Method for assessing the risk of viral pneumonia using soluble CLEC2

Abstract

To provide a simple, objective blood test-based risk assessment method which enables early prediction of aggravation of viral pneumonia, in particular COVID-19, during medical examination.SOLUTION: A risk assessment method disclosed herein comprises a step of measuring the level of soluble CLEC2 concentration in blood collected from a patient.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for risk assessment of viral pneumonia using soluble CLEC2.

Background Art

[0002] Pneumonia is one of the most common causes of death worldwide and is a major cause of death among children in developing countries and the elderly in developed countries. In Japan, the number of pneumonia deaths in 2016 reached 119,300, ranking third among the causes of death after malignant neoplasms and heart diseases. In the United States, it is estimated that 2 to 3 million people develop pneumonia every year and 60,000 people die, making it the eighth leading cause of death in the United States.

[0003] Pneumonia is caused by various microorganisms such as bacteria, viruses, mycobacteria, fungi, and parasites. However, pneumonia caused by bacteria or viruses is much more common than pneumonia caused by mycobacteria, fungi, or parasites, and specifically, the pathogen that causes it varies. Viral community-acquired pneumonia occurs approximately 200 million cases per year (100 million cases in children and 100 million cases in adults), and one-third of community-acquired pneumonia in adults is thought to be caused by viruses.

[0004] Viruses known to cause pneumonia include influenza virus, varicella-zoster virus, measles virus, respiratory syncytial virus, adenovirus, human metapneumovirus, cytomegalovirus, coronavirus (types 229E, OC43, NL63, HKU1, SARS, MERS, SARS-CoV-2), enterovirus, rhinovirus, human bocavirus, parainfluenza virus, hantavirus, parechovirus, Epstein-Barr virus, human herpesvirus, mimivirus, etc.

[0005] As of the end of May 2021, the SARS-CoV-2 infection (COVID-19) has become a very great threat to humanity, with more than 140 million people infected and more than 3.2 million people dead worldwide, and it has also had an extremely large impact on the world economy.

[0006] COVID-19 presents a very diverse clinical spectrum ranging from asymptomatic to severe cases leading to death. It is said that about 30% of cases are asymptomatic, and among the symptomatic patients, 80% are mild or moderate cases I who recover within about a week. Additionally, about 15% are moderate cases II that require oxygen inhalation for about a week, and it is said that approximately 5% are severe cases that need intensive care after the 10th day.

[0007] As risk factors for disease progression, in addition to advanced age, having underlying diseases such as diabetes, hypertension, chronic kidney disease, cerebrovascular disorders, chronic lung diseases, heart diseases, and cancer have been reported. Currently, new variants of SARS-CoV-2 have emerged, and it has become said that even young people without such risk factors are at risk of disease progression.

[0008] To assess the risk of disease progression, in addition to vital signs and blood oxygen saturation, lung imaging examinations such as CT scans and various blood tests have been proposed. As factors contributing to the progression of the overall condition, high-grade inflammation, thrombosis, and organ failure are considered major elements. As markers of inflammation, CRP, interferon γ, interleukin 6, interleukin 8, TNFα, soluble interleukin 2 receptor are mentioned, and as markers of enhanced coagulation, D-dimer, FDP are mentioned. Also, as markers of organ failure, ferritin, lactate dehydrogenase (LDH), etc. have been proposed. However, since many of these blood biomarkers increase in cases of disease progression, although it is possible to confirm disease progression, many of them cannot be used for predicting disease progression. Since rapid disease progression occurs in COVID-19, there is a need for biomarkers that can predict disease progression from the mild stage in advance.

[0009] Viral pneumonia can not only cause pneumonia, but also thrombosis and thromboembolism, which requires attention. For example, it has been reported that among acute cytomegalovirus infections, thrombosis was complicated in 6.4% of cases. Thrombosis may sometimes have few symptoms, and there are many cases where thrombosis has not been detected in infectious disease patients. It is considered that in fact, more cases of complication are seen. In addition, there are reports of thrombosis caused by herpes simplex virus type 1, type 2 and varicella-zoster virus, which belong to the same herpes virus genus (Non-Patent Document 5).

[0010] In addition, in COVID-19 caused by SARS-CoV2, another virus genus, a severe thrombosis is developed due to abnormal activation of the coagulation system, and as a result, a mechanism is known in which acute respiratory distress syndrome and other organ failures occur and the condition deteriorates. In COVID-19, due to platelet activation and coagulation system activation, pathological conditions such as Pulmonary Intravascular Coagulation (PIC) and Thrombotic Microagiopathy (TMA) in which small thromboses are formed in the lungs are observed, and all types of thrombosis such as arterial thrombosis like myocardial infarction and cerebral infarction, and venous thrombosis like deep vein thrombosis and pulmonary thromboembolism have been reported. That is, if abnormal thrombus formation can be predicted in advance in COVID-19 patients, it is expected that the severity rate and mortality can be reduced by taking corresponding measures.

[0011] In normal thrombus formation when a blood vessel ruptures, first platelets are activated and aggregate to form a primary thrombus, and then the coagulation system is activated there, and water-soluble fibrinogen becomes an insoluble fibrin network to cover and form a strong secondary thrombus. In actual hemostatic thrombi, the platelet system and the coagulation system activate each other, and a hemostatic thrombus formed by platelets, fibrin, and red blood cells and the like trapped therein is observed. Even in pathological thrombi, the thrombus is formed by both the platelet system and the coagulation system. Arterial thrombi are mainly composed of platelets, and antiplatelet drugs are used for treatment. Venous thrombi are mainly composed of the coagulation system (fibrin system), and anticoagulants are used for treatment. Known antiplatelet drugs include oral drugs such as aspirin and clopidogrel and intravenous drugs such as oxalrel. Known anticoagulants include oral drugs such as warfarin and DOAC and intravenous drugs such as heparin and argatroban. For the prevention of thrombosis in COVID-19, in Japan, it is recommended to use heparin for patients with moderate cases II or above (Non-Patent Document 3), and antiplatelet drugs are not recommended. However, according to Non-Patent Document 4, it has been reported that COVID-19 patients who were taking low-dose aspirin (an antiplatelet drug) for reasons other than COVID-19 had significantly fewer deaths than non-taking COVID-19 patients. That is, it can be understood that when a person who was taking an antiplatelet drug for secondary prevention of thrombosis in the heart or brain, etc. regardless of COVID-19 and whose platelet activation was suppressed was infected with SARS-CoV2, they were probably less likely to develop thrombosis, and as a result, the mortality rate decreased.

[0012] C-type lectin-like receptor 2 (CLEC2) has been identified on platelets as the receptor for the platelet-activating snake venom rhodocytin. CLEC2 is expressed almost specifically in platelets and megakaryocytes in humans and can be said to be a molecule specific to platelets. It has been reported that this CLEC2 is released into the blood as soluble CLEC2 (hereinafter abbreviated as sCLEC2) when platelets are activated (Patent Document 1, Non-Patent Document 1). Since the blood sCLEC2 concentration increases with platelet activation, it is suggested that the blood sCLEC2 concentration may be related to the formation of thrombosis, and it is also described that it can be used for the diagnosis of cerebral infarction and myocardial infarction (Patent Document 1). In addition, sCLEC2 is known to have a high value in diseases with pathological thrombus formation such as disseminated intravascular coagulation syndrome (DIC), thrombotic microangiopathy (TMA) (Non-Patent Document 2), and deep vein thrombosis, etc. (Patent Document 2; unpublished). That is to say, a high blood sCLEC2 value can suggest that a thrombus has been formed or is being formed somewhere in the body. Also, while the degradation products of formed thrombi (fibrin) such as D-dimer and FDP increase after the thrombus has formed, sCLEC2, which is a platelet activation marker, increases reflecting platelet activation before the thrombus is formed, so it can be expected to be used as a predictor of thrombus formation, that is, a predictor of disease severity.

[0013] Severe cases similar to COVID-19 are also known in pneumonia caused by viruses other than SARS-CoV2, and similar mechanisms are assumed in the progression to severe disease in these viral pneumonias.

Prior Art Documents

Patent Documents

[0014]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0015]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Problems to be Solved by the Invention

[0016] COVID-19 is a serious disease with a high mortality rate, and even if patients survive, they often experience severe complications and sequelae. It is extremely important to predict patients who will deteriorate early and provide preventive treatment. If the risk of deterioration can be diagnosed at the stage of mild or moderate type I, anticoagulant therapies such as heparin, warfarin, and DOAC, and antiplatelet therapies such as aspirin and clopidogrel can be used to reduce the probability of deterioration and also reduce the mortality rate.

[0017] Attempts have been made to predict early deterioration, and various markers such as D-dimer, interleukin-6, interferon-γ, interferon-λ3, etc. in the blood have been used. However, since many markers increase after deterioration, they are only useful for confirming deterioration and cannot be considered predictive markers. Also, although there are some markers that increase shortly before deterioration, in COVID-19, deterioration often occurs rapidly, so in practice, they are often not in time for prediction. In addition, scores such as COVID-GRAM, 4C Mortality Score, and A-DROP have been proposed as means to predict early deterioration in patients. However, all of these require information not only from blood test findings but also from patient background, presence or absence of underlying diseases, and vital signs. Therefore, they are not simple to predict and the evaluation of whether they can be applied to COVID-19 patients worldwide has not been sufficient.

[0018] Accordingly, an object of the present invention is to provide a simple and objective risk assessment method by blood test that enables early prediction of deterioration in the diagnosis and treatment of viral pneumonia (especially COVID-19). [Means for Solving the Problems]

[0019] The present inventors conducted intensive studies to solve the above problems. As a result, it was found that the soluble CLEC2 concentration in the blood of COVID-19 patients was already significantly increased at the mild stage compared to healthy subjects. Furthermore, it was found that the concentration increased as the disease progressed, leading to the completion of the present invention.

[0020] That is, the present invention provides the following: [1] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, the method comprising the step of measuring the concentration of soluble CLEC2 present in the blood collected from the patient. [2] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, (1) The step of providing a blood sample derived from the patient, (2) Determining the soluble CLEC2 concentration in the sample; (3) Correlating the soluble CLEC2 concentration with the severity of viral pneumonia in the patient; The method according to [1], comprising the above steps. [3] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, wherein the step of correlating the soluble CLEC2 concentration with the severity of viral pneumonia in the patient includes evaluating based on changes in the soluble CLEC2 concentration. The method according to [2]. [4] The method according to any one of [1] to [3], wherein the risk assessment is a method for assisting in predicting the exacerbation of viral pneumonia. [5] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, wherein in the step of correlating the soluble CLEC2 concentration with the severity of viral pneumonia, the cut-off value of the soluble sCLEC2 concentration is 382 - 605 pg / mL. The method according to any of [2] to [4]. [6] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, wherein in the step of correlating the soluble CLEC2 concentration with the severity of viral pneumonia, the severity is classified as follows: mild when the oxygen saturation is 96% or more and there are no respiratory symptoms or only cough without dyspnea symptoms and no pneumonia findings in any clinical state; moderate I when the oxygen saturation is 93% - 96% and there is no respiratory failure, dyspnea, or pneumonia findings are observed in the clinical state; moderate II when the oxygen saturation is 93% or less and there is respiratory failure and oxygen administration is required in the clinical state; severe when the clinical state requires admission to the intensive care unit or mechanical ventilation. The method according to any of [2] to [5]. [7] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, comprising: (1) Providing a blood sample derived from the patient; (2) Determining the soluble CLEC2 concentration in the sample; (3) Measuring the number of platelets in the sample; (4) Dividing the soluble CLEC2 concentration by the number of platelets; (5) Correlating the value obtained by dividing the soluble CLEC2 concentration by the number of platelets with the presence or absence of viral pneumonia and the likelihood of outcome in the patient; A method according to any one of [1] to [6], comprising the above steps. [8] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, wherein the step of correlating the value obtained by dividing the soluble CLEC2 concentration by the number of platelets with the severity of viral pneumonia in the patient is evaluated based on a change in the value obtained by dividing the soluble CLEC2 concentration by the number of platelets. The method of [7]. [9] The method according to [7] or [8], wherein the risk assessment is a method for assisting in predicting the aggravation of viral pneumonia.

[10] A method for risk assessment of a patient suspected of having viral pneumonia or diagnosed with viral pneumonia, wherein in the step of correlating the value obtained by dividing the soluble CLEC2 concentration by the number of platelets with the severity of viral pneumonia, the severity is defined as follows: mild when the oxygen saturation is 96% or more and there are no respiratory symptoms or only cough without dyspnea symptoms and no pneumonia findings in any clinical state; moderate I when the oxygen saturation is 93% - 96% and there is no respiratory failure, dyspnea, or pneumonia findings are observed in the clinical state; moderate II when the oxygen saturation is 93% or less and there is respiratory failure in the clinical state and oxygen administration is required; severe when the clinical state requires admission to the intensive care unit or mechanical ventilation. The method according to any one of [7] to [9].

[11] The method according to any one of [1] to

[10] , wherein the step of determining the soluble CLEC2 concentration is a highly sensitive immunoassay method, such as chemiluminescence immunoassay, electrochemiluminescence immunoassay, or fluorescence immunoassay.

Advantages of the Invention

[0021] By measuring the concentration of sCLEC2 present in the blood of patients with viral pneumonia (especially COVID-19), which is the method of the present invention, it becomes possible to easily predict the severity of patients with viral pneumonia (especially COVID-19). Also, in the monitoring of preventive treatment for patients with viral pneumonia (especially COVID-19), determination of the treatment effect is expected.

Brief Description of the Drawings

[0022]

Figure 1

Figure 2

Figure 3

Modes for Carrying Out the Invention

[0023] Hereinafter, as an embodiment of the method for performing risk assessment of viral pneumonia patients, the embodiment of the present invention will be described in detail using, as an example, the method for assessing the risk of severity of viral pneumonia patients caused by the coronavirus, but the embodiments of the usage method are not limited thereto. For example, the present invention includes A method for performing risk assessment of viral pneumonia by measuring (or determining) the concentration of soluble CLEC2 in a sample (or the value obtained by dividing the concentration of soluble CLEC2 by the platelet count); A method for assisting in the risk assessment of viral pneumonia by measuring (or determining) the concentration of soluble CLEC2 in a sample (or the value obtained by dividing the concentration of soluble CLEC2 by the platelet count); A method for measuring (or determining) the concentration of soluble CLEC2 in a sample (or the value obtained by dividing the concentration of soluble CLEC2 by the platelet count) for the risk assessment of viral pneumonia; A method for in vitro risk assessment of viral pneumonia, characterized by measuring (or determining) the soluble CLEC2 concentration in a sample (or the value obtained by dividing the soluble CLEC2 concentration by the platelet count); Use of an antibody capable of detecting the soluble CLEC2 concentration in the manufacture of a kit for risk assessment of viral pneumonia; A method for measuring (or determining) the soluble CLEC2 concentration in a sample (or the value obtained by dividing the soluble CLEC2 concentration by the platelet count) to provide information necessary for risk assessment of viral pneumonia; is included.

[0024] The disease targeted in this specification is viral pneumonia, but the causative viruses of viral pneumonia can include viruses that infect humans and cause pneumonia. For example, influenza virus, varicella-zoster virus, measles virus, RSV, adenovirus, human metapneumovirus, cytomegalovirus, coronavirus (types 229E, OC43, NL63, HKU1, SARS, MERS, SARS-CoV-2), enterovirus, rhinovirus, human bocavirus, parainfluenza virus, hantavirus, parechovirus, Epstein-Barr virus, human herpesvirus, mimivirus, etc., but are not limited thereto. These virus species also include mutant strains having mutations in the nucleic acid encoding the protein constituting the virus. Mutant strains are not distinguished by the number of mutations, sequence homology, or the function of the protein derived from the nucleic acid, and in this specification, mutant strains are understood as the same virus. The same virus can be considered to include all those classified as the same species of virus according to the classification system defined by the International Committee on Taxonomy of Viruses. In this specification, a COVID-19 patient means a patient confirmed to be infected with SARS-CoV-2 by the PCR (Polymerase Chain Reaction) method. Onset refers to the appearance of any symptoms, even if it is slight, such as fever, chills, malaise, dry cough, loss of appetite, muscle pain, diarrhea, sputum, olfactory disorder, taste disorder, etc.

[0025] In this specification, deterioration refers to a state where symptoms worsen, and it can be judged based on indicators such as an increase in the rate of breathing and pulse, and a blurred consciousness. The classification of mild, moderate, and severe cases of COVID-19 patients can be appropriately judged by those skilled in the art according to general classifications based on vital signs such as the patient's clinical condition and oxygen saturation. For example, it is possible to make a judgment with reference to the guidelines for the diagnosis and treatment of novel coronavirus issued by the Ministry of Health, Labour and Welfare. As a rough guideline, when the oxygen saturation is 96% or higher, and there are no respiratory symptoms or only coughing without dyspnea in the clinical condition, and no findings of pneumonia are recognized in any clinical condition, it is classified as a mild case. Moderate cases, which are more severe than mild cases, are divided into stage I and stage II. Moderate case I is defined as those without respiratory failure, with an oxygen saturation of 93% - 96% (excluding 93% and 96%), and corresponding to cases where dyspnea and findings of pneumonia are recognized as clinical symptoms. Moderate case II is defined as those with respiratory failure, with an oxygen saturation of 93% or lower, and corresponding to cases where oxygen administration is required as a clinical symptom. Severe cases, which are considered to be more severe than moderate case II, refer to a clinical condition where admission to the intensive care unit (ICU) is required or a ventilator is required. Even for patients classified as mild cases, since the possibility of symptom aggravation and the emergence of new symptoms cannot be denied, careful attention must be paid to the care of COVID-19 patients.

[0026] For grasping the degree of progression of factors related to deterioration and poor prognosis and lung lesions, a decrease in the number of lymphocytes, CRP, ferritin, D-dimer, LDH, KL-6, etc. may be used. Furthermore, when the symptoms further deteriorate and are classified as severe, it is the case when admission to the intensive care unit is required or a ventilator is required. In addition, in the case of children, most cases are asymptomatic or only have mild symptoms, but the risk of developing a condition called multisystem inflammatory syndrome in children (MIS-C) has been reported. This is said to be characterized by persistent fever, malaise, sore throat, headache, abdominal pain, and vomiting, with abnormalities occurring in multiple organs (heart, gastrointestinal tract, kidney, blood, skin, nerves), progressing rapidly to shock and organ failure. Therefore, the criteria for MIS-C published by the CDC may be used. In that case, it is determined based on fever, clinical laboratory findings suggesting inflammation (CRP, erythrocyte sedimentation rate, fibrinogen, D-dimer, ferritin, lactate dehydrogenase, interleukin 6, or high neutrophil / lymphocyte ratio, low albumin), as well as abnormalities in multiple (two or more) organs (heart, kidney, respiratory system, blood, gastrointestinal tract, skin, nerves), clinical symptoms requiring hospitalization, etc.

[0027] In addition, as used herein, "CLEC2" is a platelet activation receptor belonging to the C-type lectin family, which is normally present on the platelet membrane but is released into the blood upon platelet activation. As used herein, the term "soluble CLEC2 (sCLEC2)" means CLEC2 or a CLEC2-derived molecule that is released from such platelets and detected in the blood (or in the buffer when incubated in the buffer).

[0028] sCLEC2 is said to contain proteins with a molecular weight of approximately 40 kDa, proteins with a molecular weight of approximately 32 kDa, proteins with a molecular weight of approximately 25 kDa, etc. in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (Non-Patent Document 1). The proteins with a molecular weight of approximately 40 kDa and the proteins with a molecular weight of approximately 32 kDa are present on the platelet membrane surface and are presumed to be released in a state contained in microparticles produced upon platelet activation. It is considered that sugar chains are added to these. On the other hand, the protein with a molecular weight of approximately 25 kDa is considered to be cleaved by protease upon platelet activation and released from the platelets. In the present invention, the amount of sCLEC2 as described above is measured. sCLEC2 may detect the proteins with a molecular weight of approximately 40 kDa, the proteins with a molecular weight of approximately 32 kDa, and the proteins with a molecular weight of approximately 25 kDa together, or may detect only the protein with a molecular weight of approximately 25 kDa.

[0029] In the present invention, the sCLEC2 concentration used may be only the sCLEC2 concentration, or a value obtained by dividing the sCLEC2 concentration by the platelet count may be used. In this specification, unless otherwise specified, the sCLEC2 concentration is interpreted as a concept including both the case where the sCLEC2 concentration is used and the case where the sCLEC2 concentration is divided by the platelet count.

[0030] The sample used for measurement is preferably derived from humans, but samples derived from animals other than humans may be used for understanding the pathological conditions of experimental animals, etc. The experimental animals are not particularly limited, and examples include guinea pigs, rats, mice, dogs, etc.

[0031] The method for detecting the presence of sCLEC2 is not particularly limited, but an immunological method using an antibody that recognizes sCLEC2 (hereinafter sometimes referred to as "anti-sCLEC2 antibody") is preferred. As methods for immunologically detecting proteins, for example, immunoassays using labeled antibodies such as enzyme immunoassay (ELISA), chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescence immunoassay, radioimmunoassay, immunochromatography, or commonly used methods known per se such as Western blotting, latex agglutination method, immunonephelometry, etc. can be used. However, in a simple immunoassay, the measurement sensitivity may not be sufficient to measure the sCLEC2 concentration in healthy individuals, so a measurement method with sufficient sensitivity to measure blood sCLEC2 in healthy individuals should be used. For this purpose, a highly sensitive measurement method is desirable, and chemiluminescent immunoassay, electrochemiluminescent immunoassay, fluorescence immunoassay, etc. are particularly preferably used.

[0032] A sample is collected from a subject to be tested (especially a patient), for example, using a blood collection tube for plasma collection. Usually, a citrate-containing blood collection tube with few residual platelets is preferred, but those containing heparin or EDTA are also possible. An EDTA-containing blood collection tube is used for measuring the blood platelet count, but if it is a simultaneous blood collection, separate blood collection tubes may be used for each. The sCLEC2 concentration in plasma is measured, for example, using plasma centrifuged at about 2000 g for about 20 minutes, but the centrifugation conditions are not limited to this, and a measurement system using whole blood may also be used. Hereinafter, the measurement of the sCLEC2 concentration in plasma will be described as an example, but it is not limited thereto.

[0033] Regarding the correlation between the sCLEC2 concentration in the measured patient-derived sample and the possibility of COVID-19 exacerbation, a threshold value may be appropriately set and used from the comparison between the sCLEC2 concentration in the patient-derived sample and the sCLEC2 concentration in the sample from a healthy individual, or when a significant change in the sCLEC2 concentration is detected from the time-course record of the sCLEC2 concentration in the same patient, the risk of exacerbation may be evaluated.

[0034] Regarding the setting of the threshold value, if there is a statistical significant difference among mild, moderate I, moderate II, and severe COVID-19 patients in the measurement of sCLEC2, it can be used as a method to appropriately set the threshold value between them and make predictions based on the correlation with disease progression. For example, it is also possible to set a cut-off value between mild + moderate I and moderate II + severe as a criterion for disease progression. It is preferable to select any value between 382 pg / mL and 605 pg / mL as the cut-off value for disease progression. More preferably, it can be set at 490 pg / mL and used for predicting disease progression. As will be described later, since the treatment guidelines for patients classified as moderate I and moderate II are significantly different, such as recommended drug treatments, it is preferable that these predictions of disease progression can be implemented quickly and easily.

[0035] Taking a specific example of risk assessment from time-course records, for patients diagnosed with COVID-19, the blood sCLEC2 concentration can be measured over time from multiple blood samplings for the purpose of assessing the risk of disease progression, and it can be determined that the risk of disease progression is high if the value is higher compared to that of healthy individuals etc. It is also possible to use it to predict whether disease progression is occurring based on the change in the measured sCLEC2 concentration. If the value is high, it is useful for considerations in determining treatment guidelines, such as changing to different types of drugs or adding additional doses.

[0036] For example, if the value of the sCLEC2 concentration in COVID-19 patients is higher compared to healthy individuals, it can be said that there is a high possibility of disease progression in the near future or that a thrombus has already formed and the patient has become severely ill. Based on such comparisons, the sCLEC2 concentration can also be used for predicting the risk of COVID-19 disease progression.

[0037] Specifically, for COVID-19 patients, if the blood sCLEC2 concentration is measured and the value or the sCLEC2 / platelet ratio is higher than that of healthy individuals, etc., it can be judged that there is a high possibility of deterioration in the near future or that the patient is already in the process of deteriorating. Further examinations can be carried out, and based on the risk assessment, antiplatelet drugs such as aspirin and clopidogrel or anticoagulants such as heparin, warfarin, and DOAC can be prophylactically administered. Additionally, after administering antiplatelet drugs or anticoagulants, sCLEC2 concentration measurement can be performed, and if the value is high, it is also possible to consider changing to different types of drugs or adding additional doses.

[0038] Moreover, the treatment for COVID-19 patients differs depending on whether they are mild, moderate, or severe. For example, in the case of mild cases, home care is the main approach. However, when classified as moderate type I, in addition to administering antiviral drugs, symptomatic treatment for fever, respiratory symptoms, and underlying diseases is the main focus, and steroid drugs should not be used for patients who do not require oxygen administration. When classified as moderate type II, early use of steroid drugs such as dexamethasone and ciclesonide is strongly recommended while considering the lung condition, and the use of antiviral drugs such as remdesivir, favipiravir, camostat, and nelfinavir is also considered. Thus, the treatment method changes accordingly. When it comes to severe cases, airway management by tracheal intubation, the use of ventilators, extracorporeal membrane oxygenation (ECMO), blood purification therapy, etc. will be selected. As such, the treatment of COVID-19 varies significantly depending on the symptoms. Since the sCLEC2 concentration used in the present invention begins to increase relatively early in SARS-CoV2 infection and changes sensitively according to the severity of the symptoms, by performing the risk assessment of deterioration according to the present invention, a treatment plan can be promptly established and patient care can be provided, which is preferable.

[0039] In COVID-19, thrombosis often develops with disease severity. However, it was surprising that blood sCLEC2 levels were already elevated even in mild cases where thrombosis was not yet presumed to have occurred. Since blood sCLEC2 reflects platelet activation, an elevated sCLEC2 level in mild cases means that platelet activation has already occurred and the body has entered the preparatory stage for thrombosis. That is, even if a patient is currently mild and not suffering from thrombosis, a high sCLEC2 level suggests a high risk of future thrombosis and disease progression. Furthermore, when the risk of disease progression is high at the mild or moderate stage I, antithrombotic therapy with antiplatelet drugs such as aspirin and clopidogrel, or anticoagulants such as heparin, warfarin, and DOACs, is suggested to prevent thrombosis and reduce the risk of disease progression.

[0040] Alternatively, the measured sCLEC2 level, excluding platelets, may be used for risk assessment. Treatment that reduces the sCLEC2 level indicates that platelet activation is being suppressed. However, if the sCLEC2 level persists at a high level or increases, reconsideration, addition, or increase of antithrombotic drugs may be considered. In this specification, the sCLEC2 level divided by the platelet count is referred to as the C2PAC index. Even if the sCLEC2 level is described without explicitly referring to the C2PAC index in the diagnosis of viral pneumonia, it is possible to replace the sCLEC2 level with the C2PAC index for all implementations.

[0041] When using the sCLEC2 level divided by the platelet count for the diagnosis of viral pneumonia, platelet counts are usually measured using an automated hematology analyzer (hemocytometer), but it is also possible to count using a hemocytometer and microscope.

[0042] The sCLEC2 level in plasma is expressed, for example, in pg / mL, and the blood platelet count is expressed, for example, as 1000 cells / mm 3It is preferable to use the C2PAC index expressed by and calculate the sCLEC2 concentration / platelet count. Here, the concentration of sCLEC2 used may be in any unit such as ng / mL or ng / L, and the platelet count may also be in any unit such as 10,000 cells / mm 3 3 and any unit such as 3 can be used, but unified units should be used for comparison. By using various units, the sCLEC2 concentration / platelet count can take various values, but they are essentially the same concept.

[0043] It can be expected that the ratio calculation is often performed using the measured value from the clinical test instrument for measuring the sCLEC2 concentration and the measured value from the hemocytometer for measuring the platelet count. This calculation is preferably automatically performed on the hospital's test system, the hospital's system, or a system such as an electronic medical record that is connected to both measuring instruments in daily medical practice. However, a system for connecting the data of the two measuring instruments may be constructed, or a machine that can simultaneously measure the sCLEC2 concentration and the platelet count may be constructed. Also, manual calculation using both data may be performed.

[0044] Those indicating the correlation between the sCLEC2 concentration in plasma, or the sCLEC2 concentration / platelet count and the degree of platelet activation and various diseases may be used, for example, as original data or statistical processing data for calculating a determination threshold (cut-off value).

[0045] sCLEC2 is released into the blood along with platelet activation. Existing platelet activation markers, such as PF4 and βTG, have the problem that granules are stimulated by the physical pressure during blood collection and cause non-specific release. However, sCLEC2 has a signal transduction-dependent release mechanism that induces platelet activation and can be a marker that more accurately reflects the activation of platelets in vivo. Also, since CLEC2 is almost exclusively expressed in platelets and megakaryocytes in humans, it becomes a platelet-specific marker with few false positives. Therefore, it becomes possible to diagnose the activation state of platelets at an early stage by measuring sCLEC2, and it can be used for the risk diagnosis of COVID-19 patients.

Example

[0046] Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

[0047] <<Example 1: Measurement of sCLEC2 in human plasma>> In this example, the concentration of sCLEC2 in plasma was measured according to the following procedure. (Preparation of measurement reagents and test samples) · Sample diluent: Using 0.1 mol / L HEPES buffer (pH 7.5) containing a preservative, sodium octanoate 2% and n-octyl-β-D-glucoside (OG) 0.5% were combined to prepare a sample diluent. The antibody contained in the reagent was the antibody described in the examples of Japanese Patent No. 6078845 and was prepared as follows. · First antibody solution: A mouse monoclonal antibody (11D5) that recognizes sCLEC2 was bound to magnetic latex particles (JSR), and dispersed in 0.01 mol / L MES buffer (pH 6.0) containing a preservative. · Second antibody solution: Another mouse monoclonal antibody (11E6) that recognizes sCLEC2 was labeled with alkaline phosphatase (ALP) by the maleimide method and dispersed in 0.01 mol / L MES buffer (pH 6.5) containing a preservative. · Luminescent substrate solution: 2-chloro-5-(4-methoxypyro{1,2-dioxetane-3,2´-(5´-chloro)-tricyclo[3.3.1.13,7]decane}-4-yl)-1-phenyl phosphate disodium (CDP-Star (registered trademark): Applied Biosystems) was used. · B / F washing solution: A buffer containing 0.1 mol / L citric acid (pH 6.5), 0.15 mol / L NaCl, and 0.1% Triton X-100 was used. ·Test sample: The test sample 1 was the recombinant hCLEC2 protein diluted with a buffer solution (0.025 mol / L HEPES, 0.14 mol / L NaCl, 0.1% sodium octanoate, 0.3% BSA), and the test sample 2 was the citrate plasma diluted with citrate plasma.

[0048] (Measurement by measurement reagent) For the measurement, an automatic clinical examination system STACIA (registered trademark, manufactured by LSI Medience Corporation) was used. The prepared sample diluent, the first antibody solution (magnetic latex reagent), and the second antibody solution (enzyme-labeled antibody reagent) were filled into the STACIA dedicated bottle, respectively, and set in the apparatus. Subsequently, the measurement was performed according to the operation method of the said apparatus. Specifically, 40 μL of the sample diluent was added to 10 μL of the sample, and after heating at 37°C for several minutes, 25 μL of the first antibody solution (magnetic latex reagent) was added and heated at 37°C for several minutes. Next, B / F separation was performed, 50 μL of the second antibody solution (enzyme-labeled antibody reagent) was added, heated at 37°C for several minutes, and after performing B / F separation again, 100 μL of the luminescent substrate solution was added. After reacting at 37°C for several minutes, the signal intensity (count) was measured. Figure 1 shows the standard curve prepared using the hsCLEC2 protein as a standard.

[0049] 《Example 2: Measurement of sCLEC2 in plasma specimens of COVID-19 patients and healthy subjects》 Forty-two patients infected with SARS-COV2 were classified into 14 mild cases, 9 moderate cases I, 15 moderate cases II, and 4 severe cases according to the definition in Table 1 (Non-Patent Document 4).

[0050]

Table 1

[0051] The average, standard deviation, median, and 25 - 75% interval of the sCLEC2 measurement values of healthy subjects, mild cases, moderate cases I, moderate cases II, and severe patients were as shown in Table 2.

[0052]

Table 2

[0053] The P-values when analyzing between groups by the non-parametric method (Mann-Whitney U test) were as shown in Table 3.

[0054]

Table 3

[0055] COVID-19 patients showed significantly higher values compared to healthy individuals regardless of severity, and moderate II and severe cases showed significantly higher values compared to mild and moderate I cases.

[0056] The plot of the actual measured values is shown in Figure 2. When considering healthy individuals as 1, mild cases as 2, moderate I as 3, moderate II as 4, and severe cases as 5, the Spearman rank correlation coefficient with sCLEC2 showed a significant correlation with severity (0.837, P < 0.001). Also, excluding healthy individuals, the Spearman rank correlation coefficient of severity among only COVID-19 patients was 0.464, still showing a significant correlation (P = 0.002).

[0057] As described above, the blood sCLEC2 value increases just by being infected with SARS-COV2. That is, platelets are activated. The value increases with severity, and a significant increase was observed especially from moderate II cases where the blood oxygen saturation is 93% or less and oxygen inhalation is required. When analyzing the discrimination between mild + moderate I and moderate II + severe cases using the ROC curve, a discrimination ability with an area under the curve = 0.784 was shown. The cut-off value where both sensitivity and specificity exceed 60% was 382 - 605 pg / mL. When setting the cut-off value at 490 pg / mL, the positive rates in each symptom group were as shown in Table 4.

[0058]

Table 4

[0059] <<Comparative Example 1: Measurement of sCLEC2 in Plasma Specimens of Patients with Bacterial Infections>> To compare with COVID-19 patients, sCLEC2 in patients with pneumonia, lower respiratory tract inflammation, and sepsis considered to be caused by bacterial infections was measured. As shown in Figure 3, the COVID-19 patient group showed significantly higher values than those with bacterial infections. It was suggested that viral lung infections have a higher degree of platelet activation than bacterial infections.

Industrial Applicability

[0060] As described above, the measurement of blood sCLEC2 and sCLEC2 / platelet ratio in the present invention can be a clinical test that can be used for the risk diagnosis of the aggravation of viral pneumonia, and the sCLEC2 measurement reagent can be a clinical test diagnostic agent for COVID-19.

Claims

1. A method for risk assessment of a patient suspected of having SARS-CoV-2 infection or diagnosed with SARS-CoV-2 infection, wherein the risk assessment is a method for assisting in predicting the severity of SARS-CoV-2 infection, and the method includes a step of measuring the concentration of soluble CLEC2 present in the blood collected from the patient.

2. The method according to claim 1, wherein the step of determining the concentration of soluble CLEC2 is a highly sensitive immunoassay selected from the group consisting of chemiluminescence immunoassay, electrochemiluminescence immunoassay, and fluorescence immunoassay.

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