Use of s100a8 / a9 in assessing severity of infectious diseases

Abstract

A use of S100A8 / A9 in assessing the severity of infectious diseases, and a use of a biomarker (S100A8 / A9) and / or a substance for detecting the biomarker in the preparation of a product for assessing the severity of infectious diseases. By detecting the level of S100A8 / A9 in serum or plasma, the severity of infectious diseases is rapidly and accurately assessed, thereby simplifying an assessment process and improving accuracy, and the present invention is suitable for various infectious diseases. The biomarker exhibits high sensitivity and good specificity; by assessing the severity of diseases, the present invention provides decision-making support for clinicians, and provides more precise treatment schemes for patients, thereby improving therapeutic efficacy and the quality of life of the patients; and additionally, optimal disease management is achieved, the medical burden on patients' families and society is reduced, and the present invention has favorable and broad clinical application value.

Description

Application of S100A8 / A9 in assessing the severity of infectious diseases Technical Field

[0001] This invention belongs to the field of biomedical diagnostic technology, specifically relating to the application of S100A8 / A9 in assessing the severity of infectious diseases. Background Technology

[0002] Infectious diseases, as a class of diseases caused by pathogens invading the human body and causing pathological changes, are diverse in type and have complex transmission routes, posing a serious threat to human health. These diseases not only affect an individual's quality of life but can also trigger epidemics, causing significant impacts on the socio-economic system. The pathogens of infectious diseases are wide-ranging, including but not limited to viruses, bacteria, Mycoplasma pneumoniae, Mycobacterium tuberculosis, fungi, and parasites. Viruses such as influenza virus, SARS-CoV-2, adenovirus, and respiratory syncytial virus spread rapidly through the air and can cause large-scale infections in populations; bacteria such as Mycobacterium tuberculosis, Mycoplasma pneumoniae, and Streptococcus pneumoniae spread through respiratory routes, causing infections and regional epidemics; fungal and parasitic infections are mostly related to personal hygiene and environmental sanitation. During the development of infectious diseases, pathogens interact with the body's immune system, leading to a series of complex pathophysiological changes. These changes not only affect the site of infection but may also affect multiple systems throughout the body, such as the respiratory, digestive, and nervous systems. Therefore, the clinical manifestations of infectious diseases are diverse, ranging from mild fever and cough to severe respiratory failure and multiple organ failure.

[0003] In the diagnosis and treatment of infectious diseases, accurately assessing whether an infection has occurred and the severity of the disease is crucial for developing appropriate and effective treatment plans, predicting disease progression, and evaluating prognosis. Traditionally, physicians rely primarily on a patient's clinical symptoms and signs, laboratory tests (such as complete blood count and C-reactive protein), and imaging examinations (such as X-rays and CT scans) to make a comprehensive judgment. However, these methods may have limitations in certain situations, such as atypical early infection symptoms and individual differences in response, thus affecting the accuracy of the assessment.

[0004] Therefore, researching and developing biomarkers that can more sensitively and specifically reflect the immune response and pathophysiological changes in the body during infection and accurately assess the severity of infectious diseases is one of the urgent problems to be solved in clinical practice. This can provide strong support for the diagnosis and treatment of infectious diseases and is of great significance for guiding personalized and standardized treatment of infectious diseases and improving treatment outcomes.

[0005] Invention Overview

[0006] The purpose of this invention is to provide an accurate, specific, and sensitive biomarker for assessing the severity of infectious diseases. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0007] To achieve the above objectives, the present invention first provides the use of biomarkers and / or substances for detecting said biomarkers in the preparation of products having any of the following functions:

[0008] A1) Used to assess the severity of infectious diseases;

[0009] A2) Used to predict or assist in predicting the progression of infectious diseases;

[0010] The biomarker may be S100A8 / A9.

[0011] In the above applications, the substances used to detect the biomarkers may include reagents and / or instruments for detecting the expression levels of S100A8 / A9 using Western blotting, immunofluorescence, radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), double-antibody sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay (EIA), flow cytometry (FACS), chromatography, mass spectrometry, polyacrylamide gel electrophoresis, capillary electrophoresis, near-infrared spectroscopy, immunochemiluminescence, colloidal gold immunochromatography (GIC), colloidal gold immunochromatography (GICA), fluorescence immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin technology.

[0012] In the above applications, the substance used to detect the biomarker may include reagents for detecting the expression level (or content) of S100A8 / A9.

[0013] In the above applications, the reagent may include antibodies, peptides, proteins, or nucleic acid molecules that specifically bind to S100A8 / A9.

[0014] Furthermore, the antibody may be a monoclonal antibody, polyclonal antibody, bispecific antibody, multispecific antibody, genetically engineered antibody, humanized antibody (including chimeric antibody, CDR transplanted antibody and SDR transplanted antibody), or fully human antibody. It may also be an antigen-binding fragment capable of specifically binding to the target antigen, such as Fab, Fab′, F(ab′)2, antibody variable region (Fv), disulfide bond-stabilized Fv (dsFv), single-chain antibody (ScFv), single-domain antibody (sdAb, i.e., nanobody), minibody, or minimum recognition unit (MRU), but is not limited thereto.

[0015] In the above applications, the infectious diseases may include those caused by infections with viruses, bacteria, fungi, mycoplasma, chlamydia, rickettsia, spirochetes, and parasites.

[0016] Furthermore, the infectious diseases may include those caused by viral, bacterial, mycoplasma, or chlamydia infections.

[0017] In the above applications, the infectious disease can be a lower respiratory tract infection and / or pneumonia caused by various pathogens.

[0018] In the above applications, the products may include, but are not limited to, reagents, kits, detection chips (such as protein chips), test strips (such as immunochromatographic diagnostic test strips), test cards, and immunosensors.

[0019] Furthermore, the application may include assessing the severity of an infectious disease based on the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases.

[0020] Furthermore, the application may include establishing and validating a disease severity assessment model based on the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases, combined with the patients' clinical information (including but not limited to symptoms, signs, laboratory tests, etc.), and classifying and assessing the disease condition of patients with infectious diseases.

[0021] Furthermore, the method for assessing the severity of an infectious disease may include the following steps:

[0022] (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases;

[0023] (2) The expression level of S100A8 / A9 is compared with the assessment threshold. When the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is greater than or equal to the assessment threshold, the severity of the infectious disease is predicted to be high. When the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is less than the assessment threshold, the severity of the infectious disease is predicted to be low.

[0024] The assessment threshold can be obtained through the Receiver Operating Characteristic (ROC) curve.

[0025] The assessment threshold can be modeled using bioinformatics methods combined with clinical information. For example, the optimal cut-off value for serum or plasma S100A8 / A9 levels that can be used to assess the severity of infectious diseases can be determined based on ROC curves. The optimal cut-off value can be the point with the maximum slope in the ROC curve or the value that maximizes sensitivity and specificity (i.e., the expression level of S100A8 / A9 in serum or plasma corresponding to the maximum sensitivity and specificity). Exceeding this threshold indicates a high degree of disease severity in the patient.

[0026] Furthermore, the diagnostic criteria for assessing the severity of infectious diseases can be as follows: when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is greater than or equal to 5.196 μmol / L, the severity of the infectious disease is predicted to be high; when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is less than 5.196 μmol / L, the severity of the infectious disease is predicted to be low.

[0027] The present invention also provides a kit that may include any of the substances described herein for detecting the biomarkers, and the kit may have at least one of the following uses:

[0028] B1) Used to assess the severity of infectious diseases;

[0029] B2) Used to predict or assist in predicting the progression of infectious diseases;

[0030] B3) Used to assist in the diagnosis of whether a subject has an infectious disease;

[0031] B4) is used to assess the improvement of symptoms in patients with infectious diseases.

[0032] Furthermore, the test sample for the kit can be serum or plasma.

[0033] Specifically, the kit may include an antibody that specifically binds to S100A8 / A9 (anti-S100A8 / A9 antibody) or its antigen-binding fragment.

[0034] The anti-S100A8 / A9 antibody or its antigen-binding fragment may also be labeled with a detectable marker. The detectable markers include, but are not limited to, enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridine esters, acridine sulfonamides, luminol and its derivatives, ruthenium derivatives), fluorescent dyes (e.g., AMCA, FITC, PE, PI, PerCP-Cy5.5, PE-Cy7, APC, APC-H7, BV421, V500, Alexa700, BV605), near-infrared dyes (e.g., cyanine dyes, BODIPY dyes, rhodamine dyes, squaric acid dyes, porphyrin dyes), radionuclides (e.g., 124I, 18F, 11C, 99mTc, 123I), biotin, magnetic resonance imaging nanoparticles, magnetic resonance imaging quantum dots, magnetic materials (e.g., magnetic beads, gadolinium-containing nanoparticles, superparamagnetic iron oxide nanoparticles), and colloidal gold.

[0035] The kit may be a chemiluminescent immunoassay kit, enzyme-linked immunosorbent assay kit, immunoprecipitation assay kit, immunoblotting assay kit, immunochromatographic assay kit, flow cytometry assay kit, immunohistochemistry assay kit, colloidal gold immunoassay kit, or fluorescent immunoassay kit, but is not limited thereto.

[0036] Furthermore, the kit may also include reagents required for immunoassay, such as labeled antibodies or antigens, magnetic microparticles, blocking solution, diluent, washing solution, chromogenic solution, stop solution, etc., but not limited to these.

[0037] The various reagent components of the kit may be present in separate containers, or may be pre-assembled into a reagent mixture, either wholly or partially.

[0038] The components of the kit may be provided in solution form, such as an aqueous solution. When present in aqueous solution, the concentration or content of these components can be readily determined by those skilled in the art according to different needs. For example, for storage purposes, the components may be present at a higher concentration, which can be reduced to the working concentration by diluting the higher concentration solution when in operation or for use.

[0039] The kit may also contain, for example, buffers, preservatives, or protein stabilizers. The kit may also contain components necessary for detecting the detectable marker, such as enzymes or substrates. The kit may also contain one or a series of control samples, which can be measured and compared to the test sample. The kit may have written product information on or included with the kit container. The written product description explains how to use the reagents included in the kit and instructs the user on how to assess the severity of an infectious disease by detecting serum S100A8 / A9 levels.

[0040] The present invention also provides an apparatus for assessing the severity of infectious diseases, the apparatus comprising a data receiving module, a data processing module, and an output module; the data receiving module is used to receive data on the expression levels of S100A8 / A9 in the serum or plasma of patients with infectious diseases; the data processing module is used to assess the severity of infectious diseases based on the detection results of S100A8 / A9 expression levels; and the output module is used to display the assessment results, indicating the severity of the infectious disease.

[0041] Furthermore, the data processing module can establish a disease severity assessment model based on the detection results of serum or plasma S100A8 / A9 expression levels, combined with the patient's clinical information (including but not limited to symptoms, signs, laboratory tests, etc.), to grade and assess the disease status of patients with infectious diseases, and then use it to predict or assist in predicting the progression of infectious diseases.

[0042] Furthermore, the data processing module may employ bioinformatics methods, such as using receiver operating characteristic (ROC) curve analysis, to determine the optimal cut-off value for serum or plasma S100A8 / A9 levels, which can then be used to differentiate patients with infectious diseases of different severity.

[0043] Furthermore, the device may also include a computer-readable storage medium that can be used to store and update serum or plasma S100A8 / A9 level detection data and corresponding clinical information of patients with infectious diseases, in order to support the continuous optimization and validation of the evaluation model.

[0044] The present invention also provides the use of biomarkers and / or substances for detecting said biomarkers in the preparation of products having any of the following functions:

[0045] C1) is used to assist in the diagnosis of whether a subject has an infectious disease;

[0046] C2) is used to assess the improvement of symptoms in infectious diseases;

[0047] The biomarker may be S100A8 / A9.

[0048] The S100A8 / A9 described in this article is a heterodimeric complex formed by the S100A8 protein (GenBank accession number: P05109) and the S100A9 protein (GenBank accession number: P06702).

[0049] The S100A8 / A9 mentioned in this article can also be a heterodimeric complex formed by S100-A8 protein (i.e., S100A8 protein, UniProtKB / Swiss-Prot:P05109.1, 24-JUL-2024) and S100-A9 protein (i.e., S100A9 protein, UniProtKB / Swiss-Prot:P06702.1, 24-JUL-2024).

[0050] The expression levels of S100A8 / A9 described in this article were significantly different between patients with infectious diseases (such as pneumonia) and healthy controls, as well as between patients with mild and severe infectious diseases. Furthermore, the expression level of S100A8 / A9 was significantly upregulated in patients with infectious diseases (such as pneumonia), and the expression level of S100A8 / A9 was significantly correlated with the severity of the infectious disease.

[0051] The expression levels of S100A8 / A9 described in this study were significantly higher than those in healthy children and children with non-infectious diseases (both P<0.05), and can be used to assist in the diagnosis of whether subjects have infectious diseases.

[0052] The serum S100A8 / A9 expression level in patients with infectious diseases (such as pneumonia patients, specifically community-acquired pneumonia patients) is significantly lower after treatment than before treatment, which can be used to assess the improvement of infectious disease conditions.

[0053] The biomarkers described herein are applicable to routine protein detection experiments using ELISA. While the embodiments provided in this invention exemplarily employ the ELISA method to detect the biomarker S100A8 / A9 in serum or plasma samples, this invention is not limited to this specific detection method. Those skilled in the art can use any other suitable method known to them (such as Western blotting, immunofluorescence, radioimmunoassay, immunoprecipitation, immunohistochemistry, double-antibody sandwich ELISA, fluorescence-linked immunosorbent assay, enzyme immunoassay, flow cytometry, chromatography, mass spectrometry, polyacrylamide gel electrophoresis, capillary electrophoresis, near-infrared spectroscopy, immunochemiluminescence, colloidal gold immunochromatography, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance, immuno-PCR, or biotin-avidin technology, etc.) to detect the expression level of S100A8 / A9 in serum or plasma samples. These alternative methods do not depart from the scope of this invention, and this invention should include these alternative methods.

[0054] The infectious diseases described in this article can be lower respiratory tract infections and / or pneumonia (e.g., community-acquired pneumonia) caused by various pathogens.

[0055] Furthermore, the infectious diseases described in this article may be pneumonia caused by infections such as Mycoplasma pneumoniae, bacteria, and / or viruses.

[0056] The present invention also provides a method for assessing the severity of an infectious disease, the method comprising assessing the severity of the infectious disease based on the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases.

[0057] Furthermore, the method may include the following steps:

[0058] (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases;

[0059] (2) Compare the expression level of S100A8 / A9 with the evaluation threshold, and evaluate the severity of infectious disease based on the comparison results.

[0060] Furthermore, the assessment of the severity of infectious diseases based on the comparison results can be as follows: when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is greater than or equal to the assessment threshold, the severity of the infectious disease is predicted to be high; when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is less than the assessment threshold, the severity of the infectious disease is predicted to be low.

[0061] The present invention also provides a method for predicting or assisting in the prediction of the progression of infectious diseases, the method comprising predicting or assisting in the prediction of the progression of infectious diseases based on the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases.

[0062] Furthermore, the method may include the following steps:

[0063] (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases;

[0064] (2) Compare the expression level of S100A8 / A9 with the evaluation threshold, and predict or assist in the prediction of the progression of infectious diseases based on the comparison results.

[0065] Furthermore, the prediction or auxiliary prediction of the progression of infectious diseases based on the comparison results can be as follows: when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is greater than or equal to the assessment threshold, the progression of infectious diseases to severe pneumonia is predicted; when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is less than the assessment threshold, the progression of infectious diseases to mild pneumonia is predicted.

[0066] The present invention also provides a method for assisting in the diagnosis of whether a subject has an infectious disease, the method comprising assisting in the diagnosis of whether a subject has an infectious disease based on the expression level or content of S100A8 / A9 in the subject's serum or plasma.

[0067] Furthermore, the method may include the following steps:

[0068] (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of the subjects;

[0069] (2) Compare the expression level of S100A8 / A9 with the assessment threshold, and make an auxiliary diagnosis on whether the subject has an infectious disease based on the comparison results.

[0070] Furthermore, based on the comparison results, the auxiliary diagnosis of whether the subject has an infectious disease can be made as follows: when the S100A8 / A9 expression level in the subject's serum or plasma is greater than or equal to the assessment threshold, it is judged that the subject has an infectious disease or is highly likely to have an infectious disease; when the S100A8 / A9 expression level in the subject's serum or plasma is less than the assessment threshold, it is judged that the subject does not have an infectious disease or is very unlikely to have an infectious disease.

[0071] The present invention also provides a method for assessing the improvement of an infectious disease, the method comprising assessing the improvement of an infectious disease based on the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases before and after treatment.

[0072] Furthermore, the method may include the following steps:

[0073] (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases before and after treatment;

[0074] (2) Compare the S100A8 / A9 expression levels after treatment with those before treatment, and evaluate the improvement of the infectious disease based on the comparison results.

[0075] Furthermore, based on the comparison results, the improvement of the infectious disease can be assessed as follows: when the S100A8 / A9 expression level after treatment is significantly lower than before treatment, the infectious disease condition is considered to have improved.

[0076] The assessment threshold may be a reference value that allows or is sufficient to assess the severity of an infectious disease. This reference value is obtained in advance from subjects with known severity of infectious disease. The assessment threshold can be measured and an appropriate reference value selected based on techniques known to those skilled in the art. For example, the assessment threshold can be obtained using a receiver operating characteristic (ROC) curve. The assessment threshold may also be the median of S100A8 / A9 expression levels.

[0077] The assessment threshold may be a reference value that allows or is sufficient to predict or assist in predicting the progression of an infectious disease. This reference value is obtained in advance from subjects with known progression of infectious diseases.

[0078] The assessment threshold may be a reference value that is permissible or sufficient to aid in the diagnosis of whether a subject has an infectious disease. This reference value is obtained in advance from subjects known to have an infectious disease. The assessment threshold may also be a reference value that is permissible or sufficient to assess the improvement of an infectious disease. This reference value is obtained in advance from treated subjects whose improvement of an infectious disease is known.

[0079] The assessment threshold (also referred to as the threshold) can be measured and selected using techniques known to those skilled in the art, and an appropriate reference value can be selected. For example, the assessment threshold can be obtained from a receiver operating characteristic (ROC) curve. The assessment threshold can also be the median of the S100A8 / A9 expression level.

[0080] Furthermore, the expression level or content of S100A8 / A9 can be detected by Western blotting, immunofluorescence, radioimmunoassay, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), double-antibody sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay, flow cytometry, chromatography, mass spectrometry, polyacrylamide gel electrophoresis, capillary electrophoresis, near-infrared spectroscopy, immunochemiluminescence, colloidal gold immunochromatography, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SIP), immuno-PCR, or biotin-avidin technology.

[0081] Furthermore, the infectious diseases include those caused by viral, bacterial, fungal, mycoplasmal, chlamydial, rickettsial, spirochetal, and parasitic infections.

[0082] Furthermore, the infectious disease may be a lower respiratory tract infection and / or pneumonia caused by various pathogens.

[0083] In the above method, the serum or plasma of the patient with the infectious disease can be in vitro or ex vivo serum or plasma.

[0084] S100A8 / A9 or a composition containing S100A8 / A9 is used for in vitro assessment of the severity of infectious diseases and / or for in vitro prediction or assistance in predicting the progression of infectious diseases.

[0085] S100A8 / A9 or compositions containing S100A8 / A9 are used for in vitro assessment of the severity of infectious diseases and / or for in vitro prediction or aiding prediction of the progression of infectious diseases.

[0086] The beneficial effects of this invention are:

[0087] (1) Rapid and accurate: By detecting the level of S100A8 / A9 in serum or plasma, the severity of infectious diseases can be rapidly and accurately assessed, avoiding the complexity and subjectivity of traditional assessment methods.

[0088] (2) Wide range of clinical applications: The biomarkers developed in this invention can be applied to the assessment of the condition and prediction of prognosis of a variety of infectious diseases, and have broad application prospects.

[0089] (3) Guiding treatment: By assessing the severity of the disease, it provides decision support for clinicians, provides more precise treatment plans for patients, guides the selection and adjustment of treatment plans, helps improve treatment effectiveness and patients' quality of life, achieves optimal disease management, and reduces the medical burden on patients' families and society, which has great clinical application value.

[0090] In summary, this invention presents a novel method for assessing the severity of infectious diseases using serum or plasma S100A8 / A9 levels. By detecting S100A8 / A9 concentrations in serum or plasma using ELISA and combining this with clinical information, an assessment model is established to achieve rapid and accurate grading of disease severity. This method simplifies the assessment process, improves accuracy, and is applicable to a variety of infectious diseases, providing strong support for clinical treatment decisions. Furthermore, it provides supporting reagent kits and systems (devices) to support detection and assessment. Its accuracy and reliability have been validated by ROC curve analysis, demonstrating broad application prospects and significant implications for precision and personalized medicine in infectious diseases. Attached Figure Description

[0091] Figure 1 shows the standard curves plotted based on the concentration and absorbance of the standards in the S100A8 / A9 ELISA kit.

[0092] Figure 2 is a box plot comparing all pneumonia groups with the normal control group, as well as the mild pneumonia group and the severe pneumonia group.

[0093] Figure 3 shows the ROC curves plotted to illustrate the predictive value of S100A8 / A9 for pneumonia severity.

[0094] Figure 4 shows the detection results of S100A8 / A9 used in Example 3 for the auxiliary diagnosis of infectious diseases.

[0095] Figure 5 shows the ROC curve of serum S100A8 / A9 levels and traditional inflammatory markers for assessing the severity of CAP in children.

[0096] Figure 6 shows the ranking of variable importance in the random forest model.

[0097] Figure 7 shows the detection results of the S100A8 / A9 serum level changes in Example 5 as an indicator for assessing the improvement of infectious disease conditions. Embodiments of the present invention

[0098] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0099] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0100] The S100A8 / A9 ELISA kit used in the following examples was purchased from Abcam (catalog number: ab267628) and includes standards, antibodies, enzyme-labeled secondary antibodies, substrate solutions, etc.

[0101] Example 1: Obtaining S100A8 / A9 as a biomarker for assessing the severity of infectious diseases

[0102] 1. Sample Information

[0103] [Revised from Rule 26, March 2025, 2019] Clinical information (see Table 1) of 534 Chinese patients diagnosed with pneumonia who visited the Beijing Children's Hospital (Baoding Branch) affiliated with Capital Medical University between 2021 and 2023 was collected, including clinical diagnosis, symptoms, signs, laboratory test results, and imaging results. According to the Guidelines for the Diagnosis and Treatment of Community-Acquired Pneumonia in Children (2019 Edition), the pneumonia patients were divided into a mild pneumonia group (n=390) and a severe pneumonia group (n=144). A control group of 93 healthy individuals who underwent health checkups at the hospital during the same period was also included.

[0104] Inclusion criteria for pneumonia patients: meeting the diagnostic criteria for community-acquired pneumonia, having clear respiratory symptoms such as fever, cough, and sputum production, and chest X-ray or CT scan showing inflammatory changes in the lungs.

[0105] Exclusion criteria for pneumonia patients: those who do not meet the inclusion criteria or whose clinical data is incomplete; those with immune deficiency or diseases that may affect immune function.

[0106] Table 1. Information on 534 samples

[0107] 2. Diagnostic criteria for the severity of infectious diseases

[0108] According to the Guidelines for the Diagnosis and Treatment of Community-Acquired Pneumonia in Children (2019 Edition), pneumonia patients are divided into mild and severe cases. The diagnostic criteria are as follows: Severe pneumonia can be diagnosed if any one of the following criteria is met: (1) poor general condition; (2) altered mental status; (3) hypoxemia: cyanosis; tachypnea, RR ≥ 70 breaths / min (infants), RR ≥ 50 breaths / min (>1 year old); assisted breathing (groaning, nasal flaring, three-recession sign); intermittent apnea; oxygen saturation < 92%; (4) hyperpyrexia, persistent high fever > 5 days; (5) dehydration / refusal to eat; (6) ≥ 2 / 3 lung infiltration, multi-lobar infiltration, pleural effusion, pneumothorax, atelectasis, lung necrosis, lung abscess; (7) serious extrapulmonary complications (heart failure, myocarditis, gastrointestinal bleeding, central nervous system infection, severe electrolyte imbalance, etc.). Patients who meet any one of the above criteria are diagnosed with severe pneumonia, and those who do not meet the criteria are diagnosed with mild pneumonia. The collected pneumonia patients were divided into a mild group and a severe group according to the above diagnostic criteria, with 390 cases in the mild group and 144 cases in the severe group.

[0109] 3. S100A8 / A9 Concentration Detection and Calculation

[0110] All children with pneumonia had 2-3 mL of fasting venous blood collected within 24-48 hours of admission (acute phase). The blood was centrifuged at 3000 rpm for 10 min, and the serum or plasma was collected and stored at -80℃ for later testing. The S100A8 / A9 level in serum or plasma was detected by enzyme-linked immunosorbent assay (ELISA).

[0111] According to the instructions for the S100A8 / A9 ELISA kit, thaw and dilute the standards, antibodies, enzyme-labeled secondary antibodies, and other reagents to the working concentration in advance.

[0112] Set up standard wells, sample wells, and blank control wells in a 96-well plate. Add standard solutions of different concentrations to the standard wells, add the serum samples to be tested to the sample wells, and add buffer solution to the blank control wells.

[0113] Add an appropriate amount of capture antibody to each well, seal the plate, and incubate it on a shaker at room temperature for 2.5 hours.

[0114] After incubation, wash away unbound antibodies, add detection antibodies (specific antibodies against S100A8 / A9), incubate again, and wash.

[0115] Add enzyme-labeled secondary antibody, incubate, wash, then add substrate solution, and incubate in the dark for a period of time until color development occurs.

[0116] The reaction was terminated by adding a stop solution, and the absorbance of each well was measured at 450 nm using an ELISA reader.

[0117] Calculate the average absorbance of each replicate of standards, controls, and samples. Subtract the mean zero standard density. Plot a standard curve on a log-logarithmic scale, with the x-axis representing standard concentration and the y-axis representing absorbance. Draw the best-fit straight line through the standard points and use the equation to calculate the concentration of S100A8 / A9 in the samples.

[0118] 4. Results Analysis

[0119] Figure 1 shows a standard curve plotted based on the concentration and absorbance of the standards in the S100A8 / A9 ELISA kit. The horizontal axis (X-axis) represents the standard concentration, ranging from 0 to 3500 pg / ml, showing the range of concentration variation. The vertical axis (Y-axis) represents absorbance, ranging from 0 to 1.6 in Abs, reflecting the degree of absorbance variation. The equation of the straight line in the graph is y = 0.0004x + 0.0269, which describes the mathematical relationship between the standard concentration (x) and absorbance (y). By comparing the absorbance of the actual sample with the values ​​on the standard curve, the concentration of S100A8 / A9 in the actual sample can be calculated, allowing for subsequent research and analysis.

[0120] Figure 2 shows a box plot comparing all pneumonia groups with the normal control group, as well as the mild pneumonia group and the severe pneumonia group. The results indicate that the S100A8 / A9 level in the pneumonia group was significantly higher than that in the healthy control group (P<0.001); the S100A8 / A9 level in the severe pneumonia group was significantly higher than that in the mild pneumonia group (P<0.001).

[0121] Statistical methods were used to analyze the correlation between serum S100A8 / A9 levels and disease severity, employing receiver operating characteristic (ROC) curves. Serum S100A8 / A9 levels were used as the test variable, and the actual severity of the patient's disease (classified according to the guidelines for the diagnosis and treatment of community-acquired pneumonia in children) was used as the state variable.

[0122] ROC curves were plotted using SPSS 27.0 and GraphPad Prism 10.0 statistical software. During plotting, the software automatically calculated the sensitivity and specificity at different S100A8 / A9 level thresholds and generated ROC curves. The shape and position of the ROC curves were observed, particularly the area under the curve (AUC). A value closer to 1 indicates higher accuracy of the S100A8 / A9 level in predicting or assessing the severity of pneumonia. The point closest to the top left corner of the ROC curve was identified; this point corresponds to the S100A8 / A9 level threshold, which is the cut-off value. This cut-off value balances sensitivity and specificity, minimizing the risk of misdiagnosis and missed diagnosis.

[0123] Figure 3 shows the ROC curve of the predictive value of S100A8 / A9 for pneumonia severity. The AUC value is 0.8311, indicating that S100A8 / A9 has good specificity and is suitable for relevant clinical or research applications. The cutoff value is 5.196 pg / ml; when the concentration of S100A8 / A9 exceeds 5.196 pg / ml, the model predicts that the patient's pneumonia may be more severe. This suggests that when the serum concentration of S100A8 / A9 exceeds 5.196 pg / ml, the patient's pneumonia may be severe and appropriate treatment should be given. Below this cutoff value, the predicted severity of the disease may be lower. This cutoff value was chosen to balance the model's sensitivity and specificity to achieve the best predictive effect.

[0124] The screening and analysis results above indicate that S100A8 / A9 can be used as a biomarker to assess the severity of infectious diseases. S100A8 / A9 is a heterodimeric complex formed by the S100A8 protein (GenBank accession number: P05109) and the S100A9 protein (GenBank accession number: P06702).

[0125] Example 2: Validation of the assessment value of S100A8 / A9 in assessing the severity of infectious diseases.

[0126] 1. Sample Information

[0127] Clinical information (not duplicated from the sample in Example 1, see Table 2) of 177 patients diagnosed with pneumonia who visited Beijing Children's Hospital Baoding Branch, affiliated with Capital Medical University, between 2021 and 2023 was collected. This included diagnosis, symptoms, signs, and laboratory test results. Inclusion and exclusion criteria were the same as in Example 1. The 177 pneumonia patients included served as a validation group to test the value of the S100A8 / A9 model in Example 1 for assessing the severity of infectious diseases.

[0128] Table 2. Information on 177 samples

[0129] 2. The methods for detecting and calculating the concentration of S100A8 / A9 were carried out in accordance with the steps in Example 1 above.

[0130] 3. Based on the critical value of 5.196 pg / ml in the ROC curve assessment model in Example 1, the S100A8 / A9 concentration data in the serum samples of all validation groups were divided into a mild group (n=115) and a severe group (n=62). The distribution of actual disease severity in these two groups after assessment according to the 2019 edition of the Guidelines for the Diagnosis and Treatment of Community-Acquired Pneumonia in Children was observed. In the mild pneumonia group, 107 cases had true negative results and 8 cases had false negative results; in the severe pneumonia group, 39 cases had true positive results and 23 cases had false positive results. As shown in Table 3, the ROC curve assessment model for determining the severity of pneumonia in Example 1 had a specificity of 82%, a sensitivity of 83%, and an accuracy of 82%.

[0131] Table 3. Validation of the specificity, sensitivity, and accuracy of S100A8 / A9 levels in assessing the severity of pneumonia.

[0132] The results in summary indicate that S100A8 / A9 has good accuracy, high sensitivity and specificity in assessing the severity of infectious diseases, and has high clinical diagnostic value.

[0133] Example 3: S100A8 / A9 used to assist in the diagnosis of infectious diseases

[0134] 1. Sample Information

[0135] We collected data from 1459 hospitalized children diagnosed with community-acquired pneumonia (CAP) at Beijing Children's Hospital Baoding Branch, affiliated with Capital Medical University, between September 2021 and December 2024. During the same period, we included 208 hospitalized children with non-infectious diseases at Beijing Children's Hospital Baoding Branch, including 58 cases of autoimmune diseases such as Kawasaki disease, systemic lupus erythematosus, allergic purpura, and juvenile idiopathic arthritis; 99 cases of metabolic diseases such as short stature, hypothyroidism, hyperthyroidism, and vitamin D deficiency; 51 cases of neurological diseases such as epilepsy and facial nerve palsy; and 188 healthy children who underwent physical examinations and had no respiratory infections within the previous month, serving as the control group. General characteristics of the CAP group and the control group are shown in Table 4.

[0136] Table 4. General Information of the CAP Group and the Control Group

[0137] Inclusion criteria for pneumonia patients: meeting the diagnostic criteria for community-acquired pneumonia, having clear respiratory symptoms such as fever, cough, and sputum production, and chest X-ray or CT scan showing inflammatory changes in the lungs.

[0138] Exclusion criteria for pneumonia patients: CAP exclusion criteria: patients with underlying cardiopulmonary diseases such as congenital bronchopulmonary dysplasia, congenital heart disease, and bronchial asthma; patients with immune deficiencies or diseases that may affect immune function; children with pneumonia complicated by fungal infection; patients who do not meet the inclusion criteria or whose clinical data are incomplete.

[0139] Inclusion criteria for children with non-infectious diseases: no infectious symptoms within one month prior to enrollment; normal laboratory test results (such as complete blood count, C-reactive protein, procalcitonin, etc.); a clearly diagnosed non-infectious disease (such as autoimmune diseases such as Kawasaki disease, systemic lupus erythematosus, allergic purpura and juvenile idiopathic arthritis, short stature, hypothyroidism, hyperthyroidism and vitamin D deficiency, etc., neurological diseases such as epilepsy and facial nerve palsy, etc.), and no secondary infection.

[0140] 2. The methods for detecting and calculating the concentration of S100A8 / A9 were performed in accordance with the steps in Example 1 above.

[0141] 3. Results Analysis

[0142] The results are shown in Table 5 and Figure 4. The serum S100A8 / A9 expression levels were compared among the three groups of children with CAP, children with non-infectious diseases, and normal healthy children. The median S100A8 / A9 levels in the three groups were 4.258 ug / ml, 0.339 ug / ml, and 0.296 ug / ml, respectively. The S100A8 / A9 levels in the CAP group were significantly higher than those in the children with non-infectious diseases and normal healthy children (P<0.05).

[0143] Table 5. Serum S100A8 / A9 levels in each group of children

[0144] Example 4: Comparing the efficacy of serum S100A8 / A9 levels with traditional inflammatory markers in assessing the severity of CAP in children.

[0145] 1. Sample Information

[0146] We collected data on 1459 hospitalized children diagnosed with community-acquired pneumonia (CAP) at Beijing Children's Hospital Baoding Branch, affiliated with Capital Medical University, from September 2021 to December 2024. According to the 2019 edition of the Guidelines for the Diagnosis and Treatment of Community-Acquired Pneumonia in Children, these patients were divided into 942 mild cases and 517 severe cases. The demographic and clinical characteristics of mild and severe CAP patients are shown in Table 6.

[0147] According to the Guidelines for the Diagnosis and Treatment of Community-Acquired Pneumonia in Children (2019 Edition), pneumonia patients are divided into mild and severe cases. The diagnostic criteria are as follows: Severe pneumonia can be diagnosed if any one of the following criteria is met: (1) poor general condition; (2) altered mental status; (3) hypoxemia: cyanosis; tachypnea, RR ≥ 70 breaths / min (infants), RR ≥ 50 breaths / min (>1 year old); assisted breathing (groaning, nasal flaring, three-recession sign); intermittent apnea; oxygen saturation < 92%; (4) hyperpyrexia, persistent high fever > 5 days; (5) dehydration / refusal to eat; (6) ≥ 2 / 3 lung infiltration, multi-lobar infiltration, pleural effusion, pneumothorax, atelectasis, lung necrosis, lung abscess; (7) serious extrapulmonary complications (heart failure, myocarditis, gastrointestinal bleeding, central nervous system infection, severe electrolyte imbalance, etc.). Patients who meet any one of the above criteria are diagnosed with severe pneumonia, and those who do not meet the criteria are diagnosed with mild pneumonia.

[0148] Table 6. Demographic and clinical characteristics of mild and severe CAP patients.

[0149] Note: Data are baseline measurements taken near the time of blood collection after admission. P < 0.05 indicates statistical significance. Some cases of mild pneumonia had only a small amount of pleural effusion, not meeting the criteria for severe pneumonia, and were therefore diagnosed as mild pneumonia.

[0150] 2. The methods for detecting and calculating the concentration of S100A8 / A9 were performed in accordance with the steps in Example 1 above.

[0151] 3. Results Analysis

[0152] The ability of serum S100A8 / A9 levels and traditional inflammatory markers to assess the severity of CAP in children was analyzed. The results are shown in Table 7 and Figure 5. The AUC of S100A8 / A9 in assessing CAP severity was 84.5%, significantly better than NEU (67.5%), CRP (61.7%), and WBC (58.1%). Random forest model ranking of variable importance (Figure 6) showed that S100A8 / A9 had the highest weight (46.8%), better than NEU score (20.2%), CRP score (17.7%), and WBC score (15.3%), highlighting its crucial role in diagnosing CAP severity. S100A8 / A9 can be used as an inflammatory marker to assist in the diagnosis of infectious diseases and to assess their severity.

[0153] Table 7. Efficacy of serum S100A8 / A9 levels in assessing the severity of CAP in children using traditional inflammatory markers.

[0154] Example 5: Changes in S100A8 / A9 serum levels were used to assess the improvement of infectious disease conditions.

[0155] 1. Sample Information

[0156] Of the 1,459 children with CAP included, serum samples were collected from 145 children after treatment, and serum S100A8 / A9 levels were measured.

[0157] 2. The methods for detecting and calculating the concentration of S100A8 / A9 were performed in accordance with the steps in Example 1 above.

[0158] 3. Results Analysis

[0159] The results of comparing serum S100A8 / A9 levels before and after treatment in children with pneumonia are shown in Table 8 and Figure 7. The median serum S100A8 / A9 levels before and after treatment were 8.327 ug / mL and 2.727 ug / mL, respectively. The serum S100A8 / A9 levels in children with CAP were significantly lower after treatment than before treatment.

[0160] Table 8. Comparison of S100A8 / A9 serum levels before and after treatment in children with pneumonia.

[0161] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Industrial applicability

[0162] The method for assessing the severity of infectious diseases provided by this invention is rapid and accurate, with wide clinical applications. It is suitable for various infectious diseases, providing strong support for clinical treatment decisions, offering more precise treatment plans to patients, guiding the selection and adjustment of treatment plans, and achieving optimal disease management. Simultaneously, this invention provides supporting reagent kits and systems (devices) to support detection and assessment, and can be widely applied in precision medicine and personalized medicine for infectious diseases.

[0163] Cross-references to related applications

[0164] This application claims priority to Chinese Patent Application No. 202411528598.1, filed on October 30, 2024, entitled "Application of S100A8 / A9 in assessing the severity of infectious diseases", the entire contents of which are incorporated herein by reference.

Claims

1. The use of biomarkers and / or substances that detect said biomarkers in the preparation of products having any of the following functions: A1) Used to assess the severity of infectious diseases; A2) Used to predict or assist in predicting the progression of infectious diseases; The biomarker is S100A8 / A9.

2. The application according to claim 1, characterized in that, The substances used to detect the biomarkers include reagents and / or instruments for detecting the expression levels of S100A8 / A9 using methods such as Western blotting, immunofluorescence, radioimmunoassay, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), double-antibody sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay, flow cytometry, chromatography, mass spectrometry, polyacrylamide gel electrophoresis, capillary electrophoresis, near-infrared spectroscopy, immunochemiluminescence, colloidal gold immunochromatography, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immuno-PCR, or biotin-avidin technology.

3. The application according to claim 1 or 2, characterized in that, The substances used to detect the biomarkers include reagents for detecting the expression levels of S100A8 / A9.

4. The application according to claim 3, characterized in that, The reagents include antibodies, peptides, proteins, or nucleic acid molecules that specifically bind to S100A8 / A9.

5. The application according to any one of claims 1-4, characterized in that, The infectious diseases mentioned include those caused by viral, bacterial, fungal, mycoplasmal, chlamydial, rickettsial, spirochetal, and parasitic infections.

6. The application according to any one of claims 1-5, characterized in that, The infectious diseases mentioned are lower respiratory tract infections and / or pneumonia caused by various pathogens.

7. The application according to any one of claims 1-6, characterized in that, The products include reagents, kits, detection chips, test strips, test cards, and immunosensors.

8. A reagent kit, characterized in that, The kit comprises the substance for detecting the biomarker as described in any one of claims 1-4, and the kit has at least one of the following uses: B1) Used to assess the severity of infectious diseases; B2) is used to predict or assist in predicting the progression of infectious diseases.

9. The reagent kit according to claim 8, characterized in that, The test sample for the kit is serum or plasma.

10. A device for assessing the severity of an infectious disease, characterized in that, The device includes a data receiving module, a data processing module, and an output module; the data receiving module is used to receive data on the expression levels of S100A8 / A9 in the serum or plasma of patients with infectious diseases; the data processing module is used to assess the severity of infectious diseases based on the detection results of S100A8 / A9 expression levels; and the output module is used to display the assessment results.

11. The use of biomarkers and / or substances that detect said biomarkers in the preparation of products having any of the following functions: C1) is used to assist in the diagnosis of whether a subject has an infectious disease; C2) is used to assess the improvement of symptoms in infectious diseases; The biomarker is S100A8 / A9.

12. A method for assessing the severity of infectious diseases, characterized in that, The method includes assessing the severity of infectious diseases based on the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases.

13. The method according to claim 12, characterized in that, The method includes the following steps: (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases; (2) Compare the expression level of S100A8 / A9 with the evaluation threshold, and evaluate the severity of infectious disease based on the comparison results.

14. The method according to claim 13, characterized in that, The assessment of the severity of infectious diseases based on the comparison results is as follows: when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is greater than or equal to the assessment threshold, the severity of the infectious disease is predicted to be high; when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is less than the assessment threshold, the severity of the infectious disease is predicted to be low.

15. A method for predicting or assisting in predicting the progression of an infectious disease, the method comprising predicting or assisting in predicting the progression of an infectious disease based on the expression level or content of S100A8 / A9 in the serum or plasma of patients with an infectious disease.

16. The method according to claim 15, characterized in that, The method includes the following steps: (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases; (2) Compare the expression level of S100A8 / A9 with the evaluation threshold, and predict or assist in the prediction of the progression of infectious diseases based on the comparison results.

17. The method according to claim 16, characterized in that, The prediction or auxiliary prediction of the progression of infectious diseases based on the comparison results is as follows: when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is greater than or equal to the assessment threshold, the progression of infectious diseases to severe pneumonia is predicted; when the expression level of S100A8 / A9 in the serum or plasma of patients with infectious diseases is less than the assessment threshold, the progression of infectious diseases to mild pneumonia is predicted.

18. A method for assisting in the diagnosis of whether a subject has an infectious disease, the method comprising assisting in the diagnosis of whether a subject has an infectious disease based on the expression level or content of S100A8 / A9 in the subject's serum or plasma.

19. The method according to claim 18, characterized in that, The method includes the following steps: (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of the subjects; (2) Compare the expression level of S100A8 / A9 with the assessment threshold, and make an auxiliary diagnosis on whether the subject has an infectious disease based on the comparison results.

20. The method according to claim 19, characterized in that, The auxiliary diagnosis of whether a subject has an infectious disease based on the comparison results is as follows: when the expression level of S100A8 / A9 in the subject's serum or plasma is greater than or equal to the assessment threshold, it is judged that the subject has an infectious disease or is highly likely to have an infectious disease; when the expression level of S100A8 / A9 in the subject's serum or plasma is less than the assessment threshold, it is judged that the subject does not have an infectious disease or is very unlikely to have an infectious disease.

21. A method for assessing the improvement of a disease condition in an infectious disease, the method comprising assessing the improvement of a disease condition based on the expression level or content of S100A8 / A9 in the serum or plasma of a patient with an infectious disease before and after treatment.

22. The method according to claim 21, characterized in that, The method includes the following steps: (1) Detect the expression level or content of S100A8 / A9 in the serum or plasma of patients with infectious diseases before and after treatment; (2) Compare the S100A8 / A9 expression levels after treatment with those before treatment, and evaluate the improvement of the infectious disease based on the comparison results.

23. The method according to claim 22, characterized in that, The assessment of the improvement of infectious disease condition based on the comparison results is as follows: when the S100A8 / A9 expression level after treatment is significantly lower than before treatment, the infectious disease condition is considered to have improved.

24. The method according to any one of claims 11-23, characterized in that, The expression level or content of S100A8 / A9 was detected by Western blotting, immunofluorescence, radioimmunoassay, immunoprecipitation, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), double-antibody sandwich ELISA, fluorescence-linked immunosorbent assay (FLISA), enzyme immunoassay, flow cytometry, chromatography, mass spectrometry, polyacrylamide gel electrophoresis, capillary electrophoresis, near-infrared spectroscopy, immunochemiluminescence, colloidal gold immunochromatography, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SIP), immuno-PCR, or biotin-avidin technology.

25. The method according to any one of claims 11-24, characterized in that, The infectious diseases mentioned include those caused by viral, bacterial, fungal, mycoplasmal, chlamydial, rickettsial, spirochetal, and parasitic infections.

26. The application according to any one of claims 11-25, characterized in that, The infectious diseases mentioned are lower respiratory tract infections and / or pneumonia caused by various pathogens.

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