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Evaluation of functional outcome measures after fampridine treatment in patients with multiple sclerosis - An interventional follow-up study

  • Author Footnotes
    # These authors contributed equally to this work.
    Cecilie Dollerup Skov
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Department of Neurology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark
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  • Author Footnotes
    # These authors contributed equally to this work.
    Christina Borgen Sørensen
    Footnotes
    # These authors contributed equally to this work.
    Affiliations
    Department of Neurology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark
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  • Maria Thorning
    Affiliations
    Department of Neurology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark

    Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern, J.B. Winsloewsvej 21, st. C, Odense 5000, Denmark

    BRIDGE - Brain Research – Inter Disciplinary Guided Excellence, Department of Clinical Research, J.B. Winsloewsvej 19, 3. C, Odense 5000, Denmark

    Department of Orthopaedics and Traumatology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark

    Orthopaedic Research Unit, Department of Clinical Research, University of Southern Denmark, Denmark
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  • Kate Lykke Lambertsen
    Affiliations
    Department of Neurology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark

    Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern, J.B. Winsloewsvej 21, st. C, Odense 5000, Denmark

    BRIDGE - Brain Research – Inter Disciplinary Guided Excellence, Department of Clinical Research, J.B. Winsloewsvej 19, 3. C, Odense 5000, Denmark
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  • Lars Henrik Frich
    Affiliations
    Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern, J.B. Winsloewsvej 21, st. C, Odense 5000, Denmark

    Orthopaedic research Unit, Department of Regional Health Research, University of Southern Denmark
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  • Henrik Boye Jensen
    Affiliations
    Department of Brain and Nerve Diseases, Sygehus Lillebaelt, Kolding, Denmark

    Department of Regional Health Research, University of Southern Denmark, Denmark
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  • Anders Holsgaard-Larsen
    Affiliations
    Department of Orthopaedics and Traumatology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark

    Orthopaedic Research Unit, Department of Clinical Research, University of Southern Denmark, Denmark
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  • Helle H Nielsen
    Correspondence
    Corresponding author at: Department of Neurology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark.
    Affiliations
    Department of Neurology, Odense University Hospital, J.B. Winsloewsvej 4 C, Odense 5000, Denmark

    Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern, J.B. Winsloewsvej 21, st. C, Odense 5000, Denmark

    BRIDGE - Brain Research – Inter Disciplinary Guided Excellence, Department of Clinical Research, J.B. Winsloewsvej 19, 3. C, Odense 5000, Denmark
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  • Author Footnotes
    # These authors contributed equally to this work.
Open AccessPublished:July 04, 2022DOI:https://doi.org/10.1016/j.msard.2022.104034

      Highlights

      • EDSS level does not influence the effect of fampridine treatment on hand and gait function.
      • Combination of self-reported walking capacity (MSWS-12) and walking endurance (2MWT) provides a good marker detecting clinically relevant improvement after fampridine treatment.
      • The self-reported outcome measurements MSWS-12 did not differ between EDSS groups.
      • Objective walking measurements correlate well with EDSS groups.

      Abstract

      Objective

      The purpose of this interventional study on participants with multiple sclerosis (MS) with walking disability was to evaluate changes in functional hand and walking measurements after fampridine treatment, after stratifying by the Expanded Disability Status Scale (EDSS). We furthermore wanted to investigate different functional measurements to evaluate their ability to detect responders to fampridine with a clinically relevant improvement.

      Methods

      Patients were recruited from the MS Clinic at Odense University Hospital and were classified into two disability groups based on their EDSS score (moderate EDSS (EDSSMod) 4.5–5.5 [n = 19] and severe EDSS (EDSSSev) 6.0–7.0 [n = 14]). At baseline (visit 1) they completed the Timed 25-Foot Walk (T25FW), 2-Minute Walk Test (2MWT), Nine Hold Peg Test (9HPT), 12-item Multiple Sclerosis Walking Scale (MSWS-12), and the Six Spot Step Test (SSST). Participants were given 10 mg twice daily fampridine for 14 days before retested (visit 2). For each measurement, cut-off values were used to define responders with a clinically relevant improvement to treatment. The measurements were evaluated separately and in combination.

      Results

      Of the 33 participants, 25 (75.8%) were identified as having a clinically relevant improvement (CRI). For all patients combined (EDSSAll), all five measurements showed significant functional improvement after treatment. For the individual measurements, the highest participant response rates after 14 days of fampridine treatment were seen on the MSWS-12 (57.6%) and 2MWT (42.4%). The 2MWT also showed the largest performance improvement (18.5%) from visit 1 to visit 2. For patients with severe disability (EDSSSev), no significant improvement was seen after fampridine treatment on the T25FW, and most of the responders to T25FW had moderate disability (EDSSMod, 71.5%). Conversely for the SSST, most responders were EDSSSev (83.3%). No participants had a clinically relevant improvement on the 9HPT. The combination of T25FW, SSST, and MSWS-12 was less sensitive in distinguishing responders from non-responders, whereas the combination of 2MWT and MSWS-12 identified the same responders and could better distinguish fampridine responders from non-responders.

      Conclusion

      EDSS level did not influence the effect of fampridine treatment on functional hand and walking measures and the responsiveness of the measurements differed only a little between moderate and severe EDSS levels. The combination of self-reported walking capacity (MSWS-12) and walking endurance (2MWT) was better than T25FW, SSST, and MSWS-12 at detecting clinically meaningful improvement after fampridine treatment, which could prove useful in the clinical monitoring of walking disabilities in MS during fampridine treatment.

      Keywords

      1. Introduction

      Multiple sclerosis (MS) is a chronic and progressive neurological disease that is associated with a wide range of clinical and pathological features (
      • Lucchinetti C.
      • Bruck W.
      • Parisi J.
      • Scheithauer B.
      • Rodriguez M.
      • Lassmann H.
      Heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination.
      ). Up to 70% of individuals with MS with walking impairment describe it as the single main challenge of having MS and can have a substantial influence on quality of life (
      • Larocca N.G.
      Impact of walking impairment in multiple sclerosis: perspectives of patients and care partners.
      ). Interventions to improve walking in MS are therefore in demand, as are high-quality measurements that can identify individuals with MS who may benefit from these interventions.
      Fampridine (4-aminopyridine) is a medical treatment for individuals with MS who display an Expanded Disability Status Scale (EDSS) score between 4 and 7 (
      • van Munster C.E.
      • Uitdehaag B.M.
      Outcome measures in clinical trials for multiple sclerosis.
      ), where 30–40% of patients have been reported to improve their walking ability during treatment (
      • Rodriguez-Leal F.A.
      • Haase R.
      • Thomas K.
      • Eisele J.C.
      • Proschmann U.
      • Schultheiss T.
      • et al.
      Fampridine response in MS patients with gait impairment in a real-world setting: need for new response criteria?.
      ;
      • Jensen H.B.
      • Ravnborg M.
      • Dalgas U.
      • Stenager E.
      4-Aminopyridine for symptomatic treatment of multiple sclerosis: a systematic review.
      ;
      • Goodman A.D.
      • Brown T.R.
      • Krupp L.B.
      • Schapiro R.T.
      • Schwid S.R.
      • Cohen R.
      • et al.
      Sustained-release oral fampridine in multiple sclerosis: a randomised, double-blind, controlled trial.
      ). While the EDSS is the gold standard for clinical assessment of MS disability (
      • Bethoux F.
      • Bennett S.
      Evaluating walking in patients with multiple sclerosis: which assessment tools are useful in clinical practice?.
      ;
      • Goldman M.D.
      • Motl R.W.
      • Rudick R.A.
      Possible clinical outcome measures for clinical trials in patients with multiple sclerosis.
      ), it is less suited to evaluate the effect of fampridine (
      • Hobart J.
      • Freeman J.
      • Thompson A.
      Kurtzke scales revisited: the application of psychometric methods to clinical intuition.
      ). The EDSS scoring scale is non-linear, and the scores between 4 and 7 are based strictly on coarse measures of walking distance and walking aid requirements  (Schwid, 1997), thus limiting its application to detect more subtle differences obtained from fampridine treatment.
      The effect of fampridine is typically evaluated through functional measurements, like the  Timed-25 Foot Walk (T25FW), 2-Minute Walk Test (2MWT), Nine Hole Peg Test (9HPT), 12-item Multiple Sclerosis Walking Scale (MSWS-12), and Six Spot Step Test (SSST) (
      • Nieuwenhuis M.M.
      • Van Tongeren H.
      • Sorensen P.S.
      • Ravnborg M.
      The six spot step test: a new measurement for walking ability in multiple sclerosis.
      ;
      • Hobart J.C.
      • Riazi A.
      • Lamping D.L.
      • Fitzpatrick R.
      • Thompson A.J.
      Measuring the impact of MS on walking ability: the 12-Item MS Walking Scale (MSWS-12).
      ;
      • Fischer J.S.
      • Rudick R.A.
      • Cutter G.R.
      • Reingold S.C.
      The Multiple Sclerosis Functional Composite Measure (MSFC): an integrated approach to MS clinical outcome assessment. National MS Society Clinical Outcomes Assessment Task Force.
      ;
      • Gijbels D.
      • Eijnde B.O.
      • Feys P.
      Comparison of the 2- and 6-minute walk test in multiple sclerosis.
      ). The Danish guidelines for continuous fampridine treatment are based on a 20% improvement in the T25FW and/or a 4-point improvement in MSWS-12 (

      Jensen PVRoHB. PRÆPARATER TIL BEHANDLING AF MS – SYMPTOMATISK: dansk Neurologisk Selvskab; 2015 [Available from: https://neuro.dk/wordpress/nnbv/praeparater-ved-symptomatisk-ms-behandling/#overskrift6.

      ) after 14 days of treatment. The three other measurements have been tested with varying results (
      • Bethoux F.
      • Bennett S.
      Evaluating walking in patients with multiple sclerosis: which assessment tools are useful in clinical practice?.
      ;
      • Gijbels D.
      • Eijnde B.O.
      • Feys P.
      Comparison of the 2- and 6-minute walk test in multiple sclerosis.
      ;
      • Goldman M.D.
      • LaRocca N.G.
      • Rudick R.A.
      • Hudson L.D.
      • Chin P.S.
      • Francis G.S.
      • et al.
      Evaluation of multiple sclerosis disability outcome measures using pooled clinical trial data.
      ;
      • Pavsic K.
      • Pelicon K.
      • Ledinek A.H.
      • Sega S.
      Short-term impact of fampridine on motor and cognitive functions, mood and quality of life among multiple sclerosis patients.
      ;
      • Scalzitti D.A.
      • Harwood K.J.
      • Maring J.R.
      • Leach S.J.
      • Ruckert E.A.
      • Costello E.
      Validation of the 2-minute walk test with the 6-minute walk test and other functional measures in persons with multiple sclerosis.
      ;
      • Jensen H.
      • Ravnborg M.
      • Mamoei S.
      • Dalgas U.
      • Stenager E.
      Changes in cognition, arm function and lower body function after slow-release Fampridine treatment.
      ), but few studies have focused on how differences in disability level can affect the responsiveness to fampridine using the different measurements.
      The aims of the current study were (i) to evaluate changes in hand function and walking meassurements after 14 days of fampridine treatment in patients with walking disability due to MS stratified according to EDSS level, and (ii) to investigate the ability of five functional measurements to detect individuals with a clinically relevant improvement and thus responders to fampridine treatment.

      2. Materials and methods

      2.1 Study design

      This study was a single arm interventional follow-up study performed on MS participants with walking disability. The study presents data from a larger explorative, prospective observational cohort study (the MUST study) (ClinicalTrials.gov Identifier: NCT03847545), which was conducted from December 2018 to October 2021 at Odense University Hospital. Oral and written consents were obtained from all participants prior to participation. The MUST study was approved by the National Committee on Health Research Ethics (S-20170203) and the Danish Data Protection Agency (2012–58–0018) and was conducted in accordance with the Declaration of Helsinki. This manuscript follows the guidelines in the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement (
      • von Elm E.
      • Altman D.G.
      • Egger M.
      • Pocock S.J.
      • Gotzsche P.C.
      • Vandenbroucke J.P.
      • et al.
      The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement: guidelines for reporting observational studies.
      ).

      2.2 Participants and study outline

      Eligible participants were identified at the MS Clinic, Odense University Hospital. The inclusion criteria in the MUST study were: (1) a diagnosis of MS according to the McDonald 2010 criteria, (2) age >18 years,) (3) an Expanded Disability Status Scale (EDSS) score between 4 and 7, and() (4) fulfillment of the guidelines for receiving fampridine based on clinical symptoms and neurological evaluations. The exclusion criteria in the MUST study were:() (1) history of epilepsy, ()(2) MS attacks or acute decrease of functional capacity within the past 60 days, (3) change in immunomodulatory treatment within the past 60 days, (4) cancer within the past five years, (5) clinically significant systemic disease, and (6) concomitant treatment with cimetidine, carvedilol, propranolol, or metformin. Of the 71 participants invited to participate in the MUST study, 16 declined participation and 8 did not meet the inclusion criteria. The study group thus comprised 47 participants
      For the current study, three further exclusion criteria were applied. These were (1) absence from visit 2; (2) fampridine noncompliance and (3) already on fampridine at inclusion.

      2.3 Baseline (visit 1)

      Participants were examined at baseline by a trained clinician to determine their EDSS score and were then classified into two disability groups: An EDSS score of 4.5–5.5 was considered moderate (the EDSSMod group) while an EDSS score of 6.0–7.0 was considered severe (the EDSSSev group). Five measurements were administered during visit 1: The T25FW, 2MWT, 9HPT, MSWS-12, and SSST (Fig. 1).
      Fig. 1
      Fig. 1Schematic representation of the study design. T25FW, Timed-25 Foot Walk; 2MWT, Two Minute Walk Test; 9HPT, Nine-Hole Peg Test; MSWS-12, 12-item Multiple Sclerosis Walking Scale; SSST, Six Spot Step Test.

      2.4 Intervention

      In the 14 days between visit 1 and visit 2, participants were asked to take 10 mg oral fampridine twice daily, i.e. one tablet every 12 h.

      2.5 Follow-up (visit 2)

      After 14 days, the participants completed the same five measurements in the same manner as at visit 1. Walking aids were kept identical at both time points.

      2.6 Outcome measures

      2.6.1 Timed 25-Foot Walk (T25FW)

      The T25FW is a quantitative performance test to assess mobility and leg function and is based on a timed walk. Participants were instructed to walk 25 feet along a clearly marked course as quickly as possible, but safely. The time was calculated from the instruction to “Start” and ended when the participant had reached the 25-foot mark. The task was immediately repeated  (
      • Fischer J.S.
      • Rudick R.A.
      • Cutter G.R.
      • Reingold S.C.
      The Multiple Sclerosis Functional Composite Measure (MSFC): an integrated approach to MS clinical outcome assessment. National MS Society Clinical Outcomes Assessment Task Force.
      ). Results are given as mean value of the two tests.

      2.6.2 2-Minute Walk Test (2MWT)

      The 2MWT is a standardized measure of endurance and self-paced walking ability. Participants were instructed to walk for 2 min on a 20-m lane turning around cones at each end, and the total distance while walking was measured. Participants were permitted to slow down or stop if necessary (
      • Gijbels D.
      • Eijnde B.O.
      • Feys P.
      Comparison of the 2- and 6-minute walk test in multiple sclerosis.
      ).

      2.6.3 Nine_Hole Peg Test (9-HPT)

      The 9HPT is a standardized quantitative test of upper extremity function. Participants sat at a table where there stood a small, shallow container holding nine pegs and a plastic block containing nine empty holes. Participants were asked to pick up the nine pegs one at a time and place them in the nine holes and then to remove the pegs again one at a time and replace them into the shallow container. The participants were instructed to do this as quickly as possible and were timed throughout the whole procedure. Both the dominant and the non-dominant hand were tested twice (
      • Fischer J.S.
      • Rudick R.A.
      • Cutter G.R.
      • Reingold S.C.
      The Multiple Sclerosis Functional Composite Measure (MSFC): an integrated approach to MS clinical outcome assessment. National MS Society Clinical Outcomes Assessment Task Force.
      ). Results are given as the mean value of the four tests.

      2.6.4 12-item Multiple Sclerosis Walking Scale (MSWS-12)

      The MSWS-12 is a validated self-report measure of the impact of MS on the individual's walking ability (
      • Hobart J.C.
      • Riazi A.
      • Lamping D.L.
      • Fitzpatrick R.
      • Thompson A.J.
      Measuring the impact of MS on walking ability: the 12-Item MS Walking Scale (MSWS-12).
      ). The 12 items concern different aspects of walking function and quality and are rated on a scale ranging from 1 (not at all) to 5 (extremely). Individual item scores are summed to achieve a total score ranging from 12 to 60. (
      • Hobart J.C.
      • Riazi A.
      • Lamping D.L.
      • Fitzpatrick R.
      • Thompson A.J.
      Measuring the impact of MS on walking ability: the 12-Item MS Walking Scale (MSWS-12).
      ).

      2.6.5 Six Spot Step Test (SSST)

      The SSST is a quantitative assessment of lower extremity function (
      • Nieuwenhuis M.M.
      • Van Tongeren H.
      • Sorensen P.S.
      • Ravnborg M.
      The six spot step test: a new measurement for walking ability in multiple sclerosis.
      ). Participants were instructed to walk from one end to the other of a rectangular field measuring 1 × 5 m, while criss-crossing across the area to kick five cylindrical blocks out of five circles marked on the floor. Participants were asked to perform the test as fast as possible without running. Each participant was asked to do the test four times (i.e. twice for each leg). Each test was timed, and the mean value of the four runs was calculated (
      • Nieuwenhuis M.M.
      • Van Tongeren H.
      • Sorensen P.S.
      • Ravnborg M.
      The six spot step test: a new measurement for walking ability in multiple sclerosis.
      ).
      For a more comprehensive comparison of the functional measurements used in this study, see Supplementary Table 1

      2.7 Responder criteria

      Cut-off values for a clinically relevant improvement (CRI) with fampridine treatment were determined according to existing literature (
      • Goodman A.D.
      • Brown T.R.
      • Krupp L.B.
      • Schapiro R.T.
      • Schwid S.R.
      • Cohen R.
      • et al.
      Sustained-release oral fampridine in multiple sclerosis: a randomised, double-blind, controlled trial.
      ;
      • Callesen J.
      • Richter C.
      • Kristensen C.
      • Sunesen I.
      • Naesby M.
      • Dalgas U.
      • et al.
      Test-retest agreement and reliability of the Six Spot Step Test in persons with multiple sclerosis.
      ;
      • Schwid S.R.
      • Goodman A.D.
      • McDermott M.P.
      • Bever C.F.
      • Cook S.D.
      Quantitative functional measures in MS: what is a reliable change?.
      ). For the performance-based measurements (T25FW, 2MWT, 9HPT, and SSST), the cut-off for a CRI was set at 20% between visit 1 and 2 (this was arbitrary in the case of 2MWT as no valid cut-off has been described, to our knowledge). For the patient-reported MSWS-12, the cut-off for a clinically relevant improvement was set at 4 points between visit 1 and visit 2.
      In accordance with local and national guidelines, a participant was defined as a responder to fampridine (FAMR) if they met the cut-off in at least one of the following tests: T25FW, SSST and/or MSWS-12. Participants who did not meet any of the cut-off values, were classified as a non-responder to fampridine (FAMNR).
      To compare the ability of the outcome measures to detect FAMR, we further tested this responder criterion against a composite responder criterion (Comp). Comp was defined as an improvement of(1) 20% in the 2MWT and/or (2) 4 points in MSWS-12. If participants met one of these criteria, they were considered responders to fampridine (CompR); if not, they were considered non-responders (CompNR).

      2.7.1 Statistical analysis

      Data distributions were checked using q–q plots, histograms, and the Shapiro-Wilk test. Descriptive statistics are reported as mean values and standard deviations (SD) or median values and interquartile range (IQR). Student's paired t-test (parametric) and Wilcoxon signed-rank test (non-parametric) were used to detect changes between visit 1 and visit 2.
      Wilcoxon rank sum test (non-parametric) and non-paired t-test were used to compare means of EDSS groups, FAMR andFAMNR as well as CompR and CompNR. Spearman's correlation was used to test correlations between measurements. Data were analyzed using STATA-BE version 17.0 (StataCorp LLC, College Station, TX, USA) software package. P-values of 0.05 or less were considered statistically significant.

      3. Results

      3.1 Study group description before and after fampridine treatment

      Of the 47 participants in the MUST study, 14 did not fulfill the criteria for the current study, leaving 33 participants for analysis (Fig. 2). No imputation on missing data was performed and thus, uncompleted measurements were not included in the statistical analysis. The missing data were as follows; two participants did not complete the 9HPT due to tremors, one did not complete the 2MWT due to fatigue and two did not answer the MSWS-12.
      Fig. 2
      Fig. 2Flowchart of the participant disposition. T25FW, Timed-25 Foot Walk; 2MWT, Two Minute Walk Test; 9HPT, Nine Hole Peg Test; MSWS-12, 12-item Multiple Sclerosis Walking Scale; SSST, Six Spot Step Test; EDSS, Expanded Disability Status Scale.
      There were similar numbers of men and women, and mean age was 54.7 years (±11.41) (Table 1). Most participants had progressive MS (19.6% PPMS, 35.5% SPMS, 45.2% RRMS), which was reflected in the mean disease duration of 18.3 years (±8.65). The EDSS score at inclusion was 5.5 (± 0.74), with 57.6% of participants in the EDSSMod group and 42.4% in the EDSSSev group.
      Table 1Demographic and clinical features of participants.
      EDSSAll (n = 33)CRI (n = 25)No-CRI (n = 8)
      Sex
      Female16 (48.5%)14 (56.0%)2 (25.0%)
      Male17 (51.5%)11 (44.0%)6 (75.0%)
      Age54.7 (±11.41)54.7 (±11.05)54.9 (±13.26)
      EDSS score5.5 (± 0.74)5.5 (±0.81)5.7 (±0.46)
      EDSSMod19 (57.6%)14 (56.0%)5 (62.5%)
      EDSSSev14 (42.4%)11 (44.0%)3 (37.5%)
      Type of MSB
      RRMS14 (45.2%)10 (40.0%)4 (50.0%)
      PPMS6 (19.6%)4 (16.0%)2 (25.0%)
      SPMS11 (35.5%)9 (36.0%)2 (25.0%)
      MS-duration (years)18.3 (±8.65)17.92 (±7.69)19.5 (±11.70)
      Data are presented as mean (SD) or number (%). BTwo missing data. EDSS, Expanded Disability Status Scale;  MS, Multiple Sclerosis; RRMS, Relapsing-Remitting MS; PPMS, Primary Progressive MS; SPMS, Secondary progressive MS; EDSSAll, all participants included; EDSSMod, Expanded Disability Status Scale score of 4.0–5.5; EDSSSev, Expanded Disability Status Scale score of 6.0–7.0; CRI, relevant improvement, change of 20% in T25FW, 2MWT, 9HPT, SSST or 4 points in MSWS-12; No-CRI, no clinical relevant improvement, change of <20% in 25FW, 2MWT, 9HPT, SSST and <4 points in MSWS-12.
      Of the 33 participants, 25 (75.8%) were classified as CRI. Age, sex, duration of MS, EDSS score, and type of MS did not differ significantly between CRi and No-CRI.

      3.2 Comparison of the measurements by EDSS level

      Prior to fampridine treatment, the EDSSSev group performed significantly worse on the T25FW, 2MWT, and SSST compared to the EDSSMod group (^; p<0.05) but showed no significant differences on the 9HPT or MSWS-12 (p0.125, Table 2).
      Table 2Functional measurements before and after 14 days of fampridine treatment.
      OutcomeVisit 1Visit 2Mean changeResponders (n)
      EDSSAll, (n = 33)
       T25FW, seconds7.4 (2.7)6.6 (± 2.5)−11.0 (± 13.7)*7 (21.2%)
      2MWTA, meter118.6 (36.9)141.3 (± 45.3)18.5 (± 14.2)*14 (42.4%)
       9HPTB, seconds28.1 (8.1)26.5 (± 7.2)−5.5 (± 7.2)*0
      MSWS-12B, points43.3 (7.92)36.9 (± 9.2)−6.6 (± 7.5)*19 (57.6%)
       SSST, second13.8 (6.1)11.8 (± 4.7)−12.5 (± 11.6)*6 (18.2%)
      EDSSMod(n = 19)
       T25FW, seconds6.0 (± 1.2)5.1 (± 1.0)−13.3 (± 11.0)*5 (26.3%)
      2MWT, meter139.7 (± 26.8)165.1 (± 36.4)18.1 (± 11.5)*8 (42.1%)
       9HPTB, seconds27.0 (± 6.9)25.6 (± 5.7)−4.8 (± 6.9)*0
      MSWS-12, points41.5 (± 6.7)36.2 (± 7.9)−5.4 (± 7.2)*10 (52.6%)
       SSST, seconds10.7 (± 2.6)9.3 (± 2.1)−11.9 (± 6.8)*1 (5.3%)
      EDSSSev(n = 14)
       T25FW, seconds9.4 (± 2.8)^8.6 (± 2.6)−7.8 (± 16.7)*2 (14.3%)
      2MWTA, meter90.1 (± 28.9)^106.7 (± 33.5)19.2 (± 18.0)*6 (42.9%)
       9HPT, seconds29.5 (± 9.5)27.5 (± 8.8)−6.4 (± 7.8)*0
      MSWS-12B, points45.9 (± 9.1)38.0 (± 11.0)−8.5 (± 8.0) *9 (64.3%)
       SSST, seconds18.0 (± 7.0)^15.0 (± 5.3)−13.4 (± 16.2)*5 (35.7%)
      Data are presented as mean (SD) and numbers n (%). Improvement is indicated by negative change scores on T25FW, MSWS12, SSST and 9HPT, but positive change in scores on 2MWT.
      Mean change for T25FW, 2MWT, 9HPT and SSST are given in percentage. Mean change for MSWS-12 is in point. *; p < 0.05 by Wilcoxon's matched-pairs signed-ranks test applied at change between visit 1 and visit 2. ^; p<0.05 by non-paired t-test or Mann-Whitney test applied between the EDSSMod and EDSSSev at baseline and at percent change. AOne missing data. BTwo missing data. T25FW, Timed-25 Foot Walk; 2MWT, Two Minute Walk Test; 9HPT, Nine-Hole Peg Test; MSWS-12, 12-item Multiple Sclerosis Walking Scale; SSST, Six Spot Step Test; EDSS, Expanded Disability Status Scale; EDSSMod, Expanded Disability Status Scale score of 4.0–5.5; EDSSSev, Expanded Disability Status Scale score of 6.0–7.0.
      After 14 days of fampridine treatment, all patient groups (the EDSSMod group, the EDSSSev group, and EDSSAll) showed significantly improved mean scores between visit 1 and visit 2 (*; Table 2). However, there were no significant differences in change scores between the EDSSMod group and the EDSSSev group (p0.26, Table 2).
      The 2MWT and MSWS-12 demonstrated the largest proportion of participants with CRI with respectively 42.4% and 57.6% (Table 2). No responders were identified using 9HPT. Five out of seven (71.4%) of the responders to the T25FW were in the EDSSMod group. Conversely, five out of six (83.3%) responders to the SSST were in the EDSSSev group.

      3.3 Comparison of the measurements by responder criteria

      As our results indicated that 2MWT and MSWS-12 might be more sensitive in detecting fampridine responders, we defined a new composite responder criterion (CompR) and compared this with the FAMR responder criterion.
      According to the FAMR criteria, treatment responders (FAMR) showed significantly greater improvement than non-responders (FAMNR) on the 2MWT and MSWS-12 (^; p<0.05, Table 3) but there was no significant difference between FAMR and FAMNR on the T25FW, 9HPT, and SSST (p0.0817, Table 3). Furthermore, non-responders (FAMNR) showed significant improvement on the 2MWT between visit 1 and visit 2 (*; p = 0.032, Table 3).
      Table 3Functional measurements compared by responder criteria.
      Mean changeFAMR (n = 23)FAMNR (n = 10)
      EDSSAll: (n = 33)

      −4.7 (18.1)

      11.3
       T25FW−13.7 (10.7)*(14.1)*^

      −5.4 (7.9)

      −0.6
      2MWT21.8 (13.3)*(2.9)^

      −7.1 (15.6)
       9HPTB−5.5 (7.1)*
      MSWS-12B−9.0 (7.5)*

      −14.9 (8.7)*
       SSST
      Mean changeCompR (n = 25)CompNR (n = 8)
      EDSSAll (n = 33)
       T25FW−13.7 (10.3)*−2.7 (19.9)
      2MWT21.8 (12.7)*

      - 5.0 (7.7)*

      −8.5 (7.4)*

      −15.1 (8.3)*
      8.6 (14.7)^

      −7.0 (5.3)*

      0.1 (3.0)^

      −4.7 (16.7)^
       9HPTB
      MSWS-12
       SSST
      ata are presented as mean (SD). Mean change for T25FW, 2MWT, 9HPT and SSST are in percent. Mean change for MSWS-12 is in point. *; p< 0.05 by Wilcoxon's matched-pairs signed-ranks test or paired t-test applied at change between visit 1 and visit 2. ^; p<0.05 by non-paired t-test or Mann-Whitney test applied at percent change between the FAMR and FAMNR and between CompR and CompNR. B; Two missing data. T25FW, Timed-25 Foot Walk; 2MWT, 2-Minute Walk Test; 9HPT, Nine Hole Peg Test; MSWS-12, 12-item Multiple Sclerosis Walking Scale; SSST, Six Spot Step Test; FAMNR, non-responders to fampridine; FAMR, responders to fampridine; responders to the composite criteria; CompNR, non-responders to the composite criteria.
      According to the Comp criteria, treatment responders (CompR) showed significantly greater improvement than non-responders (CompNR) on the 2MTW, MSWS-12, and SSST (^; p0.047, Table 3).
      According to the FAMR criteria, 23 participants were classified as responders to fampridine (Table 4). According to the Comp criteria, however, an additional two participants were classified as responders to fampridine, resulting in 25 participants (Table 4).
      Table 42 × 2 table of the agreement between FAMR-criteria and the Comp-criteria.
      FAMRFAMNRTotal
      CompR23225
      CompNR088
      Total231033
      Data are presented as numbers. FAMNR, non-responders to fampridine; FAMR, responders to fampridine; Responders to the composite criteria; CompNR, Non-responders to the composite criteria.

      3.4 Correlations of change

      Finally, we investigated possible correlations between the measurements (Table 5). The changes in the 2MWT correlated positively with changes in SSST (ρ=0.48, p = 0.006) and T25FW (p = 0.78, p<0.0001, Table 5). None of the other measurements correlated with MSWS-12 (Table 5).
      Table 5Correlation of change scores between the included measurements after 14 days of fampridine treatment.
      T25FW2MWT9HPTMSWS-12SSST
      T25FW1
      2MWT0.791
      9HPT0.01−0.121
      MSWS-120.000.13−0.221
      SSST0.360.470.180.061
      Data are presented ρ-values. T25FW, Timed-25 Foot Walk; 2MWT, Two Minute Walk Test; 9HPT, Nine-Hole Peg Test; MSWS-12, 12-item Multiple Sclerosis Walking Scale; SSST, Six Spot Step Test.

      4. Discussion

      The main objective of this study was to evaluate the effect of fampridine on the T25FW, 2MWT, 9HPT, MSWS-12, and SSST according to disability level in participants with MS. Furthermore, to investigate the measurements’ ability to detect responders to fampridine (FAMR).
      We found that treatment with fampridine improved both hand and walking function in participants with MS with walking disability. However, the improvement in hand function was not sufficient for any participants to be classified as a CRI. We found no significant differences in improvement between patients classified as having moderate EDSS disability and those having severe EDSS disability, although there was a trend for the SSST to be more sensitive for treatment responders with severe disability (EDSSSev-FAMR) and the T25FW to be more sensitive for treatment responders with moderate disability (EDSSMod-FAMR). Furthermore, using 2MWT and MSWS-12 as response criteria (Comp) lowered the mean improvement for non-responders (FAMNR), thereby increasing the distinction between responders (FAMR) and non-responders (FAMNR). The 2MWT detected the same FAMR as the T25FW and SSST.

      4.1 Study population

      The present study demonstrated no significant differences between participants with CRI and No-CRI in terms of age, gender, EDSS level, type of MS, or duration of MS. Similar observations were made by other studies (
      • Rodriguez-Leal F.A.
      • Haase R.
      • Thomas K.
      • Eisele J.C.
      • Proschmann U.
      • Schultheiss T.
      • et al.
      Fampridine response in MS patients with gait impairment in a real-world setting: need for new response criteria?.
      ;
      • Goodman A.D.
      • Brown T.R.
      • Edwards K.R.
      • Krupp L.B.
      • Schapiro R.T.
      • Cohen R.
      • et al.
      A phase 3 trial of extended release oral dalfampridine in multiple sclerosis.
      ). This suggests that all individuals with MS with walking disability have the potential to profit from the use of fampridine and the degree to which an individual will benefit from the treatment depends on other factors.

      4.2 Evaluation of outcome measurements according to EDSS level

      For all patients (EDSSAll), 21% of the fampridine-treated participants were categorized as treatment responders (FAMR) based on an improvement ≥20% in T25FW at visit 2; this is consistent with previous findings (
      • Jensen H.
      • Ravnborg M.
      • Mamoei S.
      • Dalgas U.
      • Stenager E.
      Changes in cognition, arm function and lower body function after slow-release Fampridine treatment.
      ;
      • Goodman A.D.
      • Brown T.R.
      • Edwards K.R.
      • Krupp L.B.
      • Schapiro R.T.
      • Cohen R.
      • et al.
      A phase 3 trial of extended release oral dalfampridine in multiple sclerosis.
      ). However, 71% of these treatment responders were in the moderate disability group (EDSSMod). This is contrary to the general perception that T25FW has poor sensitivity for MS individuals with mild to moderate disability (
      • Goldman M.D.
      • Motl R.W.
      • Rudick R.A.
      Possible clinical outcome measures for clinical trials in patients with multiple sclerosis.
      ;
      • Goldman M.D.
      • LaRocca N.G.
      • Rudick R.A.
      • Hudson L.D.
      • Chin P.S.
      • Francis G.S.
      • et al.
      Evaluation of multiple sclerosis disability outcome measures using pooled clinical trial data.
      ;
      • Allart E.
      • Benoit A.
      • Blanchard-Dauphin A.
      • Tiffreau V.
      • Thevenon A.
      • Zephir H.
      • et al.
      Sustained-released fampridine in multiple sclerosis: effects on gait parameters, arm function, fatigue, and quality of life.
      ).
      Multiple studies suggest using the SSST as an alternative measurement for improvement in walking ability (
      • Nieuwenhuis M.M.
      • Van Tongeren H.
      • Sorensen P.S.
      • Ravnborg M.
      The six spot step test: a new measurement for walking ability in multiple sclerosis.
      ;
      • Jensen H.
      • Ravnborg M.
      • Mamoei S.
      • Dalgas U.
      • Stenager E.
      Changes in cognition, arm function and lower body function after slow-release Fampridine treatment.
      ;
      • Learmonth Y.C.
      • Dlugonski D.D.
      • Pilutti L.A.
      • Sandroff B.M.
      • Motl R.W.
      The reliability, precision and clinically meaningful change of walking assessments in multiple sclerosis.
      ). In the present study, only 18.2% of participants were categorized as responders to fampridine treatment (FAMR) based on improvement in SSST from visit 1 to visit 2. This was considerably lower than Jensen et al. who detected a responder rate of 48.6% for the SSST (
      • Jensen H.
      • Ravnborg M.
      • Mamoei S.
      • Dalgas U.
      • Stenager E.
      Changes in cognition, arm function and lower body function after slow-release Fampridine treatment.
      ). When comparing the study populations, however, our participants demonstrated superior baseline scores (13.8 s vs 18.9 s). indicating less severe walking disabilities; it would have been difficult, therefore, for our study to achieve the same change. This is underlined by the 83.3% response rate in the EDSSSev group. These results indicate that MS individuals with a higher level of disability are more likely to improve measured by the SSST than individuals with a lower level of disability. Very few studies have categorized participants by EDSS level, however, which limits comparisons of effect. The 2MWT showed the largest mean improvement of 18.5% between visit 1 and visit 2, which is comparable to previous findings (
      • Rodriguez-Leal F.A.
      • Haase R.
      • Thomas K.
      • Eisele J.C.
      • Proschmann U.
      • Schultheiss T.
      • et al.
      Fampridine response in MS patients with gait impairment in a real-world setting: need for new response criteria?.
      ). Mean improvement and responder rates were similar in the EDSSMod and EDSSSev groups, indicating that the 2MWT is well suited for monitoring treatment effects within an EDSS score range from 4 to 7.
      The use of patient-reported outcome measurements has previously been recommended as a reliance solely on clinical criteria may underestimate the number of MS individuals that benefit from fampridine treatment (
      • Brambilla L.
      • Rossi Sebastiano D.
      • Aquino D.
      • Torri Clerici V.
      • Brenna G.
      • Moscatelli M.
      • et al.
      Early effect of dalfampridine in patients with MS: a multi-instrumental approach to better investigate responsiveness.
      ). In the present study, the MSWS-12 showed the highest responder rate with 57.6%. The mean improvement and the responder rate were similar between EDSS groups. The mean improvement of −5.5 points in the MSWS-12 following fampridine treatment was comparable to the improvement reported by Allert et al. (
      • Allart E.
      • Benoit A.
      • Blanchard-Dauphin A.
      • Tiffreau V.
      • Thevenon A.
      • Zephir H.
      • et al.
      Sustained-released fampridine in multiple sclerosis: effects on gait parameters, arm function, fatigue, and quality of life.
      ), which was the only one, to our knowledge, that also used the clinical practice range of 0–60. Therefore, careful consideration is warranted when comparing studies using MSWS-12 in the 0–100 range.
      In the current study, no significant difference in the MSWS-12 baseline score was detected between the moderate and severe EDSS groups, suggesting that the perception of walking disability is independent of the physical disability. This is contrary to the objective walking measurements included in the present study, where participants with severe disability (EDSSSev) showed inferior performances. This questions the relationship between the EDSS and MSWS-12 although earlier studies reported good correlations, especially for lower EDSS scores (
      • Bethoux F.
      • Bennett S.
      Evaluating walking in patients with multiple sclerosis: which assessment tools are useful in clinical practice?.
      ).
      In accordance with other studies, we demonstrated a significant improvement in hand function on the 9HPT after fampridine treatment (
      • Rodriguez-Leal F.A.
      • Haase R.
      • Thomas K.
      • Eisele J.C.
      • Proschmann U.
      • Schultheiss T.
      • et al.
      Fampridine response in MS patients with gait impairment in a real-world setting: need for new response criteria?.
      ;
      • Pavsic K.
      • Pelicon K.
      • Ledinek A.H.
      • Sega S.
      Short-term impact of fampridine on motor and cognitive functions, mood and quality of life among multiple sclerosis patients.
      ). Despite this, no participant met the response criteria of 20%. However, our study population was sampled by EDSS levels (EDSS 4–7) that neglect hand function. Furthermore, the value of 9HPT was based on the average for the right and left hand, and this could potentially have obscured a positive effect on one of the hands.

      4.3 The ability of the measurements to detect treatment responders

      Previous studies have raised the question about the use of longer walking measurements for testing fampridine treatment (
      • Pavsic K.
      • Pelicon K.
      • Ledinek A.H.
      • Sega S.
      Short-term impact of fampridine on motor and cognitive functions, mood and quality of life among multiple sclerosis patients.
      ;
      • Allart E.
      • Benoit A.
      • Blanchard-Dauphin A.
      • Tiffreau V.
      • Thevenon A.
      • Zephir H.
      • et al.
      Sustained-released fampridine in multiple sclerosis: effects on gait parameters, arm function, fatigue, and quality of life.
      ;
      • Baert I.
      • Freeman J.
      • Smedal T.
      • Dalgas U.
      • Romberg A.
      • Kalron A.
      • et al.
      Responsiveness and clinically meaningful improvement, according to disability level, of five walking measures after rehabilitation in multiple sclerosis: a European multicenter study.
      ). We found that the improvement on the 2MWT was significantly higher for responders (FAMR) than for non-responders (FAMNR). However, the FAMNR did also show a significant improvement, indicating the 2MWT could be more sensitive in detecting responders compared to the T25FW and SSST. This phenomenon has also been described in other studies (
      • Pavsic K.
      • Pelicon K.
      • Ledinek A.H.
      • Sega S.
      Short-term impact of fampridine on motor and cognitive functions, mood and quality of life among multiple sclerosis patients.
      ;
      • Learmonth Y.C.
      • Dlugonski D.D.
      • Pilutti L.A.
      • Sandroff B.M.
      • Motl R.W.
      The reliability, precision and clinically meaningful change of walking assessments in multiple sclerosis.
      ).
      Studies differ when it comes to the most sensitive measurement to detect the effect of fampridine treatment. While one study found the T25FW and MSWS-12 to be the most sensitive (
      • Rodriguez-Leal F.A.
      • Haase R.
      • Thomas K.
      • Eisele J.C.
      • Proschmann U.
      • Schultheiss T.
      • et al.
      Fampridine response in MS patients with gait impairment in a real-world setting: need for new response criteria?.
      ), another found the SSST to be more responsive to fampridine treatment than the T25FW (
      • Jensen H.
      • Ravnborg M.
      • Mamoei S.
      • Dalgas U.
      • Stenager E.
      Changes in cognition, arm function and lower body function after slow-release Fampridine treatment.
      ). In our study, neither the SSST nor T25FW showed a significant difference in the improvement between FAMR and FAMNR. This discrepancy may be due to the MSWS-12 having the highest responder rate of all the measurements. It indicates that patients who respond to the MSWS-12 do not necessarily show great improvement on the SSST or T25FW. This is supported by the ρ-coefficient that showed poor correlation between change scores for the MSWS-12 compared to those for the T25FW and SSST. This is in line with other studies (
      • Allart E.
      • Benoit A.
      • Blanchard-Dauphin A.
      • Tiffreau V.
      • Thevenon A.
      • Zephir H.
      • et al.
      Sustained-released fampridine in multiple sclerosis: effects on gait parameters, arm function, fatigue, and quality of life.
      ).
      When we changed the response criteria from FAMR to CompR, the mean SSST improvement for treatment responders (CompR) became significantly higher for than for non-responders (the CompNR), even though the SSST was not a part of the Comp criteria. This indicates that inclusion of the 2MWT in the response criteria will better differentiate the responders with a clinically relevant improvement from the non-responders.
      Furthermore, the Comp criteria not only detected the same responders as the FAMR, but also detected an additional two responders. This suggests that the 2MWT is more responsive to the fampridine effect than the SSST and T25FW, and that the 2MWT in our population can replace both the SSST and the T25FW. Several studies have advocated for the use of longer walking measurements in evaluating fampridine treatment, including the 2MWT (
      • Rodriguez-Leal F.A.
      • Haase R.
      • Thomas K.
      • Eisele J.C.
      • Proschmann U.
      • Schultheiss T.
      • et al.
      Fampridine response in MS patients with gait impairment in a real-world setting: need for new response criteria?.
      ;
      • Pavsic K.
      • Pelicon K.
      • Ledinek A.H.
      • Sega S.
      Short-term impact of fampridine on motor and cognitive functions, mood and quality of life among multiple sclerosis patients.
      ;
      • Allart E.
      • Benoit A.
      • Blanchard-Dauphin A.
      • Tiffreau V.
      • Thevenon A.
      • Zephir H.
      • et al.
      Sustained-released fampridine in multiple sclerosis: effects on gait parameters, arm function, fatigue, and quality of life.
      ). To our knowledge, ours is the first study incorporating T25FW, 2MWT, 9HPT, MSWS-12, and SSST that identifies the 2MWT as being the most sensitive.

      4.4 Limitations and strengths

      The current study is not without its limitations. Firstly, the sample size was small, and a larger number of participants would have added greater certainty to our findings. Secondly, we did not include a control group, so the functional improvements cannot be directly assigned to fampridine treatment. Our response rate of 21.7% measured by T25FW was comparable to other studies (
      • Goodman A.D.
      • Brown T.R.
      • Krupp L.B.
      • Schapiro R.T.
      • Schwid S.R.
      • Cohen R.
      • et al.
      Sustained-release oral fampridine in multiple sclerosis: a randomised, double-blind, controlled trial.
      ;
      • Jensen H.
      • Ravnborg M.
      • Mamoei S.
      • Dalgas U.
      • Stenager E.
      Changes in cognition, arm function and lower body function after slow-release Fampridine treatment.
      ;
      • Motl R.W.
      • Snook E.M.
      Confirmation and extension of the validity of the Multiple Sclerosis Walking Scale-12 (MSWS-12).
      ), however, which suggests a relevant and genuine clinical effect. Thirdly, different cut-offs are used in the literature for the 2MWT, limiting the comparability of our results with other studies (
      • Allart E.
      • Benoit A.
      • Blanchard-Dauphin A.
      • Tiffreau V.
      • Thevenon A.
      • Zephir H.
      • et al.
      Sustained-released fampridine in multiple sclerosis: effects on gait parameters, arm function, fatigue, and quality of life.
      ;
      • Baert I.
      • Freeman J.
      • Smedal T.
      • Dalgas U.
      • Romberg A.
      • Kalron A.
      • et al.
      Responsiveness and clinically meaningful improvement, according to disability level, of five walking measures after rehabilitation in multiple sclerosis: a European multicenter study.
      ). We defined 20% improvement on 2MWT as a clinically relevant improvement; this cut-off was transferred from the other walking measurement and thus seemed relevant.
      The current study also has several strengths. We included multiple outcome measurements related to walking, hand function, and patient-reported outcomes, and thus we extend the knowledge about fampridine beyond its known impact on walking speed in individuals with MS. Furthermore, the EDSS level of our participants was assessed by the same trained clinician, in contrast to other studies that have used self-reported EDSS scores (
      • Learmonth Y.C.
      • Dlugonski D.D.
      • Pilutti L.A.
      • Sandroff B.M.
      • Motl R.W.
      The reliability, precision and clinically meaningful change of walking assessments in multiple sclerosis.
      ;
      • Motl R.W.
      • Snook E.M.
      Confirmation and extension of the validity of the Multiple Sclerosis Walking Scale-12 (MSWS-12).
      ). Moreover, our analyses were performed across a wide range of the EDSS levels, leading to information about fampridine treatment at different levels of disability, which is rarely seen in studies of fampridine.

      5. Conclusion

      In this study, we found that EDSS level did not influence the effect of fampridine, and that the sensitivity of the functional measurements differentiated only a little between disability levels. We provide evidence that self-reported walking capacities as well as walking endurance were better measurements to detect MS individuals with clinically relevant improvements to fampridine. With further research this could prove useful in the clinical monitoring of walking disabilities in MS during fampridine treatment.

      Conflict of interest and funding statement

      The MUST Study is supported financially by the Danish Multiple Sclerosis Society, The region of Southern Denmarks Research Foundation, The Foundation for MS patients in Fyen, Lily Benthine Lunds Foundation, The Lounkær Foundation and The Foundation for Promotion of Medical Science.
      HHN and HBJ has received financial compensation for travels, lectures and advisory boards from Biogen Idec, Novartis Healthcare, Teva, Merck and Sanofi Genzyme. The all authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

      CRediT authorship contribution statement

      Cecilie Dollerup Skov: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. Christina Borgen Sørensen: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. Maria Thorning: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing. Kate Lykke Lambertsen: Conceptualization, Methodology, Validation, Writing – review & editing. Lars Henrik Frich: Conceptualization, Methodology, Writing – original draft, Writing – review & editing. Henrik Boye Jensen: Conceptualization, Methodology, Writing – review & editing. Anders Holsgaard-Larsen: Conceptualization, Methodology, Validation, Writing – review & editing. Helle H Nielsen: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing.

      Acknowledgments

      The authors wish to thank all the participants who provided data for this study. Simon Bang Mohr Kristensen is acknowledged for statistical assistance and Claire Gudex for linguistic revisions.

      Appendix. Supplementary materials

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