Classifications

• • 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
  • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
  • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/52—Assays involving cytokines
  • G01N2333/54—Interleukins [IL]
  • G01N2333/55—IL-2
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/902—Oxidoreductases (1.)
  • G01N2333/906—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7)
  • G01N2333/90605—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4)
  • G01N2333/90611—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1) in general
  • G01N2333/90616—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1) in general with a definite EC number (1.4.1.-)

Definitions

• the present invention relates to a method for diagnosing or determining the status of tuberculosis infection in an individual afflicted with or suspected to be afflicted with tuberculosis infection.
• a method for the stratification of the therapeutic regimen of an individual with tuberculosis infection is provided as well as a method for predicting a clinical outcome or determining treatment course in an individual afflicted with tuberculosis infection.
• the present invention provides a method for monitoring the change from latent into active status of tuberculosis infection or vice versa in an individual.
• the present invention relates to a kit for use in diagnosing or detecting the status of tuberculosis infection as well as to Mycobacterium tuberculosis alanine dehydrogenase for use in specifically differentiating latent status from active diseases status of tuberculosis in an individual.
• IG RAs Mycobacterium tuberculosis specific interferon- ⁇ release assays
• IGRAs have a number of advantages: they are minimally influenced by previous Bacille Calmette-Guerin (BCG) vaccination or infection by non-tuberculosis mycobacteria, do not cause booster effect, do not necessitate a double access to health care facility , and interpretation of results is not operator-dependent (Chiappini E. et al., Clin Ther. 201 2 Apr 1 6).
• IG RAs In adults IG RAs have been reported to be more specific and at least as sensitive as TST and are currently included in diagnostic algorithms in adult guidelines (Mazurek GH. et al., United States, 201 0, MMWR Recomm Rep. 201 0 ; 59 :1 -25.). However, reported IG RAs sensitivity and specificity largely vary among studies in paediatric populations and caution is recommended regarding their use in children because of poor and contrasting data (Machingaidze S. et. al. , Pediatr Infect Dis J. 201 1 ;30 :694-700; Sun L. et. al., FEMS Immunol. Med. Microbiol.
• diagnoses in children are "probable "diseases, based on TST and IGRA results, clinical symptoms and signs, radiological findings, personal history, response to TB therapy, and physicians' experience in this field.
• a positive IG RA in a child with signs suggestive of active TB does not allow the definitive diagnosis especially if the child comes from a high TB prevalence area.
• the positive IG RA result may be due to TB disease as well as LTBI, which may be incidental to the disease causing the symptoms or signs under investigation. In this situation, a test that accurately distinguishes active tuberculosis from latent TB infection would be valuable.
• no success has been achieved in studies using ELISpot assays based on cytokines other than I FN-Y (i.e. :IL-1 0 or I L-2) (Amanatidou V. et. al. , Eur J. Clin Microbiol Infect Dis. 201 2 Jan 4) or exploring immune response to other mycobacterial antigens besides ESAT-6, CFP-1 0 and TB7.7 which are currently included in the commercially available IG RAs.
• the present invention aims for providing new methods and assays particularly useful in the issues described above.
• the present invention relates to a method for diagnosing or determining the status of tuberculosis infection in an individual afflicted with or suspected to be afflicted with tuberculosis infection, comprising : a) determining the level or amount of cytokines released or produced by mononuclear cells after stimulation, whereby said mononuclear cells are obtained from said individual ; and
• AlDH mycobacterial Alanine Dehydrogenase
• the present invention relates to a method for the stratification of the therapeutic regimen of an individual with tuberculosis infection comprising :
• mononuclear cells are stimulated in the presence of mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis.
• AlDH Alanine Dehydrogenase
• the present invention relates to a method for predicting a clinical outcome or determining the treatment course in an individual afflicted with tuberculosis infection comprising :
• the stimulation includes cultivation of said mononuclear cells with mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis.
• AlDH mycobacterial Alanine Dehydrogenase
• Another embodiment of the present invention relates to a method for monitoring the change from latent to active status of tuberculosis infection or vice versa in an individual comprising : a) determining the level or amount of cytokines released or produced from mononuclear cells from said individual after stimulation at a first point in time;
• step c) comparing the level or amount of cytokines determined in step a) to the level or amount determined in step b) or to a reference value whereby an increase in the level or the amount relative to a reference value or to the level or amount determined in step a) is indicative for a transition from latent to active status and a decrease in the level or the amount relative to a reference value or to the level or amount determined in step a) is indicative for a transition from active to latent status,
• the mononuclear cells are stimulated with mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis.
• AlDH mycobacterial Alanine Dehydrogenase
• the mycobacterial in particular, the Mycobacterium tuberculosis Alanine Dehydrogenase (AlaDH) represents a suitable stimulant for mononuclear cells obtained from the individuals afflicted with or suspected to be afflicted with tuberculosis infection and, subsequently, determining the amount or level of cytokines released or produced by said mononuclear cells after stimulation, thus, allowing to differentiate between latent and active status of tuberculosis infection in said individual, accordingly.
• AlaDH results in cytokine release or production in active TB state only while during latent TB state cytokine release or production is significantly lower. This is particularly true for the cytokine IL-2.
• the methods described herein are typically in vitro methods.
• the mononuclear cells are stemming from the individual to be tested. Said cells are obtained before from said individual whereby the step of taking a sample including the cells from the individual, e.g. taking a blood sample, is typically not part of the method according to the present invention.
• the methods as described herein include embodiments wherein whole blood samples are used for stimulation or where the mononuclear cells are isolated in advance from the whole blood sample. Unless otherwise indicated herein, the term ""monouclear cells" include the embodiment of whole blood samples. In a preferred
• the mononuclear cells are isolated mononuclear cells isolated e.g. from whole blood.
• the present invention relates to the use of a kit for diagnosing or determining the status of tuberculosis infection, or for predicting a clinical outcome or determining the treatment course in an individual afflicted with or suspected to be afflicted with tuberculosis infection, or for the stratification of the therapeutic regimen of an individual with tuberculosis infection or for monitoring the progression of tuberculosis infection, or for determining the transition from latent to active status of tuberculosis infection in an individual
• said kit comprises means for determining the level or amount of cytokines released or produced from mononuclear cells after stimulation, whereby said mononuclear cells are obtained from said individual, a stimulating agent which is mycobacterial, e.g. of Mycobacterium tuberculosis, AlaDH and instructions on how to use said test kit for a method according to the present invention.
• the present invention relates to the use of mycobacterial Alanine Dehydrogenase, in particular, of Mycobacterium tuberculosis, as a stimulant allowing to differentiate between latent and active status of
• tuberculosis infection in a individual afflicted with or suspected to be afflicted with tuberculosis infection.
• composition comprising mycobacterial AlaDH, in particular, of Mycobacterium tuberculosis, in combination ESAT-6 and CFP-1 0 is provided for use in determining tuberculosis infection in an individual, in particular for use as stimulants, e.g. in whole blood or in cell-based assays.
• Figure 1 Figure 2 shows ROC illustrating sensitivity and specificity of
• the present invention relates in a first aspect to a method for diagnosing or determining the status of tuberculosis infection in an individual afflicted with or suspected to be afflicted with tuberculosis infection, comprising :
• AlDH mycobacterial Alanine Dehydrogenase
• the present inventors recognised that depending on the level or amount of cytokines released or produced by mononuclear cells after stimulation whereby said mononuclear cells are obtained from the individual afflicted with or suspected to be effected with tuberculosis infection, it is possible to diagnose or determine the status of tuberculosis infection in said individual when nuclear cells are stimulated with mycobacterial Alanine Dehydrogenase, in particular, of Mycobacterium tuberculosis, for cytokine release or production.
• the assays and methods described herein allows to differentiate between the latent and active status of tuberculosis infection.
• the differentiation between the two different types of status is important for determining the treatment course and the risk of the course of tuberculosis infection as well as for predicting the clinical outcome.
• tuberculosis infection in said individual It has been shown that when using AlaDH as stimulant, the cytokine pattern released or produced from the mononuclear cells stimulated with the AlaDH molecule is different between individuals having latent TB infection and individuals having active TB infection. In contrast, the molecules of commercially available IG RAS allow to
• the term "individual" in the context of the present invention is typically a mammal, in particular, the mammal is a human, a non- human primate or other mammals susceptible for mycobacterial infection.
• the individual can be male or female.
• the individual can be one who has been previously diagnosed with or identified as suffering from tuberculosis infection and, optionally, but need not to have already undergone treatment for tuberculosis infection.
• An individual can also be one who has been diagnosed with or identified as suffering from tuberculosis infection but who has shown improvement in the disease as a result of receiving one or more treatments for the infection, accordingly.
• the individual may also be one who has not been previously diagnosed or identified as having tuberculosis infection.
• determining refers to assessing the presence, absence, quantity, level or amount of either a given substance within a clinical or subject derived sample, including quality and/or quantitative concentration levels of substances otherwise evaluating values or categorisation of a subject's clinical parameter.
• AlaDH comprises antigenic fragments or the whole polypeptide of AlaDH.
• the AlaDH is an antigenic oligopeptide or polypeptide derived from the peptide of Seq I D No. 1 .
• the mycobacterial AlaDH is preferably the ALaDH of TB with the NCBI- reference No. N P_21 7296.1 or the TubercuList database No. AlaDH-Rv2780.
• suitable fragments e.g. as described in EP 0966544 being incorporated herein by reference.
• the term “AlaDH” comprises antigenic fragments or the whole polypeptide of AlaDH.
• the AlaDH is an antigenic oligopeptide or polypeptide derived from the peptide of Seq I D No. 1 .
• the mycobacterial AlaDH is preferably the ALaDH of TB with the NCBI- reference No. N P_21 7296.1 or the TubercuList database No. AlaDH-Rv2780.
• the term “latent” includes the latent status of TB infection (LTBI) as well as the cured status after successful treatment unless otherwise indicated.
• the term "mononuclear cells obtained from ...an individual” refers to cells obtained before from said individual.
• the cells are stemming from the indivudal but the method according to the present invention typically does not include the sampling step. Rather, the cells are provided as a sample.
• the sample may be whole blood or a sample containing the cells after isolation steps from the sample from said individual.
• the present invention relates to method for the
• stratification of the therapeutic regimen of an individual with tuberculosis infection comprising :
• mononuclear cells are stimulated in the presence of mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis.
• AlDH Alanine Dehydrogenase
• AlaDH represents a valuable tool, namely, a valuable stimulant for whole blood samples, in particular, mononuclear cells obtained from an individual afflicted with or suspected to be afflicted with tuberculosis infection for diagnosing or
• a method for monitoring the change from latent to active status of tuberculosis infection or vice versa in an individual comprising : a) determining the level or amount of cytokines released or produced from mononuclear cells from said individual after stimulation at a first point in time;
• step b) determining the level or amount of cytokines released or produced from mononuclear cells after stimulation, whereby said mononuclear cells are obtained from said individual at a second point in time; and c) comparing the level or amount of cytokines determined in step a) to the level or amount determined in step b) or to a reference value whereby an increase in the level or the amount relative to a reference value or to the level or amount determined in step a) is indicative for a transition from latent to active status and an decrease in the level or the amount relative to a reference value or to the level or amount determined in step a) is indicative for a transition from active to latent status
• the mononuclear cells are stimulated with mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis.
• AlDH mycobacterial Alanine Dehydrogenase
• the term "reference value” refers to an index value, a value derived from one or more computer indices, a value derived from a subject with known active or latent tuberculosis infection, in particular, with a cohort of the same.
• the mononuclear cells are obtained from a sample of said individual.
• Said sample may be blood, a blood derived sample or other samples containing mononuclear cells suitable for allowing cytokine release assays or assays determining the amount of cytokines both on protein and nucleic acid level.
• the sample is a blood sample or is derived from other body fluids including Bronchoalveolar lavage and urine and tissues.
• the mononuclear cells may be obtained from blood or other body fluids and, optionally, are isolated by common techniques.
• AlaDH represents a suitable stimulant in a whole blood assay and in a cell-based assay for a method according to the present invention.
• the cytokine level or amount is determined by immuno based assays.
• immunoassays such as western blot, ELISA and ELIspot. It is particularly desired that the cytokines are determined at protein level with ELIspot or ELISA technique.
• the cytokine levels can also be measured by determining the mRNA levels corresponding to the said cytokines, accordingly.
• the skilled person is well aware of suitable methods for determining the same on nucleic acid level including PCR techniques.
• the cytokine determined is at least one selected from I L-2 or interferon- ⁇ . It is particularly desired that the IL-2 amount or level is determined released or produced by said mononuclear cells after stimulation with mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis.
• AlDH mycobacterial Alanine Dehydrogenase
• IL-2 ELIspot and/or I FN- ⁇ ELIspot based ELIspot assays allow to differentiate between latent and active status of tuberculosis infections in either children or adults.
• the reference value for the latent status of tuberculosis infection is below 25, preferably below 1 0, while the reference value for the active status of tuberculosis infection is above 50, preferably above 1 00, for I L-2 ELIspot.
• the first sample from the individual is preferably obtained at a first time point while the second sample obtained after a, preferably predetermined, time point, e.g. after undergoing treatment of tuberculosis infection or undergoing treatment for autoimmune diseases.
• a time point e.g. after undergoing treatment of tuberculosis infection or undergoing treatment for autoimmune diseases.
• latent status TB infection may proceed to active status. That is, the present invention allows to determine the treatment course in said individual or predicting the clinical outcome by identifying the actual status of tuberculosis infection in said individual.
• the method according to the present invention allows for identifying or monitoring or supervising individuals afflicted with tuberculosis infection to determine a change from latent to active status of tuberculosis or vice versa.
• the relative or absolute level or amount of cytokines increases, it is indicative for a transition from the latent to active status while a decrease in the relative or absolute level or amount is indicative for a transition from active to latent status.
• ELIspot or ELISA assays as stimulants allow to differentiate between TB infection and non TB infection only, e.g. the well known mycobacterial molecules ESAT-6, CFP-1 0 or other molecules.
• compositions comprising mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis, and ESAT-6 as well as CFP-1 0 allows to increase the sensitivity and specificity of tests allowing to differentiate between uninfected and infected individuals, in particular, tests wherein these components are used as stimulants. That is, the components of said composition may be used in separate assays or may be used in combination.
• AlDH mycobacterial Alanine Dehydrogenase
• ESAT-6 as well as CFP-1 0
• the present invention relates to the use of a kit for diagnosing or determining the status of tuberculosis infection, or for predicting a clinical outcome or determining the treatment course in an individual afflicted with or suspected to be afflicted with tuberculosis infection, or for the stratification of the therapeutic regiment of an individual with tuberculosis infection or for monitoring the progression of tuberculosis infection, or for determining the transition from latent to active status of tuberculosis infection in an individual
• said kit comprises means for determining the level or amount of cytokines released or produced from mononuclear cells after stimulation, whereby said mononuclear cells are obtained from said individual, a stimulating agent which is mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis, and instructions on how to use said test kit for a method according to the present invention.
• a stimulating agent which is mycobacterial Alanine Dehydrogenase (AlaDH), in particular
• said kit is an ELIspot or ELISA allowing for determining the level or amount of the cytokine. It is particular preferred that the ELIspot or ELISA is an assay allowing determination of the amount or level of IL-2 or interferon- ⁇ , in particular, I L-2.
• the kit is a PCR kit with means suitable for analysing cytokine expression on nucleic acid level.
• the present invention relates to the use of mycobacterial Alanine Dehydrogenase (AlaDH), in particular, of Mycobacterium tuberculosis, in differentiating latent status and active status of tuberculosis infection in an individual afflict with or suspected to be afflicted with tuberculosis infection.
• AlDH mycobacterial Alanine Dehydrogenase
• the mycobacteria Alanine Dehydrogenase is particularly useful for
• the present invention provides assays useful for performing or conducting the method according to the present invention. Said assay
• the assay comprises means for culturing mononuclear cells and AlaDH as stimulant in a form suitable to stimulate said mononuclear cells when added to a culture thereof.
• the assay may contain means for determining the amount or level of the cytokine to be detected.
• said assay is based on an immunoassay, in particular, said assay is an EliSpot or ELISA. Detection is effected by antibodies detecting specifically the desired cytokine.
• the assay is a PCR-based assay.
• Consecutive children at risk for TB infection referred to the Infectious Disease Unit, Department of Department of Sciences for Man and Child's Health University of Florence, Italy, were prospectively enrolled between 2009 and 201 0. Study children were those with clinical suspicion of TB disease and/or in close contact with recently diagnosed cases of contagious TB disease and/or internationally adopted or recently immigrated children coming from countries with a high-prevalence of TB. A maximum of two years was
• TST was administered according to the Mantoux method by injecting intradermally 5 tuberculin units (in 0.1 ml_) of purified protein derivative
• a positive TST was defined as an induration size > 5 mm for children in close contact with known or suspected contagious case of TB disease or for children suspected to have TB disease (based on clinical evidence and/or chest radiograph) and > 1 0 mm for children born in countries with a high prevalence of TB and recently immigrated.
• the QFT-IT test was performed according to the manufacturer's instructions as described previously (Millington KA, et. al. , Proc Natl Acad Sci USA.201 1 ;1 08 :5730-5. Vilaplana C, et. al., Scand J I mmunol 2008 ;67:61 0-7). After subtracting the value from the negative control, the result was positive if, the antigen-dependent response was >0.35 I U, negative if the mitogen-induced response was >0.5 l U/mL and the antigen-dependent response was ⁇ 0.35 l U/mL, and indeterminate if both mitogen-induced and antigen-dependent responses were below cut-off or mitogen-induced response >8 l U/mL.
• M. tuberculosis antigens. Briefly, T cell blasts (1 05 cells) of each patients line were stimulated with M. tuberculosis antigen in the presence of irradiated autologous APCs (5 ⁇ 1 04 cells) and seeded in triplicate in 96-well plates coated with anti-I FN- ⁇ or anti-I L-2 antibody. Cells stimulated with medium alone served as negative controls. Cells stimulated with PHA served as positive controls. I FN- ⁇ and I L-2 ELISPOT microplates were then incubated at 37 °C in 5% C02 for 24 hours.
• Cases of active TB were defined according to two categories: definite TB, children with Mycobacterium tuberculosis cultured or detected by microscopy or molecular methods from sputum or gastric aspirate culture, and probable TB: absence of microbiological confirmation but presence of all of the following criteria: (A) clinical symptoms and signs of active TB, (B) abnormal radiography and/or CT scan consistent with lung TB, (C) response to TB therapy plus, (D) either a history of TB contact or travel to a TB-endemic country within the last 24 months (Amanatidou V. et. al, see above).
• definite TB disease was made in 5 children. All these children presented with symptoms compatible with TB (e.g. persistent cough, fever, night sweats or weight loss) and/or pulmonary disease documented by chest X-ray and confirmed by CT scan results and Mycobacterium tuberculosis was detected from the cultures and/or microscopy and/or polymerase chain reaction. Probable TB was diagnosed in 20 children. All these children underwent at least three gastric aspirate or sputum examination for
• Table 1 shows the BCG vaccination status, the TST size and the QFT-G-IT result according to final diagnosis.
• Table 1 BCG vaccination status, TST size and QFT- G-IT result according to final diagnosis of the study children
• BCG Bacille Calmette-Guerin
• TST tuberculin skin test
• IGRA interferon- ⁇ release assay
• the antigens currently included in commercial available IG RAs like ESAT-6 and CFP-1 0 allows to differentiate between uninfected and infected individuals only.
• a mixture or composition of mycobacterial Mycobacterium tuberculosis AlaDH and ESAT-6 as well as CFP-1 0 allows to determine tuberculosis infection in an individual with higher sensitivity and specificity.
• sensitivity of discriminating a latent and active status reached 1 00 % while specificity was 96 % by the use of I L-2 based ELIspot assay.
• the performance of the interferon- ⁇ ELIspot assay using AlaDH antigen was not as good as the IL-2 based ELIspot assay but still allow to differentiate between the two different types of status.
• the methods and assay systems according to the present invention are particularly useful for discrimination of children with active from those with latent TB when using AlaDH antigen.

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