How to diagnose atopic dermatitis

Abstract

To find the CC type of Staphylococcus aureus, which is specifically detected in patients with atopic dermatitis in Asians such as Japanese, and clarify its relationship with atopic dermatitis; then, by using the CC-type Staphylococcus aureus as an index, to make it possible to detect the risk of developing atopic dermatitis or predict the prognosis (severity); and, in addition, to provide a method of screening candidate compounds for a preventive agent or a therapeutic agent for atopic dermatitis, with the CC-type Staphylococcus aureus as a new therapeutic target.SOLUTION: It is found that, in the lesion site skin of Japanese patients with atopic dermatitis, unlike Western patients with atopic dermatitis, Staphylococcus aureus having CC type (CC188 type, CC8 type, CC97 type, CC20 type, CC12 type) is predominant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for diagnosing atopic dermatitis, more particularly to a method for determining the risk of developing or the prognosis of atopic dermatitis in Asians. The present invention also relates to a method for screening candidate compounds for preventive or therapeutic agents for atopic dermatitis. [Background technology]

[0002] Atopic dermatitis (AD) is a multifactorial disease that presents a wide variety of clinical manifestations in different patients. In the past, it was primarily studied from an immunological perspective, but since around 2010, the importance of the skin barrier has become widely recognized, and in recent years, the involvement of the skin microbiota in the pathogenesis of atopic dermatitis has attracted attention.

[0003] A wide variety of bacterial flora exists on the surface of human skin, and different flora are formed depending on the location on the skin and the environment in which they grow. The skin flora contributes to maintaining our skin homeostasis by preventing harmful microorganisms from colonizing the skin and by influencing the regulation of the skin's immune system. Recent advances in sequencing technology have made it possible to comprehensively analyze the microbiome (the collective genome of the bacterial species that make up the microbiome), and the relationship between the pathology of various diseases and the microbiome is rapidly being elucidated.

[0004] It is known that Staphylococcus aureus is frequently detected in the lesions of atopic dermatitis, but the involvement of the skin bacterial flora, including S. aureus, in the pathogenesis of the condition remains unknown. Recent microbiome research has reported that the proportion of S. aureus increases in the lesions of atopic dermatitis, resulting in a dysbiosis, a structural abnormality in the bacterial species composition. While there is debate as to whether dysbiosis is the cause or the result of inflammation, the effectiveness of antibacterial treatments, such as sodium hypochlorite bath therapy (bleach bath therapy), has been demonstrated in clinical settings, and the skin bacterial flora is currently attracting attention as a therapeutic target for atopic dermatitis.

[0005] In recent years, it has become possible to analyze the diversity of Staphylococcus aureus at the strain level, suggesting that functional differences between strains may contribute to the onset of atopic dermatitis and the formation of diverse pathologies. Staphylococcus aureus strains are phylogenetically determined by multilocus sequence typing (MLST). Analysis using a bacterial classification method that defines strains that share five or more of seven housekeeping genes as the same gene group (Clonal Complex (CC)) has shown that CC30 type is commonly observed in healthy individuals, while CC1 type is frequently detected in the skin of patients with atopic dermatitis, as reported in analyses using Western patients (Non-Patent Documents 1 and 2). [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Fleury, OM et al., Infect.Immun. 2017, 85, e00994-16 [Non-patent document 2] Clausen, ML et al., BrJ Dermatol., 2017, 177, 1394-1400 Summary of the Invention [Problem to be solved by the invention]

[0007] In the present invention, we have identified a CC type of Staphylococcus aureus that is specifically detected in Asian patients with atopic dermatitis, such as Japanese, and have clarified its association with atopic dermatitis. Using this CC type of Staphylococcus aureus as an indicator, we are able to detect the risk of developing atopic dermatitis or predict its prognosis (worsening). Another objective of the present invention is to provide a method for screening candidate compounds for preventive or therapeutic drugs for atopic dermatitis, which targets the CC type of Staphylococcus aureus as a new therapeutic target. [Means for solving the problem]

[0008] As a result of extensive research to achieve the above-mentioned objective, the present inventors have found that Staphylococcus aureus with phylotypes (CC188, CC8, CC97, CC20, and CC12) different from those found in Western patients with AD predominate in the lesional skin of Japanese patients with atopic dermatitis (AD). Based on this, it has been discovered that bacteria with these CC types can be used as an indicator to determine the risk of developing AD, and that these bacteria can be new therapeutic targets for AD patients.

[0009] Furthermore, among these CC types, AD patients in whom CC188 type was detected tended to have more severe symptoms and higher blood test values ​​known as AD biomarkers, such as LDH, TARC, and total IgE, compared to patients in whom the bacteria was not detected. Furthermore, the present inventors also found cases in which CC20 type predominates as AD becomes more severe, leading to the completion of the present invention.

[0010] That is, the present invention relates to a method for determining the risk of developing or the prognosis of atopic dermatitis in Asians. The present invention also relates to a method for screening candidate compounds for preventive or therapeutic drugs for atopic dermatitis, and more specifically provides the following: <1> A method for determining a risk of developing atopic dermatitis, comprising: detecting Staphylococcus aureus in a sample isolated from an Asian subject; determining that the subject is at risk of developing atopic dermatitis when the presence of at least one CC type of Staphylococcus aureus selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 types is detected in the above step; A method comprising: <2> A method for determining the prognosis of atopic dermatitis, comprising: detecting Staphylococcus aureus in a sample isolated from an Asian subject; determining that the subject has a risk of atopic dermatitis becoming severe when the presence of at least one CC type of Staphylococcus aureus selected from the group consisting of CC188 type and CC20 type is detected in the step; A method comprising: <3> A method for screening a candidate compound for a preventive or therapeutic agent for atopic dermatitis, comprising: culturing Staphylococcus aureus of at least one CC type selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 in the presence and absence of a test compound, and measuring the number of the Staphylococcus aureus; determining that the test compound is a candidate compound for a preventive or therapeutic agent for atopic dermatitis when the number of Staphylococcus aureus detected in the above step after culturing in the presence of the test compound is reduced compared to the number of Staphylococcus aureus after culturing in the absence of the test compound; A method comprising: [Effects of the Invention]

[0011] According to the present invention, it is possible to determine the risk of developing atopic dermatitis or the prognosis (risk of aggravation) in Asians. If a subject is determined to be at high risk of developing atopic dermatitis or at high risk of atopic dermatitis atopic dermatitis, appropriate treatment can be administered to the subject, or daily care that suppresses the onset or aggravation of atopic dermatitis can be provided.

[0012] Furthermore, according to the present invention, it is possible to provide candidate compounds for preventive or therapeutic drugs for atopic dermatitis through screening, and further to provide a new method for treating atopic dermatitis. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing sample collection sites for analysis. [Figure 2] This graph shows the detection rate of Staphylococcus aureus in patients with atopic dermatitis ("AD" in the figure) and healthy individuals. "〈2 x 2 cm〉" and "〈5 x 5 cm〉" in the figure indicate the size of each sample collection site. The denominator for the detection rate is the number of people. [Figure 3]This graph compares the detection rate of Staphylococcus aureus by site between atopic dermatitis (AD) patients (lesioned and non-lesioned areas) and healthy controls. "B," "G," "C," and "V" in the figure indicate the sample collection sites: midline of the upper back, glabella, cubital fossa, and volar forearm, respectively. The denominator for the detection rate is the number of swabs. The size of the sample collection site in atopic dermatitis patients is 2 x 2 cm, while that in healthy controls is 5 x 5 cm, which is different. [Figure 4] This is a graph showing the phylogenetic types and percentages of Staphylococcus aureus detected in patients with atopic dermatitis. The size of the sample collection site in the patients with atopic dermatitis was 2 x 2 cm. [Figure 5] This is a heat map showing the results of an analysis of the association between Staphylococcus aureus phylotypes typical of patients with atopic dermatitis and general blood test results / disease biomarkers and symptom scores. [Figure 6] FIG. 1 is a schematic diagram showing the time course of predominant Staphylococcus aureus phylotypes and severity (SCORAD score) in a patient with atopic dermatitis (patient K042). DETAILED DESCRIPTION OF THE INVENTION

[0014] <Method for diagnosing atopic dermatitis> As shown in the Examples below, identification of the phylogenetic types of Staphylococcus aureus isolated and cultured from Japanese patients with atopic dermatitis (AD) resulted in the detection of predominantly CC188, CC8, CC20, CC97, and CC12 types of Staphylococcus aureus. This indicates that these are phylogenetic types of Staphylococcus aureus specific to Asian AD patients, such as Japanese, and that these CC types of Staphylococcus aureus are involved in the onset of AD in Asians.

[0015] Therefore, the present invention provides a method for determining a risk of developing atopic dermatitis, comprising: detecting Staphylococcus aureus in a sample isolated from an Asian subject; determining that the subject is at risk of developing atopic dermatitis when the presence of at least one CC type of Staphylococcus aureus selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 types is detected in the above step; The present invention provides a method comprising:

[0016] Furthermore, as shown in the Examples below, the present inventors have found that AD patients in whom the CC type, particularly the CC188 type, has been detected tend to have more severe symptoms and higher blood test values ​​known as AD biomarkers, such as LDH, TARC, and total IgE, compared to patients in whom the bacterium is not detected. They have also found cases in which the CC20 type predominates as AD becomes more severe.

[0017] Therefore, there is provided a method for determining the prognosis of atopic dermatitis, comprising: detecting Staphylococcus aureus in a sample isolated from an Asian subject; determining that the subject has a risk of atopic dermatitis becoming severe when the presence of at least one CC type of Staphylococcus aureus selected from the group consisting of CC188 type and CC20 type is detected in the step; The present invention provides a method comprising:

[0018] In the present invention, "atopic dermatitis" refers to a disease accompanied by inflammation of the skin associated with an allergic reaction. The "risk of developing" or "risk of aggravation" of an atopic disease refers to the onset of symptoms or the aggravation of the symptoms.

[0019] Symptoms of atopic dermatitis include, for example, rash, dermatitis, redness, scaling, eczema, erythema, papules, nodules, lichen, oozing, crusting, itching, and erosion.

[0020] The severity of atopic dermatitis can be determined, for example, according to the atopic dermatitis severity classification, the Severity Scoring of Atopic Dermatitis (SCORAD) Eczema Area and Severity Index (EASI). More specifically, in the case of SCORAD, the score of skin symptoms is calculated by adding up the area of ​​the rash (percentage of body surface area) and the severity (0 = none, 1 = mild, 2 = moderate, 3 = severe) of each rash: erythema, infiltration / papules, exudates / crusts, excoriation, lichenification, and dryness. Furthermore, the subject's subjective symptoms are taken into account to calculate the SCORAD score, and the symptoms of atopic dermatitis can be evaluated based on this. For example, a score of 25 to 50 is considered moderate, and a score above 50 is considered severe (maximum score is 103).

[0021] The severity of atopic dermatitis can also be assessed based on biomarkers, such as serum LDH, serum TARC (thymus and activation regulated chemokine), total IgE (serum IgE), specific IgE, peripheral blood eosinophil count (WBC), serum creatinine (CRTNN), and serum SCCA2.

[0022] Furthermore, the symptoms and severity of atopic dermatitis can be defined, for example, according to the "2008 Guidelines for the Treatment of Atopic Dermatitis by the Ministry of Health, Labour and Welfare's Research Group on Atopic Dermatitis" without relying on the scores or biomarkers. Specifically, according to the severity criteria described in the guidelines, the severity is assessed as mild (only mild rash is observed regardless of the area), moderate (rash accompanied by severe inflammation is observed on less than 10% of the body surface area), severe (rash accompanied by severe inflammation is observed on 10% to 30% of the body surface area), or extremely severe (rash accompanied by severe inflammation is observed on 30% or more of the body surface area). The more severe the symptoms, the more severe the atopic dermatitis is judged to be. Here, "mild rash" refers to lesions mainly characterized by mild erythema, dryness, and desquamation, and "rash accompanied by severe inflammation" refers to lesions accompanied by erythema, papules, erosions, infiltration, lichenification, etc.

[0023] In the present invention, the "subject" is not particularly limited as long as it is an Asian, and typically includes an Asian person who may be suffering from atopic dermatitis. Such an Asian person who may be suffering from atopic dermatitis may be an Asian person who has previously suffered from atopic dermatitis and is at risk of experiencing a recurrence of symptoms, or may be an Asian person who is suspected of having atopic dermatitis before being diagnosed or determined to have atopic dermatitis by a doctor or the like. Furthermore, when the method of the present invention is a method for determining prognosis, Asian people who are suffering from atopic dermatitis are also included.

[0024] In the present invention, "Asians" refers to ethnic groups living in Asia and people of Asian ethnic origin. Examples include ethnic groups living in East Asia and people of East Asian ethnic origin (so-called Northern Mongoloids, Orientals), and more specifically, Japanese (Yamato and Ryukyuan), Ainu, Korean, Chinese (Han, etc.), Mongolian, Uilta, and Nivkh peoples. "Asians" also includes ethnic groups living in Southeast Asia and people of Southeast Asian ethnic origin (Burmese, Hmong, Thai, Miao, Malay, etc.), ethnic groups living in South Asia and people of South Asian ethnic origin, ethnic groups living in Central Asia and people of Central Asian ethnic origin, and ethnic groups living in West Asia and people of West Asian ethnic origin.

[0025] The "sample" isolated from an Asian subject may be any material in which Staphylococcus aureus can be present, and typically, the target is skin (skin tissue, skin cells). The "sample" may also be skin secretions (sweat, sebum, suppuration, exudate, etc.), skin wipes, or skin washes, or even cultures of skin or their derivatives. The site of the skin from which the sample is collected is not particularly limited, but examples include the area between the eyebrows, the upper back (mid-upper back), the cubital fossa, and the palmar side of the forearm. Among these, the area between the eyebrows is preferred from the viewpoint of ease of detection of Staphylococcus aureus, regardless of whether the area is a lesion or non-lesion of atopic dermatitis. The area of ​​the skin from which the sample is collected is not particularly limited, but as shown in the Examples below, a 15cm area is preferred from the viewpoint of conditions in which Staphylococcus aureus is difficult to detect in healthy individuals.2 Less than (e.g., 14 cm 2 , 13cm 2 , 12cm 2 , 11cm 2 ), more preferably 10 cm 2 Less than (e.g., 9cm 2 , 8cm 2 , 7cm 2 , 6cm 2 ), more preferably 5 cm 2 Less than (e.g., 4 cm 2 , 3cm 2 , 2cm 2 , 1cm 2 )

[0026] "Staphylococcus aureus," also known as Staphylococcus aureus or S. aureus, is a facultatively anaerobic, Gram-positive cocci (staphylococcus). "CC188, CC8, CC20, CC97, and CC12" are phylotypes (clonal complexes) determined by multilocus sequence typing (MLST). More specifically, strains are considered to belong to the same phylotype if at least five (preferably six, more preferably seven) of the seven housekeeping genes (arcC, aroE, glpF, gmk, pta, tpi, and yqiL) share the same sequence. The sequence patterns of the seven genes that serve as the basis for each CC type are shown in Table 1 below.

[0027] [Table 1]

[0028] Among the seven genes, arcC (ARCC) is a carbamate kinase gene, aroE (AROE) is a shikimate dehydrogenase gene, glpF (GLPF) is a glycerokinase gene, gmk (GMK) is a guanylate kinase gene, pta (PTA) is a phosphate acetyltransferase gene, tpi (TPI) is a triosephosphate isomerase gene, and yqiL (YQIL) is an acetyl-CoA acetyltransferase gene.

[0029] Furthermore, the "allele numbers" shown in Table 1 refer to numbers assigned according to the sequence of each gene registered on the MLST website (http: / / www.mlst.net) on the Internet (the nucleotide sequence specified by each allele number is shown in each SEQ ID NO: in the sequence listing). "Sequence type (ST)" indicates the same genotype where all seven allele number patterns registered on the website match.

[0030] "Detection of Staphylococcus aureus" can be performed by analyzing the nucleotide sequence specific to the CC188, CC8, CC97, CC20, or CC12 type present in the sample. Those skilled in the art can prepare nucleotides (genomic DNA, cDNA, mRNA, etc.) from the sample and analyze their sequences according to standard methods.

[0031] To detect phylotype-specific nucleotide sequences, genetic polymorphism analysis is usually used, for example, by sequence analysis, which involves direct sequencing using known methods such as the dideoxy method and the Maxam-Gilbert method.

[0032] Those skilled in the art can input the determined sequence information into the MLST website to identify the phylogenetic type of Staphylococcus aureus. Furthermore, the phylogenetic type to which Staphylococcus aureus present in a sample belongs can also be determined using eBURST (http: / / eburst.mlst.net / ).

[0033] Polymorphisms can also be analyzed by hybridization methods using probes specific to genetic polymorphisms, or by various methods using primers specific to genetic polymorphisms.

[0034] More specifically, examples of methods for analyzing gene polymorphisms include next-generation sequencing (sequencing-by-synthesis, for example, sequencing using a Solexa genome analyzer or Hiseq (registered trademark) 2000 manufactured by Illumina), pyrosequencing (for example, sequencing using a sequencer GSLX or FLX manufactured by Roche Diagnostics (454) (so-called 454 sequencing)), and ligase reaction. Examples of suitable sequencing methods include reactive sequencing (for example, sequencing using SoliD (registered trademark) or 5500xl manufactured by Life Technologies), PCR, NASBA, LCR, SDA, LAMP, methods using restriction fragment length polymorphism (RFLP), denaturing gradient gel electrophoresis (DGGE), methods using chemical cleavage of mismatch sites (CCM), primer extension (TaqMan (registered trademark) method), PCR-SSCP, single strand conformation polymorphism (SSCP), Invader, single nucleotide primer, SNaPshot, MassArray, SNP-IT, BeadArray, Scorpion, MADI-TOF / MS, DNA microarray analysis, Northern blotting, and Southern blotting.

[0035] Furthermore, "detection of Staphylococcus aureus" can be carried out according to known immunological techniques using antibodies that recognize amino acid sequences specific to CC188, CC8, CC97, CC20, or CC12 types present in the sample.

[0036] Such immunological techniques include, for example, ELISA, immunoblotting, antibody array analysis, flow cytometry, imaging cytometry, radioimmunoassay, immunoprecipitation, and immunohistochemical staining.

[0037] Furthermore, the CC20 and other types of Staphylococcus aureus detected in the present invention may have the ability to form a biofilm, which can be evaluated by those skilled in the art using a method such as that described in the Examples below.

[0038] Furthermore, the above-mentioned diagnosis of atopic dermatitis is usually performed by a physician (including a person under the physician's instructions), and the above-mentioned data on the protein amount, etc. is useful for the physician's diagnosis, including determining the timing of treatment, etc. Therefore, the method of the present invention can also be described as a method for collecting data on the presence or absence of detection of Staphylococcus aureus for the physician's diagnosis, a method for presenting the data to the physician, or a method for assisting the physician's diagnosis.

[0039] <Drugs for diagnosing atopic dermatitis> As shown in the Examples below, it has been shown that at least one CC type of Staphylococcus aureus selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 types is involved in atopic dermatitis in Asians. In particular, it has been shown that at least one CC type of Staphylococcus aureus selected from the group consisting of CC188 and CC20 types contributes to the aggravation of atopic dermatitis in Asians.

[0040] Therefore, the present invention provides the following composition for testing atopic dermatitis.

[0041] A composition for detecting the risk of developing atopic dermatitis in Asians, comprising a polynucleotide for detecting a nucleotide sequence specific to at least one CC type of Staphylococcus aureus selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 types.

[0042] A composition for detecting the risk of atopic dermatitis becoming severe in Asians, comprising a polynucleotide for detecting a nucleotide sequence specific to at least one CC type of Staphylococcus aureus selected from the group consisting of CC188 type and CC20 type.

[0043] A composition for detecting the risk of developing atopic dermatitis in Asians, comprising an antibody that specifically recognizes at least one CC type of Staphylococcus aureus selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 types.

[0044] A composition for detecting the risk of atopic dermatitis becoming severe in Asians, comprising an antibody that specifically recognizes at least one CC type of Staphylococcus aureus selected from the group consisting of CC188 type and CC20 type.

[0045] In the present invention, the "polynucleotide for detecting a nucleotide sequence specific to Staphylococcus aureus such as CC188 type" is not particularly limited, as long as it detects a sequence specific to the bacterium, and examples thereof include polynucleotides having a chain length of at least 15 nucleotides and being any of the polynucleotides described in (a) to (b) below. (a) a polynucleotide that is a pair of primers designed to flank the specific nucleotide sequence; (b) A polynucleotide that is a primer or probe that hybridizes to a nucleotide sequence containing the specific nucleotide sequence.

[0046] The polynucleotides of the present invention have a base sequence complementary to the nucleotide sequence of Staphylococcus aureus, such as the CC188 strain. Here, "complementary" does not necessarily mean perfect complementarity, as long as they hybridize. These polynucleotides typically have 80% or more, preferably 90% or more, more preferably 95% or more, and particularly preferably 100% homology to the nucleotide sequence.

[0047] The "chain length" of the polynucleotide of the present invention, when used as a primer, is generally 15 to 100 nucleotides, preferably 17 to 30 nucleotides, and more preferably 20 to 25 nucleotides, and when used as a probe, is generally 15 to 1000 nucleotides, and preferably 20 to 100 nucleotides.

[0048] The polynucleotide of the present invention may be DNA or RNA, or may have nucleotides substituted in part or in whole with artificial nucleic acids such as LNA (registered trademark, bridged nucleic acid), ENA (registered trademark, 2'-O,4'-C-Ethylene-bridged nucleic acids), GNA (glycerol nucleic acid), TNA (threose nucleic acid), or PNA (peptide nucleic acid). The polynucleotide of the present invention can be chemically synthesized using a commercially available automatic nucleotide synthesizer or the like.

[0049] The polynucleotide used in the test of the present invention may be a polynucleotide bound to a labeling substance. The labeling substance is not particularly limited as long as it can be bound to the polynucleotide and can be detected by chemical or optical methods, and examples include fluorescent dyes (DEAC, FITC, R6G, TexRed, Cy5, etc.), chromogens other than fluorescent dyes such as DAB, enzymes, and radioactive substances.

[0050] The testing composition of the present invention may contain, in addition to the polynucleotide described above, other pharmacologically acceptable components, such as buffers, emulsifiers, suspending agents, stabilizers, preservatives, and physiological salts.

[0051] In addition to the above-mentioned testing composition, a test kit for atopic dermatitis can be prepared by combining other preparations, such as substrates necessary for detecting the labeled substance attached to the polynucleotide, positive and negative controls, sample collection tools (such as swabs), buffer solutions used for diluting and washing the sample, media for culturing Staphylococcus aureus, polynucleotide extraction reagents, and tubes or plates used for reacting the sample with the polynucleotide of the present invention. Furthermore, such a test kit for atopic dermatitis can include instructions for use of the kit.

[0052] Furthermore, the testing composition of the present invention can be combined with a device for detecting a nucleotide sequence specific to S. aureus such as CC188 type. Examples of such a device include a PCR device, a sequencer, and a microarray.

[0053] In the present invention, an "antibody that specifically recognizes CC-type S. aureus, such as CC188-type" may be a polyclonal or monoclonal antibody, as long as it is capable of specifically recognizing the bacterium. It may also be a functional fragment of an antibody (e.g., Fab, Fab', F(ab')2, variable region fragment (Fv), disulfide-linked Fv, single-chain Fv (scFv), sc(Fv)2, diabody, multispecific antibody, or polymer thereof). Polyclonal antibodies of the present invention can be obtained by immunizing an animal with an antigen (e.g., S. aureus, such as CC188-type, polypeptides, polynucleotides, sugar chains, lipids, etc., derived from the bacterium) and purifying the antiserum by conventional means (e.g., salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be produced by hybridoma or recombinant DNA techniques.

[0054] Furthermore, the antibody used in the test of the present invention may be an antibody bound to a labeling substance. By detecting the labeling substance, it is possible to directly measure the amount of antibody bound to the bacterium or a substance derived from the bacterium. The labeling substance is not particularly limited as long as it can be bound to the antibody and can be detected by chemical or optical methods, and examples of the labeling substance include fluorescent dyes (e.g., GFP), enzymes (e.g., HRP), and radioactive substances.

[0055] The testing composition of the present invention can contain, in addition to the antibody component, other components acceptable for use as a composition. Examples of such other components include carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, antiseptics, physiological salts, labeled substances, and secondary antibodies. In addition to the testing composition, other components can be combined, such as substrates necessary for detecting labeled substances, positive and negative controls, or sample collection tools (such as swabs), buffer solutions used for diluting and washing samples, media for culturing Staphylococcus aureus, and tubes or plates used for reacting samples with the antibodies of the present invention, to create a testing kit for atopic dermatitis. When an unlabeled antibody is used as the antibody preparation, a labeled substance (e.g., secondary antibody, protein G, protein A, etc.) that binds to the antibody can be combined. Such a testing kit for atopic dermatitis can also include instructions for use of the kit.

[0056] Furthermore, the testing composition of the present invention can be combined with a device for detecting the antibody of the present invention. Examples of such a device include a flow cytometry device and a microplate reader.

[0057] <Treatment methods for atopic dermatitis> As described above, the present invention makes it possible to determine the risk of developing atopic dermatitis or the prognosis (risk of atopic dermatitis becoming severe) in Asians. If a subject is determined to be at high risk of developing atopic dermatitis or at high risk of atopic dermatitis becoming severe, it becomes possible to administer treatment to the subject at an early stage to suppress the onset or aggravation of atopic dermatitis.

[0058] Therefore, the present invention can also provide a method for treating atopic dermatitis in a subject who has been determined by the method of the present invention to be at risk of developing atopic dermatitis or to have a poor prognosis (worsening of the condition).

[0059] The method for treating atopic dermatitis is not particularly limited, and known methods can be appropriately adopted, including, for example, drug therapy, ultraviolet light therapy, and sodium hypochlorite bath therapy.

[0060] Examples of therapeutic agents used in drug therapy include moisturizing topical preparations (heparinoid-containing preparations, urea preparations, white petrolatum, zinc oxide ointment, etc.), topical disinfectants (povidone-iodine solution, etc.), steroids (glucocorticoids or their derivatives, etc.), calcineurin inhibitors (tacrolimus, pimecrolimus, etc.), cyclosporine, methotrexate (MTX), azathioprine (AZA), and antihistamines (fexofenadine, etc.).

[0061] More specifically, the following examples of steroids are given depending on the strength of their efficacy and the severity of symptoms.

[0062] Strongest: clobetasol propionate, diflorasone acetate, etc. Very Strong: Betamethasone dipropionate, betamethasone butyrate propionate, diflucortolone valerate, fluocinonide, amcinonide, mometasone furoate, difluprednate, hydrocortisone butyrate propionate, etc. Strong: halcinonide, betamethasone valerate, fluocinolone acetonide, beclomethasone propionate, dexamethasone propionate, dexamethasone valerate, deprodone propionate, etc. Medium: Clobetasone butyrate, hydrocortisone butyrate, triamcinolone acetonide, alclometasone propionate, dexamethasone, prednisolone valerate acetate, etc. Weak: Prednisolone, hydrocortisone acetate, etc. From the Japanese Dermatological Association's Atopic Dermatitis Treatment Guidelines (2009).

[0063] The method of administering a therapeutic agent varies depending on the type and dosage form of the therapeutic agent, and the age, weight, sex, etc. of the subject to be administered, but can be administered by any of the following administration routes: oral administration, parenteral administration (e.g., topical administration (application to the skin, etc.), intravenous administration, or arterial administration). A person skilled in the art can adjust the dosage as appropriate depending on the type and dosage form of the therapeutic agent, and the age, weight, sex, health condition, etc. of the subject to be administered.

[0064] Furthermore, the present invention specifies the method of use (subject to be administered, administration timing) of such a therapeutic agent. Thus, the present invention also provides a therapeutic agent for atopic dermatitis to be administered to a subject determined by the method of the present invention to be at risk of developing atopic dermatitis or to have a poor prognosis (worsening of the condition).

[0065] <Screening method> A wide variety of bacterial flora exists on the surface of human skin, and different flora are formed depending on the skin site and environment. The skin flora contributes to maintaining skin homeostasis by preventing the colonization of harmful microorganisms on the skin and by influencing the regulation of the skin immune system.

[0066] Therefore, if it were possible to eliminate only the harmful bacteria involved in atopic dermatitis without affecting the immune system, etc., which is controlled by the skin bacterial flora, this would greatly contribute to the development of preventive drugs, therapeutic drugs, prevention methods, and treatment methods for atopic dermatitis with few side effects.

[0067] As described above, the present invention has demonstrated that at least one CC type of Staphylococcus aureus selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 types is involved in atopic dermatitis in Asians, suggesting that these specific phylogenetic types of Staphylococcus aureus may be effective therapeutic targets for atopic dermatitis.

[0068] Therefore, the present invention provides A method for screening a candidate compound for a preventive or therapeutic agent for atopic dermatitis, comprising: culturing Staphylococcus aureus of at least one CC type selected from the group consisting of CC188, CC8, CC97, CC20, and CC12 in the presence and absence of a test compound, and measuring the number of the Staphylococcus aureus; determining that the test compound is a candidate compound for a preventive or therapeutic agent for atopic dermatitis when the number of Staphylococcus aureus detected in the above step after culturing in the presence of the test compound is reduced compared to that in the absence of the test compound; A method including Also provides.

[0069] The "test substance" to be subjected to the method is not particularly limited, and examples thereof include synthetic low molecular weight compounds, antibodies, polypeptides, polynucleotides, lipids, sugars (monosaccharides, disaccharides, oligosaccharides, sugar chains, etc.), phages and libraries composed of these substances, extracts and cultures (culture supernatants, etc.) of cells (bacteria, plant cells, animal cells), bacterial secretion products, bacterial metabolic products, marine organisms, extracts derived from plants or animals, soil, and phage display.

[0070] The "S. aureus of at least one CC type selected from the group consisting of CC188, CC8, CC97, CC20, and CC12" is as described above. Suitable bacterial strains (deposited strains) for each of the S. aureus CC188, CC8, and CC20 types are listed in Table 1. All of these strains were isolated and cultured by the present inventors from lesions of Japanese patients with atopic dermatitis. The allele number patterns of all deposited strains are identical for all seven corresponding CC(ST) genes. All bacterial strains were deposited at the National Institute of Technology and Evaluation (NITE) on May 29, 2019, Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan. The details are as follows:

[0071] (1) Identification: SA0041 (2) Receipt number: NITE ABP-02959 (Accession number: NITE BP-02959) (3) Date of receipt: May 29, 2019 (4) Receiving organization: National Institute of Technology and Evaluation.

[0072] (1) Identification: SA0079 (2) Receipt number: NITE ABP-02962 (Accession number: NITE BP-02962) (3) Date of receipt: May 29, 2019 (4) Receiving organization: National Institute of Technology and Evaluation.

[0073] (1) Identification: SA0080 (2) Receipt number: NITE ABP-02960 (Accession number: NITE BP-02960) (3) Date of receipt: May 29, 2019 (4) Receiving organization: National Institute of Technology and Evaluation.

[0074] (1) Identification: SA0338 (2) Receipt number: NITE ABP-02963 (Accession number: NITE BP-02963) (3) Date of receipt: May 29, 2019 (4) Receiving organization: National Institute of Technology and Evaluation.

[0075] (1) Identification: SA0475 (2) Receipt number: NITE ABP-02961 (Accession number: NITE BP-02961) (3) Date of receipt: May 29, 2019 (4) Receiving organization: National Institute of Technology and Evaluation.

[0076] (1) Identification: SA0762 (2) Receipt number: NITE ABP-02964 (Accession number: NITE BP-02964) (3) Date of receipt: May 29, 2019 (4) Receiving organization: National Institute of Technology and Evaluation.

[0077] (1) Identification: SA1353 (2) Receipt number: NITE ABP-02965 (Accession number: NITE BP-02965) (3) Date of receipt: May 29, 2019 (4) Receiving organization: National Institute of Technology and Evaluation.

[0078] Furthermore, the CC20 or other Staphylococcus aureus strains used in the screening of the present invention may have the ability to form biofilms. Those skilled in the art can evaluate the biofilm-forming ability using a method such as that described in the Examples below. An example of such Staphylococcus aureus strain having the ability to form biofilms is the SA1353 strain, as described in the Examples below.

[0079] Those skilled in the art can "cultivate" Staphylococcus aureus using a suitable known medium. Examples of such media include staphylococcal selective medium, mannitol salt medium, egg yolk-added mannitol salt medium, Staphylococcal medium No. 110, Baird-Parker medium, buffered peptone water, LB medium, heart infusion medium, brain heart infusion medium, tryptic soy medium, standard agar medium, nutrient agar medium, and blood agar medium. Those skilled in the art can also appropriately determine the culture conditions (culture temperature, culture time, etc.).

[0080] Furthermore, the culture may be carried out not only in a medium but also on the skin of a non-human animal. Such non-human animals are not particularly limited as long as they are capable of maintaining Staphylococcus aureus on their skin, and examples thereof include mice, rats, rabbits, monkeys, chimpanzees, pigs, cows, horses, sheep, goats, dogs, cats, hamsters, and chickens. Furthermore, such non-human animals may be model animals for atopic dermatitis.

[0081] In the present invention, the "measurement" of the number of Staphylococcus aureus can be performed by a person skilled in the art based on an appropriate known method. For example, the number of single colonies after culture, the turbidity of the medium after culture, or the like can be used as an indicator. Furthermore, in the present invention, the "number" of Staphylococcus aureus includes not only the number of bacteria but also the amounts of nucleotides, proteins, etc. derived from the bacteria that reflect the number. The amounts of nucleotides, proteins, etc. can also be analyzed by a person skilled in the art using the above-mentioned genetic polymorphism analysis method or immunological method. [Example]

[0082] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. The examples were carried out using the methods shown below.

[0083] (Human skin swab collection for Staphylococcus aureus strain analysis) As shown in Table 2 below, swabs were collected from 139 patients with atopic dermatitis (AD) and 75 healthy individuals at four basic collection sites: between the eyebrows, cubital fossa, inner forearm, and midline of the upper back (see Figure 1 for collection sites).

[0084] [Table 2]

[0085] If there was a rash (or a characteristic rash) near each site, samples were taken from the same site in addition to the non-lesional area (minimum four sites, maximum eight sites). The sample area was 2x2cm per site from 139 AD patients and 29 healthy controls, and 5x5cm from 71 healthy controls. For 25 of the healthy controls, samples were taken simultaneously from both the left and right sampling sites, covering 2x2cm and 5x5cm. The swabs were rubbed with a sterile cotton swab soaked in PBS for 30 seconds, then placed in an Eppendorf tube containing 1ml of PBS and stored. 50μl of PBS was used for S. aureus isolation and culture.

[0086] (Isolation and culture of Staphylococcus aureus and identification of the bacterial species) Skin swabs from AD patients, patients with skin diseases other than AD, and healthy individuals were inoculated onto selective staphylococcal medium and cultured at 37°C for 48 hours. Up to 10 single colonies were selected from each sample and replated onto tryptic soy agar. The resulting colonies were confirmed to be S. aureus by PCR using primers specific to the femA and femB genes. Glycerin stocks were made from colonies identified as S. aureus.

[0087] (Analysis of virulence factor-related genotypes by PCR) Colonies identified as S. aureus were suspended in 100 μl of CS buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) containing lysostaphin and DNase, incubated at 37°C for 1 hour, and then lysed at 95°C for 5 minutes. Using this DNA template, coagulase typing, agr typing, and enterotoxin genes were examined by PCR to analyze the genotype of each strain.

[0088] (Extraction of genomic DNA from Staphylococcus aureus) Each S. aureus strain was cultured in 1 ml of tryptic soy broth (TSB) at 37°C for 24 hours, then centrifuged to collect the cells. The cells were then suspended in 400 μl of CS buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0) containing lysostaphin and DNase and incubated at 37°C for 1 hour. 10 μl of 10 mg / ml Proteinase K and 50 μl of 10% SDS were added and incubated at 55°C for at least 3 hours. 400 μl of saturated phenol was added, mixed, centrifuged, and the supernatant was decanted. 400 μl of phenol / chloroform was then added, mixed, centrifuged, and the supernatant was decanted. 1 ml of 99% ethanol was added, mixed, and centrifuged to precipitate DNA. 400 μl of 70% ethanol was added and centrifuged to remove the DNA. The precipitated DNA was dried and dissolved in 50 μl of sterile Milli-Q. DNA concentration was measured using nanodrop (Thermo Fisher Scientific).

[0089] (Staphylococcus aureus genome sequencing and information analysis) A library sample for each strain was prepared using 100 ng of purified genomic DNA with an Enzymatics NGS library preparation kit. The pooled library sample was accurately concentrated by qPCR. Sequence data (fastq files) were obtained using the Illumina MiSeq system with the MiSeq Sequence Reagent Kit v2 (600 cycles) (MS-102). The output fastq files were assembled using SPAdes v3.13.0 and auto-annotated using Prokka v1.11. SNP analysis was performed using CLC Genomics Workbench v9. The resulting genome sequences were uploaded to the MLST database (http: / / www.mlst.net) to identify allele numbers for seven housekeeping genes and sequence type (ST) numbers to determine the phylogenetic type (CC type). eBURST analysis was also performed.

[0090] (Severity scoring (SCORAD), blood test data) For AD patients, SCORAD (SCORing Atopic Dermatitis) was recorded on the same day as sampling. Some AD patients underwent blood tests for medical purposes while visiting the Keio University Dermatology Outpatient Clinic. Total IgE test data collected within 28 days before and after sampling was used, and blood test data other than total IgE, collected within 14 days before and after sampling, were used to analyze correlations with the CC type of Staphylococcus aureus obtained from the subjects.

[0091] (Staphylococcus aureus biofilm measurement method) Each strain of Staphylococcus aureus was precultured in 3 ml of TSB at 37°C for 24 hours and then diluted 100-fold with TSB. 100 μl of TSB or TSB containing 1% glucose was placed in a 96-well plate, and 10 μl of each 100-fold diluted bacterial solution was added and cultured at 37°C for 24 hours. After culture, the bacterial solution was removed and washed three times with 300 μl of phosphate-buffered saline (PBS). Biofilm-forming bacteria were stained with 1% crystal violet for 15 minutes. After removing the crystal violet, the plate was washed ten times with tap water. 200 μl of 33% acetic acid was added and incubated at room temperature for 15 minutes to extract the stained biofilm-forming bacteria. After treatment, the sample was diluted 10-fold with PBS and measured at OD590nm.

[0092] The results obtained by the analysis using the above method are shown in FIGS.

[0093] As shown in Figure 2, when Staphylococcus aureus was isolated and cultured from skin swab samples (at least four locations) collected from the skin (2 x 2 cm area) of 139 AD patients, Staphylococcus aureus was detected in 64.7% of cases. On the other hand, Staphylococcus aureus was rarely detected in the culture of swab samples collected from the skin (2 x 2 cm area) of 29 healthy subjects (only 3.4% were detected).

[0094] To compare the characteristics of S. aureus strains from AD patients with those from healthy individuals, samples were also taken from a 5x5cm area of ​​skin from healthy individuals. S. aureus was detected in 45.1% of the isolated cultures from 71 healthy individuals who had their samples expanded to a 5x5cm area.

[0095] The detection rate of S. aureus from each site (glabellar, cubital fossa, inner forearm, upper back) isolated from skin swab samples taken from AD patients (2 x 2 cm area) is shown in a graph, separated into lesional and non-lesional data, as shown in Figure 3. For comparison, the detection rate of S. aureus from each site (glabellar, cubital fossa, inner forearm, upper back) isolated from skin swab samples taken from healthy subjects (5 x 5 cm area) is shown on the right.

[0096] As a result, in AD patients, the rate at which Staphylococcus aureus was detected was high between the eyebrows, regardless of whether the area was lesional or non-lesional (see "G" next to "AD patients" in the figure). The detection rate of Staphylococcus aureus was higher in lesional than non-lesional areas of AD patients.

[0097] Next, we identified the phylotypes of S. aureus isolated from AD patients. As shown in Figure 4, 13 phylotypes were identified in S. aureus isolated from 82 AD patients. Among the 82 patients, CC188, CC8, and CC20 were detected in 37.8%, 29.2%, and 13.4%, respectively. Meanwhile, CC1, which has been reported to be the most characteristic type of AD in Western countries, was detected in 7.3% of the 82 AD patients. Thus, CC188, CC8, CC20, CC97, and CC12 were identified as phylotypes specific to Asian AD patients, including Japanese. These CC types suggest that S. aureus may be involved in the development of AD in Asians and that these staphylococci may be therapeutic targets for AD in Asians.

[0098] Next, the correlation between the typical Staphylococcus aureus phylotypes detected in AD patients and symptom scores (SCORAD) and general blood test results / disease progression markers was analyzed using multiple regression analysis when compared with patients in whom Staphylococcus aureus was not detected.

[0099] As shown in Figure 5, AD patients in whom the CC188 strain was detected tended to have more severe symptoms and higher blood test values ​​known as AD biomarkers, such as LDH, TARC, and total IgE, compared to patients in whom S. aureus was not detected. Furthermore, AD patients in whom the CC97 strain was detected also tended to have higher LDH and TARC levels. These findings suggest that the CC188 and CC97 strains of S. aureus are particularly associated with the pathology of AD.

[0100] Next, we focused on the case and phylogenetic type of S. aureus in a certain patient (hereinafter referred to as patient K042) and performed an analysis. As shown in Figure 6, in patient K042, it became clear that the CC20 type became dominant as the symptoms became more severe.

[0101] When a string test (a test to measure hyperviscosity) was performed on S. aureus isolated from patient K042, the SA1353 strain isolated from this patient showed a positive result. While S. aureus is often string-test negative, a strain as strongly positive as the SA1353 strain is extremely rare. The string test was performed using a method similar to that described by Fang C. T. et al., J. Exp. Med., 2004, Vol. 199, pp. 697-705, with modifications for S. aureus. Furthermore, when the SA1353 strain isolated from patient K042 was cultured in liquid, a biofilm-like pellicle was observed.

[0102] Thus, it was suggested that CC20 Staphylococcus aureus is involved in the aggravation of AD. [Industrial Applicability]

[0103] As described above, according to the present invention, it is possible to determine the risk of developing or the prognosis (risk of aggravation) of atopic dermatitis in Asians using the phylogenetic type of Staphylococcus aureus specific to AD as an indicator.

[0104] Furthermore, the present invention also makes it possible to provide a method for screening candidate compounds for preventive or therapeutic agents for atopic dermatitis, which target Staphylococcus aureus strains specific to Asian atopic dermatitis.

[0105] Therefore, the present invention is extremely useful in the development of diagnostic and therapeutic methods for atopic dermatitis.

Claims

1. A method for presenting data for determining a risk of developing atopic dermatitis, comprising: detecting Staphylococcus aureus in a sample isolated from an Asian subject; presenting data indicating that the subject is at risk of developing atopic dermatitis when the presence of at least one CC type of Staphylococcus aureus selected from the group consisting of CC8, CC97, and CC12 types is detected in the step; wherein the Asian is Japanese.

2. A method for presenting data for determining the prognosis of atopic dermatitis, comprising: detecting Staphylococcus aureus in a sample isolated from an Asian subject; and presenting data indicating that the subject is at risk of atopic dermatitis becoming severe when the presence of at least one CC type of Staphylococcus aureus selected from the group consisting of CC188 type and CC20 type is detected in the step. wherein the Asian is Japanese.

3. A method for screening candidate compounds for preventive or therapeutic drugs for atopic dermatitis in Asians, comprising: a step of culturing at least one CC type of Staphylococcus aureus selected from the group consisting of CC8, CC97, and CC12 in the presence and absence of a test compound, and measuring the number of the Staphylococcus aureus; determining that the test compound is a candidate compound for a preventive or therapeutic agent for atopic dermatitis in Asians when the number of Staphylococcus aureus after culture in the presence of the test compound detected in the step is reduced compared to the number of Staphylococcus aureus after culture in the absence of the test compound; wherein the Asian is Japanese.

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