Methods and kits for diagnosing covid-19 disease

Abstract

This invention relates to a method for diagnosing COVID-19 disease (preferably acute post-COVID-19 syndrome) in subjects by detecting persistent abnormal (amyloid) clots in samples using fluorescence or microscopic detection methods, wherein the presence of persistent abnormal (or amyloid) clots (particularly clots resistant to fibrinogen hydrolysis) in the samples is an indication of acute COVID-19 disease or acute post-COVID-19 syndrome in the subjects. The invention also relates to diagnostic kits for diagnosing acute COVID-19 disease (particularly acute post-COVID-19 syndrome) in subjects based on the disclosed methods.

Description

Background Technology

[0001] This invention relates to methods for diagnosing COVID-19 disease or post-acute COVID-19 syndrome in subjects using detection methods, said detection methods including fluorescence or microscopic detection methods or any other methods for detecting the presence of persistent abnormal (amyloid) clots in a sample, wherein the presence of persistent abnormal (or amyloid) clots in the sample is an indication of acute COVID-19 disease or post-acute COVID-19 syndrome in the subject. The invention also relates to diagnostic kits for diagnosing acute COVID-19 disease or post-acute COVID-19 syndrome in subjects, comprising a fluorescent dye or fluorescent probe for detecting the presence of persistent clots in a sample, wherein the presence of persistent clots in the sample is an indication of COVID-19 disease or post-acute COVID-19 syndrome in the subject.

[0002] Acute COVID-19 is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Symptoms of COVID-19 are diverse, including fever, cough, fatigue, difficulty breathing, and loss of smell and taste. SARS-CoV-2 is primarily transmitted through person-to-person contact, and the range of infection can be mild to fatal. In addition to a variety of respiratory symptoms, COVID-19 has been shown to cause various coagulopathy disorders.

[0003] COVID-19 has had a devastating impact on our society, especially for patients with multiple cardiovascular comorbidities, including cancer. Commonalities between patients with COVID-19 and cardiovascular disease include elevated circulating inflammatory biomarkers and significant coagulation abnormalities. Abnormal coagulation (clot) is known to occur under inflammatory conditions. Severe pathological coagulation is present during acute COVID-19 infection. Patients with COVID-19 and those with severe cardiovascular disease may also develop thrombocytopenia, which can be life-threatening.

[0004] In addition to acute COVID-19 infection, patients may also suffer from long-term health problems associated with COVID-19, recently termed LongCOVID or acute post-COVID-19 syndrome. LongCOVID manifests as a range of lingering symptoms that persist for 90 days or longer after a patient has recovered, regardless of the severity of the infection, and can persist in an estimated 25% to 35% of patients; even after a patient no longer tests positive for the SARS-CoV-2 virus.

[0005] To date, testing for acute COVID-19 has primarily focused on detecting the presence of the SARS-CoV-2 virus or the presence of antibodies produced in response to SARS-CoV-2 infection. Nucleic acid tests amplify different parts of the SARS-CoV-2 viral genome and thus detect the presence of viral RNA. Antigen tests have also been developed to detect these viral proteins in a sample using antibodies that bind to viral proteins. Antibodies are typically detectable about 14 days after the onset of viral infection, forming the basis for antibody testing, which provides an indication of prior SARS-CoV-2 infection. However, none of these testing methods can be used to diagnose LongCOVID. Furthermore, the standard method for detecting the presence and severity of coagulopathy in COVID-19 patients is based on CT chest scans for pneumonia, which has high sensitivity but low specificity and is neither inexpensive nor widely available. Additionally, thromboelastography (TEG) (a point-of-care technique) has been used to determine the extent of coagulopathy in acute COVID-19. The pathology laboratory also determined the concentration levels of D-dimer and fibrinogen to correlate these concentrations with the severity of coagulation dysfunction during acute COVID-19.

[0006] The inventors of this invention have developed a method for diagnosing COVID-19, and particularly LongCOVID or acute post-COVID-19 syndrome, based on abnormal coagulation, abnormal fibrinolysis, and pathological and abnormal clots that may persist in patients who have had acute COVID-19 but have clinically recovered and are no longer infectious. Invention Overview

[0008] This invention relates to a method for diagnosing COVID-19 disease or acute post-COVID-19 syndrome in subjects by detecting the presence of persistent abnormal (amyloid) clots in blood (or plasma) samples using fluorescence or microscopic detection methods, wherein the presence of such clots in the blood (or plasma) sample is an indicator of COVID-19 disease or acute post-COVID-19 syndrome in the subject. The invention also relates to a diagnostic kit for diagnosing COVID-19 disease or acute post-COVID-19 syndrome in subjects, comprising a fluorescent dye or fluorescent probe for detecting the presence of amyloid clots in a sample, wherein the presence of persistent abnormal (amyloid) clots in the sample is an indicator of COVID-19 disease or acute post-COVID-19 syndrome in the subject. The method according to the invention is particularly suitable for point-of-care diagnosis of COVID-19 disease, especially acute post-COVID-19 syndrome.

[0009] According to a first aspect of the invention, a method for diagnosing acute post-COVID-19 syndrome in a subject is provided, comprising:

[0010] (i) providing a sample obtained from the subject, preferably a blood sample; and

[0011] (ii) Detect the presence of persistent abnormal (amyloid) clots in the sample using fluorescence detection or microscopy.

[0012] The presence of persistent abnormal (amyloid) clots in the samples is an indicator of acute post-COVID-19 syndrome in the subjects.

[0013] In a first embodiment of a method for diagnosing acute post-COVID-19 syndrome in subjects, the method may further include the step of preparing platelet poor plasma (PPP) from a blood sample.

[0014] According to a second embodiment of the method for diagnosing acute post-COVID-19 syndrome, the method may further include treating the sample with fibrinolytic enzymes prior to the step of detecting the presence of persistent abnormal (amyloid) clots in the sample using a fluorescence or microscopic detection method.

[0015] In another embodiment of the method for diagnosing acute post-COVID-19 syndrome, the fibrinolytic enzyme may be an enzyme selected from trypsin, protein kinase, plasmin, aniplasmase, deamexase, streptokinase, urokinase, nattokinase, lumbrokinase, serrata peptide, papain, DNase, bromelain, and honokiol.

[0016] In one embodiment of a method for diagnosing acute post-COVID-19 syndrome, the method may include treating a sample with a fluorescent dye or fluorescent probe and detecting the fluorescent dye or probe. Those skilled in the art will understand that many fluorescent dyes or probes are known in the art. Preferably, the fluorescent dye or probe is a labeled dye or probe that binds to a protein, particularly an abnormal protein and / or a plasma coagulation protein. A non-limiting example of such a protein is thioflavin T (ThT). The fluorescent dye or probe may also be a labeled antibody that binds to a plasma coagulation protein.

[0017] According to another implementation of the method for diagnosing acute post-COVID-19 syndrome, the fluorescence or microscopy detection method may be fluorescence microscopy, confocal microscopy, fluorescence spectrofluorometry, or flow cytometry.

[0018] According to a second aspect of the present invention, a method for diagnosing COVID-19 disease in an individual is provided, comprising:

[0019] (i) Provide samples obtained from the object, preferably blood samples;

[0020] (ii) Optionally, treat the sample with fibrinogen hydrolase; and

[0021] (iii) Use fluorescence or microscopy to detect the presence of persistent abnormal (amyloid) clots in the sample.

[0022] The presence of persistent abnormal (amyloid) clots in the samples is an indication of acute COVID-19 disease or acute post-COVID-19 syndrome in the subjects.

[0023] In a first embodiment of the method for diagnosing COVID-19 disease in subjects, COVID-19 disease may be acute COVID-19 disease, but preferably acute post-COVID-19 syndrome.

[0024] According to a second embodiment of the method for diagnosing COVID-19, the method may further include the step of preparing anemic platelet plasma (PPP) from a blood sample.

[0025] In one embodiment of a method for diagnosing COVID-19, the fibrinolytic enzyme may be an enzyme selected from trypsin, protein kinase, plasmin, anipase, deampicase, streptokinase, urokinase, nattokinase, lumbrokinase, serrata peptide, papain, DNase, bromelain, and magnolol.

[0026] In another embodiment of a method for diagnosing COVID-19, the fluorescence detection method may include treating a sample with a fluorescent dye or fluorescent probe and detecting the fluorescent dye or probe. Those skilled in the art will understand that many fluorescent dyes or probes are known in the art. Preferably, the fluorescent dye or probe is a labeled dye or probe that binds to a protein, particularly an abnormal protein and / or a plasma coagulation protein. A non-limiting example of such a protein is thioflavone T (ThT). The fluorescent dye or probe may also be a labeled antibody that binds to a plasma coagulation protein.

[0027] According to another implementation of the method for diagnosing COVID-19, the fluorescence or microscopy detection method may be fluorescence microscopy, confocal microscopy, fluorescence spectroscopy, or flow cytometry.

[0028] According to a third aspect of the invention, a diagnostic kit for diagnosing acute post-COVID-19 syndrome in subjects is provided, comprising:

[0029] (i) A fluorescent dye or fluorescent probe used to detect the presence of persistent abnormal (amyloid) clots in a sample obtained from an object;

[0030] The presence of persistent abnormal (amyloid) clots in the samples is an indicator of acute post-COVID-19 syndrome in the subjects.

[0031] In a first embodiment of the diagnostic kit for diagnosing acute post-COVID-19 syndrome, the sample may be a blood sample. Preferably, the sample is a platelet-poor plasma (PPP) sample.

[0032] According to a second embodiment of the diagnostic kit for diagnosing acute post-COVID-19 syndrome, the diagnostic kit may also contain fibrinolytic enzymes for processing samples.

[0033] In another embodiment of the diagnostic kit for diagnosing acute post-COVID-19 syndrome, the fibrinolytic enzyme may be selected from trypsin, protein kinase, plasmin, anipase, deampicase, streptokinase, urokinase, nattokinase, lumbrokinase, serrata peptide, papain, DNase, bromelain, and magnolol.

[0034] In another embodiment of the diagnostic kit for diagnosing acute post-COVID-19 syndrome, the fluorescent dye or probe may be a fluorescent dye or probe that is detected using fluorescence microscopy, confocal microscopy, fluorescence spectroscopy, or flow cytometry. Those skilled in the art will understand that many fluorescent dyes or probes are known in the art. Preferably, the fluorescent dye or probe is a labeled dye or probe that binds to proteins, particularly abnormal proteins and / or plasma coagulation proteins. A non-limiting example of such a protein is thioflavone T (ThT). The fluorescent dye or probe may also be a labeled antibody that binds to plasma coagulation proteins.

[0035] In a fourth aspect of the invention, a diagnostic kit for diagnosing COVID-19 disease in a subject is provided, comprising:

[0036] (i) fibrinolytic enzymes for processing samples obtained from the object; and

[0037] (ii) Fluorescent dyes or probes used to detect the presence of persistent abnormal (amyloid) clots in a sample;

[0038] The presence of persistent abnormal (amyloid) clots in the samples is an indicator of COVID-19 disease in the subjects.

[0039] In a first embodiment of a diagnostic kit for diagnosing COVID-19 disease in subjects, COVID-19 disease can be acute COVID-19 disease or acute post-COVID-19 syndrome.

[0040] According to a second embodiment of the diagnostic kit for diagnosing COVID-19, the sample may be a blood sample. Preferably, the sample is a platelet-poor plasma (PPP) sample.

[0041] In a second embodiment of the diagnostic kit for diagnosing COVID-19, the fibrinolytic enzyme may be selected from trypsin, protein kinase, plasmin, anipase, deampicase, streptokinase, urokinase, nattokinase, lumbrokinase, serrata peptide, papain, DNase, bromelain, and magnolol.

[0042] According to another embodiment of a diagnostic kit for diagnosing COVID-19, the fluorescent dye or probe may be a fluorescent dye or probe that is detected using fluorescence microscopy, confocal microscopy, fluorescence spectroscopy, or flow cytometry. Those skilled in the art will understand that many fluorescent dyes or probes are known in the art. Preferably, the fluorescent dye or probe is a labeled dye or probe that binds to proteins, particularly abnormal proteins and / or plasma coagulation proteins. A non-limiting example of such a protein is thioflavin T (ThT). The fluorescent dye or probe may also be a labeled antibody that binds to plasma coagulation proteins.

[0043] According to another aspect of the invention, a diagnostic kit as defined herein is provided for a method of diagnosing COVID-19 disease in subjects. In particular, the diagnostic kit can be used for a method of diagnosing acute post-COVID-19 syndrome in subjects.

[0044] In another aspect of the invention, the use of a diagnostic kit as defined herein in a method for diagnosing COVID-19 disease in a subject, particularly in a method for diagnosing acute post-COVID-19 syndrome in a subject, is provided.

[0045] In another aspect of the invention, a method is provided for treating a subject diagnosed with acute post-COVID-19 syndrome according to the method of the invention, said method being performed by administering an anticoagulant and / or an antiplatelet drug to the subject. Those skilled in the art will understand that several anticoagulants and antiplatelet drugs are known. For example, anticoagulants may be selected from warfarin, rivaroxaban, dabigatran, apixaban, edoxaban, and heparin. In a non-limiting embodiment, a therapeutic (non-prophylactic) dose of fondaparinux, unfractionated heparin, or low molecular weight (LMW) heparin may be used as an anticoagulant.

[0046] Antiplatelet drugs can be selected from aspirin, clopidogrel, cangrelox, prasugrel, ticagrelor, and ticlopidine. In addition, antiplatelet drugs can be glycoprotein (GP) IIβ / IIIα inhibitors, such as abciximab, eptifibatide, and / or tirofiban.

[0047] In another embodiment, the method of treating the subject may include administering dual antiplatelet therapy (DAPT) to the subject. Specifically, DAPT may include administering one or more antiplatelet drugs and an anticoagulant selected from LMW heparin, fondaparin, and direct oral anticoagulants (DOACs). More specifically, DAPT may be determined for each subject based on individual circumstances. Brief description of the attached diagram

[0049] Some non-limiting embodiments of the invention will now be described by way of example only and with reference to the following figures:

[0050] Figure 1 Representative photomicrographs of plasma smears from: (A) healthy patients; (B) diabetic patients; and (C) patients with acute COVID-19, following fibrin hydrolysis.

[0051] Figure 2 Representative photomicrographs of plasma smears from four patients who had recovered from acute COVID-19, following fibrinolysis treatment with trypsin fibrinolysis. These patients contracted LongCOVID two months after recovering from acute COVID-19.

[0052] Figure 3 Representative photomicrographs from the following plasma smears: (A) a healthy patient before COVID-19 infection; (B) the same patient who developed LongCOVID symptoms 2 months after acute COVID-19; and (C) a second instance of an individual who developed LongCOVID 2 months after COVID-19. Invention Details

[0054] The invention will now be described more fully below with reference to the accompanying drawings, which illustrate some, but not all, embodiments of the invention.

[0055] The invention described herein should not be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the invention. Although specific terminology is used herein, it is used in a general and descriptive sense only and not for limiting purposes.

[0056] Unless the context clearly indicates otherwise, nouns without quantifiers used throughout this specification and in the following claims include one / a kind or more / a kind.

[0057] The terms and wording used herein are for descriptive purposes and should not be considered limiting. The terms “comprising,” “containing,” “having,” and “including,” and their variations, as used herein, are intended to cover the items listed thereafter, their equivalents, and any additional items.

[0058] This invention relates to methods for diagnosing COVID-19, including acute COVID-19 and LongCOVID, or post-acute COVID-19, based on the presence of abnormal blood clots in a sample, particularly abnormal blood clots resistant to fibrinolysis. The invention also relates to kits for diagnosing post-acute COVID-19 containing a fluorophore that binds to a persistent abnormal (amyloid) clot (with primitive plasma clotting proteins) before or after treatment with fibrinolytic enzymes, or to kits for diagnosing acute COVID-19 containing fibrinolytic enzymes and a fluorophore that binds to a persistent abnormal (amyloid) clot (with primitive plasma clotting proteins).

[0059] The inventors have demonstrated that abnormal coagulation, abnormal fibrinolysis, and pathological clots may persist in patients who have had acute COVID-19 but have clinically recovered and are no longer infectious. Such abnormal coagulation in patients with both COVID-19 and LongCOVID may be resistant to fibrinolysis. The presence of these persistent low-fibrinolytic clots can be used as a diagnostic tool for both acute and LongCOVID.

[0060] The inventors have developed a method for diagnosing COVID-19, including acute COVID-19 and LongCOVID, or post-acute COVID-19, based on the presence of abnormal blood clots in samples, particularly abnormal blood clots resistant to fibrinogen hydrolysis. This method is reproducible and successfully distinguishes between healthy plasma samples, type 2 diabetes, and acute COVID-19 and LongCOVID.

[0061] The inventors of this invention have discovered that both acute COVID-19 and LongCOVID plasma samples contain large, persistent abnormal clots with amyloid properties. The presence of these clots is known to cause severe coagulation pathology in acute COVID-19. Furthermore, these clots are present in the plasma in LongCOVID both before and after fibrinolysis, but are absent in the plasma after fibrinolysis in healthy individuals or those with inflammatory diseases (such as type 2 diabetes). Moreover, this method does not require the addition of coagulation factors such as thrombin to induce coagulation. The presence of these persistent abnormal (amyloid) clots can be used to diagnose LongCOVID, which can be further confirmed by fibrinolysis—because persistent abnormal (amyloid) clots are resistant to fibrinolysis. Furthermore, the presence of fibrinolytically hydrolyzed persistent abnormal (amyloid) clots can serve as a basis for diagnosing acute COVID-19.

[0062] Furthermore, the discovery of these persistent abnormal (amyloid) clots in LongCOVID patients led the inventors to investigate the use of known anticoagulants to treat patients diagnosed with LongCOVID, which has a positive effect on their symptoms.

[0063] The term “COVID-19 disease” as used in this article refers to the infectious disease caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), and includes both “acute COVID-19 disease” and “acute post-COVID-19 syndrome” (also known as “LongCOVID”).

[0064] As used in this article, “acute COVID-19 illness” refers to COVID-19 illness characterized by one or more of the following symptoms: fever, dry cough, fatigue, aches and discomfort, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash, discoloration of fingers or toes, difficulty or shortness of breath, chest pain or tightness, and loss of speech or movement, and which usually lasts from 5 days to 4 weeks.

[0065] As used in this article, the terms “acute post-COVID-19 syndrome” and “LongCOVID” refer to COVID-19 illness characterized by long-term complications of SARS-CoV-2 infection or delayed and / or persistent symptoms lasting more than 4 weeks from the onset of symptoms. Acute post-COVID-19 syndrome can also be divided into two categories: (1) subacute or persistent symptomatic COVID-19, which includes symptoms and abnormalities that appear 4 to 12 weeks after acute COVID-19; and (2) chronic or post-COVID-19 syndrome, which includes symptoms and abnormalities that persist or appear more than 12 weeks after the onset of acute COVID-19 and are not attributable to an alternative diagnosis.

[0066] As used herein, the term "persistent abnormal (amyloid) clot" refers to an aggregate of plasma clotting proteins with amyloid properties, ranging in diameter from 1 to 1000 μm, or from 5 to 1000 μm. Amyloid proteins are aggregates of proteins characterized by a fibrillary morphology with a diameter of 7 to 13 nm, a β-sheet secondary structure, and the ability to be stained by specific dyes that bind to plasma clotting proteins. Typically, such abnormal clots are a series of plasma clotting proteins primarily composed of fibrin(ogen), but may also contain various other known clotting proteins (e.g., prothrombin, thrombin, factor XIII) and a variety of proteins and particles that may originate from endothelial cells, platelets, erythrocytes, or leukocytes.

[0067] As used herein, the term "sample" refers to a sample obtained from a biological source, including "biological samples," such as blood, plasma, lymph, tissue, or urine samples. In one non-limiting embodiment, the sample is a plasma sample.

[0068] As used herein, the term "detection" includes the qualitative and / or quantitative detection or measurement of the level of persistent aberrant (amyloid) clots, with or without a reference control. Generally, aberrant (amyloid) clots can be detected by a "fluorescent detection method" or by observing the clots with an optical microscope. Suitable fluorescent detection methods may include fluorescence microscopy, confocal microscopy, fluorescence spectroscopy, or flow cytometry of the sample. Generally, detection involves contacting the sample with a selective reagent such as a probe, dye, or other ligand, thereby detecting the presence of a target protein in the sample or measuring the level or amount of the target protein present in the sample. Contact can be performed under any conditions suitable for detecting complexes formed between the reagent and the nucleic acid or protein of the sample. In one embodiment, the sample may be treated with a dye or probe, particularly a fluorescent dye or probe that, when bound to the aberrant (amyloid) clot, can be detected by one or more of the above-described fluorescent detection methods. In another embodiment, the binding of the fluorescent dye or probe to the aberrant (amyloid) clot can be observed visually.

[0069] If fluorescence microscopy is used to detect persistent aberrant (amyloid) clots, a thresholding method can be used to calculate the percentage of aberrant (amyloid) clots. This method allows for the measurement of the area of ​​the amyloid signal. RGB images are opened in ImageJ or any similar program, and each image is calibrated by setting a scale (calculated using the known size of the image pixel size and scale bar). Each image is then converted to black and white (8-bit, adjusted under the image type setting). The background intensity is then adjusted to white (255), and the black amyloid signal is thresholded. Huang's settings can be used during thresholding. Huang's method is an optimization approach that finds the optimal threshold by minimizing ambiguity measurements. The black aberrant (amyloid) clot region is analyzed using the analysis particle settings (where the measured particle size is from 1 to infinity). The particle size setting allows for the exclusion of any background signals that may not be true amyloid signals. Statistical analysis can be performed using Graphpad Prism or any similar statistical program known in the art.

[0070] In one embodiment of the invention, the method of the invention may include the step of treating a sample with fibrinogenase, and the diagnostic kit of the invention may include fibrinogenase for treating the sample.

[0071] As used herein, the term "fibrinogenase" refers to any enzyme capable of stimulating the dissolution of blood clots, and the term "fibrinogenase" refers to the enzymatic breakdown of blood clots by the action of such fibrinogenases. Examples of such fibrinogenases include trypsin, protein kinase, plasmin, aniplasmin, deampicase, streptokinase, urokinase, nattokinase, lumbrokinase, serratakinase, papain, DNase, bromelain, and magnolol. According to a non-limiting embodiment, the fibrinogenase is preferably trypsin or protein kinase.

[0072] The method of the present invention may further include the step of comparing the biomarker level with a control reference or with a reference value.

[0073] As used herein, the term "control" refers to a healthy subject, or a subject who has not suffered from COVID-19 disease or acute post-COVID-19 syndrome. A control may also be a subject with known or baseline levels of abnormal (amyloid) clots. A control may also be a subject that does not have detectable persistent abnormal (amyloid) clots after treatment with fibrinolytic enzymes. Preferably, the control is a healthy subject without systemic chronic inflammation. Typically, amyloid clots in control samples from healthy subjects range from less than 1 μm, while amyloid clots in control samples from subjects with inflammatory conditions range from 1 to 5 μm.

[0074] Generally, "reference values" are determined experimentally, empirically, or theoretically. Preferably, the reference value is a known or baseline level of aberrant (amyloid) clots in healthy individuals or populations of healthy individuals, where the term "healthy individual" means an individual known to be healthy, i.e., who has not had and has never had COVID-19 disease or acute post-COVID-19 syndrome. Alternatively, the reference value may be correlated with the level of aberrant (amyloid) clots in known disease states and is the average level of aberrant (amyloid) clots in sick individuals or populations of sick individuals, where the term "sick individual" means an individual known to have or have had COVID-19 disease or acute post-COVID-19 syndrome, or as an alternative to having another disease that causes aberrant (amyloid) clots in the plasma.

[0075] In some embodiments, the kit according to the invention may be provided together with the instruction manual.

[0076] The following examples are provided by way of illustration rather than by way of limitation.

[0077] Example 1

[0078] Plasma fibrin hydrolysis and fluorescence microscopy

[0079] Patient samples

[0080] Ten acute COVID-19 samples, six LongCOVID samples, five type 2 diabetes samples, and five healthy age-matched samples were analyzed.

[0081] LongCOVID patients are diagnosed with LongCOVID symptoms at least two months after a negative PRC test confirms their recovery from acute COVID-19.

[0082] Both COVID-19 and LongCOVID patients (when they were initially diagnosed with acute COVID-19) were identified as COVID-positive through PRC testing in a pathology laboratory.

[0083] Clinicians confirm that patients with type 2 diabetes have had diabetes for at least 6 months prior to the collection of blood samples.

[0084] The pathological results of blood biomarkers confirmed that the healthy individual did not suffer from any long-term chronic systemic inflammation.

[0085] Fibrin hydrolysis

[0086] Plasma samples were diluted 10-fold in 10 mM ammonium bicarbonate buffer, and protein concentrations were determined at 280 nm using a nanodrop (Thermo) spectrophotometer. Samples were adjusted to the minimum concentration (5 mg / mL), and equal volumes of each sample (40 μL) were digested. 4 μg of trypsin was added to each sample in a total of 10 μL, resulting in a final volume of 50 μL, with an enzyme-to-substrate ratio of 1:50. Plasma protein digestion was allowed to proceed at 37°C for 18 hours. After 18 hours, the samples were removed from the heating block and centrifuged at 14000 × g for 30 minutes at ambient temperature to precipitate the resistant portion. The supernatant was then removed, leaving 10 μL of sample at the bottom of the tube.

[0087] Plasma samples from healthy controls and diabetic individuals were completely digested using the digestion method described above. The digested plasma was a clear liquid with no visible sediment. In contrast, all COVID-19 and LongCOVID samples contained insoluble sediment at the bottom of the tube after trypsin digestion.

[0088] Fluorescence microscopy

[0089] After digestion, 0.5 μL of thioflavin T (ThT) to a final concentration of 5 mmol was added to 10 μL of plasma and incubated for 30 minutes. Subsequently, 3 μL of sample was placed on a microscope slide, incubated for 30 seconds, and a coverslip was placed on top of the sample. The sample was observed using a Zeiss Axio Observer 7 fluorescence microscope (Carl Zeiss Microscopy, Munich, Germany) with a Plan-Apochromat 63x / 1.4Oil DIC M27 objective. The excitation wavelength for ThT was set from 450 nm to 488 nm, and the emission wavelength was set from 499 nm to 529 nm.

[0090] The percentage of abnormal (amyloid) coagulation can be calculated using a threshold method. However, this method was not used in this analysis because the sample size and the fluorescence signal in the micrographs were considered too significant to be necessary.

[0091] Figure 1 A through C show representative photomicrographs of plasma smears from healthy individuals, diabetic patients, and COVID-19 patients, respectively, after fibrinogen hydrolysis treatment with trypsin fibrinogen hydrolysis.

[0092] Figure 2A through D show representative photomicrographs of plasma smears from four of six patients who had recovered from acute COVID-19, following fibrinogenolysis treatment with trypsin fibrinogenolysis. These patients contracted LongCOVID two months after their recovery from acute COVID-19.

[0093] Fluorescence microscopy revealed very little abnormal (amyloid) fluorescence signal in plasma samples processed using the above method in individuals with type 2 diabetes and healthy individuals. In contrast, using the same method, COVID-19 and LongCOVID plasma samples contained large, insoluble fluorescent deposits. This indicates that trypsin did not completely digest plasma samples from COVID-19 or LongCOVID patients.

[0094] Example 2

[0095] Fluorescence microscopy of plasma (performed before trypsin digestion).

[0096] Patient samples

[0097] All six LongCOVID patients showed abnormal (amyloid) clots prior to trypsin digestion.

[0098] Figure 3 (A) and (B) show plasma samples from the same patient exposed to ThT. Figure 3 (A) shows a stored plasma sample from a patient collected in 2018 prior to an acute COVID-19 infection. Figure 3 (B) shows a plasma sample from a patient two months after recovering from acute COVID-19—the patient now has LongCOVID. Figure 3 (C) shows a plasma sample from a second patient who has now developed LongCOVID, two months after recovering from acute COVID-19.

[0099] LongCOVID patients were diagnosed with acute COVID-19 and were confirmed as COVID-positive by PRC testing in the pathology laboratory.

[0100] Furthermore, all LongCOVID plasma samples showed abnormal clots prior to fibrinolytic digestion with trypsin. This indicates that this method can be used to detect LongCOVID with or without the addition of fibrinolytic enzymes.

[0101] Fluorescence microscopy without fibrin hydrolysis

[0102] After preparing anemic platelet-rich plasma from whole blood, 0.5 μL of thioflavin T (ThT) at a final concentration of 5 mmol was added to 10 μL of plasma and allowed to stand for 30 minutes. Subsequently, 3 μL of the sample was placed on a microscope slide, left to stand for 30 seconds, and then a coverslip was placed on top of the sample. The sample was observed using a Zeiss Axio Observer 7 fluorescence microscope (Carl Zeiss Microscopy, Munich, Germany) with a Plan-Apochromat 63x / 1.4Oil DIC M27 objective. The excitation wavelength of ThT was set from 450 nm to 488 nm, and the emission wavelength was set from 499 nm to 529 nm.

[0103] Figure 3 A is a photomicrograph of a comparative sample from one of the patients before COVID-19 (the plasma was collected in 2018, before the emergence of SARS-CoV-2), and Figure 2 B is a photomicrograph of a sample collected from the same patient after recovery from acute COVID-19 but during the period of long-term symptoms indicating LongCOVID. Figure 3 C shows a representative photomicrograph of a plasma smear from a second patient who had recovered from acute COVID-19. Persistent abnormal (amyloid) clots were present in their plasma even before fibrin hydrolysis.

Claims

1. Use of a fluorescent dye or fluorescent probe for detecting the presence of persistent abnormal amyloid clots in the preparation of a diagnostic kit for diagnosing acute post-COVID-19 syndrome in subjects, wherein the diagnostic kit is formulated for: (i) Provide a sample obtained from the object; (ii) treating the sample with a fluorescent dye or fluorescent probe; and (iii) Detect the presence of persistent abnormal amyloid clots in the sample by using fluorescence or microscopy detection methods to detect the fluorescent dye or fluorescent probe. The presence of persistent abnormal amyloid clots in the sample is an indication of acute post-COVID-19 syndrome in the subject.

2. The use according to claim 1, wherein the sample is a blood sample.

3. The use according to claim 2, wherein the diagnostic kit is further formulated for preparing anemic platelet-rich plasma (PPP) from the blood sample.

4. The use of claim 1, wherein the diagnostic kit is formulated for treating the sample with fibrinolytic enzyme prior to the step of detecting the presence of persistent abnormal amyloid clots in the sample using the fluorescence or microscopy detection method.

5. The use according to claim 4, wherein the fibrinolytic enzyme is selected from trypsin, protein kinase, plasmin, anipase, deampicase, streptokinase, urokinase, nattokinase, lumbrokinase, serrata peptide, papain, DNase, bromelain, and magnolol.

6. The use according to claim 1, wherein the fluorescence or microscopy detection method is fluorescence microscopy, confocal microscopy, fluorescence spectroscopy, or flow cytometry.

Images