (Sh eggs in urine), including one with a concomitant positive urine ShPCR (the urine ShPCR was not performed for the second one).

In the group of proven schistosomiasis, all patients having a positive microscopy had a positive WB, except in one case. The patient missed by the WB-serology had a positive serum ShPCR, an ELISA index value in grey zone and a positive egg detection in vesical biopsy, associated to urinary symptoms (macroscopic hematuria and micturition disorders for 6 months).

Overall, the sensitivity of serum PCR significantly exceeded the microscopy, leading to a low agreement percentage and kappa coefficient of 77.8% and 0.472 respectively (Table 4). Indeed, in patients with negative microscopy, the serum PCR positivity rate reached 76.7% (23/30) and 48.6% (17/35), respectively in "probable" and "strictly serological schistosomiasis" groups (Table 3). However, the level of circulating Schistosoma DNA was not significantly The global agreement of serum PCR with Western Blot was higher than with microscopy (85%; Kappa = 0.702, Table 4). Interestingly, among WB-positive patients, the occurrence of a positive serum PCR was found to be correlated with ELISA and HA results (Table 5, p = 0.003). PCR was positive in only 1/5 sera with both negative ELISA and HA (20.0%), whereas serum PCR was positive in 17/18 (94.4%) sera with both positive tests. Lastly, 22/32 (68.8%) sera yielded positive PCR together with discordant ELISA and HA. ELISA indexes and HA titers were significantly higher when serum PCR was positive, with median values of 2.59 [1.91;3.29] versus 1.50 [0.91;1.90] (p<0.001), and 300 [0;320] versus 48 [0;160] (p = 0.04).

Both Sm and Sh PCRs were simultaneously positive in 6 serum samples (Table 6). For 2 patients (1 & 2) the presence of the two species was confirmed by microscopic examination of stools and urine. In patients 3 & 4 there was evidence of Sh infection in the bladder biopsy and urine, but stool analysis was not performed. In total, S. mansoni was unexpectedly detected in 3 serum specimens from patients with clinical symptoms evocative of urogenital schistosomiasis (patients 3, 5, and 6).

Comparison of PCR performances on serum and excreta

Among the 25 patients whose sera and urine were simultaneously screened for Sh, the positivity rate of PCR was slightly higher in serum compared to urine, 11/60 (18.3%) versus 8/60 (13.3%), (not statistically significant). For Sm detection, serum PCR yielded roughly similar results to stool PCR, detecting Sm in 21/48 (43.8%) and in 20/48 (41.7%), respectively (not statistically significant).

Value of PCR for post-treatment follow-up

The last part of this study aimed at following the kinetics of a positive PCR on serum after treatment, to assess whether it could be a relevant tool for treatment monitoring, compared to serological techniques. Twenty-three patients benefited from follow-up serum samples (1 to 3) collected at different times after praziquantel therapy (14 to 455 days). One month after treatment, all but one serum remained positive by PCR (15/16, 93.8%), of which 9 were from proven cases (Fig 2). The patient who became negative as early as day 30 had a very low parasite burden before treatment (Ct-value for Sm PCR >40). The level of circulating Schistosoma DNA appeared to increase significantly within the first 60 days as shown in out of the 12 patients followed for more than 1 year after treatment (378 days) remained positive in serum.

By contrast, the positivity rate of serological tests ELISA and HA did not significantly decrease and remained constant within the first year after treatment. As shown in Fig 2, among the 96% of patients having a positive ELISA at day 0, up to 83% remained positive after 1 year. The HA positivity rate increased within the first four months after treatment, from 47% at day 0 to 86% at day 120, and remained at 50% after 1 year.

Such persistence of specific antibody detection, as well as DNA detection could not be linked with obvious treatment failure, since excreta remained positive with eggs for only 1/15 patients at day 30 and no further evidence of clinical relapse could be noticed.

Follow-up by PCR on stools was performed in two patients and did not detect persisting DNA two weeks after treatment, whereas PCR on serum was still positive at day 52 and day 83 post-treatment, respectively. By contrast, DNA burden in urine samples from 3 patients was strongly lowered but remained positive at day 60 (n = 3), and persisted until day 316 for one of them.

Discussion

This study presents an evaluation of the performance of two Schistosoma PCR assays on excreta and serum samples, compared to microscopy and serology-based conventional methods for the diagnosis of imported intestinal and urogenital schistosomiasis.

Serum Schistosoma PCRs were previously shown to be mainly contributive for the diagnosis of early infections, as up to 90% of patients in acute invasion phase were positive, before detectable levels of eggs were excreted [START_REF] Wichmann | Prospective Europeanwide multicentre study on a blood based real-time PCR for the diagnosis of acute schistosomiasis[END_REF]. However, data on their clinical relevance are scarce, and the few existing reports referred to small patient cohorts. Hence, the strength of this work is to focus on a large cohort of chronically-infected patients and demonstrate the relevance of Schistosoma PCRs in sera as well as urine and stools.

Specificity of both PCR assays was firstly explored in excreta and sera and proved to be excellent (100%) in our cohort of subjects who never experienced any stay in known endemic areas. The analytical specificity of the ShPCR was previously demonstrated by Cnops et al., on a panel of 23 stool samples containing various intestinal parasites other than Schistosoma [START_REF] Cnops | A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants[END_REF]. Similarly, the absence of cross-reaction with SmPCR was verified in a previous work [START_REF] Wichmann | Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma[END_REF]. Here, we confirmed the high specificity of both PCRs as none of the tested blood and intestinal parasites were cross-reactive with the schistosome tests.

In our study, PCRs assays greatly improved the detection of Schistosoma in urine and fecal samples, increasing the number of positive results by a factor of 2.6 and 1.4 for Sh and Sm respectively, compared to microscopy. This is consistent with previous studies which underlined the excellent performance of both Dra ShPCR in urine (PCR sensitivity of 36% versus 25% with microscopy on 401 samples [START_REF] Ibironke | Validation of a New Test for Schistosoma haematobium Based on Detection of Dra1 DNA Fragments in Urine: Evaluation through Latent Class Analysis[END_REF]) and Sm1-7 PCR in stool samples (PCR sensitivity of 9.6% versus 0.9% by microscopy, on 572 samples [START_REF] Espı ´rito-Santo | Evaluation of real-time PCR assay to detect Schistosoma mansoni infections in a low endemic setting[END_REF]).

These two highly sensitive real-time PCRs have been the most used for clinical diagnosis, because of the huge number of gene copies in the Schistosoma genome. A few clinical evaluations have been reported on real-time PCRs targeting the ribosomal internal transcribed spacer 2 region (ITS 2), but these PCRs detect Schistosoma sp. without discrimination between S. mansoni and S. haematobium. This gene also displays interesting performances, as the PCR sensitivity was shown to range from 89-100% for Sh detection [START_REF] Obeng | Application of a circulating-cathodic-antigen (CCA) strip test and real-time PCR, in comparison with microscopy, for the detection of Schistosoma haematobium in urine samples from Ghana[END_REF][START_REF] Vinkeles Melchers | Diagnostic Performance of Schistosoma Real-Time PCR in Urine Samples from Kenyan Children Infected with Schistosoma haematobium: Day-to-day Variation and Follow-up after Praziquantel Treatment[END_REF] and from 94-97% for Sm detection [START_REF] Schunk | Use of Occult Blood Detection Cards for Real-Time PCR-Based Diagnosis of Schistosoma Mansoni Infection[END_REF][START_REF] Meurs | Diagnosing Polyparasitism in a High-Prevalence Setting in Beira, Mozambique: Detection of Intestinal Parasites in Fecal Samples by Microscopy and Real-Time PCR[END_REF], compared to microscopy. The use of the 28S rDNA and mitochondrial regions such as cox1 gene, have also been anecdotally described, but to a lesser extent [START_REF] Cnops | Schistosoma real-time PCR as diagnostic tool for international travellers and migrants[END_REF][START_REF] Ten Hove | Multiplex real-time PCR for the detection and quantification of Schistosoma mansoni and S. haematobium infection in stool samples collected in northern Senegal[END_REF].

All the specimens positive for Schistosoma eggs in urine or stools were confirmed by PCR assays. Conversely, among samples with an isolated positive PCR (negative microscopy), falsepositivity of a PCR result was very unlikely, as all samples were from patients with epidemiological risk factors and positive WB-serology, consistent with Schistosoma infection. Additionally, the huge advantage of PCR on stools is to bypass the daily variation in egg excretion, which allows analyzing a single fecal sample, rather than 3 consecutive samples, as usually recommended. This also applies to urine samples, which can be collected from a unique micturition and not necessarily during a 24h-time period. Lastly, SmPCR was validated on biopsy samples with a concordance of 100% with microscopy, although it could only be done on a small number of specimens.

This work also presents a contribution of detection of circulating Schistosoma DNA to the current conventional diagnostic arsenal, based on serology and microscopic examination. A positive serum-based PCR is consistent with the parasite localization in blood vessels, the significant size of adult worms, and thus the expected high level of Schistosoma DNA in blood.

Here, in our cohort constituted mainly of African migrants, the PCR yield in serum exceeded 92% in patients with Schistosoma-positive urine or stools. Despite 2 false-negative results within the proven group, the global sensitivity of serum PCRs remained higher than that observed for microscopic techniques, as it was able to detect 77% and 49% of "probable active cases" and "strictly serological cases", respectively.

The WB-based serology is a reference method for the serologic screening of schistosomiasis because of its sensitivity [START_REF] Beltrame | Accuracy of parasitological and immunological tests for the screening of human schistosomiasis in immigrants and refugees from African countries: An approach with Latent Class Analysis[END_REF]. Surprisingly, when compared to serum PCR, WB yielded falsenegative results in two patients, for whom the possibility of recent infection was excluded (African migrants who left endemic areas at least 4 months before). The first patient had a doubtful ELISA index, but a concomitant bladder biopsy containing living Sh eggs, and thus was a proven case. The other one had a positive serum Sm PCR together with a positive ELISA assay, but in the absence of excreta microscopy, the case was classified as excluded (Table 3), although true infection was likely.

Among WB-positive sera, the frequency of positive Schistosoma DNA detection in serum was found to be higher when the ELISA and HA tests were concomitantly positive. Among the serological approaches for the diagnosis of schistosomiasis, these two serological tests appear to yield more frequent positive antibody detections in case of active infection, compared to WB. Indeed, most former schistosomiasis patients with positive ELISA and HA assays are expected to turn negative [START_REF] Hinz | Serological approaches for the diagnosis of schistosomiasis-A review[END_REF][START_REF] Zhu | Immunodiagnosis and its role in schistosomiasis control in China: a review[END_REF] or to have a significantly lower antibody titer [START_REF] Mott | Collaborative study on antigens for immunodiagnosis of schistosomiasis[END_REF] within a variable period after an effective treatment. By contrast, LDBIO Schistosoma WB IgG is known to persist positive after treatment for a longer period than ELISA [START_REF] Ldbiodiagnostics | Schistosoma Western Blot IgG, LDBIO. Instructions for use[END_REF], probably for several years, as already observed with other parasitic WB assays based on IgG detection [START_REF] Kumar Tenguria | Application of Western Blotting for the Post-Treatment Monitoring of Human Cystic Echinococcosis[END_REF][START_REF] Rubinsky-Elefant | Potential immunological markers for diagnosis and therapeutic assessment of toxocariasis[END_REF].

The great advantage of serum PCR is to provide species identification even when microscopic examination is negative, allowing targeted clinical work-up to identify complications of long-term infections.

Direct species identification by serum PCR allowed diagnosis of mixed infection in 6 patients. Positive results by the 2 PCR assays were unlikely to be due to cross-reaction between the 2 species, as previous studies demonstrated the species-specificity of these assays [START_REF] Wichmann | Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma[END_REF][START_REF] Cnops | A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants[END_REF]. Two hypotheses can be considered to explain this dual positivity. First, we can assume that these patients, all coming from Africa, were simultaneously infected with Sh and Sm, as frequently observed in several endemic African countries, with up to 50% in a Senegalese study from Meurs et al. [START_REF] Meurs | Epidemiology of mixed Schistosoma mansoni and Schistosoma haematobium infections in northern Senegal[END_REF]. Unfortunately, this hypothesis could be confirmed for two patients only, for whom both Sm and Sh eggs were detected in excreta. For the 4 remaining patients, stool and urine samples were not available for simultaneous screening.

Secondly, the positivity of both targets in serum could also be related to an infection involving a hybrid between S. mansoni and S. haematobium. Besides, two urine samples from the same patient also yielded both positive Sm and Sh PCRs, at low Ct values (17 and 18 for Sh and SmPCR, respectively), whereas only Sh eggs were detected by microscopy. The detection of DNA from both species in urine suggested a Sm-Sh hybrid infection in this African patient. Moreover, recent works also demonstrated the emergence of such schistosome hybrids in regions where the parasite burden is high, either between cattle and human schistosomes [START_REF] Boissier | Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study[END_REF][START_REF] Boon | Barcoding hybrids: heterogeneous distribution of Schistosoma haematobium × Schistosoma bovis hybrids across the Senegal River Basin[END_REF], or between S. mansoni and S. haematobium [START_REF] Huyse | Hybridisation between the two major African schistosome species of humans[END_REF][START_REF] Govic | Schistosoma haematobium-Schistosoma mansoni Hybrid Parasite in Migrant Boy, France, 2017[END_REF]. Thus, the impact of hybrid species on performances of diagnostic tools should be addressed, to determine whether they could generate cross-reaction with both PCRs assays. Schistosoma eggs from one of the 3 patients with a previous stay in Corsica were genotyped as S. haematobium-S. bovis hybrids [START_REF] Boissier | Outbreak of urogenital schistosomiasis in Corsica (France): an epidemiological case study[END_REF][START_REF] Ramalli | Persistence of schistosomal transmission linked to the Cavu river in southern Corsica since 2013[END_REF]. For 2 of the patients coming from Corsica, including the one with the hybrid, ShPCR was positive in urine and/or serum, demonstrating the performance of this PCR for hybrid species detection.

Apart from assessing the diagnostic performances of serum PCRs, this study also evaluated their relevance for follow-up after treatment, and ability to discriminate between ongoing or previous infections, that cannot be performed with WB-based assays. In this study, the analysis of sequential sera from praziquantel-treated patients strikingly demonstrated the potentially long persistence of Schistosoma circulating DNA without any link with clinical or biological failure. Indeed, up to 30% remained positive at 6 months, and 8% one year after an adequate cure. In addition, none of these patients were treated during the invasion stage, during which schistosomula are known to be less susceptible to praziquantel [START_REF] Pica-Mattoccia | Sex-and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment[END_REF][START_REF] Aragon | Towards an understanding of the mechanism of action of praziquantel[END_REF], and which could have explained a therapeutic failure.

Several hypotheses could explain why Schistosoma DNA is still detected a long time after treatment. Firstly, the parasite DNA could continue to be released from slowly degenerating eggs that are trapped in tissues, or from killed worms. Therefore, after treatment, no eggs are detected or at least no living eggs, but parasite DNA is not totally cleared and might remain detectable by high sensitive Schistosoma real-time PCR assays. Secondly, the incomplete cure may result from the sub-curative effect of praziquantel when used at usual doses [START_REF] Doenhoff | Is Schistosomicidal Chemotherapy Sub-curative? Implications for Drug Resistance[END_REF]. Finally, a recent review from Lu et al. [START_REF] Lu | Single-sex schistosome infections of definitive hosts: Implications for epidemiology and disease control in a changing world[END_REF] raises the possibility of single-sex schistosome infection that could partly explain why antigen and/or DNA detection could occur without egg detection. Indeed, after a single-sex cercarial exposure, male or female worms will be able to persist within the host without producing eggs, and thus cause asymptomatic infection. While reports on the emergence of low sensitive Schistosoma strains to praziquantel are still scarce [START_REF] Melman | Reduced Susceptibility to Praziquantel among Naturally Occurring Kenyan Isolates of Schistosoma mansoni[END_REF], single-sex schistosomes, especially females, have been demonstrated to be largely refractory to praziquantel compared to paired ones, in both in vitro and in vivo murine models [START_REF] Pica-Mattoccia | Sex-and stage-related sensitivity of Schistosoma mansoni to in vivo and in vitro praziquantel treatment[END_REF]. That's why, despite an appropriate praziquantel treatment, the persistence of single-sex schistosomes less susceptible to this anti-parasitic drug, could still generate a positive PCR, without any egglaying or clinical symptoms.

Other clinical studies reported long-lasting PCR positivity after treatment in a large mixed cohort studied by Wichmann et al [START_REF] Wichmann | Diagnosing Schistosomiasis by Detection of Cell-Free Parasite DNA in Human Plasma[END_REF], and in an urogenital case, until more than 300 days after the first praziquantel treatment [START_REF] Kato-Hayashi | Use of Cell-Free Circulating Schistosome DNA in Serum, Urine, Semen, and Saliva To Monitor a Case of Refractory Imported Schistosomiasis Hematobia[END_REF]. These studies also emphasized the contrasting results of persistent DNA detection, compared to antigen detection which has been shown to become negative in serum or urine within a few days or weeks after treatment [START_REF] De Clercq | Assessment of cure by detection of circulating antigens in serum and urine, following schistosomiasis mass treatment in two villages of the Office du Niger, Mali[END_REF][START_REF] Kildemoes | Rapid clearance of Schistosoma mansoni circulating cathodic antigen after treatment shown by urine strip tests in a Ugandan fishing community-Relevance for monitoring treatment efficacy and re-infection[END_REF]. Cnops et al. also reported that ShPCR remained positive for at least 3 months (n = 8 patients) with a decreasing signal after treatment (higher Ct-values), and they pointed out PCR's potential to monitor treatment [START_REF] Cnops | A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants[END_REF].

Here, an evaluation of Schistosoma serum PCR to monitor treatment has been made by comparison to serological assays. During a follow-up period limited to 1 year, the serum PCR slowly turned negative in most patients, whereas ELISA and HA-based antibody detection remained above the positivity cut-off. These results suggest that at early times post-treatment (within the first months), there is no relevant serum biomarker for follow-up and excreta analysis remains essential. Nevertheless, more than one year after treatment, serum PCR could possibly be an indicator of effective treatment, better than conventional serological tests.

In conclusion, our work emphasized the high performance of targeted Schistosoma PCRs in excreta, greatly improving the diagnosis of both urogenital and intestinal schistosomiasis. Besides, combining serum PCR with conventional serological assays contributes added value to confirming diagnosis and providing species identification to guide further clinical exploration. Finally, Schistosoma serum PCR could also be useful to monitor the outcome when performed much later than an anti-Schistosoma treatment (about one year), as a negative serum PCR may demonstrate a cured infection.

Table 1 .

 1 Performance of PCR and microscopic examination for the detection of S. mansoni and S. haematobium in urine, stool and biopsy samples. S. mansoni PCR; ShPCR: S. haematobium PCR a 5/5 positive-microscopy samples yielded positive PCR b Two samples were simultaneously positive for SmPCR and ShPCR; p<0.001 compared to microscopy c 29/29 positive-microscopy samples yielded positive PCR d p<0.001 compared to microscopy e Rectum (n = 3); colon (n = 2); rectum/colon (n = 1); perineal abscess (n = 1) and liver (n = 1) f Rectum (n = 1) colon (n = 1) collected from the same patient. 2/2 positive-microscopy samples yielded positive PCR https://doi.org/10.1371/journal.pntd.0007711.t001 stay was unknown for the 3 remaining patients. As for the visited endemic areas, most patients visited Africa (n = 92; 93%); others visited Corsica (n = 3; 3%) and other areas (n = 4; 4%).

Fig 1 .

 1 Fig 1. Flow chart for serum PCR evaluation. PNE: polynuclear eosinophil blood count. https://doi.org/10.1371/journal.pntd.0007711.g001

  5 [17.4; 21.3] versus 21.6 [19.8; 26.0] (p = 0.019) for Sm detection in stools, 17.1 [15.7; 17.8] versus 21.5 [20.7; 26.8] (p = 0.003) for Sh detection in urine.

Fig 3 .

 3 After 3, 4, and 6 months respectively, the positivity rate gradually declined to 69.0, 63.6 and 27.3% (Fig 2). One

Fig 2 .

 2 Fig 2. Kinetic of serum PCR, ELISA and HA positivity rates after treatment (n = 23 patients). Bars represent the percentage of positive samples which remained positive at different times post-treatment. The table presents the number of positive sera/number of analyzed sera, at each time (n/N (%)). a One serum was positive for both Sm and ShPCR at diagnosis. https://doi.org/10.1371/journal.pntd.0007711.g002

Fig 3 .

 3 Fig 3. Relative value of serum DNA load after treatment compared to the initial load (day 0), according to the number of days post-treatment (n = 23 patients). https://doi.org/10.1371/journal.pntd.0007711.g003

Table 2 . Results of microscopic detection and Schistosoma PCR in iterative excreta samples.

 2

	Patient	Matrix	Number of positive samples n/N
			Microscopy	PCR
	1	Urine	1/2	2/2
	2	Stool	1/3	3/3
	3	Stool	2/3	3/3
	4	Stool	1/3	3/3
	5	Stool	1/3	2/3
	6	Stool	0/3	3/3
	7	Stool	0/2	2/2
	8	Stool	2/2	2/2

https://doi.org/10.1371/journal.pntd.0007711.t002

Table 3 . Rate of positive serum PCRs in the various clinical categories, according to Schistosoma species (n = 194 patients). Diagnosis Number of positive PCR in serum n/N (%) Median Ct value for both PCRs SmPCR and/or ShPCR ShPCR SmPCR

 3 2/35 were positive for Sm and Sh PCR https://doi.org/10.1371/journal.pntd.0007711.t003

	Schistosomiasis (N = 99)	72 a /99 (72.7)	22/99 (22.2)	56/99 (56.6)	34.1 [32.0; 36.1]
	Proven N = 34	32/34 (94.1)	13/34 (38.2)	22/34 (64.7)	32.9 [30.9; 35.1]
	Urogenital N = 13	11/13 (84.6)	11/13 (84.6)	1 b /13 (7.7)
	Intestinal N = 18	18/18 (100.0)	0/18 (0)	18/18 (100)
	Mixed infection N = 2 c	2/2 (100)	2/2 (100)	2/2 (100)
	Undetermined N = 1 d	1/1 (100)	0/1 (0)	1/1 (100)
	Probable active N = 30	23 e /30 (76.7)	5/30 (16.7)	19/30 (63.3)	34.7 [32.3; 36.6]
	Strictly serological N = 35	17 f /35 (48.6)	4/35 (11.4)	15/35 (42.9)	35.0 [32.2; 36.2]
	Excluded schistosomiasis N = 95	1/95 (1.1)	0/95 (0)	1/95 (1.1)	NA
	NA: not applicable
	a 6/72 patients had simultaneously positive Sm and ShPCR
	b One serum sample simultaneously yielded positive results for both Sm and ShPCR

c Two patients had a concomitant detection of Sm in stool and Sh in urine d One patient had a positive Sm PCR whereas eggs of Sh or S. intercalatum were microscopically detected in the rectal biopsy e 1/30 was positive for Sm and Sh PCR f

Table 4 . Sensitivity and agreement of serum PCR against microscopy and WB-based serology. Reference method Schistosoma serum PCR sensitivity (%)

 4

	Kappa coefficient	% Agreement	p a

a p-value, Fisher's exact test https://doi.org/10.1371/journal.pntd.0007711.t004

Table 5 . Serum PCR performance according to combined ELISA and HA serological tests among patients with positive WB (N = 55 a ). ELISA and HA results PCR results n(%) p b Negative Positive

 5

	Both negative tests (n = 5)	4 (80.0%)	1 (20.0%)	0.003
	Discordant tests (n = 32)	10 (31.2%)	22 (68.8%)
	Both positive tests (n = 18)	1 (5.6%)	17 (94.4%)
	a HA and ELISA data were simultaneously available in 55/99 WB-positive sera.
	b p-value, Chi-square test
	https://doi.org/10.1371/journal.pntd.0007711.t005

Table 6 . Characteristics of patients with simultaneously positive Sm and Sh PCRs in serum. Patient No Sex. age (yrs) Visited endemic area Length of stay (yrs) Clinical symptoms Clinical group Ct values for serum PCR Microscopy result PCR in excreta

 6

	1	M. 55	South Africa.	ND (traveler)	None	proven	Sh: 35.46	U: Sh	U: ND
			Mali				Sm: 34.70	S: Sm	S: Sm
	2	M. 20	Guinea-Conakry	19	Hematuria	proven	Sh: 33.97	U: Sh+Si	U: ND
							Sm: >40	S: Sm	S: Sm
	3	M. 18	Cameroon	12	Renal colic	proven	Sh: 36.26	Bladder biopsy:
								Schistosoma sp.
							Sm: 37.04	U, S: ND	ND
	4	M. 25	Sudan	24	None	probable	Sm: 36.12	U: ND
							Sh: 33.57	S: -	ND
	5	M. 15	Mali	15	Hematuria, micturition	serological	Sh: 39.87	U: -	U: Sh
					disorders		Sm: 34.70	S: ND	S: ND
	6	M. 15	Guinea	ND (migrant)	Abdominal pain,	serological	Sm: 32.13	U: -	U: -
					hematuria		Sh: 30.64	S: -	S: ND

ND: not determined; U: urine; S: stools; -: negative; Sh: S. haematobium; Sm: S. mansoni: Si: S. intercalatum https://doi.org/10.1371/journal.pntd.0007711.t006

PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0007711 September 11, 2019

Acknowledgments

The authors gratefully acknowledge the staff of the Molecular Unit of the Parasitology-Mycology department. The proofreading has been carried out by a professional translator, native English speaker, specialized in the medical field.

This study was supported in part by the French "Agence Nationale de la Recherche" (ANR 18 CE35 0001 03) (AB, JF, JB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All relevant data are within the manuscript and its Supporting Information files.

Supporting information

S1 STARD Checklist. (DOCX)

Author Contributions

Conceptualization: Antoine Berry.

Formal analysis:


Nicolas Bazoge
email: nicolas.bazoge@univ-rennes2.fr

Ronan Gaugne
email: ronan.gaugne@irisa.fr

Florian Nouviale
email: florian.nouviale@irisa.fr

Valérie Gouranton
email: valerie.gouranton@irisa.fr

Bruno Bossis
email: bruno.bossis@univ-rennes2.fr

Expressive potentials of motion capture in the Vis Insita musical performance

Keywords: Motion capture, control interface, musical performance CCS Concepts, Human centered computing → Sound based input/output, Gestural input, • Applied computing → Sound and music computing

INTRODUCTION

Since the early 2000s and the industrial development of optical motion capture systems, several studies [START_REF] Bevilacqua | 3d motion capture data: Motion analysis and mapping to music[END_REF][START_REF] Bevilacqua | Virtual dance and music environment using motion capture[END_REF][START_REF] Camurri | Multimodal analysis of expressive gesture in music and dance performances[END_REF][START_REF] Dobrian | Gestural control of music: Using the vicon 8 motion capture system[END_REF] have been carried out on the use of such systems in musical applications, particularly within the NIME community. However, partly due to the significant financial investment that a professional motion capture (MoCap) system represents, few musical performances based on this technology have yet been created [START_REF] Bevilacqua | Virtual dance and music environment using motion capture[END_REF][START_REF] Jensenius | Microinteraction in Music/Dance Performance[END_REF].

Moreover, because of MoCap's predisposition to capture the movements of the human body, we can see that a large majority of the work that has been done concerns the field of dance or the sonification of gesture.

This research-creation project was therefore mainly interested in the problems of performing electronic music live, and the potential of MoCap's technologies to stage this music and make it heard through plastic elements.

In order to approach our subject as exhaustively as possible, we included it in a bibliographical research on how art historians, artists and researchers question the way in which live electronic music is perceived by the audience and interpreted by the performer-musicians [START_REF] Auslander | Liveness: Performance in a Mediatized Culture[END_REF][START_REF] Baytas | The perception of live-sequenced electronic music via hearing and sight[END_REF][START_REF] Butler | Playing with something that runs: technology, improvisation, and composition in DJ and laptop performance[END_REF].

However, the study described in this article focused on practical and experimental research through the creation of a musical performance that attempts to implement expressive and poetic forms of musical interpretation. The objective was multiple: to analyse the possibilities and limits of this instrumental interface under real conditions of presentation, to make a video recording of the performance1 in order to keep an analyzable trace, and to collect the feedback of spectators.

We relied on the NIME community, in particular on the research of the University of Oslo about the results of the experiment with the SoundSaber [START_REF] Nymoen | Soundsaber -a motion capture instrument[END_REF], an instrument designed on a similar principle of motion capture. Unlike the Soundsaber, which requires continuous manipulation to be kinetically active (except in the case where it is launched by the performer), the tangible interfaces we imagined had to be able to behave autonomously after being manipulated because of their own energy (kinetic energy) and bring a contingent dimension to the performance.

From an artistic point of view, we wanted the aesthetics of these kinetic objects to visually arouse the viewer's curiosity and attention. We had thus imagined very early giving them a very strong plastic dimension, as much in their shape, their colour, their material, their lines, as in their own lighting, with as reference and inspiration, the kinetic art of the 50s and 60s [START_REF] Arnauld Pierre | Nicolas Schöffer Space, Light, Time[END_REF][START_REF] Borchardt-Hume | Alexander Calder Performing Sculpture[END_REF][START_REF] Bourdages | Les Sculptures Cinétiques de Jean Tinguely[END_REF].

The term "motion capture instrument" (MCI) used in this article will refer to the instrumental interface as a whole, including both hardware and software. Proposed by K. Nymoen et al. [START_REF] Nymoen | Soundsaber -a motion capture instrument[END_REF], it well reflects both the idea of a musical instrument and the technology from which it is designed. This article will propose a reflection on these two aspects: the hardware -the motion capture system and the objects captured -as well as the software that had to be developed for digital audio use, MoCap systems being generally designed for applications in the field of video games, cinema or biomechanical research.

Finally, we will attempt to report on the points that seemed important and original to us at the end of a complete creative process that goes as far as the public presentation of the performance.

METHODOLOGY

The project was carried out in two stages at the University of Rennes: (1) a research, experimentation and development phase of approximately six months in Immersia, a virtual reality platform equipped with motion capture systems; (2) a two-week artistic residency in the Pôle Numérique Rennes-Villejean (PNRV), a university building equipped with digital tools and dedicated to the creation of shows and interactive installations.

The performance was shown four times to the public during the JACES2 in the University of Rennes2, a one-week group exhibition dedicated to digital art productions, as well as once for the Journée Science et Musique3 , a general public event organized by the IRISA computer science research laboratory. These performances gave rise to valuable exchanges with the audience, contributing to the analysis presented here.

In the virtual reality platform

The preliminary research and experiments in the Immersia platform were carried out in a space of approximately 20m 2 equipped with an ARTTrack24 motion capture system. A first phase focused on the detailed operation of the MoCap system made it possible to consider the applications that would have to be developed in the light of the project's objectives. These applications (ARTtoOSC and µZYX ) will be presented in detail later in this paper.

The second phase concerned the design of kinetic objects in order to prepare their subsequent manufacture in the fablab of the University of Rennes2 equipped with the necessary tools (laser cutting machine, power tools, 3D printer, etc.). During this phase, work was made on both the functional and technical aspects of these objects (grip, strength, etc.), while keeping good aesthetics.

Intermediate period

An intermediate period of a few weeks before the artistic residency made it possible to finalize the prototyping of six infrared targets, one kinetic object (a light pendulum), and to create an Ableton Live session in preparation for the mapping of the traditional instruments used on stage. This included the import of different virtual synthesizers and audio processing plugins that we wanted to be able to control during the performance. Finally, we established a lightplot with the projectors that we intended to control with the MCI.

Artistic residency

Following this preliminary research and validation of the technical viability of the system, a two-week residency period was held at the PNRV, during which the performer worked independently to compose the music, develop the staging and dramatic construction of the performance and create the lighting.

THE KINETIC OBJECTS

This section will describe the design of the pendulum used for the Vis Insita performance, as well as the infrared targets necessary for a kinetic object to be detected.

The luminous pendulum

In order to make it easy to read for the audience and implement it into our study, we imagined from the outset an object that could be handed in several ways and capable of occupying the entire stage space.

A simple solution was to suspend an infrared target from the ceiling. However, the target was not heavy enough to swing for a long enough time, so we had to weigh it down. From there came the idea of making a kinetic object inspired by a pendulum.

As the object was intended to be observed by the public, it was important to also think about its aesthetic aspect. Poetically inspired by the inner force of matter (Vis Insita) defined by Isaac Newton [START_REF] Newton | Principes mathématiques de la philosophie naturelle[END_REF], we started with the aesthetic idea of an "energy ball" (Figure 1). The pendulum consists of thin white PVC plates assembled in the shape of a sphere using threaded rods, inside which we have placed a 24V-50W incandescent lamp that can be dimmed using a lighting application installed on a laptop computer, and a dimmer controlled via a USB/DMX interface.

The target for tracking the pendulum was attached to the cable at the top with a metal clamp, where the MoCap system has less occlusions during the performance.

Passive infrared targets

Unlike an isolated marker, a passive infrared target (IrT) composed of several markers is a robust way to track the objects in space: the multi-marker geometry allows, after a recognition phase by the MoCap system, an indexing and thus a re-detection in case of partial or total momentary occlusion. It also allows a capture of rotation, which is impossible with a single spherical marker.

However, in order for the system to discriminate different targets present on stage, it was necessary to give them differentiating enough geometries to avoid any ambiguity.

During the first tests with the targets supplied with the MoCap system, we quickly realized that we would have to manufacture our own ones.

First, from a functional point of view, the manufacturer's targets were only designed to be attached to the human body, including wrist and ankle joints, with an adjustable elastic band. This system prevented us from attaching them to the moving objects we imagined. On the other hand, they did not correspond to the general aesthetics we wanted to give to our kinetic objects, and it was not technically possible to modify them.

Considering these targets are the basic element of our MCI, we approached their design by taking inspiration from the atom, the latter being made up of a nucleus around which elementary particles gravitate.

However, these handmade IrT had to be able to withstand potential shocks and be firmly attached to kinetic objects. The IrT nucleus was carved from a piece of raw wood (beech) dense enough to add wooden inserts in order to securely screw threaded rods, to the opposite end of which the infrared markers were attached.

In order to standardize and stiffen the entire IrT, we covered it with a thermosetting polymer paste and then covered it with a white acrylic paint.

The markers were made from commercially available beech logs to which we applied retro-reflective tape. This tape was laser cut to optimize the quantity and cleanliness of the contours.

SOFTWARE IMPLEMENTATION

The software implementation is based on two main components, as shown in Figure 2: (1) the ARTtoOSC component is dedicated to converting raw MoCap system data into OSC data via a VRPN interface; (2) the µZYX component is dedicated to managing these OSC data for use in music and lighting applications.

The ARTtoOSC application

Natively, the ARTTrack2 system sends angular and spatial data from the optical capture to its embedded DTrack2 application in a proprietary format. It was therefore necessary, as a first step, to convert these data into usable data for our project. The choice was quickly made on the Open Sound Control (OSC) [START_REF] Freed | Features and future of open sound control version 1.1 for nime[END_REF] protocol, which is fast, open and versatile. For this purpose, we coded in C# an application called ARTtoOSC.

In order to allow portability to MoCap systems from different manufacturers, we chose to code this application using the set of VRPN5 (Virtual Reality Peripheral Network) classes and servers. This open-source system, designed to implement a transparent interface between software applications and the many physical devices used in a virtual reality system, was perfectly suited to the devices of the ART system.

The ARTtoOSC application returns two types of OSC messages:

-The target identifier (integer) and its position in the three-dimensional reference frame (Cartesian coordinates x, y and z expressed in metres with respect to the reference point determined during the calibration of the MoCap system), in format body/identifier/pos

[x][y][z].
-The target identifier (integer) and its three rotation angles (Euler angles rx, ry and rz expressed in degrees on a scale from -180

• to 180 • ) in format body/identifier/rot [rx][ry][rz].
The OSC data from ARTtoOSC is then sent to µZYX via UDP in order to speed up the communication (to the detriment of lost packets).

The µZYX interface

The main functions of the µZYX interface are to format the spatial and rotational data from IrTs into MIDI data compatible with digital audio space, as well as to calibrate and route them.

As our MCI must potentially be able to control other heterogeneous parameters such as light parameters for example, we have opted for a second possibility of conversion to OSC, a communication protocol that is also very widespread among applications encountered in the performing arts.

The µZYX application was developed in Max 8 6 , on the one hand because this programming environment is completely prescribed for the realization of patches requiring OSC and MIDI data management, and on the other hand because it allowed to quickly realize a graphical interface essential to an efficient data management in scenic conditions, both in repetition and presentation.

This graphical interface consists of a main module and as many target modules as necessary (Figure 3). The main module allows selecting the OSC data input port, saving the configuration of all target modules as presets, disabling the general MIDI output, choosing the MIDI input port for remote patch control and pre-configuring the dimensions of the playing area.

The target module allows (according to a reading from left to right) the calibration to the digital audio space of the physical data of the IrT specified in the upper left corner, the calibration, routing, visualization and smoothing of this data to a MIDI or OSC output port.

Additional options enhance these features, such as the ability to specify an offset for each of the six input parameters and initialize them in a position in the physical space different from the zero point of the MoCap system.

The central window of the target module also offers various options for setting MIDI signals, such as disabling them or inverting the range. It also has an arpeggiator that discretizes incoming continuous signals into MIDI notes. The range, mode and tone of this arpeggiator are adjustable. The velocity and duration of the notes can be associated with a gestural parameter within this window.

THE VIS INSITA PERFORMANCE 5.1 Creation

The creation of the performance followed our preliminary research and took place over a period of two weeks in a PNRV plateau of about 60m 2 in which we installed the device. This stage could accommodate approximately thirty people per performance, for one hundred and twenty people out of the four performances given.

The device consisted of several standard instruments (electric bass guitar, MIDI keyboard, electronic percussion kit) connected to a laptop computer via a sound card. The solo performer sampled live these instruments in the Ableton Live software using a MIDI pedalboard to trigger both recording and playback of the samples. This management of live sampling was supported in Live by MIDI remote scripts.

The pendulum was suspended in the centre of the playing area, as presented in Figure 4. It was equipped with an IrT whose movements were captured by the eight cameras of the MoCap system, four hung on the technical grid, and four placed on the ground. The displacements and rotations of this IrT acted during the first quarter of the performance on the tone, height and distortion of a synth pad. In the final part, it modulated dozens of sound processing parameters (glitch, delay, reverb, phasing, pan, etc.) on the main audio output bus, and controlled the horizontal and vertical movements of the automated LED projector located above.

This switch of the parameters controlled by the same target was made possible thanks to the preset system of the µZYX interface. These presets, accessible in the main window of the interface (Figure 3, at the top) and remotely selectable in MIDI, instantly recalled a set of different configurations for the target concerned, but also for all the targets used on stage.

For a greater variety of movements and instrumental controls, we decided to implement two other IrTs. The first was fixed on the head of the electric bass guitar (Figure 5, left), modulating in real time the sound of the latter according to its movement on stage, as well as the luminous intensity of the pendulum. The guitar became therefore an augmented instrument. The second Irt appeared in the last third of the performance, when the performer played the electronic percussion kit (Figure 5, right). It was fixed on his left wrist and then modulated different sound treatments that altered the sounds of these percussions according to the three-dimensional movements of the wrist. These percussions were themselves connected directly in MIDI to the lighting software in order to turn on some projectors in a synchronized way with the drum strikes.

Presentation

The public presentation for the Journée Science et Musique event was performed in front of an audience of about one hundred and fifty people, and lasted about twenty minutes. For this event, the Company ART provided us with the latest generation equipment (ARTTrack5) equipped with POE (Power Over Ethernet) technology, reducing the system's assembly time, and providing a higher refresh rate (150Hz instead of 60Hz with the ARTTRACK2 system), improving the quality of tracking and reducing overall latency by about 10ms.

DISCUSSIONS 6.1 MCIs in scenic context

One of the main technical unknowns before the field tests was to know to what extent an MoCap system designed for the fields of virtual reality, animation cinema or biomechanical research, would be adaptable to an environment for which it was not planned at all: the stage.

First, since the system was de facto sensitive to infrared radiation, the first question was whether it would be affected by incandescent light sources installed on stage, which, in addition to the visible spectrum, also emit in infrared wavelengths (850nm). After tests and adjustments, it turned out that this did not affect the quality of the tracking, the system being robust enough to recognize the geometry of the targets despite the presence of many parasitic detection points.

If MCIs in live conditions offer a number of expressive and artistic perspectives that we will see later, they raise spatial questions for the performer that do not exist for a standard instrument.

In the case of an MCI, it is necessary to clearly define the two universes to which we have to deal: the real universethe room, the stage, the physical place of the presentationand the computer universe, which has its own size ranges according to the transfer protocol considered (from 0 to 127 in MIDI and from 0 to 1 in OSC in the case of µZYX ). These two universes are not isomorphic since different calibrations for each dimension as well as non-linear transfer curves (exponential, logarithmic, inverted, etc.) can occur.

One of the main tasks in designing the µZYX software interface was therefore to allow an easy transposition from the physical to the digital universe. We used six horizontal sliders (Figure 3, bottom), three for positions and three for rotations, allowing both to visualize the modulations of the input signal with the black cursor and to set an output range (in cyan blue).

These essential spatial data calibration operations can be tedious and daunting because they require the performer to be present on stage to position and move the IrTs while having to work on the µZYX interface. In order to make the MCIs viable in stage conditions, a lot of work had to be done on the creation of a clear, light and intuitive graphical interface, as well as on the possibilities of controlling this interface wirelessly and remotely via a Bluetooth alphanumeric keyboard.

Beyond this problem, MCIs seem to offer interesting potential in terms of space. Indeed, the evolution zone of musicians is often constrained by the fixed nature of the electronic instruments and control interfaces they use, especially in electronic music [START_REF] Butler | Playing with something that runs: technology, improvisation, and composition in DJ and laptop performance[END_REF]. Most of the time, they remain standing behind them, and have very little places to move. Although the MCIs are spatially limited by the detection zone of infrared cameras, we can see that the evolutionand therefore expression -zone can become much larger and act on macroscopic levels [START_REF] Jensenius | Microinteraction in Music/Dance Performance[END_REF] order of several meters.

Autonomous movements

As we saw in the introduction, the most widespread examples of musical applications of MoCap are particularly relevant to dance, with technological and aesthetic issues in this field becoming well known and analysed.

Of the three targets used during the performance, the target of the wrist is certainly the one that most closely resembles a case of body capture (in this case, a part of the body) as found in dance. On the other hand, the pendulum represents a different scenario since the capture takes place on an object that is both manipulated by the performer (and therefore dependent on his movements) and also has autonomous movements.

In both cases, the activation energy source remains the performer. But in the case of the pendulum, the movements are also related to physical forces during the autonomous phases (for example when the pendulum makes large circular trajectories throughout the stage space) such as gravity, rebound, elasticity, etc., giving it very specific movement qualities that the performer (and even a dancer) would most certainly have great difficulty reproducing.

What seems important to us here is therefore the propensity of the pendulum -and kinetic objects in general -to generate natures of movement (and therefore parameter control) that are difficult to obtain in any other way, whether in their regularity, scale, complexity, velocity, or regular damping, for example.

This opens up interesting interpretation perspectives for a performer: the influence on sound parameters is no longer directly linked to his/her gestures; it is mediated by an interface with its own physical and kinetic characteristics that he/she can creatively exploit. This type of relationship involves a sonification of movement and an intentionality on the part of the performer in the trajectories he/she imprints on the object. In return, the object's behaviour influences the performer's actions.

The work of learning beforehand becomes essential: like a juggler with clubs or a puppeteer with articulated characters, by working at length with the object and taking over its kinematics, the performer ends up knowing the slightest reactions and can play with them in a very subtle way. There is then interaction between him/her, the object and the music. If it is a dance, then it is neither a solo nor a duo, but a dynamic and creative trio, using both improvisation and an important kinesthetic mastery.

In addition, the dissociation of the sound generator (virtual synthesizers and sound engine) from the action device (e.g. the pendulum) offers the possibility of adapting the latter to both the morphology and the proprioception of the musician. The interface is configurable and customizable regardless of the nature of the sound to be generated.

A relational interface

We have seen that the choice of manufacture of the pendulum, with its fins entangled in each other, was to allow easy gripping in different ways.

If this was the case during the performance, it turned out that the affordance of the object was not satisfactory, the number of plates and the rotation of the pendulum on its vertical axis sometimes making it difficult to grasp and anticipate. This difficulty was felt by some spectators: even without having had the opportunity to manipulate the pendulum, they talked, for example, about their feeling of "fear" that the object would fall out of the performer's hands, or that he/she would not be able to catch it.

This phenomenon, called "motor resonance" by perception neurophysiologists [START_REF] Rizzolatti | Mirrors in the Brain: How Our Minds Share Actions and Emotions[END_REF], describes the empathic relationship that occurs in an individual's mind through his/her mirror neurons, when he/she watches another person performs a movement and mentally simulates the same movement within his/her own body.

Thus, in the case of the pendulum, the unstable and dynamic relationship between the object and the performer created a palpable tension on stage. The emotion transmitted was not only through musical expression but also through anticipation and physical appropriation of the artist's gestures.

On the other hand, one of the problems frequently encountered during a concert in the field of live electronic music is the doubt that the spectator may sometimes have between what he/she sees of the performer's actions and the real consequences on the sound [START_REF] Butler | Playing with something that runs: technology, improvisation, and composition in DJ and laptop performance[END_REF]. Feedback regarding our performance has shown that if the movements are partly autonomous, even random, and the sounds heard are perceived as being related to their movements, then the viewer has offered confidence in this relationship, since he/she considers that it cannot be pre-programmed, or planned in advance. Just like the string of a guitar that can break at any moment, creating the random aspect of the interface on stage would therefore be a way to restore a relationship of trust between the performer's actions and what is shown and heard by the spectator.

Plasticity of interfaces

Among the various existing motion capture technologies, optical MoCap is distinguished by the non-invasive aspect of electronics. The captured part of the MCI then becomes constitutable of any material and can take on almost any shape or appearance, the only constraint being that it must be equipped with an IrT to be detected.

This has the effect of promoting a design based not only on a technical, functional or technological approach, but also on plastic and aesthetic considerations.

To think of an instrumental interface with regard to its own movements is above all to think of an object in the space it occupies, in its materiality, in its relationship to the one who manipulates it, but also to the one who looks at it. It means considering this object in its spatial qualities, its geometry, its lines, its balance, its dynamics, and in the astonishment and questioning it arouses in the viewer. The history of visual arts sheds light on a trend that has insatiably sought an aesthetic of movement in the object: kinetic sculpture.

Particularly active in Europe and the United States in the 1950s and 1960s, this movement, led by artists such as Jean Tinguely, Nicolas Schöffer and Alexander Calder, has constantly challenged the spectator's perceptive faculties. Could the instrumental interface not thus tend towards this fully-fledged art object, capable of combining movement, sound and visual?

In the case of the Vis Insita performance, we have seen earlier that the pendulum has been designed to be easily gripped, to be readable by the public, and to have certain luminous aspects. However, it has also been based on artistic intent: creating a poetry of movement and musical interpretation on stage and shaking up the spectator's usual perception in order to encourage multi-sensory listening to live music.

Through technologies and new interfaces, the role of musicians is shifting: not only are they becoming luthiers of their own instruments, but also their field of relational potentialities with the spectator is expanding. The instrumental interface is no longer only used to generate or control a sound; it becomes an artistic element in itself, an integral part of an overall creative process.

CONCLUSION AND FUTURE WORK

Our initial intention was mainly to evaluate the expressive and instrumental potential of a MCI designed from a kinetic object that can be manipulated by a performer, beyond the capture of his/her own body itself.

There are many tracks and they will deserve to be further developed technologically, especially with regard to the µZYX interface (improvement of the graphic user interface, preset management, implementation of remote control via smartphone, etc.).

We would like to conclude this article by focusing on how the creation of performance has evolved the conceptual approach of our MCI throughout our research.

Faced with the constraints imposed by the challenge of a real presentation in public, we have indeed gradually become aware of the possibilities of MCIs in terms of multimodal interaction and simultaneous management of heterogeneous parameters, with synthesis and sound processing parameters, but also light parameters.

The prospects for applications of this type of research can therefore extend well beyond the Vis Insita project and are addressed not only to digital musicians, but also to any artist (visual artists, dancers, choreographers, etc.) working on interactive installations requiring tangible, plastic and original means of human-machine interaction, and even human-human interaction.

The consideration of the place of presentation and the proximity to the public led us to have a global reflection on the design of the interface with an ecological approach [START_REF] Gurevich | Expression and its discontents: Toward an ecology of musical creation[END_REF].

We believe that this type of practical approach by confronting concrete situations of meeting the public in less and less conventional places will deserve to be developed in the future in order to cross-fertilize experiences and to bring new theoretical elements to the design of NIMEs.

Figure 1 :

 1 Figure 1: The light pendulum equipped with its passive infrared target.

Figure 2 :

 2 Figure 2: Software implementation diagram.

Figure 3 :

 3 Figure 3: Graphical interface of µZYX : main module (top) and one target module (bottom).

Figure 4 :

 4 Figure 4: Overview of the Vis Insita performance.

Figure 5 :

 5 Figure 5: Left: target on bass guitar; Right: target on performer's wrist.

https://vimeo.com/328923793

https://https://journees-arts-culture-sup.fr/

https://jsm.irisa.fr/

from the German manufacturer A.R. Tracking

https://github.com/vrpn/vrpn/wiki

https://cycling74.com/

ACKNOWLEDGMENTS

We would like to thank the Company ART for the ART-TRACK5 system loans, the organizers of the Journée Science et Musique 2018, and Marin Esnault for his support.