METHOD FOR DIAGNOSIS OF LYME ARTHRITIS, METHOD FOR DIFFERENTIAL DIAGNOSIS OF LYME ARTHRITIS, LYSOPHOSPHATIDYLETHANOLAMINE FOR USE AS BIOMARKER, KIT FOR DIAGNOSIS OF LYME ARTHRITIS AND KIT FOR DIFFERENTIAL DIAGNOSIS OF LYME ARTHRITIS

Abstract

The subject matter of the invention relates to a method for in vitro diagnosis of Lyme disease and a method for in vitro differential diagnosis of Lyme arthritis versus rheumatoid arthritis, in which methods, in a sample from a subject, the level of lysophosphatidylethanolamine comprising myristic acid (LysoPE(14:0)) is determined and such determined level of lysophosphatidylethanolamine is compared with the level of lysophosphatidylethanolamine comprising myristic acid in a reference sample; in wherein the level of lysophosphatidylethanolamine comprising myristic acid which is higher than the level in the said reference sample indicates that the subject suffers from Lyme disease. The subject matter of the invention further relates to lysophosphatidylethanolamine comprising myristic for use as a biomarker of Lyme disease, as a biomarker of Lyme arthritis, as a biomarker for differential diagnosis of Lyme arthritis versus rheumatoid arthritis, as a biomarker of neuroborreliosis. The subject matter of the invention also relates to a kit for in vitro diagnosis of Lyme disease and a kit for in vitro differential diagnosis of Lyme arthritis, which kits comprise a means for determining the level of lysophosphatidylethanolamine comprising myristic acid and instructions for carrying out the methods for diagnosis according to the invention.

Claims

1. A method for in vitro diagnosis of Lyme disease in a subject, characterized in that: a) in a sample from a subject, a level of lysophosphatidylethanolamine comprising myristic acid (LysoPE(14:0)) is determined, and b) the level of lysophosphatidylethanolamine determined in step a) is compared with the level of lysophosphatidylethanolamine comprising myristic acid in a reference sample; wherein the level of lysophosphatidylethanolamine comprising myristic acid which is higher than the level in the said reference sample indicates that the subject suffers from Lyme disease.

2. The method for in vitro diagnosis of Lyme disease according to claim 1, characterized in that the sample from the subject is a sample selected from the group consisting of whole blood, plasma, serum and cerebrospinal fluid.

3. The method for in vitro diagnosis of Lyme disease according to claim 2, characterized in that the sample is plasma.

4. The method for in vitro diagnosis of Lyme disease according to claim 1, characterized in that the subject is a human subject.

5. The method for in vitro diagnosis of Lyme disease according to claim 1, characterized in that the level of lysophosphatidylethanolamine comprising myristic acid is determined by liquid chromatography coupled with mass spectrometry (LC-MS) method.

6. The method for in vitro diagnosis of Lyme disease according to claim 1, characterized in that the Lyme disease is Lyme arthritis or neuroborreliosis.

7. A method for in vitro differential diagnosis of Lyme arthritis versus rheumatoid arthritis, characterized in that: a) in a sample from a subject, a level of lysophosphatidylethanolamine comprising myristic acid (LysoPE(14:0)) is determined, and b) the level of lysophosphatidylethanolamine determined in step a) is compared with a reference level of lysophosphatidylethanolamine in a reference sample; wherein if the level of lysophosphatidylethanolamine in the sample from the subject is higher than the reference level of lysophosphatidylethanolamine in the said sample indicates that the subject is diagnosed with Lyme arthritis.

8. The method for in vitro differential diagnosis according to claim 7, characterized in that the sample from the subject is a sample selected from the group consisting of whole blood, plasma, serum and cerebrospinal fluid.

9. The method for in vitro differential diagnosis according to claim 8, characterized in that the sample is plasma.

10. The method for in vitro differential diagnosis according to claim 7, characterized in that the subject is a human subject.

11. The method for in vitro differential diagnosis according to claim 7, characterized in that the level of lysophosphatidylethanolamine comprising myristic acid is measured by liquid chromatography-mass spectrometry (LC-MS) method.

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. A kit for in vitro diagnosis of Lyme disease, characterised in that it comprises a means for determining the level of lysophosphatidylethanolamine comprising myristic acid and instructions for carrying out the method for in vitro diagnosis of Lyme disease according to claim 1.

17. A kit for in vitro differential diagnosis of Lyme arthritis, characterised in that it comprises a means for determining the level lysophosphatidylethanolamine comprising myristic acid and instructions for carrying out the method for in vitro differential diagnosis of Lyme arthritis versus rheumatoid arthritis according to claim 7.

18. The method for in vitro diagnosis of Lyme disease according to claim 5, wherein the liquid chromatography coupled with mass spectrometry (LC-MS) method is a LC-MS/MS method.

19. The method for in vitro differential diagnosis according to claim 11, wherein the liquid chromatography-mass spectrometry (LC-MS) method is a LC-MS/MS method.

Description

DESCRIPTION OF FIGURES

[0052] FIG. 1—shows the structural formula of lysophosphatidylethanolamine comprising myristic acid.

[0053] FIG. 2—FIG. 2A shows a representative Total Ion Chromatogram (TIC) in negative ionisation mode for blood plasma of a patient with rheumatoid arthritis (RA) (the top chromatogram); Extracted Ion Chromatogram (EIC) for m/z 424.6424 in negative ionisation mode (the bottom chromatogram); FIG. 2B—shows a representative mass spectrum MS of the peak at RT=12.06 min (the top spectrum); the fragmentation mass spectrum MS/MS of the ion having m/z 424.2442 corresponding to the molecular ion of LPE (14:0) (the bottom spectrum)—the m/z 227.2016 signal confirms the presence of myristic acid (14:0) in the structure of the fragmented compound.

[0054] FIG. 3—shows a box-and-whisker plot which presents graphically changes in the relative content of LPE (14:0) in healthy persons, patients with rheumatoid arthritis and patients with Lyme arthritis. (p<0.0001****). LA—Lyme arthritis; RA—rheumatoid arthritis; C—control.

[0055] FIG. 4—shows representative Total Ion Chromatograms (TICs), as a phospholipid profile in negative ionization mode: plasma from a patient with Lyme arthritis—panel A; plasma from a healthy person—panel B; plasma from a patient with rheumatoid arthritis—panel C.

[0056] FIG. 5—shows representative Extracted Ion Chromatograms (EICs) in negative ionization mode for m/z 424.6424 corresponding to the molecular ion of LPE(14:0), retention time RT=12 min: plasma from a patient with Lyme arthritis—panel A; plasma from a healthy person—panel B; plasma from a patient with rheumatoid arthritis—panel C.

[0057] FIG. 6—shows mass spectrum MS of the peak at RT=12.06 min (the top panel); the fragmentation mass spectrum MS/MS of the ion having m/z 424.2442 corresponding to the molecular ion of LPE (14:0) (the bottom panel)—the m/z 227.2016 signal confirms the presence of myristic acid (14:0) in the structure of the fragmented compound.

[0058] FIG. 7—shows representative overlaid-mode Extracted Ion Chromatograms (EICs) in negative ionization mode for m/z 424.6424 corresponding to the molecular ion of LPE(14:0), retention time RT=12 min: LA—plasma from a patient with Lyme arthritis; C—plasma from a healthy person; RA—plasma from a patient with rheumatoid arthritis.

EXAMPLE—DETERMINATION OF THE LPE (14:0) LEVEL

Test Material

[0059] The biological material used for the analyses was plasma obtained from the venous blood of persons suffering from rheumatoid arthritis (RA), persons suffering from Lyme arthritis (LA) and healthy persons. LA group consisted of 9 patients with diagnosed (tick-borne) Lyme arthritis (2 women and 7 men) aged from 22 to 81 years (the mean age was 49 years) hospitalized in the Department of Infectious Diseases and Neuroinfection of the University of Bialystok. The illness was diagnosed on the basis of the presence of anti-Borrelia burgdorferi specific IgM and/or IgG antibodies in blood serum, detected by ELISA method. RA group consisted of 9 patients with active RA (2 women and 7 men) aged 23 to 79 years (the mean age was 48 years). The assessment of activity of the disease was based on the four-variable DAS28-CRP parameter. The control group consisted of 9 healthy persons (2 women and 7 men) aged from 24 to 71 years (the mean age was 47 years). Venous blood was obtained from both the patients and healthy persons to heparinized test tubes. The samples were centrifuged at 2000×g, in 4° C. for 20 minutes in order to obtain plasma. After adding buthylhydroxytoluene (BHT), acting as an antioxidant, the samples were kept at −80° C. until analysis. The study was started after obtaining the consent of the Bioethics Committee of the Medical University of Bialystok and a written consent of each person participating in the study.

[0060] The compound was identified in both the plasma from patients and the plasma from healthy persons by a highly specialized analytical technique LC-MS using hydrophilic interaction liquid chromatography (HILIC), after previous isolation of lipid fraction by the solvent-solvent extraction method as described in detail below.

Extraction of Lipids from Blood Plasma

[0061] Extraction was carried out in glass test tubes previously washed with chloroform (CHCl.sub.3) in order to remove any impurities. 200 μl of plasma and 1.5 ml of methanol (MeOH) cooled to −20° C. was placed in each test tube. The mixture obtained was vortexed for 10 minutes. Then, 3 ml of CHCl.sub.3 cooled to −20° C. was added and was again vortexed for 3 minutes. In the next stage, extracted samples were incubated in ice for one hour. During incubation, from time to time, the content of the test tubes was vortexed for a moment. After incubation, in order to initiate separation of phases, 1.25 ml of ultrapure (Milli-Q) water was added and the samples were again left in ice for 10 minutes, their content being mixed in Vortex shaker from time to time. Then, the samples were centrifuged at 2500×g for 10 minutes. After centrifuging, the bottom organic phase was transferred to a new glass test tube and another extraction was carried out by adding 2 ml of CHCl.sub.3/MeOH (2:1, v/v) mixture cooled to −20° C. The next obtained portion of chloroform layer was combined with the previously collected portion and evaporated to dry residue in a nitrogen atmosphere. The extracts obtained were kept in −80° C. until analysis.

Determination of the Profile of Phospholipids in Blood Plasma Using the LC-MS-QTOF System

[0062] To obtain the profile of phospholipids, the obtained extracts of blood plasma lipids from the patients with rheumatoid arthritis, patients with Lyme arthritis and healthy persons were analysed using LC-MS-QTOF in data dependent MS/MS mode (auto MS/MS). Separation of phospholipids classes was carried out with the use of hydrophilic interaction liquid chromatography (HILIC) using Ascentis Si HPLC Pore column, 15 cm×1.0 mm, 3 mm; (Sigma-Aldrich) and gradient elution using a combination of two mobile phases A and B. Mobile phase A contained 25% water, 50% acetonitrile, and 25% v/v methanol and 10 mM addition of salt in the form of ammonium acetate. Mobile phase B contained 60% acetonitrile, 40% methanol and 10 mM addition of ammonium acetate. The gradient applied at 40 ml/min mobile phase flow used a mixture having the following composition: [0063] 0% mobile phase A and 100% mobile phase B [0 min] [0064] 100% mobile phase A and 0% mobile phase B [20 min] [0065] 100% mobile phase A and 0% mobile phase B [35 min] [0066] 0% mobile phase A and 100% mobile phase B [45 min]

[0067] Mobile phase B was used as a solvent for samples of extracts comprising 20 μg of phospholipids. 5 μl of such prepared samples were injected into the chromatographic column.

[0068] Samples of the extracts were analysed in negative ionisation mode using electrospray ionisation (ESI) sources.

[0069] Agilent Technologies ultra-performance liquid chromatograph (UPLC), series 1290; Agilent Technologies QTOF mass detector, 6540, equipped with an electrospray ionization (ESI) source; Peak Scientific LC-MS 20 nitrogen generator; Ascentis Si HPLC Pore chromatographic column, 15 cm×1.0 mm, 3 mm, Sigma-Aldrich were used for the diagnosis by the method according to the invention. However, the methods according to the invention may be carried out using any other devices, systems and analytical kits which enable quantitative determination of lysophosphatidylethanolamine comprising myristic acid.

Statistical Analysis

[0070] The results obtained were subjected to statistical analysis in the following way. Fold change of relative LPE (14:0) content in healthy persons, patients with rheumatoid arthritis and patients with Lyme arthritis was analysed. The statistical analysis was performed based on one-way ANOVA analysis. Relative amount of LPE (14:0) was calculated by dividing the peak area of LPE (14:0) obtained on the basis of Extracted Ion Chromatograms (EICs) by the peak area of PC (28:0) being an Internal Standard (ISTD). The results were expressed as arithmetical means and mean standard errors. The comparisons between the control group and the persons suffering from rheumatoid arthritis and Lyme arthritis were made using one-way ANOVA analysis and the post hock test: Tukey's HSD test, with the use of the statistical package GraphPad Prism v.7.0, GraphPad Software, USA. The differences at p<0.05 were considered as statistically significant.

[0071] The results obtained are presented in Table 1 below and in FIGS. 2 to 7.

TABLE-US-00001 TABLE 1 Fold change of relative content of LPE (14:0) in healthy persons, patients with rheumatoid arthritis and patients with Lyme arthritis. Abbreviations: LA - Lyme arthritis; RA - rheumatoid arthritis; LPE - lysophosphatidylethanolamine; RT - retention time; ID - abbreviation of compound name; C - control. Log.sub.2 (fold change) LA RA LA versus Value versus Value versus Value m/z RT ID C p C p RA p 424.2464 12.38 LPE 3.937 <0.0001 0.691 ns −3.246 <0.0001 (14:0)

[0072] FIG. 3 shows a box-and-whisker plot which presents graphically changes in the relative content of LPE (14:0) in healthy persons (C), patients with rheumatoid arthritis (RA) and patients with Lyme arthritis (LA). (p<0.0001****). It has been demonstrated that the relative content of LPE (14:0) in patients with Lyme arthritis is statistically significantly higher compared to both healthy human subjects (control) and patients with RA.

[0073] FIG. 4 shows representative exemplary Total Ion Chromatograms (TICs) as a phospholipid profile in negative ionisation mode: plasma from a patient with Lyme arthritis—panel A; plasma from a healthy person—panel B; plasma from a patient with rheumatoid arthritis—panel C.

[0074] FIG. 5 shows representative exemplary Extracted Ion Chromatograms (EICs) in negative ionisation mode for m/z 424.6424 corresponding to the molecular ion of LPE (14:0), retention time RT=12 min: plasma for a patient with Lyme arthritis—panel A; plasma from a healthy person—panel B; plasma from a patient with rheumatoid arthritis—panel C. The difference in the obtained chromatogram profile of a patient with Lyme arthritis (panel A) can be clearly seen, which indicates the presence of LPE (14:0) and at the same time allows the diagnosis of the Lyme disease. A markedly higher peak of lysophospholipid comprising myristic acid, i.e. LPE (14:0) can be noticed in panel A as opposed to panels B and C.

[0075] FIG. 6 shows MS mass spectrum of the peak at RT=12.06 min (top panel); fragmentation MS/MS mass spectrum of an ion having m/z 424.2442 corresponding the molecular ion of LPE (14:0) (bottom panel)—the m/z 227.2016 signal confirms the presence of myristic acid (14:0) in the structure of the fragmented compound.

[0076] FIG. 7 shows representative overlaid mode Extracted Ion Chromatograms (EICs) in negative ionization mode for m/z 424.6424 corresponding to the molecular ion of LPE (14:0), retention time RT=12 min: plasma from a patient with Lyme arthritis—LA; plasma from a healthy person—C; plasma from a patient with rheumatoid arthritis—RA. It can be clearly seen that the highest peak was obtained for the patient with Lyme arthritis, which indicates that the method according to the invention using a biomarker in the form of lysophosphatidylethanolamine comprising myristic acid enables specific diagnosis of Lyme disease, in particular the diagnosis of Lyme arthritis versus a healthy person and differential diagnosis versus a person suffering from RA (but which person does not suffer from Lyme disease or Lyme arthritis or neuroborreliosis)

[0077] The data presented confirm that the methods according to to the invention enable precise, sensitive and specific differentiation between a subject suffering from Lyme disease, especially Lime arthritis, and both a healthy subject and a subject suffering from RA (but which subject does not suffer from Lyme disease or Lyme arthritis).

[0078] In summary, as it can be seen in the above Table 1 and in the presented Figures, the statistically significant differences in the content of LPE (14:0) were obtained in the studied samples from persons suffering from Lyme disease, especially Lyme arthritis versus the samples from healthy persons as well as persons suffering from rheumatoid arthritis (but without Lyme disease or Lyme arthritis or neuroborreliosis). In the samples of plasma obtained from patients with Lyme arthritis the relative content of LPE (14:0) was significantly higher than in the samples of plasma from healthy persons and persons with RA not suffering from Lyme disease/Lyme arthritis. The results obtained indicate that LPE (14:0) may be effectively used as a biomarker of Lyme disease, and in particular as a biomarker of Lyme arthritis, enabling specific diagnosis of Lyme arthritis, especially differential diagnosis of Lyme arthritis versus rheumatoid arthritis.