Ph.D Kristell Pothier
email: kpothier@univ-tours.fr

M.Sc Tudor Vrinceanu

M.Sc Brittany Intzandt

Ph.D Laurent Bosquet

Ph.D Antony D Karelis

Ph.D Maxime Lussier

T T Minh Vu

MD Anil Nigam

Ph.D Karen Z H Li

Ph.D Nicolas Berryman

MD T T Minh Vu

Ph.D Louis Bherer
email: louis.bherer@umontreal.ca

A comparison of physical exercise and cognitive training interventions to improve determinants of functional mobility in healthy older adults

Keywords: Aerobic training, Gross Motor Abilities, Computerized cognitive training, Timed-Up and Go Test, Cognitive switching abilities

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

Results: Repeated-measures ANOVAs showed a time effect for TUG performance (F (1,75) =14.92, p<.001): all groups equally improved after the intervention (Δ TUGpost-pre , in seconds, with 95% CI: AE = -.44 [-.81 to -.08]; GMA= -.60 [-1.10 to -.10]; ). No significant between group differences were observed. CRF was improved in the AE group only (Hedges' G=.27, small effect), ECW and cognitive switching improved the most in the GMA (Hedges' G= -.78, moderate effect) and COG groups (Hedges' G=-1.93, large effect) respectively. Smaller improvements in ECW were observed following AE and COG trainings (Hedges' G: AE=-.39, COG=-.36, both small effects) as well as in cognitive switching following AE and GMA training (Hedges' G: AE=-.42, GMA=-.21, both small effects).

INTRODUCTION

Mobility, defined as the ability to move within the environment [START_REF] Webber | Mobility in older adults: a comprehensive framework[END_REF], is a crucial clinical outcome in older adults. While usual gait speed, a marker for walking performance, is positively associated with functional status and overall health in aging [START_REF] Middleton | Walking speed: the functional vital sign[END_REF], the Timed-Up and Go (TUG) also represents a pertinent test to monitor older adults' mobility [START_REF] Podsiadlo | The timed "Up & Go": A test of basic functional mobility for frail elderly persons[END_REF]. It consists of everyday common motor tasks: to stand up from a chair, to walk three meters, to turn and to sit back on the chair. Individuals with slower TUG performances tend to have an increased risk of incident disability within two years [START_REF] Donoghue | Using timed up and go and usual gait speed to predict incident disability in daily activities among community-dwelling adults aged 65 and older[END_REF], an increased risk of adverse cardiovascular outcomes [START_REF] Chun | The Timed Up and Go test and the ageing heart: Findings from a national health screening of 1,084,875 community-dwelling older adults[END_REF], and an increased risk of all-cause mortality [START_REF] Bergland | Mobility as a predictor of all-cause mortality in older men and women: 11.8 year follow-up in the Tromso study[END_REF]. Preventing mobility declines should be a priority as this could delay disability and result in slower disease progression in older adults.

Although mobility is definitely a complex construct, observational studies have highlighted three key determinants of gait speed and TUG performance. Cardiorespiratory fitness (CRF), reflecting how efficient the circulatory and respiratory systems are at providing oxygen from the ambient air to the working muscles, is a strong predictor of multiple health outcomes as it integrates many physiological systems (e.g., cardiovascular, muscular, respiratory; see [START_REF] Ross | Importance of assessing cardiorespiratory fitness in clinical practice: a case for fitness as a clinical vital sign: a scientific statement from the American Heart Association[END_REF]. Higher levels of CRF are associated with faster walking and TUG performances [START_REF] Berryman | Executive functions, physical fitness and mobility in well-functioning older adults[END_REF]. The energy cost of walking (ECW), defined as the energy needed to walk through a given distance (Malatesta et al., 2003), is also associated with both gait speed and TUG performances [START_REF] Berryman | Executive functions, physical fitness and mobility in well-functioning older adults[END_REF][START_REF] Schrack | The relationship of the energetic cost of slow walking and peak energy expenditure to gait speed in mid-to-late life[END_REF]. In a longitudinal study with a sample of 457 participants aged 40 and older, those with an elevated ECW were at higher risk of developing slow gait speed [START_REF] Schrack | Rising energetic cost of walking predicts gait speed decline with aging[END_REF]. Along with physical capacities, walking also requires cognitive abilities, in particular executive functions, to adequately navigate in the surrounding environment (Yogev-Seliman et al., 2008). Interestingly, lower TUG performances seem to be significantly associated with lower executive, and especially cognitive switching abilities, in healthy older adults [START_REF] Langeard | Switching Ability Mediates the Age-Related Difference in Timed Up and Go Performance[END_REF], highlighting the involvement of cognition in walking and mobility.

In line with the World Health Organization's recommendations on healthy ageing that emphasize the importance of prevention over treatment, intervention to enhance mobility could have an important impact on older adults' overall quality of life (WHO, 2015). In order to optimize such intervention, it is essential to focus on key determinants of performance, and CRF, ECW as well as cognitive switching appear as great intervention targets. A few past intervention studies have shown that different approaches can be used to improve determinants of walking identified earlier; an aerobic training intervention can lead to improved CRF [START_REF] Henderson | Gait speed response to aerobic versus resistance exercise training in older adults[END_REF], a gross motor skills intervention to enhanced ECW [START_REF] Berryman | Multiple roads lead to Rome: combined high-intensity aerobic and strength training vs. gross motor activities leads to equivalent improvement in executive functions in a cohort of healthy older adults[END_REF], and a cognitive training focusing on executive functions have led to faster gait speed in older adults [START_REF] Pothier | A comparison of the impact of physical exercise, cognitive training and combined intervention on spontaneous walking speed in older adults[END_REF]. However, the effects of these three types of intervention have never been compared in the same intervention study.

Thus, the objective of this original study was to compare, in the same study design, the impact of three training methods (i.e., aerobic exercise, gross motor abilities and computerized cognitive training) known to improve key determinants associated with mobility in older adults (i.e., CRF, ECW and cognitive switching). It was hypothesized that all three interventions would improve both gait speed and TUG performances. Furthermore, it was expected that the improvement in mobility within each training group would be associated with the improvement in the relevant specific variable (i.e., CRF, ECW and cognitive switching).

METHODS

Study design

This is a randomized, parallel assignment, open label intervention study with a three-arm design. After a medical visit performed by a geriatrician and collection of demographic and clinical data, participants were invited to three pre-intervention visits evaluating physical and cognitive outcomes. Participants were then randomized to one of the following three training protocols: Aerobic (AE), Gross Motor Abilities (GMA) or Cognitive (COG) program (see Supplementary Figure 1). After the twelve-week training program, participants completed the same assessments in the same order as pre-tests. All participants provided written informed consent and the study was approved by the ethical review board of the Research Center of the Geriatric Hospital where the study took place. This study was conducted from May 2015 to December 2017. No study-related adverse events (e.g., injuries) have been reported in any of the groups.

Participants

One hundred and twenty-five community-dwelling participants aged 60 years and older met the inclusion criteria and were interested in this study. Inclusion criteria were to be nonsmokers, to consume less than two standard alcohol units per day, and not undergo any hormone therapy treatment (to avoid potential interaction between hormone replacement therapy and physical activity effects; [START_REF] Erickson | Interactive effects of fitness and hormone treatment on brain health in postmenopausal women[END_REF]. Participants were excluded if they: participated in a structured training program in the last year, underwent major surgery or were diagnosed with any medical illness within the previous year known to impact the primary variables (mobility, cognitive functions, or their ability to take part in any of the training programs; assessed by the geriatrician), had contraindications to perform physical activity or limited mobility, thyroid or pituitary diseases, neurological disease or early signs of dementia (Mini Mental State Examination, MMSE<26;Folstein et al., 1975), depression (Geriatric Depression Scale, GDS ≥ 11;[START_REF] Yesavage | Development and validation of a geriatric depression screening scale: a preliminary report[END_REF], major uncorrected sensory impairments, or presence of a somatic or known progressive psychiatric pathology.

Interventions

All programs lasted 12 weeks and included a total of 36 sessions of 60-minutes thrice weekly on Mondays, Wednesdays and Fridays. A certified kinesiologist supervised the AE and GMA groups and a research assistant trained in neuropsychology supervised the COG group. In addition, all participants were instructed not to change their daily habits during the research study.

 Aerobic intervention

The AE training was designed to enhance CRF [START_REF] Berryman | Multiple roads lead to Rome: combined high-intensity aerobic and strength training vs. gross motor activities leads to equivalent improvement in executive functions in a cohort of healthy older adults[END_REF]. Each training session alternated between high intensity interval exercises and moderate intensity continuous exercises. In each session, participants warmed up for ten minutes at 50% of their maximal aerobic power (MAP), which was established during the maximal graded exercise test prior to the intervention. Each high intensity interval session involved two sets of five minutes (with a two-minute resting period in-between) alternating between 15 second bouts of cycling (LifeFitness, Kinequip, St-Hubert, Quebec) at intensities corresponding to 100% of participants' MAP, with 15 seconds recovery at 60%. For the moderate intensity section, the warm-up was followed by 20 minutes continuous cycling at 65% MAP. Every session ended with a 10-minute cool-down period at 50% of their MAP. The intensity of the aerobic exercise was increased individually according to each participant's MAP by 5% after each month, with all participants increasing to 75% MAP at the end of training protocol for the moderate intensity part and 110% MAP for high intensity.

 Gross motor abilities intervention

The GMA training is based on a protocol elaborated in a previous study [START_REF] Berryman | Multiple roads lead to Rome: combined high-intensity aerobic and strength training vs. gross motor activities leads to equivalent improvement in executive functions in a cohort of healthy older adults[END_REF], intended to improve coordination, balance and agility. Participants started each session on a treadmill with a ten-minute low-intensity walking exercise (of increasing difficulty up to a speed of 4 km/h, 1% incline). Mondays' exercises prioritized locomotion and lower body coordination, Wednesdays' exercises targeted balance and Fridays' exercises prioritized handeye coordination (e.g., aiming and throwing). As the intervention progressed, exercises combining multiple skills (coordination, agility, balance) were added to increase the level of difficulty. For instance, participants had to maintain balance on one foot before throwing a ball in a box or walk sideways while holding an object in their hand. Participants did these GMA exercises for approximately 30 minutes, and then completed another ten-minute low-intensity walking period. Each session was concluded with five minutes of stretching to increase overall body flexibility and breathing exercises to allow participants to cool down.

 Cognitive intervention

The o The n-back task is a continuous performance task requiring to update information.

Stimuli (from either a group of 9 consonants or 6 symbols) were presented sequentially and participants had to indicate if the current stimuli matched the one from n steps earlier in the sequence. Stimuli were presented visually, on the screen, and were also heard in each participant's headphones. Two response buttons were displayed on the right side of the tablet.

The one presented in the top portion of the screen was used for the response "is the same" and the one in the bottom portion for "is different". Only the right thumb was used for this task. For the present study, the load factor n could vary from one to three. During the first month, only 1and 2-back were administered. At the beginning of the second month, 3-back was incorporated and for the third month of training, only 2-and 3-back were administered.

Each training session of the cognitive program was composed of approximately 20 minutes of each task, with increasing difficulty throughout the 12-week intervention. Participants

were instructed to answer as fast as possible while avoiding errors.

Outcomes

Primary Outcomes:

 Gait Speed: Walking speed was assessed by using a 10-m walking test in which participants had to walk in a straight line at their usual pace. Timing gates (TC-System, Brower Timing Systems, Draper, Utah, USA) were used to calculate walking speed in m.s-1 for each participant. Three trials were completed, and the average completion time was recorded for the analyses. Recently, in a cohort of community-dwelling older adults, [START_REF] Donoghue | Reliability, measurement error and minimum detectable change in mobility measures: a cohort study of community-dwelling adults aged 50 years and over in Ireland[END_REF] reported good test-retest reliability for this commonly used measure (SEM 95% IC in cm.s-1: 0.0703 (0.062 -0.0798); ICC 95% IC : 0.88 (0.83-0.91)). Of relevance, an important past study has shown that the best initial estimate of small meaningful change is near 0.05 m.s -1 for this test [START_REF] Perera | Meaningful change and responsiveness in common physical performance measures in older adults[END_REF].

 Timed-Up and Go test (TUG): Participants had to rise from a chair (with armrests), walk three meters, turn around a cone, walk back to the chair, and sit down [START_REF] Podsiadlo | The timed "Up & Go": A test of basic functional mobility for frail elderly persons[END_REF].

Participants were asked to walk at their usual gait speed. Three trials were administered per condition, and the average performance of the three trials (in seconds) was kept for the analyses.

Good test-retest reliability was also found for this test [START_REF] Donoghue | Reliability, measurement error and minimum detectable change in mobility measures: a cohort study of community-dwelling adults aged 50 years and over in Ireland[END_REF] with a TUG's minimal detectable change at the 95% confidence level of 2.08 s (SEM 95% IC : 0.75 (0.66 -0.85); ICC 95% IC : 0.75 (0.66-0.82)).

Secondary Outcomes:

 Cardiorespiratory fitness (CRF): Peak oxygen uptake, VO 2 Peak, was used to assess participants' CRF. The detailed protocol has previously been described [START_REF] Berryman | Executive functions, physical fitness and mobility in well-functioning older adults[END_REF].

All participants completed a maximal graded exercise test on a cycle ergometer (Lode, CORIVAL). They were equipped with an electrocardiogram to monitor heart rate and wore a mask that covered their mouth and nose for gas exchanges during the test (analyzed using the and Medisoft SA, United States of America). Once calibration was completed, participants began at a pre-defined load (women had an initial workload of 35 Watts and men began at 50 Watts), which was then increased by 15 Watts every minute (regardless of sex). Participants were required to maintain a pedaling rate of 60 to 80 revolutions per minute. Testing was completed when participants were unable to maintain the cadence or according to criteria described by the American College of Sports Medicine (ACSM, 2001). A certified exercise physiologist administered all tests. VO 2 Peak was defined as the highest volume of oxygen consumed over a 30 second interval in ml.kg -1 .min -1 . CRF is generally associated with good test-retest reliability in older adults (see Hugget et al., 2005, for a review; r=0.67-0.90).

 Energy Cost of Walking (ECW):

To measure ECW, all participants were equipped with the same mask and utilized the same metabolic cart (and the same calibration procedure) to measure the O 2 consumption and CO 2 production as during the VO 2 Peak assessment. They walked on a treadmill for six minutes at a constant speed of 4 km.h -1 . The ECW was calculated as described elsewhere (Berryman et al., 2017); briefly, the oxygen uptake of walking (representing the mean VO 2 from the last two minutes of the walking task, in ml.kg -1 .min -1 ) was divided by the walking speed (m.min -1 ) to obtain the oxygen cost of walking in ml.kg -1 .m -1 .

Thereafter, values in ml.kg -1 .m -1 were converted in L.kg -1 .m -1 . Using the respiratory exchange ratio (RER) corresponding to the last two minutes of walking, an appropriate energy equivalent of oxygen (J.L -1 ) was used to convert the previously calculated oxygen cost of walking (L.kg - 1 .m -1 ) in J.kg -1 .m -1 [START_REF] Peronnet | Table of nonprotein respiratory quotient: an update[END_REF] to obtain a suitable ECW. These procedures are in agreement with the literature for moderate intensity exercise and have the advantage of considering substrates metabolized during exercise [START_REF] Fletcher | Economy of running: beyond the measurement of oxygen uptake[END_REF][START_REF] Xu | Oxygen uptake kinetics during exercise[END_REF].

Oxygen steady-state and RER values less than 1 during the last two minutes of walking were necessary to consider measurements as being valid. Good test-retest reliability for the ECW was observed in a sample of 43 participants (including 20 older adults): ICC = 0.86 and Coefficient of Variation = 3.4% [START_REF] Gaesser | Cycling efficiency and energy cost of walking in young and older adults[END_REF].

 Cognitive switching: A Digit-Stroop task was used to evaluate executive functions, and especially cognitive switching [START_REF] Sedo | 5 digit test': a multilinguistic non-reading alternative to the Stroop test[END_REF]. It consisted of five different conditions, all presented on a touch-sensitive tablet (a 3 rd generation 9.7 inches Ipad). First, in the Familiarization part, participants had to press the button corresponding to the digit presented on the screen ("1" to "3" with their left thumb, "4" to "6" with their right thumb). Second, in the Reading condition, multiple identical digits were presented in a small group; the identity of the digit corresponded with the quantity of the digits presented (e.g., four copies of the digit "4") and participants had to press the corresponding button with their thumb. In the Counting condition, groups of one to six asterisks were presented and the participants had to report how many asterisks were present. In the Inhibition condition, multiple identical digits were presented on the screen and the digits' identity was incompatible with the quantity presented (e.g., five copies of the digit "4"). Participants were asked to identify the quantity of digits, and to avoid reporting the identity of the digits. Finally, stimuli in the Switching condition were identical to those of the Inhibition condition, except that, for some random trials, the group of digits was surrounded by a white frame, indicating a switch of goal: for those trials only, participants had to report the identity instead of the quantity of digit(s). For each condition, mean reaction times (RT, in seconds) of the correct trials were collected. For the present study, the variable of interest extracted from the digit-Stroop task was the Switching condition, due to its documented relevance in mobility [START_REF] Langeard | Switching Ability Mediates the Age-Related Difference in Timed Up and Go Performance[END_REF].

Sample Size

To estimate the correct sample size, three power analyses were done (one per intervention group). To detect a pre-to post-intervention difference on each key determinant of mobility, with an α risk of .05% and 80% power, 12 individuals were required in the COG group, 20 in the AE group and 32 in the GMA group. The higher estimation (n=32) has thus been selected.

Considering a dropout rate of approximately 25% (based on previous intervention studies conducted in our lab), a minimum of 120 participants have been be recruited.

Randomization and masking

A random sequence was generated using SPSS, then individually altered, one participant at a time, until the three training groups were equivalent for gender, age, education level, and usual gait speed at baseline (within a 95% confidence interval). The coordinator of the study was responsible for randomizing participants and kept confidential all the information linking data to participants' identity in a password-protected computerised file.

Participants were aware of the three intervention groups available and knew after the preintervention evaluations which of the three groups they were assigned to. Evaluators administering the pre-and post-intervention tests were blind to participants' assigned interventions and, overall, trainers were not involved in the pre-and post-intervention evaluations (the certified kinesiologist who supervised the AE and GMA training programs was present to guide the evaluators during the CRF and ECW pre-intervention testing).

Numbers Analyzed

Among the 125 participants who completed baseline screening, 30 ceased the program prior to or during pre-training evaluations due to exclusion criteria or scheduling conflicts. From the remaining 95 participants that were randomized (AE: 32, GMA: 32, COG 31), 17 abandoned the intervention voluntarily before its completion (6 from AE: study too demanding = 1, sickness/health issues = 2, no longer interested = 3; 6 from COG: study too demanding = 1, sickness/health issues = 2, no longer interested = 2, too many absences = 1; 5 from GMA: study too demanding = 1, sickness/health issues = 2, no longer interested = 1, involved in another parallel study = 1), leaving a total of 78 healthy older adults in the final sample (see Supplementary Figure 2). Participants were required to attend at least 75% of the training sessions to be included in the final analysis. Only one participant (COG) met this exclusion criteria. All the statistics were performed with the data from the 78 participants.

Statistical Analysis

Data distribution was checked by probing the kurtosis and skewness of all variables, and, to decrease the impact of outliers, data were winsorized at 3 SD away from the mean [START_REF] Tabachnick | Using multivariate statistics[END_REF]. Less than 1.5% of the data was missing, and Little's MCAR test using expectation maximization showed that the data was missing at random (X 2 = 1452.548, p = 1.00). As a result, the missing data was replaced with an average obtained from five multiple imputations using the SPSS built-in Mersenne Twister Random Number Generator, following the Markov chain Monte Carlo method. All statistical analyses were performed using IBM SPSS Statistics software v20 for Windows (IBM, Inc., Chicago, IL).

To investigate the effect of the three training interventions on our primary outcomes (gait speed and TUG performances), and on secondary outcomes (CRF, ECW, and cognitive switching), repeated measures ANOVAs were used with time (pre-post) as the within-subjects factor, and group (AE, COG, GMA) as the between-subjects factor. Where necessary, Bonferroni post-hoc tests were used. All reported p-values are two-tailed, and the significance level was set to .05. Effect sizes were calculated using Hedges' G (see [START_REF] Dupuy | Effect of overreaching on cognitive performance and related cardiac autonomic control[END_REF]. As proposed by Cohen [START_REF] Cohen | Statistical power analysis for the behavioral sciences[END_REF], the magnitude of the effect was considered small (.2 ≤ ES < .5), moderate (.5 ≤ ES < .8), or large (ES  .8). Correlations were used to evaluate potential associations between the primary (potential change in gait speed and TUG performances) and secondary outcomes (changes in the training related variables within each group (CRF, ECW, and cognitive switching).

RESULTS

Baseline data

There were no group differences observed at baseline for any of the general characteristics (see Table 1).

Training Effects on the primary outcomes

The gait speed did not differ between groups and did not significantly change after the training (all p > .05; Hedges' G: AE=.05; GMA=.10; COG=-.07).

The TUG showed a significant time effect (F (1,75) =14.92, p<.001) but no group*time interaction (F (2,75) <.44, p=.645), which indicates that all groups significantly improved TUG performances after the intervention (see Figure 1; Δ Post-Pre , in seconds, with 95% CI: AE = -.44 [-.81 to -.08]; GMA= -.60 [-1.10 to -.10]; ). The magnitude of the pre-topost difference was considered small for each group (Hedges' G: AE=-.31; GMA=-.39; COG=-.

22).

Training Effects on secondary outcomes

Cardiorespiratory Fitness: While there was no significant time effect (F (1,75) =1.12, p=.293), the group*time interaction on CRF was significant (VO 2Peak values: F (2,75) =7.13, p=.001). The AE group was the only one to show improvement (F (1,25) =9.18, p=.006): Δ Post-Pre , in ml.kg -1 .min -1 , with 95% CI: AE= 1.932 [618 to 3.245]; GMA= -329 [-1.144 to .486]; COG= -.639 [-1.710 to .432]). The magnitude of the pre-to-post difference was considered small (Hedges' G=.27) for the AE group (Hedges' G: GMA=-.06; COG=-.11) (see Figure 1).

Energy Cost of Walking:

The gross ECW revealed a significant time effect (F (1,74)= 23.18, p<.001) but no significant group*time interaction (F (2,74) =.70, p=.502). All groups improved after the intervention: Δ Post-Pre , in J.kg -1 .m -1 , with 95% CI: ; ; ). The effect sizes suggest a difference between groups though, with a numerically higher relative change in magnitude for GMA (Hedges' G= -.78 [moderate effect]), compared to AE and COG groups (Hedges' G: AE=-.39, COG=-.36 [both small effects]); see Figure 1). The correlations between changes in TUG and changes in the training related variables within each group only revealed a significant positive association in the GMA group: the improvement in mobility (TUG change) is associated with the improvement in ECW for these participants (r=.461, n=27, p=.016). The other correlations are non-significant (p > .05 for all correlations; see Supplementary Table 1).

DISCUSSION

The objective of the present study was to compare the impact of three specific physical and cognitive interventions on determinants of mobility performance in a cohort of healthy older adults. As expected, results revealed similar benefits induced by AE, GMA and COG interventions on TUG performances. Interestingly, and as hypothesized, each intervention improved key determinants associated with mobility: CRF, ECW and cognitive switching abilities, although these specific improvements were only partially correlated to changes in TUG performances.

In contrast to our main hypothesis, the three training programs did not induce gait speed improvements in this cohort. Intervention studies have previously demonstrated the positive impact of aerobic, gross motor and even cognitive exercises on older adults' walking performance [START_REF] Boyne | Factors influencing the efficacy of aerobic exercise for improving fitness and walking capacity after stroke: a meta-analysis with meta-regression[END_REF][START_REF] Cadore | Effects of different exercise interventions on risk of falls, gait ability, and balance in physically frail older adults: a systematic review[END_REF][START_REF] Henderson | Gait speed response to aerobic versus resistance exercise training in older adults[END_REF][START_REF] Intzandt | The effects of exercise on cognition and gait in Parkinson's disease: A scoping review[END_REF]. However, these past studies have focused on improving gait speed in older adults at risk (post-stroke or frail patients, obese individuals, or older adults with Parkinson's disease). In addition, when included populations were healthy older adults, previous intervention studies showing mobility improvements used more complex walking tests such as pathways with longer distances and/or a curved path, similar to the TUG test [START_REF] Hortobágyi | Effects of three types of exercise interventions on healthy old adults' gait speed: a systematic review and meta-analysis[END_REF][START_REF] Pothier | A comparison of the impact of physical exercise, cognitive training and combined intervention on spontaneous walking speed in older adults[END_REF]. Moreover, the population included in the present study was relatively young (m=69.28 ± 4.85 yo), highly educated (m=16.35 ± 3.82 years of education) and with a mean gait speed at baseline above normal values (m=1.38 ± .17 m.s -1expected values being around 1.29 m.s -1 for this age; see [START_REF] Bohannon | Normal walking speed: a descriptive metaanalysis[END_REF]. Therefore, the absence of a gait speed pre-to-post change in this study could be explained by a ceiling effect due to the use of a too simple walking test considering our fit sample.

The present study reports an overall pre-to-post change related to a more complex assessment of mobility (higher TUG performance, a crucial determinant of functional mobility) in older adults without significant health or mobility deficits. Of importance, this improvement, following three different training programs targeting key mobility-related mechanisms (CRF, ECW, cognitive switching), is equivalent among training groups. Due to the multi-determined design of the TUG test, reflecting a sum rather than a single entity of physical abilities [START_REF] Benavent-Caballer | Physical factors underlying the Timed "Up and Go" test in older adults[END_REF], it is not surprising to observe improvements in mobility following both AE and GMA trainings in our population. These results are in line with previous studies showing a positive impact of aerobic, strength, or balance training programs on functional mobility [START_REF] Steadman | A randomized controlled trial of an enhanced balance training program to improve mobility and reduce falls in elderly patients[END_REF][START_REF] Hess | Effect of high-intensity strength-training on functional measures of balance ability in balance-impaired older adults[END_REF][START_REF] Freiberger | Preventing falls in physically active community-dwelling older people: a comparison of two intervention techniques[END_REF][START_REF] Coetsee | The effect of three different exercise training modalities on cognitive and physical function in a healthy older population[END_REF]. Conversely, this is one of the very few studies in healthy older adults demonstrating TUG improvements after a computerized cognitive intervention focused on EF (see also [START_REF] Smith-Ray | Impact of Cognitive Training on Balance and Gait in Older Adults[END_REF]. Previous intervention studies reported better TUG scores after multicomponent training programs such as cognitive-motor dual-task exercises [START_REF] Vaillant | Balance, aging, and osteoporosis: effects of cognitive exercises combined with physiotherapy[END_REF] or computerized sport games [START_REF] Pichierri | Cognitive and cognitive-motor interventions affecting physical functioning: a systematic review[END_REF].

Results reported here extend previous findings by demonstrating that seated cognitive stimulation alone, targeting executive functions, can help improve older adults' functional mobility. Although the observed TUG improvement must be interpreted with caution since it is most likely within the TUG's minimal detectable change, it should nevertheless be highlighted that such small changes might be quite relevant in a highly functional population with a much smaller margin for improvements compared to participants with mobility limitations. From a clinical standpoint, reinforcing that multiple roads (i.e., AE, GMA, and COG trainings) can be followed to maintain or improve such a crucial outcome is of great interest. Older adults could therefore choose their own program (different physical or cognitive exercises) based on their motivation, abilities, or the available facilities and human resources, and expect similar improvements in functional mobility, and therefore in daily autonomy. The current findings are in line with the WHO's recommendations on healthy ageing (WHO, 2015) promoting better preventive care (rather than reacting to a disease) by supporting specific interventions enhancing healthy older adults' intrinsic capacities over time. In addition, healthcare professionals (e.g. As predicted, each intervention used in the present studies also improved specific variables associated with mobility, showing higher CRF, ECW, and switching performances following respectively the AE, GMA, and COG training programs. Interestingly, results also showed crossed improvements; while CRF was the only variable to show an exclusive improvement following the AE training, switching performances improved the most in the COG group followed by smaller, but significant, improvements in GMA and AE groups. Similarly, although the largest improvement in ECW was observed in the GMA group, smaller significant improvements were also detected in the two other groups. While cognitive benefits following exercise interventions have been regularly reported [START_REF] Colcombe | Fitness effects on the cognitive function of older adults: a meta-analytic study[END_REF][START_REF] Ludyga | Systematic review and metaanalysis investigating moderators of long-term effects of exercise on cognition in healthy individuals[END_REF][START_REF] Northey | Exercise interventions for cognitive function in adults older than 50: a systematic review with metaanalysis[END_REF], the present study confirms this phenomenon by showing that both AE and GMA were able to enhance switching abilities. Since the two physical groups targeted different training components (aerobic vs. gross motor functions), the cognitive switching improvement is likely linked to different mechanisms. It has been hypothesized that the cognitive benefit of AE training would be associated with production changes of neurotrophic factors, whereas the improvements observed in GMA training could rely on specific neurocognitive mechanisms, such as a higher activation in the visual-spatial network (see Bherer and Pothier, 2021 for review). Similarly, in addition to the GMA group, the ECW was improved in AE participants and, more surprisingly, in COG group. Considering the strong links between mobility performance and executive functioning (Yogev-Seliman et al., 2008), one could hypothesize that specific cognitive exercises would increase specific brain areas that are also involved in ECW performance, and enhance compensatory mechanisms [START_REF] Li | Cognitive involvement in balance, gait and dual-tasking in aging: a focused review from a neuroscience of aging perspective[END_REF] to further observe lower ECW values following an efficient cognitive training program centered on executive functions. Although more studies are required to better understand this relationship, the current results expand the available literature by showing that COG training can also improve walking efficiency. We hypothesized that TUG improvements would have been observed through modifications in key secondary outcomes. Results showed a significant positive association between TUG and ECW changes in the GMA group, supporting the role of ECW in complex mobility. However, the absence of significant correlations between primary and secondary outcomes in the other groups, despite observed crossed improvements, support the notion that mobility is a truly complex construct, potentially involving a cascade of other secondary changes that would need to be investigated. Some limitations associated to this intervention study should also be pointed out. First, regarding the lack of participants' motivation measurement; as is the case with all training intervention studies, it is possible that participants that were more motivated to take part in the study could have benefited more from the training program [START_REF] Ferrand | Exploring motivation for exercise and its relationship with health-related quality of life in adults aged 70 years and older[END_REF]. Second, it would be interesting to extend these findings to more accurate and precise outcomes, specifically reagarding mobility. However, a recent study compared the accuracy of gait testing in natural setting with with gaitmat or electric walk mats and showed the reliability of measuring usual gait speed with stopwatch [START_REF] Montero-Odasso | Dual-task gait speed assessments with an electronic walkway and a stopwatch in older adults. A reliability study[END_REF]. In addition, the fact that no study, to our knowledge, has evaluated the psychometric properties of the digit-stroop task could also be reported as a limitation. Previous RCTs have used the same task and reported a positive impact of physical interventions on executive functioning in older adults (see [START_REF] Esmail | Effects of dance/movement training vs. aerobic exercise training on cognition, physical fitness and quality of life in older adults: a randomized controlled trial[END_REF] but further research studies would be needed to fully understand the impact that the intervention has versus the test-retest variation in the tests used. Third, and although the present study employed a similar intervention structure, length and intensity based on other successful protocols (e.g., [START_REF] Berryman | Multiple roads lead to Rome: combined high-intensity aerobic and strength training vs. gross motor activities leads to equivalent improvement in executive functions in a cohort of healthy older adults[END_REF][START_REF] Pothier | A comparison of the impact of physical exercise, cognitive training and combined intervention on spontaneous walking speed in older adults[END_REF], it still remains unclear if those values are truly comparable. A recent perspective article [START_REF] Herold | Dose-response matters!-a perspective on the exercise prescription in exercise-cognition research[END_REF] emphasized the fact that the observed interindividual heterogeneity in response to physical exercising could be addressed with an adapted exercise prescription (making doses comparable across the individuals). This improved understanding of dose-response relationships could help designing more efficient training programs approaches against age-related declines. Finally, this study was not considered as an RCT considering the absence of a control group with no exercise at all. Therefore, it was not preregistered, which could be considered as another limit. However, there was no other study comparing in parallel those interventions and it seemed necessary to perform this first study before moving forward with a more comprehensive RCT.

The present results do strengthen the idea that multiple approaches can be followed to maintain functional mobility outcomes in older adults. In addition, the current results show that three types of intervention programs, using aerobic and gross motor exercise or cognitive training can be used to improve mobility in healthy older adults. The specific mechanisms supporting these efficient interventions remain to be elucidated and require further investigation,

o

  COG intervention was performed in a seated position, on an individual tablet through a dedicated web-based computerized neuropsychological battery. The training was composed of three different tasks centered on executive functions: Dual-task, Stroop and N-Back. The tasks involved instruction and difficulty manipulations and individualized feedback to maximize performances. The Dual-Task paradigm (Lussier et al., 2020) involved performing two discrimination tasks alone or concurrently. Participants had to discriminate three different stimuli by pressing the appropriate button with their left thumb or/and to discriminate three others with their right thumb. Stimuli (fruits vs. means of transportation, letters vs. numbers, or sounds vs. beeps) were presented visually or auditorily. After two training sessions, participants were asked to prioritize one hand over the other during the dual-task condition, according to instructions. This was done in order to increase the level of difficulty and maximize training effects. o The Stroop task involved the same five conditions used at pre-and postintervention assessments (familiarization, reading, counting, inhibition, switching; see below), but used different stimuli (tasks are identical but included letters and symbols instead of numbers, to avoid training on the pre-and post-tests stimuli).

  Figure1).

  kinesiologists, neuropsychologists) could consider one of these three interventions as an option during the implementation of an older individual's training program.

Figure 1 :

 1 Figure 1: Pre-to-Post Changes for all groups in Timed-Up and Go (TUG) test, and in each training relevant variables.

Table 1 .

 1 Baseline descriptive values (means or percentage, and standard deviations). Body Mas Index; Handgrip: kg force, sum of both hands; Sit-to-Stand: in seconds; MMSE = Mini Mental State Examination; GDS = Geriatric Depression Scale; Cardiovascular Risk = total number of concurrent conditions analysed by the geriatrician (high blood pressure, diabetes, high cholesterol, coronary artery disease, congestive heart failure, arrhythmia, valvular heart disease)

	Characteristic	All sample	AE	GMA	COG	F or χ 2	p
		N=78	n=26	n=27	n=25
	Age	69.98 (5.56)	69.28 (4.85)	70.21 (5.86)	70.46 (6.07)	F= .32	.73
	BMI (kg/m 2 )	26.10 (4.32)	26.46 (4.60)	25.25 (3.62)	26.63 (4.74)	F= .80	.46
	Handgrip (kg)	53.21 (18.41)	52.58 (16.93)	53.72 (21.17)	53.32 (18.41)	F= .03	.98
	Education Level (years)	16.05 (3.62)	16.35 (3.82)	16.26 (3.73)	15.50 (3.62)	F= .41	.66
	Female (%)	65.4	73.1	74.1	48.0	X 2 =4.92	.09
	Attendance (%)	91.99 (5.46)	92.73 (4.79)	90.84 (6.10)	92.44 (5.41)	F= .92	.40
	MMSE	28.49 (1.16)	28.92 (.89)	28.26 (1.29)	28.28 (1.17)	F=2.90	.06
	GDS	5.09 (5.66)	3.31 (3.97)	5.74 (5.03)	6.24 (7.33)	F=2.04	.14
	Cardiovascular Risk	.73 (1.24)	.97 (1.20)	.70 (1.32)	.60 (1.22)	F= .31	.74
	Abbreviations: BMI:

Table 2 .

 2 Pre and post mean values (standard deviation) of walking speeds, and all secondary outcomes, for each training group.

	Variables		AE		GMA		COG	Group*Time
									Interaction
		Pre β	Post	Pre β	Post	Pre β	Post	F	p
	Gait Speed (m.s -1 )	1.43 (.23)	1.43 (.21)	1.34 (.13)	1.36 (.12)	1.37 (.17)	1.36 (.15)	.292	.748
	Timed-Up and Go, in sec	8.54 (1.45)	8.10 (1.30)	8.93 (1.65)	8.33 (.96)	8.86 (1.48)	8.53 (1.35)	.441	.645
	Cardiorespiratory Fitness	21.58 (6.43) 23.51 (7.23) 20.26 (5.32) 19.93 (4.82) 20.25 (5.95) 19.61 (5.67) 7.128	.001*
	(VO 2Peak in ml.kg -1 .min -1 )
	Energy Cost of Walking	3.74 (.48)	3.54 (.50)	3.91 (.44)	3.60 (.32)	3.93 (.57)	3.73 (.44)	.695	.502
	gross values (J.kg -1 .m -1 )
	Cognitive switching (ms)	1646.95	1527.50	1593.75	1517.71	1691.56	1191.12	42.231 <.000*
		(263.43)	(280.72)	(354.74)	(297.24)	(268.84)	(196.32)

Abbreviations: TUG: Timed Up-and-Go test; CRF: CardioRespiratory Fitness; ECW: Energy Cost of Walking. * p value<.05. β : no significant difference between the three groups for primary and secondary outcomes

ACKNOWLEDGMENTS

The authors would also like to thank all the participants involving in this study. Authors are also thankful for the precious help of all the research assistants, and members of LESCA laboratory during this research study. This study was not preregistered. Data, analytic methods, and study materials can be made available upon request.

FUNDING

This work was supported by the Canadian Institutes of Health Research (Grant #136859). KP was supported by a Postdoctoral research fellowship from The Fonds de Recherche du Québec -Nature et Technologies (#200437). TV was supported by a Canada Graduate Scholarship -Masters offered by the Canadian Institutes of Health Research. LB was supported by the Canada Research Chair Program. Funding sources had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

CONFLICT OF INTEREST

The authors declare that they have no competing interests.


MD Claire Leblanc
email: claire.leblanc@aphp.fr

MD Sophie Brun

PhD Olivier Bouchaud

PhD Arezki Izri

PhD Vichita Ok

MD Marion Caseris

MD Fréderic Sorge

MD Luu-Ly Pham

MD André Paugam

PhD Luc Paris

PhD Stéphane Jaureguiberry

PhD Coralie Bloch-Queyrat

PhD Marouane Boubaya

MD Albert Faye

PhD Patricia Mariani

PhD Loïc De Pontual

Imported schistosomiasis in Paris region of France: A multicenter study of prevalence and diagnostic methods

Keywords: imported schistosomiasis, prevalence, children, diagnostic methods ICT: Immunochromatographic tests IS: imported schistosomiasis IHA: Indirect hemagglutination assay LCA: Latent class analysis NPV: Negative predictive value OR: odds ratio POC

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

Introduction

Schistosomiasis is a neglected parasitic disease that affects about 250 million people worldwide [START_REF] De Pontual | Schistosomiasis[END_REF]. The prevalence of imported schistosomiasis (IS) among children has not been extensively characterized [START_REF] Redditt | Health status of newly arrived refugees in Toronto, Ont: Part 1: infectious diseases[END_REF], [START_REF] Mutch | Tertiary paediatric refugee health clinic in Western Australia: analysis of the first 1026 children[END_REF]. This parasitosis is a public health issue in pediatric populations, since the prevalence in endemic countries is highest among young adolescents [START_REF] Colley | Human schistosomiasis[END_REF] and the later the diagnosis, the greater the risk of developing long-term comorbidities (such as urinary tract fibrosis, liver cirrhosis, and cancer) [START_REF] Abdou | Bilharziose urinaire : une série française[END_REF]. Children are often asymptomatic complicating the diagnosis for pediatricians [START_REF] Lingscheid | Schistosomiasis in European Travelers and Migrants: Analysis of 14 Years TropNet Surveillance Data[END_REF].

International guidelines recommend that adult migrants from endemic areas should be systematically screened for schistosomiasis, regardless of whether they are symptomatic or not [START_REF] Chaves | The Australasian Society for Infectious Diseases and Refugee Health Network of Australia recommendations for health assessment for people from refugee-like backgrounds: an abridged outline[END_REF],[8], [START_REF] Gray | Diagnosis and management of schistosomiasis[END_REF]. However, the lack of studies of pediatric IS means that there are no specific guidelines on screening and diagnostic methods in children.

Furthermore, the lack of a "reference standard" diagnostic test for schistosomiasis is associated with underdiagnosis of this disease, especially in individuals with a low worm burden [START_REF] Ogongo | Diagnosis of schistosomiasis mansoni: an evaluation of existing methods and research towards single worm pair detection[END_REF], [START_REF] Toor | Are We on Our Way to Achieving the 2020 Goals for Schistosomiasis Morbidity Control Using Current World Health Organization Guidelines?[END_REF]. Combining several laboratory assays can nevertheless increase the reliability of diagnosis. According to the current French guidelines, a diagnosis of schistosomiasis should be confirmed by combining serologic assays (screening by an ELISA and/or an indirect hemagglutination (IHA) assay and confirmation by a Western blot (WB) assay if one (or two) of the previous test is positive) and microscopic detection of eggs in urine or feces samples [START_REF]Haute Autorité de Santé -Actualisation des actes de biologie médicale relatifs au diagnostic de la schistosomose (bilharziose)[END_REF]. A urine rapid diagnostic test for the circulating cathodic antigen (CCA) produced by adult Schistosoma has been used in endemic areas for several years (Point of care (POC) -CCA) [START_REF] El-Ghareeb | Circulating cathodic antigen cassette test versus haematuria strip test in diagnosis of urinary schistosomiasis[END_REF]. These POC-CCAs give immediate results, are inexpensive, and require little analytical material but their accuracy is very controversial depending on the studies, with a low specificity for IS [START_REF] Marti | Specificity of the POC-CCA urine test for diagnosing S. mansoni schistosomiasis[END_REF], [START_REF] Neumayr | Performance of the point-of-care circulating cathodic antigen (POC-CCA) urine cassette test for followup after treatment of S. mansoni infection in Eritrean refugees[END_REF]. Moreover, it has been reported that rapid immunoglobulin G and M immunochromatographic tests (ICTs) of serum samples are a highly sensitive tool and help not to miss cases of IS [START_REF] Coulibaly | A new rapid diagnostic test for detection of anti-Schistosoma mansoni and anti-Schistosoma haematobium antibodies[END_REF], [START_REF] Nausch | Field evaluation of a new antibody-based diagnostic for Schistosoma haematobium and S. mansoniat the point-of-care in northeast Zimbabwe[END_REF]. It appears to be a suitable screening tests, even when used alone but its lower specificity compared to microscopy cannot confirm diagnosis [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]. Finally, the value of molecular biology techniques (such as real-time polymerase chain reaction (RT-PCR) assays) relative to other diagnostic tools is currently being studied but has not yet been clearly determined [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], [START_REF] Guegan | Real-time PCR for diagnosis of imported schistosomiasis[END_REF].

In view of the lack of (i) epidemiological and clinical data on IS in children in Europe and (ii) data on the performance and feasibility of the various diagnostic tests in this population, we performed a prospective and multicenter study of confirmed and suspected cases, the prevalence of IS and the diagnosis test performance using latent class method (LCA)in an atrisk pediatric population in Paris area, France.

II.

Methods

Study design

Between June 16 th , 2017, and June 5 th , 2018, children were prospectively included during general pediatric consultation or hospitalization for a different pathology by four hospitals in the greater Paris region of France (Delafontaine, Jean Verdier , Necker Enfant Malades and Robert Debré hospitals). Clinicians screened every patient for study inclusion criteria during consultation or hospitalization. These centers were selected because they treat a large number of immigrants or people having visited friends and relatives in regions where schistosomiasis is endemic.

Inclusion and exclusion criteria

The main inclusion criteria were (i) age over 3 and under 18, and (ii) arrival within the previous 12 months from an endemic area (defined by the 2012 World Health Organization (WHO) Schistosomiasis World Map) or, for children born in France, return from a stay of more than 3 months in an endemic area with at least one skin contact with freshwater during the trip. The exclusion criteria were (i) previous diagnosis and treatment of schistosomiasis in France and no subsequent at-risk fresh water contacts after treatment (for children born in France), (ii) lack of consent or (iii) missing clinical or diagnostic data. Due to ethical considerations, children who did not need a blood sample could not be included in the study.

Collection of clinical and laboratory data

When corresponding to inclusion criteria, demographic and clinical data (gender, age, comorbidities, country of origin or endemic regions visited, and hematuria) were collected anonymously on an online case report form. Weight and height were recorded and compared to the WHO growth standards. Four mL of venous blood was collected in an anticoagulantfree, and 7 mL in an EDTA tube for a blood count. An eosinophil count above 0.5 G/L was considered abnormal.

For each child, the parents or legal guardians were also asked to collect (i) three samples of fresh feces several days apart and (ii) a 24-hour urine sample or a post-exercise full bladder urine sample. All samples were sent to the Parasitology and Mycology Laboratory at Avicenne Hospital (Bobigny, France) for centralized analysis. After sedimentation or centrifugation of the 24-hour or full-bladder urine sample, the whole pellet was examined under the microscopic. Schistosoma eggs in feces samples were detected by direct microscopic observation after centrifugation in merthiolate-iodine-formaldehyde saline reagent . Serologic assays were carried out according to the respective manufacturer's instructions: an IHA assay (Fumouze Diagnostics, Signes, France), an ELISA assay (Bordier Affinity products, Crissier, Switzerland), a serum immunochromatographic assay for anti-Schistosoma IgG/IgM (LDBIO Diagnostics, Lyon, France), a WB assay for anti-Schistosoma IgG (SCHISTO II, LDBIO Diagnostics) and a cassette-based POC-CCA (Rapid Medical Diagnostics, Pretoria, South Africa). The results of the POC-CCA were scored as "negative", "trace" or "positive" when the test line was absent, faint or a clear, respectively. DNA from one gram of feces or from 1 ml of urine was extracted with the MagNA Pure Compact Nucleic Acid Isolation Kit I -Large Volume (Roche), after incubation with proteinase K and Bacteria Lysis Buffer (Roche) for 10 min at 65°C and then 10 min at 95°C.

A genus-specific RT-PCR assay targeted the 28S ribosomal RNA gene of the most common Schistosoma species in humans. The forward and reverse primers were those described previously by 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]. An internal positive control (Taqman Exogenous IPC, Life Technologies) was used to detect the possible presence of PCR inhibitors. The reaction was run on a QuantStudio7™ system (Thermo Fisher Scientific).

Ethics statement

Except in the case of unaccompanied minor refugees (UMRs), each child's parents or legal guardians gave their written, informed consent to the child's participation in the study. The children also gave their verbal, informed consent. Assistance from a translator was provided when necessary. Given the absence of parents or legal guardians for UMRs, the latter were considered to be emancipated minors and so were asked to give their written, informed consent directly.

In line with the French legislation on non-interventional, observational research, the study was registered with the French National Data Protection Commission (Commission nationale de l'informatique et des libertés (Paris, France); reference: 2043391) and approved by the local institutional review board (CLEA, Avicenne Hospital, Bobigny, France; reference: CLEA-2015-020). Personal data were anonymized by assigning a unique identifier to each study participant. The laboratory data were anonymized after the child and/or the parents or legal guardians had been informed of the diagnosis. Children with positive serology and/or positive microscopy were treated with praziquantel (40-60mg per kg) and a follow up was planned according to the French guidelines (microscopy and ultra-sound follow up every 3 months for at least a year) [START_REF]Haute Autorité de Santé -Actualisation des actes de biologie médicale relatifs au diagnostic de la schistosomose (bilharziose)[END_REF].

Study primary and secondary endpoints

The study's primary endpoint was to estimate prevalence of imported schistosomiasis in children using the LCA. The secondary endpoints were to describe children suspected cases (positive ELISA and WB serologic assays) and confirmed cases (urine or feces microscopic analysis with positive egg search) [START_REF] Marchese | Schistosomiasis in immigrants, refugees and travellers in an Italian referral centre for tropical diseases[END_REF] of schistosomiasis and describe the diagnostic performance (sensitivity, specificity, negative predictive value (NPV), and positive predictive value (PPV)) of the various tests: the WB, ELISA, IHA, and immunochromatographic serologic assays, the egg microscopy analysis of urine and feces samples, and the urine POC-CCA. In all cases, blood and urine samples were also screened for eosinophilia and hematuria, respectively.

Sample size calculation

We expected to see a prevalence rate of 10% for IS. For a 95% confidence interval (CI) with a precision of ± 3.5%, it is necessary to include 300 children using exact Clopper Pearson Method. [START_REF] Clopper | The Use of Confidence or Fiducial Limits Illustrated in the Case of the Binomial[END_REF].

Statistical analysis

The data collected were described using the number and the percentage (%) for the qualitative variables. In the other cases, the mean and standard error (SD) or the median and interquartile range (IQR) were used for the quantitative variables. With a view to identifying clinical factors associated with IS, we performed a logistic regression. All factors with a p-value <0.25 were included in the multivariate model with stepwise selection based on the Akaike information criterion (AIC).

In the absence of a reference standard, a statistical model based on latent class analysis (LCA) was used to combine multiple test results and prevalence (the WB, ELISA, and IHA assays, the urine microscopic analysis, the POC-CCA, and the ICT) in order to construct a reference standard outcome [START_REF] Rutjes | Evaluation of diagnostic tests when there is no gold standard. A review of methods[END_REF], [START_REF] Reitsma | A review of solutions for diagnostic accuracy studies with an imperfect or missing reference standard[END_REF]. Feces microscopic analysis, feces and urine RT-PCR were excluded due to the large proportion of missing data. Three models presented a variable containing 2, 3 and 4 latent classes. The latent two-class model was selected using the AIC and Bayesian Information Criterion (BIC). Then for each of the diagnostic tests, we calculated sensitivity, specificity, positive and negative predictive values (PPV and NPV).

All tests were two-tailed, and the results were considered statistically significant when p< 0.05. All analyses were performed using R software (version 3.3.3, The R Foundation for Statistical Computing [START_REF] Development | R: A language and environment for statistical computing[END_REF]).

III. Results

Study population characteristics

A total of 123 children of 300 planned were included. Nine children were excluded, due to missing clinical or laboratory data. The study population's demographic and clinical characteristics are summarized in Table 1.

One hundred and eight of the 114 children (95%) had been born outside of France. The main countries of birth were Mali (n=34, 31.6%), Republic of Guinea (n=21, 19.3%), Côte d'Ivoire (n=16, 14.9%) and Democratic Republic of Congo (n=14, 13.1%). For the six patients born in France, the at-risk areas visited were Benin, Democratic Republic of Congo, Côte d'Ivoire, Mali, and Senegal. Male to female sex ratio was 2.9.

Forty-eight children (42.1%) were UMRs; on average, the UMR were older than the non-UMRs (mean age: 15.5 ± 1.73 vs. 11.6 ± 4.46, respectively; p<0.001). New arrivals were asked if they had already been treated with praziquantel (in mass treatment campaigns) but the answers were often uncertain and could not be reliably analyzed.

Parasitological and biological data

The patients' laboratory data are summarized in Table 2. Of the nine patients with a positive urine microscopy assay for Schistosoma haematobium, six had a negative POC-CCA, two had a positive POC-CCA, and the ninth patient was not tested with a POC-CCA). Of the three patients with a positive Schistosoma feces microscopic analysis, 2 had a positive POC-CCA test (one positive microscopy for Schistosoma mansoni and the other for Schistosoma haematobium). In 15 patients, the feces microscopic analysis revealed the presence of other parasites (Giardia intestinalis, Entamoeba histolytica/dispar, Entamoeba. coli, Endolimax nanus, Ankylostomidea, Hymenolepsis nana, Blastocystis hominis). Twenty-nine of the 114 patients had a positive WB assay. Twenty-two patients had positive results for two or more serologic tests: 15 were positive according to the WB, the IHA and the ELISA assays, and 13 were positive according to the WB and the ELISA assays only. Thirty-seven patients had a positive ICT, 20 corresponding to a "trace" result (considered as positive). Only five patients had a negative ICT and a positive WB assay. Twenty-four children had a positive ICT, WB and ELISA assays. One child was tested positive for S. haematobium in microscopy, with positive ELISA, IHA and ICT assays but with a negative WB assay.

Prevalence and burden of schistosomiasis

There were 12 confirmed cases (12/114, 10%) and 13 (13/114, 11.4%) suspected cases.

The estimated prevalence of IS using LCA analysis was 24.3% (23/93, 95CI%: 16.4-34.8).

Our initial screening target (n=300) was based on a hypothetical prevalence of 10%.

However, the final analysis revealed a prevalence above 20% meaning the precision of this prevalence is now wider (± 8.5%) with 114 children included [START_REF] Clopper | The Use of Confidence or Fiducial Limits Illustrated in the Case of the Binomial[END_REF]. The clinical data on schistosomiasis-positive (confirmed and suspected patients) are summarized in Table 3. Nine out of the 25 positive patients (36.0%) had underlying conditions -mostly infectious diseases, such as active tuberculosis, malaria, typhoid and scabies. The child born in France had been infected for schistosomiasis in Mali. Thirteen of the 25 children (52%) were asymptomatic.

3.3.a. Confirmed IS

Of the nine patients with positive urine microscopic analyses [9/111], two were asymptomatic. Three of the 5 patients with macroscopic hematuria had a positive urine dipstick test for hematuria. The two patients with negative dipstick for hematuria had intermittent macroscopic hematuria. Two patients had abdominal pain, one had a positive urine dipstick test for hematuria and the other was negative.

Three out of 66 tested patients had a positive feces microscopic analysis (two for S. mansoni and one for S. haematobium). The two patients harboring S. mansoni were asymptomatic, and the patient harboring S. haematobium had abdominal pain and a negative urine dipstick test.

3.3.b Suspected/confirmed IS

Fifteen schistosomiasis suspected/confirmed cases out of 25 (60%) had undergone an ultrasound assessment after diagnosis, the rest being lost to follow up. Eight of the 15 tested patients (53.3%) had an abnormal abdominal ultrasound result (bladder calcification or hepatomegaly). It is of interest to note that 3 asymptomatic patients had an abnormal abdominal ultrasound result (including a confirmed case positive for S. mansoni).

The results of bivariate and multivariable logistic regression analyses comparing schistosomiasis suspected/confirmed cases with negative cases are summarized in Table 4.

Diagnostic test performance

Ninety-three of 114 patients (81.6%) had available data for six diagnostic tests (the WB, ELISA, and IHA assays, the urine microscopic analysis, the POC-CCA, and the ICT), and were included in the LCA (Table 5). The two latent classes are composed of 23 patients determined to be positive and 70 patients determined to be negative.

ICT had the highest sensitivity (100%), microscopy and IF had the highest specificity (100%).WB and ELISA assays had the equivalent performances.

IV. Discussion

The present prospective study is the first to highlight a high prevalence of IS (24.3%) in atrisk children in the greater Paris region of France, according to the LCA method. Twenty-four of the 25 patients diagnosed with schistosomiasis were boys (96%). This very strong male predominance was not found in other studies but might be due (at least in part) to selection bias, with the inclusion of a high proportion of UMRs [START_REF] Herbinger | Spectrum of imported infectious diseases among children and adolescents returning from the tropics and subtropics[END_REF]. In 2017, it was estimated that UMRs in France were predominantly boys (95.8%) from sub-Saharan, north and east Asia [27]. The UMRs were older than the non-refugees in the study; an older age was found to be a confounding factor and not a risk factor. Most of the children diagnosed with schistosomiasis in the present study were from Mali or Republic of Guinea -both of which are schistosomiasis high-prevalence countries [START_REF] Lai | Spatial distribution of schistosomiasis and treatment needs in sub-Saharan Africa: a systematic review and geostatistical analysis[END_REF]. Of the three patients previously treated for schistosomiasis, two still had one or more positive serologic tests, but a negative microscopy. These two children were still symptomatic even though they reported previous treatment in the endemic country. Neither of the two patients was able to specify the date or the name of the treatment received in the endemic area. One can hypothesize that treatment in the endemic zone was ineffective (depending on the number of doses received) [START_REF] King | Utility of repeated praziquantel dosing in the treatment of schistosomiasis in high-risk communities in Africa: a systematic review[END_REF], that children were reinfected after treatment (since they were symptomatic), or that children retained a "serological scar". We consider that all patients with a history of schistosomiasis and previous treatment in an endemic area should be screened for recontamination or relapse and should be provided with appropriate care.

In line with the literature data [START_REF] Lingscheid | Schistosomiasis in European Travelers and Migrants: Analysis of 14 Years TropNet Surveillance Data[END_REF], [START_REF] Coltart | Schistosomiasis in non-endemic countries[END_REF], our results showed that of the patients with suspected/confirmed IS were asymptomatic. Four confirmed out of 12 and nine suspected cases out of 13 were asymptomatic. Children who had positive diagnostic tests were nevertheless more symptomatic than those with negative tests (adjusted OR for IS = 3.32).

One can hypothesize that children were asymptomatic because they did not have the time to develop symptoms or because they were ancient cases and not active schistosomiasis (especially for adolescents), even though this second theory seams less plausible since three asymptomatic patients had an abnormal ultrasound. Among confirmed IS with signs and symptoms, hematuria was the most common feature (5 out of 8 cases) but did not always match with a positive urine dipstick, since hematuria can be intermittent. Clinicians should be aware that macroscopic hematuria is not the only sign of S. haematobium infection. Though, in the study, the urinary dipstick test for hematuria showed to be a useful predictor of IS (adjusted OR = 10.47) and clinicians should use it easily. Considering all children included, there were 13 out 114 with hematuria but only 6 were confirmed/suspected of IS, though 2 of the 7 considered negative for IS had a positive WB and immunochromatography assay, but were negative for the rest of the tests. These results show how difficult is the diagnosis of IS even when children are symptomatic. Given the high prevalence of IS observed in children at risk in the greater Paris region, systematic praziquantel treatment of all at-risk patients (even without screening) could be considered, since the treatment is well tolerated and cheap (€ 4 per course). However, the fact that three asymptomatic patients (including one tested positive for S. mansoni) had abnormal ultrasound results confirms the importance of systematic screening and regular follow-up of at-risk patients. In order to avoid missing complicated, asymptomatic forms of schistosomiasis, pediatricians should screen children at risk of schistosomiasis, especially UMRs who present an adjusted OR for IS of 3.09 in our study.

In the literature, the prevalence of IS varies markedly from one study to another, depending on the diagnostic method used [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], [START_REF] Chernet | Accuracy of Diagnostic Tests for Schistosoma mansoni Infection in Asymptomatic Eritrean Refugees: Serology and Point-of-Care Circulating Cathodic Antigen Against Stool Microscopy[END_REF], [START_REF] Chernet | Prevalence rates of six selected infectious diseases among African migrants and refugees: a systematic review and meta-analysis[END_REF]. Our initial screening target was 300 children with a hypothetical prevalence of 10% but unfortunately we were able to include 114 children and the study found a greater prevalence of 23%. The reasons of this lower inclusion number were mainly because most blood tests were not planned (exclusion criteria) for the child outside the study or parental refusal of consent. UMRs were the most included patients because as new arrivals, a blood test was planned to screen at least for tuberculosis, HIV, hepatitis B and C. The high prevalence of IS found here is slightly higher than the literature value for serologic studies, probably because of a selection bias with a large proportion of UMRs [START_REF] Theuring | Screening for infectious diseases among unaccompanied minor refugees in Berlin, 2014-2015[END_REF]. When considering microscopic analyses alone, the prevalence here would have been 8% for urine samples and 4.5% for feces samples, in line with the literature data. One of the limitations of the present study was that some of the included patients had not undergone all the diagnostic tests (primarily the feces microscopy analysis, and the urine and feces RT-PCR assays). This study highlights the difficulties of feces collection for children and the need for easier diagnostic methods. We were therefore only able to perform the LCA on 93 patients (81.6%) with data from the other 6 diagnostic tests.

In a recent study, the use of RT-PCR improved Schistosoma detection rate in urine and fecal samples by a factor of 2.6 for S. haematobium and of 1.4 for S. mansoni, relative to microscopy [START_REF] Guegan | Real-time PCR for diagnosis of imported schistosomiasis[END_REF]. In the present study, a molecular diagnosis of IS using RT-PCR was not relevant. Relatively few of the 114 patients were tested with this technique (51 for feces and 77 for urine), and the RT-PCR assay did not revealed any additional cases (relative to the serologic or microscopy analysis). This RT-PCR assay should be improved by studying a larger cohort of patients and performing an extraction control.

The POC-CCA gave rather disappointing results in our study, even though the literature data clearly show that POC-CCA is less effective at detecting S. haematobium than S. mansoniprobably because S. mansoni secretes more CCA [START_REF] Colley | Schistosomiasis is more prevalent than previously thought: what does it mean for public health goals, policies, strategies, guidelines and intervention programs?[END_REF]. This diagnostic method is probably effective for the detection of S. mansoni in endemic areas [START_REF] Danso-Appiah | Accuracy of point-of-care testing for circulatory cathodic antigen in the detection of schistosome infection: systematic review and meta-analysis[END_REF] but is not well suited to the detection of IS [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], [START_REF] Grenfell | Suitability of commercially available POC-CCA tests for schistosomiasis: Considerations for efficiency, reproducibility and decision making criteria for field application in areas of low endemicity[END_REF]. It has been reported that a lateral flow test for CAA was highly sensitive for diagnosing low-level schistosomiasis, such as IS [START_REF] Van Grootveld | Improved diagnosis of active Schistosoma infection in travellers and migrants using the ultra-sensitive in-house lateral flow test for detection of circulating anodic antigen (CAA) in serum[END_REF]. However, this recently developed technique was not available at the time of our study.

Our serologic assay results showed that ELISA and WB assays achieved the same level of diagnostic performance. Beltrame et al. found that WB assay was more sensitive but that ELISA assay had higher PPV and specificity; the researchers were unable to account for this finding [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]. In Europe, the WB assay main disadvantage is its price (€48.60 per test, vs.

€13.50 for an ELISA assay). In our study, the IHA assay had a very low sensitivity (48%) but a very high specificity making this method less interesting. The serological approach is a valuable screening tool but its interpretation is only possible in a relatively late stage of infection and does not allow the differentiation between acute and previous infections or between persisting infection and re-infection and cannot be used for the follow up [START_REF] Hinz | Serological approaches for the diagnosis of schistosomiasis -A review[END_REF]. Only the combination of diagnostic techniques (e.g. serologic assays with microscopic analyses) increases the sensitivity/specificity of schistosomiasis screening but also increases the cost.

In the present study, the serum ICT gave the best results in terms of sensitivity and the NPV.

Beltrame et al. found similar results [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]. This method has many advantages, such as its low cost, rapidity (with a result in 30 minutes) and lack of requirement for material. The relatively low specificity observed in the present study was probably caused by the qualitative nature of the test result (i.e. positive or negative only). The test was difficult to read because the test line was faintly positive for 20 of the 37 tested patients -making the interpretation readerdependent. However, the ICT gave promising results for mass screening for new diagnostic of schistosomiasis and for a low-cost, and should be considered for use in non-endemic areas.

Other interesting, rapid point-of-care diagnostic techniques (such as blood immunochromatography) are currently being developed [START_REF] Buonfrate | Diagnostic study on an immunochromatographic rapid test for schistosomiasis: comparison between use on serum and on blood spot from fingerprick[END_REF].

V. Conclusion

This study showed a high prevalence of IS in at-risk children in the greater Paris region of France. Moreover, this is the first prospective study to have compared ten diagnostic methods for IS in at-risk children. The serum ICT appears to be an attractive screening method in terms of high sensitivity, low cost, and short turnaround time; it might usefully replace less sensitive methods such as IHA and help the clinicians to provide personalized care, rather than mass treatment, to children at risk of schistosomiasis in non-endemic areas.

Table 1 .

 1 Demographic and clinical characteristics of the children population studied *surgical, auto-immune diseases (lupus, sclerodermia), traumatic(fracture,burn), scabies,

	nephrotic syndrome, typhoid

Table 2 :

 2 Laboratory test data

	Diagnostic method	N (%)
	WB
	Tests performed	114 (100%)
	Positive	29 (25.4%)
	ELISA
	Tests performed	114 (100%)
	Positive	28 (25.0%)
	IHA
	Tests performed	112 (98.2%)
	Positive	15 (13.4%)
	Serum ICT
	Tests performed	105 (92.1%)
	Positive	37 (35.2%)
	Feces microscopy analysis
	Tests performed	66 (57.9%)
	Positive for parasites	18 (27.3%)
	S. mansoni	2 (11%)
	S. haematobium	1 (5.6%)
	Other	15 (83.4%)
	Urine microscopy analysis
	Tests performed	111 (97.4%)
	Positive (S. haematobium)	9 (8.1%)
	POC-CCA
	Tests performed	102 (89.5%)
	Positive	14 (13.7%)
	Feces RT-PCR
	Tests performed	51 (44.7%)
	Positive	2 (3.9%)
	Urine RT-PCR
	Tests performed	77 (67.5%)
	Positive	1 (1.3%)
	Eosinophilia
	Tests performed	114 (100%)
	Positive (>500/mm3)	14 (12.3%)
	Mean ± SD	1,270 ± 1,011.2

Acknowledgements

We are very grateful to Dr Denis Limonne (LD-BIO Diagnostics, Lyon, France) for donating the Western blot and serum immunochromatographic assays, and to Chantel Lamon (Rapid Medical Diagnostics, Pretoria, South Africa) for donating the POC-CCA.

We would like to thank Maya Husain, Alice Bergevin, Simon Escoda, Alexis Mandelcwajg for all their help with the acquisition of data.