Application of IL-33 as biomarker in preparation of reagent, kit or system for diagnosing diseases

Abstract

The invention discloses application of IL-33 as a biomarker in preparation of a reagent, a kit or a system for diagnosing diseases. The reagent, the kit or the system is used for diagnosing whether an ACLF patient suffers from IPA or not. The IL-33 is used as the biomarker, IPA in ACLF patients can be effectively recognized, and high diagnosis accuracy is achieved. Between an IPA group and a non-IPA group of ACLF, IL-33 is used for diagnosing an area under an IPA subject working characteristic curve (AUC) of 0.883, and good diagnosis efficiency is shown; in patients with ACLF combined with pulmonary infection, the area under working characteristic curve (AUC) of subjects for diagnosing IPA by IL-33 is 0.871, good diagnosis efficiency is shown, and the excellent differential diagnosis capability of IL-33 on pulmonary aspergillus infection and bacterial infection is further confirmed.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical technology, and particularly relates to the use of IL-33 as a biomarker in the preparation of reagents, kits or systems for diagnosing diseases. BACKGROUND

[0002] Acute-on-chronic liver failure (ACLF) is a clinical syndrome characterized by acute liver function deterioration on the basis of chronic liver disease, accompanied by multiple organ dysfunction and short-term high mortality. Hepatitis B virus (HBV) infection is one of the main causes of chronic liver disease. Infection is one of the most common complications of ACLF, significantly increasing the short-term mortality of patients.

[0003] Invasive pulmonary aspergillosis (IPA) is a serious infection caused by Aspergillus fungi, mainly occurring in patients with low immune function. Due to factors such as inherent immune dysfunction, multiple hospitalizations, invasive surgery, and long-term use of broad-spectrum antibiotics and immunosuppressive drugs, ACLF patients are at high risk for IPA. However, the early symptoms of IPA are often occult or atypical, making it difficult to distinguish from bacterial infection, and are prone to misdiagnosis or missed diagnosis, further increasing the mortality risk of ACLF patients, with a mortality rate of over 70%.

[0004] Timely diagnosis of IPA is crucial for improving the prognosis of ACLF patients. Currently, the diagnosis of IPA mainly relies on imaging techniques, microbiological detection, and mycological laboratory indicators. However, these traditional diagnostic tools have many limitations in ACLF patients: imaging techniques lack specificity and are difficult to distinguish between fungal infection and other types of lung lesions; routine immune detection (such as GM detection) is prone to false negative or false positive results, especially in immunosuppressed states; and metagenomic next-generation sequencing (mNGS) technology is limited in its widespread application in ACLF patients due to its high cost, strict requirements for fungal load, complex results interpretation, and frequent need for bronchoscopic sampling.

[0005] In summary, the existing diagnostic methods have many limitations, and there is an urgent need for a new diagnostic tool to improve the diagnostic accuracy of IPA. Based on this need, the present application proposes a detection method using IL-33 as a biomarker, aiming to provide a rapid, accurate, and non-invasive diagnostic means for IPA in ACLF patients, thereby improving clinical outcomes and optimizing treatment regimens. SUMMARY

[0006] In order to conveniently, quickly and accurately identify the patients with ACLF combined with IPA in clinic, realize individualized precise treatment, and save medical resources, the application provides a detection method which is objective, feasible and high in repeatability, mainly uses the cytokine Interleukin (IL)-33 as a biomarker to realize rapid diagnosis of IPA. The purpose of the application is to provide the use of IL-33 as a biomarker in preparation of a reagent, a kit or a system for diagnosing diseases.

[0007] In order to achieve the above purpose, the application adopts the following technical solutions:

[0008] In the first aspect, the application provides the use of IL-33 as a biomarker in preparation of a reagent, a kit or a system for diagnosing diseases, which is used for diagnosing whether the patients with ACLF have IPA.

[0009] In the above technical solution, the diagnosis comprises the following steps:

[0010] S1: collecting a biological sample of the patient with ACLF;

[0011] S2: determining the level of IL-33 in the biological sample;

[0012] S3: comparing the determined level of IL-33 with a preset threshold value to determine whether the patient has IPA.

[0013] In the above technical solution, in S1, the biological sample comprises one of blood, bronchoalveolar lavage fluid or a tissue sample.

[0014] In the above technical solution, in S1, the ACLF diagnostic criteria are as follows:

[0015] A1: the age is 18-75 years old;

[0016] A2: the cause of chronic liver disease is hepatitis B;

[0017] A3: the ACLF diagnostic criteria formulated by the Chinese Research Group of Severe Hepatitis B in 2017 are met.

[0018] In the above technical solution, in S2, the IL-33 determination method is selected from any one or a combination of multiple items:

[0019] B1: multiplex immunoassay;

[0020] B2: flow cytometry;

[0021] B3: enzyme-linked immunosorbent assay;

[0022] B4: RT-qPCR;

[0023] B5: Immunofluorescence staining;

[0024] B6: Mass spectrometry analysis;

[0025] B7: Western blotting;

[0026] B8: ELISpot.

[0027] In the above technical solution, in S3, the preset threshold is defined as follows:

[0028] C1: IL-33 level ≥ 66.97 pg / mL, the ACLF patient can be diagnosed as IPA;

[0029] C2: IL-33 level < 66.97 pg / mL, then it cannot be diagnosed as IPA.

[0030] In a second aspect, the application provides use of a reagent for detecting a biomarker level in the preparation of a kit or diagnostic agent for diagnosing a disease, the kit or diagnostic agent being used for diagnosing whether an ACLF patient has IPA, the biomarker being IL-33.

[0031] In a third aspect, the application provides a kit for diagnosing whether an ACLF patient has IPA, the kit comprising a reagent for detecting IL-33 level.

[0032] In a fourth aspect, the application provides a detection system for diagnosing whether an ACLF patient has IPA, the detection system comprising

[0033] a detection device for detecting IL-33 level in a biological sample of an ACLF patient;

[0034] a comparison device for comparing the measured IL-33 level with a preset threshold to determine whether the patient has IPA.

[0035] In the above technical solution, the preset threshold is defined as follows:

[0036] C1: IL-33 level ≥ 66.97 pg / mL, the ACLF patient can be diagnosed as IPA;

[0037] C2: IL-33 level < 66.97 pg / mL, then it cannot be diagnosed as IPA.

[0038] The application has the following beneficial effects:

[0039] (3) High accuracy: the application uses IL-33 as a biomarker to effectively identify IPA in ACLF patients, with high diagnostic accuracy.

[0040] (4) Excellent diagnostic performance: The area under the receiver operating characteristic curve (AUC) of IL-33 for diagnosing IPA between IPA group and non-IPA group in ACLF was 0.883, showing good diagnostic performance; The area under the receiver operating characteristic curve (AUC) of IL-33 for diagnosing IPA in ACLF patients with pulmonary infection was 0.871, showing good diagnostic performance, further confirming its excellent differential diagnostic ability for pulmonary aspergillus infection and bacterial infection.

[0041] (5) Accurate and fast diagnosis: When detecting IL-33 levels in patient plasma samples using Luminex method, in the identification of IPA and non-IPA in ACLF, when IL-33 ≥ 66.97 pg / mL, the sensitivity for diagnosing IPA was 93.80%, and the specificity was 82.80%. In the identification of whether there is aspergillus infection (IPA) in ACLF patients with pulmonary infection, when IL-33 ≥ 66.97 pg / mL, the sensitivity for diagnosing IPA was 93.80%, and the specificity was 84.40%.

[0042] (6) Reduce patient pain: The present application can use blood samples for analysis, compared with traditional culture or invasive detection methods, significantly reducing the pain and risk of patients, thereby improving the acceptance of detection.

[0043] (7) Wide applicability: The present application is suitable for critically ill patients with ACLF, especially for high-risk groups with impaired immune function, and has wide applicability and application potential.

[0044] (8) Simple operation and low cost: The detection method has wide applicability, simple operation and low cost, and is suitable for promotion and use in different medical levels.

[0045] (9) Promote early intervention: The detection method of the present application can promote early intervention, and timely identification of IPA will enable clinicians to quickly take appropriate treatment measures, thereby reducing the risk of death and improving survival rate of patients.

[0046] In summary, the present application provides a new diagnostic biomarker, which helps to promote the diagnosis of IPA in ACLF patients and clinical decision-making, avoid misdiagnosis and missed diagnosis, and reduce treatment delay. DETAILED DESCRIPTION

[0047] In order to better illustrate the purpose, technical scheme and advantages of the present application, the present application will be further described below in combination with specific embodiments. The present application can be implemented in many different forms, and should not be understood as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided to make the present disclosure thorough and complete, and to fully convey the concept of the present application to those skilled in the art, and the present application will be limited only by the claims.

[0048] Unless otherwise specified, the following examples were performed under conventional conditions or according to the manufacturer's recommendations. Unless otherwise specified, the reagents or instruments used were conventional products available on the market.

[0049] The embodiment of the present application provides a detection method for diagnosing invasive pulmonary aspergillosis (IPA) in patients with acute-on-chronic liver failure (ACLF), and the specific steps are as follows:

[0050] 1. Collecting biological samples

[0051] Collect biological samples from patients who meet the diagnostic criteria for ACLF. The sample types include but are not limited to blood, bronchoalveolar lavage fluid or tissue samples. For blood samples, perform venous blood collection and centrifuge as soon as possible after collection to obtain serum or plasma.

[0052] The diagnostic criteria for ACLF are as follows:

[0053] A1: Age 18-75 years old;

[0054] A2: The cause of chronic liver disease is hepatitis B;

[0055] A3: Meets the diagnostic criteria for ACLF developed by the 2017 Chinese Severe Hepatitis B Research Group.

[0056] 2. Determining IL-33 levels

[0057] The Luminex method is used to determine the IL-33 level in the sample. The specific operation steps can be performed according to the standard operating procedures of the determination method to ensure the accuracy and repeatability of the experimental results.

[0058] 3. Comparing IL-33 levels with preset threshold

[0059] Compare the measured IL-33 level with the preset threshold (such as X pg / mL):

[0060] (1) If the IL-33 level is ≥X pg / mL, it is determined that the patient has IPA;

[0061] (2) If the IL-33 level is <X pg / mL, it is determined that the patient does not have IPA.

[0062] 4. Data analysis and interpretation

[0063] In the present application, we statistically analyzed the IL-33 level data in multiple patient samples, calculated the relevant sensitivity and specificity indicators to ensure the accuracy of the diagnosis. The specific method is as follows:

[0064] We included 16 IPA patients and matched 32 patients with pulmonary bacterial infection and 32 patients without infection according to age, sex, underlying diseases and severity of liver failure (see Table 1).

[0065] In ACLF patients, we calculated the area under the curve (AUC) and the cut-off value of IL-33 between IPA group and non-IPA group. When the cut-off value was set at 66.97 pg / ml, the AUC was 0.883, showing significant diagnostic accuracy, with a sensitivity of 93.80% and a specificity of 82.80% (see Table 2).

[0066] Next, in ACLF patients with pulmonary infection (pulmonary infection was divided into bacterial infection and aspergillosis), we calculated the AUC and cut-off value of IL-33 between IPA group and pulmonary bacterial infection group. When the cut-off value was set at 66.97 pg / mL, the AUC was 0.871, also showing significant diagnostic accuracy, with a sensitivity of 93.80% and a specificity of 84.40% (see Table 3).

[0067] Table 1. Characteristics and clinical features of ACLF patients in IPA group, pulmonary bacterial infection group and non-infection group

[0068]

[0069]

[0070] Table 2. Value of IL-33 in distinguishing IPA and non-IPA patients in ACLF patients

[0071]

[0072] Table 3. Value of IL-33 in distinguishing IPA and bacterial infection patients in ACLF patients with pulmonary infection

[0073]

[0074] Based on the above analysis results, the criteria for using IL-33 levels to diagnose whether ACLF patients have IPA are as follows:

[0075] (1) If IL-33 is ≥ 66.97 pg / mL, the ACLF patient can be diagnosed as IPA;

[0076] (2) If IL-33 is < 66.97 pg / mL, it cannot be diagnosed as IPA.

[0077] Similarly, the diagnostic criteria for using IL-33 levels to identify whether ACLF patients with pulmonary infection have IPA are as follows:

[0078] (1) If IL-33 is greater than or equal to 66.97 pg / mL, the patient with ACLF complicated with pulmonary infection has IPA;

[0079] (2) If IL-33 is less than 66.97 pg / mL, the patient with ACLF complicated with pulmonary infection does not have IPA (i.e. simple pulmonary bacterial infection).

[0080] In summary, the method for detecting invasive pulmonary aspergillosis (IPA) in patients with acute-on-chronic liver failure (ACLF) using IL-33 has wide applicability, excellent performance, controllable cost, simple operation, and can early identify the infection and infection type of patients, thereby achieving precision medicine and precision prevention and treatment.

[0081] Obviously, the above embodiments are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. Use of IL-33 as a biomarker in the manufacture of a reagent, kit or system for the diagnosis of a disease, characterized in that: The reagent, kit or system is used for diagnosing whether an ACLF patient has IPA.

2. Use according to claim 1, characterized in that: The diagnosis comprises the following steps: S1: collecting a biological sample of an ACLF patient; S2: determining the level of IL-33 in the biological sample; S3: comparing the measured IL-33 level with a preset threshold to determine whether the patient has IPA.

3. Use according to claim 2, characterized in that: In S1, the biological sample comprises one of blood, bronchoalveolar lavage fluid or a tissue sample.

4. Use according to claim 2, characterized in that: In S1, the ACLF diagnostic criteria are as follows: A1: the age is 18-75 years old; A2: the cause of chronic liver disease is hepatitis B; A3: the ACLF diagnostic criteria formulated by the Chinese Severe Hepatitis B Research Group in 2017 are met.

5. Use according to claim 2, characterized in that: In S2, the IL-33 determination method is selected from any one or a combination of the following: B1: multiplex immunoassay; B2: flow cytometry; B3: enzyme-linked immunosorbent assay; B4: RT-qPCR; B5: immunofluorescence staining; B6: mass spectrometry; B7: Western blotting; B8: ELISpot.

6. Use according to claim 2, characterized in that: In S3, the preset threshold is defined as follows: C1: if the IL-33 level is ≥66.97 pg / mL, the ACLF patient can be diagnosed as having IPA; C2: if the IL-33 level is <66.97 pg / mL, the patient cannot be diagnosed as having IPA.

7. Use of a reagent for detecting the level of a biomarker in the manufacture of a kit or diagnostic agent for the diagnosis of a disease, characterized in that: The kit or diagnostic agent is used for diagnosing whether an ACLF patient has IPA, and the biomarker is IL-33.

8. A kit for diagnosing whether an ACLF patient is suffering from IPA, characterized in that: The kit comprises a reagent for detecting the level of IL-33.

9. A detection system for diagnosing whether an ACLF patient is suffering from IPA, characterized by: The detection system comprises a detection device for detecting the level of IL-33 in a biological sample of an ACLF patient; a comparison device for comparing the measured IL-33 level with a preset threshold to determine whether the patient has IPA.

10. The detection system of claim 9, wherein: the preset threshold is defined as follows: C1: if the IL-33 level is ≥66.97 pg / mL, the ACLF patient can be diagnosed as having IPA; C2: if the IL-33 level is <66.97 pg / mL, the patient cannot be diagnosed as having IPA.

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