Biomarker for identifying sepsis induced by infectious pneumonia and non-infectious lung disease and application thereof

Abstract

The invention belongs to the technical field of diagnosis, and particularly relates to a biomarker for identifying sepsis and lung diseases induced by infectious pneumonia and application of the biomarker. The invention discloses application of a detection agent or component for acquiring marker molecule level in a sample in preparation of a product for identifying sepsis and non-infectious lung diseases induced by infectious pneumonia, the marker molecule comprises at least one of ITGA7, CDKN2C and PCOLCE2, and sepsis and non-infectious lung diseases induced by infectious pneumonia can be effectively identified. The method is further used for guiding use of antibiotics.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311369798.2 filed with the Chinese Patent Office on October 20, 2023, entitled “Differentiation of Sepsis Induced by Infectious Pneumonia and Non-infectious Lung Disease”, the entire contents of which are incorporated into this application by application. Technical Field

[0003] The present application relates to the field of diagnosis, and in particular to biomarkers for distinguishing sepsis induced by infectious pneumonia and non-infectious lung diseases and their uses. Background Art

[0004] Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated response to infection. Clinical manifestations of sepsis are hypotension, tachycardia, fever, and leukocytosis. As the condition worsens, signs of shock (such as cool skin and cyanosis) and signs of organ dysfunction (such as oliguria, acute kidney injury, and altered mental status) appear. According to the Global Burden of Disease Study, there were an estimated 48.9 million new cases of sepsis in 2017. There were approximately 11 million deaths, accounting for 19.7% of the total global deaths. Research data from 2020 showed that the incidence of ICU sepsis was 20%, and in a multicenter study of ICUs, the mortality rate of sepsis patients was 35.5%. A sepsis epidemiological survey found that 1.02 million patients died of sepsis nationwide in 2015, accounting for 12.6% of the total mortality rate.

[0005] It is very important for patients with sepsis induced by infectious pneumonia to receive adequate and broad-spectrum anti-infective treatment in the early stage. In order to identify patients with sepsis outside the ICU early, a simplified version of the SOFA score, the rapid sequential organ failure assessment (qSOFA), is proposed based on the following three criteria: altered consciousness, systolic blood pressure ≤100 mmHg, and respiratory rate ≥22 times / min; patients who meet 2 of the 3 criteria should be included in the category of sepsis. However, the clinical manifestations of sepsis induced by non-infectious lung disease and pneumonia are similar, and it is very easy to misdiagnose sepsis induced by pneumonia: the manifestations vary depending on the condition. If the area of ​​lesions in the lungs is relatively large, hypoxia symptoms will occur, and symptoms such as increased breathing, decreased blood pressure, chest tightness and shortness of breath will appear. About more than 20% of patients with sepsis induced by infectious pneumonia with qSOFA>2 have non-infectious diseases and need to be excluded through infection detection, but infection source detection often takes 48-72 hours. In order to control the disease as early as possible, clinical practice often gives empirical antibiotic treatment to all patients with qSOFA>2, leading to the abuse of antibiotics in patients with non-infectious lung diseases.

[0006] Therefore, continuing to explore potential diagnostic targets for sepsis, exploring the mechanism of action of targets, and promoting new drug development and clinical transformation will be of great significance to the treatment of sepsis.

[0007] In view of this, this application is filed. SUMMARY OF THE INVENTION

[0009] In order to solve the above technical problems, the present application screens the genes or their proteins that are differentially expressed in peripheral blood monocytes of patients with sepsis induced by infectious pneumonia and patients with non-infectious lung diseases, and finds indications for distinguishing sepsis induced by infectious pneumonia and non-infectious lung diseases, which are then used to guide the use of antibiotics and solve the pain points of clinical antibiotic abuse and misuse.

[0010] Therefore, this application has at least the following purposes:

[0011] The purpose of this application is to seek an application for distinguishing sepsis and non-infectious lung diseases induced by infectious pneumonia, as well as the use of guiding antibiotic therapy, and related products or methods.

[0012] To achieve the above objectives, this application proposes the following specific technical solutions:

[0013] The present application provides the use of a detection agent or component for obtaining the level of a marker molecule in a sample in the preparation of a product for distinguishing sepsis induced by infectious pneumonia and non-infectious lung disease, wherein the marker molecule is at least one of ITGA7, CDKN2C, and PCOLCE2.

[0014] The present application provides the use of a detection agent or component for obtaining the level of a marker molecule in a sample in the preparation of a product for evaluating whether to perform antibiotic therapy on a patient, wherein the marker molecule is at least one of ITGA7, CDKN2C, and PCOLCE2.

[0015] The present application provides the use of marker molecules in distinguishing sepsis induced by infectious pneumonia and non-infectious lung diseases; the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2.

[0016] The present application provides the use of marker molecules in assessing whether to perform antibiotic therapy on a patient; the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2.

[0017] The present application provides a product for distinguishing sepsis and non-infectious lung diseases induced by infectious pneumonia, comprising a detection agent or component for obtaining the level of marker molecules in a sample, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2.

[0018] The present application provides a product for evaluating whether to perform antibiotic therapy on a patient, comprising a detection agent or component for obtaining the level of marker molecules in a sample, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2.

[0019] In some embodiments, the product includes, but is not limited to, a kit form, a system device form, a computer readable medium, or a computer system form.

[0020] In some embodiments, the marker molecule level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease. For example, in some embodiments, ITGA7 level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease, in some embodiments, CDKN2C level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease, and in some embodiments, PCOLCE2 level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease.

[0021] In some embodiments, the patient's qSOFA>2 is evaluated for antibiotic therapy.

[0022] In some embodiments, the level includes nucleic acid level or protein level, such as nucleic acid expression level of a marker molecule, or protein expression level, to determine whether the subject has sepsis induced by infectious pneumonia or non-infectious lung disease.

[0023] In some embodiments, the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein; in some embodiments, it may also include the abundance or concentration of mutations such as nucleic acid or protein.

[0024] In some embodiments, the nucleic acid level is obtained or detected by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0025] In some embodiments, the nucleic acid level is obtained or detected by gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry, single molecule detection or liquid chromatography.

[0026] In some embodiments, the reagent for detecting the nucleic acid level includes specific primers and / or probes for detecting the nucleic acid. It should be noted that specific primers generally include primers designed according to the target nucleic acid, such as primers for gene sequencing, primers for polymerase chain reaction, primers for isothermal amplification reaction, or other primers capable of detecting the target nucleic acid; specific probes generally include probes designed according to the target nucleic acid, such as real-time fluorescence quantitative PCR probes, gene chip detection probes, specific probes for probe hybridization, or other probes capable of detecting the target nucleic acid.

[0027] In some embodiments, the nucleic acid includes the gene of the biomarker molecule, a DNA fragment extracted from the gene of the biomarker molecule, or RNA corresponding to the gene of the biomarker molecule or its DNA fragment, such as mRNA.

[0028] In some embodiments, the protein level is obtained or detected by sequencing technology, immunoassay technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0029] In some embodiments, the protein level is measured by amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein profiling, immuno-PCR, single molecule detection, or liquid chromatography.

[0030] In some embodiments, the reagent for detecting the level of the protein comprises detecting a ligand for the protein.

[0031] In some embodiments, the ligand comprises at least one of an antibody, an antigen binding fragment, a single-chain antibody, and a nucleic acid aptamer.

[0032] In some embodiments, the product also includes detection agents or components for acquiring levels of other marker molecules.

[0033] In some embodiments, the other marker molecules include but are not limited to one or more of PCT, CRP, HBP, and IL-6.

[0034] In some embodiments, the product also includes a sample processing reagent, and the sample processing reagent depends on the specific sample. For example, more specifically, the sample processing reagent includes at least one of a sample lysis reagent, a sample purification reagent or a sample extraction reagent.

[0035] In some embodiments, the product further comprises at least one of a standard, a calibrator, a control, or a buffer.

[0036] In some embodiments, the product of the present application can be supplemented with tubes for mixing the components, microplates, and instruction materials recording the method of use, etc. as needed.

[0037] In some embodiments, the sample of the present application includes tissue, cells, urine, saliva, pleural effusion, ascites, feces, synovial fluid or various body fluids. Wherein, body fluid refers to intracellular fluid and extracellular fluid, including tissue fluid, plasma, blood (such as whole blood, serum, or plasma), lymph, cerebrospinal fluid, semen, aqueous humor, etc. In some embodiments, the sample includes peripheral blood mononuclear cells.

[0038] It is understood that conventional reagents or components corresponding to specific detection methods can be purchased commercially or integrated into the applications, methods or products of the present application.

[0039] The present application also provides a method for identifying sepsis induced by infectious pneumonia, comprising the step of obtaining the level of marker molecules in a sample of a subject, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2. In some embodiments, the method is an in vitro method.

[0040] In some embodiments, the method comprises the following steps: (a) detecting or determining the level of marker molecules in the sample of the subject, and (b) comparing with a set threshold absolute amount; wherein the threshold absolute amount can distinguish between sepsis induced by infectious pneumonia and non-infectious lung disease; in some embodiments, the level of marker molecules in samples of patients with sepsis induced by infectious pneumonia is higher than that in patients with non-infectious lung disease;

[0041] Alternatively; (b) using the marker molecule level in the subject's sample as an input value and operating in an algorithm for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease.

[0042] In some embodiments, the marker molecule level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease. For example, in some embodiments, ITGA7 level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease, in some embodiments, CDKN2C level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease, and in some embodiments, PCOLCE2 level is used as an independent indicator for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease.

[0043] In some embodiments, the method further comprises the step of obtaining or detecting the levels of other marker molecules in the subject's sample. In some embodiments, the other marker molecules include but are not limited to one or more of PCT, CRP, HBP, and IL-6.

[0044] The present application also provides a method for evaluating whether to perform antibiotic therapy on a patient, comprising the step of obtaining the level of marker molecules in a patient sample, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2;

[0045] In some embodiments, the method comprises the steps of: (i) detecting or determining the level of a marker molecule in the sample of the subject, and (ii) comparing it with a set threshold absolute amount; in some embodiments, antibiotic therapy is performed on patients whose marker molecule level is higher than the threshold absolute amount.

[0046] In some embodiments, the method further comprises the step of obtaining the level of other marker molecules in the patient sample. In some embodiments, the other marker molecules include but are not limited to one or more of PCT, CRP, HBP, and IL-6.

[0047] In some embodiments, the patient has qSOFA>2.

[0048] In some embodiments, the level comprises a nucleic acid level or a protein level.

[0049] In some embodiments, the nucleic acid level is the expression level of DNA, RNA or mRNA. In some embodiments, the nucleic acid includes the gene of the marker molecule, a DNA fragment intercepted from the gene of the biomarker molecule, the RNA corresponding to the gene of the biomarker molecule or its DNA fragment, such as mRNA.

[0050] In some embodiments, the nucleic acid level or protein level includes but is not limited to the abundance or concentration of nucleic acid or protein; in some embodiments, it may also include the abundance or concentration of mutations such as nucleic acid or protein.

[0051] In some embodiments, the nucleic acid level is obtained or detected by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0052] In some embodiments, the nucleic acid level is obtained or detected by gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip, probe hybridization, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry, single molecule detection or liquid chromatography.

[0053] In some embodiments, the protein level is obtained or detected by sequencing technology, immunoassay technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology.

[0054] In some embodiments, protein levels are obtained or detected by amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein profiling, immuno-PCR, single molecule detection, or liquid chromatography.

[0055] In some embodiments, the reagent for detecting the protein level includes a ligand for detecting the protein. In some embodiments, the ligand includes at least one of an antibody, an antigen binding fragment, a single-chain antibody, and a nucleic acid aptamer.

[0056] The samples of the present application include tissues, cells, urine, saliva, pleural effusion, ascites, feces, synovial fluid or various body fluids. Among them, body fluid refers to intracellular fluid and extracellular fluid, including tissue fluid, plasma, blood (such as whole blood, serum, or plasma), lymph, cerebrospinal fluid, semen, aqueous humor, etc. In some embodiments, the sample includes peripheral blood mononuclear cells.

[0057] In some embodiments, the subject or patient is preferably a human.

[0058] In some embodiments, the method can be used for disease diagnosis or non-disease diagnosis.

[0059] The present application also provides a method for treating sepsis or non-infectious lung disease induced by infectious pneumonia, the method comprising the following steps:

[0060] (1) obtaining the level of at least one marker molecule among ITGA7, CDKN2C, and PCOLCE2 in the subject sample and comparing it with the level in the control sample;

[0061] (2) comparing with a set threshold absolute amount, wherein the threshold absolute amount can distinguish between sepsis induced by infectious pneumonia and non-infectious lung disease; optionally, the level of marker molecules in samples of patients with sepsis induced by infectious pneumonia is higher than that in patients with non-infectious lung disease;

[0062] or;

[0063] (2) using the level of at least one marker molecule among ITGA7, CDKN2C, and PCOLCE2 in the subject sample as an input value and operating in an algorithm for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease;

[0064] (3) If the subject suffers from sepsis induced by infectious pneumonia, the subject will be treated with anti-infective drugs; if the subject suffers from non-infectious lung disease, the subject does not need to be treated with anti-infective drugs.

[0065] In some embodiments, the anti-infective drugs include, but are not limited to, one or more of antibiotic drugs, antiviral drugs, antifungal drugs, and antiparasitic drugs.

[0066] In some embodiments, if the subject suffers from sepsis induced by infectious pneumonia, the subject is treated with antibiotics.

[0067] In some embodiments, the antibiotic drugs include, but are not limited to, one or more of penicillins, cephalosporins, macrolides, fluoroquinolones, and nitroimidazoles.

[0068] In this application, non-infectious lung disease refers to lung diseases caused by non-infectious causes, common causes include exposure to air irritants at work or in hobbies. In addition, certain cancer treatments and dozens of drugs can also cause non-infectious lung disease. Dyspnea is usually accompanied by a dry cough (without sputum) is the most common symptom of non-infectious lung disease. Diagnostic methods for non-infectious lung disease include: blood tests, chest X-rays or imaging tests such as CT, pulmonary function tests, bronchoscopy, surgical lung biopsy, etc.

[0069] In some embodiments, non-infectious lung diseases include but are not limited to aspiration pneumonia, interstitial lung disease, or alveolar hemorrhage syndrome.

[0070] Beneficial technical effects of this application:

[0071] The present application screens genes or their proteins that are differentially expressed in peripheral blood monocytes of patients with sepsis induced by infectious pneumonia and patients with non-infectious lung diseases, discovers indications for distinguishing sepsis induced by infectious pneumonia and non-infectious lung diseases, and then uses them to guide the use of antibiotics and solve the pain points of clinical antibiotic abuse and misuse. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0073] Figure 1 , the discovery set showed that the expression of patients with infectious pneumonia-induced sepsis (Cap_sepsis) in peripheral blood mononuclear cells was higher than that of patients with non-infectious lung disease (No_Cap);

[0074] Figure 2 , the validation set showed that the expression of patients with sepsis induced by infectious pneumonia (Cap_sepsis) in peripheral blood mononuclear cells was higher than that of patients with non-infectious lung disease (No_Cap);

[0075] Figure 3 ,The discovery set shows the ROC curves of ITGA7, CDKN2C, and PCOLCE2;

[0076] Figure 4 ,The validation set shows the ROC curves of ITGA7, CDKN2C, and PCOLCE2;

[0077] Figure 5 , found the ROC curve of the centralized ITGA7+PCOLCE2 joint test;

[0078] Figure 6 , found the ROC curve of the centralized ITGA7+CDKN2C joint test;

[0079] Figure 7 , found the ROC curve of the centralized CDKN2C+PCOLCE2 joint test;

[0080] Figure 8 , found the ROC curve of the centralized ITGA7+CDKN2C+PCOLCE2 joint test;

[0081] Fig. 9 , the difference in ITGA7 expression levels between clinical samples of sepsis induced by infectious pneumonia and clinical samples of non-infectious lung diseases;

[0082] Fig.10 , CDKN2C expression level difference between clinical samples of sepsis induced by infectious pneumonia and clinical samples of non-infectious lung diseases;

[0083] Fig.11 , The difference in PCOLCE2 expression levels between clinical samples of sepsis induced by infectious pneumonia and clinical samples of non-infectious lung diseases. DETAILED DESCRIPTION OF THE INVENTION

[0085] The content for which protection is requested in this application can be realized by those skilled in the art with reference to the content of this article. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The methods and applications of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the preparation methods and applications herein without departing from the content, spirit and scope of this application to realize and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which this application belongs.

[0086] The following basic terms or definitions are provided only to help understand the application. These definitions should not be construed as having a scope less than that understood by those skilled in the art. Unless defined otherwise hereinafter, the meaning of all technical terms and scientific terms used in the specific embodiments of the application is intended to be the same as that commonly understood by those skilled in the art. Although it is believed that the following terms are well understood by those skilled in the art, the following definitions are still set forth to better explain the application.

[0087] As used in this application, the terms "comprises", "comprising", "having", "containing" or "involving" are inclusive or open-ended and do not exclude other unrecited elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a group is defined below as comprising at least a certain number of embodiments, this should also be understood to disclose a group that optionally consists of only these embodiments.

[0088] When referring to a singular noun an indefinite or definite article e.g. "a" or "an", "the" or "an" is used, this includes a plural of that noun.

[0089] The terms "approximately" and "substantially" in this application represent the accuracy range that can be understood by those skilled in the art to still ensure the technical effect of the feature in question. The term usually represents ±10% deviation from the indicated value, preferably ±5%.

[0090] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessarily required to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances, and the embodiments described in this application can be implemented in other sequences than those described or illustrated in this application.

[0091] References to embodiments of the present application will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present application. It will be apparent to those skilled in the art that various modifications and variations may be made to the present application without departing from the scope or spirit of the present application. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment. Therefore, it is intended that the present application covers such modifications and variations that fall within the scope of the appended claims and their equivalents. Other objects, features, and aspects of the present application are disclosed in or apparent from the following detailed description. It will be appreciated by those of ordinary skill in the art that this discussion is merely a description of exemplary embodiments and is not intended to limit the broader aspects of the present application.

[0092] As used herein, the term "differentiation" refers to determining which disease a patient with one or more similar symptoms suffers from or is most likely to suffer from from two or more diseases based on analysis of clinical data. In some embodiments, it can be determined or assisted in determining or inferring whether a patient suffers from sepsis induced by infectious pneumonia or non-infectious lung disease based on the level of marker molecules.

[0093] As used herein, the terms "sepsis" and "Sepsis" are interchangeable. Sepsis is a systemic inflammatory response syndrome caused by the invasion of bacteria and other pathogenic microorganisms into the body. In addition to the manifestations of systemic inflammatory response syndrome and primary infection lesions, critically ill patients often have manifestations of organ insufficiency. Generally including previous sepsis and sepsis. According to the severity of sepsis, it can be divided into sepsis, severe sepsis and septic shock. Severe sepsis refers to sepsis accompanied by organ dysfunction, poor tissue perfusion or hypotension. Septic shock refers to severe sepsis accompanied by persistent hypotension that cannot be corrected after adequate fluid resuscitation, and is also considered a special type of severe sepsis.

[0094] Sepsis has a high incidence rate. There are more than 18 million cases of severe sepsis worldwide each year, and 750,000 cases of sepsis in the United States each year, and this number is increasing at a rate of 1.5% to 8.0% per year. Sepsis is a serious disease with a high mortality rate. About 14,000 people die from its complications every day worldwide, and about 215,000 people die in the United States each year. According to foreign epidemiological surveys, the mortality rate of sepsis has exceeded that of myocardial infarction and has become the main cause of death in non-cardiac patients in intensive care units. In recent years, despite great progress in anti-infection treatment and organ function support technology, the mortality rate of sepsis is still as high as 30% to 70%. Sepsis treatment is expensive and consumes a lot of medical resources, which seriously affects the quality of human life and has posed a huge threat to human health. Therefore, in 2001, the European Society of Intensive Care Medicine, the American Society of Critical Care Medicine, and the International Sepsis Forum launched the "Surviving Sepsis Campain" (Sepsis C). In 2002, multiple organizations in Europe and the United States jointly launched and signed the "Barcelona Declaration", and further developed and continuously updated the Sepsis C Guidelines based on evidence-based medicine research on sepsis to improve the treatment of sepsis and reduce the mortality of sepsis. The Sepsis C Guidelines were first formulated in 2003 and revised again in 2008.

[0095] The term "pneumonia" as used herein includes infectious pneumonia and non-infectious lung disease. Infectious pneumonia refers to pneumonia caused by pathogenic microorganisms; pathogenic microorganisms include bacteria, viruses, chlamydia, mycoplasma, rickettsia, parasites, etc., among which pneumonia caused by bacteria is the most common, and bacterial pneumonia is a type of infectious pneumonia. Common symptoms are cough, sputum, or aggravation of existing respiratory symptoms, and the appearance of purulent sputum or bloody sputum, with or without chest pain. Patients with large lesions of pneumonia may have dyspnea and respiratory distress. Most patients have fever. There are no obvious abnormalities in early lung signs, and severe patients may have increased respiratory rate, nasal flaring, and cyanosis. There are typical signs of pulmonary consolidation, such as dullness on percussion, increased vocal fremitus, and bronchial breath sounds, and moist rales can also be heard. In patients with concurrent pleural effusion, the affected side of the chest has dullness on percussion, weakened vocal fremitus, and weakened breath sounds. Non-infectious lung disease means that the inflammation of the lungs is not caused by the invasion of microorganisms such as bacteria or viruses. It is more common in lung trauma, drowning, smoke inhalation, including pneumonia caused by autoimmune diseases, and even pneumonia caused by allergic reasons. The manifestations vary depending on the condition. If the area of ​​lesions in the lungs is relatively large, symptoms of hypoxia will appear, with increased breathing, even chest tightness, shortness of breath, and difficulty breathing, accompanied by cyanosis of the lips and nail beds, which is a more serious hypoxia. Others may cause an increase in heart rate and a decrease in blood pressure, which varies according to different situations. In addition, there are manifestations of the primary disease that causes the disease itself, such as autoimmune diseases, which may cause bone and joint deformation or rash, etc.

[0096] As used herein, the term "ITGA7" means that the protein encoded by this gene belongs to the integrin alpha chain family. Integrins are heterodimeric integral membrane proteins composed of alpha and beta chains. They mediate a wide range of cell-cell and cell-matrix interactions and therefore play a role in cell migration, morphological development, differentiation and metastasis. This protein is a receptor for the basement membrane protein laminin-1. It is primarily expressed in skeletal and cardiac muscle and may be involved in the differentiation and migration processes of myogenesis. Defects in this gene are associated with congenital myopathies. Splicing transcript variations encoding different isoforms of this gene have been found.

[0097] The protein encoded by the gene "CDKN2C" as used herein is a member of the INK4 family of cyclin-dependent kinase inhibitors. The protein has been shown to interact with CDK4 or CDK6 and prevent the activation of CDK kinases, thus acting as a cell growth regulator that controls the G1 progression of the cell cycle. Ectopic expression of this gene has been shown to inhibit the growth of human cells in a manner that appears to be related to the presence of wild-type RB1 function. Studies on knockout mice have shown a role for this gene in regulating spermatogenesis as well as suppressing tumorigenesis. Two alternatively spliced ​​transcript variants of this gene, encoding the same protein, have been reported.

[0098] As used herein, the term "PCOLCE2" encodes a protein that enhances collagen binding activity; heparin binding activity; and peptidase activator activity. The protein is predicted to be involved in the positive regulation of peptidase activity. It is predicted to act on the upstream or intracellular response of leukemia inhibitory factor. It is expected to be located in the extracellular region.

[0099] As used herein, "level" is understood in the broadest sense, including qualitative or quantitative levels, including but not limited to content, abundance, concentration, degree, ratio, multiple, etc.

[0100] The term antibiotic therapy and antibiotic treatment are used interchangeably. According to the present invention, patients with sepsis induced by confirmed or suspected infectious pneumonia can receive antibiotic treatment, while patients with confirmed non-infectious lung disease do not need to receive antibiotic treatment, thereby evaluating or guiding whether the patient needs antibiotic therapy.

[0101] As used herein, "treatment" includes any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) alleviating the disease, i.e., causing partial or complete improvement of the disease. The therapeutic agent may be administered before, during, or after the onset of the disease or injury.

[0102] The terms "individual," "subject," "subject," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly a human, in need of diagnosis, treatment, or therapy.

[0103] The terms "sample", "sample", "test sample", "subject sample" and the like as used herein include various sample types obtained from a patient, individual or subject and can be used for diagnosis or monitoring determination. Patient samples can be obtained from healthy subjects, sick patients or patients with sepsis (e.g., sepsis induced by community-acquired pneumonia) related symptoms. In addition, the sample obtained from the patient can be divided into parts, and only a part can be used for diagnosis. In addition, the sample or a part thereof can be stored under the condition of keeping the sample for subsequent analysis. This definition specifically includes other liquid samples of blood and biological origin (including but not limited to tissue, cells (such as PMBC cells, T cells, etc.), body fluids, serum, plasma, whole blood, urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid, etc.). In a specific embodiment, the sample includes a blood sample. In another embodiment, a serum sample is used. In a specific embodiment, the sample includes peripheral blood mononuclear cells. This definition also includes samples that are manipulated in any way after the sample is obtained, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washing or enrichment of certain cell populations. These terms also include clinical samples, and also include cells in culture, cell supernatants, tissue samples, organs, etc. Samples can also include fresh frozen and / or formalin-fixed, paraffin-embedded tissue blocks, such as blocks prepared from clinical or pathological biopsies, prepared for pathological analysis or research by immunohistochemistry. Samples can be tested immediately after collection, stored at RT, 4 degrees Celsius, -20 degrees Celsius, or -80 degrees Celsius, and tested after storage for 24 hours, 1 week, 1 month, 1 year, 10 years, or up to 30 years.

[0104] The term "ROC" or "ROC curve" as used herein may refer to a receptor operating characteristic curve. A ROC curve may be a graphical representation of the performance of a binary classifier system. For any given method, a ROC curve may be generated by plotting sensitivity for specificity under a variety of threshold settings. In addition, as long as at least one of the three parameters (e.g., sensitivity, specificity, and threshold settings) is provided, and the ROC curve may determine the value or expected value of any unknown parameter. Unknown parameters may be determined using a curve fit for the ROC curve.

[0105] As used herein, the term "AUC" or "ROC-AUC" generally refers to the area under the receiver operating characteristic curve. This metric takes into account the sensitivity and specificity of the method and can provide a measured value of the diagnostic utility of the method. Typically, ROC-AUC is in the range of 0.5 to 1.0, where a value closer to 0.5 indicates that the method has limited diagnostic utility (e.g., lower sensitivity and / or specificity), and a value closer to 1.0 indicates that the method has greater diagnostic utility (e.g., higher sensitivity and / or specificity). See, e.g., Pepe et al., "Limitations of the Odds Ratio in Gauging the Performance of a Diagnostic, Prognostic, or Screening Marker", Am. J. Epidemiol 2004, 159 (9): 882-890, which is incorporated herein by reference in its entirety. Other approaches to characterizing diagnostic utility using likelihood functions, odds ratios, information theory, predictive values, calibration (including goodness of fit), and reclassification measures are summarized in Cook, "Use and Misuse of the Receiver Operating Characteristic Curve in Risk Prediction), Circulation 2007, 115:928-935, which is incorporated herein by reference in its entirety.

[0106] The detection agent or component for obtaining the level of marker molecules in the sample can be understood to include the detection reagent for directly obtaining the level of marker molecules in the sample, or the component for indirectly obtaining the level of marker molecules in the sample (such as a computer program directly obtaining an indicator of the level of marker molecules in a sample that has been detected).

[0107] According to some embodiments of the present application, marker molecule level indicators can be obtained at nucleic acid level, protein level, etc. The detection method is not limited, and any method that can be used to directly or indirectly evaluate marker molecules or other marker molecule nucleic acid or protein levels is suitable for this application.

[0108] As used herein, "nucleic acid level" should be understood in a broad sense, including both DNA level and RNA level (such as free RNA); the horizontal level includes but is not limited to methylation, copy number, variation, and expression level.

[0109] It can be understood that there are many ways to detect nucleic acids in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography. It can be understood that nucleic acids include DNA or RNA levels.

[0110] For example, in some embodiments, the gene sequencing method can be transcriptome sequencing or genome sequencing. Genome sequencing can adopt first-generation Sanger sequencing, second-generation sequencing ("NGS") or third-generation sequencing reagents, etc. Second-generation sequencing ("NGS") is different from "Sanger sequencing" (first-generation sequencing), which is based on the electrophoretic separation of chain termination products in a single sequencing reaction. NGS is a revolutionary change to traditional Sanger sequencing technology, and can sequence hundreds of thousands to millions of nucleic acid molecules at a time. The sequencing platform of NGS that can be used for the present application is commercially available, including but not limited to Roche / 454FLX, Illumina / Solexa GenomeAnalyzer and Applied Biosystems SOLID system, etc. Transcriptome sequencing can also quickly and comprehensively obtain almost all transcripts and gene sequences of a certain species' specific cells or tissues in a certain state through the second-generation sequencing platform, which can be used to study gene expression, gene function, structure, alternative splicing and new transcript prediction, etc. In some other embodiments of the present application, the nucleic acid sequencing method can be single-molecule real-time sequencing. Single-molecule DNA sequencing technology is a new generation of sequencing technology developed in the past 10 years, also known as the third-generation sequencing technology, including single-molecule real-time sequencing, true single-molecule sequencing, single-molecule nanopore sequencing and other technologies.

[0111] For another example, in some specific embodiments, the polymerase chain reaction method includes primers for marker molecule amplification, wherein the primer refers to a primer that can specifically amplify the marker molecule gene, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least part of the template sequence and serve as the starting point for synthesizing primer extension products. In some specific embodiments, the probe hybridization method includes a probe for the marker molecule, wherein the probe refers to a probe that can specifically identify the marker molecule gene or the transcript of the gene, which is a molecule that can bind to a specific sequence or subsequence or other part of another molecule, and generally refers to a nucleic acid probe that binds to another nucleic acid (i.e., a specific sequence of the marker molecule as a target nucleotide) through complementary base pairing. In some specific embodiments, the gene chip refers to a composite structure formed by an array of the aforementioned probes fixed on a substrate material (specifically including but not limited to polymers such as nylon membranes, nitrocellulose membranes, and glass, etc.). In some embodiments, when RNA needs to be detected, cDNA needs to be obtained by reverse transcription, and then the detection is implemented by the primers for marker molecule amplification.

[0112] The terms "probe" and "primer" used in this application refer to oligonucleotides complementary to the target position of the target nucleic acid, preferably single-stranded deoxyribonucleotides, including naturally occurring dNMPs (dAMP, dGMP, dCMP and dTMP), deformed nucleotides or non-natural nucleotides, and may also contain ribonucleotides. Of course, the "probe" and "primer" can be modified or sequences unrelated to the target nucleic acid can be added as needed; for example, in real-time fluorescence quantitative PCR amplification, the 5' end of the probe can be modified with a fluorescent group and the 3' end can be modified with a fluorescent quenching group; for example, in the sequencing process, a label sequence can be added to the 5' end of the primer or a corresponding adapter sequence can be added according to the sequencing platform.

[0113] In the present application, the meaning of "complementary" refers to the sufficient complementarity that a primer or a probe selectively hybridizes with a target nucleic acid sequence under hybridization conditions, and has the meaning of including both substantially complementary and perfectly complementary, preferably perfectly complementary. The term "substantially complementary sequences" used in the present specification includes not only completely identical sequences, but also sequences that can function as primers on a specific target sequence and are partially inconsistent with the sequence to be compared.

[0114] The sequences of gene probes and primers do not need to have sequences that are completely complementary to a portion of the sequence of the template, as long as they have sufficient complementarity within the range that they can hybridize with the template to exert their inherent effects. Therefore, the gene probes and primers in the present invention do not need to have sequences that are completely complementary to the above-mentioned nucleotide sequences as templates, as long as they have sufficient complementarity within the range that they can hybridize with the template to exert their inherent effects. The design of primers and gene probes is a skill mastered by those skilled in the art, for example, a primer design program (e.g., PRIMER 3 program) can be utilized.

[0115] The term "ligand" used in this application refers to a molecule that a target protein specifically binds to, and the ligand can be a nucleic acid (RNA or DNA), a polypeptide, a peptide or a compound.

[0116] The term "antibody" as used herein is well known in the art and refers to a specific immunoglobulin for an antigenic site. Antibodies can be made according to conventional methods in the art. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, humanized, human, chimeric, synthetic, recombinant, heterozygous, mutated, transplanted, and in vitro produced antibodies. Antibodies can include a constant region or a portion thereof, such as κ, λ, α, γ, δ, ε, and μ constant region genes. For example, various isotypes of heavy chain constant regions can be used, including: IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, and IgE. For example, a light chain constant region can be κ or λ. In certain embodiments, the term "antibody" can also refer to antibody derivatives, such as antibody-based fusion proteins (e.g., including regions equivalent to the Fc region of an immunoglobulin) or further modified to contain additional non-protein moieties, such as water-soluble polymers, such as antibodies of polyethylene glycol (PEG).

[0117] The terms "antigen binding domain" and "antigen binding fragment" refer to a part of an antibody molecule that contains amino acids responsible for specific binding between an antibody and an antigen. For some antigens, an antigen binding domain or antigen binding fragment may bind only a portion of the antigen. The portion of the antigen that is specifically recognized and bound by an antibody is called an "epitope" or "antigenic determinant". Antigen binding domains and antigen binding fragments include Fab; F(ab')2 fragments (a bivalent fragment having two Fab fragments linked by a disulfide bond in the hinge region); Fv fragments; single-chain Fv fragments (scFv); Fd fragments (having two VH and CH1 domains); single-domain antibodies (sdAb; consisting of a single VH domain) and other antibody fragments that retain antigen binding function. The Fab fragment has VH-CH1 and VL-CL domains covalently linked by disulfide bonds between the constant regions. The Fv fragment is smaller and has non-covalently linked VH and VL domains. scFv contains a flexible polypeptide connecting (1) the C-terminus of VH to the N-terminus of VL, or (2) the C-terminus of VL to the N-terminus of VH. sdAbs include heavy chain antibodies that naturally lack light chains and single domain antibodies derived from conventional four-chain antibodies. These antigen binding domains and fragments are obtained using conventional techniques known to those skilled in the art, and their functions are evaluated in the same manner as intact immunoglobulins.

[0118] The term "aptamer" as used herein refers to a polynucleotide composed of a specific type of single-stranded nucleic acid (DNA, RNA or modified nucleic acid), which has a stable tertiary structure and has the property of being able to bind to a target molecule with high affinity and specificity. As described above, since aptamers can specifically bind to antigenic substances like antibodies, but are more stable than proteins and have a simple structure, and are composed of polynucleotides that are easy to synthesize, they can be used instead of antibodies.

[0119] It can be understood that there are many ways to detect proteins in the art, including but not limited to sequencing technology, immune technology, electrophoresis technology, biomass spectrometry technology or chromatography technology, all of which can be used in the present application; in some specific embodiments of the present application, including but not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry, immune PCR, single molecule detection or liquid chromatography.

[0120] For example, in some embodiments, the method for quantitatively detecting marker molecules at the protein level includes specific antibodies. Among them, specific antibodies refer to antibodies that can specifically recognize proteins encoded by marker molecules, specifically including but not limited to monoclonal antibodies (such as full-length or complete monoclonal antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (such as bispecific antibodies with expected biological activity), nanobodies, or at least one of certain antibody fragments. It is understood that the antibody can be a human, humanized and / or affinity matured antibody.

[0121] It is understandable that in order to further enhance the identification efficiency, the biomarker molecules detected by the reagents can also be other biological materials that can be used as marker molecules, such as nucleic acid fragments, proteins, metabolites, etc. with diagnostic effects that are well known to those skilled in the art. In this way, the combined use of multiple biomarker molecules can be achieved, and they cooperate with each other to achieve better identification of sepsis and non-infectious lung diseases induced by infectious pneumonia, thereby better guiding the use of antibiotics. For example, in some specific embodiments, diagnostic reagents for detecting other genes or proteins are also included, such as PCT, CRP, HBP, IL-6, etc. mentioned in this application, and the combined detection can be a combination of any one or more of them.

[0122] In some embodiments of the present application, the test samples described in the present application can be selected from tissues, cells, body fluids serum, plasma, whole blood (peripheral blood), urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, feces and synovial fluid; in an optional embodiment, the sample is selected from any one of tissues, cells, serum, plasma, whole blood, and cerebrospinal fluid.

[0123] As used herein, "algorithms" include any equation, model, mathematical formula, algorithmic, analytical or programmed process or statistical technique or classification analysis that takes one or more input data or parameters (whether continuous or categorical) and calculates an output value, index, index value or score. Examples of algorithms include, but are not limited to, ratios, sums, regression operators such as indices or coefficients, biomarker molecule value conversion and standardization (including but not limited to standardized models based on clinical parameters such as age, gender, race, etc.), rules and criteria, statistical classification models, and neural networks trained on populations. In some embodiments, such as in the case of inputting the biomarker molecule level of the subject, the algorithm can be a linear or nonlinear equation and statistical classification analysis of the relationship between the subject's marker molecule level and disease diagnosis, identification, etc., and the algorithm can achieve the output value obtained based on the input value, and determine whether the subject is in a disease state based on the output value.

[0124] In some embodiments, the "level of a marker molecule" herein includes but is not limited to the abundance or concentration of a marker molecule nucleic acid or protein, such as in some specific embodiments, the expression abundance or concentration of the corresponding protein.

[0125] It is understandable that the control sample can be selected according to actual needs. For example, in disease diagnosis, the control sample is a normal population sample, while when used for prognosis assessment, it can be a control sample of different prognostic conditions.

[0126] In some specific embodiments, a set value for the expression level of a marker molecule can be given, and the set value can be determined based on the expression level of the marker molecule in patients with sepsis induced by infectious pneumonia and patients with non-infectious lung disease. For example, the average value or median value of the expression level of the marker molecule in the sample of a suitable number of samples, or other reasonable values ​​or ratios, when the expression level of the marker molecule of the subject is higher than the set value, it is judged or suspected to be sepsis induced by infectious pneumonia; or, when the expression level of the marker molecule of the subject is lower than the set value, it is judged or suspected to be non-infectious lung disease. In some specific embodiments, in the use of guiding the use of antibiotics, when the expression level of the marker molecule of the subject is higher than the set value, it is evaluated to be antibiotic therapy for the patient. It can be understood that the set value determined based on the average value, median value or other suitable values ​​or ratios needs to have good classification significance, and the known samples can be tested by using commonly used statistical test methods through the classification based on the set value. When the result is statistically significant, it indicates that the set value can be used as a judgment standard.

[0127] The "threshold" described herein is based on the common understanding in the art. In the process of disease diagnosis and identification, a set identification cut-off is given to achieve a series of diagnosis and identification of diseases. For example, some thresholds are set with reference to AUC.

[0128] The level of a marker molecule refers to a value derived from direct measurement or further indirect measurement of the biomarker molecule of a subject, which is usually derived at least in part from the abundance or concentration of the biomarker molecule in the subject's sample. The indirect derived value includes the value derived by applying a function to the measured value of the biomarker molecule. Directly measured values ​​include, but are not limited to, the value of a biomarker molecule determined by at least one of sequencing, hybridization, mass spectrometry, immunoassay, immunofluorescence, flow cytometry, etc.

[0129] Products, according to the core use or method of the present disclosure, can be configured into corresponding product forms for the process of detecting the level of marker molecules, for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease; or; for assessing whether to treat patients with antibiotics. The product includes reagents or components for detecting marker molecules. It is understood that such product forms are diverse, including but not limited to kit forms, system devices, computer-readable media or computer system forms.

[0130] In some embodiments of the present application, a kit for detecting or analyzing marker molecule levels to distinguish between sepsis and non-infectious lung disease induced by infectious pneumonia; or; for evaluating whether to treat a patient with antibiotics is also disclosed herein. Such a kit may include reagents for detecting the level of one or more marker molecules and instructions for distinguishing between sepsis and non-infectious lung disease induced by infectious pneumonia; or; for evaluating whether to treat a patient with antibiotics based on the detected levels.

[0131] A kit can be a broad definition, and can be a reagent, or include a group of reagents, or a large package of reagents, or even a reagent raw material that clearly defines the purpose of the present application. In some embodiments, the kit includes a group of reagents for generating a data set by at least one determination. The group of reagents can detect the level of quantitative marker molecules. The group of reagents can also further detect the level of one or more other marker molecules. In some aspects, the reagents are detected at the level of nucleic acids or proteins.

[0132] When the detection agent is detected at the nucleic acid level, it can be understood that there are many ways of nucleic acid detection in the art, including but not limited to sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology, all of which can be used in this application. In some specific embodiments of the present application, it includes but is not limited to any of the following specific methods: gene sequencing, polymerase chain reaction, isothermal amplification reaction, gene chip method, probe hybridization method, gel electrophoresis, Northern blotting, nucleic acid mass spectrometry or liquid chromatography.

[0133] Thus, for example, in some embodiments, the reagent may be a sequencing reagent, such as a first-generation Sanger sequencing reagent, a second-generation sequencing ("NGS") reagent, a third-generation sequencing reagent, etc. In other embodiments, the reagent is a PCR primer reagent, wherein the primer refers to a primer capable of specifically amplifying a marker molecule gene, such as a polynucleotide of a certain length, such as a primer of about 35 nucleotides or longer, which can hybridize with at least a portion of the template sequence and serve as a starting site for synthesizing primer extension products. In other embodiments, the reagent is a probe reagent, wherein the probe refers to a probe capable of specifically identifying a marker molecule gene or a transcript of the gene, which is a molecule capable of binding to a specific sequence or subsequence or other portion of another molecule, generally referring to a nucleic acid probe that binds to another nucleic acid (i.e., a specific sequence of a marker molecule as a target nucleotide) through complementary base pairing.

[0134] When the detection agent is used for detection at the protein level, it can be understood that there are many methods for protein detection in the art, including but not limited to sequencing technology, immunoassay, electrophoresis technology, biomass spectrometry technology or chromatography technology, all of which can be used in the present application; in some specific embodiments of the present application, including but not limited to any of the following specific methods: amino acid sequencing, enzyme-linked immunosorbent assay, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrometry or liquid chromatography.

[0135] Thus, for example, in some embodiments, the reagent is a specific antibody, wherein a specific antibody refers to an antibody that can specifically recognize a protein encoded by a marker molecule, specifically including but not limited to a monoclonal antibody (e.g., a full-length or complete monoclonal antibody), a polyclonal antibody, a multivalent antibody, a multispecific antibody (e.g., a bispecific antibody with expected biological activity), or at least one of certain antibody fragments. It is understood that the antibody can be a human, humanized, and / or affinity matured antibody.

[0136] In some embodiments, such a kit may include a carrier, package or container that is compartmentalized to receive one or more containers, such as vials, tubes, etc., each container containing one of the individual elements to be used in the method. The kit of the present application may include a container as described above and one or more other containers containing materials required from the standpoint of a commercial end user, including buffers, diluents, filters, and package inserts with instructions for use.

[0137] In some embodiments, the kit further includes a sample processing reagent, which may include at least one of a sample lysis reagent, a sample purification reagent, and a sample extraction reagent.

[0138] In some embodiments, the product further comprises at least one of a standard, a calibrator, a reference substance, and a buffer. The reference substance is a control substance for testing the effectiveness of the experiment and serves as a control for judging the results. The buffer can be any solution known in the art that can provide appropriate buffer conditions during the detection process.

[0139] The kit may include instructions for use of a set of reagents. For example, the kit may include instructions for performing at least one assay, such as an immunoassay, a protein binding assay, an antibody-based assay, an antigen binding protein-based assay, a protein-based array, an enzyme-linked immunosorbent assay (ELISA), flow cytometry, a protein array, blots, Western blots, nephelometry, turbidimetry, chromatography, mass spectrometry, enzyme activity, and an immunoassay selected from RIA, immunofluorescence, immunochemiluminescence, immunoelectrochemiluminescence, immunoelectrophoresis, competitive immunoassay, and immunoprecipitation.

[0140] In addition to the above components, the kit will further include instructions for implementing the subject method. These instructions can be present in the subject kit in a variety of forms, one or more of which can be present in the kit. One form in which these instructions can be present is as printed information on a suitable medium or substrate, for example, one or more sheets of paper on which the information is printed, the paper being in the packaging of the kit in the form of a package insert, etc.

[0141] System Devices

[0142] In some embodiments of the present application, the present invention also discloses a system for detecting or analyzing the level of marker molecules to distinguish between sepsis induced by infectious pneumonia and non-infectious lung disease; or; for evaluating whether to treat a patient with antibiotics. Such a system may include a set of reagents for detecting the level of one or more marker molecules; a device configured to receive a mixture of one or more reagents and a test sample obtained from a subject to measure the level of the marker molecules; and a computer system that is coupled to the device in communication to obtain the measured level and score the subject for sepsis or non-infectious lung disease induced by infectious pneumonia; or; score the patient for antibiotic treatment accordingly.

[0143] The device is configured to detect the marker molecule level in the mixture of reagent and test sample. For example, the device can determine the marker molecule level by immunoassay or the assay for nucleic acid detection. The mixture of reagent and test sample can be provided to the device by various containers, and the examples of these containers include holes, vials or tubes of orifice plates (for example, 96 orifice plates). Because of this, the device can have an opening (for example, a slot, a cavity, an opening, a sliding tray), and the opening can receive the container that holds the reagent test sample mixture and read to generate the quantitative expression value of a soluble medium. The example of the device includes an ELISA reader (for example, a luminescent ELISA reader, an absorbent ELISA reader, a fluorescent ELISA reader), a spectrometer and a spectrophotometer.

[0144] The computer system communicates with the device to receive the quantitative expression value of the soluble medium. The computer system analyzes the quantitative expression value by applying a model and determines whether the subject has sepsis induced by infectious pneumonia or non-infectious lung disease; or; is used to evaluate whether to treat the subject with antibiotics.

[0145] Computer system or computer readable medium

[0146] In some embodiments of the present application, the methods or uses of the present application can be performed on a computer, including detecting or analyzing marker molecule levels by computer to determine whether the subject has sepsis induced by infectious pneumonia or non-infectious lung disease; or; for evaluating whether to treat the subject with antibiotics.

[0147] For example, the construction and execution of the model for generating a score (e.g., LFPI score) can be implemented in hardware or software or a combination of the two. In one embodiment, a readable storage medium is provided, such as a medium including a data storage material encoded with machine-readable data, when using a machine programmed with instructions utilizing the data, the data storage material can display any data set of the prediction model of the present application and execution and results. Such data can be used for a variety of purposes, such as identification, treatment considerations, etc. The embodiment of the above method can be implemented in a computer program executed on a programmable computer, and the computer includes a processor, a data storage system, a graphics adapter, a network adapter, at least one input device and at least one output device, etc. The display is coupled to a graphics adapter. Program code is applied to input data to perform the above functions and generate output information. Output information is applied to one or more output devices in a known manner. The computer can be, for example, a personal computer, a microcomputer, or a workstation of traditional design.

[0148] Each program can be implemented in a high-level program or object-oriented programming language to communicate with a computer system. However, if desired, the program can be implemented in an assembly or machine language. In any case, the language can be a compiled language or an interpreted language. Each such computer program is preferably stored in a storage medium or device (e.g., ROM or disk) readable by a general or special programmable computer, for configuring and operating a computer to perform the program described herein when the computer reads the storage medium or device. The system can also be considered to be implemented in the form of a computer-readable storage medium configured with a computer program, wherein the storage medium so configured causes the computer to operate in a specific and predefined manner to perform the functions described herein.

[0149] Feature patterns and their databases can be provided in various media to facilitate their use. "Medium" refers to a manufactured article containing feature pattern information of the present application. The database of the present application can be recorded on a computer-readable medium, such as any medium that can be directly read and accessed by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy disks, hard disk storage media, and magnetic tapes; optical storage media, such as CD-ROMs; electronic storage media such as RAM and ROM; and hybrids of these categories, such as magnetic / optical storage media. Those skilled in the art can easily understand how any currently known computer-readable medium can be used to produce a record containing current database information. "Record" refers to the process of storing information on a computer-readable medium using any such method known in the art. Based on the method used to access the stored information, any convenient data storage structure can be selected. A variety of data processor programs and formats can be used for storage, such as word processing text files, database formats, etc.

[0150] The implementation scheme of the present application is described in detail below with reference to examples.

[0151] Dataset

[0152] Example 1. Expression analysis in peripheral blood mononuclear cells (PBMC) of patients with sepsis induced by infectious pneumonia and patients with non-infectious lung disease

[0153] In this example, an expression matrix file of peripheral blood mononuclear cell samples of sepsis patients was downloaded from the Gene Expression Omnibus (GEO) database, which is GSE65682. Among them, the GSE65682 data set contains 108 patients with sepsis induced by infectious pneumonia and 33 patients with non-infectious lung diseases. After the GSE65682 data is normalized, all samples are allocated as a discovery set and a validation set in a ratio of 7:3.

[0154] This example uses 22 non-infectious lung disease samples and 62 infectious pneumonia-induced sepsis samples as metadata cohort analysis. Figure 1 The results showed that ITGA7, CDKN2C, and PCOLCE2 were differentially expressed independently (P < 0.05);

[0155] At the same time, 11 non-infectious lung disease samples and 46 sepsis samples induced by infectious pneumonia were used as validation sets. Figure 2 The results also confirmed that ITGA7, CDKN2C, and PCOLCE2 were independently differentially expressed (P < 0.05).

[0156] Thus, it was confirmed that ITGA7, CDKN2C, and PCOLCE2 can be used independently to distinguish between infectious pneumonia-induced sepsis and non-infectious lung diseases.

[0157] Example 2, ROC evaluation

[0158] This example plotted the receiver operating characteristic (ROC) curves of ITGA7, CDKN2C, and PCOLCE2 in the discovery set. The results showed that the AUC values ​​of ITGA7, CDKN2C, and PCOLCE2 in the discovery set were 0.784, 0.735, and 0.779, respectively (see Figure 3 ), and the AUC values ​​of ITGA7, CDKN2C, and PCOLCE2 in the validation set were 0.760, 0.835, and 0.711 (see Figure 4 ).

[0159] Example 3, joint test

[0160] The above marker molecules were randomly combined in the discovery set and validation set, and the receiver operating characteristic (ROC) curve was plotted. Figure 5 It was found that the AUC of ITGA7+PCOLCE2 joint detection was 0.821. Figure 6 It was found that the AUC of ITGA7+CDKN2C joint detection was 0.809. Figure 7 The results showed that the AUC of CDKN2C+PCOLCE2 joint detection was 0.818. Figure 8 It was shown that the AUC of the ITGA7+CDKN2C+PCOLCE2 triple test in the discovery set was 0.864; Figure 5-Figure 8 The test results confirmed that the combined detection of two or three of ITGA7, CDKN2C, and PCOLCE2 can improve the effectiveness of distinguishing sepsis induced by infectious pneumonia from non-infectious lung diseases.

[0161] Example 4: Clinical sample testing

[0162] In this embodiment, 60 samples of human whole blood were collected, including 30 samples of sepsis (induced by infectious pneumonia) and 30 samples of non-infectious lung disease. After collecting all the whole blood specimens, peripheral blood mononuclear cells were separated, dissolved with Trizol (Beyotime, R-0016), and stored at -80°C. Total RNA was isolated from peripheral blood mononuclear cells according to the manufacturer's instructions. The mRNA was reverse transcribed using the transcript all-in-one-first-strand cDNA synthesis superMix for qPCR (One-step gDNA removal) kit (TransGen Biotech, AT341-01). qPCR detection was performed using the PerfectStart Green qPCRSuperMix kit (TransGen Biotech, AQ601-02). Commercial products were used as detection primers, and the relative gene expression was calculated using the 2-ΔCT method. p value <0.05 was statistically significant. Fig. 9 , Fig.10 and Fig.11 The test results showed that there were statistical differences in the expression levels of ITGA7, CDKN2C, and PCOLCE2 between sepsis induced by infectious pneumonia and non-infectious lung disease samples.

[0163] At the same time, the protein in the sample was extracted and detected by western blotting. For example, the Bradford method was first used to configure the standard curve protein sample. Then, SDS-PAGE electrophoresis was performed and transfected onto a PVDF membrane. Finally, the transfected membrane was blocked and incubated overnight with a specific monoclonal antibody (commercial product) for the protein to be tested, washed and then incubated with an HRP-labeled secondary antibody for 1 hour, and DAB was used to observe the brown protein color band. The results showed that there were significant differences in the expression levels of ITGA7, CDKN2C, and PCOLCE2 between sepsis induced by infectious pneumonia and non-infectious lung disease samples.

[0164] The above clinical results indicate that ITGA7, CDKN2C, and PCOLCE2 can be used as biomarkers to differentiate between infectious pneumonia-induced sepsis and non-infectious lung diseases.

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments can still be modified, or some or all of the technical features therein can be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. Use of a detection agent or component for obtaining the level of marker molecules in a sample in the preparation of a product for distinguishing sepsis induced by infectious pneumonia and non-infectious lung disease; the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2.

2. Use of a detection agent or component for obtaining the level of a marker molecule in a sample in the preparation of a product for evaluating whether to administer antibiotic therapy to a patient; the marker molecule comprises at least one of ITGA7, CDKN2C, and PCOLCE2.

3. A product for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease, wherein: It comprises a detection agent or component for obtaining the level of marker molecules in a sample, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2.

4. A product for evaluating whether to administer antibiotic therapy to a patient, wherein: It comprises a detection agent or component for obtaining the level of marker molecules in a sample, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2.

5. The use according to claim 2 or the product according to claim 4, wherein: The patient had qSOFA>2.

6. The use according to any one of claims 1, 2 or 5 or the product according to any one of claims 3 to 5, wherein: The level includes nucleic acid level or protein level; Optionally, the nucleic acid level is obtained or detected by sequencing technology, nucleic acid amplification technology, nucleic acid hybridization technology, electrophoresis technology, biological mass spectrometry technology or chromatography technology; Optionally, the nucleic acid level is obtained or detected by gene sequencing method, polymerase chain reaction method, isothermal amplification reaction method, gene chip method, probe hybridization method, gel electrophoresis method, Northern blotting method, nucleic acid mass spectrometry or liquid chromatography; Optionally, the reagent for detecting the nucleic acid level includes specific primers and / or probes for detecting the nucleic acid; Optionally, the nucleic acid includes the gene of the marker molecule, a DNA fragment extracted from the gene of the biomarker molecule, and the RNA corresponding to the gene of the biomarker molecule or its DNA fragment; Optionally, the protein level is obtained or detected by sequencing technology, immunoassay technology, electrophoresis technology, biomass spectrometry technology or chromatography technology; optionally, the protein level is obtained or detected by amino acid sequencing, enzyme-linked immunosorbent assay, chemiluminescence, immunochromatography, radioimmunoassay, immunohistochemistry, immunoblotting, flow cytometry, gel electrophoresis, protein spectrum or liquid chromatography; optionally, the reagent for detecting the protein level includes a ligand for detecting the protein; optionally, the ligand includes at least one of an antibody, an antigen-binding fragment, a single-chain antibody and a nucleic acid aptamer.

7. The use according to any one of claims 1, 2 or 5 or the product according to any one of claims 3 to 5, wherein: The product also includes detection agents or components for obtaining the levels of other marker molecules; optionally, the other marker molecules include but are not limited to one or more of PCT, CRP, HBP, and IL-6.

8. The use according to any one of claims 1, 2 or 5 or the product according to any one of claims 3 to 5, wherein: The sample includes one or more of tissue, cells, body fluid, serum, plasma, whole blood, urine, semen, saliva, pleural effusion, ascites, cerebrospinal fluid, stool or synovial fluid.

9. A method for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease, wherein: The method comprises the step of obtaining the level of marker molecules in the subject sample, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2; Optionally, the method comprises the following steps: (a) detecting or determining the level of a marker molecule in a sample from the subject, (b) comparing with a set threshold absolute amount; wherein the threshold absolute amount can distinguish between sepsis induced by infectious pneumonia and non-infectious lung disease; optionally, the level of marker molecules in samples of patients with sepsis induced by infectious pneumonia is higher than that in patients with non-infectious lung disease; or; (b) using the marker molecule level in the subject's sample as an input value and operating in an algorithm for distinguishing sepsis induced by infectious pneumonia from non-infectious lung disease; Optionally, the method further comprises the step of acquiring the levels of other marker molecules in the subject's sample. Optionally, the other marker molecules include but are not limited to one or more of PCT, CRP, HBP, and IL-6.

10. A method for evaluating whether to administer antibiotic therapy to a patient, wherein: The method comprises the steps of obtaining the level of marker molecules in the patient sample, wherein the marker molecules include at least one of ITGA7, CDKN2C, and PCOLCE2; Optionally, the method comprises the following steps: (i) detecting or determining the level of marker molecules in a sample from the subject, (ii) comparing with a set threshold absolute amount; optionally, administering antibiotic therapy to patients whose marker molecule levels are higher than the threshold absolute amount; Optionally, the method further comprises the step of obtaining the levels of other marker molecules in the patient sample, and optionally, the other marker molecules include but are not limited to one or more of PCT, CRP, HBP, and IL-6; Optionally, the patient has qSOFA>2; Optionally, the marker molecule level includes a nucleic acid level or a protein level; optionally, the nucleic acid level is an expression level of DNA, RNA or mRNA.

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