Application of CD45 molecular expression relative change of monocyte and lymphocyte in diagnosis of infection and inflammation

Abstract

The invention discloses application of CD45 molecular expression relative change of mononuclear cells and lymphocytes in diagnosis of infection and inflammation, and creatively discovers that the index of the CD45 molecular expression relative change of the mononuclear cells and the lymphocytes can be used for quickly and accurately judging whether a patient is accompanied with infection or not for the first time. According to the present invention, the method has characteristics of high specificity and high specificity, can effectively distinguish virus infection and bacterial infection, has characteristics of high diagnosis efficiency, high AUC value, high sensitivity and high specificity, can be used for guiding clinical treatment decision and antibiotic use, can be used for early screening, prevention, treatment and post-treatment monitoring of infection, and has important conversion significance.

Description

Technical Field

[0001] The invention belongs to the field of biomedical technology, and in particular, relates to the application of relative changes in the expression of CD45 molecules of monocytes and lymphocytes in diagnosing infection. Background Art

[0002] In recent years, the prevalence of drug-resistant bacteria and viral infections such as the new coronavirus, influenza virus, respiratory syncytial virus, and adenovirus has increased significantly. With the development of globalization, the speed and scope of bacterial and viral transmission are also expanding. Judging infection and effectively distinguishing between bacterial and viral infections is crucial to reducing the abuse of antibiotics, the occurrence of clinical drug-resistant bacteria, and the effective treatment of infectious diseases. Traditional detection methods, including culture, microscopy, biochemical identification, serological detection, etc., are of great significance in pathogen detection, but due to their shortcomings such as long time consumption, low sensitivity, and insufficient specificity, they are gradually replaced by modern molecular biology techniques (such as PCR and high-throughput sequencing). Although the use of PCR and high-throughput sequencing technology has greatly improved the sensitivity and specificity of pathogen detection, and the detection speed has also been greatly accelerated. However, due to the diversity of virus types and the high requirements for laboratories to carry out PCR detection, PCR technology still has some limitations. There is an urgent need for a simple and rapid detection method to distinguish the types of infection to assist clinical treatment decisions and antibiotic use.

[0003] The CD45 antigen, also known as PTPRC, is encoded by the PTPRC gene in humans. PTPRC was originally called leukocyte common antigen (LCA) and is a receptor-type protein tyrosine phosphatase (PTP) that is ubiquitously expressed in all nucleated hematopoietic cells and accounts for about 10% of lymphocyte surface proteins. The CD45 glycoprotein plays a crucial role in lymphocyte development and antigen signaling and is an important regulator of the Src family kinases. Due to alternative splicing, the CD45 protein exists in multiple isoforms that differ in the extracellular domain but have the same transmembrane and cytoplasmic domains. CD45RA is an isoform of the CD45 complex with restricted expression in different subtypes of lymphoid cells. CD45 isoforms differ in their ability to translocate to sphingolipid-rich membrane domains, and their expression depends on the cell type and the physiological state of the cell. CD45 has been shown to be an important regulator of T cell and B cell antigen receptor signaling and inhibits JAK kinases to regulate cytokine receptor signaling. CD45 also plays an important role in promoting cell survival by regulating integrin-mediated signal transduction pathways, DNA fragmentation during apoptosis, and inhibiting or upregulating various immune functions.

[0004] At present, there are no studies or reports on using the relative changes in the expression of CD45 molecules on monocytes and lymphocytes as an indicator for diagnosing infection and distinguishing between bacterial and viral infections. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide the art with the application of the relative changes in the expression of CD45 molecules in monocytes and lymphocytes in the diagnosis of infection and inflammation. The present invention uses the ratio of the expression levels of CD45 molecules in peripheral blood monocytes and lymphocytes to quickly and effectively determine whether the patient is infected, and effectively distinguish whether it is a viral infection or a bacterial infection, which can be used to guide clinical treatment decisions and the use of antibiotics.

[0006] The present invention adopts the following technical solutions to achieve the above-mentioned invention objectives:

[0007] The first aspect of the present invention provides the use of a reagent for detecting the relative expression levels of CD45 molecules of monocytes and lymphocytes in a sample in the preparation of a product for diagnosing infection or identifying the type of infection.

[0008] Further, the reagent includes:

[0009] Reagents for detecting the relative expression levels of CD45 protein in monocytes and lymphocytes in a sample;

[0010] A reagent that detects the relative levels of CD45 DNA in monocytes and lymphocytes in a sample; or

[0011] A reagent that detects the relative levels of CD45 RNA in monocytes and lymphocytes in a sample.

[0012] Furthermore, the relative expression levels of CD45 molecules in monocytes and lymphocytes include the ratio of CD45 molecule expression levels in monocytes and lymphocytes, the ratio of CD45 molecule expression levels in lymphocytes and monocytes, the difference in CD45 molecule expression levels in monocytes and lymphocytes, and / or the difference in CD45 molecule expression levels in lymphocytes and monocytes.

[0013] Further, the reagent for detecting the relative expression level of monocyte and lymphocyte CD45 protein in the sample includes affinity proteins that specifically bind to monocyte and lymphocyte CD45 protein;

[0014] Optionally, the affinity protein includes an antibody, an antibody functional fragment, an agglutinating agent, a receptor and / or a coupled antibody that specifically binds to monocyte and lymphocyte CD45 protein;

[0015] Optionally, the reagent for detecting the relative DNA level of monocyte and lymphocyte CD45 in the sample includes a reagent for detecting the relative DNA level, DNA methylation level and / or DNA phosphorylation level of monocyte and lymphocyte CD45 in the sample;

[0016] Optionally, the reagent for detecting the relative level of DNA of monocytes and lymphocytes CD45 in the sample includes a reagent for detecting the relative level of DNA by sequencing technology;

[0017] Optionally, the reagent for detecting the relative RNA level of monocyte and lymphocyte CD45 in the sample includes a reagent for detecting the relative expression level of mRNA and / or miRNA of monocyte and lymphocyte CD45 in the sample;

[0018] Optionally, the reagent for detecting the relative level of monocyte and lymphocyte CD45 RNA in the sample includes primers that specifically amplify monocyte and lymphocyte CD45 and / or probes that specifically recognize monocyte and lymphocyte CD45.

[0019] Further, the diagnosis of infection is diagnosing whether the subject has an infection, diagnosing whether the subject has a viral infection, or diagnosing whether the subject has a bacterial infection;

[0020] Optionally, the identification of infection type is to identify and differentiate between viral infection and bacterial infection.

[0021] Furthermore, the sample is a peripheral blood sample, tissue sample, serum sample, plasma sample, cell sample, urine sample and / or exosome sample derived from a subject.

[0022] In the present invention, as long as the indicator of the relative expression level of CD45 molecules in monocytes and lymphocytes as described above is used to diagnose infection or identify the type of infection, such methods fall within the protection scope of the present invention, and are not limited to the specific method of using the indicator of the relative expression level of CD45 molecules in monocytes and lymphocytes as described above. As long as the indicator of the relative expression level of CD45 molecules in monocytes and lymphocytes as described above can achieve or basically achieve the purpose of diagnosing infection or identifying the type of infection, it will fall within the protection scope of the present invention.

[0023] In a specific embodiment of the present invention, monocytes and lymphocytes are selected from peripheral blood samples by flow cytometry analysis, and the mean fluorescence intensity (MFI) or Geometric Mean of the CD45 molecules of the monocytes and lymphocytes are respectively counted using software such as FlowJo, and then the relative expression levels of the CD45 molecules of the monocytes and lymphocytes (CD45 MFI monocytes / CD45 MFI lymphocytes) are calculated.

[0024] In some embodiments, the software for counting the mean fluorescence intensity (MFI) of CD45 molecules of monocytes and lymphocytes includes but is not limited to: FlowJo, ImageJ, Kaluza, SPSS, BD FACSDiva or CellProfiler.

[0025] In some embodiments, the method for detecting the relative expression level of CD45 molecules in monocytes and lymphocytes as described above in the present invention is not limited to the flow cytometry used in the specific embodiments of the present invention. Other techniques for distinguishing monocytes and lymphocytes, and combining fluorescence, chemiluminescence, immunohistochemistry, flow cytometry, protein immunoblotting, enzyme-linked immunosorbent assay and other methods to detect the expression of CD45 molecules in monocytes and lymphocytes also fall within the scope of protection of the present invention.

[0026] In some embodiments, the other techniques for distinguishing monocytes from lymphocytes include but are not limited to: morphological analysis techniques (e.g., optical microscopy observation, electron microscopy observation), cytochemical staining techniques (e.g., peroxidase staining, nonspecific esterase staining, acid phosphatase staining), immunoanalysis techniques (e.g., immunohistochemistry, immunofluorescence), and molecular biology techniques (e.g., gene expression analysis, DNA methylation analysis).

[0027] In the present invention, the monocytes originate from hematopoietic stem cells in the bone marrow. In the bone marrow, they develop into mature monocytes through the promonocyte and immature monocyte stages, and then are released into the blood circulation. The functions of the monocytes include: phagocytosis, antigen presentation, and cytokine secretion.

[0028] In the present invention, the lymphocytes originate from bone marrow hematopoietic stem cells. A portion of the hematopoietic stem cells develop into B lymphocytes in the bone marrow; another portion of the hematopoietic stem cells migrate to the thymus with the blood flow and develop into T lymphocytes in the thymus. The lymphocytes are divided into T lymphocytes, B lymphocytes and natural killer cells (NK cells).

[0029] In some embodiments, the primers and amplification primers refer to nucleic acid fragments comprising 5-100 nucleotides. In preferred embodiments, the primers or amplification primers contain 15-30 nucleotides that can initiate an enzymatic reaction (e.g., an enzymatic amplification reaction). In a specific embodiment of the present invention, the primers refer to primers that specifically amplify monocyte and lymphocyte CD45.

[0030] In some embodiments, the probe refers to a molecule that can bind to a specific sequence or subsequence or other part of another molecule. In a specific embodiment of the present invention, the probe refers to a probe that specifically recognizes monocytes and lymphocytes CD45. Unless otherwise indicated, a probe generally refers to a polynucleotide probe that can bind to another polynucleotide (often referred to as a target polynucleotide) through complementary base pairing.

[0031] Depending on the stringency of the hybridization conditions, the probe can bind to a target polynucleotide that lacks complete sequence complementarity with the probe. Hybridization methods include, but are not limited to, solution phase, solid phase, mixed phase, or in situ hybridization assays. Exemplarily, the probes include gene-specific DNA oligonucleotide probes, such as microarray probes fixed to a microarray substrate, quantitative nuclease protection test probes, probes linked to molecular barcodes, and probes fixed to beads.

[0032] The stringency of hybridization reaction can be easily determined by those of ordinary skill in the art, and is usually calculated empirically based on probe length, washing temperature and salt concentration. Generally speaking, longer probes require higher temperatures to anneal correctly, while shorter probes require lower temperatures. Hybridization usually depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below its melting temperature. The higher the degree of expected homology between the probe and the hybridizable sequence, the higher the relative temperature that can be used. As a result, it is inferred that higher relative temperatures will tend to make the reaction conditions more stringent, while lower temperatures are also less stringent.

[0033] In some embodiments, the reagents that specifically bind to monocyte and lymphocyte CD45 protein include, but are not limited to, antibodies, affinity proteins, and also include peptides, aptamers and / or compounds that specifically bind to monocyte and lymphocyte CD45 protein.

[0034] Further, the antibody is well known in the art and refers to a specific immunoglobulin for an antigenic site. The antibody described in the present invention refers to an antibody that specifically binds to the monocyte and lymphocyte CD45 protein described in the present invention, and the antibody can be prepared according to conventional methods in the art. The form of the antibody includes polyclonal antibodies or monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2 and Fv fragments), single-chain Fv (scFv) antibodies, multispecific antibodies (such as bispecific antibodies), monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising the antigen binding site of the antibody, and any other modified immunoglobulin molecules comprising the antigen binding site, as long as the antibody exhibits the desired biological binding activity.

[0035] In some embodiments, the peptide has the ability to highly bind to the target substance (monocyte and lymphocyte CD45 protein of the present invention) and will not be denatured during heat treatment or chemical treatment. Moreover, due to its small size, it can be used as a fusion protein by attaching it to other proteins. Specifically, because it can be specifically attached to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery substance.

[0036] In some embodiments, the aptamer 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 the target molecule (the monocyte and lymphocyte CD45 protein of the present invention) with high affinity and specificity. As described above, since the aptamer can specifically bind to antigenic substances like an antibody, but is more stable than a protein, has a simpler structure, and is composed of a polynucleotide that is easy to synthesize, it can be used instead of an antibody.

[0037] In some embodiments, the sample includes any sample (subject source) collected and sampled from any cell, tissue or body fluid, wherein the sample includes but is not limited to: the source of the tissue or cell sample can be from a solid tissue of a fresh, frozen and / or preserved organ or tissue sample, or a biopsy or aspirate, blood or any blood component; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid. The tissue sample can be a primary or in vitro cultured cell or cell line. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. The tissue sample may contain compounds naturally mixed with the tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or similar compounds.

[0038] In some embodiments, the sample includes, but is not limited to, tissue, blood, tissue-derived cells, blood-derived cells, serum, plasma, lymph, synovial fluid, exosomes, cell extracts, feces, urine, saliva, sputum, joint cavity fluid, pleural effusion, serous cavity effusion, lymph, cerebrospinal fluid, uterine cavity fluid, digestive fluid, bile, alveolar bronchial washings, organs, and any combination thereof. In a preferred embodiment, the sample is a peripheral blood sample derived from a subject.

[0039] In the present invention, it is verified by the real clinical samples collected by the present invention that the relative expression level of the monocyte and lymphocyte CD45 molecules has a very high diagnostic efficacy for diagnosing infection or distinguishing the type of infection, and the accuracy, AUC value, sensitivity and specificity are all high. Among them, the diagnostic efficacy is verified by the receiver operating characteristic curve (ROC), and the area under the curve (Area under the curve, AUC) refers to the area under the ROC curve well known to those skilled in the art. The determination of the area under the curve (AUC) helps to compare the accuracy of the classifier through the overall data range.

[0040] A classifier with a larger area under the curve (AUC) has a greater ability to accurately classify unknowns between two groups of interest (e.g., disease samples and normal or control samples). The receiver operating characteristic curve (ROC) is useful for graphically representing the performance of a particular feature (e.g., the relative expression level of CD45 molecules on monocytes and lymphocytes described in the present invention and / or any item of additional biomedical information) in terms of distinguishing between two populations (e.g., disease group and healthy control group).

[0041] Typically, based on a single feature value, the above-mentioned feature data of the entire population (e.g., disease group and control group) are arranged in ascending order. Then, for each value of the above-mentioned feature, the true positive rate and false positive rate for the data are calculated. The above-mentioned true positive rate is determined by dividing the number of cases by the total number of cases after calculating the number of cases above the value for its characteristic. The above-mentioned false positive rate is determined by dividing the number of controls by the total number of controls after calculating the number of controls above the value for its characteristic. Although this definition refers to the situation where the characteristics of the patient group are high relative to the control group, this definition is also applicable to the situation where the characteristics of the patient group are low relative to the control group (in this case, the number of samples with values ​​lower than the above-mentioned characteristics can be calculated).

[0042] The receiver operating characteristic curve (ROC) can be generated for other single calculations and for single characteristics. For example, two or more characteristics can be mathematically combined (e.g., addition, subtraction, multiplication, etc.) to provide a single sum value, which can be represented by the receiver operating characteristic curve (ROC). Additionally, the combination of multiple characteristics that can derive a single calculation value can be drawn with the receiver operating characteristic curve (ROC). These characteristic combinations can constitute a test. The above-mentioned receiver operating characteristic curve (ROC) is a graph representing the true positive rate (sensitivity) of the test relative to the false positive rate (1-specificity) of the test.

[0043] A second aspect of the invention provides a diagnostic product for diagnosing an infection or identifying the type of infection.

[0044] Furthermore, the diagnostic product comprises the reagent described in the first aspect of the present invention.

[0045] Furthermore, the diagnostic product is a detection kit, a detection chip and / or a detection test strip.

[0046] In some embodiments, the detection kit includes primers, probes, chips or specific binding proteins that specifically bind to monocyte and lymphocyte CD45. In some embodiments, the detection kit may also include: containers, instructions for use, positive controls, negative controls, buffers, adjuvants or solvents. For example, one or more of a solution for suspending or fixing cells, a detectable label or marker, a solution for lysing cells, etc.

[0047] In some embodiments, the detection kit may also be accompanied by instructions for use of the detection kit, which record how to use the detection kit provided by the present invention to perform detection, and how to use the test results to determine whether the subject is infected and the type of infection.

[0048] In some embodiments, the detection chip includes a solid phase carrier, a probe that specifically recognizes monocyte and lymphocyte CD45 attached to the solid phase carrier, or a specific antibody or ligand for monocyte and lymphocyte CD45 molecules. In some embodiments, the solid phase carrier for preparing the detection chip of the present invention includes various commonly used materials in the field of gene chips or protein chips, including but not limited to: nylon membrane, glass slide or silicon slide modified with active groups (such as aldehyde groups, amino groups, etc.), unmodified glass slide, plastic sheet, etc. In some embodiments, the detection chip includes a gene chip or a protein chip.

[0049] In some embodiments, the gene chip includes a solid phase carrier; and oligonucleotide probes fixed in order on the solid phase carrier, and the oligonucleotide probes specifically correspond to part or all of the sequences shown in monocyte and lymphocyte CD45. The protein chip includes a solid phase carrier, and specific antibodies or ligands for monocyte and lymphocyte CD45 molecules fixed on the solid phase carrier. The solid phase carrier can be made of various commonly used materials in the chip field, including but not limited to: plastic products, microparticles, membrane carriers, etc.

[0050] In some embodiments, the test strip comprises a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad.

[0051] Exemplarily, the sample pad is usually made of materials such as glass fiber, and is used to drip the biological sample to be tested (for example, peripheral blood) so that it can be quickly absorbed at this site and diffuse to other areas. The conjugate pad generally contains markers, such as colloidal gold, fluorescent microspheres, etc., which are pre-bound to specific antibodies against the CD45 molecules of monocytes and lymphocytes. When the sample passes through the conjugate pad, the CD45 molecules therein will specifically bind to the markers marked with antibodies. The nitrocellulose membrane is the core part of the test strip, and a test line (T line) and a control line (C line) are usually drawn on it. Antibodies that can specifically bind to CD45 molecules are fixed on the test line, and substances that can bind to the markers of the labeled antibodies are fixed on the control line. When the sample solution containing the markers that bind to the CD45 molecules of monocytes and lymphocytes flows to the detection line, the CD45 molecules will be captured by the antibodies on the detection line, causing the markers to aggregate and color at the detection line; the control line is used to verify the validity of the test strip. Regardless of whether the sample contains CD45 molecules, the markers will bind to the substances at the control line and color. The absorbent pad is generally made of absorbent material and is located at the end of the test strip. Its function is to absorb the liquid that passes through the nitrocellulose membrane, so that the liquid can continue to flow in one direction on the test strip to ensure the smooth progress of the detection process.

[0052] The third aspect of the present invention provides the use of a reagent for detecting the relative expression levels of CD45 molecules of monocytes and lymphocytes in a sample in the preparation of a system and / or device for diagnosing infection or identifying the type of infection.

[0053] In the present invention, the system and / or device is a method for distinguishing different components, elements, parts, parts or assemblies at different levels. However, if other words can achieve the same purpose, the words can be replaced by other expressions. It is well known to those skilled in the art that the present invention can be implemented as an apparatus, method or computer program product. Therefore, the content disclosed in the present invention can be specifically implemented in the following forms, that is, it can be complete hardware, it can be complete software (including firmware, resident software, microcode, etc.), and it can also be a combination of hardware and software. In addition, in some specific embodiments, the present invention can also be implemented in the form of a computer program product in one or more computer-readable media, and the computer-readable medium contains a computer-readable program code.

[0054] Any combination of one or more computer-readable media may be used. The computer-readable medium may be a computer-readable signal medium or a computer-readable storage medium. The computer-readable storage medium may be, for example, but not limited to, an electrical, magnetic, optical, electromagnetic, infrared, or semiconductor system, device or device, or any combination thereof. In the present invention, the computer-readable storage medium may be any tangible medium containing or storing a program that may be used by or in conjunction with an instruction execution system, device or device.

[0055] In addition, the present invention also provides a method for diagnosing infection or identifying the type of infection, the method comprising:

[0056] Obtain data on the relative expression levels of CD45 molecules in monocytes and lymphocytes in the sample to be tested;

[0057] Classification prediction is performed based on the relative expression level data of monocyte and lymphocyte CD45 molecules to obtain a classification result of whether the sample to be tested is accompanied by infection or the type of infection.

[0058] The classification result is obtained based on the prediction model;

[0059] The method for constructing the prediction model includes: obtaining the relative expression level data of the monocyte and lymphocyte CD45 molecules of the training set samples and the clinical characteristics corresponding to the samples, the clinical characteristics including healthy people, infected patients (including virally infected patients and bacterially infected patients), virally infected patients, and bacterially infected patients, extracting the relative expression level data of the monocyte and lymphocyte CD45 molecules in the training set and inputting them into the machine learning model to construct the prediction model, obtaining the constructed prediction model, and generating the corresponding threshold value.

[0060] In some embodiments, the machine learning model includes a linear regression model, a logistic regression model, a random forest model, a Lasso regression model, a neural network model, a decision tree model, a perceptron model, a support vector machine model and / or a naive Bayes model.

[0061] Furthermore, the relative expression levels of CD45 molecules in monocytes and lymphocytes include the ratio of CD45 molecule expression levels in monocytes and lymphocytes, the ratio of CD45 molecule expression levels in lymphocytes and monocytes, the difference in CD45 molecule expression levels in monocytes and lymphocytes, and / or the difference in CD45 molecule expression levels in lymphocytes and monocytes.

[0062] Furthermore, the sample to be tested is a peripheral blood sample, tissue sample, serum sample, plasma sample, cell sample, urine sample and / or exosome sample derived from a subject.

[0063] A fourth aspect of the present invention provides a system for diagnosing an infection or identifying the type of infection.

[0064] Further, the system includes a processor, an input module, and an output module;

[0065] Among them, the processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the relative expression levels of monocytes and lymphocytes CD45 molecules in the subject's sample, and contains a computer-readable medium of instructions, which, when executed by the processor, executes an algorithm on the input relative expression levels of monocytes and lymphocytes CD45 molecules; the output module is used to output whether the subject is infected, the type of infection or the risk of infection.

[0066] In some embodiments, the system comprises:

[0067] Data acquisition unit: acquiring data on the relative expression levels of CD45 molecules of monocytes and lymphocytes in the sample to be tested;

[0068] Analysis and prediction unit: Based on the relative expression level data of CD45 molecules of monocytes and lymphocytes, classification prediction is performed to obtain a classification result of whether the sample to be tested is accompanied by infection or the type of infection.

[0069] The classification result is obtained based on the prediction model;

[0070] The method for constructing the prediction model includes: obtaining the relative expression level data of the monocyte and lymphocyte CD45 molecules of the training set samples and the clinical characteristics corresponding to the samples, the clinical characteristics including healthy people, infected patients (including virally infected patients and bacterially infected patients), virally infected patients, and bacterially infected patients, extracting the relative expression level data of the monocyte and lymphocyte CD45 molecules in the training set and inputting them into the machine learning model to construct the prediction model, obtaining the constructed prediction model, and generating the corresponding threshold value.

[0071] In some embodiments, the machine learning model includes a linear regression model, a logistic regression model, a random forest model, a Lasso regression model, a neural network model, a decision tree model, a perceptron model, a support vector machine model and / or a naive Bayes model.

[0072] Furthermore, the relative expression levels of CD45 molecules in monocytes and lymphocytes include the ratio of CD45 molecule expression levels in monocytes and lymphocytes, the ratio of CD45 molecule expression levels in lymphocytes and monocytes, the difference in CD45 molecule expression levels in monocytes and lymphocytes, and / or the difference in CD45 molecule expression levels in lymphocytes and monocytes.

[0073] Furthermore, the sample to be tested is a peripheral blood sample, tissue sample, serum sample, plasma sample, cell sample, urine sample and / or exosome sample derived from a subject.

[0074] In addition, the present invention also provides an electronic device, which includes a memory and a processor; the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, it is used to execute the steps of the method for diagnosing infection or identifying infection type as described above.

[0075] In addition, the present invention also provides a computer-readable storage medium, on which a computer program is stored. When the computer program is executed by a processor, the steps of the method for diagnosing an infection or identifying an infection type as described above are implemented.

[0076] In addition, the present invention also provides an indicator for diagnosing whether a subject is accompanied by infection, the type of infection accompanied by infection, or the risk of being accompanied by infection, wherein the indicator is the relative expression level of CD45 molecules in monocytes and lymphocytes.

[0077] Furthermore, the relative expression levels of CD45 molecules in monocytes and lymphocytes include the ratio of CD45 molecule expression levels in monocytes and lymphocytes, the ratio of CD45 molecule expression levels in lymphocytes and monocytes, the difference in CD45 molecule expression levels in monocytes and lymphocytes, and / or the difference in CD45 molecule expression levels in lymphocytes and monocytes.

[0078] In addition, the present invention also provides a method for diagnosing whether a subject has an infection, the type of infection, or the risk of an infection, the method comprising the following steps:

[0079] (1) Collect samples from subjects in need;

[0080] (2) detecting the relative expression levels of CD45 molecules on monocytes and lymphocytes in samples derived from the subject;

[0081] (3) Determining whether the subject has an infection, the type of infection, or the risk of infection based on the relative expression levels of CD45 molecules on monocytes and lymphocytes in the sample obtained from the subject.

[0082] Furthermore, the relative expression levels of CD45 molecules in monocytes and lymphocytes include the ratio of CD45 molecule expression levels in monocytes and lymphocytes, the ratio of CD45 molecule expression levels in lymphocytes and monocytes, the difference in CD45 molecule expression levels in monocytes and lymphocytes, and / or the difference in CD45 molecule expression levels in lymphocytes and monocytes.

[0083] Furthermore, whether there is an infection is whether there is a bacterial or viral infection; the type of associated infection is whether the associated infection is a bacterial infection or a viral infection; the risk of associated infection refers to the risk of associated bacterial or viral infection, the risk of associated infection being a bacterial infection, or the risk of associated infection being a viral infection.

[0084] In some embodiments, the subject refers to any animal, including humans and non-human animals. Non-human animals include all vertebrates, for example, mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cattle, and any livestock or pets; and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human.

[0085] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0086] (1) The present invention creatively discovered for the first time that the ratio of CD45 molecule expression levels of peripheral blood monocytes and lymphocytes can be used to quickly and effectively determine whether a patient has an infection, and to effectively distinguish whether the infection is viral or bacterial. The present invention verified the relative change of CD45 molecule expression of monocytes and lymphocytes in collected clinical samples and found that the indicator has a high diagnostic efficacy for determining whether a patient has an infection or distinguishing whether the infection is viral or bacterial, with an AUC value of up to 0.9994, and high sensitivity and specificity. It can be used to guide clinical treatment decisions and antibiotic use, and can be used in early screening, prevention, treatment and post-treatment monitoring of infection, and has important translational significance.

[0087] (2) The present invention can determine whether a subject has infection inflammation and effectively distinguish bacterial infection from viral infection by only using the ratio of the expression levels of CD45 molecules of monocytes and lymphocytes. The operation is simple and fast and easy to automate. Compared with the conventional CD64 infection index of the same type used by those skilled in the art, it is more effective in distinguishing infection types, especially in determining the infection type of patients in fever clinics, and can assist clinicians in quickly and accurately determining infection and infection type and guiding the use of antibiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 : The results of gating the peripheral blood monocytes and lymphocytes of healthy physical examination patients, bacterial and viral infection patients according to CD45 / SSA;

[0089] Figure 2 : Changes in CD45 protein expression in peripheral blood monocytes and lymphocytes in healthy patients, patients with bacterial and viral infections;

[0090] Figure 3 : Changes of CD45 MFI of peripheral blood monocytes in patients with bacterial and viral infections in the screening cohort and their ROC curve results;

[0091] Figure 4 : Changes of CD45 MFI in peripheral blood lymphocytes of patients with bacterial and viral infections in the screening cohort and their ROC curve results;

[0092] Figure 5 : In the exploration cohort, the changes of CD45 MFI monocyte / lymphocyte ratio in the exploration cohort of patients with bacterial and viral infections and the ROC curve results;

[0093] Figure 6 : Changes in CD45 MFI monocyte / lymphocyte ratio in the validation cohort of patients with bacterial and viral infections and ROC curve results;

[0094] Figure 7 : Changes in peripheral blood neutrophil CD64 infection index in patients with bacterial and viral infections and ROC curve results. DETAILED DESCRIPTION

[0095] The present invention is further described below in conjunction with specific embodiments. The following specific embodiments are only used to explain the present invention and cannot be understood as limiting the present invention. It can be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and purpose of the present invention, and the scope of the present invention is defined by the claims and their equivalents.

[0096] The reagents and raw materials used in the present invention are easily obtained by those of ordinary skill in the art, and can be obtained from commercial sources unless otherwise specified. The experimental methods for which specific conditions are not specified in the present invention are usually tested under conventional conditions or under conditions recommended by the manufacturer. In particular, the following examples are only used to illustrate the present invention and should not limit the scope of the present invention in any way. It should be noted that the experimental conditions and results described in the following examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0097] Example Screening and validation of biomarkers (relative changes in CD45 molecule expression in monocytes and lymphocytes) for diagnosis of infection and inflammation

[0098] 1. Experimental Materials

[0099] (1) Antibodies:

[0100] PE / Cyanine7 Anti-Human CD45 Antibody[HI30](Elabscience, Catalog No.: E-AB-F1137H), the antibody with clone number HI30 recognizes all subtypes of human CD45 molecules;

[0101] Pacific Blue TM anti-human CD14 antibody (Biolegend, Catalog No.: 367122);

[0102] APC Anti-human CD64 antibody[10.1](Elabscience, Cat. No.: E-AB-F1082E).

[0103] (2) Red blood cell lysis solution: OptiLyse C Lysing Solution (Beckmann, A11895).

[0104] (3) Flow cytometer: DxFLEX (Beckmann) or BD Lyric.

[0105] 2. Experimental methods

[0106] Healthy people undergoing physical examinations, patients with viral infections (including influenza A virus, influenza B virus, coronavirus, respiratory syncytial virus, adenovirus, parainfluenza virus type I-III, rhinovirus, herpes virus (Epstein-Barr virus, cytomegalovirus, etc.), enterovirus (norovirus, rotavirus, etc.), and human papillomavirus infection, with positive nucleic acid test or virus copy number exceeding the threshold), and patients with bacterial infections (culture positive, including Gram-positive and Gram-negative bacteria) were included. Peripheral whole blood anticoagulated with 100 μL of EDTA or sodium citrate was collected and PE / Cyanine7 Anti-Human CD45 antibody, APC Anti-human CD64 antibody, and Pacific Blue TM2 μL of anti-human CD14 antibody was added, and the mixture was shaken and mixed. Incubated at room temperature in the dark for 20 minutes. After adding 2 mL of red blood cell lysis buffer, the mixture was shaken and mixed. Incubated in the dark for 15 minutes. Centrifuged at 2000 rpm for 3 minutes, the supernatant was removed and the cells were resuspended. 2 mL of phosphate buffer or saline was added and centrifuged at 2000 rpm for 3 minutes. The supernatant was discarded and the cell pellet was resuspended in phosphate buffer and analyzed by flow cytometer (Beckman DxFLEX). In the flow cytometric analysis process, leukocytes are selected by setting a gate according to the FSC / SSC scatter plot, lymphocytes, monocytes and neutrophils are selected by setting a gate according to the SSC / CD45 scatter plot, or monocytes are selected by setting a gate according to the SSC / CD14 scatter plot, and the mean fluorescence intensity (MFI) or Geometric Mean of the CD45 molecules of monocytes and lymphocytes are respectively counted using software such as FlowJo, and the neutrophil CD64 infection index and the CD45 index (CD45 MFI monocyte / CD45 MFI lymphocyte) of the present invention are calculated.

[0107] In this embodiment, the peripheral blood samples from healthy examinees, the peripheral blood samples from patients with bacterial infection and the peripheral blood samples from patients with viral infection collected by the present invention are randomly divided into a screening cohort, an exploration cohort and a verification cohort. In the screening cohort, the healthy examinees: the patients with bacterial infection: the patients with viral infection = 50 cases: 109 cases: 227 cases; in the exploration cohort, the healthy examinees: the patients with bacterial infection: the patients with viral infection = 40 cases: 61 cases: 80 cases; in the verification cohort, the healthy examinees: the patients with bacterial infection: the patients with viral infection = 77 cases: 96 cases: 193 cases.

[0108] 3. Experimental results

[0109] The results of gating the peripheral blood monocytes and lymphocytes of healthy physical examination patients, bacterial and viral infection patients according to CD45 / SSA are as follows Figure 1 As shown in Figure 2, the changes in CD45 protein expression in peripheral blood monocytes and lymphocytes in healthy patients, patients with bacterial and viral infections are shown in Figure 2. Figure 2 shown.

[0110] In the screening cohort, the changes in CD45 MFI of peripheral blood monocytes in patients with bacterial and viral infections and the ROC curve results are shown in Figure 2. Figure 3The results showed that compared with the CD45 MFI of peripheral blood monocytes in healthy subjects, the CD45 MFI of peripheral blood monocytes in patients with bacterial and viral infections was significantly higher than that in patients with MFI showed significant differential expression, and it had high diagnostic efficacy as a biomarker for diagnosing bacterial infection, viral infection, infected patients or distinguishing infection types (bacterial infection or viral infection). The AUC values ​​were all high, and it had high sensitivity and specificity. Among them, the AUC value of peripheral blood monocyte CD45 expression level for diagnosing bacterial infection (healthy physical examination VS bacterial infection) was as high as 0.8679, with a sensitivity of 80.8% and a specificity of 92.9%; the AUC value of peripheral blood monocyte CD45 expression level for diagnosing viral infection (healthy physical examination VS viral infection) was as high as 0.9219, with a sensitivity of 86.6% and a specificity of 96.4%; the AUC value of peripheral blood monocyte CD45 expression level for diagnosing infected patients (healthy physical examination VS infected patients) was as high as 0.9064, with a sensitivity of 86.4% and a specificity of 92.9%.

[0111] In the screening cohort, the changes in CD45 MFI of peripheral blood lymphocytes in patients with bacterial and viral infections and the ROC curve results are shown in Figure 2. Figure 4 As shown, the results show that compared with the peripheral blood lymphocyte CD45 MFI of healthy subjects, the peripheral blood lymphocyte CD45 MFI of patients with bacterial and viral infections showed differential expression. However, its diagnostic efficacy as a biomarker for diagnosing bacterial infection, viral infection, infected patients or distinguishing the type of infection (bacterial infection or viral infection) alone is low, and the AUC values ​​are all less than 0.7000. It can be seen that the expression level of peripheral blood lymphocyte CD45 alone cannot be used as an effective biomarker for diagnosing bacterial infection, viral infection, infected patients or distinguishing the type of infection (bacterial infection or viral infection). Whether it has a better diagnostic efficacy when combined with peripheral blood mononuclear cell CD45 for diagnosing bacterial infection, viral infection, infected patients or distinguishing the type of infection (bacterial infection or viral infection), and how to combine it with peripheral blood mononuclear cell CD45 for diagnosing bacterial infection, viral infection, infected patients or distinguishing the type of infection (bacterial infection or viral infection) to have better diagnostic efficacy are all unexpected by those skilled in the art based on the prior art.

[0112] In the exploration cohort, the changes in the CD45 MFI monocyte / lymphocyte ratio in the exploration cohort of patients with bacterial and viral infections and the ROC curve results are shown in Figure 2. Figure 5The results showed that compared with the CD45 MFI monocyte / lymphocyte ratio of healthy subjects, the CD45 MFI monocyte / lymphocyte ratio of patients with bacterial and viral infections showed significant differential expression, and as a biomarker for diagnosing bacterial infection, viral infection, infected patients or distinguishing infection types (bacterial infection or viral infection), it had a high diagnostic efficacy, a high AUC value, and high sensitivity and specificity. Among them, the AUC value of the CD45 MFI monocyte / lymphocyte ratio of peripheral blood for diagnosing bacterial infection (healthy physical examination VS bacterial infection) was as high as 0.9734, with a sensitivity of 91.8% and a specificity of 95.0%; the AUC value of the CD45 MFI monocyte / lymphocyte ratio of peripheral blood for diagnosing viral infection (healthy physical examination VS viral infection) was as high as 0.9994, with a sensitivity of 97.5% and a specificity of 100%; When the MFI monocyte / lymphocyte ratio was used to identify the infection type (bacterial infection VS viral infection), the AUC value was as high as 0.918, with a sensitivity of 85.0% and a specificity of 90.2%. When the peripheral blood CD45 MFI monocyte / lymphocyte ratio was used to diagnose infected patients (healthy physical examination VS infected patients), the AUC value was as high as 0.9881, with a sensitivity of 95.0% and a specificity of 97.5%.

[0113] Among them, the data distribution and optimal cut-off value of CD45 MFI monocyte / lymphocyte ratio in the exploration cohort are shown in Tables 1 and 2, respectively.

[0114] Table 1 Exploration cohort data distribution

[0115] Group Health Checkup Bacterial infection Viral infection Number of samples 40 61 80 Minimum 0.3434 0.4871 0.751 Maximum 0.7929 1.586 2.212 Mean 0.4776 0.8543 1.304 Standard Deviation (Std) 0.08344 0.1967 0.2662

[0116] Table 2 Optimal cut-off value for the exploration cohort

[0117] Comparison Items Optimal cut-off value Health Checkup vs Bacterial Infection 0.589 Health Checkup vs Viral Infection 0.818 Bacterial vs Viral Infections 1.025 Healthy checkup vs infected patients (bacteria or viruses) 0.606

[0118] In the validation cohort, the changes in the CD45 MFI monocyte / lymphocyte ratio in the validation cohort of patients with bacterial and viral infections and the ROC curve results are shown in Figure 2. Figure 6The results showed that compared with the CD45 MFI monocyte / lymphocyte ratio of healthy subjects, the CD45 MFI monocyte / lymphocyte ratio of patients with bacterial and viral infections showed significant differential expression, and as a biomarker for diagnosing bacterial infection, viral infection, infected patients or distinguishing infection types (bacterial infection or viral infection), it had a high diagnostic efficacy, a high AUC value, and high sensitivity and specificity. Among them, the AUC value of the CD45 MFI monocyte / lymphocyte ratio of peripheral blood for diagnosing bacterial infection (healthy physical examination VS bacterial infection) was as high as 0.9556, with a sensitivity of 96.1% and a specificity of 89.6%. The AUC value of the CD45 MFI monocyte / lymphocyte ratio of peripheral blood for diagnosing viral infection (healthy physical examination VS viral infection) was as high as 0.9988, with a sensitivity of 100% and a specificity of 99.5%. The AUC value of the MFI monocyte / lymphocyte ratio for distinguishing infection types (bacterial infection VS viral infection) was as high as 0.8933, with a sensitivity of 82.3% and a specificity of 82.9%. The AUC value of the peripheral blood CD45 MFI monocyte / lymphocyte ratio for diagnosing infected patients (healthy physical examination VS infected patients) was as high as 0.9845, with a sensitivity of 100% and a specificity of 94.1%.

[0119] Among them, the data distribution of the CD45 MFI monocyte / lymphocyte ratio in the validation cohort and the diagnostic efficacy evaluation results of the optimal cut-off value of the exploration cohort in the validation cohort are shown in Tables 3 and 4, respectively. The results once again prove that the CD45 MFI monocyte / lymphocyte ratio provided by the present invention can be used as an effective biomarker for diagnosing bacterial infection, viral infection, infected patients or distinguishing infection types (bacterial infection or viral infection), with high accuracy, sensitivity and specificity.

[0120] Table 3. Validation cohort data distribution

[0121] Group Health Checkup Bacterial infection Viral infection Number of samples 77 96 193 Minimum 0.3311 0.4342 0.5666 Maximum 0.6404 1.472 4.642 Mean 0.5164 0.8406 1.246 Standard Deviation (Std) 0.0662 0.2006 0.34

[0122] Table 4 Evaluation of diagnostic efficacy of the optimal cut-off value of the exploratory cohort in the validation cohort

[0123]

[0124] Comparative Example The relative change of CD45 molecule expression in monocytes and lymphocytes provided by the present invention is compared with the CD64 infection index commonly used as a biomarker of pathogen infection in the prior art for diagnosing infection.

[0125] It is well known in the art that the Neutrophil CD64 Index is a sensitive indicator for evaluating infection or inflammatory response based on the expression of CD64 receptors on the surface of neutrophils. CD64 is a high-affinity Fcγ receptor on the surface of neutrophils, and its expression is significantly upregulated during infection and inflammatory response. Therefore, the expression level of CD64 is often used as a biomarker for pathogen infection.

[0126] Principle of CD64 infection index: CD64 receptor is an immune receptor on the surface of neutrophils. Under normal circumstances, its expression level is low, but in bacterial infection or immune activation, the expression of CD64 will increase significantly. This upregulated CD64 level helps neutrophils identify and eliminate pathogens. In the infected state, the upregulation of CD64 not only enhances the phagocytic ability of neutrophils, but also activates the function of neutrophils by binding to immune complexes, including the release of cytokines and chemokines.

[0127] Calculation method for neutrophil CD64 index: Substitute the mean fluorescence intensity data of CD64 expression of neutrophils, monocytes and lymphocytes in adult samples into the following formula to calculate the neutrophil CD64 index of the sample, and finally obtain the test result based on the reference range.

[0128]

[0129] In order to further verify that the relative change of monocyte and lymphocyte CD45 molecule expression provided by the present invention can be used for accurate diagnosis of infection, this comparative example verifies the diagnostic efficacy of the conventional indicator for diagnosing infection, namely, neutrophil CD64 infection index, in the screening cohort collected by the present invention. The changes of peripheral blood neutrophil CD64 infection index in patients with bacterial and viral infections and the ROC curve results are shown in Figure 2. Figure 7As shown, the results show that compared with the peripheral blood neutrophil CD64 infection index of healthy subjects, there is no significant difference in the peripheral blood neutrophil CD64 infection index of virally infected patients, and it cannot be used as an effective biomarker for diagnosing viral infection and infected patients. The AUC values ​​are low, 0.4691 and 0.6452, respectively, and the sensitivity and specificity are also low. In addition, the AUC value of using it as a biomarker for diagnosing bacterial infection or distinguishing the type of infection (bacterial infection or viral infection) is also significantly lower than the relative change of monocyte and lymphocyte CD45 molecule expression provided by the present invention. The above results show that the relative change of monocyte and lymphocyte CD45 molecule expression provided by the present invention can be used for the accurate diagnosis of infection, and its diagnostic efficacy is significantly better than the neutrophil CD64 infection index disclosed in the prior art, which is a conventional indicator for infection diagnosis, and a technical effect that is unexpected by a person skilled in the art based on the content disclosed in the prior art has been achieved.

Claims

1. Use of a reagent for detecting the relative expression level of CD45 molecules of monocytes and lymphocytes in a sample in the preparation of a product for diagnosing infection or identifying the type of infection.

2. The use according to claim 1, characterized in that: The reagents include: Reagents for detecting the relative expression levels of CD45 protein in monocytes and lymphocytes in a sample; A reagent that detects the relative levels of CD45 DNA in monocytes and lymphocytes in a sample; or A reagent that detects the relative levels of CD45 RNA in monocytes and lymphocytes in a sample.

3. The use according to claim 1, characterized in that: The relative expression levels of CD45 molecules in monocytes and lymphocytes include the ratio of CD45 molecule expression levels in monocytes and lymphocytes, the ratio of CD45 molecule expression levels in lymphocytes and monocytes, the difference in CD45 molecule expression levels in monocytes and lymphocytes and / or the difference in CD45 molecule expression levels in lymphocytes and monocytes.

4. The use according to claim 2, characterized in that: The reagent for detecting the relative expression level of monocyte and lymphocyte CD45 proteins in the sample comprises an affinity protein that specifically binds to monocyte and lymphocyte CD45 proteins; Optionally, the affinity protein includes an antibody, an antibody functional fragment, an agglutinating agent, a receptor and / or a coupled antibody that specifically binds to monocyte and lymphocyte CD45 protein; Optionally, the reagent for detecting the relative DNA level of monocyte and lymphocyte CD45 in the sample includes a reagent for detecting the relative DNA level, DNA methylation level and / or DNA phosphorylation level of monocyte and lymphocyte CD45 in the sample; Optionally, the reagent for detecting the relative level of DNA of monocytes and lymphocytes CD45 in the sample includes a reagent for detecting the relative level of DNA by sequencing technology; Optionally, the reagent for detecting the relative RNA level of monocyte and lymphocyte CD45 in the sample includes a reagent for detecting the relative expression level of mRNA and / or miRNA of monocyte and lymphocyte CD45 in the sample; Optionally, the reagent for detecting the relative level of monocyte and lymphocyte CD45 RNA in the sample includes primers that specifically amplify monocyte and lymphocyte CD45 and / or probes that specifically recognize monocyte and lymphocyte CD45.

5. The use according to claim 1, characterized in that: The diagnosis of infection is to diagnose whether the subject has an infection, diagnose whether the subject has a viral infection, or diagnose whether the subject has a bacterial infection; Optionally, the identification of infection type is to identify and differentiate between viral infection and bacterial infection.

6. The use according to claim 1, characterized in that: The sample is a peripheral blood sample, tissue sample, serum sample, plasma sample, cell sample, urine sample and / or exosome sample derived from a subject.

7. A diagnostic product for diagnosing an infection or identifying the type of infection, characterized in that: The diagnostic product comprises the reagent of any one of claims 1-6.

8. The diagnostic product according to claim 7, characterized in that The diagnostic product is a detection kit, a detection chip and / or a detection test strip.

9. Use of a reagent for detecting the relative expression level of CD45 molecules in monocytes and lymphocytes in a sample in the preparation of a system and / or device for diagnosing infection or identifying the type of infection.

10. A system for diagnosing an infection or identifying the type of infection, characterized in that: The system comprises a processor, an input module, and an output module; Among them, the processor is used to perform logical operations on the input information using bioinformatics methods; the input module is used to input the relative expression levels of monocytes and lymphocytes CD45 molecules in the subject's sample, and contains a computer-readable medium of instructions, which, when executed by the processor, executes an algorithm on the input relative expression levels of monocytes and lymphocytes CD45 molecules; the output module is used to output whether the subject is infected, the type of infection or the risk of infection.

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