Method of testing or assessing risk of developing severe symptoms from covid-19 or influenza viruses, and kit for use therein

Abstract

To provide a method of testing or assessing the risk of developing severe symptoms from COVID-19 or influenza viruses using midkine, a causal agent of NETosis, as a marker, and to provide a kit for use therein.SOLUTION: The present invention relates to: a method of testing or assessing the risk of developing severe symptoms from COVID-19 or influenza viruses, the method comprising quantifying the midkine level in urine collected from a subject, where a result of the quantification is used for the test or assessment; and a kit for use in the method of testing or assessing the risk of developing severe symptoms from COVID-19 or influenza viruses, the kit including a substance for quantifying the midkine level.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for testing or evaluating the risk of COVID-19 and influenza virus becoming severe using midkine, a causative agent of NETosis, as a marker, and a kit for use therein. [Background technology]

[0002] Since the first cases of COVID-19 were reported in China at the end of 2019, it has caused a pandemic around the world, and infections continue to spread today due to the emergence of various mutant strains. The death toll from COVID-19 has surpassed six million worldwide, calling for an urgent response. Meanwhile, NETosis is believed to be involved in the worsening of COVID-19. NETosis is a type of cell death in which neutrophils release net-like DNA (NETs) outside the cell. Excessive release of NETs stimulates the coagulation system, leading to the formation of blood clots. This is thought to result in the development and severity of arteriovenous thrombosis-related diseases such as pulmonary embolism, cerebral infarction, and deep vein thrombosis.

[0003] The inventors suspected that the substance that triggers NETosis might be midkine (MK), and investigated the possibility that elevated midkine levels in plasma and urine increase the risk of severe COVID-19 through NETosis. Midkine is a heparin-binding secreted protein that was discovered as a gene product whose expression is induced during the differentiation of embryonic tumor cells. Its functions can be broadly divided into neural survival and differentiation, cancer initiation and progression, and tissue remodeling. Midkine is a low molecular weight molecule with a molecular weight of 13 kDa and is not filtered out by the kidney, so it can be detected in urine. Urine is easier to collect than blood, can reduce the contact time between medical staff and COVID-19 infected patients, and can also be collected by the COVID-19 infected patients themselves. Therefore, in the future, the development of simple test kits that can be used by COVID-19 infected patients is expected.

[0004] For example, Patent Documents 1 and 2 disclose a method for examining the risk of severe illness in COVID-19 infected patients using urine as a specimen, a test kit therefor, a companion diagnostic agent, and a severe illness risk marker therefor. According to this invention, urine that can be collected without being exposed to droplets containing SARS-CoV-2 can be used as a specimen, and the risk of severe illness in COVID-19 can be examined early by a non-invasive method. In addition, it is also possible to classify the risk of severe illness at an early stage and evaluate the risk of recurrence after discharge of COVID-19 infected patients waiting for discharge.

[0005] However, it has not been known until now that an increase in the midkine level increases the risk of severe illness in COVID-19 and influenza virus through NETosis, including Patent Documents 1 and 2.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] Therefore, an object of the present invention is to provide a method for examining or evaluating the risk of severe illness in COVID-19 and influenza virus using midkine, which is a causative substance of NETosis, as a marker, and a kit for use therein. [Means for solving the problem]

[0008] The method for testing the risk of COVID-19 becoming severe according to the present invention comprises: Quantifying the midkine level in urine or blood collected from a subject, The result of the quantification is Used to test for the risk of developing severe COVID-19 It is characterized by:

[0009] The method for testing the risk of COVID-19 becoming severe according to the present invention comprises: The midkine level is Midkine levels in urine collected from subjects on the 7th day after infection. It is characterized by:

[0010] The method for testing the risk of COVID-19 becoming severe according to the present invention comprises: The midkine level is Midkine levels in the blood collected from subjects 1 to 3 days after infection. It is characterized by:

[0011] The method for assessing the risk of COVID-19 worsening according to the present invention comprises: Midkine levels in urine or blood collected from subjects were used to Assessing the risk of severe illness from COVID-19 It is characterized by:

[0012] The method for assessing the risk of COVID-19 worsening according to the present invention comprises: The evaluation Midkine levels in urine collected from subjects on the 7th day after COVID-19 infection If it exceeds 5.83ng / ml, Assessing that the risk of severe illness from COVID-19 is high, It is characterized by:

[0013] The method for evaluating the risk of severe COVID-19 according to the present invention is, wherein the evaluation is, when the midkine value in the blood collected from a subject on the 1st to 3rd day after being infected with COVID-19 is more than 1.11 ng / ml, it is evaluated that the risk of severe illness due to COVID-19 is high, which is characterized by.

[0014] The test or evaluation marker for the risk of severe COVID-19 according to the present invention is, a marker to be quantified for the test or evaluation of the risk of severe illness due to COVID-19, wherein the marker is, the midkine value in urine or blood collected from a subject, which is characterized by.

[0015] The kit used for the test or evaluation of the risk of severe illness due to COVID-19 according to the present invention is, characterized by containing a substance for quantifying the midkine value. which is characterized by.

[0016] The kit used for the test or evaluation of the risk of severe illness due to COVID-19 according to the present invention is, characterized by having a reactant pre-coated with an antibody for quantifying the midkine value. which is characterized by.

[0017] The kit used for the test or evaluation of the risk of severe COVID-19 according to the present invention is, wherein the midkine value is, the midkine value in urine collected from a subject on the 7th day after infection, which is characterized by.

[0018] The kit used for the test or evaluation of the risk of severe COVID-19 according to the present invention is, wherein the midkine value is, Midkine levels in the blood collected from subjects 1 to 3 days after infection. It is characterized by:

[0019] The method for testing the risk of influenza virus aggravation according to the present invention comprises: quantitating the midkine level in blood collected from a subject, The result of the quantification is Used to test for the risk of developing severe symptoms due to influenza viruses, It is characterized by:

[0020] The method for testing the risk of influenza virus aggravation according to the present invention comprises: The midkine level is Midkine levels in the blood collected from subjects 3 to 5 days after infection. It is characterized by:

[0021] The method for assessing the risk of influenza virus aggravation according to the present invention comprises: Based on the midkine levels in the blood collected from the subjects, Assessing the risk of severe illness due to influenza virus It is characterized by:

[0022] The method for assessing the risk of influenza virus aggravation according to the present invention comprises: The evaluation The midkine levels in the blood collected from subjects 3 to 5 days after influenza virus infection were If it exceeds 2.89ng / ml, The risk of developing severe symptoms due to influenza virus is assessed to be high. It is characterized by:

[0023] The markers to be quantified for testing or evaluating the risk of severe illness caused by influenza virus according to the present invention are: The marker is Midkine levels in the blood collected from the subjects It is characterized by:

[0024] The kit of the present invention used for testing or evaluating the risk of severe illness caused by influenza virus comprises: Contains a substance for quantifying midkine levels, It is characterized by:

[0025] The kit of the present invention used for testing or evaluating the risk of severe illness caused by influenza virus comprises: A reactant pre-coated with an antibody for quantifying midkine level. It is characterized by:

[0026] The kit of the present invention used for testing or evaluating the risk of severe illness caused by influenza virus comprises: The midkine level is Midkine levels in the blood collected from subjects 3 to 5 days after infection. It is characterized by: [Effects of the Invention]

[0027] The present invention can reduce the amount of contact time between medical professionals and patients infected with COVID-19 or influenza virus, and can also provide a method for testing or assessing the risk of COVID-19 or influenza virus becoming severe, and a kit for use therein, which allows patients infected with COVID-19 or influenza virus to collect samples themselves. [Brief explanation of the drawings]

[0028] [Figure 1] Graph showing the relationship between plasma midkine levels and mv [Figure 2] Graph showing the relationship between urinary midkine levels and mv [Figure 3] Graph showing plasma midkine levels and analysis results for each item [Figure 4] Graph showing urinary midkine levels and analysis results for each item [Figure 5]Graph showing the change in midkine value according to the number of days elapsed after infection [Figure 6] Graph for determining the midkine level criterion for disease progression based on the midkine value in urine on the 7th day after hospitalization for moderate cases [Figure 7] Graph showing the relationship between the midkine value on the 7th day and the number of days of oxygen administration [Figure 8] Figure explaining the concept of classification of the "hospital" group and the "better" group [Figure 9] Graph comparing the expression levels of midkine genes on the first day of hospitalization [Figure 10] Graph comparing inflammatory cytokines in each group on the first day of hospitalization [Figure 11] Graph showing the change in midkine value for each number of days elapsed after infection [Figure 12] Graph for determining the judgment value for predicting disease progression based on the expression level of midkine gene in blood on the first day of hospitalization [Figure 13] Graph for determining the judgment value for predicting disease progression based on the expression level of midkine gene in blood 3 days after infection

Mode for Carrying Out the Invention

[0029] A method for testing or evaluating the risk of severe COVID-19 using midkine, which is a causative substance of NETosis according to the present invention, as a marker, and a kit used therefor will be described as follows.

[0030] <Example 1> 1. Materials Used in the Experiment (1) Specimens 71 plasma specimens and 25 urine specimens from COVID-19 moderate cases (hereinafter simply referred to as "patients") who were being treated at Nagoya University Hospital and its 16 affiliated facilities were used. (2) Kits The "IP test 'Midkine'" (Immuno-Probe Co., Ltd.), an ELISA kit for quantifying the midkine concentration in human blood, was used.

[0031] 2. Experimental Method (1) Preparation of cleaning solution A 10-fold diluted washing solution was prepared by adding 50 ml of 10x washing solution to 450 ml of purified water. (2) Preparation of standards 1 ml of sample diluent was added to 8 ng of midkine standard to prepare a solution of 8000 pg / ml, and 300 μl of this solution was diluted (2-fold) with 300 μl of sample diluent to prepare a concentration of 4000 pg / ml. Furthermore, 300 μl of sample dilution solution was added to the sample to prepare solutions with concentrations of 2000, 1000, 500, 250, and 125 pg / ml. A solution with a concentration of 0 pg / ml was prepared using only 300 μl of the sample diluent. Using the above procedure, standards with concentrations of 4000, 2000, 1000, 500, 250, 125, and 0 pg / ml were prepared. The standard prepared at each concentration was transferred from the 1.5 ml tube to an open tube, 220 μl per concentration. (3) Sample preparation In a safety cabinet, 120 μl of each of the 71 plasma samples and 25 urine samples was placed in an open tube, and diluted (2-fold) with 120 μl of sample dilution solution to prepare the samples. Of the 71 plasma samples, samples with high values that did not fall on the midkine calibration curve were diluted 4 to 10 times with sample dilution solution to prepare samples. (4) Preparation of standard antibodies 120 μl of the labeled antibody was added to 12 ml of the sample dilution solution to dilute (100-fold), and the mixture was kept in the dark until use.

[0032] 3. Measurement (1) 100 μl of the prepared standard was added to a 96-well plate. (2) 100 μl of each of the prepared samples was added to a 96-well plate. (3) Both (1) and (2) were allowed to stand at room temperature for 1 hour. (4) Both (1) and (2) were washed three times with 300 μl / well of washing solution. (5) The prepared standard antibodies were added to each well of (1) and (2) at 100 μl each. (6) Both (1) and (2) were left standing at room temperature for 1 hour. (7) Both (1) and (2) were washed 5 times with 300 μl / well of the washing solution. (8) The chromogenic solution was added to each well of (1) and (2) at 100 μl each. (9) Both (1) and (2) were shielded from light at room temperature and left standing for 20 minutes. (10) The reaction stop solution was added to each well of (1) and (2) at 100 μl each. (11) Measurements were taken using a plate reader at a main wavelength of around 450 nm and a sub-wavelength of around 650 nm.

[0033] 4. Analysis (1) Items used in the analysis Among the clinical data provided by the Nagoya University Hospital, the items used in the analysis (hereinafter referred to as "each item") are as follows. age: Age death: Whether dead male: Gender covid: (a) Normal oxygen administration (b) More advanced respiratory assistance therapy (c) Invasive mechanical ventilation management (d) Those who underwent ECMO management mv: Whether invasive mechanical ventilation management was performed oxy_data: Number of days of oxygen administration Based on the patient's treatment method, the severity was quantified as "covid", where "(a) Normal oxygen administration" represents mild symptoms and "(d) Those who underwent ECMO management" represents severe symptoms. Also, "mv" represents whether "(c) Invasive mechanical ventilation management" was performed, and this is the treatment method for determining whether a patient with moderate symptoms is mild or severe. (2) Analysis method Based on each item of the clinical data and the quantified mid-cytokine values, calculations of the correlation coefficient, display of box-and-whisker plots, tests by the Mann-Whitney U test, and principal component analysis were performed using Python.

[0034] 5. Results (1) Correlation Based on each item of clinical data and the quantified midkine value, the correlation coefficient was calculated and shown in Tables 1 to 3 below. Table 1 shows the correlation coefficients between the midkine values in plasma and each item, Table 2 shows the correlation coefficients between the midkine values in serum and each item, and Table 3 shows the correlation coefficients between the midkine values in urine and each item. No correlation was found between the midkine values in plasma and each item. Correlation was found between the midkine values in urine and "age", "covid", and "mv". Since it was considered that serum would show the same results as plasma, no further analysis was performed after that.

[0035] [Table 1]

[0036] [Table 2]

[0037] [Table 3]

[0038] (2) Box plot The relationship between the quantified midkine value and the item "mv" indicating whether "(b) Invasive mechanical ventilation management" was performed is shown in FIGS. 1 and 2. (3) Mann-Whitney U test Using the quantification of severity based on the patient's treatment method, "(a) Normal oxygen administration" and "(b) More advanced respiratory support therapy" were classified as "mild", and "(c) Invasive mechanical ventilation management" and "(d) Those who underwent ECMO management" were classified as "severe", respectively. The Mann-Whitney U test was performed on these two groups of "mild" and "severe". The null hypothesis was "There is no difference in the population of mid - cytokine values between mild and severe patients", and the p - value was calculated. In the case of mid - cytokine values in plasma, the p - value was 0.0945, which is not less than 0.05. Thus, the result was that no significant difference was found. However, for mid - cytokine values in urine, the p - value was 0.0346, which is less than 0.05. Thus, the result was that a significant difference was found. (4) Principal component analysis The results of principal component analysis performed on the quantified mid - cytokine values and some items of clinical data are shown in Figures 3 and 4. The PC1 on the horizontal axis of the figure was evaluated as "a factor related to severity", and the PC2 on the vertical axis of the figure was evaluated as "a factor related to life and death". The contribution rate of mid - cytokine values in plasma was 35.3% for PC1 and 24.2% for PC2. The contribution rate of mid - cytokine values in urine was 42.0% for PC1 and 24.6% for PC2. (5) Relationship between mid - cytokine and the number of days of oxygen administration Figure 5 shows a graph of the change in mid - cytokine values according to the number of days elapsed after COVID - 19 infection. The reason why "the number of days after infection" varies depending on the sample is that there is a difference in the collection date among patients.

[0039] 6. Discussion From the relationship between the result of calculating the correlation coefficient (the absolute value of the correlation coefficient) and the following "interpretation", no correlation was found between the mid - cytokine values in plasma and the severity indicators "covid" and "mv", but a correlation was found for mid - cytokine values in urine. <Absolute value of correlation coefficient> <Interpretation> 0.0 - 0.2 Almost no correlation 0.2 - 0.4 Slightly correlated 0.4 - 0.7 Considerably correlated 0.7 - 1.0 Strongly correlated (2) From the box-and-whisker plots of FIGS. 1 and 2, the midkine values of those who performed "mv" tended to be higher than those who did not perform "mv", and the urinary midkine values showed more significant results. (3) From the results of the Mann-Whitney U test, a significant difference was observed in the urinary midkine values. (4) From the results of the principal component analysis of FIGS. 3 and 4, in PC1, which is a factor related to severity, the midkine values had a larger negative value compared to other items, indicating a high possibility of involvement in disease progression. (5) From these results, it is considered that midkine in body fluids is involved in the progression of moderate COVID-19 patients to severe cases. Also, it was suggested that urinary midkine values are more useful than plasma midkine values as an indicator of disease progression. (6) From the results showing the change in midkine values according to the number of days after COVID-19 infection in FIG. 5, on the 3rd day after infection, the midkine values increased in most patients, while on the 7th day, there were patients with increasing midkine values and patients with decreasing midkine values. Also, on the 14th day, the midkine values of most patients had decreased. As shown in FIG. 6, when discriminating the midkine values on the 7th day with "oxy_>=15d-MK at day7", 62.5% of the moderate patients who progressed to severe cases were included at the mean + 2SD (5.83). Therefore, when the urinary midkine value collected from the subjects on the 7th day after COVID-19 infection exceeds 5.83 ng / ml, it can be evaluated that the risk of progression to severe COVID-19 is high. Therefore, it was found that midkine can be a marker for evaluating the risk of progression to severe COVID-19. (7) From this, it was considered that the midkine value on the 7th day might affect the future condition of the patient, and the correlation with the "number of days of oxygen administration" was examined. The "number of days of oxygen administration" is the number of days of oxygen administration treatment, and it was judged to have recovered when the oxygen administration ended. In other words, a long "number of days of oxygen administration" is considered to be synonymous with worsening of the condition, and the relationship between the midkine level on day 7 and the number of days of oxygen administration is shown in Figure 7. The correlation coefficient was calculated to be 0.461, confirming the correlation. (8) Based on the above results, the midkine level in urine collected from subjects can be used as an indicator to evaluate the risk of severe illness after COVID-19 infection. In particular, the midkine level on the 7th day after infection can be an important indicator to evaluate the risk of severe illness after COVID-19 infection. Therefore, quantifying the midkine level in urine collected from subjects is an effective method for testing the risk of severe illness after COVID-19. (9) Furthermore, in many cases in which urinary midkine levels were high on the 7th day after infection, the levels were already high on the 3rd day after infection (Figure 5). Therefore, in order to determine the risk of COVID-19 becoming severe at an early stage, it is desirable to test the risk of COVID-19 becoming severe by quantifying midkine levels in urine collected from subjects early, such as on the third day after infection.

[0040] <Example 2> 1. Data used for analysis Bulk RNA-Seq (GSE212041, LaSalle TJ, et al. Cell Rep Med. 2022. PMID: 36208629) was used. We used RNA-Seq data from neutrophils from 304 COVID-19-infected patients who were tested and found to be infected with COVID-19 among patients who visited the Emergency Department of Massachusetts General Hospital complaining of COVID-19 symptoms, and 76 non-COVID-19 patients who were tested and found not to be infected with COVID-19 (hereinafter, COVID-19-infected patients and non-COVID-19 patients are collectively referred to as "patients"), as well as eight hospitalized non-COVID-19 patients (controls) who were not infected with COVID-19 and were hospitalized at the same hospital. For the following analysis, only data from 380 patients were used; data from 8 hospitalized non-infected individuals (controls) was not used. 2.Analysis results The expression levels of the midkine gene on the first day of hospitalization (Day 0) were compared between the "hospital" group (patients whose condition worsened or continued hospitalization on Days 3 and 7 of hospitalization) and the "better" group (patients whose condition improved on Day 3 of hospitalization), as shown in Figure 8. As a result, as shown in FIG. 9, the expression level of the midkine gene was significantly lower in the "better" group than in the "hospital" group. Furthermore, when the gene expression levels of other pro-inflammatory cytokines in the "better" and "hospital" groups were analyzed, the difference in the expression level of the midkine gene was the most significant, as shown in Figure 10.

[0041] Example 3 1. Purpose of the experiment Aging is known to alter the host response to influenza virus infection. To confirm whether midkine could be a marker for assessing the risk of severe illness due to influenza virus, we used bulk RNA sequencing (bulk RNA-seq) to examine cellular changes in the lungs of young (16-week-old) and aged (80-week-old) mice infected with influenza virus using RNA-seq data. 2. Experimental Procedure Mice were infected with influenza A / PR8 / 34 (approximately 50 PFU / mouse) to establish acute infection. Infection was performed by intranasal (in) administration under anesthesia. Mice were euthanized after infection, and the right ventricle was perfused with 10 mL of cold DPBS (Corning). For bulk RNA-seq, a portion of the right lung was harvested after ventricular perfusion and flash-frozen on dry ice. RNA was extracted using the RNAEasy Mini Kit (Qiagen). Reverse transcription was performed, and the resulting cDNA was enzymatically fragmented and indexed using the Nextera XT DNA Library Preparation Kit (Illumina). The barcoded cDNA library was sequenced on an Illumina NextSeq 500 instrument using the NextSeq 500 / 550 High Output Kit v2 (75 cycles) (20024906, Illumina), using the following cycle numbers: 100 bp, ... 37 (Lead 1) 8 (index 1) 8 (index 2) 37 (Lead 2) Demultiplexing was performed using bcl2fastq (v2.20, Illumina). Alignment was performed using Salmon (v1.4.0) ( Patro et al., 2017 ) and the GRCm39 reference genome (Ensembl) with default settings. 3.Analysis Analyses were performed using R with DESeq2 (v1.28.1) ( Love et al., 2014 ). The Mdk values (TPM corrected) were compared for 16-week-old and 80-week-old mice on the first day of infection (Day 0), 3 days after infection (Day 3), and 9 days after infection (Day 9). As shown in Figure 11, on Day 0, no significant difference was observed between 16-week-old and 80-week-old mice, but on Day 3, the Mdk level was significantly increased compared to Day 0 and Day 9. As shown in Figure 12, the average midkine level in the blood of healthy individuals is 0.15 ng / ml, and 0.15 x 7.39 = 1.11. Therefore, if the midkine level in the blood collected from a subject 1 to 3 days after infection with COVID-19 exceeds 1.11 ng / ml, the risk of developing severe COVID-19 can be assessed as high. As shown in Figure 13, a blood midkine gene expression level corrected for TPM on day 3 after influenza infection exceeding 5.78 was considered to be influenza positive (worsening). Furthermore, assuming that 1 unit of midkine gene expression correlates with 1 unit of midkine protein expression, and the cutoff value for blood midkine levels in healthy individuals is 0.50 ng / ml, a blood midkine level 5.78 times higher than this, or exceeding 2.89 ng / ml on days 3 to 5 after influenza virus infection, can be assessed as indicating a high risk of severe illness due to influenza virus. Therefore, midkine was found to be a potential marker for assessing the risk of severe illness caused by influenza virus.

[0042] Example 4 Example of a test kit using midkine as a marker to assess the risk of severe COVID-19 and influenza virus infection The test kit can be a variety of test, analytical, and diagnostic kits containing a substance for quantifying midkine levels.

[0043] (1) For example, a test tube with fins (e.g., a tube with four fins, similar to those on the rear end of a surfboard, attached to the tip of a rod extending from the cap of the tube) can be used, to which an antibody for quantifying midkine levels has been applied in advance. The sample and buffer are placed in this test tube and allowed to react for about 30 minutes, after which the sample and buffer are discarded. Next, a secondary antibody (for example, an antibody obtained by immunizing a chicken with human midkine and labeling the resulting antibody with peroxidase (POD)) is appropriately diluted with buffer and added to the test tube. If the sample contains midkine, the fin, which has been pre-coated with antibodies, will react with the adsorbed midkine through an antigen-antibody reaction, and color will develop due to the action of the color-developing reagent that is added afterwards. By measuring the color tone of this developed color, it is possible to determine whether the test is normal, false positive, or positive.

[0044] (2) Other test kits, such as Sandwich ELISA, can also be used. An antibody for quantifying midkine levels is immobilized on a microplate, which can be used by reacting a test sample, a primary antibody, and a secondary antibody (and optionally a detection substrate). In addition to microplates, IC strips, filters for urine pretreatment, and fluorescently labeled secondary antibodies can also be used.

[0045] (3) The results of these test kits can be judged, for example, by comparing them with a color chart that shows pre-set colors for normal, false positive, and positive results. By using still images or videos taken using the camera function of a mobile phone such as a smartphone, a third party in a remote location can compare the color with this color chart, or the color can be determined automatically using an app on the mobile phone such as the smartphone that took the photo. A specific method for making a judgment using still images and videos is, for example, to take a picture of a white piece of paper (white No. 0) with a camera and use this as a reference.The difference in color tone between this and the next sample photographed is converted into a numerical value, and the size of the numerical value is used to determine whether the result is normal, a false positive, or a positive result. Alternatively, the reacted reaction solution can be directly quantified using a cuvette or an equivalent device, using a measuring instrument such as an absorbance meter or a plate reader.

Claims

1. comprising a step of quantifying the midkine value in urine or blood collected from a subject, wherein the result of the quantification is used for testing the risk of severe illness due to COVID-19, A method for testing the risk of severe illness due to COVID-19, characterized by the above.

2. The midkine value is the midkine value in urine collected from a subject on the 7th day after infection, The testing method according to claim 1, characterized by the above.

3. The midkine value is the midkine value in blood collected from a subject on the 1st to 3rd day after infection, The testing method according to claim 1, characterized by the above.

4. Evaluating the risk of severe illness due to COVID-19 based on the midkine value in urine or blood collected from a subject, An evaluation method for the risk of severe illness due to COVID-19, characterized by the above.

5. When the evaluation is such that the midkine value in urine collected from a subject on the 7th day after infection with COVID-19 is more than 5.83 ng / ml, evaluating that the risk of severe illness due to COVID-19 is high, The evaluation method according to claim 4, characterized by the above.

6. When the evaluation is such that the midkine value in blood collected from a subject on the 1st to 3rd day after infection with COVID-19 is more than 1.11 ng / ml, evaluating that the risk of severe illness due to COVID-19 is high, The evaluation method according to claim 4, characterized by the above.

7. A marker for quantification for testing or evaluating the risk of severe illness due to COVID-19, wherein the marker is the midkine value in urine or blood collected from a subject, A testing or evaluation marker for the risk of severe illness due to COVID-19, characterized by the above.

8. A kit for use in testing or evaluating the risk of severe illness due to COVID-19, comprising a substance for quantifying the midkine value, A kit characterized by the above.

9. A kit for use in testing or evaluating the risk of severe illness due to COVID-19, having a reactant pre-coated with an antibody for quantifying the midkine value, A kit characterized by the above.

10. The midkine value is the midkine value in urine collected from a subject on the 7th day after infection, The kit according to claim 8 or 9, characterized by the above.

11. The midkine value is the midkine value in blood collected from a subject on the 1st to 3rd day after infection, The kit according to claim 8 or 9, characterized by the above.

12. ​ A process for quantifying the midkine value in blood collected from a subject, wherein the result of the quantification is used for testing the risk of severe influenza virus infection, characterized by a method for testing the risk of severe influenza virus infection.

13. The midkine value is the midkine value in blood collected from a subject on the 3rd to 5th day after infection, characterized by the testing method according to claim 12.

14. Evaluating the risk of severe influenza virus infection based on the midkine value in blood collected from a subject, characterized by a method for evaluating the risk of severe influenza virus infection.

15. When the evaluation shows that the midkine value in blood collected from a subject on the 3rd to 5th day after infection with influenza virus exceeds 2.89 ng / ml, it is evaluated that the risk of severe influenza virus infection is high, characterized by the evaluation method according to claim 14.

16. A marker for quantification for testing or evaluating the risk of severe influenza virus infection, wherein the marker is the midkine value in blood collected from a subject, characterized by a marker for testing or evaluating the risk of severe influenza virus infection.

17. A kit for testing or evaluating the risk of severe influenza virus infection, characterized by containing a substance for quantifying the midkine value.

18. A kit for testing or evaluating the risk of severe influenza virus infection, characterized by having a reactant pre-coated with an antibody for quantifying the midkine value.

19. The midkine value is the midkine value in blood collected from a subject on the 3rd to 5th day after infection, characterized by the kit according to claim 17 or 18. ​ ​ ​

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