Application of NMI protein as a biomarker in the evaluation of the severity and prognosis of adult sepsis

Abstract

The present application relates to the field of biotechnology, in particular to the field of immunodiagnosis of biomarkers, and more particularly to the application of a molecular marker in the evaluation of the severity and prognosis of adult sepsis. The biomarker of the present application is NMI, which is found to have significant expression difference in the peripheral blood serum of sepsis patients through ELISA detection, and its expression amount is related to the severity and prognosis of the patients. ROC curve analysis shows that NMI has high efficiency in evaluating the severity and prognosis of sepsis, and is better than the currently available indicators. In clinical application, only the peripheral blood of the patient needs to be extracted, and the severity and prognosis of the patient can be evaluated, which is less traumatic and easy to operate, and is more likely to become an effective means for evaluating the severity and prognosis of sepsis patients.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to the application of NMI protein as a biomarker in the assessment of sepsis severity and prognosis. Background Technology

[0002] Sepsis and septic shock are significant issues in clinical medicine, particularly in critical care. Currently, the definitions of sepsis and septic shock primarily follow the definition and diagnostic criteria of Sepsis 3.0, jointly published in 2016 by the Society of Critical Care Medicine (SCCM) and the European Society of Intensive Care Medicine (ESICM). Sepsis is defined as life-threatening organ dysfunction caused by a dysregulation of the host response to infection. For patients with infection or suspected infection, a diagnosis of sepsis is made when the Sequential Organ Failure Assessment (SOFA) score increases by ≥2 points from baseline. Septic shock is defined as a subset of sepsis, specifically, in patients with sepsis who, despite adequate fluid resuscitation, still require vasoactive drugs to maintain a mean arterial pressure ≥65 mmHg and a blood lactate concentration ≥2 mmol / L. This latest publication has removed the definition of severe sepsis. Sepsis remains one of the leading causes of death worldwide, placing a severe health and economic burden on healthcare systems.

[0003] Due to the significant heterogeneity among sepsis patients and the incomplete understanding of the pathophysiological mechanisms of sepsis, early identification and assessment of sepsis severity are crucial for early intervention and reducing mortality. Currently, the main methods and tools routinely used to evaluate sepsis severity and clinical prognosis are scoring systems, including the Acute Physiology and Chronic Health Evaluation II (APACHE-II) and SOFA. To improve the simplicity and speed of sepsis assessment, quick SOFA (qSOFA) was proposed; however, its accuracy in assessing severity is affected by the removal of many molecules that need to be measured. Furthermore, biomarkers also play an important role in the early assessment of sepsis or septic shock patients. Traditional inflammatory factors, such as C-reactive protein (CRP) and procalcitonin (PCT), while having some sensitivity, have poor specificity. Elevated levels of other pro-inflammatory factors, such as interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor (TNF), have been shown to be associated with sepsis mortality, but evidence of their correlation with disease severity is weaker, making it difficult to distinguish between sepsis and septic shock. Therefore, there is an urgent need for specific biomolecules to assist in assessing the severity of sepsis and its clinical prognosis.

[0004] NMI, an N-Myc-STAT interacting protein, was first discovered in 1996. Its gene is located on chromosome 2q23, has a molecular weight of 38 kDa, and its structure can be divided into three parts: Coiled-Coil, NID1, and NID2. It is typically located in the cytoplasm and is one of the proteins that indirectly influence specific transcriptional events. In humans, NMI protein expression has been detected in all fetal tissues except the brain, with the highest expression levels found in the adult spleen, liver, and kidneys. As an adapter molecule, NMI is widely involved in transcriptional regulation and signal transduction, exhibiting high expression after interferon induction and playing a variety of important biological functions.

[0005] Currently, studies have reported on the important role of NMI protein in the diagnosis and prognostic assessment of various diseases. For example, an article published in 2022 by Zhang et al. (DOI: 10.1186 / s12931-022-02139-x) used NMI to predict the severity and prognosis of community-acquired pneumonia; a 2019 paper by Xiahou et al. (DOI: 10.1038 / s41467-017-00930-9) reported that NMI is released by activated macrophages in lipopolysaccharide-induced septic shock, promotes the release of inflammatory factors by activating the TLR4 signaling pathway, and that knockout of the NMI gene can reduce the inflammatory response and mortality rate in a mouse model of sepsis. However, there are no clinical studies on the role of NMI in assessing the severity and prognosis of sepsis.

[0006] Biomarkers can be used to assist in the early assessment of the severity of sepsis patients and to evaluate their prognosis, guide clinicians in developing treatment plans, improve the timeliness of clinical treatment for sepsis, increase patient survival rates, and improve patients' quality of life. Summary of the Invention

[0007] This invention addresses the shortcomings of existing technologies by providing an application of NMI protein as a biomarker in the assessment of sepsis severity and prognosis. It can be used for early auxiliary assessment of the severity and prognosis of adult sepsis patients, guiding clinicians in developing treatment plans, improving clinicians' ability to identify severe sepsis and assess its severity, thereby improving patient survival rates and prognosis.

[0008] The application of NMI protein as a biomarker in the development of kits for assessing the severity and prognosis of sepsis in adults. NMI protein is an N-Myc and STAT interacting protein. Peripheral venous serum was collected from the subjects being assessed.

[0009] Preferably, peripheral venous serum is collected within 48 hours of the subject's admission or onset of illness.

[0010] More preferably, a peripheral venous serum NMI protein concentration ≥110.85 pg / ml indicates a high risk of death and poor prognosis; a peripheral venous serum NMI protein concentration ≥100 pg / ml indicates a 30-day survival rate of less than 60%; and a peripheral venous serum NMI protein concentration ≥150 pg / ml indicates a 30-day survival rate of less than 30%. The prognostic endpoint is the 30th day after the subject's admission, and the evaluation criterion is whether the subject survives.

[0011] Preferably, the detection method for NMI protein is enzyme-linked immunosorbent assay (ELISA). The concentration of NMI in peripheral venous serum is detected using ELISA, and the obtained data are used to plot the recipient operating characteristic curve (ROC). The area under the curve (AUC), sensitivity, specificity, and Youden index (sensitivity + specificity - 1) are calculated to evaluate its efficacy in assessing the severity of NMI.

[0012] Preferably, the subjects being evaluated are patients who have developed sepsis in its early stages. Early symptoms are relatively mild, making diagnosis relatively difficult, thus using the NMI protein indicator for auxiliary diagnosis has greater practical value.

[0013] Preferably, the subjects being evaluated are adults aged >18 years.

[0014] Preferably, when using NMI protein as a biomarker to assess the severity and prognosis of sepsis in adults, combining it with existing methods can further improve the assessment effectiveness. Specifically, it can be used in conjunction with the APACHE-II, SOFA, or qSOFA scoring systems.

[0015] The invention is characterized by: 1. The expression level of NMI in the peripheral venous blood serum of sepsis patients who died within 30 days was significantly higher than that of sepsis patients who survived within 30 days (p<0.001). The expression level of NMI in the peripheral venous blood serum of patients who developed septic shock was significantly higher than that of sepsis patients (p<0.001).

[0016] 2. The AUC value of peripheral venous blood serum NMI for assessing mortality within 30 days was 0.92 (0.89-0.96), with a sensitivity of 86.4%, a specificity of 88.9%, a Youden index of 0.75, and a cutoff value of 80.45 pg / ml.

[0017] 3. The AUC value of peripheral venous blood serum NMI for assessing the occurrence of septic shock was 0.86 (0.82-0.90), with a sensitivity of 76.4%, a specificity of 85.7%, a Youden index of 0.62, and a cutoff value of 110.85 pg / ml.

[0018] 4. The AUC value of the new scoring system incorporating NMI was significantly higher than that of the original scoring systems (APACHE-II; SOFA; qSOFA). The AUC value of NMI combined with APACHE-II was 0.94 (APACHE-II's AUC value was 0.75), with a sensitivity of 77.8%, a specificity of 97.6%, and a Youden index of 0.75; the AUC value of NMI combined with SOFA was 0.93 (SOFA's AUC value was 0.77), with a sensitivity of 84.7%, a specificity of 92.9%, and a Youden index of 0.78; the AUC value of NMI combined with qSOFA was 0.93 (qSOFA's AUC value was 0.67), with a sensitivity of 88.1%, a specificity of 88.1%, and a Youden index of 0.76.

[0019] 5. Significant differences were found in the curves predicting 30-day mortality and clinical severity among sepsis patients with different peripheral venous blood serum NMI concentration groups (<25; 10-50; 50-100; >100 pg / ml) (p<0.001). The mortality rate for sepsis patients with peripheral venous blood serum NMI concentrations >100 pg / ml was close to 40%, and the mortality rate for sepsis patients with NMI concentrations >150 pg / ml reached nearly 70%.

[0020] 6. Comparing patients with septic shock and those with sepsis, the peripheral venous serum NMI concentration was significantly higher in the former (p<0.001).

[0021] 7. Internal validation confirmed the stability of NMI prediction performance.

[0022] Beneficial effects of this invention: This invention is the first to discover that peripheral venous blood serum NMI concentration is related to the severity and prognosis of sepsis, providing important reference value for early assessment of the condition of sepsis patients and prediction of prognosis.

[0023] This invention provides a molecular marker, NMI, for assessing the severity and prognosis of sepsis. The expression level of NMI in peripheral blood serum within 48 hours of admission / onset of illness is detected by ELISA, enabling early assessment of sepsis severity and prognosis. In clinical application, only peripheral venous blood needs to be drawn from sepsis patients to assess the severity and prognosis of their condition. This method is minimally invasive, easy to perform, and has the potential to become an effective means of assessing the severity and prognosis of sepsis patients, facilitating timely intervention by physicians and significantly reducing patient mortality and medical costs. Attached Figure Description

[0024] Figure 1 Differences in serum NMI levels among different groups. Figure 1a): Comparison between the control group, patients with sepsis, and patients with septic shock. Figure 1 b): Comparison between control group, survivors and non-survivors. Samples below the lower limit were uniformly quantified to half of the minimum detection threshold (11.72 pg / ml).

[0025] Figure 2 Comparison of NMI with other serum markers and scores. Comparison of ROC curves between NMI and other serum parameters for predicting septic shock. Figure 2 a) and 30-day mortality rate ( Figure 2 The development of c) in the NMI. ROC curves between NMI and other clinical scores are used to predict septic shock ( Figure 2 (b) and 30-day mortality rate ( Figure 2 The occurrence of d) in.

[0026] Figure 3 Kaplan-Meier survival curves are created using NMI critical values ​​to predict survival rates. Figure 3 a): Kaplan-Meier survival curves created using the critical value (110.85 pg / ml). Figure 3 b): Kaplan-Meier survival curves created by NMI concentration stratification (<50 pg / ml, 50-100 pg / ml, 100-150 pg / ml, >150 pg / ml).

[0027] Figure 4 The added value of NMI in predicting septic shock and 30-day mortality. A comparison of ROC curves between APACHE-II and a new scoring system incorporating NMI for predicting sepsis severity. Figure 4 a) and the result ( Figure 4 (d)). The ROC curve between SOFA and the new scoring system including NMI is used to predict the severity of sepsis. Figure 4 b) and the result ( Figure 4 (e)). The ROC curve between qSOFA and the new scoring system including NMI is used to predict the severity of sepsis. Figure 4 c) and the result ( Figure 4 f in the middle).

[0028] Figure 5 Internal validation of NMI in predicting the occurrence of septic shock ( Figure 5 a) and the mortality rate within 30 days ( Figure 5 b) Stability of performance. Detailed Implementation

[0029] The implementation of the present invention will be described in detail below with reference to the accompanying drawings and embodiments. The following embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0030] All reagents and raw materials used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer.

[0031] In the following examples, serum samples from sepsis patients were all obtained from the Second Affiliated Hospital of Zhejiang University School of Medicine.

[0032] The inventors collected serum samples from 399 sepsis patients between 2024 and 2025, of whom 302 were in the training group and 97 were in the validation group. All patients were from the Second Affiliated Hospital of Zhejiang University School of Medicine, and the study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University School of Medicine (Approval No.: 2017-016). Follow-up ended on April 1, 2025. The implementation is described in further detail below with reference to the accompanying drawings.

[0033] Example 1: Specimen Collection and Basic Information Collection According to the diagnostic criteria for sepsis 3.0 jointly published by the Society of Critical Care Medicine (SCCM) and the European Society of Intensive Care Medicine (ESICM) in 2016, 302 sepsis patients aged ≥18 years were collected, and patients with rheumatic immune diseases, hematological diseases, and other non-infectious febrile diseases that may cause elevated PCT (procalcitonin) and CRP (C-reactive protein) were excluded.

[0034] Collect 5 ml of peripheral venous serum from the patient within 48 hours of admission, incubate at room temperature for 2 hours or overnight at 4°C, then centrifuge at 1000×g for 20 minutes and collect the supernatant.

[0035] Basic information about the patient upon admission is collected: age, gender, and peripheral venous blood data including absolute white blood cell count (WBC), absolute neutrophil count, neutrophil percentage (NCP), procalcitonin (PCT), C-reactive protein (CRP), interleukin (IL-6), lactate, length of hospital stay, whether the patient was admitted to the ICU, length of ICU stay, and whether the patient died within 30 days.

[0036] Example 2: Determination of serum NMI in peripheral venous blood of patients with sepsis Main instruments and equipment: The main instruments and equipment include a microplate reader (Biotek ELX-808), a small benchtop centrifuge (Eppendorf 5418), a centrifuge (SBC140-2 handheld), a vortex mixer (Vortex-2), an ultrapure water system (Cascuda 1), and a biochemical incubator (MIR-162).

[0037] ELISA method for detecting serum NMI concentration in peripheral venous blood of sepsis patients: (1) Preparation of working solutions: Prepare the required standard working solutions, washing solutions, biotinylated antibody working solutions, and enzyme conjugation working solutions in advance according to the NMI ELISA kit instructions.

[0038] (2) Sample addition: Add the standard working solution sequentially to the first two rows of wells, with two wells of each concentration added side by side, 100 μL per well. Add the serum to be tested to the other wells, 100 μL per well. After covering with a membrane, incubate at 37°C for 100 minutes.

[0039] (3) Discard the liquid, spin dry, and do not wash. Add 100 μL of biotinylated antibody working solution to each well, mix well, cover with a membrane, and incubate at 37°C for 1 hour.

[0040] (4) Shake off the liquid in the wells, add 200 μL of washing buffer to each well, soak for 1-2 minutes, aspirate or shake off the liquid in the microplate, and pat dry on thick absorbent paper. Repeat this washing step 3 times.

[0041] (5) Add 100 μL of enzyme conjugate working solution to each well, cover with membrane and incubate at 37°C for 60 minutes.

[0042] (6) Discard the liquid in the hole, spin dry, and wash the plate 5 times.

[0043] (7) Add 90 μL of substrate solution (TMB) to each well, cover with a membrane, and incubate at 37°C in the dark for about 15-30 minutes. Terminate the reaction when a clear gradient appears in the standard wells.

[0044] (8) Add the stop solution in the same order as the substrate solution, adding 50 μL of stop solution to each well to terminate the reaction.

[0045] (9) Immediately measure the optical density (OD value) of each well using an ELISA reader at a wavelength of 450 nm.

[0046] (10) NMI determination: Calculate the average OD value of each replicate well. Subtract the OD value of the blank well from the average OD value of each standard as the correction value. Plot the standard curve on logarithmic graph paper with concentration on the x-axis and OD value on the y-axis. Substitute the measured OD correction value of the sample to be tested into the standard curve to determine the NMI concentration in the sample to be tested.

[0047] Example 3: Basic Clinical Characteristics of Patients with Sepsis Statistical analysis of basic information of sepsis patients: For normally distributed data, the mean ± standard deviation is used; for non-normally distributed data, the median [interquartile range] is used; for non-numerical variables, the number (percentage) is used. The statistical results are shown in Table 1.

[0048] Table 1: Statistical table of clinical characteristics of patients with sepsis

[0049] Note: Data is described using median (interquartile range) or number (percentage).

[0050] Example 4: Assessment of NMI's ability to identify the severity and prognosis of adult sepsis patients Statistical analysis was performed on the differences in peripheral venous blood serum NMI, PCT, CRP, WBC, absolute neutrophil count, and IL-6 between patients who died within 30 days and those who did not, and between patients with sepsis and septic shock. The rank-sum test was used to analyze the differences between the two groups. ROC curves were plotted to predict the 30-day mortality rate and incidence of septic shock in sepsis patients using serum NMI, PCT, CRP, WBC, NLR, and lactate levels. The optimal cutoff values ​​and diagnostic efficacy indicators—area under the curve (AUC), sensitivity, specificity, and Youden's index—were calculated.

[0051] Table 2: Efficacy of serum NMI, PCT, CRP, WBC, IL-6, NLR, lactate, APACHE-II, SOFA, and qSOFA in assessing 30-day mortality and septic shock in sepsis patients.

[0052] *：p<0.05，** : p<0.01, ***: p<0.001, compared with serum NMI respectively.

[0053] like Figure 1 As shown, the expression level of NMI in the peripheral venous blood serum of sepsis patients who died within 30 days was significantly higher than that of sepsis patients who survived within 30 days (p<0.001), and the expression level of NMI in the peripheral venous blood serum of septic shock patients was significantly higher than that of sepsis patients (p<0.001).

[0054] As shown in Table 2, comparing the predictive efficacy of peripheral venous serum NMI, PCT, CRP, WBC, IL-6, NLR, lactate, and APACHE-II, SOFA, and qSOFA in assessing the occurrence of septic shock in patients revealed that the AUC value of peripheral venous serum NMI concentration in assessing the occurrence of septic shock was 0.92 (0.89-0.96), with a sensitivity of 86.4%, a specificity of 88.9%, and a Youden index of 0.75. NMI's ability to comprehensively predict the severity of sepsis is superior to other clinical indicators and scoring systems. This indicates that NMI can help in the early assessment of the severity of sepsis in patients, providing some guidance for clinical diagnosis and treatment.

[0055] As shown in Table 2 and Figure 2 As shown, by comparing the predictive efficacy of peripheral venous serum NMI, PCT, CRP, WBC, IL-6, NLR, lactate, and APACHE-II, SOFA, and qSOFA on 30-day mortality in sepsis patients, it was found that the AUC value of peripheral venous serum NMI concentration in assessing 30-day mortality was 0.86 (0.82-0.90), with a sensitivity of 76.4%, a specificity of 85.7%, and a Youden index of 0.62. NMI's ability to comprehensively predict 30-day mortality in sepsis patients is superior to other clinical indicators and scoring systems. This indicates that NMI can help in the early assessment of the clinical prognosis of sepsis and provide guidance for early clinical intervention.

[0056] Example 5: Comparison of 30-day Kaplan-Meier survival curves of sepsis patients stratified by peripheral venous serum NMI levels. Statistical analysis of 30-day Kaplan-Meier survival curves of peripheral venous serum NMI at different concentrations in sepsis patients: The log-rank test was used to verify the differences in survival curves at different concentrations. Figure 3 As shown, among sepsis patients with different peripheral venous blood serum NMI concentration groups (<50; 50-100; 100-150; >150 pg / ml), the mortality rate of sepsis patients with peripheral venous blood serum NMI concentration >150 pg / ml was close to 70%. This indicates that the higher the serum NMI concentration, the higher the 30-day mortality rate, thus demonstrating that serum NMI concentration can assess the clinical prognosis of sepsis patients.

[0057] Example 6: The effect of NMI on the predictive performance of APACHE-II, SOFA, and qSOFA scoring systems The effectiveness of NMI measurements in improving the performance of existing commonly used scoring systems was further evaluated: (e.g.) Figure 4As can be seen, the AUC of the APACHE-II score with added NMI for predicting septic shock was 0.94 (0.91-0.97), with a sensitivity of 77.8%, a specificity of 97.6%, and a Youden index of 0.75. For predicting 30-day mortality, the AUC was 0.88 (0.84-0.92), with a sensitivity of 78.0%, a specificity of 85.7%, and a Youden index of 0.64, significantly higher than the AUC of the original score. C (0.75 (0.69-0.80), 0.79 (0.74-0.84)); The SOFA score with added NMI had an AUC of 0.93 (0.90-0.96) for predicting septic shock, with a sensitivity of 84.7% and a specificity of 92.9%, and a Youden index of 0.78. For predicting 30-day mortality, the AUC was 0.87 (0.84-0.92), with a sensitivity of 79.5% and a specificity of [missing value]. The AUC of the NMI-added qSOFA score for predicting septic shock was 0.93 (0.89-0.96), with a sensitivity of 88.1% and a specificity of 88.1% and a Youden index of 0.76. For predicting 30-day mortality, the AUC was 0.86 (0.82-0.91), with a sensitivity of 76.4% and a specificity of 85.7% and a Youden index of 0.62, significantly higher than the AUC of the original score (0.67 (0.61-0.73) and 0.70 (0.61-0.73)). These data indicate that the NMI, when used alone, not only has good predictive efficacy for the clinical prognosis and severity of sepsis but also significantly improves the predictive efficacy of existing scoring systems.

[0058] Example 7: Internal validation of the stability of NMI in predicting the incidence of septic shock and 30-day mortality in sepsis patients To verify the stability of NMI prediction performance, the remaining 97 patients out of 399 who were not in the training group were used as the validation group. The results are as follows: Figure 5 As shown, in the validation group, the AUC for predicting septic shock was 0.86, the sensitivity was 92.0%, the specificity was 69.0%, and the Youden index was 0.61. The AUC for predicting 30-day mortality was 0.92, the sensitivity was 89.0%, the specificity was 83.0%, and the Youden index was 0.72. These data validated the stability of the NMI obtained in the training group (302 cases) in predicting the occurrence of septic shock and 30-day mortality in sepsis patients.

Claims

1. The application of a reagent for detecting NMI protein as a biomarker in the preparation of a prognostic assessment kit for adult sepsis, characterized in that, NMI is an N-Myc and STAT interacting protein, and the biological sample used to assess adult subjects was peripheral venous serum. A peripheral venous blood serum NMI protein concentration ≥110.85 pg / ml indicates a high risk of death and poor prognosis; when the concentration is ≥100 pg / ml, the 30-day survival rate is less than 60%; and when it is ≥150 pg / ml, the 30-day survival rate is less than 30%. The adult subjects assessed were patients who had early-stage sepsis.

2. The application according to claim 1, characterized in that, Peripheral venous serum was collected within 48 hours of the subject's admission or onset of illness.

3. The application according to claim 1, characterized in that, The endpoint for predicting the prognosis was the 30th day after the subject's admission, and the evaluation criterion was whether the subject was alive.

4. The application according to claim 1, characterized in that, The detection method for NMI is enzyme-linked immunosorbent assay (ELISA).

5. The application according to claim 1, characterized in that, The subjects being evaluated were adults aged >18 years.

6. The application according to claim 1, characterized in that, Used in conjunction with APACHE-II, SOFA, or qSOFA scoring systems.

Images