Marker combination for sepsis-related encephalopathy and application

Abstract

The invention relates to the technical field of diagnostics, and particularly discloses a marker combination for sepsis-related encephalopathy and application. The peripheral blood biomarker combination is based on joint detection of various markers such as CitH3, MPO-DNA, MPO, NE, NT-ProCNP, S100B and the like, and efficient diagnosis of sepsis-related encephalopathy can be realized. Compared with the prior art, the marker combination has the advantage of simple components, more or more complex biomarkers do not need to be introduced, the diagnosis accuracy can be remarkably improved only through pairwise combination or combination forms of the markers, and the problem that an existing detection technology is poor in diagnosis accuracy is effectively solved.

Description

Technical Field

[0001] The present application relates to the field of diagnostic technology, and in particular to a combination of markers for sepsis-related encephalopathy and their application. Background Art

[0002] In clinical practice, the diagnosis of sepsis-associated encephalopathy (SAE) is achieved in patients who meet the diagnostic criteria for sepsis (Sepsis-3) through a combination of mental status assessment (such as coma and delirium) and auxiliary examinations (including electroencephalograms, imaging studies, and laboratory tests). However, the diagnosis of SAE primarily relies on a diagnostic strategy of exclusion, and factors such as metabolic disorders, medication effects, other central nervous system infections, and structural lesions often interfere with the diagnosis of SAE.

[0003] Studies have shown that levels of calcium-binding protein (S100B), amino-terminal pro-C-type natriuretic peptide (NT-proCNP), and neuron-specific enolase (NSE) are significantly elevated in the peripheral blood of patients with sepsis. These elevated levels reflect neuronal damage and blood-brain barrier dysfunction, and are therefore considered potential diagnostic markers for SAE. However, the sensitivity and specificity of single-agent tests remain insufficient. Furthermore, studies have shown that elevated levels of myeloperoxidase-deoxyribonucleic acid (MPO-DNA) in peripheral blood are significantly associated with mortality and the severity of organ dysfunction in patients with septic shock. Studies in mouse sepsis models have also demonstrated a positive correlation between the formation of neutrophil extracellular traps (NETs) and the progression of SAE. However, NETs contain multiple components, including citrullinated histone H3 (CitH3), neutrophil elastase (NE), MPO, and MPO-DNA. Whether the components released into the peripheral blood can serve as diagnostic markers for SAE and their clinical value remains to be determined.

[0004] In view of this, the present invention aims to explore new SAE diagnostic markers to improve the detection efficiency of SAE and provide a theoretical basis and practical support for early clinical intervention. Summary of the Invention

[0005] In view of this, the purpose of this application is to provide a marker combination and application of sepsis-related encephalopathy to solve the problem of poor diagnostic accuracy of existing detection technologies.

[0006] To achieve the above technical objectives, the present application provides a marker combination for sepsis-related encephalopathy, including any one of the following combinations:

[0007] Combination 1: The marker combination includes at least S100B and NT-ProCNP;

[0008] Combination 2: The marker combination includes at least S100B and CitH3;

[0009] Combination 3: The marker combination includes at least S100B and MPO-DNA;

[0010] Combination 4: The marker combination includes at least S100B and MPO;

[0011] Combination 5: The marker combination includes at least NT-ProCNP and CitH3;

[0012] Combination 6: The marker combination includes at least NT-ProCNP and MPO-DNA.

[0013] Furthermore, composition 1 also includes at least one NETs marker.

[0014] Furthermore, the NETs marker includes at least one of CitH3, NE, MPO-DNA and MPO.

[0015] The present application provides an application of a marker combination for preparing a product for screening and diagnosing sepsis-related encephalopathy.

[0016] The present application provides a product for diagnosing sepsis-related encephalopathy, the product comprising at least one of a reagent, a kit, a device, and a system, and the product comprising a marker combination.

[0017] The present application provides an application of a product for diagnosing sepsis-related encephalopathy, which is used for screening and diagnosis of sepsis-related encephalopathy.

[0018] In summary, the present invention proposes a marker combination for sepsis-associated encephalopathy. This combination, based on the combined detection of multiple markers including CitH3, MPO-DNA, MPO, NE, NT-ProCNP, and S100B, can achieve efficient and accurate diagnosis of SAE.

[0019] Compared to existing technologies, the marker combination used in this application has the advantage of simplicity. Without the need to introduce more or more complex biomarkers, the accuracy of diagnosis can be significantly improved by simply combining two or more of the above specific markers. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0021] Figure 1The distribution of peripheral blood biomarker expression levels of Sepsis-nonE patients and SAE patients provided in the examples of this application; wherein, Figure 1 A is a schematic diagram of patient grouping. Figure 1 B is the expression level distribution diagram of NT-ProCNP and S100B, Figure 1 C is the distribution diagram of the expression levels of CitH3, NE, MPO-DNA, and MPO;

[0022] Figure 2 The ROC curves for detecting SAE using the markers provided in the Examples and Comparative Examples of the present application alone or in combination are shown; wherein, Figure 2 A is the ROC curve of MPO-DNA, CitH3, MPO, and NE alone for detecting SAE. Figure 2 B is the ROC curve of S100B and NT-ProCNP alone and in combination for detecting SAE. Figure 2 C is the ROC curve of S100B combined with CitH3 / MPO / MPO-DNA / NE for SAE detection. Figure 2 D is the ROC curve of NT-ProCNP combined with CitH3 / MPO / MPO-DNA / NE for SAE detection. Figure 2 E is the ROC curve of SAE detected by the combination of S100B, NT-ProCNP and CitH3 / MPO / MPO-DNA / NE. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions of the embodiments of this application in conjunction with the accompanying drawings. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this specification, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection requested by this application.

[0024] In the description of this application, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely to facilitate the description of this application and simplify the description. They do not indicate or imply that the devices or components referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on this application. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0025] Unless otherwise specified or limited, the terms "mounted," "connected," and "connected" should be interpreted broadly. For example, they can refer to fixed, removable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediary; and internal communication between two components. Those skilled in the art will understand the specific meanings of these terms in this application.

[0026] The sources of all raw materials in the present invention are not particularly limited and can be purchased on the market or prepared according to conventional methods known to those skilled in the art.

[0027] The present invention provides a combination of markers for sepsis-related encephalopathy, including any one of the following combinations:

[0028] Combination 1: The marker combination includes at least S100B and NT-ProCNP;

[0029] Combination 2: The marker combination includes at least S100B and CitH3;

[0030] Combination 3: The marker combination includes at least S100B and MPO-DNA;

[0031] Combination 4: The marker combination includes at least S100B and MPO;

[0032] Combination 5: The marker combination includes at least NT-ProCNP and CitH3;

[0033] Combination 6: The marker combination includes at least NT-ProCNP and MPO-DNA.

[0034] In some embodiments, Composition 1 further comprises at least one of the NETs markers.

[0035] In some embodiments, the NETs marker comprises at least one of CitH3, NE, MPO-DNA, and MPO.

[0036] The present application provides an application of a marker combination for preparing a product for screening and diagnosing sepsis-related encephalopathy.

[0037] An embodiment of the present application provides a product for diagnosing sepsis-related encephalopathy, the product including at least one of a reagent, a kit, a device, and a system, and the product including a marker combination.

[0038] The present application provides an application of a product for diagnosing sepsis-related encephalopathy, which is used for screening and diagnosis of sepsis-related encephalopathy.

[0039] The applicant further provides the following reference specific embodiments to describe the present invention. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0040] The manufacturers of the kits used in the following examples and comparative examples are as follows: the ELISA kits for detecting the four markers MPO-DNA, MPO, NE, and CitH3 were purchased from ELISA Company; the ELISA kits for detecting the two markers NT-ProCNP and S100B were purchased from Kanglang Biotechnology Company.

[0041] Example 1

[0042] This example provides a marker combination and detection method for sepsis-related encephalopathy:

[0043] In this example, before using a biomarker combination to diagnose a patient, the expression levels of relevant biomarkers in sepsis patients were first detected to clarify the characterization role of each marker in sepsis-related encephalopathy, and the biomarker combination was determined based on its expression level. The detection methods and results are as follows:

[0044] See also Figure 1 A. First, based on the results of consciousness assessment, electroencephalogram (EEG) and magnetic resonance imaging (MRI), sepsis patients were divided into sepsis-non-encephalopathy (Sepsis-nonE) group (47 cases) and sepsis-associated encephalopathy (SAE) group (28 cases). Peripheral blood samples were collected from the two groups of patients, and the expression levels of six biomarkers, including CitH3, MPO-DNA, MPO, NE, NT-ProCNP and S100B, in the peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). Among them, Figure 1 B shows the expression levels of NT-ProCNP and S100B in the peripheral blood of the two groups of patients; Figure 1 C shows the expression levels of CitH3, NE, MPO-DNA, and MPO in the peripheral blood of the two groups of patients. The results showed that the above six biomarkers can characterize sepsis patients to a certain extent. Compared with the Sepsis-nonE group, the levels of MPO-DNA, CitH3, NT-ProCNP, and S100B in the peripheral blood of patients in the SAE group were significantly increased (p < 0.05).

[0045] Based on the above test results, this embodiment sets the peripheral blood biomarker combination to S100B and NT-ProCNP, and uses this peripheral blood biomarker combination to jointly diagnose SAE, the steps are as follows:

[0046] Step S1: Sample collection and reagent preparation

[0047] Sample source: Peripheral blood samples from Sepsis-nonE patients (47 cases) and SAE patients (28 cases) were collected using EDTA anticoagulant tubes, centrifuged at 1800 rpm for 5 minutes, and the supernatant was discarded to obtain plasma;

[0048] Prepare washing solution: dilute 30 times concentrated washing solution with distilled water at a ratio of 1:30 and set aside;

[0049] Step S2: Divide the wells of the ELISA plate into a blank control group, a standard group, and a sample group: No blood sample or enzyme-labeled reagent is added to the blank control group, and the remaining steps are the same as those for the sample group; the standard group is operated according to the standard sample and operation method provided by the ELISA kit; 40 μL of diluent is first added to the sample group, followed by 10 μL of blood sample; Note: When adding the sample, the liquid should be added dropwise to the bottom of the ELISA plate well to avoid touching the well wall. After adding the sample, gently shake the ELISA plate to mix the liquid;

[0050] Step S3, incubation: Seal the ELISA plate with a sealing film and then incubate the ELISA plate at 37°C for 30 minutes;

[0051] Step S4, washing: remove the sealing film, discard the liquid in the wells of the ELISA plate, add an appropriate amount of diluted washing solution to each well, let it stand for 30 seconds, then discard the washing solution, repeat this operation 5 times, and finally turn the ELISA plate upside down on absorbent paper and pat dry;

[0052] Step S5, adding enzyme labeling reagent: add 50 μL of enzyme labeling reagent to each well, except for the blank control well, and repeat the incubation and washing steps;

[0053] Step S6, color development: first add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake the plate to mix the solution thoroughly, and then place it in a dark environment at 37°C for 10 minutes for color development;

[0054] Step S7, stop the reaction: add 50 μL of stop solution to each well to stop the color reaction. At this time, the color of the solution immediately changes from blue to yellow;

[0055] Step S8, absorbance measurement: Use the blank control group to adjust to zero, and measure the absorbance (OD value) of each well in turn at a wavelength of 450 nm. All measurements must be completed within 15 minutes after adding the stop solution;

[0056] Step S9, data analysis: based on the measured absorbance, draw the ROC curve diagram, the result is as follows Figure 2 As shown in B.

[0057] Examples 2 to 10

[0058] The marker combinations used in Examples 2 to 10 correspond to the following combinations 2 to 10, respectively. The detection steps are the same as in Example 1. The results are shown in Figure 2 C~2E:

[0059] Combination 2: marker combination includes S100B and CitH3;

[0060] Combination 3: marker combination includes S100B and MPO-DNA;

[0061] Combination 4: marker combination including S100B and MPO;

[0062] Combination 5: marker combination includes NT-ProCNP and CitH3;

[0063] Combination 6: marker combination including NT-ProCNP and MPO-DNA;

[0064] Combination 7: marker combination including S100B, NT-ProCNP, and CitH3;

[0065] Combination 8: marker combination including S100B, NT-ProCNP, and MPO-DNA;

[0066] Combination 9: marker combination including S100B, NT-ProCNP, and MPO;

[0067] Combination 10: The marker combination includes S100B, NT-ProCNP, and NE.

[0068] Comparative Examples 1 to 6

[0069] The marker combinations used in Comparative Examples 1 to 6 were S100B, NT-ProCNP, CitH3, MPO-DNA, MPO, and NE, respectively. The detection steps were the same as those in Example 1. The results were obtained by referring to Figure 2 A~2B.

[0070] Comparative Examples 7-9

[0071] The marker combinations used in Comparative Examples 7 to 9 correspond to the following comparative combinations 1 to 3, respectively. The detection steps are the same as those in Example 1. The results are shown in Figure 2 C~2D:

[0072] Comparison combination 1: marker combination includes S100B and NE;

[0073] Comparison combination 2: marker combination including NT-ProCNP and MPO;

[0074] Comparison combination 3: The marker combination includes NT-ProCNP and NE.

[0075] Figure 2The ROC curves obtained when the biomarkers provided in the above examples and comparative examples are used alone or in combination for diagnosing SAE patients are analyzed, and the following conclusions can be drawn:

[0076] Figure 2 A shows that the area under the curve (AUC) of MPO-DNA was 0.721, while the AUCs of CitH3, MPO, and NE were all lower than 0.7; Figure 2 Figure B shows that the AUC of NT-ProCNP is 0.652, and the AUC of S100B is 0.647, but the AUC of the combined detection of the two is 0.711.

[0077] Figure 2 C shows that the AUC of S100B alone is 0.647, which increases to 0.649 after the combined detection of S100B and CitH3, to 0.652 after the combined detection of S100B and MPO, and to 0.762 after the combined detection of S100B and MPO-DNA, significantly improving the diagnostic efficacy of SAE.

[0078] Figure 2 D shows that the AUC of NT-ProCNP alone is 0.652, and the AUC is increased to 0.658 after the combined detection of NT-ProCNP and CitH3; the AUC of NT-ProCNP and MPO-DNA combined detection is 0.758, which also improves the diagnostic efficiency.

[0079] Figure 2 E shows that the detection of any one of the markers of NT-ProCNP, S100B and neutrophil extracellular traps (NETs) can effectively improve the detection efficiency; among them, the combined detection of NT-ProCNP, S100B and MPO-DNA has the highest AUC, which can reach 0.825.

[0080] In summary, the peripheral blood biomarker combinations provided in Examples 1 to 10 of the present application have significantly better detection accuracy than single marker detection, and also better than the combined detection of biomarker combinations provided in Comparative Examples 7 to 9. This shows that the biomarker combinations provided in this application have a synergistic detection effect, which can achieve more accurate detection results.

[0081] The above are only preferred embodiments of the present application and are not intended to limit the present invention. Although the present application has been described in detail with reference to examples, those skilled in the art can still modify the technical solutions described in the aforementioned examples or make equivalent replacements for some of the technical features therein. However, any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application should be included in the scope of protection of the present application.

Claims

1. A marker combination for sepsis-related encephalopathy, characterized in that: Comprising any one of the following compositions: Composition 1: The marker combination includes S100B and NT-ProCNP; Composition 2: The marker combination includes S100B and CitH3; Composition 3: The marker combination includes S100B and MPO-DNA; Composition 4: The marker combination includes S100B and MPO; Composition 5: The marker combination includes NT-ProCNP and CitH3; Composition 6: The marker combination includes NT-ProCNP and MPO-DNA.

2. The marker combination for sepsis-related encephalopathy according to claim 1, characterized in that: The composition 1 also includes at least one of the NETs markers.

3. The marker combination for sepsis-related encephalopathy according to claim 2, characterized in that: The NETs marker includes at least one of CitH3, NE, MPO-DNA and MPO.

4. A use of the marker combination according to any one of claims 1 to 3, characterized in that: Used to prepare products for screening and diagnosis of sepsis-related encephalopathy.

5. A product for diagnosing sepsis-related encephalopathy, characterized in that: The product comprises at least one of a reagent, a kit, a device, and a system, and the product comprises the marker combination according to any one of claims 1 to 3.

6. Use of the product for diagnosing sepsis-related encephalopathy according to claim 5, characterized in that: The product is used for screening and diagnosis of sepsis-related encephalopathy.

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