KYNURENINE: USEFUL BIOMARKER IN ACUTE COVID-19 AND LONG COVID

Abstract

An in vitro method for the detection of inflammation caused by an acute COVID-19 infection, long COVID and/or PIMS. The level of kynurenine in a body fluid is determined and the value of kynurenine measured in the patient to be diagnosed is compared with the average value obtained from a comparable cohort of persons who do not suffer from said diseases, whereby the value of kynurenine in patients is increased.

Claims

1. An in vitro method for the detection of inflammation caused by an acute COVID-19 infection, long COVID and/or PIMS, wherein the level of kynurenine in a body fluid is determined and wherein the value of kynurenine measured in the patient to be diagnosed is compared with the average value obtained from a comparable cohort of persons who do not suffer from said diseases, whereby the value of kynurenine in patients is increased.

2. The in vitro method according to claim 1, wherein the determination of kynurenine in a body fluid is used for therapy control.

3. The in vitro method according to claim 1, wherein the determination of kynurenine in a body fluid is used for monitoring acute COVID-19 infection, long COVID and/or PIMS and/or the recovery of a patient.

4. The in vitro method according to claim 1, wherein the body fluid is serum, saliva or cerebro-spinal fluid (csf).

5. The in vitro method according to claim 1, wherein the body fluid is saliva.

6. The in vitro method according to claim 1, wherein the level of kynurenine is at least two times higher in a patient than in the control group.

7. The in vitro method according to claim 1, wherein acute COVID-19 infection is a contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

8. The in vitro method according to claim 1, wherein long COVID is a condition characterized by long-term consequences persisting or appearing after the typical convalescence period of COVID-19.

9. The in vitro method according to claim 1, wherein PIMS is a systemic paediatric illness involving persistent fever and extreme inflammation following exposure to SARS-CoV-2.

10. The in vitro method according to claim 1, wherein it is an ELISA test, or a lateral flow immunochromatographic assay, or a microfluidic test, or a colorimetric test, or an immunoblot.

11. A use of kynurenine as a biomarker in the in vitro detection of inflammation caused by an acute COVID-19 infection, long COVID and/or PIMS.

12. The use of kynurenine according to claim 11, wherein the level of kynurenine is at least 3 μM in serum, at least 1 μM in saliva, and at least 1 μM in CSF.

13. A test kit with which the level of kynurenine in a body fluid is determined by using the method according to claim 1.

14. A test kit according to claim 13, wherein is an ELISA test kit, or a lateral flow immunochromatographic assay test kit, or a microfluidic test kit, or a colorimetric test kit, or an immunoblot test kit.

15. The test kit according to claim 13, wherein the body fluid is serum, saliva or cerebro-spinal fluid (csf).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0080] FIG. 1 shows a schematic overview of the kynurenine pathway, the major route of the tryptophan degradation in higher eukaryotes. Enzymes are indicated in italics.

[0081] FIG. 2 shows a correlation of kynurenine concentrations measured in μM either in saliva (x-axis) or in serum (y-axis).

[0082] FIG. 3 shows the level of kynurenine in normal controls (n=302) vs. COVID-19 patients in the early acute stage (1st week, n=85). The difference was significant (p<0.001).

[0083] FIG. 4 shows a comparison of different entities of infections with the disease of long COVID. There is a significant difference between infections (comprising pneumonia, urinary tract infections, and severe wound infections) and patients in the early phase of long COVID (p<0.027). Patients after renal transplantation with CMV-reactivation/disease (n=34) under immunosuppressive medication showed an increase of kynurenine levels too, but significantly lower compared to patients with long COVID.

[0084] FIG. 5 shows a measurement of kynurenine in serum and saliva in normal controls (n=302) and in patients with an acute COVID-19 infection (n=9). Both values (serum and saliva) in the COVID-19+ were significantly higher (p<0.001).

[0085] FIG. 6 shows a comparison of kynurenine in serum in the follow up of Covid-19+ patients, either with (n=9) or without (n=28) an existing long COVID. Patients with the syndrome are currently under therapeutic management.

[0086] FIG. 7 shows a follow up of CRP-measurement in patients after COVID-19 infection and with long COVID. As can be seen, CRP was not a useful biomarker for follow-up in patients with long COVID.

[0087] FIG. 8 shows the level of kynurenine measured in serum in the 3rd month after positive PCR-testing: either cured or with a long-COVID syndrome for 3 months or more than 5 months. Kynurenine is still elevated significantly, whereas the values of the cured patients are in a normal range.

[0088] FIG. 9 shows the demographic and biochemical data of three different cohorts of patients (A, B and C) that were included in the study. Cohort A represents normal control patients without an infection by SARS-CoV-2. Cohort B represents long COVID-patients in the acute phase of the disease, either treated on the infection-ward or in the ICU. Cohort C represents patients under the diagnosis of long COVID disease. All three cohorts were comparable concerning age and gender distribution. There was a significant difference concerning kynurenine between the normal controls and patients with acute COVID-19 and long COVID.

[0089] N=number of individuals tested

[0090] n.s.=not significant

[0091] n.a.=not available

[0092] n.d.=not determined

[0093] The present invention is further illustrated by the following examples which are, however, not limiting the scope of the present invention.

EXAMPLE 1

[0094] Kynurenine test for the detection of inflammation caused by an acute COVID-19 infection, long COVID, and/or PIMS.

1.1 General Used Technique of Colorimetric Assay

[0095] The tryptophan metabolites via kynurenine can be quantitatively determined in biologic fluids by color reactions which are known since many decades. In general, a detection method via the formation of a colored reaction product can be performed by standard methods.

[0096] Microplate Readers are laboratory instruments designed to detect biological, chemical or physical events of samples in microtiter plates. They are widely used in research, drugdiscovery, bioassay validation, quality control as well as manufacturing processes in the pharmaceutical and biotechnological industry and academic organizations. Sample reactions can be assayed in 6-1536 well format microtiter plates. The most common microplate format used in academic research laboratories or clinical diagnostic laboratories is a 96-well (8 by 12 matrix) with a typical reaction volume between 100 and 200 μL per well.

[0097] Common detection modes for microplate assays are absorbance, fluorescence intensity, luminescence, time-resolved fluorescence, and fluorescence polarization. Absorbance detection has been available in microplate readers for more than 3 decades, and is used for assays such as ELISA assays, protein and nucleic acid quantification or enzyme activity assays. A light source illuminates the sample using a specific wavelength (selected by an optical filter, or a monochromator), and a light detector located on the other side of the well measures how much of the initial (100%) light is transmitted through the sample: the amount of transmitted light will typically be related to the concentration of the molecule of interest.

1.2. Description of the Test

[0098] This test was developed as a modified method.

[0099] Saliva withdrawn for PCR-Analysis for detection of COVID-19 antibodies (n=9) of the patients was normally withdrawn every Monday, Wednesday and Friday between 7:00 and 8:00 o'clock a.m. After measurement of antibodies, the remaining saliva was stored at −30° C. and served as a pool for longitudinal en bloc kynurenine measurements.

[0100] A color reagent was prepared and a dilution of a standard solution of kynurenine was also prepared. The color reaction is performed with a so-called “Ehrlich-Reagenz” which results in a yellow color. A solution comprising 2% by weight dimethylaminobenzaldehyde dissolved in 20% HCl is designated as “Ehrlich-Reagenz”. Said coloring reagent serves for the detection of primary amino groups, pyrrole and indole derivatives as well. The colorimetric determination of the concentration is performed with monochromatic light. The standard solution of kynurenine was prepared by using L-kynurenine sulfate.

[0101] Equal amounts of sample were mixed with 100 μl trichloroacetic acid (30%) thoroughly. After centrifugation the supernatant was measured. The absorbents of each sample at 492 nm were compared with the absorbents at 650 nm or 690 nm of the same sample. Then the absorbents of the controls (average of 5 wells) were subtracted from the absorbents of each well. By preparing a standard curve, the concentration of kynurenine in each sample could be determined.

EXAMPLE 2

[0102] Serum values of kynurenine were determined as follows:

[0103] Blood for routine monitoring of the patients was normally withdrawn every Monday, Wednesday and Friday between 7:00 and 8:00 o'clock a.m. After measurement of routine parameters the remaining serum was stored at −30° C. and served as a pool for longitudinal en bloc kynurenine measurements.

[0104] The determination of the level of kynurenine in serum was performed as described under example 1.2.

[0105] As shown earlier, there exists a linear correlation between the measurement of kynurenine in serum and in saliva. The relation is 3,3 (serum):1 (saliva) (FIG. 2).

Statistical Analysis

[0106] Descriptive data analysis and analysis of variance methods were used to characterize the data. All p-values are two-sided and considered to be descriptive. For a formal statement of descriptive significance a nominal type I error level of α=0.05 (two-sided) was assumed.

[0107] The exact Mann-Whitney U test was performed for comparison of two groups with not normally distributed continuous variables.

Results

[0108] In a pilot-study, a cohort (A) of normal control patients (n=302, range of age: 18-75 years, mean 47,1 y; gender 144 f/158 m), and a second cohort (B) of patients (n=85, range of age: 19-90 years, mean 63.1 y; gender 27 f/58 m) treated either on the infection ward (n=67) or the ICU (n=18) were analyzed.

[0109] The third cohort (C) of patients (n=66, range of age: 17-90 years, mean 66.6 y; gender 22 f/44 m) were treated either on the ICU (n=6) or the normal ward (59). This group of patients was investigated mainly as part of a proof of concept to study long COVID. Patients were PCR negative and either under weaning at the ICU (n=6) or the normal ward (n=59).

[0110] In a group of n=85 patients infected with COVID-19, (mean age 52.8 ±30 years) the inventors estimated kynurenine first in serum later also in saliva (n=9). Kynurenine was significantly elevated in COVID-19 patients (cohort B, n=85) compared to normal controls (cohort A, n=302), (10.81+8.8 μM vs. 2.5+0.4 μM; p<0.001) (FIG. 3). Samples in cohort B were taken in the first week starting treatment. For better graphical demonstration, two results in the COVID-19+group (58 and 43 μM), with a hyperinflammatory syndrome were left out.

[0111] Compared to other types of infections, like cytomegalovirus, CMV, -reactivation/-disease or bacterial infections, there was a typical range of kynurenine elevation for each entity. All ranges were significantly different. Both types of virus infections, CMV and COVID-19, showed a significant rise of kynurenine levels, much more pronounced in the COVID-19 group. The picture of hyperinflammation with extraordinary high levels of kynurenine was solely found in COVID-19 patients (up to 57.91 μM, not shown for better graphical demonstration) (FIG. 4).

[0112] Kynurenine was measured not only in serum. In FIG. 5, the inventors compared the measurements of kynurenine of normal controls and Covid-19+ patients (n=9) either measured in serum or saliva. It could be shown that kynurenine levels in COVID-19+ patients are increased both in serum and saliva compared to levels of kynurenine in individuals without a SARS-CoV-2 infection (normal controls).

[0113] Looking in the follow-up of a starting group of patients (n=9) with the diagnosis of long COVID, the inventors could demonstrate the sustained elevated level of kynurenine compared to normal controls from month 2 until month 4 (FIG. 6). This was not found for CRP (FIG. 7). 9 patients with signs of long COVID and 28 patients after a COVID-19+ infection without signs of long COVID were compared. Also, after more than 5 months, the level of kynurenine in long COVID patients was still elevated (FIG. 8). CRP, described in the literature as a good biomarker in COVID-19 patients, was not useful in this study to identify long COVID. CRP at 2 months after positive testing with COVID-19 was near normal (normal range>5 mg/L or 5 μg/ml) (FIG. 7)

[0114] Kynurenine in COVID-19+ patients (n=9) could be measured additionally in saliva and showed comparable results (FIG. 5). Kynurenine levels of patients compromised by virus-infections (CMV vs. COVID-19) are comparable (FIG. 4).

[0115] In total, it could be demonstrated by the present invention, that kynurenine is useful as a biomarker for conditions in COVID-19, thus, it is a useful biomarker in detecting inflammation caused by and monitoring of acute COVID-19, long COVID and/or PIMS. Kynurenine is able to depict the inflammatory and hyperinflammatory character of a SARS-CoV-2 disease. It was further shown by the present invention, that kynurenine is able to detect the chronic subclinical systemic inflammation seen in long COVID. This demonstrates also that kynurenine can be used for therapeutical monitoring.

[0116] Furthermore, measurements of kynurenine in saliva as shown in the present invention for the first time regarding an acute COVID-19 infection, long COVID and PIMS, open the opportunity for self-monitoring of the patient and a kind of therapy control.