WO2019158746A1 - Method for detecting fatigue syndromes in an individual - Google Patents

Method for detecting fatigue syndromes in an individual Download PDF

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WO2019158746A1
WO2019158746A1 PCT/EP2019/053941 EP2019053941W WO2019158746A1 WO 2019158746 A1 WO2019158746 A1 WO 2019158746A1 EP 2019053941 W EP2019053941 W EP 2019053941W WO 2019158746 A1 WO2019158746 A1 WO 2019158746A1
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biomarkers
status
individual
previous
syndrome
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French (fr)
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Nathalie VAN BRUAENE
Tine DE RYCK
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Laboratorium M. Nuytinck Bv Bvba
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/70—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving creatine or creatinine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/84—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving inorganic compounds or pH
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/88—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving prostaglandins or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/90—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving iron binding capacity of blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the invention pertains to the technical field of an in vitro method and/or a diagnostic kit for detecting the onset and/or the existence of fatigue syndromes or fatigue conditions such as non-functional overreaching, overtraining syndrome, burn-out, chronic stress, chronic fatigue and/or depression in an individual using biosignatures.
  • Fatigue conditions or fatigue syndromes such as burn-out, overtraining, chronic stress, chronic fatigue and depression disorders are becoming more common in our society.
  • the occurrence of these syndromes is often linked to an evolution in lifestyle, where the pressure to perform has increased considerably in the last decades.
  • the pressure to perform has increased considerably in the last decades.
  • individuals In addition to the pressure that individuals experience at work, they are also faced with a pressure to perform outside the working environment. For example, many individuals put a lot of pressure on themselves to be a good partner or a good parent, to be a good friend, etc.
  • it is also becoming a trend to excel at personal hobbies further adding to the pressure individuals put on themselves.
  • Fatigue syndromes are, in general, associated with irritability, disturbed sleep patterns, decreased appetite, changes in hormone levels, increased vulnerability to infections and general malaise.
  • Overtraining syndrome is considered a stage of fatigue combined with maladaptation of various physiological mechanisms to the intensified training, resulting in chronic performance reduction. Athletes suffering from OTS present symptoms such as excessive fatigue, exhaustion, and decreased performance levels. Depending on the level of overtraining it can take years for the athlete to recover.
  • NFO non-functional overreaching
  • NFO and OTS have hitherto depended on monitoring the performance of the athlete and inferring fatigue, overreaching or overtraining on the basis of a deterioration in the athlete's performance. Using this technique, however, it is only possible to detect these conditions when the athlete has already passed his peak condition.
  • the invention aims to provide a reliable and objective method for detecting the onset and / or existence of fatigue syndromes such as burn-out syndrome, non- functional overreaching, overtraining syndrome and chronic fatigue syndrome in both humans and animals. This will help in the early detection and also in the follow-up measurements of an intervention.
  • the invention provides a method according to Claim 1. More in particular, in a first aspect the invention provides an in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers (biosignature) and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker panel status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from Table 1.
  • a method according to an embodiment of the invention is particularly suitable to detect the onset and/or existence of overtraining syndrome, burn-out syndrome and/or depression disorder as it is based on accurate, objective and reproducible parameters, which have been established based on clinical data. Additionally, the method according to an embodiment of the invention provides an objective early screening procedure for these fatigue conditions.
  • the present invention provides a screening method according to claim 10. More in particular, the invention provides a method for screening a group of individuals via the in vitro method according to embodiments of the previous method.
  • the screening method of the invention can avoid huge economic losses caused by individuals suffering from undiagnosed fatigue syndromes present in, for example, a group of employees.
  • the present invention provides a diagnostic kit according to claim 12. More in particular, the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising means to carry out a method according to any one of claims 1-11.
  • the present invention provides a diagnostic kit according to claim 13. More in particular the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising a sample collection and/or storage means and an assay to determine the status of one or more, preferably two or more, biomarkers in the sample, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from Table 1.
  • a diagnostic kit according to the invention is particularly suitable to monitor the fatigue status of an individual in a simple, objective and efficient manner. It further constitutes an easy to use and rapid mode of detection, and eliminates the need for additional instrumentation.
  • the present invention relates to an assay according to claim 19. More in particular, the invention provides an assay to determine the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of an individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from Table 1.
  • Figure 1 Relation between the fatigue condition and the corresponding BIRIX.
  • Figure 2 Comparison of ROC curves of LAS and two commonly used univariate biomarkers.
  • Figure 3 Boxplot representation of the LAS score in athletes.
  • Figure 4 LAS profile of an athlete during his training for an IM
  • Figure 5 Boxplot representation of the MAS score.
  • Figure 7 Boxplot representation of the TAS score in team sport athletes.
  • Figure 8 Team overview of the TAS profiles of a pro-league soccer team
  • Figure 9 Evaluation of the LAS in practice by medical sport doctors
  • Figure 10 Implementation of the BIRIX in work environment
  • the present invention concerns an in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn- out and/or depression can be concluded from said biomarker status in said sample.
  • OTS Overtraining syndrome
  • Non-functional overreaching can develop as a consequence of intensified training and constitutes the earliest stage or onset of OTS. At this stage, adequate rest can prevent the development of OTS.
  • Burn-out syndrome or burn-out refers to the exhaustion of physical or emotional strength or motivation usually as a result of long-term stress or frustration. Burn-out syndrome is often characterized by a reduced self-esteem of the individual at its workplace. Most characteristics of burn-out syndrome are shared with overtraining syndrome. Additionally, burn-out syndrome often leads to the development of depression disorder.
  • Chronic fatigue syndrome (CFS) or myalgic encephalomyelitis (ME) refers to a disorder characterized by long-term fatigue and extreme tiredness that do not go away with rest and result in the limitation of a person's ability to carry out ordinary daily activities.
  • athlete or “sportsperson” refers to a person that is skilled or trained in exercises, sports, or games requiring physical strength, agility, endurance and/or stamina.
  • professional athlete or “elite athlete” refers to an athlete that receives payment for his or her performance.
  • revisional athlete or “amateur athlete” or “hobby” athlete refers to an athlete that trains during his or her spare time, mainly to relax.
  • sport refers to a physical activity.
  • employee refers to a person who is handling commissioned by an "employer” or “firm”.
  • the job of the employee can be broad, going from physically active jobs, like but not limited to military training, cleaning managers or masons to people with a sedentary job like, but not limited to managers or manager assistants.
  • Biomarker refers to a naturally occurring indicator such as a molecule, gene, metabolite, protein, cell type, vitamin, trace element, feature or characteristic which can be measured accurately, objectively and reproducibly, by which a particular pathological or physiological condition, such as but not limited to a process, disease, syndrome or disorder, can be identified.
  • An example of a feature biomarker is the erythrocyte sedimentation rate.
  • the status of a biomarker refers to the outcome of measurement of said biomarker and comprises the presence, absence and/or amount of said biomarker, wherein the "amount" of said biomarker includes changes in the amount of said biomarker.
  • variable refers to a single variable, which can be related to just one biomarker, or the combination of two or more biomarkers, as for example a ratio of 2 biomarkers.
  • Biosignature signifies the result of measures of a set of biomarkers relevant for the early detection of disease and prediction of treatment effectiveness
  • BIRIX biologicalmarker imbalance risk index
  • LAS load adaptation score
  • MAS or “mental adaptation score” reflects the BIRIX of the mental adaption to external and internal stress factors.
  • TAS training adaptation score
  • sample refers to any sample that is taken from the body of an individual (e.g., a human, such as an athlete or an animal, such as a horse) and contains one or more biomarkers.
  • the term "individual” as used herein refers to a human individual as well as a mammalian individual.
  • condition herein refers to "medical condition” as a broad term that includes all diseases and disorders, but can include injuries and normal health situations, such as pregnancy, that might affect a person's health, benefit from medical assistance, or have implications for medical treatments.
  • fatigue condition or “fatigue syndrome” refers to one or more medical conditions chosen from the group of non- functional overreaching (NFO), overtraining syndrome (OTS), burn-out syndrome, chronic fatigue syndrome (CFS) , chronic stress and/or depression disorder.
  • NFO non- functional overreaching
  • OTS overtraining syndrome
  • CFS chronic fatigue syndrome
  • testosterone found in an individual is bound to protein, with a large amount bound to sex hormone-binding globulin (SHBG) and a smaller amount bound weakly to albumin.
  • the biomarker "free testosterone” refers to unbound testosterone.
  • the biomarker “bio-available testosterone” refers to testosterone bound to albumin and free testosterone.
  • the biomarker “total testosterone” refers to both bound and unbound testosterone.
  • cortisol-binding globulin CBG
  • albumin cortisol-binding globulin
  • the invention provides an in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn- out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from the group comprising ferritin, haptoglobin, hemoglobin, hemoglobin in reticulocytes, iron, transferrin, transferrin saturation, hematocrit, plasma viscosity, 3- hydroxyanthranilic acid, 3-hydroxyisobutyrate, 3-hydroxykynurenine, 4-pyridoxic
  • the method of the invention is based on a combination of biomarkers, a biosignature, whose status can be accurately, objectively and reproducibly measured. As such, the methodology is objective.
  • biomarkers can be used in the method according to the invention, usually biomarkers are classified on their clinical applications and, in the case of the invention described herein, include but are not limited to:
  • Biomarkers such as genomic biomarkers, transcriptomic biomarkers, proteomic biomarkers and metabolic biomarkers.
  • Cellular biomarkers allow cells to be isolated, sorted, quantified and characterized by their morphology and physiology. Cellular biomarkers can discriminate between a large sample of cells based on their antigens
  • Feature or characteristics biomarkers include indicators with complex characteristics that can be influenced by multiple factors but whose status can be objectively, reproducibly and accurately determined.
  • Characteristics biomarkers used in the method according to the current invention include, but are not limited to, for example the erythrocyte sedimentation rate.
  • biomarkers can be measured using various different methods known to the person skilled in the art. These methods include, but are not limited to, immunological assays, mass spectrometry-based methodologies and biochemical assays. Proteomic biomarkers and peptide hormones are in most cases measured using methods based on immunoassays or on mass spectrometry. Genetic biomarkers and transcriptomic biomarkers are in most cases measured using PCR-based methods or biochemical assays. Metabolic biomarkers are most often measured using biochemical assays and mass-spectrometry based methods.
  • fluorescence- based assays - based on the detection of fluorescent dyes - are often used in the detection of all kinds of biomarkers and are especially suited for the detection of cellular biomarkers. It goes without saying that the list of methods given here is a non- exhaustive list of examples and that the method according to the invention may implement other assays, known to the skilled person, suited to measure the status of the biomarkers as currently disclosed.
  • the method of the invention provides an early screening procedure that may be carried out on a sample obtained from an individual, before the individual presents symptoms or before he is aware of any symptoms.
  • the method thus allows for detection of the earliest stages of NFO/OTS, burn-out and/or depression, allowing an adaptation of the training, work-load or lifestyle to avoid the development of NFO/OTS, the worsening of NFO/OTS or the development of CFS or burn-out which could eventually result in depression.
  • early diagnosis of these syndromes/disorders results in a better standard of living as they can then be adequately treated before they have a more detrimental effect on the individual's health.
  • Another advantage of the method according to the invention is that early diagnosis in professional athletes can be used to prevent disappointing performance by introducing more recovery time in between trainings, resulting in a positive effect on the athlete's career.
  • the method of the invention therefore, provides the opportunity for athletes, and their coaches, to monitor training effects and performance such that early intervention may be made to the athletes' training schedules before the development of OTS and the serious and long term symptoms associated with it.
  • the method according to the current invention also offers the advantage that reliable and accurate diagnosis of NFO/OTS, burn-out syndrome and/or depression disorder avoids the need of running a large number of other medical tests in order to exclude other causes.
  • the method may be particularly useful in the screening of employees for e.g. the detection of the onset of burnout.
  • the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual is concluded by determining the status of at least two biomarkers, preferably at least three biomarkers in a sample obtained from the body of the individual.
  • the inventors have found that combining the status of two or three biomarkers leads to an unexpected increase in accuracy and sensitivity of the detection of onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual. This allows to infer an unambiguous conclusion related to the onset and/or the existence of said conditions in an individual based on the determination of the status of individual biomarkers which may not be informative on their own.
  • one or more biomarkers are selected from the group comprising ferritin, haptoglobin, hemoglobin, hemoglobin in reticulocytes, iron, transferrin, transferrin saturation, hematocrit, plasma viscosity, 3-hydroxyanthranilic acid, 3-hydroxyisobutyrate, 3-hydroxykynurenine, 4-pyridoxic acid, anandamide, anthranilic acid, beta-endorphins, calcitonin, cotinine, cystathionine, epidermal growth factor (EGF), flavin mononucleotide, free tryptophan, g-amuniobutyric acid (GABA), glutamate receptor, isoleucine, kynurenic acid (KYNA), leucin, L-kynurenine, Nl- methylnicotinamide, nicotinamide, nicotinic acid, picolinic acid, prolactin, pyridoxal, pyridoxal 5'
  • At least one of said biomarkers is selected from the group consisting of 3-hydroxyanthranilic acid, 3- hydroxyisobutyrate, 3-hydroxykynurenine, 4-pyridoxic acid, adiponectin, alanine aminotransferase (ALT), albumin, alkaline phosphatase, alpha-amylase, anthranilic acid, aspartate aminotransferase (AST), basophils, bio-available testosteron (TST), calcium, cotinine, C-reactive protein (CRP), creatine kinase, creatinine, estimated glomerular filtration rate (GFR), cystathionine, dehydroepiandrosterone sulfate (DHEA- S), eosinophils, erythrocyte sedimentation rate, erythrocytes, ferritin, flavin mononucleotide, folic acid, free cortisol, free testosterone , gamma-glutamyl trans
  • At least one of said biomarkers is selected from the group consisting of 3-hydroxyanthranilic acid, alanine aminotransferase (ALT), alpha-amylase, anthranilic acid, total cortisol, free cortisol, cotinine, dehydroepiandrosterone sulfate (DHEA-S), ferritin, growth hormone, IGF-1, IGF-BP3, picolinic acid, quinolinic acid (QUIN), resistin, riboflavin, secretory Immunoglobulin A (slgA), total testosterone, free testosterone, bio-available testosterone, thiamine, TNF-a, triglycerides , vascular endothelial growth factor A (VEGF-A) and Vitamin D.
  • ALT alanine aminotransferase
  • DHEA-S dehydroepiandrosterone sulfate
  • ferritin ferritin
  • growth hormone IGF-1, IGF-BP3, picolinic acid,
  • one or more, preferably two or more, biomarkers are selected from Table 1.
  • said one or more, preferably two or more, biomarkers are selected from Table 2.
  • Table 2 list of key biomarkers
  • the status of at least two and even more preferably at least three of said biomarkers is determined, preferably markers chosen from Table 1 or Table 2. More by preference, the status of at least 4, even more by preference, at least 5 of said biomarkers is determined. Even more by preference, the status of at least 6, more by preference, at least 7 of said biomarkers is determined. Most by preference, the status of at least 8 to 10 of said biomarkers is determined.
  • the method according to the present invention additionally comprises determining the status of one or more additional parameters selected from Table 3, in combination with at least one marker chosen from Table 1 or 2.
  • the markers of Table 3 will always be used in combination with at least one or more, such as two, markers from Table 1 or 2.
  • the status of at least two biomarkers of Table 1 or 2, and one marker of Table 3, is determined.
  • the status of at least 2 biomarkers of Table 1 or 2, and at least two markers of Table 3, is determined.
  • the status of at least 1 to 3 biomarkers of Table 1 or 2, and at least 1 to 3 markers of Table 3, is determined.
  • the status of at least 1 to 4 biomarkers of Table 1 or 2, and at least 1 to 4 markers of Table 3 is determined.
  • the status of at least 1 to 5 biomarkers of Table 1 or 2, and at least 1 to 5 markers of Table 3 is determined.
  • the status of at least 1 to 10 biomarkers of Table 1 or 2, and at least 1 to 10 markers of Table 3, is determined
  • the status of at least two biomarkers for determining the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual is concluded wherein at least one biomarker is selected from a first group of biomarkers consisting of group 1, group 2, group 3 or group 4, and at least one additional biomarker is selected from a second group of biomarkers consisting of group 1, group 2, group 3 or group 4 which is different from the first group of biomarkers, wherein group 1 consists of alanine aminotransferase (ALT), albumin, alkaline phosphatase, aspartate aminotransferase (AST), calcium, creatine kinase, creatinine, estimated glomerular filtration rate (GFR), erythrocyte sedimentation rate, ferritin, folic acid, gamma-glutamyl trans
  • ALT
  • the status of at least one additional biomarker is determined which is selected from a third group of biomarkers consisting of group 1, group 2, group 3 or group 4, wherein the third group is different from the first and second group.
  • the status of at least one additional biomarker is determined which is selected from a fourth group of biomarkers consisting of group 1, group 2, group 3 or group 4, wherein the fourth group is different from the first, the second and the third group. Detection of at least two biomarkers belonging to different groups according to the grouping described above enriches the diagnostic performance of the biomarkers resulting in enhanced diagnostic accuracy.
  • an analysis is performed on a body fluid sample.
  • Said body fluid sample is chosen from whole blood (optionally diluted), a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory, intestinal and genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy.
  • samples are obtained through collection of the fluid in a container (e.g. urine or saliva) or absorbing material or by inserting a needle into the body cavity and aspirating with a syringe a portion of the fluid (e.g. blood, lymph fluid).
  • Isolated blood cells include white blood cells such as leukocytes, more in particular neutrophils, basophils, monocytes, or lymphocytes, as well as red blood cells or erythrocytes.
  • the current methodology is compatible with various sorts of body fluid samples, including blood, serum, saliva and urine.
  • body fluid samples including blood, serum, saliva and urine.
  • serum instead of whole blood allows the sample to be stored and analyzed later.
  • urine or saliva instead of blood represents a truly non-invasive sample type and allows for e.g. home- testing and shipment of the sample to the test lab. This is obviously an additional advantage compared to other sample obtaining methods such as drawing blood.
  • the sample can also be obtained from venous blood collected in the normal way. In the case of whole blood, it can be collected with an anticoagulant present in order to prevent the normal clotting process. It may also be used immediately, for example, a small amount of blood can be diluted with assay buffer.
  • Sport coaches and sport scientists working with elite or professional athletes are challenged to find a balance between the hard training required to optimize performance and the introduction of sufficient resting and recovery periods necessary for the prevention of physiological maladaptation and the occurrence of fatigue syndromes.
  • These syndromes are likely to occur in high performance or elite level athletes because these athletes are driven towards achieving success in competitions at world class level and, as such, may ignore early warning signs of overtraining in the pursuit of their competitive goal.
  • the method according to any of the previous embodiments is therefore especially well- suited when the individual is an athlete or a sportsperson trained in one or more sports including, but not limited to, archery, badminton, baseball, basketball, bowling, canoeing, car racing, cheerleading, climbing, combat sports such as aikido, jujutsu, judo, wrestling, boxing, capoeira, karate, kickboxing, taekwondo and fencing, cricket, cycling, dancing, decathlon, discus throwing, diving, football, golf, gymnastics, hammer throwing, handball, heptathlon, high jumping, hiking, hockey, horse racing, ice hockey, ice skating, kayaking, kitesurfing, long jumping, military, motorcycle racing, mountaineering, parachuting, paragliding, polo, racketlon, racquetball, rafting, rope jumping, rowing, rugby, running, sailing, skateboarding, skating, skiing, snowboarding, soccer, squash, surfing, swimming, table tennis
  • the individual is a runner, a rower, a cycler, a swimmer, a triathlon athlete or a soccer player.
  • the current method allows sport coaches and sport scientists to objectively monitor the fatigue status of the athlete, thus introducing an objective criterion on which they can rely for developing/adapting the athlete's training program in order to achieve an optimal balance between training intensity and recovery.
  • information provided by the athlete or employee such as his or her training or work scheme
  • the measurement of biometric variables e.g. hearth rhythm
  • indicators derived from self-reported questionnaires are used to identify time-points or moments when the probability to develop a fatigue condition is increased.
  • This information is subsequently used to prompt the individual to use the in vitro method for detecting the onset and/or the existence of a fatigue condition according to the current invention.
  • the method of the current invention can be used to detect the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in a human or in a non-human mammal.
  • the method of the invention thus, for the first time, enables care-takers to objectively detect and diagnose depression disorder in animals, avoiding the need for complicated medical tests in order to exclude other medical reasons and allowing for an adequate therapy to be prescribed.
  • the method of the invention allows screening of employees on a regular basis, therefore providing a method to objectively monitor the chronic stress levels and the fatigue status of the employees.
  • chronic stress and fatigue puts the employees at high risk for the development of burn-out.
  • the annual global cost of burn- out is huge.
  • burn-out has a significant impact on the individual himself, especially affecting social aspects of his personal life.
  • work load can be adapted in time, resulting in huge benefits, both for the employee and the company. Therefore, in a further embodiment, the method according to any of the previous embodiments, is especially well-suited when the individual is an employee.
  • the invention provides a method for screening a group of individuals via the in vitro method according to any one of the previous embodiments.
  • the group of individuals are employees. Screening of employees using the method according to the invention can avoid huge economical losses caused by continued pressure/performance of undiagnosed employees suffering from burn-out syndrome or depression disorder. Finally, preventing the development of more serious forms of the syndrome or disorder owing to early diagnosis can have a profound positive effect on the individual's emotional/cognitive health in addition to his physical health.
  • a group of individuals, as used in the context of the current invention refers to more than one single individual.
  • the group of individuals is a team of sportspersons such as, but not limited to, a hockey team, a soccer team, a football team, a basketball team, a rowing team, a swimming team or a relay team.
  • the method allows to screen for the most fatigued team member and thus allows the person responsible of assembling the team, to adapt the training on an individual base or to avoid including this weak link in the team when the team ought to perform at its best, for example during a competition.
  • the individuals in the group of individuals can be mammals such as, but not limited to, dogs or horses.
  • the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising a means to carry out a method as described above.
  • the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising a sample collection and/or storage means and an assay to determine the status of one or more, preferably two or more, biomarkers in the sample, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from the markers as discussed above.
  • said one or more, preferably two or more, biomarkers are selected from Table 1 or Table 2, and optionally in combination with those from Table 3. Further possible combinations are mentioned above.
  • kits allows individuals to monitor their fatigue status in a simple and efficient manner.
  • the kit can be used by the individual himself or by a different individual (e.g. coach, trainer, employer, friends) to provide the individual with information about his current health condition, which enables them to make informed decisions relating to for example training load or workload.
  • Detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit provided by the invention constitutes an easy to use and rapid mode of detection. Since the kit contains all components needed for detection, there is no need for additional instrumentation.
  • the kit generates an outcome value which is submitted in an online tool such as, but not limited to, a website or a mobile application, to generate or calculate a prognostic value for the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual.
  • the online tool allows the data to be stored and to be consulted at different time points.
  • the diagnostic kit directly provides a prognostic value without the need of an external online tool and/or calculation.
  • sample collection means refers to a device to collect a body fluid sample from an individual, and includes invasive sample collection means (e.g. a needle to withdraw blood), home-collection kits (finger prick or small disposable device to simplify blood sampling) as well as non-invasive sample collection means (e.g. a tube to collect saliva).
  • invasive sample collection means e.g. a needle to withdraw blood
  • home-collection kits finger prick or small disposable device to simplify blood sampling
  • non-invasive sample collection means e.g. a tube to collect saliva.
  • Non-invasive sampling includes for example, but is not limited to, respiratory secretions, saliva, buccal swabs, oral fluid, urine, feces, sweat, tears, sputum, ear flow and/or smears.
  • Invasive sample types include but are not restricted to whole blood (optionally diluted), a blood fraction, isolated blood cells, serum, plasma, secretions of the intestinal and/or genitourinary tracts, lymph fluid and/or tissue
  • sample storage means refers to a device especially designed to store samples for diagnostic assays in a way as to maintain the informative status of the biomarkers to be measured in the sample as long as required, which in this case is until the status of one or more specific biomarkers in that sample have been determined.
  • the sample collection and/or storage means of the diagnostic kit comprises a sample inlet site for introduction of a body fluid sample of the individual.
  • the kit comprises a sample collection means that is easy to operate and does not require special training in order to be able to perform sample collection using the kit.
  • the main advantage of this type of sample collection means is that it enables self or assisted sample collection thereby avoiding the need to consult a physician and considerably increasing the user friendliness of the diagnostic kit.
  • the diagnostic kit provided by the invention allows at-home diagnostic testing which may provide an individual with valuable information and enable him to seek medical intervention earlier, preventing further medical complications.
  • the sample is chosen from whole blood, a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory, intestinal and genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy.
  • detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit is concluded from the status of at least one, preferably at least two, more preferably at least three biomarkers selected from the markers as listed in Table 1 or 2.
  • detection of the onset and/or existence of non- functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit is concluded from the status of at least two biomarkers selected from Table 1 or 2, optionally in combination with one or more markers from Table 3.
  • the inventors have found that combining the status of two or three biomarkers leads to an increase in accuracy and sensitivity of the detection of onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn- out and/or depression in an individual.
  • the inventors have additionally found that the status of biomarkers belonging to the groups listed above are the most informative for drawing conclusions on the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression. This allows to infer an unambiguous conclusion related to the onset and/or the existence of said fatigue conditions in an individual based on the determination of the status of individual biomarkers which may not be informative on their own.
  • the diagnostic kit according to the invention is provided as a collection of such kits which is suited for the screening of a group of individuals, preferably of a group of employees.
  • the term collection as used herein refers to a quantity of at least two. This kit thus enables for example the employer with a fast, easy and objective means to monitor the fatigue status of its employees.
  • the invention provides an assay to determine the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of an individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from the markers as discussed above.
  • said one or more biomarkers are selected from Table 1 or Table 2, and optionally in combination with those from Table 3. Further possible combinations are mentioned above.
  • the assay is portable.
  • the assay can be performed on a body fluid sample that can comprise whole blood, a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory/intestinal/genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy.
  • the assay is used to determine the status of more than one biomarker in the sample, preferably of at least two biomarkers and more preferably of at least of three biomarkers.
  • Example 1 Selection of biomarkers to detect onset and/or existence of fatigue syndromes
  • Biomarkers related to NFO/OTS/burn-out syndrome, CFS or depression disorder have been described in the past, however, they have often been tested only on a moderate number of patients or have often generated conflicting results and are therefore not considered reliable biomarkers for the detection of these conditions. Furthermore, up to now, researchers and medical doctors are mainly interpreting the biomarkers one by one. The inventors combine single biomarkers in a multidimensional approach and selected a number of factors to be tested as potential biomarkers for the onset and/or existence of the fatigue syndromes and / or disorders using standard sampling and assay methods in a clinically relevant setup. The markers were tested during a scientific experiment in a subpopulation of mostly recreational athletes with potential to develop NFO/OTS and/or burnout.
  • the experiment consisted of 300 endurance sports athletes who were monitored during the course of one training season.
  • a training season was subdivided in different phases starting with a maintenance phase, then a basic training phase, a development phase, a peak phase, a tapering phase and eventually a challenge phase.
  • a maintenance phase was subdivided in different phases starting with a maintenance phase, then a basic training phase, a development phase, a peak phase, a tapering phase and eventually a challenge phase.
  • the participants of the study also have a professional occupation and often care of their family.
  • the fatigue and stress status of the athletes was estimated during the course of the training season using three different indicators.
  • athletes reported on their psychological and physical mood on a daily basis by filling in a diary.
  • the intensity of the training session (if any) of that day was recorded by the patient as the rating of perceived exertion (RPE), measured according to the Borg RPE scale, which is known to the skilled person, together with the duration of the training.
  • RPE rating of perceived exertion
  • the psychological status of the athlete was recorded on a daily basis by answering 7 questions in the diary.
  • the athletes completed the "Profile of Mood States” (POMS) questionnaire on a weekly basis.
  • POMS is a simple psychological rating scale used to assess transient, distinct mood states.
  • the stress and recovery of the athletes was recorded at sampling points (see next paragraph) using the "Recovery- Stress Questionnaire for Athletes” (RESTQ-Sport) which allows the simultaneous assessment of stress and recovery of the athlete.
  • blood and saliva samples were analyzed at sampling points spread over the course of the training season.
  • two additional blood and saliva samples were taken : one before the introduction and one after termination of an additional recovery period. All samples were taken after a 36 hour rest period or a 36 hour period with light training. More than 100 biomarkers were analyzed in the blood and saliva samples.
  • the discriminative performance of each individual variable was quantified using the area under the receiver operating curve (AUC) and positive and negative predictive values (respectively PPV and NPV).
  • AUC area under the receiver operating curve
  • PPV and NPV positive and negative predictive values
  • PLS-DA Partial Least Squares - Discriminant Analysis
  • Models were selected corresponding to models that have the best statistically significant prognostic performance for the outcome within the subgroup studied.
  • Table 4 AUC statistical analysis of univariate versus multivariate (LAS, MAS) approach
  • biomarkers biosignatures
  • the inventors found a unique and surprising correlation between the statuses of several of the biomarkers (biosignatures) tested and the onset and/or existence of fatigue syndromes such as, but not limited to NFO, OTS, burn-out, CFS or depression disorder.
  • fatigue syndromes such as, but not limited to NFO, OTS, burn-out, CFS or depression disorder.
  • a different BIRIX score could be calculated based on the measures of a specific set of biomarkers (biosignatures), which is specific for gender and age.
  • a load adaptation score (LAS), which explains how an individual reacts upon physical training stress and provides a risk index for the onset of NFO/overtraining (example 2); the mental adaptation score (MAS), which defines how an individual reacts upon mental stressors, such as work environment (example 3); and training adaptation score (TAS), which defines how team athletes are coping with the external training load taking into account their personal life, their individual characteristics and the groups and organizational pressure (example 4).
  • LAS load adaptation score
  • MAS mental adaptation score
  • TAS training adaptation score
  • biomarkers included in this dataset are disclosed in Tables 1-3 and/or groups 1-4.
  • biomarker panels or combinations of biomarkers whose status can be used to detect the onset and/or the existence of fatigue syndromes including NFO, OTS, burn-out syndrome, CFS and depression disorder.
  • the panels of biomarkers are combined in specific BIRIX formulae. An example of the formulae is provided below. Nevertheless, other variants of the formulae combining different biomarkers or biosignatures are possible. sqrt((f(a * f ( ⁇ og[Biomarkerl ] 10, time ) + b * f( ⁇ og[Biomarker2 ] 10, age ) + g
  • Example 2 Detecting the onset of overtraining syndrome in an individual by measuring the load adaptation score
  • the fatigue status of a recreational athlete was monitored using a method according to an embodiment of the invention, more precisely the LAS (load adaptation score).
  • the prognostic value of the LAS pointed towards an increased risk for non-functional overreaching. Due to the result of the test the athlete, the medical doctor, coach and athlete agreed to lower the amount of training volume for 1.5 week. After accomplishing a deadline at work, the stress level of the athlete was reduced, and the training intensity was subsequently increased again. They agreed that indeed the current work load combined with the training was too intensive. The results of the next follow-up sample 1 month later showed a lowered LAS score together with a better recovery of the athlete. Thanks to the biomarker analysis the development of overtraining was prevented and the athlete managed to perform at his personal best during the Iron Man.
  • Example 3 Screening of employees for the onset of burnout syndrome by measuring the mental adaptation score
  • a group of 35 employees at a firm was monitored during 12 months using a method according to an embodiment of the invention, more precisely the MAS, in order to detect the onset and/or existence of fatigue syndromes, particularly of burn-out.
  • the fatigue status of the employees was measured every three months by analyzing saliva and/or serum samples of each individual. Furthermore a brief two- weekly questionnaire was provided to assure close follow-up and to enable introducing extra sampling points when needed.
  • the human resources department of the firm which was responsible for the screening received overviews of the objective MAS-scores in the different departments to identify those departments where extra coaching is needed.
  • the MAS profile of an employee was shown.
  • the prognostic value of the test pointed towards an increased risk for fatigue syndromes such as burn-out in one of the employees.
  • This employee was notified and he contacted his medical doctor for a more elaborated diagnosis (e.g. mental burnout score).
  • the medical doctor decided that the results of these test pointed towards an increase wrought-out of the employee.
  • the employee's medical doctor and the human resources department decided to reorganize the tasks of the employee, such as more structured deadlines and clear communication with his supervisor. This resulted in a decrease in the stress pattern of the employee and after one month, the normal workload was restored.
  • the results of the next sampling also pointed towards the recovery of the employee.
  • Example 4 follow up of the training adaptation in a professional soccer team
  • the training efficiency in team sport athletes was monitored using a method according to an embodiment of the invention, more precisely the TAS (training adaptation score).
  • TAS training adaptation score
  • team sports training sessions are often in groups, hardly differentiated depending on the training capacity and adaptation possibilities of the athlete. Nevertheless, none of the athletes in a team are the same. They all have a different mental and physical load during games and their home situations are often different as some of them are living apart from their family in a foreign country, while others have a partner and children to look after.
  • a pro-league soccer team was monitored during one season. Every 6 weeks the TAS of all team players was determined by analyzing their blood and saliva samples. An overview of the BIRIX was provided to the physical coach who was able to personalize the intensive training sessions to reach the maximal potential of every player.
  • defender 2 showed an increase in the TAS score.
  • a mental questionnaire which was filled at the sampling time showed an increase in fatigue and loss of concentration.
  • the sport psychologist further discussed the answers on the questionnaire with the athlete. It became clear that the care of a sick child had a huge demand of the player. The nights were not as they used to be, resulting in a build-up of sleep shortage. The player did not notify the physical coach and trainer as he was afraid to be excluded from the starting team. The medical staff discussed this case and decided to give the defender extra credits to miss the early morning sessions. His personal feedback moments with the coach/medical doctor/psychologist were rescheduled, allowing the player to be more relaxed in the morning. By this small interference, the athlete was more relaxed and his fatigue feeling was restored, resulting in a better concentration and performance at match days.
  • the pro league team was able to objectively identify those athletes that are in need of an individual approach. In this way, the athletes were able to perform at their personal best during the matches, resulting in very good results and gain against their most feared opponents.
  • Example 5 Evaluation of the value of the BIRIX for non-functional overreaching in the medical sport doctor's practice
  • biomarkers play an important role as they can be a disease predictor before the symptoms appear.
  • specific risk factors can be calculated (BIRIX) ( Figure 10).
  • BIRIX specific risk factors
  • a first measure of the BIRIX should be done upon the start of a health program. As most of the people have wearables or cell phones which registers many data, these data can also be incorporated in a health survey system that is patient or consumer centered. This additional information can help to adjust the personalized advices based on the BIRIX outcomes (eg. MAS). Employees at risk will receive personal follow up and practical actions using chat bots or private sessions with a psychologist or coach.
  • extra MAS analysis can be performed to measure the impact of the actions taken by the employee. In case no improvement was detected, the employee was send to his medical doctor. In the future certain data coming from wearables will be more reliable and will be able to monitor changes in behavior. They will be used as monitoring system after the first BIRIX analysis. When certain alarms in those data appear, the employee can be notified to perform an extra MAS analysis. This enables a cost effective follow-up of the employee. As the employer is not aware of which employees are performing analysis, it is not in violation of the privacy legislation. Nevertheless the human resources department and prevention department receive overviews of the profiles subdivided in the different departments.

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Abstract

The current invention concerns an in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of one or more, preferably two or more,biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn- out and/or depression can be concluded from said biomarker status in said sample.

Description

METHOD FOR DETECTING FATIGUE SYNDROMES IN AN INDIVIDUAL
TECHNICAL FIELD
The invention pertains to the technical field of an in vitro method and/or a diagnostic kit for detecting the onset and/or the existence of fatigue syndromes or fatigue conditions such as non-functional overreaching, overtraining syndrome, burn-out, chronic stress, chronic fatigue and/or depression in an individual using biosignatures.
BACKGROUND
Fatigue conditions or fatigue syndromes such as burn-out, overtraining, chronic stress, chronic fatigue and depression disorders are becoming more common in our society. The occurrence of these syndromes is often linked to an evolution in lifestyle, where the pressure to perform has increased considerably in the last decades. In addition to the pressure that individuals experience at work, they are also faced with a pressure to perform outside the working environment. For example, many individuals put a lot of pressure on themselves to be a good partner or a good parent, to be a good friend, etc. On the other hand it is also becoming a trend to excel at personal hobbies further adding to the pressure individuals put on themselves.
The impact of fatigue syndromes in economic terms is huge. The global costs of each of these syndromes are estimated to exceed hundreds of billions of US dollars annually. In addition, these syndromes have a significant impact on the individual himself, especially affecting social aspects of his personal life. Fatigue syndromes are, in general, associated with irritability, disturbed sleep patterns, decreased appetite, changes in hormone levels, increased vulnerability to infections and general malaise.
In work environment, chronic stress and insufficient recovery can lead to physical and emotional exhaustion, resulting in increased absenteeism. This comes with a huge economic cost apart from the extra workload for the remaining employees. As prevention is better than cure, companies are integrating prevention programs and risk analysis in favor for the wellbeing of their employees. Most tools are based on questionnaires integrated in apps providing advice, nevertheless, the compliance to fill questionnaire completely on a regular base is low. In addition, most people susceptible for mental illness or malaise do not recognize the signals and symptoms by themselves, resulting in a wrong image based on questionnaires. Therefore there is a huge need for evidence based objective tools to prevent their employees to develop chronic fatigue symptoms such as burn out or depression and to measure the effect of wellness programs in companies.
In sport, intensified training results in performance changes such as short-term overreaching (functional overreaching), extreme overreaching (non-functional overreaching), or overtraining syndrome. Overtraining syndrome (OTS) is considered a stage of fatigue combined with maladaptation of various physiological mechanisms to the intensified training, resulting in chronic performance reduction. Athletes suffering from OTS present symptoms such as excessive fatigue, exhaustion, and decreased performance levels. Depending on the level of overtraining it can take years for the athlete to recover.
The onset stage of OTS is referred to as non-functional overreaching (NFO). Adequate rest during the onset stage of OTS (NFO), however, can prevent the development of OTS.
Detection of NFO and OTS has hitherto depended on monitoring the performance of the athlete and inferring fatigue, overreaching or overtraining on the basis of a deterioration in the athlete's performance. Using this technique, however, it is only possible to detect these conditions when the athlete has already passed his peak condition.
Furthermore, fatigue syndromes such as mental disorder and OTS also affect animals. Diagnosis thereof is almost impossible due to lack of verbal communication with the animal patient.
There is a need for a technique which can objectively determine the fatigue status (NFO, OTS, burn-out) of an individual. Due to the huge impact of these fatigue syndromes, there is a further need to diagnose the onset thereof in order to prevent development of more serious forms of the syndromes. Nowadays a lot of health programs try to prevent fatigue status, however as there is no real objective tool to measure the effect of these programs, the impact is unknown.
The invention thereto aims to provide a reliable and objective method for detecting the onset and / or existence of fatigue syndromes such as burn-out syndrome, non- functional overreaching, overtraining syndrome and chronic fatigue syndrome in both humans and animals. This will help in the early detection and also in the follow-up measurements of an intervention. SUMMARY OF THE INVENTION
The present invention thereto provides a method according to Claim 1. More in particular, in a first aspect the invention provides an in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers (biosignature) and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker panel status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from Table 1.
A method according to an embodiment of the invention is particularly suitable to detect the onset and/or existence of overtraining syndrome, burn-out syndrome and/or depression disorder as it is based on accurate, objective and reproducible parameters, which have been established based on clinical data. Additionally, the method according to an embodiment of the invention provides an objective early screening procedure for these fatigue conditions.
In a second aspect, the present invention provides a screening method according to claim 10. More in particular, the invention provides a method for screening a group of individuals via the in vitro method according to embodiments of the previous method. The screening method of the invention can avoid huge economic losses caused by individuals suffering from undiagnosed fatigue syndromes present in, for example, a group of employees.
In a third aspect, the present invention provides a diagnostic kit according to claim 12. More in particular, the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising means to carry out a method according to any one of claims 1-11.
In a fourth aspect, the present invention provides a diagnostic kit according to claim 13. More in particular the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising a sample collection and/or storage means and an assay to determine the status of one or more, preferably two or more, biomarkers in the sample, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from Table 1.
A diagnostic kit according to the invention is particularly suitable to monitor the fatigue status of an individual in a simple, objective and efficient manner. It further constitutes an easy to use and rapid mode of detection, and eliminates the need for additional instrumentation.
In a fifth and final aspect, the present invention relates to an assay according to claim 19. More in particular, the invention provides an assay to determine the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of an individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from Table 1.
DESCRIPTION OF FIGURES
Figure 1 : Relation between the fatigue condition and the corresponding BIRIX.
Figure 2: Comparison of ROC curves of LAS and two commonly used univariate biomarkers.
Figure 3 : Boxplot representation of the LAS score in athletes.
Figure 4: LAS profile of an athlete during his training for an IM Figure 5: Boxplot representation of the MAS score.
Figure 6: MAS profile of an employee
Figure 7: Boxplot representation of the TAS score in team sport athletes.
Figure 8: Team overview of the TAS profiles of a pro-league soccer team Figure 9: Evaluation of the LAS in practice by medical sport doctors Figure 10: Implementation of the BIRIX in work environment
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns an in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn- out and/or depression can be concluded from said biomarker status in said sample.
Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the present invention.
As used herein, the following terms have the following meanings:
Overtraining syndrome (OTS) refers to a stage of fatigue combined with maladaptation of various physiological mechanisms to the intensified training that results in chronic performance reduction. OTS is also referred to by athletes as "staleness".
Non-functional overreaching (NFO) can develop as a consequence of intensified training and constitutes the earliest stage or onset of OTS. At this stage, adequate rest can prevent the development of OTS.
Burn-out syndrome or burn-out refers to the exhaustion of physical or emotional strength or motivation usually as a result of long-term stress or frustration. Burn-out syndrome is often characterized by a reduced self-esteem of the individual at its workplace. Most characteristics of burn-out syndrome are shared with overtraining syndrome. Additionally, burn-out syndrome often leads to the development of depression disorder. Chronic fatigue syndrome (CFS) or myalgic encephalomyelitis (ME) refers to a disorder characterized by long-term fatigue and extreme tiredness that do not go away with rest and result in the limitation of a person's ability to carry out ordinary daily activities.
The term "athlete" or "sportsperson" refers to a person that is skilled or trained in exercises, sports, or games requiring physical strength, agility, endurance and/or stamina. The term "professional athlete" or "elite athlete" refers to an athlete that receives payment for his or her performance. The term "recreational athlete", "amateur athlete" or "hobby" athlete refers to an athlete that trains during his or her spare time, mainly to relax. The term "sport" refers to a physical activity.
The term "employee" refers to a person who is handling commissioned by an "employer" or "firm". The job of the employee can be broad, going from physically active jobs, like but not limited to military training, cleaning managers or masons to people with a sedentary job like, but not limited to managers or manager assistants.
"Biomarker", "marker" or "biological marker" refers to a naturally occurring indicator such as a molecule, gene, metabolite, protein, cell type, vitamin, trace element, feature or characteristic which can be measured accurately, objectively and reproducibly, by which a particular pathological or physiological condition, such as but not limited to a process, disease, syndrome or disorder, can be identified. An example of a feature biomarker is the erythrocyte sedimentation rate. The status of a biomarker refers to the outcome of measurement of said biomarker and comprises the presence, absence and/or amount of said biomarker, wherein the "amount" of said biomarker includes changes in the amount of said biomarker.
The term "univariate" refers to a single variable, which can be related to just one biomarker, or the combination of two or more biomarkers, as for example a ratio of 2 biomarkers.
"Biosignature" signifies the result of measures of a set of biomarkers relevant for the early detection of disease and prediction of treatment effectiveness
The term "BIRIX" or "biomarker imbalance risk index" is based on the biosignature and describes the risk for a fatigue condition or syndrome. It reflects the attribute, characteristic or exposure of an individual that increases the likelihood of developing a disease or injury. The term "LAS" or "load adaptation score" reflects the BIRIX for athletes when they encounter external and internal training load.
The term "MAS" or "mental adaptation score" reflects the BIRIX of the mental adaption to external and internal stress factors.
The term "TAS" or "training adaptation score" reflects the BIRIX of team athletes towards their training sessions.
The term "sample" as used herein refers to any sample that is taken from the body of an individual (e.g., a human, such as an athlete or an animal, such as a horse) and contains one or more biomarkers.
Although the examples herein concern human individuals and the language is primarily directed to human concerns, the term "individual" as used herein refers to a human individual as well as a mammalian individual.
The term "condition" herein refers to "medical condition" as a broad term that includes all diseases and disorders, but can include injuries and normal health situations, such as pregnancy, that might affect a person's health, benefit from medical assistance, or have implications for medical treatments. The term "fatigue condition" or "fatigue syndrome" refers to one or more medical conditions chosen from the group of non- functional overreaching (NFO), overtraining syndrome (OTS), burn-out syndrome, chronic fatigue syndrome (CFS) , chronic stress and/or depression disorder.
Most testosterone found in an individual is bound to protein, with a large amount bound to sex hormone-binding globulin (SHBG) and a smaller amount bound weakly to albumin. The biomarker "free testosterone" refers to unbound testosterone. The biomarker "bio-available testosterone" refers to testosterone bound to albumin and free testosterone. The biomarker "total testosterone" refers to both bound and unbound testosterone.
Similarly, most of the cortisol found in an individual is bound to a protein, with a major amount bound to cortisol-binding globulin (CBG) and a smaller amount bound to albumin. The biomarker "free cortisol" refers to unbound cortisol. The biomarker "total cortisol" refers to both bound and unbound cortisol. "A", "an", and "the" as used herein refers to both singular and plural referents unless the context clearly dictates otherwise. By way of example, "a compartment" refers to one or more than one compartment.
"Comprise", "comprising", and "comprises" and "comprised of" as used herein are synonymous with "include", "including", "includes" or "contain", "containing", "contains" and are inclusive or open-ended terms that specifies the presence of what follows e.g. component and do not exclude or preclude the presence of additional, non-recited components, features, element, members, steps, known in the art or disclosed therein.
In a first aspect, the invention provides an in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn- out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from the group comprising ferritin, haptoglobin, hemoglobin, hemoglobin in reticulocytes, iron, transferrin, transferrin saturation, hematocrit, plasma viscosity, 3- hydroxyanthranilic acid, 3-hydroxyisobutyrate, 3-hydroxykynurenine, 4-pyridoxic acid, anandamide, anthranilic acid, beta-endorphins, calcitonin, cotinine, cystathionine, epidermal growth factor (EGF), flavin mononucleotide, free tryptophan, y-amuniobutyric acid (GABA), glutamate receptor, isoleucine, kynurenic acid (KYNA), leucin, L- kynurenine, Nl-methylnicotinamide, nicotinamide, nicotinic acid, picolinic acid, prolactin, pyridoxal, pyridoxal 5'-phosphate, pyridoxine, quinolinic acid (QUIN), riboflavin, S100 calcium-binding protein B (S100B), serotonin (5-hydroxytryptamine or 5-HT ), serotonin (5-hydroxytryptamine or 5-HT) receptor, thiamine, thiamine monophosphate, trans-3'-hydroxycotinine, trigonelline, tryptophan, valine, xanthurenic acid, brain-derived neurotrophic factor, neopterin, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-Ia), calcium, potassium, magnesium, sodium, soluble CD146, E-selectin, intracellular adhesion molecule (ICAM ) family members, vascular cell adhesion protein (VCAM ) family members, vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor-C (VEGF-C), albumin, alkaline phosphatase, creatinine, estimated glomerular filtration rate (GFR), folic acid, gamma-glutamyl transpeptidase, lipase, erythrocyte sedimentation rate, serum total protein, triglycerides , thyroid-stimulating hormone (TSH), glutamate, glutamine, glycogen, adrenocorticotropic hormone (ACTH), bio-available testosteron (TST), free cortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), growth hormone, sex hormone binding globulin, total cortisol , total testosterone, free testosterone , basophils, eosinophils, erythrocytes, leukocytes, lymphocytes, monocytes, neutrophils, natural killer cells (NK cells), macrophages, prostaglandin 2, reticulocytes, serum free light chains, secretory Immunoglobulin A (slgA), thrombocytes, C-reactive protein (CRP)„ elastase, eotaxin family members, granulocytes, IL-10, IL-15, ILl-beta, IL-lra, IL-2, IL-4, IL-6, IL-7, IL-8, INF-gamma, MCP-l/CCL-2, MMP-9, myeloperoxidase (MPO), NF-kB (nuclear factor kappa-light- chain-enhancer of activated B cells), osteonectin (SPARC), osteoprotegerin, serum amyloid A (SAA), soluble tumor necrosis factor alpha receptor type II, TNF-a, resistin, adiponectin, ghrelin, IGF-1, IGF-BP3, inhibin B, insulin, insulin resistance, leptin, visfatin, apolipoprotein CIII, urea, 3-methyl histidine, 3-nitrotyrosine, 4- hydroxynonenal (4-HNE), catalase, chromium, citrate synthase, co-enzyme Q10, cholecalciferol (VitD3), F2-isoprostane, free fatty acids , glutathione (GSH), glutathione disulfide (GSSG), glutathione peroxidase (GXP), glutathione reductase activity in erythrocytes, HSP70, HSP72, malondialdehyde, nitric oxide, oxipurin, protein carbonyls, reactive oxygen species (ROS), reactive nitrogen species(RNS), skeletal troponin I, superoxide dismutase (SOD), thiobarbituric acid-reactive substances (TBARS), total antioxidant capacity, uric acid, Vitamin A, Vitamin B12, Vitamin C, Vitamin D, Vitamin E, zinc, alpha-amylase, chromogranin A, catecholamines, angiotensin-converting- enzyme (ACE) activity, alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcineurin, creatine kinase, c-terminal agrin fragment (CAF), free DNA, lactate, lactate dehydrogenase (LDH), lipoperoxyl and alkoxyl , myoglobulin, a-acid glycoprotein, alphal antitrypsin and a-macroglobulin.
In contrast to previous methods used to diagnose the syndromes and disorders referred to in the current invention, which were often based on the individuals' perception of symptoms, the method of the invention is based on a combination of biomarkers, a biosignature, whose status can be accurately, objectively and reproducibly measured. As such, the methodology is objective.
Different types of biomarkers can be used in the method according to the invention, usually biomarkers are classified on their clinical applications and, in the case of the invention described herein, include but are not limited to:
Molecular biomarkers such as genomic biomarkers, transcriptomic biomarkers, proteomic biomarkers and metabolic biomarkers. Cellular biomarkers allow cells to be isolated, sorted, quantified and characterized by their morphology and physiology. Cellular biomarkers can discriminate between a large sample of cells based on their antigens
Feature or characteristics biomarkers include indicators with complex characteristics that can be influenced by multiple factors but whose status can be objectively, reproducibly and accurately determined. Characteristics biomarkers used in the method according to the current invention include, but are not limited to, for example the erythrocyte sedimentation rate.
Depending on their nature, biomarkers can be measured using various different methods known to the person skilled in the art. These methods include, but are not limited to, immunological assays, mass spectrometry-based methodologies and biochemical assays. Proteomic biomarkers and peptide hormones are in most cases measured using methods based on immunoassays or on mass spectrometry. Genetic biomarkers and transcriptomic biomarkers are in most cases measured using PCR-based methods or biochemical assays. Metabolic biomarkers are most often measured using biochemical assays and mass-spectrometry based methods. In addition, fluorescence- based assays - based on the detection of fluorescent dyes - are often used in the detection of all kinds of biomarkers and are especially suited for the detection of cellular biomarkers. It goes without saying that the list of methods given here is a non- exhaustive list of examples and that the method according to the invention may implement other assays, known to the skilled person, suited to measure the status of the biomarkers as currently disclosed.
The method of the invention provides an early screening procedure that may be carried out on a sample obtained from an individual, before the individual presents symptoms or before he is aware of any symptoms. The method thus allows for detection of the earliest stages of NFO/OTS, burn-out and/or depression, allowing an adaptation of the training, work-load or lifestyle to avoid the development of NFO/OTS, the worsening of NFO/OTS or the development of CFS or burn-out which could eventually result in depression. Eventually, early diagnosis of these syndromes/disorders results in a better standard of living as they can then be adequately treated before they have a more detrimental effect on the individual's health.
Another advantage of the method according to the invention is that early diagnosis in professional athletes can be used to prevent disappointing performance by introducing more recovery time in between trainings, resulting in a positive effect on the athlete's career. The method of the invention, therefore, provides the opportunity for athletes, and their coaches, to monitor training effects and performance such that early intervention may be made to the athletes' training schedules before the development of OTS and the serious and long term symptoms associated with it.
Furthermore, recreational athletes who often combine their job with intensive training also benefit from the current invention. The combination of training, work and social activities is very challenging, and involves a notable amount of stress which impacts both physical performance at sport as well as performance at work. The current invention allows medical doctors, training coaches, employers and athletes themselves to objectively monitor the resilience of the athletes, enabling them to interfere with the different stressors in time, therefore avoiding the possible negative impact of a failure to accomplish a sport challenge in a satisfying manner on their future sports performances, their mental health and their performance at work.
In addition to early diagnostic screening, the method according to the current invention also offers the advantage that reliable and accurate diagnosis of NFO/OTS, burn-out syndrome and/or depression disorder avoids the need of running a large number of other medical tests in order to exclude other causes. The method may be particularly useful in the screening of employees for e.g. the detection of the onset of burnout.
In one embodiment, the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual is concluded by determining the status of at least two biomarkers, preferably at least three biomarkers in a sample obtained from the body of the individual. The inventors have found that combining the status of two or three biomarkers leads to an unexpected increase in accuracy and sensitivity of the detection of onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual. This allows to infer an unambiguous conclusion related to the onset and/or the existence of said conditions in an individual based on the determination of the status of individual biomarkers which may not be informative on their own.
According to the current invention, one or more biomarkers are selected from the group comprising ferritin, haptoglobin, hemoglobin, hemoglobin in reticulocytes, iron, transferrin, transferrin saturation, hematocrit, plasma viscosity, 3-hydroxyanthranilic acid, 3-hydroxyisobutyrate, 3-hydroxykynurenine, 4-pyridoxic acid, anandamide, anthranilic acid, beta-endorphins, calcitonin, cotinine, cystathionine, epidermal growth factor (EGF), flavin mononucleotide, free tryptophan, g-amuniobutyric acid (GABA), glutamate receptor, isoleucine, kynurenic acid (KYNA), leucin, L-kynurenine, Nl- methylnicotinamide, nicotinamide, nicotinic acid, picolinic acid, prolactin, pyridoxal, pyridoxal 5'-phosphate, pyridoxine, quinolinic acid (QUIN), riboflavin, S100 calcium- binding protein B (S100B), serotonin (5-hydroxytryptamine or 5-HT ), serotonin (5- hydroxytryptamine or 5-HT) receptor, thiamine, thiamine monophosphate, trans-3'- hydroxycotinine, trigonelline, tryptophan, valine, xanthurenic acid, brain-derived neurotrophic factor, neopterin, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-Ia), calcium, potassium, magnesium, sodium, soluble CD146, E-selectin, intracellular adhesion molecule (ICAM ) family members, vascular cell adhesion protein (VCAM ) family members, vascular endothelial growth factor A (VEGF- A), vascular endothelial growth factor-C (VEGF-C), albumin, alkaline phosphatase, creatinine, estimated glomerular filtration rate (GFR), folic acid, gamma-glutamyl transpeptidase, lipase, erythrocyte sedimentation rate, serum total protein, triglycerides , thyroid-stimulating hormone (TSH), glutamate, glutamine, glycogen, adrenocorticotropic hormone (ACTH), bio-available testosteron (TST), free cortisol, dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA-S), growth hormone, sex hormone binding globulin, total cortisol , total testosterone, free testosterone , basophils, eosinophils, erythrocytes, leukocytes, lymphocytes, monocytes, neutrophils, natural killer cells (NK cells), macrophages, prostaglandin 2, reticulocytes, serum free light chains, secretory Immunoglobulin A (slgA), thrombocytes, C-reactive protein (CRP)„ elastase, eotaxin family members, granulocytes, IL-10, IL-15, ILl-beta, IL-lra, IL-2, IL-4, IL-6, IL-7, IL-8, INF-gamma, MCP-l/CCL-2, MMP-9, myeloperoxidase (MPO), NF-kB (nuclear factor kappa-light- chain-enhancer of activated B cells), osteonectin (SPARC), osteoprotegerin, serum amyloid A (SAA), soluble tumor necrosis factor alpha receptor type II, TNF-a, resistin, adiponectin, ghrelin, IGF-1, IGF-BP3, inhibin B, insulin, insulin resistance, leptin, visfatin, apolipoprotein CIII, urea, 3-methyl histidine, 3-nitrotyrosine, 4- hydroxynonenal (4-HNE), catalase, chromium, citrate synthase, co-enzyme Q10, cholecalciferol (VitD3), F2-isoprostane, free fatty acids , glutathione (GSH), glutathione disulfide (GSSG), glutathione peroxidase (GXP), glutathione reductase activity in erythrocytes, HSP70, HSP72, malondialdehyde, nitric oxide, oxipurin, protein carbonyls, reactive oxygen species (ROS), reactive nitrogen species(RNS), skeletal troponin I, superoxide dismutase (SOD), thiobarbituric acid-reactive substances (TBARS), total antioxidant capacity, uric acid, Vitamin A, Vitamin B12, Vitamin C, Vitamin D, Vitamin E, zinc, alpha-amylase, chromogranin A, catecholamines, angiotensin-converting- enzyme (ACE) activity, alanine aminotransferase (ALT), aspartate aminotransferase (AST), calcineurin, creatine kinase, c-terminal agrin fragment (CAF), free DNA, lactate, lactate dehydrogenase (LDH), lipoperoxyl and alkoxyl , myoglobulin, a-acid glycoprotein, alphal antitrypsin and a-macroglobulin. Preferably, at least one of said biomarkers is selected from the group consisting of 3-hydroxyanthranilic acid, 3- hydroxyisobutyrate, 3-hydroxykynurenine, 4-pyridoxic acid, adiponectin, alanine aminotransferase (ALT), albumin, alkaline phosphatase, alpha-amylase, anthranilic acid, aspartate aminotransferase (AST), basophils, bio-available testosteron (TST), calcium, cotinine, C-reactive protein (CRP), creatine kinase, creatinine, estimated glomerular filtration rate (GFR), cystathionine, dehydroepiandrosterone sulfate (DHEA- S), eosinophils, erythrocyte sedimentation rate, erythrocytes, ferritin, flavin mononucleotide, folic acid, free cortisol, free testosterone , gamma-glutamyl transpeptidase, growth hormone, haptoglobin, hematocrit, hemoglobin, hemoglobin in reticulocytes, IGF-1, IGF-BP3, IL-10, ILl-beta, IL-lra, IL-6, insulin, intracellular adhesion molecule (ICAM ) family members, iron, isoleucine, kynurenic acid (KYNA), lactate, lactate dehydrogenase (LDH), leptin, leucin, leukocytes, lipase, L-kynurenine, lymphocytes, magnesium, MCP-l/CCL-2, monocytes, myeloperoxidase (MPO), Nl- methylnicotinamide, neopterin, neutrophils, nicotinamide, nicotinic acid, picolinic acid, prolactin, pyridoxal, pyridoxal 5'-phosphate, pyridoxine, quinolinic acid (QUIN), resistin, reticulocytes, riboflavin, S100 calcium-binding protein B (S100B), secretory Immunoglobulin A (slgA), serum total protein, sex hormone binding globulin, sodium, thiamine, thiamine monophosphate, thrombocytes, thyroid-stimulating hormone (TSH), TNF-o, total cortisol , total testosterone, trans-3'-hydroxycotinine, transferrin, transferrin saturation, triglycerides , trigonelline, tryptophan, urea, valine, vascular endothelial growth factor A (VEGF-A), Vitamin B12, Vitamin D and xanthurenic acid. More preferably, at least one of said biomarkers is selected from the group consisting of 3-hydroxyanthranilic acid, alanine aminotransferase (ALT), alpha-amylase, anthranilic acid, total cortisol, free cortisol, cotinine, dehydroepiandrosterone sulfate (DHEA-S), ferritin, growth hormone, IGF-1, IGF-BP3, picolinic acid, quinolinic acid (QUIN), resistin, riboflavin, secretory Immunoglobulin A (slgA), total testosterone, free testosterone, bio-available testosterone, thiamine, TNF-a, triglycerides , vascular endothelial growth factor A (VEGF-A) and Vitamin D.
In a further or other embodiment, one or more, preferably two or more, biomarkers are selected from Table 1.
Table 1: list of relevant biomarkers
Figure imgf000014_0001
Figure imgf000015_0001
Figure imgf000016_0002
In a further or other embodiment, said one or more, preferably two or more, biomarkers are selected from Table 2. Table 2: list of key biomarkers
Figure imgf000016_0001
Figure imgf000016_0003
Figure imgf000017_0001
In a further embodiment, the status of at least two and even more preferably at least three of said biomarkers is determined, preferably markers chosen from Table 1 or Table 2. More by preference, the status of at least 4, even more by preference, at least 5 of said biomarkers is determined. Even more by preference, the status of at least 6, more by preference, at least 7 of said biomarkers is determined. Most by preference, the status of at least 8 to 10 of said biomarkers is determined.
In a preferred further or other embodiment, the method according to the present invention additionally comprises determining the status of one or more additional parameters selected from Table 3, in combination with at least one marker chosen from Table 1 or 2. In an embodiment, the markers of Table 3 will always be used in combination with at least one or more, such as two, markers from Table 1 or 2.
Table 3: additional parameters
Figure imgf000018_0001
In an embodiment, the status of at least two biomarkers of Table 1 or 2, and one marker of Table 3, is determined. In a further embodiment, the status of at least 2 biomarkers of Table 1 or 2, and at least two markers of Table 3, is determined. In a further or another embodiment, the status of at least 1 to 3 biomarkers of Table 1 or 2, and at least 1 to 3 markers of Table 3, is determined. By preference, the status of at least 1 to 4 biomarkers of Table 1 or 2, and at least 1 to 4 markers of Table 3, is determined. Even more by preference, the status of at least 1 to 5 biomarkers of Table 1 or 2, and at least 1 to 5 markers of Table 3, is determined. Most by preference, the status of at least 1 to 10 biomarkers of Table 1 or 2, and at least 1 to 10 markers of Table 3, is determined In an embodiment, the status of at least two biomarkers for determining the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual is concluded wherein at least one biomarker is selected from a first group of biomarkers consisting of group 1, group 2, group 3 or group 4, and at least one additional biomarker is selected from a second group of biomarkers consisting of group 1, group 2, group 3 or group 4 which is different from the first group of biomarkers, wherein group 1 consists of alanine aminotransferase (ALT), albumin, alkaline phosphatase, aspartate aminotransferase (AST), calcium, creatine kinase, creatinine, estimated glomerular filtration rate (GFR), erythrocyte sedimentation rate, ferritin, folic acid, gamma-glutamyl transpeptidase, haptoglobin, hematocrit, hemoglobin, hemoglobin in reticulocytes, iron, kynurenic acid (KYNA), lactate dehydrogenase (LDH), lipase, magnesium, serum total protein, sodium, thyroid-stimulating hormone (TSH), transferrin, transferrin saturation, triglycerides , urea, Vitamin B12 and Vitamin D; group 2 consists of 3-hydroxyanthranilic acid, 3- hydroxyisobutyrate, 3-hydroxykynurenine, 4-pyridoxic acid, anthranilic acid, cotinine, cystathionine, flavin mononucleotide, isoleucine, lactate, leucin, L-kynurenine, Nl- methylnicotinamide, nicotinamide, nicotinic acid, picolinic acid, pyridoxal, pyridoxal 5'- phosphate, pyridoxine, quinolinic acid (QUIN), riboflavin, S100 calcium-binding protein B (S100B), thiamine, thiamine monophosphate, trans-3'-hydroxycotinine, trigonelline, tryptophan, valine and xanthurenic acid; group 3 consists of adiponectin, basophils, C- reactive protein (CRP), eosinophils, erythrocytes, IL-10, ILl-beta, IL-lra, IL-6, intracellular adhesion molecule (ICAM ) family members, leukocytes, lymphocytes, MCP-l/CCL-2, monocytes, myeloperoxidase (MPO), neopterin, neutrophils, resistin, reticulocytes, thrombocytes, TNF-a and vascular endothelial growth factor A (VEGF-A); and group 4 consists of alpha-amylase, bio-available testosteron (TST), dehydroepiandrosterone sulfate (DHEA-S), free cortisol, free testosterone , growth hormone, IGF-1, IGF-BP3, insulin, leptin, prolactin, secretory Immunoglobulin A (slgA), sex hormone binding globulin, total cortisol and total testosterone. In a further embodiment, the status of at least one additional biomarker is determined which is selected from a third group of biomarkers consisting of group 1, group 2, group 3 or group 4, wherein the third group is different from the first and second group. In a further embodiment the status of at least one additional biomarker is determined which is selected from a fourth group of biomarkers consisting of group 1, group 2, group 3 or group 4, wherein the fourth group is different from the first, the second and the third group. Detection of at least two biomarkers belonging to different groups according to the grouping described above enriches the diagnostic performance of the biomarkers resulting in enhanced diagnostic accuracy. In an embodiment, an analysis is performed on a body fluid sample. Said body fluid sample is chosen from whole blood (optionally diluted), a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory, intestinal and genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy. Preferably, samples are obtained through collection of the fluid in a container (e.g. urine or saliva) or absorbing material or by inserting a needle into the body cavity and aspirating with a syringe a portion of the fluid (e.g. blood, lymph fluid). Isolated blood cells include white blood cells such as leukocytes, more in particular neutrophils, basophils, monocytes, or lymphocytes, as well as red blood cells or erythrocytes.
As mentioned before, the current methodology is compatible with various sorts of body fluid samples, including blood, serum, saliva and urine. The use of serum instead of whole blood allows the sample to be stored and analyzed later. Using urine or saliva instead of blood represents a truly non-invasive sample type and allows for e.g. home- testing and shipment of the sample to the test lab. This is obviously an additional advantage compared to other sample obtaining methods such as drawing blood.
Only a small sample is required for detection in the method, such as provided from a pin prick device or lancet rendered to the individual in order to obtain capillary blood, or from a mouth swab to obtain saliva. However, the sample can also be obtained from venous blood collected in the normal way. In the case of whole blood, it can be collected with an anticoagulant present in order to prevent the normal clotting process. It may also be used immediately, for example, a small amount of blood can be diluted with assay buffer.
Recreational athletes are usually combining sportive goals with work pressure and/or the care of their family. In order to prevent the development or worsening of fatigue syndromes such as non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression, these recreational athletes need to establish a good balance between training, work, social activities and rest. This population would highly benefit from the current invention, as numerous recreational athletes are unaware of the cumulative impact of these different stress factors and the invention enables them to objectively monitor their fatigue status.
Sport coaches and sport scientists working with elite or professional athletes are challenged to find a balance between the hard training required to optimize performance and the introduction of sufficient resting and recovery periods necessary for the prevention of physiological maladaptation and the occurrence of fatigue syndromes. These syndromes are likely to occur in high performance or elite level athletes because these athletes are driven towards achieving success in competitions at world class level and, as such, may ignore early warning signs of overtraining in the pursuit of their competitive goal.
The method according to any of the previous embodiments, is therefore especially well- suited when the individual is an athlete or a sportsperson trained in one or more sports including, but not limited to, archery, badminton, baseball, basketball, bowling, canoeing, car racing, cheerleading, climbing, combat sports such as aikido, jujutsu, judo, wrestling, boxing, capoeira, karate, kickboxing, taekwondo and fencing, cricket, cycling, dancing, decathlon, discus throwing, diving, football, golf, gymnastics, hammer throwing, handball, heptathlon, high jumping, hiking, hockey, horse racing, ice hockey, ice skating, kayaking, kitesurfing, long jumping, military, motorcycle racing, mountaineering, parachuting, paragliding, polo, racketlon, racquetball, rafting, rope jumping, rowing, rugby, running, sailing, skateboarding, skating, skiing, snowboarding, soccer, squash, surfing, swimming, table tennis, tennis, triathlon, volleyball, wakeboarding, water polo, weightlifting, windsurfing and wood chopping. Preferably, the individual is a runner, a rower, a cycler, a swimmer, a triathlon athlete or a soccer player. The current method allows sport coaches and sport scientists to objectively monitor the fatigue status of the athlete, thus introducing an objective criterion on which they can rely for developing/adapting the athlete's training program in order to achieve an optimal balance between training intensity and recovery.
In a further embodiment, information provided by the athlete or employee such as his or her training or work scheme, the measurement of biometric variables (e.g. hearth rhythm) and/or indicators derived from self-reported questionnaires are used to identify time-points or moments when the probability to develop a fatigue condition is increased. This information is subsequently used to prompt the individual to use the in vitro method for detecting the onset and/or the existence of a fatigue condition according to the current invention. By introducing additional criteria to estimate when in vitro testing would be most meaningful, unnecessary continuous monitoring is avoided which could otherwise result in a needless build-up of costs.
It is also the case that certain non-human mammals that are exercised and trained to perform in racing competition events suffer from OTS. Examples of such non-human mammals include horses, dogs (including solo runners such as greyhounds and sleddogs such as huskies and Eurohounds), and camels. Also pet owners are well aware of mood changes in their companions, however, diagnosis of clinical depression is difficult since dogs and cats cannot talk and tell a physician about their feelings. Therefore, in another further embodiment, the method of the current invention can be used to detect the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in a human or in a non-human mammal. The method of the invention thus, for the first time, enables care-takers to objectively detect and diagnose depression disorder in animals, avoiding the need for complicated medical tests in order to exclude other medical reasons and allowing for an adequate therapy to be prescribed.
The method of the invention, allows screening of employees on a regular basis, therefore providing a method to objectively monitor the chronic stress levels and the fatigue status of the employees. As described earlier, chronic stress and fatigue puts the employees at high risk for the development of burn-out. The annual global cost of burn- out is huge. In addition, burn-out has a significant impact on the individual himself, especially affecting social aspects of his personal life. Thus, by detecting the early onset of burn-out symptoms using the method of the invention, work load can be adapted in time, resulting in huge benefits, both for the employee and the company. Therefore, in a further embodiment, the method according to any of the previous embodiments, is especially well-suited when the individual is an employee.
Accordingly, and in a second aspect, the invention provides a method for screening a group of individuals via the in vitro method according to any one of the previous embodiments. Preferably, the group of individuals are employees. Screening of employees using the method according to the invention can avoid huge economical losses caused by continued pressure/performance of undiagnosed employees suffering from burn-out syndrome or depression disorder. Finally, preventing the development of more serious forms of the syndrome or disorder owing to early diagnosis can have a profound positive effect on the individual's emotional/cognitive health in addition to his physical health. A group of individuals, as used in the context of the current invention, refers to more than one single individual.
In another further embodiment, the group of individuals is a team of sportspersons such as, but not limited to, a hockey team, a soccer team, a football team, a basketball team, a rowing team, a swimming team or a relay team. The method allows to screen for the most fatigued team member and thus allows the person responsible of assembling the team, to adapt the training on an individual base or to avoid including this weak link in the team when the team ought to perform at its best, for example during a competition. Similarly, in another further embodiment, the individuals in the group of individuals can be mammals such as, but not limited to, dogs or horses.
In a third aspect the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising a means to carry out a method as described above.
In a fourth aspect, the invention provides a diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising a sample collection and/or storage means and an assay to determine the status of one or more, preferably two or more, biomarkers in the sample, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from the markers as discussed above.
In particular, said one or more, preferably two or more, biomarkers are selected from Table 1 or Table 2, and optionally in combination with those from Table 3. Further possible combinations are mentioned above.
Integration of the method of the invention into a suitable kit, allows individuals to monitor their fatigue status in a simple and efficient manner. The kit can be used by the individual himself or by a different individual (e.g. coach, trainer, employer, friends) to provide the individual with information about his current health condition, which enables them to make informed decisions relating to for example training load or workload. Detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit provided by the invention, constitutes an easy to use and rapid mode of detection. Since the kit contains all components needed for detection, there is no need for additional instrumentation.
In one embodiment of the diagnostic kits described above, the kit generates an outcome value which is submitted in an online tool such as, but not limited to, a website or a mobile application, to generate or calculate a prognostic value for the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual. The online tool allows the data to be stored and to be consulted at different time points. In another embodiment, the diagnostic kit directly provides a prognostic value without the need of an external online tool and/or calculation.
A "sample collection means" refers to a device to collect a body fluid sample from an individual, and includes invasive sample collection means (e.g. a needle to withdraw blood), home-collection kits (finger prick or small disposable device to simplify blood sampling) as well as non-invasive sample collection means (e.g. a tube to collect saliva). Non-invasive sampling includes for example, but is not limited to, respiratory secretions, saliva, buccal swabs, oral fluid, urine, feces, sweat, tears, sputum, ear flow and/or smears. Invasive sample types include but are not restricted to whole blood (optionally diluted), a blood fraction, isolated blood cells, serum, plasma, secretions of the intestinal and/or genitourinary tracts, lymph fluid and/or tissue biopsy.
A "sample storage means" refers to a device especially designed to store samples for diagnostic assays in a way as to maintain the informative status of the biomarkers to be measured in the sample as long as required, which in this case is until the status of one or more specific biomarkers in that sample have been determined.
In a further preferred embodiment, the sample collection and/or storage means of the diagnostic kit comprises a sample inlet site for introduction of a body fluid sample of the individual. In another preferred embodiment, the kit comprises a sample collection means that is easy to operate and does not require special training in order to be able to perform sample collection using the kit. The main advantage of this type of sample collection means is that it enables self or assisted sample collection thereby avoiding the need to consult a physician and considerably increasing the user friendliness of the diagnostic kit. The diagnostic kit provided by the invention allows at-home diagnostic testing which may provide an individual with valuable information and enable him to seek medical intervention earlier, preventing further medical complications.
In another further preferred embodiment of the invention, the sample is chosen from whole blood, a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory, intestinal and genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy.
In an embodiment, detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit is concluded from the status of at least one, preferably at least two, more preferably at least three biomarkers selected from the markers as listed in Table 1 or 2.
In a further or other embodiment, detection of the onset and/or existence of non- functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit is concluded from the status of at least two biomarkers selected from Table 1 or 2, optionally in combination with one or more markers from Table 3.
The inventors have found that combining the status of two or three biomarkers leads to an increase in accuracy and sensitivity of the detection of onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn- out and/or depression in an individual. The inventors have additionally found that the status of biomarkers belonging to the groups listed above are the most informative for drawing conclusions on the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression. This allows to infer an unambiguous conclusion related to the onset and/or the existence of said fatigue conditions in an individual based on the determination of the status of individual biomarkers which may not be informative on their own.
In a further embodiment, the diagnostic kit according to the invention is provided as a collection of such kits which is suited for the screening of a group of individuals, preferably of a group of employees. The term collection as used herein refers to a quantity of at least two. This kit thus enables for example the employer with a fast, easy and objective means to monitor the fatigue status of its employees.
In a fifth and last aspect, the invention provides an assay to determine the status of one or more, preferably two or more, biomarkers in a sample obtained from the body of an individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said one or more, preferably two or more, biomarkers are selected from the markers as discussed above. In particular, said one or more biomarkers are selected from Table 1 or Table 2, and optionally in combination with those from Table 3. Further possible combinations are mentioned above.
In a preferred embodiment the assay is portable. In another preferred embodiment the assay can be performed on a body fluid sample that can comprise whole blood, a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory/intestinal/genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy.
The inventors have found that combining the status of two or three biomarkers leads to an unexpected increase in sensitivity allowing to infer a conclusion related to the onset and/or the existence of said conditions in an individual based on the determination of the status of individual biomarkers which may not be informative on their own. Therefore, in a further preferred embodiment, the assay is used to determine the status of more than one biomarker in the sample, preferably of at least two biomarkers and more preferably of at least of three biomarkers.
The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended to, nor should they be interpreted to, limit the scope of the invention.
EXAMPLES
Example 1 : Selection of biomarkers to detect onset and/or existence of fatigue syndromes
Biomarkers related to NFO/OTS/burn-out syndrome, CFS or depression disorder have been described in the past, however, they have often been tested only on a moderate number of patients or have often generated conflicting results and are therefore not considered reliable biomarkers for the detection of these conditions. Furthermore, up to now, researchers and medical doctors are mainly interpreting the biomarkers one by one. The inventors combine single biomarkers in a multidimensional approach and selected a number of factors to be tested as potential biomarkers for the onset and/or existence of the fatigue syndromes and / or disorders using standard sampling and assay methods in a clinically relevant setup. The markers were tested during a scientific experiment in a subpopulation of mostly recreational athletes with potential to develop NFO/OTS and/or burnout.
The experiment consisted of 300 endurance sports athletes who were monitored during the course of one training season. A training season was subdivided in different phases starting with a maintenance phase, then a basic training phase, a development phase, a peak phase, a tapering phase and eventually a challenge phase. Apart from their sport activites the participants of the study also have a professional occupation and often care of their family.
The fatigue and stress status of the athletes was estimated during the course of the training season using three different indicators. First, athletes reported on their psychological and physical mood on a daily basis by filling in a diary. In this diary, the intensity of the training session (if any) of that day was recorded by the patient as the rating of perceived exertion (RPE), measured according to the Borg RPE scale, which is known to the skilled person, together with the duration of the training. Furthermore the psychological status of the athlete was recorded on a daily basis by answering 7 questions in the diary. Second, the athletes completed the "Profile of Mood States" (POMS) questionnaire on a weekly basis. POMS is a simple psychological rating scale used to assess transient, distinct mood states. Finally, the stress and recovery of the athletes was recorded at sampling points (see next paragraph) using the "Recovery- Stress Questionnaire for Athletes" (RESTQ-Sport) which allows the simultaneous assessment of stress and recovery of the athlete.
Additionally, four blood and saliva samples were analyzed at sampling points spread over the course of the training season. Upon indication of the onset or occurrence of non-functional overreaching based on the information provided in the diary and POMS, two additional blood and saliva samples were taken : one before the introduction and one after termination of an additional recovery period. All samples were taken after a 36 hour rest period or a 36 hour period with light training. More than 100 biomarkers were analyzed in the blood and saliva samples.
When all the information was combined, it resulted in a unique dataset, which was never before available. All data was subjected to extensive quality control to assure the correctness of the data.
To identify the participants with increased risk for NFO/overtraining/burn out/depression, the answers on the different questionnaires were interpreted as described in literature or with help from experts in the field. The samples were subdivided in 3 subgroups: a prediction, verification and validation group, to select those biosignatures that showed confident trends in all groups.
Firstly, the discriminative performance of each individual variable was quantified using the area under the receiver operating curve (AUC) and positive and negative predictive values (respectively PPV and NPV). An AUC of 0.5 or lower indicates an absence of predictive power for the outcome.
Secondly, Partial Least Squares - Discriminant Analysis (PLS-DA) models were generated for all combinations of biomarkers and possible clinical predictors as identified earlier.
The following statistical methodology was applied :
1. The concentrations and relative concentrations of analytes were log-transformed before modelling.
2. PLS-DA multivariable models were made by selecting variables as follows:
- 1 to A/p predictors.
- All possible combinations of variables.
- At most Nc clinical variables.
3. A three-fold cross validation was performed stratifying over the outcomes.
4. Models were additionally trained on the entire dataset.
5. Models were selected corresponding to models that have the best statistically significant prognostic performance for the outcome within the subgroup studied.
The statistical analysis described above showed that none of the univariate biomarkers showed sufficient power to discriminate between people with low or high risk for fatigue syndromes (AUC <0.656; Table 4).
Table 4: AUC statistical analysis of univariate versus multivariate (LAS, MAS) approach
AUC
Reduced
Risk outcome NFO/OTS mental
fitness
Leukocytes 0,548 0,532
secretory I 0,525 0,520
Iron 0,606 0,624
Transferrin s
Figure imgf000028_0001
0,558
Figure imgf000028_0002
0,636 Transferrin saturation 0,574 0,557
Ferritin 0,527 0,560
creatine kinase 0,520 0,509
Prolactin 0,527 0,656
Cortisol 0,555 0,513
DHEA-S 0,585 0,617
Testosteron 0,555 0,601
DHEA-S/cortisol 0,582 0,510
Testosteron/Cortisol 0,563 0,533
LAS 0,855 0,817
MAS
Figure imgf000029_0001
0,634
Figure imgf000029_0002
0,856
Using the statistical method as described, the best performing combinations of biomarkers (biosignatures) were selected to calculate the BIRIX.
Strikingly, the inventors found a unique and surprising correlation between the statuses of several of the biomarkers (biosignatures) tested and the onset and/or existence of fatigue syndromes such as, but not limited to NFO, OTS, burn-out, CFS or depression disorder. Depending on the outcome of the fatigue syndrome (e.g. overtraining, burnout, depression) a different BIRIX score could be calculated based on the measures of a specific set of biomarkers (biosignatures), which is specific for gender and age. As an example of the usefulness of the BIRIX, we provide 3 more examples: a load adaptation score (LAS), which explains how an individual reacts upon physical training stress and provides a risk index for the onset of NFO/overtraining (example 2); the mental adaptation score (MAS), which defines how an individual reacts upon mental stressors, such as work environment (example 3); and training adaptation score (TAS), which defines how team athletes are coping with the external training load taking into account their personal life, their individual characteristics and the groups and organizational pressure (example 4).
The biomarkers included in this dataset are disclosed in Tables 1-3 and/or groups 1-4.
Based on this dataset, the inventors have thus identified several biomarker panels or combinations of biomarkers (biosignatures) whose status can be used to detect the onset and/or the existence of fatigue syndromes including NFO, OTS, burn-out syndrome, CFS and depression disorder. The panels of biomarkers are combined in specific BIRIX formulae. An example of the formulae is provided below. Nevertheless, other variants of the formulae combining different biomarkers or biosignatures are possible. sqrt((f(a * f ( \og[Biomarkerl ] 10, time ) + b * f(\og[Biomarker2 ] 10, age ) + g
* f(\og[Biomarker3] 10 , age), gender)— s)2)
+ ((/( * / (\og[Biomarker4 / BiomarkerS] 10, age * time) + e
* f(\og[Biomarker6] 10, age))2))— t)2) The BIRIX were evaluated using ROC-curves. Therefore an AUC of at least 0.80 was accepted, with a sensitivity and specificity above 80%. In Figure 1, the ROC curve of the LAS score is compared to the ROC curves of two biomarkers that are currently used in practice to identify the risk and/or existence of NFO/overtraining. As is shown, the performance of the LAS is significantly (p<0.001) better compared to the univariate interpretation (Table 5).
Table 5: comparison of ROC curves of LAS and two commonly used univariate biomarkers
Figure imgf000030_0001
Example 2: Detecting the onset of overtraining syndrome in an individual by measuring the load adaptation score
The fatigue status of a recreational athlete was monitored using a method according to an embodiment of the invention, more precisely the LAS (load adaptation score).
In Figure 2 the relevance of the selected biomarkers and biomarker combinations (biosignature) is illustrated where, according to an embodiment of the in vitro method of the invention, the status of two or more of these biomarkers was determined in 165 samples. From the boxplot in Figure 3 it is clear that samples of individuals where no onset nor existence of a fatigue condition was detected ("0" in Figure 3) could be discriminated with a high negative predictive value form the samples obtained from individuals suffering from non-functional overreaching ("1" in Figure 3). In Figure 4, the profile of the LAS of an athlete during the training towards an Iron Man is shown. At fixed time points during his training period, saliva and serum samples were taken from the athlete and analyzed. At a regular base, a brief questionnaire was provided and the training sessions were monitored using wearables, to assure close follow-up and specific advice by the medical team during the training.
At the third fixed sampling point (8 weeks before the Iron Man), the prognostic value of the LAS pointed towards an increased risk for non-functional overreaching. Due to the result of the test the athlete, the medical doctor, coach and athlete agreed to lower the amount of training volume for 1.5 week. After accomplishing a deadline at work, the stress level of the athlete was reduced, and the training intensity was subsequently increased again. They agreed that indeed the current work load combined with the training was too intensive. The results of the next follow-up sample 1 month later showed a lowered LAS score together with a better recovery of the athlete. Thanks to the biomarker analysis the development of overtraining was prevented and the athlete managed to perform at his personal best during the Iron Man.
It is supposed that the present invention is not restricted to any form of realization described previously and that some modifications can be added to the presented example without reappraisal of the appended claims. For example, the present invention has been described referring to NFO/OTS, but it is clear that the invention can be applied to other fatigue syndromes.
Example 3: Screening of employees for the onset of burnout syndrome by measuring the mental adaptation score
A group of 35 employees at a firm was monitored during 12 months using a method according to an embodiment of the invention, more precisely the MAS, in order to detect the onset and/or existence of fatigue syndromes, particularly of burn-out.
In Figure 5 the boxplots of the MAS, the status of two or more of these biomarkers (biosignatures) as an embodiment of the in vitro method of the invention based on measurements in 92 samples, is shown. It is clear that the people with an increased stress/fatigue level as a possible precursor of burn out or depression ("1" in Figure 5) have a higher MAS score compared to the people where no onset or existence of a fatigue condition was detected ("0" in Figure 5).
More in detail, the fatigue status of the employees was measured every three months by analyzing saliva and/or serum samples of each individual. Furthermore a brief two- weekly questionnaire was provided to assure close follow-up and to enable introducing extra sampling points when needed.
The human resources department of the firm, which was responsible for the screening received overviews of the objective MAS-scores in the different departments to identify those departments where extra coaching is needed.
Furthermore, all employees received their personal MAS together with some practical advice. In case a high risk was detected, the employee received private sessions with a psychologist/mental coach. Extra blood and saliva samples were taken for the follow up of this employee.
In Figure 6, the MAS profile of an employee was shown. At the third sampling point (during the third quarter), the prognostic value of the test pointed towards an increased risk for fatigue syndromes such as burn-out in one of the employees. This employee was notified and he contacted his medical doctor for a more elaborated diagnosis (e.g. mental burnout score). The medical doctor decided that the results of these test pointed towards an increase wrought-out of the employee. The employee's medical doctor and the human resources department decided to reorganize the tasks of the employee, such as more structured deadlines and clear communication with his supervisor. This resulted in a decrease in the stress pattern of the employee and after one month, the normal workload was restored. The results of the next sampling (during the fourth quarter) also pointed towards the recovery of the employee. Thanks to the screening method, the development of burn-out in this employee was prevented and the employee was spared of a persistent fatigue condition that would otherwise have had a negative effect both on work-related aspects and on his personal life. Furthermore the costs associated with the absenteeism of the employee was avoided.
It is supposed that the present invention is not restricted to any form of realization described previously and that some modifications can be added to the presented example without reappraisal of the appended claims. For example, the present invention has been described referring to burn-out, but it is clear that the invention can be applied for instance to chronic fatigue syndrome or to depression disorder.
Example 4: Follow up of the training adaptation in a professional soccer team
The training efficiency in team sport athletes was monitored using a method according to an embodiment of the invention, more precisely the TAS (training adaptation score). In team sports training sessions are often in groups, hardly differentiated depending on the training capacity and adaptation possibilities of the athlete. Nevertheless, none of the athletes in a team are the same. They all have a different mental and physical load during games and their home situations are often different as some of them are living apart from their family in a foreign country, while others have a partner and children to look after.
In Figure 7 the relevancy of the selected biomarkers and biomarker combinations (biosignature) is illustrated where, according to an embodiment of the in vitro method of the invention, the status of two or more of these biomarkers (biosignatures) was determined in 100 samples. From the boxplot in Figure 7 it is clear that samples of individuals that benefit from the group training sessions ("0" in Figure 7) could be discriminated form the samples obtained from individuals suffering from fatigue and maladaptation towards the training sessions ("1" in Figure 7).
A pro-league soccer team was monitored during one season. Every 6 weeks the TAS of all team players was determined by analyzing their blood and saliva samples. An overview of the BIRIX was provided to the physical coach who was able to personalize the intensive training sessions to reach the maximal potential of every player.
At the second sampling point, at the beginning of the competition period one of the strikers (striker 1 in Figure 8) was flagged in the TAS. After profound interaction with the striker, it appeared that he did not respect the off-season rest-period. As he had a severe injury at the end of previous season he was convinced that he needed to have extra training sessions to reach his previous potential. Nevertheless, the lack of a rest period combined with an intensive training camp, resulted in the overload of the athlete. As a result the striker was not able to perform at his maximal capacity during sufficient time during the game. A more personalized training schedule was set up allowing the player to recover, which was also visualized in the following TAS scores.
At the 4th sampling point defender 2 showed an increase in the TAS score. A mental questionnaire which was filled at the sampling time showed an increase in fatigue and loss of concentration. The sport psychologist further discussed the answers on the questionnaire with the athlete. It became clear that the care of a sick child had a huge demand of the player. The nights were not as they used to be, resulting in a build-up of sleep shortage. The player did not notify the physical coach and trainer as he was afraid to be excluded from the starting team. The medical staff discussed this case and decided to give the defender extra credits to miss the early morning sessions. His personal feedback moments with the coach/medical doctor/psychologist were rescheduled, allowing the player to be more relaxed in the morning. By this small interference, the athlete was more relaxed and his fatigue feeling was restored, resulting in a better concentration and performance at match days.
By using the TAS analyses in the follow up of the athletes, the pro league team was able to objectively identify those athletes that are in need of an individual approach. In this way, the athletes were able to perform at their personal best during the matches, resulting in very good results and gain against their most feared opponents.
It is supposed that the present invention is not restricted to any form of realization described previously and that some modifications can be added to the presented example without reappraisal of the appended claims. For example, the present invention has been described for a soccer team, but it is clear that the invention can be applied for instance for other team sports like volleyball, handball, hockey...
Example 5: Evaluation of the value of the BIRIX for non-functional overreaching in the medical sport doctor's practice
As evaluation of the value of the Load adaptation score (LAS; BIRIX for NFO) in practice, medical sport doctors were asked to give a score for non-functional overreaching for the endurance athletes based on their insights and experience. The LAS was compared with the score given by the doctor. In half of the cases (52%) the LAS corresponded well with the evaluation of the medical doctor. In quite a lot of cases (32%) the LAS was higher than expected and in 16% of the cases was lower than expected (Figure 9).
This is not surprising as the doctors already indicated that they have no good tools to detect NFO and all had their own way of diagnosing NFO. After reporting the scores, a second interaction with the medical sport doctors was planned. In most cases (85%) the LAS score enabled them to interact with the athletes and to agree upon a new training strategy. In the other cases (15%) there was either no follow-up of the athlete or there was a skeptical attitude towards the new test (BIRIX). In 41% of the cases, the doctor would not have been able to identify the people at risk using their standard follow up methods, like lactate thresholds, anamnesis, questionnaires, etc.
Example 6: Implementation of score in work environment
The trend in medicine is to go towards prevention of diseases. For this, biomarkers play an important role as they can be a disease predictor before the symptoms appear. Based on the measures of biomarkers specific risk factors can be calculated (BIRIX) (Figure 10). A first measure of the BIRIX should be done upon the start of a health program. As most of the people have wearables or cell phones which registers many data, these data can also be incorporated in a health survey system that is patient or consumer centered. This additional information can help to adjust the personalized advices based on the BIRIX outcomes (eg. MAS). Employees at risk will receive personal follow up and practical actions using chat bots or private sessions with a psychologist or coach. During the follow up period, extra MAS analysis can be performed to measure the impact of the actions taken by the employee. In case no improvement was detected, the employee was send to his medical doctor. In the future certain data coming from wearables will be more reliable and will be able to monitor changes in behavior. They will be used as monitoring system after the first BIRIX analysis. When certain alarms in those data appear, the employee can be notified to perform an extra MAS analysis. This enables a cost effective follow-up of the employee. As the employer is not aware of which employees are performing analysis, it is not in violation of the privacy legislation. Nevertheless the human resources department and prevention department receive overviews of the profiles subdivided in the different departments.
It is supposed that the present invention is not restricted to any form of realization described previously and that some modifications can be added to the presented example without reappraisal of the appended claims. For example, the present invention has been described referring to stress monitoring, but it is clear that the invention can be applied to other fatigue syndromes.

Claims

1. An in vitro method for detecting the onset and/or the existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual, the method comprising determining the status of two or more biomarkers in a sample obtained from the body of the individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said two or more biomarkers are selected from Table 1 or Table 2.
2. The method according to the previous claim, characterized in that, said two or more biomarkers are chosen from Table 2.
3. The method according to any of the previous claims, characterized in that, said method comprises determining the status of three or more biomarkers in a sample obtained from the body of the individual, wherein said three or more biomarkers are selected from Table 1 or 2.
4. The method according to any of the previous claims, characterized in that, said method additionally comprises determining the status of one or more additional parameters selected from Table 3.
5. The method according to any one of the previous claims, characterized in that said method comprises determining the status of four or more biomarkers in a sample obtained from the body of the individual, wherein said four or more biomarkers are selected from Table 1 or 2.
6. The method according to any one of the previous claims, characterized in that said body fluid sample is chosen from whole blood, a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory, intestinal and genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy.
7. The method according to any one of the previous claims, characterized in that said individual is a human or non-human mammal.
8. The method according to any one of the previous claims, characterized in that said individual is an athlete, preferably a triathlon athlete, a cyclist, a runner, a rower or a soccer player.
9. The method according to any one of the previous claims, characterized in that said individual is an employee.
10. A method for screening a group of individuals via the in vitro method according to any one of the previous claims.
11. The method according to claim 10, characterized in that the individuals are employees.
12. A diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising means to carry out a method according to any one of previous claims 1-9.
13. A diagnostic kit for use in the in vitro detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual comprising :
- a sample collection and/or storage means and
- an assay to determine the status of two or more biomarkers in the sample, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said two or more biomarkers are selected from Table 1 or Table 2.
14. The diagnostic kit according to previous claim 13, characterized in that said two or more biomarkers are selected from Table 2.
15. The diagnostic kit according to any one of the previous claims 13-14, characterized in that, detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit is concluded from the status of at least three biomarkers selected from Table 1 or 2.
16. The diagnostic kit according to any one of the previous claims 13-15, characterized in that, detection of the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual using the diagnostic kit is concluded from the status of one or more additional parameters selected from Table 3.
17. The diagnostic kit according to any one of the previous claims 13-16, characterized in that said sample is chosen from whole blood, a blood fraction, isolated blood cells, serum, plasma, secretions of the respiratory, intestinal and genitourinary tracts, saliva, oral fluid, urine, feces, sweat, tears, sputum, ear flow, lymph fluid, swabs, smears and/or a tissue biopsy.
18. The diagnostic kit according to any one of the previous claims 13-17, characterized in that the status of at least four biomarkers is used to determine the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression in an individual.
19. An assay to determine the status of two or more biomarkers in a sample obtained from the body of an individual, wherein said status relates to the presence, absence and/or the amount of said biomarkers and wherein the onset and/or existence of non-functional overreaching, chronic fatigue syndrome, overtraining syndrome, burn-out and/or depression can be concluded from said biomarker status in said sample characterized in that said two or more biomarkers are selected from Table 1.
20. The assay according to previous claim 19, characterized in that at least two of said biomarkers are selected from Table 2.
21. The assay according to any one of the previous claims 19-20, characterized in that, said assay allows to determine the status of at least three biomarkers selected from Table 1 or 2.
22. The assay according to any one of the previous claims 19-21, characterized in that, said assay additionally allows to determine the status of one or more additional parameters selected from Table 3.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3783362A3 (en) * 2019-08-22 2021-06-09 Laboratorium M. Nuytinck Biosignatures for chronic mental stress
WO2023052261A1 (en) * 2021-09-28 2023-04-06 Société des Produits Nestlé S.A. Method for detecting and/or quantifying mood disorder and/or improvements of the mood disorder status using trigonelline as biomarker and improved methods and compositions thereof.
WO2023160800A1 (en) * 2022-02-24 2023-08-31 Laboratorium M. Nuytinck Biosignatures for fatigue syndromes
SE2230095A1 (en) * 2022-03-29 2023-09-30 Filip Larsen Method of preventing overtraining, or determining the appropriate amount of exercise

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050249699A1 (en) * 2004-05-05 2005-11-10 Stoff Jesse A Immunodynamic complexes and methods for using and preparing such complexes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB201005561D0 (en) * 2010-04-01 2010-05-19 Cambridge Entpr Ltd Biomarkers
GB201321474D0 (en) * 2013-12-05 2014-01-22 Cambridge Entpr Ltd Novel biomarkers
EP2995310B1 (en) * 2014-09-12 2021-11-03 SHI Research & Development AB Compositions for the treatment of overtraining syndrome

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050249699A1 (en) * 2004-05-05 2005-11-10 Stoff Jesse A Immunodynamic complexes and methods for using and preparing such complexes

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
A.COUTTS ET AL: "The usefulness of biochemical markers for early diagnosis of overreaching in rugby league players", JOURNAL OF SCIENCE AND MEDICINE IN S, SPORTS MEDICINE AUSTRALIA, BELCONNEN, AU, vol. 5, no. 4, 1 December 2002 (2002-12-01), pages 6, XP005355811, ISSN: 1440-2440 *
ANGST GREN H ET AL: "Patterns of depression reflected in pituitary - thyroid and pituitary - adrenal endocrine changes", PSYCHONEUROENDOCRINOLOGY, OXFORD, GB, vol. 7, no. 4, 1 January 1982 (1982-01-01), pages 309 - 327, XP024386456, ISSN: 0306-4530, [retrieved on 19820101], DOI: 10.1016/0306-4530(82)90035-X *
MICHAEL GLEESON: "Biochemical and immunological markers of over-training", JOURNAL OF SPORTS SCIENCE & MEDICINE, 1 June 2002 (2002-06-01), Turkey, pages 31 - 41, XP055202228, Retrieved from the Internet <URL:http://www.ncbi.nlm.nih.gov/pubmed/24688268> [retrieved on 20180605] *
PURVIS D ET AL: "Physiological and Psychological Fatigue in Extreme Conditions: Overtraining and Elite Athletes", PM&R, ELSEVIER, AMSTERDAM, NL, vol. 2, no. 5, 1 May 2010 (2010-05-01), pages 442 - 450, XP027065999, ISSN: 1934-1482, [retrieved on 20100501], DOI: 10.1016/J.PMRJ.2010.03.025 *
URHAUSEN A ET AL: "BLOOD HORMONES AS MARKERS OF TRAINING STRESS AND OVERTRAINING", SPORTS MEDI, ADIS PRESS, AUCKLAND, NZ, vol. 20, no. 4, 1 October 1995 (1995-10-01), pages 251 - 276, XP009067878, ISSN: 0112-1642 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3783362A3 (en) * 2019-08-22 2021-06-09 Laboratorium M. Nuytinck Biosignatures for chronic mental stress
WO2023052261A1 (en) * 2021-09-28 2023-04-06 Société des Produits Nestlé S.A. Method for detecting and/or quantifying mood disorder and/or improvements of the mood disorder status using trigonelline as biomarker and improved methods and compositions thereof.
WO2023160800A1 (en) * 2022-02-24 2023-08-31 Laboratorium M. Nuytinck Biosignatures for fatigue syndromes
SE2230095A1 (en) * 2022-03-29 2023-09-30 Filip Larsen Method of preventing overtraining, or determining the appropriate amount of exercise

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