Biomarker for diagnosing, assisting diagnosis of, determining and / or assisting determination of at least one of eosinophilic otitis media, eosinophilic paranasal sinusitis, and granulomatosis with eosinophilic polyangiitis; method associated with said biomarker; and kit

Abstract

The purpose of the present invention is to provide a biomarker, and a method, for diagnosing, assisting diagnosis of, determining and / or assisting determination of at least one of eosinophilic otitis media, eosinophilic paranasal sinusitis, granulomatosis with eosinophilic polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media, while accurately differentiating these from other forms of otitis media and disease. To meet this purpose, a biomarker that uses at least an EPO-DNA complex contained in an eosinophil extracellular trap is provided for diagnosing, assisting diagnosis of, determining and / or assisting determination of at least one of eosinophilic otitis media, eosinophilic paranasal sinusitis, and granulomatosis with eosinophilic polyangiitis and further, a method for diagnosing, assisting diagnosis of, determining and / or assisting determination of at least one of eosinophilic otitis media, eosinophilic paranasal sinusitis, and granulomatosis with eosinophilic polyangiitis is provided which involves measuring the expression level of the EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample or nasal secretion sample taken from the patient, and measuring the expression level of MPO-DNA complexes as needed.

Description

[Amendment based on Rule 91, 14.10.2022] Biomarkers for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, and methods and kits therefor

[0001] [Amendment under Rule 91 14.10.2022] The present invention relates to biomarkers for one or more of diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, as well as methods and kits therefor.

[0002] Eosinophilic otitis media is a difficult-to-treat form of otitis media characterized by a glue-like middle ear fluid with significant eosinophil infiltration. Similar to serous otitis media, serous fluid is observed early in the onset period, followed by a gradual development of glue-like ear discharge. This glue-like ear discharge is also seen in adult serous otitis media. While serous otitis media improves with treatment, eosinophilic otitis media does not improve and is difficult to treat. It is also associated with asthma, chronic sinusitis, and increased eosinophils in peripheral blood and tissues. As bone conduction hearing thresholds increase, hearing loss can progress rapidly, eventually leading to deafness. Therefore, early differentiation before the condition worsens is highly important (Non-Patent Document 1).

[0003] Eosinophilic otitis media frequently develops as a complication of eosinophilic sinusitis. Eosinophilic sinusitis is characterized by the infiltration of numerous activated eosinophils into the sinus mucosal epithelium, resulting in histological features such as epithelial cell damage, replacement with secretory cells, and basement membrane thickening. It is often accompanied by lower respiratory tract diseases such as bronchial asthma. The majority of cases are bilateral and occur in adults, and are resistant to endoscopic sinus surgery and are intractable.

[0004] Furthermore, eosinophilic granulomatosis with polyangiitis (EGPA) presents clinical symptoms similar to those of eosinophilic otitis media and eosinophilic sinusitis. This disease is characterized by a marked increase in peripheral blood eosinophils, allergic diseases such as bronchial asthma, and histologically, granulomas accompanied by eosinophilic infiltration around small and medium-sized blood vessels. Early clinical symptoms in the otorhinolaryngological field not only progress to hearing loss, but also rapidly progress to a systemic form, leading to a fatal condition. Therefore, early diagnosis and treatment initiation are desirable (Non-Patent Document 2).

[0005] A common mechanism seen in these three diseases is the release of granulocyte extracellular traps during a special type of cell death called ETosis. It has already been suggested that eosinophil extracellular traps are involved in eosinophilic otitis media and eosinophilic sinusitis (Non-Patent Document 3). However, much remains unknown about eosinophil extracellular traps. While it is known that they are based on a framework composed of DNA and histones and that various cytoplasmic enzymes are contained in this complex, no candidate biomarkers for diagnosis or disease activity have been identified. Therefore, not only has a standard method for objectively and quantitatively measuring eosinophil extracellular traps not been established, but it is also completely unknown that they are useful as biomarkers for any disease.

[0006] Non-Patent Document 1: Iino Y. et al., Diagnostic criteria of eosinophilic otitis media, a newly recognized middle ear disease, Auris Nasus Larynx, 2011, Vol.38, p.456-461. Non-Patent Document 2: Harabuchi Y, Kishibe K, Tateyama K, et al., Clinical features and treatment outcomes of otitis media with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (OMAAV): A retrospective analysis of 235 patients from a nationwide survey in Japan, Mod Rheumatol, 2017, Vol.27, p.87-94. Non-Patent Document 3: Ueki S, et al., Eosinophilic Otitis Media: the Aftermath of Eosinophil Extracellular Trap Cell Death, Curr Allergy Asthma Rep., 2017,17(5):33. Non-Patent Document 4: Brinkmann V, et al., Neutrophil extracellular traps kill bacteria, Science, 2004, Vol.303, p.1532-1535.

[0007] The problem to be solved by the present invention is to provide a biomarker that accurately diagnoses, assists in diagnosis, judges, and / or assists in judgment of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media from other otitis media or diseases, and a method for performing one or more of the above-mentioned diagnosis, diagnostic aid, judges, and / or judges.Furthermore, the present invention is to provide a biomarker that accurately diagnoses, assists in diagnosis, judges, and / or assists in judgment of eosinophilic otitis media and various ANCA-associated vasculitic otitis media, both of which are intractable otitis media and difficult to distinguish from each other based on their initial symptoms, and a method therefor.

[0008] The present inventors have focused on the fact that in eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, a special type of cell death called ETosis occurs, resulting in the release of extracellular traps. They have closely examined the substances contained in these traps and found that using EPO-DNA complexes as biomarkers and their expression levels as indicators is useful for one or more of the early diagnosis, diagnostic assistance, judgment, and judgment assistance of these diseases, and have completed the present invention.

[0009] Furthermore, the inventors have discovered that by using the expression level of this EPO-DNA complex and the expression level of the MPO-DNA complex contained in neutrophil extracellular traps as biomarkers as indicators, one or more of the following can be accurately diagnosed, aided in diagnosis, judged, and aided in judgement of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media and acute otitis media which are common types of otitis media, and acute sinusitis, and have completed the present invention.

[0010] According to the present invention, highly accurate data useful for diagnosing eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitis media limited to the middle ear, otitis media with effusion, acute otitis media, and acute sinusitis can be obtained. This enables early detection of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitis media limited to the middle ear, and appropriate treatment can be performed, which contributes to preventing serious complications associated with these conditions and reducing the mortality rate. Furthermore, the present invention can prevent delays in the initiation of treatment for eosinophilic otitis media, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitis media limited to the middle ear, which can lead to deafness, and by avoiding the need for cochlear implantation, etc., this leads to an improvement in the quality of life (QOL) of patients and a reduction in medical costs.

[0011] 1 shows EPO-DNA complex levels measured by ELISA using middle ear fluid collected from patients suffering from one or more of eosinophilic otitis media, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, and otitis media with effusion. An "*" in the figure indicates a statistically significant difference (p<0.05) after one-way analysis of variance. 1 shows EPO-DNA complex levels measured by ELISA using middle ear fluid or nasal discharge collected from patients suffering from one or more of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, otitis media with effusion, acute otitis media, and acute sinusitis. An "*" in the figure indicates a statistically significant difference (p<0.05) after one-way analysis of variance. This figure shows the optimal cutoff value for the expression level of the EPO-DNA complex determined using an ROC curve. This figure shows the optimal cutoff value for the expression level of the EPO-DNA complex determined using an ROC curve. This figure shows MPO-DNA complex levels measured by ELISA using middle ear fluid collected from patients suffering from one or more of the following: eosinophilic otitis media, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, and otitis media with effusion. An "*" in the figure indicates a statistically significant difference (p<0.05) after one-way analysis of variance.

[0023] Figure 1 shows MPO-DNA complex levels measured by ELISA using middle ear fluid or nasal discharge collected from patients suffering from one or more of the following: eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, otitis media with effusion, acute otitis media, and acute sinusitis. An "*" in the figure indicates a statistically significant difference (p<0.05) after one-way analysis of variance. Figure 2 shows the optimal cutoff value for MPO-DNA complex expression level determined using an ROC curve. Figure 3 shows the optimal cutoff value for MPO-DNA complex expression level determined using an ROC curve.This is an identification table that serves as an index for diagnosing, assisting in diagnosis, determining, and assisting in determination of eosinophilic otitis media patients, eosinophilic sinusitis patients, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis, based on the expression levels of EPO-DNA complexes and MPO-DNA in a subject.

[0012] The present invention is as follows: [1] A biomarker for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, using at least an EPO-DNA complex contained in an eosinophil extracellular trap. [2] The biomarker according to [1] above, further characterized by being used in combination with a biomarker for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media, using an MPO-DNA complex contained in a neutrophil extracellular trap. [3] The method comprises the following steps (a), (c'), and (d): (a): measuring the expression level of an EPO-DNA complex contained in an eosinophil extracellular trap in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal secretion sample collected from a subject; (c'): comparing the expression level of the EPO-DNA complex measured in step (a), and, if step (b) is optionally included, the expression level of the MPO-DNA complex, with the expression level of the EPO-DNA complex in a control subject, and, if step (b) is optionally included, the expression level of the MPO-DNA complex in a control subject; (d): assessing that the subject is likely to suffer from at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, if the expression level of the EPO-DNA complex measured in step (a) is higher than that of the EPO-DNA complex in the control subject; and optionally further comprising step (b); (b): measuring the expression level of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample collected from a subject; and[4] The following steps (a), (b), (c) and (d-1): (a): measuring the expression level of EPO-DNA complex contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complex contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (c): comparing the expression levels of EPO-DNA complex and MPO-DNA complex measured in steps (a) and (b) with the expression levels of EPO-DNA complex and MPO-DNA complex in a control subject; (d-1): assessing that the subject is likely to be affected by eosinophilic granulomatosis with polyangiitis when both the expression levels of EPO-DNA complex and MPO-DNA complex measured in steps (a) and (b) are higher than the expression levels of EPO-DNA complex and MPO-DNA complex in the control subject; A method for diagnosing, assisting in diagnosis, and assisting in judgment of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, ANCA-associated vasculitic otitis media confined to the middle ear, otitis media with effusion, acute otitis media, and acute sinusitis, comprising:[5] The following steps (a), (b), (c) and (d-2): (a): measuring the expression level of EPO-DNA complex contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complex contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (c): comparing the expression levels of EPO-DNA complex and MPO-DNA complex measured in steps (a) and (b) with the expression levels of EPO-DNA complex and MPO-DNA complex in a control subject; (d-2): a step of assessing that the subject is likely to be suffering from eosinophilic otitis media and / or eosinophilic sinusitis, when the expression level of the EPO-DNA complex measured in step (a) is higher than that in the control subject but the expression level of the MPO-DNA complex measured in step (b) is unchanged from that in the control subject; and[6] The following steps (a), (b), (c) and (d-3): (a): measuring the expression level of EPO-DNA complex contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complex contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (c): comparing the expression levels of EPO-DNA complex and MPO-DNA complex measured in steps (a) and (b) with the expression levels of EPO-DNA complex and MPO-DNA complex in a control subject; (d-3): a step of evaluating the subject to be highly likely to be suffering from microscopic polyangiitis, granulomatosis with polyangiitis, or middle ear-limited ANCA-associated vasculitic otitis media, when the expression level of the EPO-DNA complex measured in step (a) is not different from that in the control subject, but the expression level of the MPO-DNA complex measured in step (b) is higher than that in the control subject;[7] The following steps (a), (b), (c) and (d-4): (a): measuring the expression level of EPO-DNA complex contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complex contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample and a nasal discharge sample collected from a subject; (c): comparing the expression levels of EPO-DNA complex and MPO-DNA complex measured in steps (a) and (b) with the expression levels of EPO-DNA complex and MPO-DNA complex in a control subject; (d-4): assessing that the subject is likely to be suffering from otitis media with effusion, acute otitis media, or acute sinusitis when both the expression levels of the EPO-DNA complex and the MPO-DNA complex measured in steps (a) and (b) are unchanged from those in the control. [8] The method according to any one of items [3] to [7] above, wherein the control is a healthy subject, a patient with otitis media with effusion, a patient with acute otitis media, or a patient with acute sinusitis. [9] A kit for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, characterized by comprising one or more specific antibodies and their labels for detecting the expression of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal secretion sample.

[10] The kit according to [9] above, further comprising one or more specific antibodies and markers thereof for detecting the expression of MPO-DNA complexes contained in neutrophil extracellular traps, for performing one or more of the following: diagnosis, diagnostic aid, judgment, and judgment aid for at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, otitis media with effusion, acute otitis media, and acute sinusitis, characterized by further comprising one or more specific antibodies and markers thereof for detecting the expression of MPO-DNA complexes contained in neutrophil extracellular traps.

[0013] Of the present invention, the method for diagnosing, assisting in diagnosis, determining, and assisting in determination as set forth in [1] to

[10] above (hereinafter, may be referred to as "the method") is a method for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis, but is not limited to the above steps as long as it includes these. The method is a method for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis, and does not include a diagnostic act by a physician.

[0014] Another embodiment of the present invention is a method for collecting data for one or more of diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis, the method comprising the above steps (a) and (c), and optionally any one or more of (b) and (d), (d-1), (d-2), (d-3), and (d-4); or The method comprises the steps (a) and (c), and optionally one or more of steps (b) and (d), (d-1), (d-2), (d-3), and (d-4), and further comprises a step of administering treatment to a subject diagnosed or determined to be highly likely to be suffering from at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, otitis media with effusion, acute otitis media, and acute sinusitis in any one or more of steps (d-1), (d-2), (d-3), and (d-4), wherein the subject is treated for the disease. Examples of the specific antibodies include specific antibodies and labeled products thereof for detecting expression of MPO-DNA complexes contained in neutrophil extracellular traps and expression of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, or a nasal discharge sample, for one or more of diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, otitis media with effusion, acute otitis media, and acute sinusitis.

[0015] As used herein, "eosinophilic otitis media" refers to a condition in which one major item of the diagnostic criteria proposed by Iino et al. is met, and two or more minor items are met: Major item) Serous otitis media or chronic otitis media with significant eosinophilic middle ear fluid; Minor items) (1) Colloidal middle ear fluid, (2) Resistance to conventional treatments for otitis media, (3) Concomitant bronchial asthma, (4) Concomitant nasal polyps.

[0016] In this specification, "eosinophilic sinusitis" is diagnosed based on the JESREC score, where (1) affected side: both sides, 3 points; (2) presence of nasal polyps, 2 points; (3) presence of predominant ethmoid sinus shadows on CT, 2 points; and (4) peripheral blood eosinophils (%): 2 < ≦ 5, 4 points; 5 < ≦ 10, 8 points; and 10 < 10 points, with a total JESREC score of 11 points or more and 70 or more eosinophils in the nasal polyp tissue (400x magnification field). Furthermore, if eosinophilic otitis media is present, the condition is classified as severe.

[0017] In this specification, "eosinophilic granulomatosis with polyangiitis" is diagnosed based on the following criteria: (1) a history of asthma, (2) eosinophilia of 10% or more in peripheral blood, (3) mono- or polyneuropathy, (4) non-fixed pulmonary infiltrates, (5) sinus abnormalities, and (6) evidence of extravascular eosinophilic infiltration on biopsy.

[0018] The common mechanism of onset of these three diseases is the release of granulocyte extracellular traps when a special type of cell death called ETosis occurs, and it is thought that eosinophil extracellular traps in particular are involved.

[0019] In particular, by examining the expression levels of EPO-DNA complexes, which are substances specific to eosinophil extracellular traps due to differences in the cytoplasmic enzymes they contain, in eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, and general otitis media, it is possible to perform one or more of the following: diagnosis, diagnostic assistance, determination, and determination assistance for at least one of these conditions. This is the first aspect of the present invention.

[0020] In contrast, other diseases that cause clinical symptoms in the otolaryngological field, which are known to similarly cause ETosis, do not produce eosinophil extracellular traps. Rather, neutrophil extracellular traps are believed to be involved in ETosis (Non-Patent Document 4). As a result, otitis media due to microscopic polyangiitis, granulomatosis with polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear are known to present with clinical symptoms in the otolaryngological field.

[0021] Microscopic polyangiitis (MPA) is a disease with a poor prognosis that presents with vasculitis and systemic symptoms such as fever, anorexia, and weight loss, in addition to rapidly progressive glomerulonephritis and interstitial pneumonia. In recent years, there have been a relatively large number of cases in which ear symptoms appear at the onset or during the course of the disease, in addition to systemic symptoms affecting the upper respiratory tract, lungs, kidneys, and heart. This has been shown to be one of the intractable otitis media that are difficult to diagnose and treat.

[0022] According to the Ministry of Health, Labor and Welfare's criteria, the diagnostic criteria for microscopic polyangiitis are: (1) major symptoms include (a) rapidly progressive glomerulonephritis, (b) alveolar hemorrhage or interstitial pneumonia, (c) organ symptoms other than the kidneys and lungs: purpura, subcutaneous bleeding, gastrointestinal bleeding, mononeuritis multiplex, etc.; (2) major tissue findings include necrosis of arterioles, capillaries, and postcapillary venules, and perivascular inflammatory cell infiltration; and (3) major laboratory findings include positive MPO-ANCA. If there are positive CRP, proteinuria / hematuria, elevated serum BUN and Cr, and chest X-rays show infiltrates or interstitial pneumonia suggestive of alveolar hemorrhage, then (4) the diagnosis is definite if two or more of the major symptoms are met and the tissue findings are positive, or if two or more of the major symptoms including (a) and (b) are met and MPO-ANCA is positive, or suspected if three of the major symptoms are met, or if one of the major symptoms and MPO-ANCA is positive.

[0023] Granulomatosis with polyangiitis (GPA) is a refractory systemic vasculitis characterized by three features: (1) necrotizing granulomatous lesions of the nose, ears, eyes, upper respiratory tract, and lungs; (2) necrotizing granulomatous vasculitis of small and medium-sized vessels throughout the body; and (3) necrotizing crescentic nephritis of the kidneys. More than 80% of cases initially present with upper respiratory tract lesions, but many cases have also been reported in which ear lesions were present at initial consultation. Granulomatosis with polyangiitis often presents with facial nerve paralysis and hypertrophic pachymeningitis, and there have been increasing reports of refractory otitis media accompanied by MPO-ANCA-positive, rapidly progressive sensorineural hearing loss, and facial nerve paralysis.

[0024] The diagnostic criteria for granulomatosis with polyangiitis, according to the Ministry of Health, Labour and Welfare criteria, are as follows: 1. Major symptoms (1) Upper respiratory tract (E) symptoms: E: nose (purulent rhinorrhea, bleeding, saddle nose), eyes (eye pain, decreased vision, exophthalmos), ears (otitis media), oral cavity / pharyngeal pain (ulcers, hoarseness, airway obstruction), (2) Lung (L) symptoms: L: bloody sputum, cough, dyspnea, (3) Kidney (K) symptoms: hematuria, proteinuria, rapidly progressive renal failure, edema, hypertension, (4) Symptoms of vasculitis: <1> Systemic symptoms: fever (38°C or higher, for more than 2 weeks), weight loss (6 kg or more within 6 months), <2> Organ symptoms: purpura, polyarthritis (pain), episcleritis, polyneuritis, ischemic heart disease (angina pectoris / myocardial infarction), gastrointestinal bleeding (vomiting blood / hemorrhage), pleurisy, Major tissue findings: <1> Necrotizing granulomatous inflammation accompanied by giant cells of E, L, and K, <2> Necrotizing crescentic nephritis without immunoglobulin deposition, <3> Necrotizing granulomatous vasculitis of small arterioles 3. Major laboratory findings: Proteinase 3-ANCA (PR3-ANCA) (cytoplasmic pattern, C-ANCA by fluorescent antibody method) is highly positive.

[0025] The results were judged as follows: (a) cases showing three or more major symptoms, including symptoms in one organ each of the upper respiratory tract (E), lungs (L), and kidneys (K); (b) cases showing two or more major symptoms of the upper respiratory tract (E), lungs (L), kidneys (K), and vasculitis, and one or more histological findings <1>, <2>, and <3>; (c) cases showing one or more major symptoms of the upper respiratory tract (E), lungs (L), kidneys (K), and vasculitis, and one or more histological findings <1>, <2>, and <3>, and C(PR3). -ANCA positive cases are considered to be definite, and suspected cases are (a) cases showing two or more of the symptoms of the upper respiratory tract (E), lungs (L), kidneys (K), and major symptoms of vasculitis, (b) cases showing one of the symptoms of the upper respiratory tract (E), lungs (L), kidneys (K), and major symptoms of vasculitis and one of the histological findings <1>, <2>, or <3>, and (c) cases showing one of the symptoms of the upper respiratory tract (E), lungs (L), kidneys (K), and major symptoms of vasculitis and C(PR3)-ANCA positive.

[0026] These microscopic polyangiitis, granulomatosis with polyangiitis, and eosinophilic granulomatosis with polyangiitis share common clinical features, are PR3-ANCA or MPO-ANCA positive, and antineutrophil cytoplasmic antibodies (ANCA) are deeply involved in the pathology, leading to the name ANCA-associated vasculitic otitis media. However, the ANCA positivity rate in eosinophilic granulomatosis with polyangiitis is only 40-50%, and they differ in that microscopic polyangiitis and granulomatosis with polyangiitis are characterized by infiltration of neutrophils and macrophages, whereas eosinophilic granulomatosis is characterized by infiltration of eosinophils.

[0027] This series of syndromes known as ANCA-associated vasculitis is an intractable, life-threatening disease that causes a variety of symptoms affecting the kidneys, lungs, nerves, skin, and eyes. It is known to develop after an upper respiratory tract infection and frequently presents with clinical symptoms in the otolaryngological field. ANCA-associated vasculitic otitis media presenting as otitis media not only progresses to hearing loss but also rapidly progresses to systemic disease, leading to a fatal condition. Therefore, early diagnosis and treatment initiation are desirable. However, many cases show negative results for ANCA on hematological testing or do not reveal characteristic vasculitis on histopathological examination, making it time-consuming to reach a definitive diagnosis. In particular, in cases of localized middle ear disease, it is difficult to diagnose or distinguish from serous otitis media or acute otitis media, which are common types of otitis media caused by Eustachian tube dysfunction, at the initial consultation.

[0028] In this specification, "ANCA-associated vasculitic otitis media" means a condition in which all of A), B), and C) of the following diagnostic criteria proposed by the "ANCA-associated vasculitic otitis media working group" of the Japan Otological Society are met: A) Clinical course (one or more of the following two items are met): 1. Otitis media that is not responsive to antibiotics or tympanostomy tubes 2. Progressive increase in bone conduction threshold B) Findings (one or more of the following items are met): 1. Already diagnosed with ANCA-associated vasculitis syndrome 2. Positive serum MPO-ANCA or serum PR3-ANCA 3. Findings consistent with vasculitis (either (1) or (2)) are seen in biopsy tissue (1) Necrotizing granulomatous inflammation with giant cells, (2) Necrotizing vasculitis of small and small arteries 4. Reference findings, complications, and sequelae (one or more of (1) to (5) applies) (1) Upper respiratory tract infection other than the ear, scleritis, pulmonary lesions, renal lesions, (2) facial nerve paralysis, (3) hypertrophic pachymeningitis, (4) mononeuritis multiplex, (5) symptoms and findings improve with corticosteroid administration but recur when the administration is discontinued. C) Differential diagnosis (the following diseases are ruled out) (1) cholesteatomatous otitis media, (2) cholesterol granuloma, (3) eosinophilic otitis media, (4) tuberculous otitis media, (5) malignant otitis externa, (6) skull base osteomyelitis, (7) neoplastic disease, (8) autoimmune disease other than ANCA-associated vasculitis syndrome.

[0029] The above-mentioned phrase "positive for MPO-ANCA or PR3-ANCA" means that the MPO-ANCA antibody titer or PR3-ANCA antibody titer in the blood is equal to or greater than the reference value. Conversely, the phrase "negative for MPO-ANCA or PR3-ANCA" means that the MPO-ANCA antibody titer or PR3-ANCA antibody titer in the serum is below the reference value. This reference value cannot be specified in general because it differs depending on the diagnostic criteria, individual facilities, and measurement methods; however, for example, it is approximately 2.0 U / mL when measured by the commonly used ELISA (Enzyme-Linked Immunosorbent Assay) method.

[0030] Furthermore, the above-mentioned "findings consistent with vasculitis in biopsy tissue" means that findings of "necrotizing granulomatous inflammation with giant cells" or "necrotizing vasculitis of small arterioles" are observed in histopathological examination.

[0031] Among the various diseases in which ETosis is involved, serous otitis media, a common type of otitis media in which exudate accumulates in the middle ear, is caused by Eustachian tube dysfunction and does not produce the two extracellular traps described above. Acute otitis media, another common type of otitis media, is a condition in which pus accumulates in the middle ear. Although extracellular traps may be produced as a defense mechanism against infection, the generated extracellular traps are quickly degraded, preventing excessive production and prolonged retention of the two extracellular traps in the body. Therefore, in the present invention, serous otitis media and acute otitis media are considered equivalent in that the expression levels of EPO-DNA complex and MPO-DNA complex are considered negative.

[0032] In view of the above-mentioned differences in the mechanisms of onset, by using a combination of the expression levels of EPO-DNA complexes contained in eosinophil extracellular traps and MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal secretion sample as an indicator, it is possible to accurately diagnose, assist in diagnosis, determine, and / or assist in determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media (common otitis media), acute otitis media, and acute sinusitis. This is the second aspect of the present invention.

[0033] Furthermore, as per the diagnostic criteria described above, both eosinophilic otitis media and eosinophilic granulomatosis with polyangiitis are accompanied by asthma, chronic sinusitis, and increased eosinophils in the peripheral blood and tissues, and therefore have similar clinical symptoms. Therefore, it is not easy to differentiate eosinophilic otitis media in the early stages from one or more of eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and middle ear-limited ANCA-associated vasculitis otitis media. In fact, even when diagnosed as eosinophilic otitis media, systemic symptoms may develop over the course of the disease, leading to a final diagnosis of eosinophilic granulomatosis with polyangiitis.

[0034] Therefore, the search for a new biomarker that can accurately differentiate between eosinophilic otitis media and eosinophilic granulomatosis with polyangiitis and enable early diagnosis would be of great clinical significance. A third aspect of the present invention is to combine the MPO-DNA complex contained in neutrophil extracellular traps and the EPO-DNA complex contained in eosinophil extracellular traps as biomarkers, thereby enabling one or more of the following: diagnosis, diagnostic assistance, determination, and determination assistance of eosinophilic otitis media and eosinophilic granulomatosis with polyangiitis.

[0035] The biomarker of the present invention for performing one or more of the diagnosis, diagnostic aid, judgment and judgment aid of eosinophilic otitis media is a biomarker for performing one or more of the diagnosis, diagnostic aid, judgment and judgment aid of eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media and acute sinusitis, which comprises an MPO-DNA complex contained in a neutrophil extracellular trap and an EPO-DNA complex contained in an eosinophil extracellular trap. There are no particular limitations on the biomarker (hereinafter sometimes referred to as the "biomarker"), provided that the expression level of the EPO-DNA complex and / or the expression level of the MPO-DNA complex in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample collected from a subject suffering from the disease is higher than the expression levels of the EPO-DNA complex and the MPO-DNA complex in a control subject, and that the biomarker performs one or more of the above-mentioned functions of diagnosis, diagnostic assistance, judgment, and judgment assistance.

[0036] As used herein, a "subject" refers to a person who may or may not be affected by eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, or ANCA-associated vasculitic otitis media limited to the middle ear. In the Examples described below, it is specifically shown that the device can accurately diagnose, assist in diagnosis, make a determination, or assist in the determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitis media limited to the middle ear, and other common otitis media such as serous otitis media, acute otitis media, and acute sinusitis. Therefore, the target patient is preferably a person who has had or currently has signs or symptoms of otitis media and is uncertain as to whether or not they are suffering from one or more of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitis media limited to the middle ear, otitis media with effusion, acute otitis media, or acute sinusitis. The above subjects also include those who have previously suffered from one or more of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media confined to the middle ear, otitis media with effusion, acute otitis media, and acute sinusitis, and have subsequently achieved remission, but it is unknown whether or not the disease has recurred.

[0037] The biological sample that can be used herein is preferably a liquid sample, such as at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample. Considering the purpose of differentiating one or more of eosinophilic otitis media, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media from common otitis media with effusion and acute otitis media, a middle ear fluid sample is the most preferred biological sample. A nasal discharge sample is the most preferred biological sample for differentiating eosinophilic sinusitis from acute sinusitis.

[0038] As used herein, a "peripheral blood sample" can be collected using a known method. Specifically, peripheral blood collected from a subject or control is mixed with a separating agent, centrifuged, and the serum is used as a sample.

[0039] As used herein, a "middle ear fluid sample" can be collected by tympanic membrane puncture, a procedure commonly performed by otolaryngologists. Specifically, middle ear fluid collected from a subject or control is centrifuged, and the supernatant is used as the sample. This procedure allows the separation of fractions containing neutrophil extracellular traps and eosinophil extracellular traps from fractions containing other impurities.

[0040] As used herein, a "nasal discharge sample" can be collected by a nasal procedure commonly performed by otolaryngologists. Specifically, nasal discharge collected from a subject or control subject is centrifuged, and the supernatant is used as the sample. This procedure allows the separation of fractions containing neutrophil extracellular traps and eosinophil extracellular traps from fractions containing other impurities.

[0041] In the present method and the present biomarker, the expression levels of EPO-DNA complexes contained in eosinophil extracellular traps and MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample can be measured using any immunoassay method known in the art. Examples include radioimmunoassays (RIA), enzyme immunoassays (EIA) such as ELISA, latex agglutination assays (LTIA), and immunochromatography. There are no particular limitations on the method, as long as it can specifically detect part or all of the MPO-DNA complex and the EPO-DNA complex.

[0042] For example, the expression levels of MPO-DNA complexes contained in neutrophil extracellular traps and EPO-DNA complexes contained in eosinophil extracellular traps can be measured using a known sandwich ELISA method that uses antibodies that specifically recognize the expression levels of MPO-DNA complexes contained in eosinophil extracellular traps. From the viewpoint of improving detection sensitivity, the sandwich method is preferred.

[0043] Other known immunological assays include immunochromatography, immunoblotting, CBA (Cytometric Bead Assay), Western blotting, and flow cytometry. From the viewpoint of simplicity and speed, immunochromatography is preferred. When immunochromatography is used, diagnosis can be performed in a short time at the bedside or while the patient is visiting the hospital.

[0044] In the present method and the present biomarker, the expression levels of EPO-DNA complexes contained in eosinophil extracellular traps and MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample may be absolute values ​​or relative values ​​using a reference sample as a comparison standard.

[0045] In step (a) of the method, the expression level of the EPO-DNA complex contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal secretion sample collected from the subject is measured; in step (c'), the expression level of the EPO-DNA complex in the subject is compared with the expression level of the EPO-DNA complex in a control; and in step (d), if the expression level of the EPO-DNA complex in the subject is higher than the expression level of the EPO-DNA complex in the control, the subject is assessed as having a high probability of suffering from at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis.

[0046] The present method comprising the above steps (a), (c'), and (d) can further comprise the step of (b) measuring the expression level of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample collected from a subject.

[0047] In this case, in steps (a) and (b), the expression levels of both the EPO-DNA complex and the MPO-DNA complex are measured, and in step (c'), the expression levels of the MPO-DNA complex and the EPO-DNA complex in the subject are compared with the expression levels of the MPO-DNA complex and the EPO-DNA complex in the control, respectively. In step (d), if the expression level of the EPO-DNA complex in the subject is higher than that in the control, it is possible to assess that the subject is highly likely to be suffering from at least one or more of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, and it is also possible to perform one or more of the following diagnosis, diagnostic assistance, judgment, and judgment assistance for the following diseases:

[0048] Specifically, by including the following steps (a), (b), (c), and (d-1), it is possible to evaluate that a subject is highly likely to be suffering from eosinophilic granulomatosis with polyangiitis. (a): measuring the expression level of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from a subject; (c): comparing the expression levels of EPO-DNA complexes and MPO-DNA complexes measured in steps (a) and (b) with the expression levels of EPO-DNA complexes and MPO-DNA complexes in a control subject; (d-1): assessing that the subject is likely to be affected by eosinophilic granulomatosis with polyangiitis when both the expression levels of EPO-DNA complexes and MPO-DNA complexes measured in steps (a) and (b) are higher than the expression levels of EPO-DNA complexes and MPO-DNA complexes in the control subject;

[0049] By including the following steps (a), (b), (c), and (d-2), it is possible to evaluate that a subject is likely to suffer from eosinophilic otitis media and / or eosinophilic sinusitis. (a): measuring the expression level of EPO-DNA complex contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complex contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from a subject; (c): comparing the expression levels of EPO-DNA complex and MPO-DNA complex measured in steps (a) and (b) with the expression levels of EPO-DNA complex and MPO-DNA complex in a control subject; (d-2): assessing that the subject is likely to be suffering from eosinophilic otitis media and / or eosinophilic sinusitis when the expression level of the EPO-DNA complex measured in step (a) is higher than that in the control subject but the expression level of the MPO-DNA complex measured in step (b) is not different from that in the control subject;

[0050] The method comprises the following steps (a), (b), (c), and (d-3), which enable a subject to be assessed as having a high likelihood of suffering from one or more of microscopic polyangiitis, granulomatosis with polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear: (a): measuring the expression level of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from the subject; (b): measuring the expression level of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from the subject; (c): comparing the expression levels of EPO-DNA complexes and MPO-DNA complexes measured in steps (a) and (b) with the expression levels of EPO-DNA complexes and MPO-DNA complexes in a control subject; (d-3): assessing that the subject is highly likely to be suffering from microscopic polyangiitis, granulomatosis with polyangiitis, or middle ear-limited ANCA-associated vasculitic otitis media, when the expression level of the EPO-DNA complex measured in step (a) is not different from that in the control subject, but the expression level of the MPO-DNA complex measured in step (b) is higher than that in the control subject;

[0051] By including the following steps (a), (b), (c), and (d-4), it is possible to evaluate whether a subject is likely to suffer from serous otitis media, acute otitis media, or acute sinusitis. (a): measuring the expression level of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample collected from a subject; (c): comparing the expression levels of EPO-DNA complexes and MPO-DNA complexes measured in steps (a) and (b) with those in a control subject; (d-4): assessing that the subject is likely to be suffering from otitis media with effusion, acute otitis media, or acute sinusitis, when both the expression levels of EPO-DNA complexes and MPO-DNA complexes measured in steps (a) and (b) are not different from those in the control subject;

[0052] The measurement and comparison of the expression levels of the EPO-DNA complex and the MPO-DNA complex in steps (a) and (b) and steps (c') and (c) above can be carried out in any order, and the desired effects of the method can be obtained regardless of the order in which they are carried out.

[0053] Any threshold (cutoff value) can be set as the threshold used to determine whether the "expression level of MPO-DNA complex and expression level of EPO-DNA complex in a subject" is higher than the "expression level of MPO-DNA complex and expression level of EPO-DNA complex in a control." Examples of such thresholds include the mean value, "mean value + standard deviation (SD)," "mean value + 2SD," "mean value + 3SD," median, "median value + SD," "median value + 2SD," "median value + 3SD," etc., of the "expression level of MPO-DNA complex or EPO-DNA complex in a control." The threshold was calculated by calculating the receiver operating characteristic curve (ROC) using statistical analysis software based on the data on "the expression level of the MPO-DNA complex or the EPO-DNA complex in the subject" and the data on "the expression level of the MPO-DNA complex or the EPO-DNA complex in the control" so as to increase the sensitivity (the rate at which one or more of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media can be correctly determined as positive) and specificity (the rate at which subjects who are not affected by one or more of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media can be correctly determined as negative). It can also be calculated using a characteristic curve.

[0054] The kit of the present invention for diagnosing, assisting in diagnosis, determining, and assisting in determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, otitis media with effusion, acute otitis media, and acute sinusitis includes a kit for detecting the expression of MPO-DNA complexes contained in neutrophil extracellular traps and eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear effusion sample, and a nasal discharge sample. The kit is not particularly limited as long as it contains a specific antibody for detecting the expression of an EPO-DNA complex contained in the kit and a labeled antibody thereof, and is used to diagnose, assist in diagnosis, determine, or assist in determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis.

[0055] The present invention relating to the kit is a use invention relating to a kit for diagnosing, assisting in diagnosis, determining, or assisting in determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, otitis media with effusion, acute otitis media, and acute sinusitis. These kits may include antibodies that specifically recognize EPO-DNA complexes or MPO-DNA complexes. The kits of the present invention may also include other reagents or materials used in immunoassays. For example, the kit of the present invention typically includes, in addition to the antibody of the present invention, components generally used in this type of diagnostic kit, such as a carrier, a pH buffer, a stabilizer, a solid phase (e.g., a strip, a plate, beads, etc.) for immunochromatography, ELISA, or latex agglutination, and reagents such as labeling substances, as well as an instruction manual and package inserts such as instructions for diagnosing, assisting in diagnosis, determining, and assisting in determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis.

[0056] The antibody in the present kit may be any of monoclonal antibodies, polyclonal antibodies, human antibodies, chimeric antibodies, humanized antibodies, etc. The class of the antibody is not particularly limited, and may be any of IgG, IgD, IgE, IgA, sIgA, IgM, etc. Antibody binding fragments (Fab, Fab', F(ab') ２ etc.) are also included in the term "antibody" as long as they specifically bind to the biomarker.

[0057] Labels used in the immunoassay include any labels used in the art. The antibody in the present kit may be labeled with a labeling substance. Examples of such labeling substances include enzymes such as peroxidase (e.g., horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase, and acetylcholinesterase; fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride, and tetramethylrhodamine isothiocyanate; fluorescent substances such as FITC; fluorescent proteins such as green fluorescent protein, cyan fluorescent protein, blue fluorescent protein, yellow fluorescent protein, red fluorescent protein, and luciferase; ３ H. １４ C. ３２ P. １４ C. ３５ S or ３ H. １２５ I, or １３１ Examples of such a substance include visualization substances such as radioisotopes such as I, metal colloids, non-metal colloids, dye sols, dye particles such as disperse dyes, latex particles, and colored microparticles; biotin, avidin, a sensitization system using avidin labeled with the label after primary labeling with biotin; and substances having affinity for a low molecular weight substance such as digoxigenin, and antibodies labeled with the label after primary labeling with the low molecular weight substance.

[0058] The present kit for confirming the expression levels of the EPO-DNA complex and the MPO-DNA complex may be a single integrated kit, or separate kits for confirming the respective expression levels may be used in combination.

[0059] The present invention will be specifically explained below with reference to examples, but the technical scope of the present invention is not limited to these examples.

[0060] [Example 1A] Confirmation that measurement of the expression level of EPO-DNA complex can provide one or more of the diagnosis, diagnostic aid, determination and determination aid of eosinophilic otitis media or eosinophilic granulomatosis with polyangiitis.

[0061] (Sample Collection) Middle ear fluid samples were collected by tympanic membrane puncture from 13 patients with eosinophilic otitis media, 11 patients with eosinophilic granulomatosis with polyangiitis, and 23 control patients with serous otitis media. The samples were centrifuged at 1500 rpm for 5 minutes, and the supernatants were frozen and stored at -80°C until measurement. For samples with a viscous nature, the supernatants were extracted after treatment with a lysis buffer to dissolve the samples, or after shredding and stirring using microscissors or a homogenizer.

[0062] (Method for measuring EPO-DNA complexes) EPO mouse monoclonal antibody (ab224506; Abcam) was adsorbed onto a 96-well microplate and blocked with 1% bovine serum albumin. The sample was added to the 96-well microplate along with a cell death detection antibody (Cell Death Detection ELISA kit; Roche) and allowed to react. After washing with phosphate-buffered saline, color development was carried out using a peroxidase substrate, and the absorbance was measured at 405 nm using a microplate reader (Bio-Rad 680; Bio-Rad Laboratories).

[0063] (Measurement results of EPO-DNA complex) The measured EPO-DNA complex level in the middle ear fluid ranged from 1.17 to 78.2 OD in patients with eosinophilic otitis media. ４０５ (median 8.74 OD ４０５), and eosinophilic granulomatosis patients had 2.33-25.8 OD ４０５ (median 8.92 OD ４０５ ), and in patients with otitis media with effusion, 0.03-0.58 OD ４０５ (median 0.19 OD ４０５ ) and the EPO-DNA complex levels in patients with eosinophilic otitis media and eosinophilic granulomatosis with polyangiitis were significantly higher than those in patients with otitis media with effusion (see Figure 1A).

[0064] (Setting the cutoff value for the expression level of the EPO-DNA complex) When an ROC curve was created based on the expression levels of the EPO-DNA complex in patients with eosinophilic otitis media, patients with eosinophilic granulomatosis with polyangiitis, and patients with otitis media with effusion, the AUC value was a high value of 0.96. The optimal cutoff value for the EPO-DNA complex level for distinguishing patients with eosinophilic otitis media and patients with eosinophilic granulomatosis with polyangiitis from patients with otitis media with effusion was set at 2.25 OD ４０５ When set to , the sensitivity was 95.7% and the specificity was 91.5% (see Figure 2A).

[0065] [Example 1B] Confirmation that measurement of the expression level of EPO-DNA complexes can provide one or more of the diagnosis, diagnostic aid, determination, and determination aid for eosinophilic otitis media, eosinophilic sinusitis, or eosinophilic granulomatosis with polyangiitis.

[0066] (Sample Collection) Samples were collected from 13 patients with eosinophilic otitis media, 13 patients with eosinophilic sinusitis, 15 patients with eosinophilic granulomatosis with polyangiitis, and 32 patients with otitis media with effusion, 14 patients with acute otitis media, 5 patients with acute sinusitis, 11 patients with granulomatosis with polyangiitis, 9 patients with microscopic polyangiitis, and 8 patients with ANCA-associated vasculitic otitis media limited to the middle ear. Middle ear fluid was collected by tympanic membrane puncture or nasal aspiration. The samples were centrifuged at 1500 rpm for 5 minutes, and the supernatants were frozen and stored at −80°C until measurement. For viscous samples, the supernatants were extracted after treatment with lysis buffer or after shredding and stirring using microscissors or a homogenizer.

[0067] (Method for measuring EPO-DNA complex) The expression level of the EPO-DNA complex was measured in the same manner as in Example 1A.

[0068] (Measurement Results of EPO-DNA Complex) The measured EPO-DNA complex levels in middle ear fluid or nasal secretions were 4.55 to 83.3 OD in patients with eosinophilic otitis media and eosinophilic sinusitis. ４０５ (median 14.4 OD ４０５ ), and in patients with eosinophilic granulomatosis with polyangiitis, 6.28-39.9 OD ４０５ (median 17.8 OD ４０５ ), and in patients with otitis media with effusion, 0.01 to 3.57 OD ４０５ (median 0.07 OD ４０５ ), 0.01 to 16.5 OD in patients with acute otitis media and acute sinusitis ４０５ (median 2.08 OD ４０５ ), and in patients with granulomatosis with polyangiitis, 0.66 to 5.85 OD ４０５ (median 2.01 OD ４０５ ), and 0.13-25.8 OD in patients with microscopic polyangiitis. ４０５ (median 1.84 OD ４０５ ), and in patients with ANCA-associated vasculitic otitis media limited to the middle ear, 1.12-15.0 OD ４０５ (median 1.67 OD ４０５ ), and the EPO-DNA complex levels in patients with eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis were significantly higher than those in patients with otitis media with effusion, acute otitis media, acute sinusitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear, respectively (see Figure 1B).

[0069] (Setting the cutoff value for the expression level of the EPO-DNA complex) An ROC curve was created based on the expression levels of the EPO-DNA complex in patients with eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, and serous otitis media, acute otitis media, acute sinusitis, granulomatosis with polyangiitis, microscopic polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media. The AUC value was as high as 0.93. The optimal cutoff value of EPO-DNA complex level for distinguishing patients with eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis from patients with otitis media with effusion, acute otitis media, acute sinusitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear was 5.62 OD. ４０５ When set to , the sensitivity was 97.6% and the specificity was 91.1% (see Figure 2B).

[0070] [Example 2A] Confirmation that measuring the expression levels of the EPO-DNA complex and the MPO-DNA complex can diagnose, aid in diagnosis, determine, or aid in determination of eosinophilic otitis media, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, or serous otitis media.

[0071] (Sample Collection) Middle ear fluid samples were collected by tympanic membrane puncture from 13 patients with eosinophilic otitis media, 11 patients with eosinophilic granulomatosis with polyangiitis, 9 patients with granulomatosis with polyangiitis, 5 patients with microscopic polyangiitis, 10 patients with ANCA-associated vasculitic otitis media limited to the middle ear, and 23 control patients with serous otitis media. The samples were centrifuged at 1500 rpm for 5 minutes, and the supernatants were frozen and stored at -80°C until measurement. For samples with a viscous nature, the supernatants were extracted after treatment with lysis buffer or after shredding and stirring using microscissors or a homogenizer.

[0072] (Method for measuring MPO-DNA complexes) MPO mouse monoclonal antibody (4A4; Bio-Rad Laboratories) was adsorbed onto a 96-well microplate and blocked with 1% bovine serum albumin. Samples were added to the 96-well microplate along with a cell death detection antibody (Cell Death Detection ELISA kit; Roche) and allowed to react. After washing with phosphate-buffered saline, color development was carried out using a peroxidase substrate, and absorbance was measured at 405 nm using a microplate reader (Bio-Rad 680; Bio-Rad Laboratories).

[0073] (Method for measuring EPO-DNA complex) The expression level of the EPO-DNA complex was measured in the same manner as in Example 1A.

[0074] (Measurement results of EPO-DNA complex) The measured EPO-DNA complex level in the middle ear fluid ranged from 1.17 to 78.2 OD in patients with eosinophilic otitis media. ４０５ (median 8.74 OD ４０５ ), and eosinophilic granulomatosis patients had 2.33-25.8 OD ４０５ (median 8.92 OD ４０５ ), and 0.14-3.17 OD in patients with granulomatosis with polyangiitis. ４０５ (median 1.72 OD ４０５ ), and 0.33 to 3.89 OD in patients with microscopic polyangiitis. ４０５ (median 1.33 OD ４０５ ), and in patients with ANCA-associated vasculitic otitis media limited to the middle ear, the OD was 1.12 to 4.92. ４０５ (median 1.43 OD ４０５ ), and in patients with otitis media with effusion, 0.03-0.58 OD ４０５ (median 0.19 OD ４０５ ), and the EPO-DNA complex levels in patients with eosinophilic otitis media and patients with eosinophilic granulomatosis with polyangiitis were significantly higher than those in patients with any one of granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, and otitis media with effusion (see Figure 1A).

[0075] (Setting the cutoff value for the expression level of the EPO-DNA complex) When an ROC curve was created based on the expression levels of the EPO-DNA complex in patients with eosinophilic otitis media, patients with eosinophilic granulomatosis with polyangiitis, patients with granulomatosis with polyangiitis, patients with microscopic polyangiosis, patients with ANCA-associated vasculitic otitis media limited to the middle ear, and patients with serous otitis media, the AUC value was as high as 0.96.

[0076] The optimal cutoff value of EPO-DNA complex level for distinguishing patients with eosinophilic otitis media and eosinophilic granulomatosis with polyangiitis from patients with granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, or otitis media with effusion was 2.25 OD. ４０５ When set to , the sensitivity was 95.7% and the specificity was 91.5% (see Figure 2A).

[0077] (Measurement Results of MPO-DNA Complex) The measured MPO-DNA complex level in the middle ear fluid was 0.01 to 0.35 OD in patients with eosinophilic otitis media. ４０５ (median 0.08 OD ４０５ ), eosinophilic granulomatosis with polyangiitis: 0.26-3.40 OD ４０５ (median 0.99 OD ４０５ ), and in patients with granulomatosis with polyangiitis, 0.17-2.08 OD ４０５ (median 1.15 OD ４０５ ), and 0.08-2.08 OD in patients with microscopic polyangiitis. ４０５ (median 1.01 OD ４０５ ), and in patients with ANCA-associated vasculitic otitis media limited to the middle ear, 0.14 to 1.70 OD ４０５ (median 0.66 OD ４０５ ), and in patients with otitis media with effusion, 0.01-0.14 OD ４０５ (median 0.08 OD ４０５ ), and the MPO-DNA complex levels in patients with one or more of eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear were significantly higher than those in patients with eosinophilic otitis media or otitis media with effusion (see Figure 3A).

[0078] (Setting the cutoff value for the expression level of the MPO-DNA complex) When an ROC curve was created based on the expression levels of the MPO-DNA complex in patients suffering from one or more of eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear, as well as in patients with eosinophilic otitis media and patients with otitis media with effusion, the AUC (area under the curve) value was a high value of 0.97.

[0079] The optimal cutoff value for MPO-DNA complex levels to differentiate patients with eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear from patients with eosinophilic otitis media and otitis media with effusion is 0.14 OD. ４０５ When set to , the sensitivity was 97.1% and the specificity was 94.4% (see Figure 4A).

[0080] [Example 2B] Confirmation that measuring the expression levels of the EPO-DNA complex and the MPO-DNA complex can diagnose, aid in diagnosis, determine, or aid in determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, or serous otitis media, acute otitis media, or acute sinusitis.

[0081] (Sample Collection) Middle ear fluid samples were collected by tympanic membrane puncture or nasal discharge samples were collected by nasal treatment from 13 patients with eosinophilic otitis media, 13 patients with eosinophilic sinusitis, 15 patients with eosinophilic granulomatosis with polyangiitis, 11 patients with granulomatosis with polyangiitis, 9 patients with microscopic polyangiitis, 8 patients with ANCA-associated vasculitic otitis media limited to the middle ear, and 32 patients with otitis media with effusion, 14 patients with acute otitis media, and 5 patients with acute sinusitis. The samples were centrifuged at 1500 rpm for 5 minutes, and the supernatants were frozen and stored at −80°C until assay. For viscous samples, the supernatants were extracted after treatment with lysis buffer or after shredding and stirring using microscissors or a homogenizer.

[0082] (Method for Measuring MPO-DNA Complex) The expression level of the MPO-DNA complex was measured in the same manner as in Example 2A.

[0083] (Method for measuring EPO-DNA complex) The expression level of the EPO-DNA complex was measured in the same manner as in Example 1A.

[0084] (Measurement Results of EPO-DNA Complex) The measured EPO-DNA complex levels in middle ear fluid or nasal secretions were 4.55 to 83.3 OD in patients with eosinophilic otitis media and eosinophilic sinusitis. ４０５ (median 14.4 OD ４０５ ), and in patients with eosinophilic granulomatosis with polyangiitis, 6.28-39.9 OD ４０５ (median 17.8 OD ４０５ ), and in patients with granulomatosis with polyangiitis, 0.66 to 5.85 OD ４０５ (median 2.01 OD ４０５ ), and 0.13-25.8 OD in patients with microscopic polyangiitis. ４０５ (median 1.84 OD ４０５ ), and in patients with ANCA-associated vasculitic otitis media limited to the middle ear, 1.12-15.0 OD ４０５ (median 1.67 OD ４０５ ), and in patients with otitis media with effusion, 0.01 to 3.57 OD ４０５ (median 0.07 OD ４０５ ), 0.01 to 16.5 OD in patients with acute otitis media and acute sinusitis ４０５ (median 2.08 OD ４０５ ), and the EPO-DNA complex levels in patients with eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis were significantly higher than those in patients with granulomatosis with polyangiitis, microscopic polyangiitis, ANCA-associated vasculitic otitis media limited to the middle ear, and otitis media with effusion, acute otitis media, and acute sinusitis (see Figure 1B).

[0085] (Setting the cutoff value for the expression level of the EPO-DNA complex) When an ROC curve was created based on the expression levels of the EPO-DNA complex in patients with eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyvascular patients, patients with ANCA-associated vasculitic otitis media limited to the middle ear and patients with serous otitis media, acute otitis media, and acute sinusitis, the AUC value was a high value of 0.93.

[0086] The optimal cutoff value of EPO-DNA complex level for distinguishing patients with eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis from patients with granulomatosis with polyangiitis, microscopic polyangiitis, patients with ANCA-associated vasculitic otitis media limited to the middle ear or patients with otitis media with effusion, acute otitis media, and acute sinusitis was 5.62 OD. ４０５ When set to , the sensitivity was 97.6% and the specificity was 91.1% (see Figure 2B).

[0087] (Measurement Results of MPO-DNA Complex) The measured MPO-DNA complex levels in middle ear fluid or nasal discharge were 0.01 to 9.80 OD in patients with eosinophilic otitis media and eosinophilic sinusitis. ４０５ (median 1.77 OD ４０５ ), and in patients with eosinophilic granulomatosis with polyangiitis, 0.11 to 34.6 OD ４０５ (median 11.0 OD ４０５ ), and in patients with granulomatosis with polyangiitis, 3.54 to 20.8 OD ４０５ (median 11.4 OD ４０５ ), and in patients with microscopic polyangiitis, 0.01 to 32.6 OD ４０５ (median 12.5 OD ４０５ ), and in patients with ANCA-associated vasculitic otitis media limited to the middle ear, 6.84 to 16.8 OD ４０５ (median 11.7 OD ４０５ ), and in patients with otitis media with effusion, 0.01 to 4.20 OD ４０５ (median 0.11 OD ４０５ ), 0.01 to 15.0 OD in patients with acute otitis media and acute sinusitis ４０５ (median 2.51 OD ４０５), and the MPO-DNA complex levels in patients suffering from one or more of eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media confined to the middle ear were significantly higher than those in patients with eosinophilic otitis media, eosinophilic sinusitis, otitis media with effusion, acute otitis media, and acute sinusitis, respectively (see Figure 3B).

[0088] (Setting the cutoff value for the expression level of the MPO-DNA complex) When an ROC curve was created based on the expression levels of the MPO-DNA complex in patients suffering from one or more of eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear, as well as in patients with eosinophilic otitis media, eosinophilic sinusitis, otitis media with effusion, acute otitis media, and acute sinusitis, the AUC (area under the curve) value was a high value of 0.92.

[0089] The optimal cutoff value for MPO-DNA complex levels to differentiate patients with eosinophilic granulomatosis with polyangiitis, granulomatosis with polyangiitis, microscopic polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear from patients with eosinophilic otitis media, eosinophilic sinusitis, otitis media with effusion, acute otitis media, and acute sinusitis was 6.71 OD. ４０５ When set to , the sensitivity was 83.7% and the specificity was 93.5% (see Figure 4B).

[0090] Using the calculated cutoff values, eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis were determined from the expression levels of the EPO-DNA complex and MPO-DNA complex in the subject by a method for performing at least one of the following: diagnosis, diagnostic assistance, judgment, and judgment assistance (see FIG. 5 ).

[0091] The sensitivity, specificity, and positive or negative predictive value from the results of Examples 1A and 2A were as follows (FIG. 5). Sixteen subjects had high levels of both the EPO-DNA complex expression level and the MPO-DNA complex expression level, and when the diagnosis of otitis media caused by eosinophilic granulomatosis with polyangiitis was made, the sensitivity was 100%, the specificity was 91.7%, the positive predictive value was 68.8%, and the negative predictive value was 100%.

[0092] Eleven subjects had high expression levels of the EPO-DNA complex and low expression levels of the MPO-DNA complex. When these subjects were diagnosed with eosinophilic otitis media, the sensitivity was 84.6%, the specificity was 100%, the positive predictive value was 100%, and the negative predictive value was 96.7%.

[0093] Twenty subjects had low expression levels of the EPO-DNA complex and high expression levels of the MPO-DNA complex. When these subjects were diagnosed with one or more of the following conditions: granulomatosis with polyangiitis, microscopic polyangiitis, or ANCA-associated vasculitic otitis media limited to the middle ear, the sensitivity was 79.2%, the specificity was 97.9%, the positive predictive value was 95.0%, and the negative predictive value was 90.2%.

[0094] Twenty-four subjects had low levels of both the EPO-DNA complex expression level and the MPO-DNA complex expression level, and when diagnosed with serous otitis media, the sensitivity was 95.7%, the specificity was 95.8%, the positive predictive value was 91.7%, and the negative predictive value was 97.9%.

[0095] On the other hand, the sensitivity, specificity, and positive or negative predictive value from the results of Examples 1B and 2B were as follows (FIG. 5). Twenty-three subjects had high levels of both the EPO-DNA complex expression level and the MPO-DNA complex expression level, and when the diagnosis of otitis media due to eosinophilic granulomatosis with polyangiitis was made, the sensitivity was 80.0%, the specificity was 89.5%, the positive predictive value was 52.2%, and the negative predictive value was 96.9%.

[0096] Twenty-six subjects had high expression levels of the EPO-DNA complex and low expression levels of the MPO-DNA complex. When these subjects were diagnosed with either eosinophilic otitis media or eosinophilic sinusitis, the sensitivity was 80.8%, the specificity was 96.8%, the positive predictive value was 87.5%, and the negative predictive value was 94.8%.

[0097] Eighteen subjects had low expression levels of the EPO-DNA complex and high expression levels of the MPO-DNA complex. When these subjects were diagnosed with one or more of the following conditions: granulomatosis with polyangiitis, microscopic polyangiitis, or ANCA-associated vasculitic otitis media limited to the middle ear, the sensitivity, specificity, positive predictive value, and negative predictive value were 100%, 100%, and 100%, respectively.

[0098] Fifty-five subjects had low levels of both the EPO-DNA complex expression level and the MPO-DNA complex expression level. When these subjects were judged to have one or more of serous otitis media, acute otitis media, and acute sinusitis, the sensitivity was 98.0%, the specificity was 92.8%, the positive predictive value was 90.9%, and the negative predictive value was 98.5%.

[0099] The above results demonstrate that by measuring the expression level of the EPO-DNA complex and using it as an index, it is possible to accurately diagnose, assist in diagnosis, determine, and / or assist in determination of eosinophilic otitis media, eosinophilic sinusitis, or eosinophilic granulomatosis with polyangiitis, and serous otitis media, acute otitis media, and acute sinusitis.

[0100] Furthermore, it has been shown that measuring the expression levels of the EPO-DNA complex and the MPO-DNA complex and combining them to use as an index can accurately diagnose, assist in diagnosis, determine, or assist in determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis.

[0101] According to the present invention, highly accurate data useful for diagnosing, assisting in the diagnosis, determining, and / or assisting in the determination of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitis otitis media, serous otitis media, and acute otitis media and acute sinusitis can be obtained. Therefore, eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and middle ear-limited ANCA-associated vasculitis otitis media can be detected early and appropriate treatment can be performed, which contributes to the prevention of serious complications associated with these conditions and a reduction in mortality rate.

[0102] Furthermore, the present invention can prevent delays in the start of treatment for ANCA-associated vasculitic otitis media, eosinophilic otitis media, and other conditions that lead to deafness, and by avoiding the need for cochlear implantation, etc., it will lead to an improvement in the quality of life (QOL) of patients and a reduction in medical costs.

Claims

1. A biomarker for performing one or more of the following: diagnosis, diagnostic assistance, determination, and determination assistance of at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, using at least an EPO-DNA complex contained in eosinophil extracellular traps.

2. The biomarker according to claim 1, further comprising an MPO-DNA complex contained in a neutrophil extracellular trap, which is used in combination with a biomarker for one or more of diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media.

3. A biomarker for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media, using an MPO-DNA complex contained in a neutrophil extracellular trap.

4. The following steps (a), (c') and (d); (a): measuring the expression level of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample collected from a subject; (c'): comparing the expression level of the EPO-DNA complex measured in step (a) with the expression level of the EPO-DNA complex in a control subject; (d): when the expression level of the EPO-DNA complex measured in step (a) is higher than the expression level of the EPO-DNA complex in the control subject, assessing that the subject is likely to suffer from at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis; A method for performing one or more of the following: diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, comprising:

5. The following steps (b), (c'') and (d'); (b): measuring the expression level of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample collected from a subject; (c''): comparing the expression level of the MPO-DNA complex measured in step (b) with the expression level of the MPO-DNA complex in a control subject; (d'): when the expression level of the MPO-DNA complex measured in step (b) is higher than the expression level of the MPO-DNA complex in the control subject, assessing that the subject is likely to be suffering from at least one of eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear; A method for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear, comprising:

6. A method for one or more of diagnosis, diagnostic assistance, judgment and judgment assistance, characterized by comprising a step of assessing that a subject is likely to suffer from at least one of eosinophilic otitis media, eosinophilic sinusitis and eosinophilic granulomatosis with polyangiitis when the expression level of EPO-DNA complex contained in eosinophil extracellular traps is higher than the expression level of EPO-DNA complex in a control subject.

7. A method for one or more of diagnosis, diagnostic assistance, judgment, and judgment assistance, comprising a step of assessing that a subject is highly likely to be suffering from at least one of eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear, when the expression level of the MPO-DNA complex contained in neutrophil extracellular traps is higher than the expression level of the MPO-DNA complex in a control subject.

8. The method comprises the following steps (a), (b), and (c), and any one of (d-1) to (d-4): (a): measuring the expression level of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample collected from a subject; (b): measuring the expression level of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal discharge sample collected from a subject; (c): comparing the expression levels of the EPO-DNA complex and the MPO-DNA complex measured in steps (a) and (b) with the expression levels of the EPO-DNA complex and the MPO-DNA complex in a control subject; (d-1): assessing that the subject is likely to be affected by eosinophilic granulomatosis with polyangiitis when both the expression levels of the EPO-DNA complex and the MPO-DNA complex measured in the steps (a) and (b) are higher than the expression levels of the EPO-DNA complex and the MPO-DNA complex in a control subject; (d-2): assessing that the subject is likely to suffer from eosinophilic otitis media and / or eosinophilic sinusitis when the expression level of the EPO-DNA complex measured in step (a) is higher than that of the EPO-DNA complex in the control subject, but the expression level of the MPO-DNA complex measured in step (b) is the same as that of the control subject; (d-3): when the expression level of the EPO-DNA complex measured in step (a) is the same as that in the control subject, but the expression level of the MPO-DNA complex measured in step (b) is higher than that in the control subject, assessing that the subject is highly likely to suffer from at least one of microscopic polyangiitis, granulomatosis with polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear; (d-4): assessing that the subject is highly likely to suffer from at least one of secretory otitis media, acute otitis media, or acute sinusitis, when both the expression levels of the EPO-DNA complex and the MPO-DNA complex measured in the steps (a) and (b) are not different from those in the control subject; A method for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis, comprising:

9. A step of assessing that the subject is highly likely to be suffering from eosinophilic granulomatosis with polyangiitis when the expression levels of both the EPO-DNA complex contained in the eosinophil extracellular traps and the MPO-DNA complex contained in the neutrophil extracellular traps are higher than the expression levels of the EPO-DNA complex and the MPO-DNA complex in a control subject; assessing that the subject is likely to suffer from eosinophilic otitis media and / or eosinophilic sinusitis when the expression level of the EPO-DNA complex contained in the eosinophil extracellular traps is higher than that of the EPO-DNA complex contained in the control subject, but the expression level of the MPO-DNA complex contained in the neutrophil extracellular traps is the same as that of the control subject; assessing that the subject is highly likely to suffer from at least one of microscopic polyangiitis, granulomatosis with polyangiitis, and ANCA-associated vasculitic otitis media limited to the middle ear, when the expression level of the EPO-DNA complex contained in the eosinophil extracellular traps is the same as that of the control subject, but the expression level of the MPO-DNA complex contained in the neutrophil extracellular traps is higher than that of the control subject; assessing that the subject is highly likely to be suffering from at least one of otitis media with effusion, acute otitis media, or acute sinusitis, when the expression levels of both the EPO-DNA complex contained in the eosinophil extracellular traps and the MPO-DNA complex contained in the neutrophil extracellular traps are not different from those of the control subject; A method for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, serous otitis media, acute otitis media, and acute sinusitis, comprising any one of the following:

10. 10. The method according to claim 4, wherein the control subject is a healthy subject, a patient with otitis media with effusion, a patient with acute otitis media, or a patient with acute sinusitis.

11. A kit for carrying out the method according to claim 6.

12. A kit for carrying out the method according to claim 7.

13. A kit for carrying out the method according to claim 9.

14. A kit for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, and eosinophilic granulomatosis with polyangiitis, characterized by comprising one or more specific antibodies and their labels for detecting the expression of EPO-DNA complexes contained in eosinophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, and a nasal secretion sample.

15. 15. The kit according to claim 14, further comprising one or more specific antibodies and markers thereof for detecting the expression of MPO-DNA complexes contained in neutrophil extracellular traps, said kit being used for one or more of diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic otitis media, eosinophilic sinusitis, eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, middle ear-limited ANCA-associated vasculitic otitis media, otitis media with effusion, acute otitis media, and acute sinusitis, said kit further comprising one or more specific antibodies and markers thereof for detecting the expression of MPO-DNA complexes contained in neutrophil extracellular traps.

16. A kit for diagnosing, assisting in diagnosis, determining, and assisting in determination of at least one of eosinophilic granulomatosis with polyangiitis, microscopic polyangiitis, granulomatosis with polyangiitis, and middle ear-limited ANCA-associated vasculitic otitis media, characterized by comprising one or more specific antibodies and their labels for detecting the expression of MPO-DNA complexes contained in neutrophil extracellular traps in at least one of a peripheral blood sample, a middle ear fluid sample, or a nasal discharge sample.