Use of HNL in the preparation of a reagent for identifying bloodstream infections

Abstract

The use of HNL in the preparation of a reagent for identifying bloodstream infection belongs to the field of precision medicine, and provides the use of HNL in a detection kit for diagnosing whether a patient has bloodstream infection. The patient is a patient with chronic hepatitis B cirrhosis. The detection kit diagnoses whether there is bacterial bloodstream infection by detecting whether the content level of HNL in a blood, plasma or serum sample taken from the patient exceeds a set cut-off value. Unexpectedly, the present invention realizes the diagnosis of bacterial bloodstream infection in patients with chronic hepatitis B cirrhosis, a special primary disease, through HNL, and the diagnostic results have excellent sensitivity of 90.6% and specificity of 100%, with great clinical value and industrialization prospects.

Description

Technical Field

[0001] The present invention relates to the field of precision medicine, specifically to the field of disease diagnosis biomarkers, and particularly to the use of HNL in the preparation of reagents for identifying bloodstream infections. Background Art

[0002] Human neutrophil lipocalin (HNL) is a secreted glycoprotein stored in the secondary granules of neutrophils and is the main component of neutrophil secondary granules. In the field of detection of bacterial infections, HNL as referred to in the industry usually specifically refers to the 45-KD homodimer of HNL. In the present invention, this industry convention is also followed, that is, HNL in the present invention specifically refers to the HNL45-KD homodimer. Since neutrophils participating in the process of acute bacterial infection specifically secrete the HNL45-KD homodimer, it has high sensitivity and specificity for the diagnosis of bacterial infections. In addition, the 25-KD monomer form of HNL widely used in clinical practice is called NGAL, which is mainly produced by renal tubular epithelial cells. When the kidney is damaged or diseased, renal tubular epithelial cells release a large amount of NGAL into urine or blood, making it an early and sensitive biomarker for kidney injury. Furthermore, the content of NGAL in urine or blood can be detected to assist in judging whether there is kidney injury or disease and the progression of the condition. This monomer form of NGAL can also polymerize with MMP-9 highly expressed in tumor patients to form a 135-KD heterodimer. Therefore, some studies also assist in judging and monitoring the progression of tumor diseases in patients by detecting and analyzing the content change of this heterodimer.

[0003] When human peripheral blood neutrophils are stimulated and activated, HNL can be released from inside the cells to the outside. When acute bacterial infection occurs, the concentration of HNL in the blood can significantly increase within 1 - 2 hours. HNL can further induce the release of intracellular granules of white blood cells, chemotactically aggregate inflammatory cells, and eliminate pathogenic microorganisms, showing an anti-inflammatory and antibacterial effect in inflammatory damage.

[0004] In view of the mechanism of action of HNL, HNL has been developed as a biomarker for the diagnosis of diseases such as sepsis and inflammation. It diagnoses corresponding diseases by detecting the content level of HNL in samples such as the serum of an individual and comparing it with the content level of HNL in normal people.

[0005] The PCT international patent application with publication number WO9529404A1 provides the use of HNL as a biomarker for diagnosing human inflammation, especially inflammation caused by bacterial infections. WO9529404A1 further discloses that the HNL level in the serum of healthy individuals is 37.95 - 190.87 μg / L, and three cut-off values, 190 μg / L, 155 μg / L, and 100 μg / L, are set to distinguish between patients with viral infections and those with bacterial infections. Among them, when the cut-off value is set at 155 μg / L, both the sensitivity and specificity are relatively acceptable.

[0006] S.Y. Xu et al. (S.Y. Xu et al. The development of an assay for human neutrophil lipocalin (HNL) to be used as a specific marker of neutrophil activity in vivo and vitro. Journal of Immunological Methods 171 (1994) 245 - 252) also established a method for determining the content of the biomarker HNL by radioimmunoassay (RIA). It discloses that the HNL levels in the serum and plasma of healthy individuals are 37.95 - 190.87 μg / L (average level 78.4 μg / L), among which the HNL level in plasma is 30.51 - 105.8 μg / L (average level 50.65 μg / L), and the HNL content level in serum is significantly higher than that in plasma.

[0007] Based on the research disclosed in the aforementioned literature, medical device products for HNL detection have been approved for marketing in China. For example, Jixiekezhun 20162400304, whose main intended uses involve clinical applications such as assisting in the early diagnosis of bacterial infections or sepsis, and differentiating between bacterial infections and viral infections. The product manual records the normal reference value for diagnosis as ≤115.55 ng / mL.

[0008] However, so far, the detection and diagnostic applications of HNL still use the content levels of healthy people as the reference. The reason is that it is still unclear whether the presence of primary diseases affects and how it affects the concentration of HNL. Related primary diseases may additionally increase the burden on the immune system, and different types of primary diseases have different impacts on the burden on the immune system. Therefore, at this time, compared with the HNL content levels after bacterial infection in the patient group without related primary diseases, the average HNL content in the patient group with related primary diseases may increase as a whole, may decrease as a whole, or may show an irregular and unstable situation where some patients increase, some patients decrease, and some patients maintain HNL content levels equivalent to those after bacterial infection in the patient group without related primary diseases. Therefore, in such a complex situation of influence, if the diagnosis of bacterial infection is still carried out according to the cut-off value standard of the healthy population, it is very likely to result in "false negative" or "false positive" diagnostic results. Even more seriously, in such patients, the HNL concentration is extremely unstable, making it impossible to apply it to such patients. A typical example is patients with combined blood diseases. Since HNL in the blood is secreted by neutrophils, the level of HNL is directly related to the number of neutrophils. Such primary diseases will cause extremely abnormal neutrophil numbers, seriously affecting the blood concentration of HNL. Therefore, in the instructions for use of approved diagnostic kits such as Jixiekezhun 20162400304, there are similar reminder records such as "When neutropenia is combined with infection, HNL does not increase or only increases slightly due to reduced release" to clarify the limitations of the marketed HNL detection kit for the diagnosis of bacterial infection in patients with combined primary diseases. This further reflects the complexity and unpredictability of using HNL as a biomarker for the diagnosis of bacterial infection in patients with combined primary diseases.

[0009] The livers of patients with liver cirrhosis have been damaged to varying degrees and there are related immune deficiencies, which are related to factors such as severe damage to the reticuloendothelial system, complement system deficiencies, T lymphocyte and B lymphocyte deficiencies, and deficiencies in the phagocytic functions of immunoglobulins and macrophages in these patients. The immune deficiencies in patients with liver cirrhosis are prone to causing granulocytopenia and systemic inflammatory responses such as reduced immunity due to various pathogens entering the body's blood system. Once bloodstream infection with bacteria occurs, the bacteria continue to multiply in the blood, producing a large amount of toxins, causing extensive tissue damage, and seriously threatening the lives of patients.

[0010] Almost all patients with liver cirrhosis are accompanied by leukopenia, which further severely affects the blood concentration change of HNL, and then affects the diagnostic accuracy of using HNL as a biomarker to diagnose bloodstream infection in such patients with liver cirrhosis complicated with primary diseases. A specific example is that in the preliminary study of the present invention, an HNL detection kit (Jilin Medical Device Approval No. 20162400304) approved by the drug regulatory authority was used to diagnose whether there is bacterial bloodstream infection in patients with liver cirrhosis. According to the cut-off value of 115.55 ng / ml provided in the kit instruction manual as the critical value for diagnosis, the sensitivity is extremely poor, and almost more than 86% of the cirrhotic bloodstream infection patients are misdiagnosed as non-infected people with "false negative".

[0011] However, that's not all. For patients with the original liver cirrhosis disease, using HNL as a biomarker for the diagnosis of bloodstream infection also faces greater challenges. As mentioned above, compared with the HNL content level in the group of patients without the relevant primary liver cirrhosis disease after bacterial infection, in the group of patients with primary liver cirrhosis disease, after bacterial infection, the average level of its HNL content may increase as a whole, may decrease as a whole, or may show an irregular and unstable situation where some patients increase, some patients decrease, and some patients maintain an HNL content level equivalent to that of the group of patients without the relevant primary liver cirrhosis disease after bacterial infection. Based on this, in view of the uncertainty of the overall change in the HNL content level after bacterial bloodstream infection in the group of patients with liver cirrhosis, it is not feasible and the diagnostic effect cannot be expected by simply adjusting the cut-off value to apply to the diagnosis of bloodstream infection in the group of patients with liver cirrhosis according to the conventional idea.

[0012] Based on the above, how to diagnose at an early stage with high specificity and high sensitivity whether a patient with liver cirrhosis has bloodstream infection, so as to conduct more targeted clinical treatments such as antibiotics and avoid the occurrence of septic shock, this is a very urgent clinical need. Summary of the Invention

[0013] In order to meet the urgent and unmet clinical needs, the present invention provides the use of the biomarker HNL in diagnosing whether there is bacterial bloodstream infection in a specific group of patients with primary diseases, specifically indicating that the specific type of primary disease is chronic hepatitis B liver cirrhosis. On this basis, the present invention also correspondingly provides a series of related uses and the corresponding HNL detection kit.

[0014] The first aspect of the present invention provides the use of HNL or its specific binding substance in preparing a detection reagent for distinguishing whether a subject has bacterial bloodstream infection, wherein the subject is a patient with chronic hepatitis B liver cirrhosis; the cut-off value of the detection reagent in the detection reaction is 50 - 59 ng / mL.

[0015] Further, the differentiation is to diagnose whether there is a bacterial bloodstream infection by detecting whether the HNL content level in the sample to be tested taken from the body fluid sample of the subject exceeds a set cut-off value.

[0016] Further, it is characterized in that the cut-off value for the detection is 54 - 56 ng / mL, preferably 55 ng / mL.

[0017] Further, the body fluid sample taken from the subject is blood, serum, plasma, preferably serum.

[0018] Further, the specific binding substance is an antibody or antibody fragment that specifically binds to HNL.

[0019] Further, the detection reagent is used in detection methods such as electrophoresis, immunofluorescence, direct competition method, indirect competition method, ELISA method, RIA method, flow cytometry method or immunochromatography method; preferably, it is the ELISA method or colloidal gold immunochromatography method.

[0020] The second aspect of the present invention is to provide the use of HNL or its specific binding substance in the preparation of a detection reagent for judging the severity of bacterial bloodstream infection in a subject to determine whether there is a bacterial bloodstream infection. The subject is a patient with chronic hepatitis B cirrhosis. The detection reagent judges the severity of bacterial bloodstream infection by detecting how much the HNL content level in the body fluid sample taken from the subject exceeds the set cut-off value. The cut-off value for the detection is 50 - 59 ng / mL.

[0021] Further, it is characterized in that the cut-off value for the detection is 54 - 56 ng / mL, preferably 55 ng / mL.

[0022] Further, the body fluid sample taken from the subject is blood, serum, plasma, preferably serum.

[0023] Further, the specific binding substance is an antibody or antibody fragment that specifically binds to HNL.

[0024] Further, the detection reagent is used in detection methods such as electrophoresis, immunofluorescence, direct competition method, indirect competition method, ELISA method, RIA method, flow cytometry method or immunochromatography method; preferably, it is the ELISA method or colloidal gold immunochromatography method.

[0025] The third aspect of the present invention is to provide the use of HNL or its specific binding agent in the preparation of a detection reagent for evaluating the prognosis of antibiotic treatment for a subject with bacterial bloodstream infection, wherein the subject is a patient with chronic hepatitis B cirrhosis, and the detection reagent diagnoses the presence of bacterial bloodstream infection by detecting that the HNL content level in the body fluid sample taken from the subject before antibiotic treatment exceeds a set cut-off value, and the cut-off value of the detection kit is 50-59 ng / mL; and the detection reagent evaluates the treatment prognosis of the administered antibiotic by detecting the HNL content level in the body fluid sample taken from the subject again after antibiotic treatment and comparing it with the detection result of the HNL content level of the patient before antibiotic treatment.

[0026] Further, it is characterized in that the cut-off value for the detection is 54-56 ng / mL, preferably 55 ng / mL.

[0027] Further, the body fluid sample taken from the subject is blood, serum, plasma, preferably serum.

[0028] Further, the specific binding agent is an antibody or antibody fragment that specifically binds to HNL.

[0029] Further, the detection methods used for the detection reagent are electrophoresis, immunofluorescence, direct competition method, indirect competition method, ELISA method, RIA method, flow cytometry method or immunochromatography method; preferably, it is ELISA method or colloidal gold immunochromatography method.

[0030] The fourth aspect of the present invention is to provide an HNL detection kit, which is characterized in that the detection kit contains a binding agent that specifically binds to HNL, a standard product, a buffer solution, and a warning line marker, and the warning line marker includes a detection result pattern corresponding to the cut-off value of the HNL content of the sample; the warning line marker also includes a prominent and eye-catching marker corresponding to the detection result when the HNL content of the sample is greater than 50-59 ng / mL.

[0031] Further, the detection methods used for the kit are electrophoresis, immunofluorescence, direct competition method, indirect competition method, ELISA method, RIA method, flow cytometry method or immunochromatography method; preferably, it is ELISA method or colloidal gold immunochromatography method;

[0032] Further, the warning line marker also includes a prominent and eye-catching marker corresponding to the detection result when the HNL content of the sample is greater than 54-56 ng / mL.

[0033] Further, the warning line marker also includes a prominent and eye-catching marker corresponding to the detection result when the HNL content of the sample is greater than 55 ng / mL.

[0034] The beneficial effects of the present invention include:

[0035] For the first time, the present invention applies HNL to the detection and differentiation of bacterial bloodstream infections in patients with chronic hepatitis B cirrhosis, providing effective support for the subdivision treatment of diseases and the evaluation of treatment effects. Specifically, the detection reagent diagnoses the presence of bacterial bloodstream infection by detecting whether the HNL content level in a blood, plasma or serum sample taken from the patient exceeds a set cut-off value. The present invention unexpectedly realizes the diagnosis of bacterial bloodstream infection in patients with chronic hepatitis B cirrhosis complicated with special primary diseases through HNL, and both the sensitivity of the diagnosis result is 90.6% and the specificity is 100%, which are at excellent levels, and have great clinical value and industrialization prospects. Brief Description of the Drawings

[0036] Figure 1 The data analysis curve graph showing whether there is bacterial bloodstream infection in 71 patients with chronic hepatitis B cirrhosis is shown, where A is the ROC curve and B is the data analysis. Detailed Embodiments

[0037] The concept of the present invention and the technical effects produced are further elaborated below in combination with specific embodiments to fully understand the purpose, features and effects of the present invention. The methods are all conventional methods unless otherwise specified. The materials can all be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0038] Example 1 Experiment on Detecting Whether There Is Bloodstream Infection in Patients with Non-Hepatitis B Cirrhosis by Using HNL Level

[0039] Unless otherwise specifically noted, the reagents and medicaments used in the test examples of the present invention are all ordinary commercially available products. The instruments used are also general equipment in the industry. The specific clinical experimental means not listed also refer to the relevant national standards and common practices in the industry. These are not elaborated in detail one by one in the present invention.

[0040] The HNL detection kit used in the test examples of the present invention can be obtained through ordinary commercial channels, for example, it can be directly obtained through ordinary commercial sales of Youboshi (Zhejiang) Biotechnology Co., Ltd. The HNL detection kit used in the test examples of the present invention can also be obtained by using the conventional preparation methods in the industry or referring to the preparation methods disclosed in relevant prior art documents. For example, it can be prepared by the method described in the Chinese patent application with the application number CN202310712274.2. When the test examples of the present invention are specifically implemented, the HNL detection kit used is obtained through ordinary commercial sales of Youboshi (Zhejiang) Biotechnology Co., Ltd.

[0041] 1. Test subjects:

[0042] 46 non - hepatitis B liver cirrhosis patients (including patients with hepatitis C liver cirrhosis, autoimmune liver cirrhosis, and alcoholic liver cirrhosis) were randomly selected from the Department of Infectious Diseases of the Second Affiliated Hospital of Chongqing Medical University.

[0043] 2. Test methods:

[0044] 1) The serum HNL content levels of 46 non - hepatitis B liver cirrhosis patients were detected respectively using the HNL detection kit. The HNL detection results were recorded in Table 1 below.

[0045] 2) Using the clinical gold standard blood culture for diagnosing bloodstream infection (combined with the patient's chronic liver disease history, symptoms, signs, and laboratory tests such as hepatitis B and C virological markers, autoimmune liver disease markers, blood routine, C - reactive protein, procalcitonin test, etc.), supplemented by imaging examinations such as liver magnetic resonance imaging and liver CT. And using a variety of clinical routine means and laboratory tests such as liver and kidney function, myocardial markers, brain damage markers, blood culture, urine culture, and stool culture, the clinical conditions of 46 non - hepatitis B liver cirrhosis patients were clinically diagnosed to determine whether there was bloodstream infection in their true conditions, and the clinical diagnosis results were recorded in Table 1 below.

[0046] 3) Combining the clinical diagnosis results of each case in Table 1 by conventional means, statistical analysis was performed on the HNL detection concentration results shown in Table 1.

[0047] Table 1

[0048]

[0049] 3. Result analysis:

[0050] Statistical analysis of the data in Table 1:

[0051] For patients with non - hepatitis B cirrhosis as the primary disease in this special combination, there is no significant statistical difference between the two groups of data of HNL test results for differentiating and diagnosing bloodstream infection and non - bloodstream infection.

[0052] Even if the diagnostic threshold (cut - off value) is set at 50 ng / mL, among 21 non - hepatitis B cirrhosis patients clinically diagnosed with bloodstream infection, only 7 cases were correctly diagnosed after HNL test, and the calculated sensitivity is 33.3%. Among 25 non - hepatitis B cirrhosis patients clinically diagnosed with non - bloodstream infection, only 19 cases were correctly diagnosed after HNL test, and the calculated specificity is 76%. Its sensitivity and specificity, especially the sensitivity, are difficult to meet the clinical needs.

[0053] Example 2: Experiment on using HNL level to detect whether there is bloodstream infection in patients with hepatitis B cirrhosis

[0054] 1. Test subjects

[0055] Randomly select 71 patients with chronic hepatitis B cirrhosis from the Department of Infectious Diseases of the Second Affiliated Hospital of Chongqing Medical University.

[0056] 2. Test methods and results

[0057] 1) Use the HNL test kit to detect the serum HNL content levels of 71 patients with chronic hepatitis B cirrhosis respectively. Take 55 ng / mL of HNL content as the diagnostic threshold. If the test result shows that the HNL content in an individual sample is greater than the above - mentioned diagnostic threshold, it is marked as bloodstream infection. If the HNL content in an individual sample is not greater than the above - mentioned diagnostic threshold, it is marked as non - bloodstream infection.

[0058] Record the HNL test results and the diagnostic marker results with 55 ng / mL as the diagnostic threshold in Table 2 below.

[0059] 2) Use blood culture, the clinical gold standard for diagnosing bloodstream infection (combined with the patient's chronic liver disease history, symptoms, signs, and laboratory tests such as hepatitis B and C virological markers, autoimmune liver disease markers, blood routine, C - reactive protein, procalcitonin test, etc.), supplemented by imaging liver magnetic resonance and liver CT examinations. And use a variety of clinical routine means and laboratory tests such as liver and kidney function, myocardial markers, brain damage markers, blood culture, urine culture, and stool culture to clinically diagnose the conditions of 71 patients with chronic hepatitis B cirrhosis, determine whether there is bloodstream infection in their true conditions, and record the clinical diagnosis results in Table 2 below.

[0060] 3) Compare the differences between the diagnostic marker results of the HNL test kit shown in Table 2 and the clinical diagnosis results of routine means, and statistically calculate the detection sensitivity and specificity.

[0061] Table 2

[0062]

[0063] 3. Result Analysis

[0064] Statistical analysis of the data in Table 2:

[0065] Among 32 clinically diagnosed patients with chronic hepatitis B cirrhosis complicated with bloodstream infection, 29 cases were correctly diagnosed by HNL detection; among 39 clinically diagnosed patients with chronic hepatitis B cirrhosis without bloodstream infection (non-bloodstream infection), 39 cases were correctly diagnosed by HNL detection.

[0066] Performing statistical analysis on the data in Table 2, the results shown in Figure 1 A were obtained. Through data analysis using SPSS software, with 55 ng / mL as the diagnostic threshold (cut-off value), it can well distinguish patients with chronic hepatitis B cirrhosis. For all 71 patients with chronic hepatitis B cirrhosis using the kit of the present application, the sensitivity and specificity for differentiating bloodstream infection after HNL detection were 90.6% and 100% respectively, and the AUC was 0.97 ( Figure 1 B), which is superior to the current methods.

[0067] Therefore, different from patients with non-hepatitis B cirrhosis, for this specific population of patients with chronic hepatitis B cirrhosis complicated with primary diseases, using HNL as a biomarker for differentiating bloodstream infection, the diagnostic detection effect is very excellent and the clinical value is prominent.

[0068] Preparation Example: Preparation of an HNL detection kit for diagnosing whether a patient with chronic hepatitis B cirrhosis has bacterial bloodstream infection

[0069] The basic composition and structure of the HNL detection kit in this preparation example can be obtained by the conventional preparation methods in the industry or by referring to the preparation methods disclosed in relevant prior art documents. For example, it can be prepared by the method described in the Chinese patent application with the application number CN202310712274.2.

[0070] The detection methods that can be optionally used for the HNL detection kit in this preparation example are electrophoresis, immunofluorescence, direct competition method, indirect competition method, ELISA method, RIA method, flow cytometry method or immunochromatography method. Usually, the ELISA method or colloidal gold immunochromatography method is preferred.

[0071] Specifically, the HNL detection kit of this preparation example includes a reference cut-off value, and the cut-off value is 55 ng / mL.

[0072] For the convenience of clinical use, a warning line mark for bloodstream infection of chronic hepatitis B cirrhosis with HNL can be further designed on the HNL detection kit of this preparation example. The warning line mark can be, for example, the detection result pattern corresponding to a sample HNL content of 55 ng / mL. In another specific preparation example, the warning line mark also includes a prominent and eye-catching mark corresponding to the detection result when the sample HNL content is 55 ng / mL.

[0073] Another use of the HNL detection kit of this preparation example can be, for example, to judge the severity of bloodstream infection in patients with chronic hepatitis B cirrhosis with bloodstream infection. This detection kit judges the severity of bloodstream infection by detecting how much the HNL content level in a blood, plasma or serum sample taken from the patient exceeds the set cut-off value.

[0074] Another use of the HNL detection kit of this preparation example can be, for example, to evaluate the prognosis of antibiotic treatment in patients with chronic hepatitis B cirrhosis with bloodstream infection. This detection kit diagnoses the presence of bloodstream infection by detecting that the HNL content level in a blood, plasma or serum sample taken from the patient before antibiotic treatment exceeds the set cut-off value. The cut-off value of this detection kit is 55 ng / mL; and this detection kit evaluates the treatment prognosis of the administered antibiotic by detecting the HNL content level in a blood, plasma or serum sample taken from the patient again after antibiotic treatment and comparing it with the detection result of the HNL content level of this patient before antibiotic treatment.

[0075] The test examples and examples involved in the above specific implementation manners only describe the preferred implementation manners of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solution of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of HNL or its specific binding substance in the preparation of a detection reagent for distinguishing whether a patient with chronic hepatitis B cirrhosis has bacterial bloodstream infection, characterized in that: The cut-off value of the detection is 55 ng / mL; the differentiation is performed by detecting the HNL content level in the patient's body fluid sample, and diagnosing whether there is a bacterial bloodstream infection based on whether it exceeds the set cut-off value; the body fluid sample is a serum sample taken from the patient.

2. Use of HNL or its specific conjugate in the preparation of a detection reagent for evaluating the prognosis of antibiotic treatment for bacterial bloodstream infection in patients with chronic hepatitis B cirrhosis, characterized in that: The detection reagent is used to detect the HNL content level in the patient's body fluid sample before antibiotic treatment and it exceeds the set cut-off value to diagnose the presence of bacterial bloodstream infection, and the cut-off value of the detection is 55 ng / mL; and the detection reagent is used to detect the HNL content level in the patient's body fluid sample again after antibiotic treatment, and compared with the HNL content level detection result of the patient before antibiotic treatment to evaluate the treatment prognosis of the administered antibiotics; the body fluid sample is a serum sample taken from the patient.

3. The use according to claim 1 or 2, characterized in that The specific binding substance is an antibody or antibody fragment that specifically binds to HNL.

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