Biomarker for diagnosing nontuberculous mycobacteria-infected lung disease and determining treatment initiation time

Abstract

The present invention relates to a biomarker for diagnosing nontuberculous mycobacteria-infected lung disease and determining a treatment initiation time. In addition, the present invention relates to a method for diagnosing nontuberculous mycobacteria-infected lung disease and determining a treatment initiation time by using a chitinase 3-like 1 (CHI3L1) protein marker in blood. The present invention provides chitinase 3-like 1 (CHI3L1) as a biomarker for diagnosing nontuberculous mycobacteria-infected lung disease and determining a treatment initiation time. The biomarker of the present invention is remarkably useful in diagnosing nontuberculous mycobacteria-infected lung disease and determining a treatment initiation time, and thus is expected to be widely used in the field of health and medicine.

Description

Biomarkers for diagnosing and determining the timing of treatment initiation in nontuberculous mycobacterial lung disease

[0001] The present invention relates to a biomarker for diagnosing nontuberculous mycobacterial lung disease and determining the timing of treatment initiation. Furthermore, the present invention relates to a method for diagnosing nontuberculous mycobacterial lung disease and determining the timing of treatment initiation using the blood-borne CHI3L1 (Chitinase 3-Like 1) protein marker.

[0002] Mycobacterium species are divided into two groups according to the degree of pathogenicity and infectivity to the host: the tuberculosis group, which includes the obligate pathogenic bacteria Mycobacterium tuberculosis and Mycobacterium leprae, and the nontuberculous mycobacteria (NTM), which are opportunistic bacteria excluding these. The above nontuberculous mycobacteria are widely distributed in nature, and the virulence that causes diseases varies depending on the species. Specifically, M. avium complex (MAC), M. abscessus (MAB), and M. kansasii have relatively high virulence, while M. fortuitum has relatively low virulence. Diseases caused by nontuberculous mycobacteria are largely classified into four characteristic clinical syndromes: pulmonary disease, lymphadenitis, skin, soft tissue, and bone infection, and disseminated disease. Among these, pulmonary disease is the most common form, accounting for more than 90% of all diseases.

[0003] Reports of lung disease caused by nontuberculous mycobacteria (NTM) have been increasing since 2000, with MAC lung disease currently the most frequently reported in most developed countries. In Korea, MAC lung disease is reported to account for approximately 70-80% of all nontuberculous mycobacterial lung disease cases, with MAB being the second most common causative agent. NSM-induced lung disease presents with symptoms such as cough, fever, hemoptysis, and sputum production. Diagnosis of NSM-induced lung disease is primarily through sputum testing. If the same type of NTM is cultured more than twice from the sputum of a patient suspected of infection, accompanied by chest X-ray findings and symptoms (cough, sputum, etc.), a diagnosis of NSM-induced lung disease is made. However, because NTM-induced lung disease is commonly found in everyday environments and can be detected in healthy individuals, accurate assessment of actual infection is crucial. Furthermore, to be diagnosed as the causative agent, the same organism must be cultured repeatedly. This necessitates repeated cultures over several months, resulting in a significant delay in diagnosis. These limitations necessitate the development of clear biomarkers for the diagnosis of nontuberculous mycobacteria.

[0004] Furthermore, patients diagnosed with nontuberculous mycobacterial lung disease often do not receive immediate treatment. Because the disease is characterized by slow changes in chest X-ray findings and sometimes without a worsening of the disease, sufficient follow-up is necessary to assess the progression of the disease once nontuberculous mycobacteria are detected in sputum. Furthermore, patients with nontuberculous mycobacterial lung disease are predominantly elderly and require long-term treatment, typically lasting more than a year. Therefore, the timing of treatment initiation must be determined by considering the potential benefits to the patient along with the potential side effects of the drug. Therefore, there is a growing need for biomarkers that can accurately determine the timing of treatment initiation after diagnosis of nontuberculous mycobacterial lung disease.

[0005] Accordingly, the present invention, conceived in response to the aforementioned technical needs, relates to a biomarker for diagnosing nontuberculous mycobacterial lung disease and determining the timing of treatment initiation. The biomarker of the present invention is expected to be of significant use in the healthcare and medical fields, as it offers significant utility in diagnosing nontuberculous mycobacterial lung disease and determining the timing of treatment initiation.

[0006] [Reference] This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2024-00405542).

[0007] The present inventors have diligently researched to discover a biomarker for highly accurate diagnosis of patients infected with nontuberculous mycobacteria (NTM), particularly Mycobacterium avium complex (MAC), a very common cause of lung disease in humans, for which the development of objective and reliable diagnostic markers is difficult, and further for determining the timing of treatment initiation. As a result, the present invention was completed by discovering the CHI3L1 (Chitinase 3-Like 1) protein, a biomarker that can clearly distinguish between patients infected with nontuberculous mycobacteria and non-infected subjects and determine the timing of treatment initiation.

[0008] Accordingly, one object of the present invention is to provide a composition for diagnosing a nontuberculous mycobacterial infection disease, including a preparation capable of measuring the expression level of the CHI3L1 protein or a gene encoding the same, and a diagnostic method using the same.

[0009] Another object of the present invention is to provide a composition for determining the time point at which treatment is started for a nontuberculous mycobacterial infection, including a preparation capable of measuring the expression level of the CHI3L1 protein or a gene encoding the same, and a method for determining the time point at which treatment is started using the same.

[0010] The objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0011] Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification do not necessarily refer to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0012] Unless otherwise specifically defined in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0013] Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

[0014] According to one aspect of the present invention, the present invention provides a composition for diagnosing a nontuberculous mycobacterial infection disease, comprising a preparation capable of measuring the expression level of a CHI3L1 protein or a gene encoding the same.

[0015] Because nontuberculous mycobacteria are commonly found in everyday environments and can be detected in healthy individuals, accurate assessment of actual infection is crucial. Furthermore, diagnosing the causative agent requires repeated cultures of the same bacteria, necessitating repeated cultures over several months, resulting in a significant delay in diagnosis. These limitations necessitate the development of a clear biomarker for the diagnosis of nontuberculous mycobacteria.

[0016] In this specification, the term “nontuberculous mycobacteria” refers to acid-fast bacteria other than tuberculosis bacilli, and specifically includes all mycobacteria that do not cause tuberculosis or leprosy. Unlike general bacteria, acid-fast bacteria refer to strains that have the ability to withstand and not dissolve even when acid is added during the staining process. The representative example of these acid-fast bacteria is Mycobacterium tuberculosis, and acid-fast bacteria other than tuberculosis bacilli are called nontuberculous mycobacteria (NTM), and new nontuberculous mycobacteria are discovered almost every year. The nontuberculous mycobacteria are widely distributed in nature, and the virulence that causes diseases varies depending on the species. Specifically, Mycobacterium avium complex (MAC), Mycobacterium abscessus (MAB), and Mycobacterium kansasii have relatively high virulence, while Mycobacterium fortuitum has relatively low virulence. Diseases caused by nontuberculous mycobacteria are broadly classified into four characteristic clinical syndromes: pulmonary disease, lymphadenitis, skin, soft tissue, and bone infections, and disseminated disease. Among these, pulmonary disease is the most common form, accounting for more than 90% of all diseases. Pulmonary disease caused by the above nontuberculous mycobacteria is characterized by symptoms such as cough, fever, hemoptysis, and sputum. Reports of pulmonary disease caused by nontuberculous mycobacteria have been increasing since 2000, and pulmonary disease caused by MAC is currently the most frequently reported causative agent in most developed countries. In Korea, MAC pulmonary disease is reported to account for approximately 70-80% of all causative agents of nontuberculous mycobacterial pulmonary disease, with MAB being the second most common causative agent.

[0017] In the present invention, the nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense. The bacterium may be selected from the group consisting of Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium scrofulaceum, Mycobacterium ulcerans, Mycobacterium simiae, and Mycobacterium xenopi, and more preferably, Mycobacterium avium, but is not limited thereto.

[0018] In the present invention, the non-tuberculous mycobacterial infection disease may be a non-tuberculous mycobacterial infection in the lungs, lymph nodes, skin, cartilage, or bone, and more preferably, a non-tuberculous mycobacterial infection in the lungs, but is not limited thereto.

[0019] The term “CHI3L1 (Chitinase 3-Like 1)” herein, also known as YKL-40, is a secreted glycoprotein belonging to the chitinase-like proteins (CLPs). This protein has a size of 40 kDa and has conserved characteristics as a chitin-binding protein. The three-dimensional crystal structure of CHI3L1 shows a typical folding structure of chitinase family proteins, but chitinase activity is not expressed due to the lack of essential amino acid residues within the enzyme domain. CHI3L1 is expressed in various cells of lung tissue (e.g., neutrophils, monocytes / macrophages, monocyte-derived dendritic cells, epithelial cells) and is closely related to various biological activities such as tissue damage, inflammatory responses, and tissue repair. CHI3L1 is also associated with various diseases. For example, high levels of CHI3L1 protein and gene expression have been observed in patients suffering from various diseases, including asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, cancer, diabetes, and arteriosclerosis.

[0020] In the present invention, the CHI3L1 may have a slightly different base sequence depending on the species of the target organism being diagnosed with a nontuberculous mycobacterial infection, but is not limited as long as it can be recognized as a CHI3L1 protein by those skilled in the art to which the present invention pertains. Furthermore, as long as it is a gene encoding a protein recognizable as CHI3L1, there is no limitation on the specific base sequence. However, when a specific polypeptide base sequence or nucleic acid base sequence is required, those skilled in the art can collect and utilize information from known databases such as the National Center for Biotechnology Information (NCBI) in the United States.

[0021] The term “diagnosis” as used herein includes determining the susceptibility of a subject to a specific disease or condition, determining whether a subject currently has a specific disease or condition (e.g., nontuberculous mycobacterial infection), determining the prognosis of a subject with a specific disease or condition, or therametrics (e.g., monitoring the condition of a subject to provide information on the efficacy of treatment). Specifically, in the spirit of the present invention, the term “diagnosis” as used herein means determining whether a subject currently has a specific disease or condition.

[0022] In this specification, the term “diagnostic composition” refers to an integrated mixture or device including a measuring means capable of measuring the expression level of the CHI3L1 protein or the gene encoding it to determine whether or not a nontuberculous mycobacterial infection disease has occurred in a subject or to predict the possibility of onset, and may be used with the same meaning as “diagnostic kit.” Since the diagnostic composition of the present invention includes a means for measuring the biomarker discovered in the present invention, the term “diagnostic composition” may also be expressed as a “quantitation device” for the CHI3L1 protein or the gene encoding it as a target biomarker.

[0023] In the present invention, the preparation for measuring the expression level of the biomarker protein is not particularly limited, but may include, for example, one or more selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the protein.

[0024] In the present invention, the "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to the biomarker protein. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibodies can be readily produced using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art, including a process of injecting an antigen of the biomarker protein into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be produced from any animal, such as a goat, rabbit, sheep, monkey, horse, pig, cow, or dog. In addition, monoclonal antibodies can be produced using hybridoma methods or phage antibody library techniques well known in the art. Antibodies produced by the above methods can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. Furthermore, the antibody of the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of the antibody molecule. A functional fragment of an antibody molecule means a fragment that possesses at least an antigen-binding function, such as Fab, F(ab'), F(ab')2, and Fv.

[0025] The "PNA (Peptide Nucleic Acid)" in the present invention refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg, and Buchardt of the University of Copenhagen, Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases its binding affinity and stability to DNA or RNA, and is therefore used in molecular biology, diagnostic analysis, and antisense therapy.

[0026] In the present invention, the “aptamer” is an oligonucleic acid or peptide molecule.

[0027] In the present invention, a preparation for measuring the expression level of a gene encoding the biomarker protein may include at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to the gene encoding the biomarker protein.

[0028] In the present invention, the "primer" refers to a fragment that recognizes a target gene sequence, and includes a pair of forward and reverse primers, but is preferably a pair of primers that provide analysis results with specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, and thus only amplifies the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be imparted.

[0029] In the present invention, the "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample, and refers to a substance that can specifically confirm the presence of the target substance in the sample through the binding. The type of probe is not limited to a substance commonly used in the art, but is preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA. More specifically, the probe includes a biomaterial derived from or similar to a living organism or manufactured in vitro, and may be, for example, an enzyme, a protein, an antibody, a microorganism, animal or plant cells and organs, nerve cells, DNA, and RNA. DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, etc.

[0030] In the present invention, the term "LNA (Locked nucleic acids)" refers to a nucleic acid analog containing a 2'-O, 4'-C methylene bridge. LNA nucleosides contain common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rule. However, due to the molecule's 'locking' caused by the methylene bridge, LNA cannot form an ideal shape in Watson-Crick binding. When LNA is included in a DNA or RNA oligonucleotide, LNA can pair with a complementary nucleotide chain more quickly, thereby increasing the stability of the double helix.

[0031] In the present invention, the term "antisense" refers to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that allows the antisense oligomer to hybridize with a target sequence within RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or approximate sequence complementarity to the target sequence.

[0032] Since the biomarker protein according to the present invention or the information on the gene encoding the same is known, a person skilled in the art will be able to easily design a primer, probe, or antisense nucleotide that specifically binds to the gene encoding the protein based on this.

[0033] In the present invention, the expression level of the CHI3L1 protein or the gene encoding it is measured in whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, It may be measured in synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0034] According to another aspect of the present invention, the present invention provides a diagnostic kit for a nontuberculous mycobacterial infection, comprising a diagnostic composition for a nontuberculous mycobacterial infection, comprising a preparation capable of measuring the expression level of a CHI3L1 protein or a gene encoding the same.

[0035] In this specification, the term "diagnostic kit" may be used with the same meaning as the "diagnostic composition" described above. The definitions of the diagnostic composition of the present invention and CHI3L1 as a biomarker overlap with those described in the diagnostic composition of the present invention, and thus are omitted hereinafter to avoid excessive clutter in the specification.

[0036] In the present invention, the onset and possibility of onset of a nontuberculous mycobacterial infection can be diagnosed at an early stage using the above diagnostic kit.

[0037] In the present invention, the kit may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0038] The diagnostic kit of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the analysis method.

[0039] For example, the diagnostic kit of the present invention may further include essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes a pair of primers specific for a gene encoding a marker protein. The primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of a control gene. In addition, the reverse transcription polymerase reaction kit may include a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0040] Additionally, the diagnostic kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or fragment thereof are attached, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include cDNA or oligonucleotides corresponding to a control gene or fragment thereof.

[0041] Additionally, the diagnostic kit of the present invention may include essential components necessary for performing an ELISA. The ELISA kit includes an antibody specific for the protein. The antibody is an antibody with high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit may also include an antibody specific for a control protein. In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.

[0042] According to another aspect of the present invention, the present invention provides a method for providing information for diagnosing a nontuberculous mycobacterial infection disease, comprising the step of measuring the expression level of a CHI3L1 protein, or a gene encoding the same, in a biological sample isolated from an individual.

[0043] In the present invention, the above “subject” refers to a target subject of diagnosis, which is an individual whose onset of a nontuberculous mycobacterial infection is uncertain.

[0044] In the method for providing information for diagnosing a nontuberculous mycobacterial infection disease of the present invention, definitions of nontuberculous mycobacteria, CHI3L1 as a biomarker, a preparation for measuring the expression level of the biomarker, and diagnosis overlap with those described in the diagnostic composition of the present invention, and thus, description thereof is omitted hereinafter to avoid excessive clutter in the specification.

[0045] In the present invention, the “biological sample isolated from an individual” includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, and joint fluid. It may be joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0046] The method for providing information for diagnosing the nontuberculous mycobacterial infection of the present invention may diagnose the target individual as being infected with nontuberculous mycobacteria when the expression level of the CHI3L1 protein or the gene encoding it is significantly higher than that of a normal control group.

[0047] In addition, the method for providing information for diagnosing the nontuberculous mycobacterial infection disease of the present invention may further include a step of considering clinical variables such as the age (Age), presence of a cavity (Cavity), increased erythrocyte sedimentation rate (ESR), or sex (Sex) of the individual. At this time, the four clinical variables may be named ACES (Age, Cavity, ESR, Sex). ACES is calculated by adding 1 point for each case in which the individual is 65 years of age or older (age ≥ 65 years), the presence of a cavity in a chest CT, the increased erythrocyte sedimentation rate (ESR) is 15 mm / h or more for males and 20 mm / h or more for females, or the gender of the target individual is male, and when the calculated value is high, it can be predicted that the progression of the MAC infection lung disease of the target individual is likely to worsen, the treatment will fail, or the mortality rate will increase.

[0048] According to another aspect of the present invention, the present invention provides a composition for determining the start time of treatment for a nontuberculous mycobacterial infection, comprising a preparation capable of measuring the expression level of a CHI3L1 protein or a gene encoding the same.

[0049] Patients diagnosed with nontuberculous mycobacterial lung disease (NTMALD) often do not receive immediate treatment. Because chest X-ray findings in this disease are slow to change, and sometimes there may be no exacerbation, sufficient follow-up is necessary to assess disease progression when NTMALs are detected in sputum. Furthermore, patients with NTMALD are predominantly elderly and require long-term treatment, typically lasting more than a year. Therefore, determining the timing of treatment initiation must consider the potential benefits to the patient alongside the potential side effects of the drug. Therefore, a biomarker that can accurately determine the timing of treatment initiation after diagnosis of NTMALD is essential.

[0050] In this specification, the term “nontuberculous mycobacteria” refers to acid-fast bacteria other than tuberculosis bacilli, and specifically includes all mycobacteria that do not cause tuberculosis or leprosy. Unlike general bacteria, acid-fast bacteria refer to strains that have the ability to withstand and not dissolve even when acid is added during the staining process. The representative example of these acid-fast bacteria is Mycobacterium tuberculosis, and acid-fast bacteria other than tuberculosis bacilli are called nontuberculous mycobacteria (NTM), and new nontuberculous mycobacteria are discovered almost every year. The nontuberculous mycobacteria are widely distributed in nature, and the virulence that causes diseases varies depending on the species. Specifically, Mycobacterium avium complex (MAC), Mycobacterium abscessus (MAB), and Mycobacterium kansasii have relatively high virulence, while Mycobacterium fortuitum has relatively low virulence. Diseases caused by nontuberculous mycobacteria are broadly classified into four characteristic clinical syndromes: pulmonary disease, lymphadenitis, skin, soft tissue, and bone infections, and disseminated disease. Among these, pulmonary disease is the most common form, accounting for more than 90% of all diseases. Pulmonary disease caused by the above nontuberculous mycobacteria is characterized by symptoms such as cough, fever, hemoptysis, and sputum. Reports of pulmonary disease caused by nontuberculous mycobacteria have been increasing since 2000, and pulmonary disease caused by MAC is currently the most frequently reported causative agent in most developed countries. In Korea, MAC pulmonary disease is reported to account for approximately 70-80% of all causative agents of nontuberculous mycobacterial pulmonary disease, with MAB being the second most common causative agent.

[0051] In the present invention, the nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense. The bacterium may be selected from the group consisting of Mycobacterium malmoense, Mycobacterium marinum, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium scrofulaceum, Mycobacterium ulcerans, Mycobacterium simiae, and Mycobacterium xenopi, and more preferably, Mycobacterium avium, but is not limited thereto.

[0052] In the present invention, the non-tuberculous mycobacterial infection disease may be a non-tuberculous mycobacterial infection in the lungs, lymph nodes, skin, cartilage, or bone, and more preferably, a non-tuberculous mycobacterial infection in the lungs, but is not limited thereto.

[0053] The term “CHI3L1 (Chitinase 3-Like 1)” herein, also known as YKL-40, is a secreted glycoprotein belonging to the chitinase-like proteins (CLPs). This protein has a size of 40 kDa and has conserved characteristics as a chitin-binding protein. The three-dimensional crystal structure of CHI3L1 shows a typical folding structure of chitinase family proteins, but chitinase activity is not expressed due to the lack of essential amino acid residues within the enzyme domain. CHI3L1 is expressed in various cells of lung tissue (e.g., neutrophils, monocytes / macrophages, monocyte-derived dendritic cells, epithelial cells) and is closely related to various biological activities such as tissue damage, inflammatory responses, and tissue repair. CHI3L1 is also associated with various diseases. For example, high levels of CHI3L1 protein and gene expression have been observed in patients suffering from various diseases, including asthma, chronic obstructive pulmonary disease (COPD), rheumatoid arthritis, cancer, diabetes, and arteriosclerosis.

[0054] In the present invention, the CHI3L1 may have a slightly different base sequence depending on the species of the target organism for determining the start time of treatment for a nontuberculous mycobacterial infection, but is not limited as long as it can be recognized as a CHI3L1 protein by those skilled in the art to which the present invention pertains. In addition, as long as it is a gene encoding a protein recognizable as CHI3L1, there is no limitation on the specific base sequence. However, when a specific polypeptide base sequence or nucleic acid base sequence is required, a person skilled in the art can collect and utilize information from a known database such as the National Center for Biotechnology Information (NCBI) in the United States.

[0055] In this specification, the term “determining the time to start treatment” refers to determining the time at which medical treatment should begin for a specific disease or condition. The medical treatment herein is, but is not limited to, antibiotic treatment. The antibiotic treatment may include: ① macrolide antibiotics such as clarithromycin (1,000 mg) or azithromycin (250 mg); ② rifampin (600 mg) or rifabutin (300 mg) antibiotics; and / or ③ ethambutol (25 mg / kg for the first 2 months, 15 mg / kg thereafter) antibiotics. The macrolide antibiotics of ① are administered as the most basic, but optionally, at least three drugs from ① to ③ are administered in combination daily. The dosage and frequency of administration of the antibiotics may be adjusted depending on the patient’s condition. For example, for patients with nodular bronchiectasis, intermittent treatment can be administered with ① clarithromycin 1,000 mg or azithromycin 500 mg; ② rifampicin 600 mg; and ③ ethambutol 25 mg / kg three times a week. Patients with fibrocavitary or severe nodular bronchiectasis can be administered with ① clarithromycin 1,000 mg once daily (or 500 mg twice daily) or azithromycin 250 mg; ② rifampicin 10 mg / kg (maximum 600 mg); and ③ ethambutol 15 mg / kg daily. Patients with severe and extensive lesions or patients undergoing retreatment can additionally receive amikacin or streptomycin injection for the initial 2-3 months.

[0056] In this specification, the term “composition for determining the time point of treatment start” refers to an integrated mixture or device including a measuring means capable of measuring the expression level of the CHI3L1 protein or the gene encoding it to determine the time point at which medical treatment of a subject should be started, and can be used with the same meaning as “kit for determining the time point of treatment start.” Since the composition for determining the time point of treatment of the present invention includes a means for measuring the biomarker discovered in the present invention, the term “composition for determining the time point of treatment start” can also be expressed as a “device for determining the time point of treatment start” of the CHI3L1 protein or the gene encoding it as a target biomarker.

[0057] In the present invention, the preparation for measuring the expression level of the biomarker protein is not particularly limited, but may include, for example, one or more selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the protein.

[0058] In the present invention, the "antibody" refers to a substance that specifically binds to an antigen and causes an antigen-antibody reaction. For the purposes of the present invention, the antibody refers to an antibody that specifically binds to the biomarker protein. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. The antibodies can be readily produced using techniques well known in the art. For example, polyclonal antibodies can be produced by methods well known in the art, including a process of injecting an antigen of the biomarker protein into an animal and collecting blood from the animal to obtain serum containing the antibody. Such polyclonal antibodies can be produced from any animal, such as a goat, rabbit, sheep, monkey, horse, pig, cow, or dog. In addition, monoclonal antibodies can be produced using hybridoma methods or phage antibody library techniques well known in the art. Antibodies produced by the above methods can be separated and purified using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity chromatography. Furthermore, the antibody of the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains, but also a functional fragment of the antibody molecule. A functional fragment of an antibody molecule means a fragment that possesses at least an antigen-binding function, such as Fab, F(ab'), F(ab')2, and Fv.

[0059] The "PNA (Peptide Nucleic Acid)" in the present invention refers to an artificially synthesized polymer similar to DNA or RNA, and was first introduced in 1991 by Professors Nielsen, Egholm, Berg, and Buchardt of the University of Copenhagen, Denmark. While DNA has a phosphate-ribose sugar backbone, PNA has a repeated N-(2-aminoethyl)-glycine backbone linked by peptide bonds, which greatly increases its binding affinity and stability to DNA or RNA, and is therefore used in molecular biology, diagnostic analysis, and antisense therapy.

[0060] In the present invention, the “aptamer” is an oligonucleic acid or peptide molecule.

[0061] In the present invention, a preparation for measuring the expression level of a gene encoding the biomarker protein may include at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to the gene encoding the biomarker protein.

[0062] In the present invention, the "primer" refers to a fragment that recognizes a target gene sequence, and includes a pair of forward and reverse primers, but is preferably a pair of primers that provide analysis results with specificity and sensitivity. When the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, and thus only amplifies the target gene sequence containing the complementary primer binding site and does not cause non-specific amplification, high specificity can be imparted.

[0063] In the present invention, the "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample, and refers to a substance that can specifically confirm the presence of the target substance in the sample through the binding. The type of probe is not limited to a substance commonly used in the art, but is preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, and most preferably PNA. More specifically, the probe includes a biomaterial derived from or similar to a living organism or manufactured in vitro, and may be, for example, an enzyme, a protein, an antibody, a microorganism, animal or plant cells and organs, nerve cells, DNA, and RNA. DNA includes cDNA, genomic DNA, and oligonucleotides, RNA includes genomic RNA, mRNA, and oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, etc.

[0064] In the present invention, the term "LNA (Locked nucleic acids)" refers to a nucleic acid analog containing a 2'-O, 4'-C methylene bridge. LNA nucleosides contain common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rule. However, due to the molecule's 'locking' caused by the methylene bridge, LNA cannot form an ideal shape in Watson-Crick binding. When LNA is included in a DNA or RNA oligonucleotide, LNA can pair with a complementary nucleotide chain more quickly, thereby increasing the stability of the double helix.

[0065] In the present invention, the term "antisense" refers to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that allows the antisense oligomer to hybridize with a target sequence within RNA by Watson-Crick base pairing, typically allowing the formation of an mRNA and RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity or approximate sequence complementarity to the target sequence.

[0066] Since the biomarker protein according to the present invention or the information on the gene encoding the same is known, a person skilled in the art will be able to easily design a primer, probe, or antisense nucleotide that specifically binds to the gene encoding the protein based on this.

[0067] In the present invention, the expression level of the CHI3L1 protein or the gene encoding it is measured in whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, It may be measured in synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0068] According to another aspect of the present invention, the present invention provides a kit for determining the time point at which treatment is started for a nontuberculous mycobacterial infection, comprising a composition for determining the time point at which treatment is started for a nontuberculous mycobacterial infection, comprising a preparation capable of measuring the expression level of a CHI3L1 protein or a gene encoding the same.

[0069] In this specification, the term "kit for determining the timing of treatment initiation" may be used with the same meaning as the "composition for determining the timing of treatment initiation" as described above. The definitions of the composition for determining the timing of treatment initiation of the present invention and CHI3L1 as a biomarker overlap with those described in the composition for determining the timing of treatment initiation of the present invention, and thus, to avoid excessive clutter in the specification, their descriptions are omitted below.

[0070] In the present invention, the time point at which medical treatment for a subject infected with nontuberculous mycobacteria should be initiated can be determined using the kit for determining the time point at which treatment is initiated.

[0071] In the present invention, the kit may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0072] The kit for determining the treatment start time of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the analysis method.

[0073] For example, the kit for determining the treatment start time of the present invention may further include essential elements necessary for performing a reverse transcription polymerase reaction. The reverse transcription polymerase reaction kit includes a pair of primers specific for a gene encoding a marker protein. The primers are nucleotides having a sequence specific for the nucleic acid sequence of the gene, and may have a length of about 7 bp to 50 bp, more preferably about 10 bp to 30 bp. It may also include a primer specific for the nucleic acid sequence of a control gene. In addition, the reverse transcription polymerase reaction kit may include a test tube or other appropriate container, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), an enzyme such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0074] Additionally, the treatment start timing determination kit of the present invention may include essential elements necessary for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or fragment thereof are attached, and reagents, preparations, enzymes, etc. for producing a fluorescently labeled probe. The substrate may also include cDNA or oligonucleotides corresponding to a control gene or fragment thereof.

[0075] Additionally, the kit for determining the treatment start time of the present invention may include the essential elements necessary for performing an ELISA. The ELISA kit includes an antibody specific for the protein. The antibody is an antibody with high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit may also include an antibody specific for a control protein. In addition, the ELISA kit may include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies), and their substrates or other substances capable of binding to antibodies.

[0076] According to another aspect of the present invention, the present invention provides a method for providing information for determining the timing of treatment initiation for a nontuberculous mycobacterial infection, comprising the step of measuring the expression level of a CHI3L1 protein, or a gene encoding the same, in a biological sample isolated from an individual.

[0077] In the present invention, the above “subject” refers to a target subject whose onset of non-tuberculous mycobacterial infection is confirmed, and is the subject of determination of the time point for starting treatment.

[0078] In the method for providing information for determining the start time of treatment for a nontuberculous mycobacterial infection of the present invention, definitions of nontuberculous mycobacteria, CHI3L1 as a biomarker, a preparation for measuring the expression level of the biomarker, and determination of the start time of treatment overlap with those described in the composition for determining the start time of treatment of the present invention, and thus, description thereof is omitted hereinafter to avoid excessive clutter in the specification.

[0079] In the present invention, the “biological sample isolated from an individual” includes whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, and joint fluid. It may be joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0080] The method for providing information for determining the timing of starting treatment for a nontuberculous mycobacterial infection of the present invention may be such that, when the expression level of the CHI3L1 protein or the gene encoding it is significantly higher than that of a normal control group, the target individual is determined to be an individual for whom medical treatment for a nontuberculous mycobacterial infection should be started.

[0081] In addition, the method for providing information for determining the timing of starting treatment for the nontuberculous mycobacterial infection of the present invention may further include a step of considering clinical variables such as the age (Age), presence of a cavity (Cavity), increased erythrocyte sedimentation rate (ESR), or sex (Sex) of the individual. At this time, the four clinical variables may be named ACES (Age, Cavity, ESR, Sex). ACES is calculated by adding 1 point for each case where the individual is 65 years of age or older (age ≥ 65 years), where a cavity exists in a chest CT, where the increased erythrocyte sedimentation rate (ESR) is 15 mm / h or more for males and 20 mm / h or more for females, or where the gender of the target individual is male, and when the calculated value is high, the target individual may be determined as an individual for whom medical treatment for nontuberculous mycobacterial infection should be started.

[0082] According to another aspect of the present invention, the present invention provides a method for diagnosing a nontuberculous mycobacterial infection disease, comprising the step of measuring the expression level of a CHI3L1 protein, or a gene encoding the same, in a biological sample isolated from an individual.

[0083] The definition of specific detailed components in the above method for diagnosing nontuberculous mycobacterial infections of the present invention overlaps with that described in the method for providing information for diagnosing nontuberculous mycobacterial infections described above, and thus, to avoid excessive clutter in the specification, the description thereof is omitted below.

[0084] According to another aspect of the present invention, the present invention provides a method for determining the time point for starting treatment of a nontuberculous mycobacterial infection disease, comprising the step of measuring the expression level of a CHI3L1 protein or a gene encoding the same in a biological sample isolated from an individual infected with a nontuberculous mycobacteria.

[0085] The definition of specific detailed components in the method for determining the starting point of treatment for a nontuberculous mycobacterial infection of the present invention overlaps with that described in the method for providing information for determining the starting point of treatment for a nontuberculous mycobacterial infection described above, and thus, to avoid excessive clutter in the specification, the description thereof is omitted below.

[0086] According to another aspect of the present invention, the present invention provides a use of a preparation capable of measuring the expression level of a CHI3L1 protein or a gene encoding the same for diagnosing a nontuberculous mycobacterial infection disease.

[0087] The definition of specific detailed components in the above-described non-tuberculous mycobacterial infection disease diagnosis use of the present invention overlaps with that described in the aforementioned composition for diagnosis of non-tuberculous mycobacterial infection disease, and thus, to avoid excessive clutter in the specification, the description thereof is omitted below.

[0088] According to another aspect of the present invention, the present invention provides a use of a preparation capable of measuring the expression level of a CHI3L1 protein or a gene encoding the same for determining the time point at which treatment of a nontuberculous mycobacterial infection is started.

[0089] The definition of specific detailed components in the use of the present invention for determining the start time of treatment for a nontuberculous mycobacterial infection is redundant with that described in the composition for determining the start time of treatment for a nontuberculous mycobacterial infection described above, and thus, to avoid excessive clutter in the specification, the description thereof is omitted below.

[0090] The present invention proposes Chitinase 3-Like 1 (CHI3L1) as a biomarker for diagnosing and determining the timing of treatment for nontuberculous mycobacterial lung disease. The biomarker of the present invention is expected to be of significant use in the healthcare field, as it offers significant utility in diagnosing nontuberculous mycobacterial lung disease and determining the timing of treatment.

[0091] Figure 1 is a result confirming that the expression level of CHI3L1 increases in the serum of patients with MAC infection lung disease compared to a healthy control group in one embodiment of the present invention.

[0092] Figure 2 is a result confirming that, in one embodiment of the present invention, a higher inflammatory response occurs in WT mice compared to CHI3L1 KO mice when infected with nontuberculous mycobacteria (M. aviumSMC#7).

[0093] FIG. 3 is a result confirming that, in one embodiment of the present invention, a higher inflammatory response occurs in CHI3L1 KO mice compared to WT mice when infected with Mycobacterium tuberculosis (H37Rv).

[0094] FIG. 4 is a result confirming that, in one embodiment of the present invention, patients with a high expression level of CHI3L1 at the time of diagnosis of MAC infection lung disease are more likely to have a disease progression and receive antibiotic treatment.

[0095] FIG. 5 is a result confirming that, in one embodiment of the present invention, patients with a high expression level of CHI3L1 at the time of diagnosis of MAC infection lung disease are more likely to exhibit rapid disease progression.

[0096] Figure 6 is a result confirming that, in one embodiment of the present invention, the expression level of CHI3L1 is higher in patients in the severe group than in patients in the mild group based on the BACES standard.

[0097] FIG. 7 is a result confirming that, in one embodiment of the present invention, it is possible to predict disease progression, treatment failure, and mortality of MAC infection lung disease using four clinical variables, namely, CHI3L1 expression level and ACES (Age, Cavity, ESR, Sex).

[0098] Figure 8 shows the results of comparing the expression levels of CHI3L1 after infection with nontuberculous mycobacteria in four types of mice with different susceptibilities in one embodiment of the present invention.

[0099] Figure 9 shows the results of comparing inflammatory lesions in lung tissue after infection with nontuberculous mycobacteria in four types of mice with different susceptibilities in one embodiment of the present invention.

[0100] Figure 10 is a result confirming that, in one embodiment of the present invention, the expression level of CHI3L1 in lung tissue lysates increases in mice with higher susceptibility to nontuberculous mycobacteria.

[0101] Wild-type (WT) and CHI3L1 knockout (CHI3L1 KO) mice were infected with M. avium SMC#7, a nontuberculous mycobacteria strain isolated from a clinical patient, by pneumonitis. All mice were necropsied 10 weeks after infection, and the bacterial load and lesion levels in the lung tissue were measured. Comparison of the two groups confirmed that the bacterial load and histopathological inflammation levels in the lung tissue of WT mice were significantly higher than those of CHI3L1 KO mice.

[0102] Meanwhile, wild-type mice (WT) and CHI3L1 knockout mice (CHI3L1 KO) were infected with the H37Rv strain, a standard strain of Mycobacterium tuberculosis, by intravenous injection. All mice were necropsied at 4 weeks post-infection, and the bacterial load and lesions in the lung tissue were measured. Comparison of the two groups confirmed that, unlike nontuberculous mycobacteria, the bacterial load and histopathological inflammation in the lung tissue of CHI3L1 KO mice were significantly higher than those of WT mice in the tuberculosis infection.

[0103] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0104] Example

[0105] Example 1. Confirmation of increased expression of CHI3L1 in the serum of patients with MAC-infected lung disease.

[0106] To confirm the potential of CHI3L1 as a diagnostic biomarker for MAC-infected lung disease, the expression levels of CHI3L1 were compared in the sera of patients with MAC-infected lung disease and healthy controls using ELISA.

[0107] Example 1-1. Collection and storage of patient and healthy control samples

[0108] From January 2012 to August 2016, 145 serum samples from patients with MAC lung disease who had not started antibiotic treatment and 29 serum samples from healthy controls were obtained with the patients' consent from Samsung Medical Center in Seoul. In addition, five additional samples were collected from the healthy control group. All samples were stored in an ultra-low temperature freezer at −80°C or lower and were completely thawed immediately prior to analysis.

[0109] Example 1-2. Confirmation of CHI3L1 expression level in serum using ELISA

[0110] The expression level of CHI3L1 was measured in serum samples using the Enzyme-Linked Immunosorbent Assay (ELISA) method using the DY2599 ELISA kit and the additional reagent kit DY008 from R&D Systems, which are commercial products developed to detect the CHI3L1 protein. During the experiment, serum samples were processed according to the instructions for the DY2599 ELISA kit, and the expression level of CHI3L1 was quantified through steps such as sample dilution and processing. Through this, the difference in the expression level of CHI3L1 between patients with MAC infection lung disease and a healthy control group was compared and analyzed.

[0111] Specifically, the capture antibody was first diluted to the specified working concentration in protein-free PBS and then 100 mL was added to each 96-well microplate. The plates were then sealed and incubated overnight at 4°C. The following day, each well was washed three times with washing solution. Each well was filled with 400 mL of washing solution, and the solution was completely removed after each wash. After the final wash, the plate was inverted on a clean paper towel to remove any remaining washing solution. Then, each well was blocked with 300 mL of assay buffer. The plates were then incubated at room temperature for 1 hour. The plates prepared in this manner were subjected to three aspiration / washing cycles as before. Next, each well was filled with 100 mL of a total of eight solutions (including a blank) prepared by diluting serum samples and standard solutions of known concentrations 1 / 2 in assay buffer. The surfaces were then covered with adhesive strips and incubated at room temperature for 2 hours. Subsequently, each well was washed three times by repeating the aspiration / washing process as before. Then, 100 mL of detection antibody diluted in assay buffer was added to each well. The well was covered with a new adhesive strip and incubated at room temperature for 2 hours. Aspiration / washing was repeated once more to complete washing. Next, 100 mL of a working concentration of streptavidin-HRP was added to each well. The plate was wrapped in foil to avoid direct light and incubated at room temperature for 20 minutes. Finally, the plate was repeatedly aspirated / washed to complete washing. In the final step, 100 mL of substrate solution was added to each well and incubated at room temperature for 20 minutes while protecting from direct light. Finally, 50 mL of stop solution was added to each well.After gently tapping the plate to mix thoroughly, the optical density of each well was measured at 450 nm using a microplate reader.

[0112] The above measurement results are shown in Fig. 1. The measurement results confirmed that the expression level of CHI3L1 was significantly higher in the serum of 145 MAC-PD patients who did not start antibiotic treatment compared to 34 healthy controls.

[0113] Example 2. Comparison of Mycobacterial Infections between Wild-Type and CHI3L1 Knockout Mice

[0114] To determine the contribution of the CHI3L1 protein to the progression of disease in mycobacterial infections, we examined disease progression in mice infected with nontuberculous mycobacteria and Mycobacterium tuberculosis.

[0115] Example 2-1. Comparison of nontuberculous mycobacterial infection in wild-type and CHI3L1 knockout mice.

[0116] Wild-type mice (WT) and CHI3L1 knockout mice (CHI3L1 KO) were infected with M. aviumSMC#7, a nontuberculous mycobacteria strain isolated from a clinical patient, by airway infection. All mice were necropsied 10 weeks after infection, and the bacterial load and lesions in the lung tissue were measured. To measure the bacterial load in the lung tissue, lung tissues were isolated from each group, ground, and lysed using sterile PBS. The lysates were diluted 1 / 100, 1 / 1000, and 1 / 10000 in PBS, and 50 ㎕ per well were added to pre-prepared 7H10 solid medium. The cells were cultured in a microbiological incubator for approximately 10 days, and the bacterial counts after 10 days were measured. Lung tissue lesions were confirmed by hematoxylin and eosin staining.

[0117] The results of the above test are shown in Fig. 2. As a result of comparing the two groups, it was confirmed that the amount of bacteria and the degree of histopathological inflammation in the lung tissue of WT mice were significantly higher than those of CHI3L1 KO mice.

[0118] Example 2-2. Comparison of tuberculosis infection in wild-type and CHI3L1 knockout mice.

[0119] Wild-type (WT) and CHI3L1 knockout (CHI3L1 KO) mice were infected with the H37Rv strain, a standard strain of Mycobacterium tuberculosis, by intravenous injection. All mice were necropsied 4 weeks after infection, and the amount of bacteria and lesions in lung tissue were measured.

[0120] The results of the above test are shown in Fig. 3. As a result of comparing the two groups, it was confirmed that, unlike nontuberculous mycobacteria, the amount of bacteria and the degree of histopathological inflammation in the lung tissue of CHI3L1 KO mice were significantly higher than those of WT mice in tuberculosis infection.

[0121] Example 3. Confirmation of the potential of CHI3L1 as a marker for predicting disease progression in patients with MAC-infected lung disease.

[0122] To confirm the potential of CHI3L1 as a biomarker indicating the starting point of treatment in patients with MAC-infected lung disease, we compared the expression levels of CHI3L1 in the serum at diagnosis between patients with MAC-infected lung disease who progressed and started antibiotic treatment after diagnosis and patients whose disease did not progress through ELISA.

[0123] Example 3-1. Collection and storage of patient samples

[0124] From January 2012 to August 2016, 89 serum samples from patients with MAC-infected lung disease at the time of diagnosis were obtained from Samsung Hospital in Seoul with the patients' consent over a period of approximately 6 years. Of these, 29 serum samples were from patients who had progressed their disease and received antibiotic treatment, and 60 serum samples were from patients who had not progressed their disease and did not receive antibiotic treatment. At this time, the antibiotic treatment was administered with ① macrolide antibiotics such as clarithromycin (1,000 mg) or azithromycin (250 mg); ② rifampin (600 mg) or rifabutin (300 mg); and / or ③ ethambutol (25 mg / kg for the first 2 months, 15 mg / kg thereafter). The above macrolide antibiotics ① are administered as the most basic, but optionally, at least three drugs from ① to ③ are administered in combination daily. The dosage and frequency of administration of the above antibiotics can be adjusted depending on the patient's condition. For example, for patients with nodular bronchiectasis type, intermittent treatment is administered by administering ① clarithromycin 1,000 mg or azithromycin 500 mg; ② rifampicin 600 mg; and ③ ethambutol 25 mg / kg three times a week. Patients with fibrocavitary type or severe nodular bronchiectasis type are administered ① clarithromycin 1,000 mg once daily (or 500 mg twice daily) or azithromycin 250 mg; ② rifampicin 10 mg / kg (maximum 600 mg); and ③ ethambutol 15 mg / kg; daily. Patients with severe, extensive disease or those undergoing retreatment may receive additional amikacin or streptomycin injections for an initial 2-3 months. Treatment should continue for at least 12 months after a negative sputum culture is confirmed.All samples were stored in an ultra-low temperature freezer below -80°C and were completely thawed before use.

[0125] Example 3-2. Confirmation of CHI3L1 expression level in serum using ELISA

[0126] The expression level of CHI3L1 was measured in the patient's serum sample using the same analysis method as in Example 1-2 using the DY2599 ELISA kit from R&D Systems.

[0127] The above measurement results are shown in Fig. 4. As a result of follow-up observation using the measurement values, it was confirmed that the CHI3L1 expression level was significantly higher in the serum samples at diagnosis of patients whose disease progressed and received antibiotic treatment (Progressor) than in the serum samples at diagnosis of patients whose disease did not progress (Non-progressor).

[0128] Example 4. Confirmation of the potential of CHI3L1 as a marker for predicting disease progression in patients with MAC-infected lung disease.

[0129] Using the DY2599 ELISA kit from R&D Systems, the expression level of CHI3L1 was compared according to the period from the date of diagnosis to the date of start of treatment in patients with disease progression using the same analysis method as in Example 1-2.

[0130] The results of the above test are shown in Fig. 5. As a result of the comparison, among the serum samples at the time of diagnosis of patients who progressed and received antibiotic treatment, the CHI3L1 expression level in the group in which treatment began within 180 days from the time of diagnosis (Progressor) was significantly higher than the CHI3L1 expression level in the group in which treatment began after 180 days. This means that the higher the expression level of CHI3L1 at the time of diagnosis, the more rapidly the disease progression may occur.

[0131] Example 5. Comparison of BACES scores and CHI3L1 expression levels in patients with MAC-infected lung disease.

[0132] We compared the BACES score of patients with the expression level of CHI3L1, using the BACES score, which has been identified as an aggravating factor of nontuberculous lung disease infection in relation to disease progression, treatment failure, and mortality in MAC-infected lung disease.

[0133] Example 5-1. Collection and storage of patient samples

[0134] From January 2012 to August 2016, 145 serum samples were obtained from patients with MAC lung disease who had not started antibiotic treatment at Samsung Medical Center in Seoul. All samples were stored in an ultra-low temperature freezer at −80°C or below and were completely thawed immediately before analysis.

[0135] Example 5-2. BACES score

[0136] The BACES score is a scoring system designed to predict mortality in patients with nontuberculous mycobacterial lung disease. It includes five clinical variables: body mass index (BMI), age, presence of cavities, elevated erythrocyte sedimentation rate (ESR), and sex. The BACES score is calculated by adding one point for each of the five variables below, and a higher score is interpreted as an increased mortality risk (Am J Respir Crit Care Med. 2021 Jan 15;203(2):230-236).

[0137] (1) BMI<18.5 kg / m 2

[0138] (2) Age ≥ 65 years old

[0139] (3) Presence of a cavity on chest CT

[0140] (4) ESR (male >15 mm / h, female >20 mm / h)

[0141] (5) Male gender

[0142] Example 5-3. Comparison of BACES scores and CHI3L1 expression levels

[0143] The BACES scores of 145 patients with MAC lung disease who had not started antibiotic treatment were calculated using the criteria specified in Example 5-2, and based on this, they were classified into two groups as follows: a mild group (BACES score 0-1) and a severe group (BACES score 2-4). The results of a comparison of the expression levels of CHI3L1 in the two groups are shown in Figure 6. The comparison results confirmed that the expression levels of CHI3L1 were significantly higher in patients in the severe group than in patients in the mild group.

[0144] In addition, the expression level of CHI3L1 in the serum of 145 patients with MAC infection lung disease who did not start antibiotic treatment was compared with the five clinical variables used to calculate the BACES score, and the results are shown in Fig. 7. As a result of the comparison, among the five clinical variables that make up the BACES, except for BMI, the expression level of CHI3L1 was higher in patients with high risk (age ≥ 65 years, presence of cavities in chest CT, ESR (male > 15 mm / h, female > 20 mm / h), male gender) than in patients who did not, confirming the significance between the clinical variables and the expression level of CHI3L1. In the case of the BMI clinical variable, there was a high risk (BMI < 18.5 kg / m 2 ) did not show a significant difference in CHI3L1 expression levels between patients with and without MAC infection. These results imply that the disease progression, treatment failure, and mortality of MAC infection lung disease can be predicted using CHI3L1 expression levels and four clinical variables, ACES (Age, Cavity, ESR, and Sex).

[0145] Example 6. Comparison of CHI3L1 expression levels in four mouse strains with different susceptibilities.

[0146] By comparing the expression levels of CHI3L1 protein in four mouse models with different susceptibilities, we evaluated the effect of CHI3L1 on susceptibility to nontuberculous mycobacterial infection and disease progression.

[0147] Example 6-1. Comparison of susceptibility to nontuberculous mycobacterial infection in four types of mice.

[0148] BALB / c, C57BL / 6, A / J, and C3H mice were infected with Mycobacterium aviumSMC#7, a nontuberculous mycobacteria strain isolated from a clinical patient, by airway infection. All mice were necropsied at 5 and 10 weeks after infection, and the bacterial load and lesions in the lung tissue were measured. To measure the bacterial load in the lung tissue, lung tissues were separated from each group, homogenized in sterile PBS, and diluted 1 / 100, 1 / 1000, and 1 / 10000. 50 ㎕ per well was inoculated onto 7H10 solid medium. After culturing in a microbiological incubator for approximately 10 days, the number of cultured bacteria was measured. Lesions in the lung tissues were confirmed by hematoxylin and eosin staining.

[0149] The results of the above test are shown in Figures 8 and 9. As a result of confirming the amount of bacteria and the degree of histopathological inflammation in the lung tissues of four types of mice, the susceptibility to nontuberculous mycobacteria was found to be high in the order of BALB / c, C57BL / 6, A / J, and C3H.

[0150] Example 6-2. Comparison of CHI3L1 expression levels in four mouse strains with different susceptibility to nontuberculous mycobacterial infection.

[0151] The expression level of CHI3L1 was measured using an enzyme-linked immunosorbent assay (ELISA) in lung tissue lysates from BALB / c, C57BL / 6, A / J, and C3H mice infected with nontuberculous mycobacteria at 10 weeks post-infection. First, the capture antibody was diluted to the concentrations specified in phosphate-buffered saline (PBS), added to each well of a 96-well microplate (100 μl) and incubated overnight at 4°C. The following day, each well was washed three times with washing solution, and 300 μl of assay buffer was added and incubated for 1 hour at room temperature. After repeated washing, serum samples, lung lysates, and standard solutions were diluted 1 / 2 and added to each well (100 μl). After incubation for 2 hours at room temperature, washing was repeated three times. After adding 100 μl of diluted detection antibody to each well, the wells were incubated at room temperature for 2 hours and washed repeatedly. Next, 100 μl of streptavidin-HRP was added to each well, wrapped in foil, and incubated at room temperature for 20 minutes. After a final wash, 100 μl of substrate solution was added to each well, and the wells were incubated at room temperature for 20 minutes, protected from light. Finally, 50 μl of stop solution was added to each well, the plate was gently shaken to mix, and the optical density was measured at 450 nm using a microplate reader.

[0152] The results of the above test are shown in Figure 10. It was confirmed that the expression level of CHI3L1 in lung tissue lysates increased in mice with higher susceptibility to nontuberculous mycobacteria. This suggests that CHI3L1 expression can be used as a diagnostic marker for nontuberculous mycobacteria.

[0153] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0154] The present invention relates to a biomarker for diagnosing nontuberculous mycobacterial lung disease and determining the timing of treatment initiation. The biomarker of the present invention is expected to be of significant use in the healthcare field, as it offers significant utility in diagnosing nontuberculous mycobacterial lung disease and determining the timing of treatment initiation.

Claims

1. A composition for diagnosing a nontuberculous mycobacterial infection disease, comprising a preparation capable of measuring the expression level of the CHI3L1 protein or a gene encoding the same.

2. In paragraph 1, The above nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, A composition selected from the group consisting of Mycobacterium marinum, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium scrofulaceum, Mycobacterium ulcerans, Mycobacterium simiae, and Mycobacterium xenopi.

3. In paragraph 1, A composition wherein the above nontuberculous mycobacterial infection disease is a nontuberculous mycobacterial infection in the lungs, lymph nodes, skin, cartilage, or bone.

4. In paragraph 1, A composition comprising at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the CHI3L1 protein, which can measure the expression level of the CHI3L1 protein.

5. In paragraph 1, A composition comprising at least one member selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to a gene encoding the CHI3L1 protein, wherein the measurable agent of the gene encoding the CHI3L1 protein is present.

6. In paragraph 1, The expression level of the CHI3L1 protein or the gene encoding it is measured in whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, A composition measured in synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

7. A diagnostic kit for nontuberculous mycobacterial infections, comprising a diagnostic composition according to any one of claims 1 to 6.

8. In paragraph 7, The above kit is a kit that is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

9. A method for providing information for diagnosing a nontuberculous mycobacterial infection disease, comprising the step of measuring the expression level of the CHI3L1 protein or a gene encoding the same in a biological sample isolated from an individual.

10. In paragraph 9, The above nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, A method, wherein the bacterium is selected from the group consisting of M. marinum, M. szulgai, M. terrae, M. scrofulaceum, M. ulcerans, M. simiae, and M. xenopi.

11. In paragraph 9, A method wherein the above non-tuberculous mycobacterial infection is a non-tuberculous mycobacterial infection in the lungs, lymph nodes, skin, cartilage, or bone.

12. In paragraph 9, A method wherein the expression level of the CHI3L1 protein is measured using at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the CHI3L1 protein.

13. In paragraph 9, A method wherein the expression level of a gene encoding the CHI3L1 protein is measured using at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to a gene encoding the CHI3L1 protein.

14. In paragraph 9, Biological samples isolated from the above-mentioned individual include whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, and joint aspirate. A method wherein the extract is an aspirate, organ secretions, cells, cell extract, or cerebrospinal fluid.

15. In paragraph 9, The method further comprises a step of considering clinical variables such as age of the subject, presence of a cavity, increased erythrocyte sedimentation rate (ESR), or sex.

16. A composition for determining the start time of treatment for a nontuberculous mycobacterial infection, comprising a preparation capable of measuring the expression level of the CHI3L1 protein or a gene encoding the same.

17. In paragraph 16, The above nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, A composition selected from the group consisting of Mycobacterium marinum, Mycobacterium szulgai, Mycobacterium terrae, Mycobacterium scrofulaceum, Mycobacterium ulcerans, Mycobacterium simiae, and Mycobacterium xenopi.

18. In paragraph 16, A composition wherein the above nontuberculous mycobacterial infection disease is a nontuberculous mycobacterial infection in the lungs, lymph nodes, skin, cartilage, or bone.

19. In paragraph 16, A composition comprising at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the CHI3L1 protein, which can measure the expression level of the CHI3L1 protein.

20. In paragraph 16, A composition comprising at least one member selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to a gene encoding the CHI3L1 protein, wherein the measurable agent of the gene encoding the CHI3L1 protein is present.

21. In paragraph 16, The expression level of the CHI3L1 protein or the gene encoding it is measured in whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, A composition measured in synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

22. A kit for determining the start time of treatment for a nontuberculous mycobacterial infection, comprising a diagnostic composition according to any one of claims 16 to 21.

23. In paragraph 22, The above kit is a kit that is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

24. A method for providing information for determining the timing of starting treatment for a nontuberculous mycobacterial infection, comprising the step of measuring the expression level of CHI3L1 protein or a gene encoding the same in a biological sample isolated from an individual.

25. In paragraph 24, The above nontuberculous mycobacteria are Mycobacterium avium, Mycobacterium abscessus, Mycobacterium flavescence, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium chelonae, Mycobacterium celatum, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium gastri, Mycobacterium haemophilum, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium malmoense, A method, wherein the bacterium is selected from the group consisting of M. marinum, M. szulgai, M. terrae, M. scrofulaceum, M. ulcerans, M. simiae, and M. xenopi.

26. In paragraph 24, A method wherein the above non-tuberculous mycobacterial infection is a non-tuberculous mycobacterial infection in the lungs, lymph nodes, skin, cartilage, or bone.

27. In paragraph 24, A method wherein the expression level of the CHI3L1 protein is measured using at least one selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the CHI3L1 protein.

28. In paragraph 24, A method wherein the expression level of a gene encoding the CHI3L1 protein is measured using at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically bind to a gene encoding the CHI3L1 protein.

29. In paragraph 24, Biological samples isolated from the above-mentioned individual include whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, and joint aspirate. A method wherein the extract is an aspirate, organ secretions, cells, cell extract, or cerebrospinal fluid.

30. In paragraph 24, The method further comprises a step of considering clinical variables such as age of the subject, presence of a cavity, increased erythrocyte sedimentation rate (ESR), or sex.

31. A method for diagnosing a nontuberculous mycobacterial infection disease, comprising the step of measuring the expression level of CHI3L1 protein or a gene encoding the same in a biological sample isolated from an individual.

32. A method for determining the time point for starting treatment for a nontuberculous mycobacterial infection, comprising the step of measuring the expression level of the CHI3L1 protein or a gene encoding the same in a biological sample isolated from an individual infected with nontuberculous mycobacteria.

33. Use of a preparation capable of measuring the expression level of CHI3L1 protein or a gene encoding it for diagnosis of nontuberculous mycobacterial infections.

34. Use of a preparation capable of measuring the expression level of CHI3L1 protein or the gene encoding it for determining the timing of treatment initiation for nontuberculous mycobacterial infections.

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