CZ2017590A3 - A method of detecting a joint disease, a device for detecting joint disease, a substance for determining the cause of a joint disease, its monitoring and the use of this method, substance and device - Google Patents

Abstract

The articular disease detection method involves mixing 100 µl of the articular exudate biological sample in a stabilizing agent with 100 µl of acetonitrile, mixing the sample with a centrifuge for 10 seconds, centrifuging the sample for 5 minutes, separating one sample of articular effusion on the HPLC column at 22 ° C using a linear gradient from 5% acetonitrile to 70% acetonitrile over 60 minutes at a flow rate of 1.0 ml / min, acidification of the mobile phases with 0.5% trifluoroacetic acid, detection of the substance under ultraviolet radiation at 220 nm and application 50 µl. The device for detecting the joint disease and / or determining the appropriate treatment of the joint apparatus consists of a sampling set, an HPLC analysis device, and further comprises a mobile phase of component A consisting of acetonitrile and water of 5:95 and a mobile phase of component B of acetonitrile and water in 70:30. The cause of the joint disease and its monitoring is formed by alpha-defensin obtained from joint exudate, and the alpha-defensin concentration was determined to determine the specific disease.

Description

Technical field

The method concerns the determination of exact concentrations of the antimicrobial peptide alpha-defensin (Human Neutrophile Peptide 1-3 / HNP1-3) as a joint disease and monitoring agent from the collected joint effusion by high-performance liquid chromatography for the diagnosis of infectious diseases in orthopedics and traumatology. Furthermore, the invention relates to a device for detecting joint disease and / or determining a suitable treatment of the joint apparatus and the use of the method, agent and device.

BACKGROUND OF THE INVENTION

The diagnosis of infectious complications in orthopedics is not always clear. Making the right and timely decision whether or not it is a joint infection is critical to the treatment strategy, especially for joint replacements. Often the basic examination methods may not give a clear diagnosis due to the course of the disease. In general, an accurate diagnosis can be made on the basis of medical history, inflammatory markers, joint punctate examination and imaging methods. Inflammatory markers, especially CRP, are highly beneficial, but may not be high in the early stage of the disease, immunosuppressed patients or low virulence of the causative agent and mitigated TEP infections. Of course, high levels of CRP may be due to other site infections, rheumatological or oncological diseases. Imaging methods such as X-rays display bone changes only at later stages as a result of infectious disease. US, MRI displays joint filling, abscess around joint, non-specific inflammatory changes in early stages. Scintigraphic examination seems to be very beneficial, but we know from practice that a negative finding of this examination does not mean that it is not an infectious disease. In addition, the time to obtain an examination result is long in most cases. Highly beneficial for diagnosis is joint puncture, of course, under sterile cautious tubes. Already a macroscopic image can tell what is happening in the affected joint. If we aspire to pus, the diagnosis is usually clear. There is a lack of clarity in the color from yellow, greenish to light brown, sometimes with fibrin attacks. The basic and very important examination is microbiological examination including examination of bacterial susceptibility to antibiotics. Furthermore, a cytological examination of the punctate, which clearly distinguishes inflammatory effusion, non-inflammatory effusion, and possibly the presence of cancer, is also appropriate. Another beneficial examination is PCR for the presence of bacterial agents. Thanks to this examination we can find out if there was an agent of infection in the affected joint, but we do not know anything about bacterial sensitivity. Moreover, the delivery of examination results is usually not fast. There are also other new methods of investigation more or less specific for the diagnosis of infection. E.g. determination of CD64 antigen expression on neutrophils. Although this marker is specific to bacterial infection in the early stages of the disease, it is not commonly available for routine diagnosis.

For rapid diagnosis of joint infectious disease, it is recommended to screen for leukocyte esterase from joint exudate. This is a very easy and fast test using a diagnostic strip. However, we do not always reliably determine a clear diagnosis. From our experience, the presence of joint infection is presented by rapid staining of the diagnostic strip to the desired color (dark purple).

SUMMARY OF THE INVENTION

The invention relates to a method for the detection of joint disease, a device for the detection of joint disease

- 1 CZ 2017 - 590 A3 diseases and / or the identification of appropriate treatment of the joint apparatus and the substance for the determination of the cause of the joint disease and its monitoring.

The object of the present invention, as a very promising diagnostic method for infectious complications in orthopedics, is a method for detecting by examining for the presence of alpha-defensin in joint replacements. The use of this method on a commercial basis is advantageous in view of the clear outcome and benefit of eliminating the hitherto poorly accurate and often complicated treatment procedure. The method is based on the detection of alpha-defensin using HPLC and a diagnostic set.

Alpha-defensin is an antimicrobial peptide produced by activated leukocytes by inflammation, primarily due to bacterial disease.

The present invention relates to a method for determining alpha-defensin levels of HNP1-3 from joint exudate using high performance liquid chromatography (HPLC). The collected joint fluid sample must be applied to a special solution that stabilizes the antimicrobial peptides.

If the examination is positive, ie the presence of alpha-defensin above the determined cut-off concentration, infectious joint replacement disease is diagnosed. The disadvantage of this method in a single examination is that false blood results in the presence of blood in the punctate. However, this examination is a valuable aid in the reimplantation of joint replacements. During surgery we can decide on the type of surgery, which is essential for the success of therapy.

With this method, accurate concentrations of alpha-defensin (human neutrophile peptide, HNP1-3) can be determined.

The advantage of this method is the possibility to determine HNP1-3 even in the presence of blood without affecting the outcome of its presence, because due to the accuracy of the method there is the possibility of disease monitoring in repeated punctures of the affected joint.

Alpha-defensin, or human neutrophile peptide (HNP), is an antimicrobial peptide produced in the region of the locomotor system only by activated leukocytes in response to microbial agents or pro-inflammatory cytokines. These are short cationic peptides with an antimicrobial effect on a wide range of gram-positive and gram-negative bacteria, as well as on some fungi and viruses. The mechanism of action of these antimicrobial peptides is not exactly known. The killing of microorganisms is believed to be due to disruption of the microbial membrane. Thanks to the polar defensin topology, with spatially separated positively charged and hydrophobic regions, AMP allows insertion into phospholipid membranes by having their hydrophobic regions embedded in the lipid portion of the membrane and their positively charged portions interact with anionic phospholipids, thereby damaging the negatively charged membranes bacteria, which ultimately leads to the lysis of microorganisms.

The diagnosis of infectious joint disease is thus based on the theory of alpha-defensin production by activated leukocytes.

The invention is based on a method for determining levels of alpha-defensin (HNP1-3) by HPLC. Based on measurements from a group of patients diagnosed with both purulent arthritis and unpululent arthritis and infected joint replacements, the authors concluded that the interpretation of the level of alphadefensin from articular effusion in TEP was more explicit. In arthritis, the level of alpha-defensin in the joint exudate is also increased in rheumatic diseases. The aim of the research was to determine the "cutoff ⁴ concentration of alpha-defensin" for infectious complications of TEP and "cut-off" concentration of alpha-defensin for purulent arthritis.

-2GB 2017 - 590 A3

Finding the optimal diagnostic method for the determination of infectious disease in orthopedics is very important. From clinical experience we know that in many cases the diagnosis of joint filling is not entirely clear, especially in joint replacements. At the same time, a timely and correct diagnosis is crucial for the patient if his or her disease is resolved. It seems that we do not yet know such a marker in orthopedics that would immediately reveal to us that it is 100% or not an infectious disease. Except for culture and PCR, all laboratory screening methods are focused on the immune system's response to the presence of the causative agent of the infectious disease. However, the immune system responds in a similar or the same way to other diseases (especially rheumatological diseases).

Recently, much work has been done to determine the presence of alpha-defensin in joint punctate to determine infectious complications in joint replacements. Long-flow immunochromatographic techniques (Synovasure®) or immunohistochemical determination of alpha-defensin levels (Dermigian et al 2015, Parvizi) are most commonly used to determine alpha-defensin levels from joint exudate.

Based on the results of our HPLC measurements, we conclude that the determination of alpha-defensin concentrations from joint exudate in joint replacements makes the diagnosis of infectious disease more explicit. In arthritis, alpha-defensin is also produced by neutrolils in rheumatological diseases and reactive arthritis.

Therefore, our “cut off” concentration for purulent arthritis is 60 mg / l higher compared to the “cut off” concentration for joint replacement infections. Furthermore, there is also a so-called “gray zone” in which all the above-mentioned diagnoses in arthritis can be included. We also verified that the more active the disease, the higher the levels of HNP1-3 are. In very rare cases, we found very high concentrations of HNP1-3 (up to 700 mg / l) in rheumatoid arthritis during its active phase. However, neutrolils appear to be most productive in the production of alphadefensine in bacterial infection. In several such cases we measured the concentration of HNP 1-3 even above 2000 mg / l from the joint exudate by the HPLC method.

According to our results, arthritic joint damage in most cases does not produce HNP 1-3 neutrophils. Only in a small number of patients with gonarthrosis, HNP1-3 was present at concentrations up to 13 mg / l.

As mentioned above, the presence of alpha-defensin in the joint exudate is a response to activated neutrolils. They appear to be most active in the presence of a bacterial infection. However, alphadefensine is present in the joint punctate also in rheumatological diseases and reactive arthritis. At very high levels of this antimicrobial peptide, the cause of its production is clear. At lower levels of HNP 1-3, diagnostics may not be unequivocal with this marker alone. Therefore, it is necessary to use other diagnostic methods for correct diagnosis. E.g. CRP in combination with HPN1-3 levels largely refines the diagnosis of the cause of joint filling. Purulent arthritis is characterized by a high concentration of alpha-defensin in the joint exudate and an increased level of CRP in the patient's blood or joint exudate. At high levels of CRP in the blood and low concentrations of HNP 1-3 in the joint exudate, the cause of the joint filling is more likely due to rheumatological disease.

In conclusion, by measuring alpha-defensin levels from the joint exudate, we can better diagnose infectious complications in orthopedics, especially in joint replacements. This has a very high benefit not only for the patient but also for the whole society. Based on our results, HPLC determination of HNP 1-3 is an important marker for the diagnosis of purulent arthritis. Moreover, the results of the measurement we introduce are not influenced by the presence of blood in the sample and we have a unique opportunity to objectively monitor the dynamics of the disease.

-3 2017 - 590 A3

List of drawings:

Giant. 1 shows a diagram of the measurement procedure by the HPLC method, respectively. chromatogram to determine the concentration of alpha-defensin.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

The method for detecting joint disease and / or determining appropriate treatment for the joint apparatus has been verified by the following procedure. There were 151 patients who had a joint puncture for various causes. Patients were selected from different ages with joint filling both with and without endoprosthesis. Sampling of the joint exudate always took place under sterile conditions, mainly in the outpatient department, but also in the operating room. Based on medical history, clinical examination, laboratory tests and imaging methods, patients were divided into groups:

- osteoarthritis (30 patients)

- clear bacterial TEP infection (17 patients),

- without TEP infection (25 patients),

- purulent arthritis (33 patients),

rheumatoid disease (28 patients), and

- reactive arthritis (18 patients).

The collected articular effusion was pipetted into the stabilizer tube (acetonitrile with HPLC water in a 1: 1 by volume with trifluoroacetic acid to 0.5% concentration in this mixture) in an accurate amount of 1 ml by pipette. . The samples prepared in this way were further processed at the Institute of Medical Chemistry and Clinical Biochemistry of the 2nd Faculty of Medicine and Charles University Hospital in Motol, where the HNP 1 -3 concentrations from the joint exudate of the patient were measured using high-performance liquid chromatography.

For the purpose of collecting and dispatching samples to the laboratory, a kit according to the present invention is provided, which is part of a device for detecting joint disease and / or determining a suitable treatment for the joint apparatus of the invention, comprising the following elements:

- 1 x test tube with 4 ml of 1: 1 acetonitrile-HPLC stabilizer with trifluoroacetic acid at a concentration of 0.5% in this mixture;

- 10 ml plastic tube with plastic screw cap;

- 1x 1 ml syringe with cone;

- 2x injection needle with 1.2 mm needle diameter;

The kit may further include a cover sheet, a biochemical examination request, and an envelope for transporting the tubes with the address of the testing laboratory.

The sample processing is as follows:

- Joint puncture under sterile cautious tubes and aspiration of the effusion with a syringe from the package (1 ml) or collection of the effusion with a 1 ml syringe from the tube.

-4GB 2017 - 590 A3

- Inject exactly 1 ml of the articular punctate from the packed syringe into a stabilizer tube (labeled "Alpha-defensin // HPLC"). If the amount of joint punctate is smaller, less can be applied - this volume must be recorded on the application for biochemical examination.

- Labeling the test tube with a “patient identification label and filling out a biochemical examination request.

- Dispatch of the labeled test tube with the completed biochemical examination request to the laboratory

Example 2

Device and method for detecting joint disease and / or determining a suitable treatment of the joint apparatus of the present invention:

The device for detecting joint disease and / or determining a suitable treatment for the joint apparatus consists of a set according to Example 1 and an Agilent 1260 Infinite HPLC system (Agilent Technologies, USA) with a diode array detector.

Separation is performed on a Vydac 218 TP C18 column (250 x 4.6 mm, 5 µm, Grace) at 22 ° C.

Alpha-defensin is detected at 220 nm. A linear gradient of mobile phases A and B at a flow rate of 1 ml / min is used for separation.

Mobile phase A consists of acetonitrile and water in a ratio of 5:95, mobile phase B consists of acetonitrile and water in a ratio of 70:30.

Trifluoroacetic acid is added to both phases to a final concentration of 0.5% (v / v).

The sample volume is 50 μΐ.

The analyzes are evaluated using OpenLab software (Chem 32, Agilent Technologies). The total analysis time is 60 minutes.

The method for detecting joint disease and / or determining a suitable treatment for the joint apparatus consists of the following steps:

- mixing 100 μΐ of the biological sample in the stabilizing agent with 100 μΐ of acetonitrile

- sample mixing (vortex, 10 s)

- centrifugation (5 minutes)

- HPLC analysis of the prepared sample according to the above conditions

The measurement results assigned to individual patients from the above mentioned groups were statistically processed.

Statistical software GraphPad Prism, version 6.01 (San Diego, CA) was used to evaluate the data. The probability level P used was less than 0.05. Names of tests used: MannWhitney Theorem U, limit values were determined by ROC analysis (ROC). Agostino D-Pearsob's data distribution normality test was used to determine normality (data distribution).

Results and amount of the substance for the determination of the cause of the joint disease and its monitoring with the suggestion of suitable treatment:

-5 CZ 2017 - 590 A3 “Cut off”, ie the lowest value, the alpha defensin concentration (HNP1-3) for joint replacement infection is 38 mg / l (sensitivity: 0.94, specificity: 1) “Cut off” alpha concentration -defensin (HNP1-3) for reactive arthritis is 8 mg / l (sensitivity 0.79, specificity 0.9) The cut-off concentration of alpha-defensin (HNP 1-3) for arthritis of rheumatoid origin is 16.5 mg / l (sensitivity 0.68, specificity 0.93) "Cut off" concentration of alpha-defensin (HNP1-3) for purulent arthritis is 98 mg / l (sensitivity: 0.97, specificity: 0.87) "GRAY ZONE for purulent arthritis is 63 - 108 mg / l.

The device for the detection of joint disease consists of a set consisting of the following parts:

- 1 x 1: 1 acetonitrile stabilizer tube with HPLC water with trifluoroacetic acid;

- 10 ml plastic tube with plastic screw cap;

- 1x syringe;

- 2x injection needle;

and an HPLC analysis apparatus comprising an HPLC system equipped with a diode array detector comprising, for the separation step, a 4.6 x 250 mm column with a particle size of 5 µm and further comprising a mobile phase of component A consisting of acetonitrile and water in a ratio of 5: 95 and a mobile phase of component B consisting of acetonitrile and water in a ratio of 70:30 and a substance for the determination of the cause of joint disease and its monitoring, consisting of alpha-defensin obtained from joint exudate and detected by HPLC analysis.

The volume of the stabilizer is 4 ml of acetonitrile with HPLC water (1: 1 by volume with trifluoroacetic acid), the concentration of which in this mixture is 0.5%.

Industrial applicability

The method, device and substance of the present invention are useful in the medical field, particularly in orthopedics and traumatology, for the treatment of joint disease detection and / or the determination of a suitable treatment of the joint apparatus, and the compound is useful for determining and monitoring the cause of joint disease.

PATENT CLAIMS

Claims (9)

-1 x syringe; -1 x acetonitrile stabilizer tube with HPLC water 1: 1 by volume with trifluoroacetic acid; - 10 ml plastic tube with plastic screw cap; A method for detecting a joint disease comprising the following steps: - mixing 100 μΐ of the biological joint exudate sample in the stabilizing agent with 100 μΐ of acetonitrile; - agitate the sample by centrifuge for 10 seconds, - centrifuging the sample for 5 minutes, -6GB 2017 - 590 A3 separating one joint exudate sample on an HPLC apparatus at 22 ° C using a linear gradient from 5% acetonitrile to 70% acetonitrile over 60 minutes at a mobile phase flow rate of 1.0 ml / min; acidifying the mobile phases with 0.5% trifluoroacetic acid; - detection of the substance under ultraviolet radiation at a wavelength of 220 nm; - administration of the centrifuged sample to the injection at 50 μΐ. - 2 x injection needle; and an HPLC analysis apparatus comprising an HPLC system equipped with a diode array detector comprising, for the separation step, a 4.6 x 250 mm column with a particle size of 5 µm and further comprising a mobile phase of component A consisting of acetonitrile and water in a ratio of 5: 95 and a mobile phase of component B consisting of acetonitrile and water in a ratio of 70:30 and a substance for the determination of the cause of joint disease and its monitoring, consisting of alpha-defensin obtained from joint exudate and detected by HPLC analysis. A device for detecting joint disease and / or determining a suitable treatment for the joint apparatus, characterized in that it comprises a set consisting of the following elements: The joint disease detection device according to claim 2, wherein the stabilizer volume is 4 ml and the stabilizer consists of acetonitrile with HPLC water in a 1: 1 by volume mixture with trifluoroacetic acid, the concentration of which in this mixture is 0.5%. A joint disease detection device according to claim 2, characterized in that the volume of the syringe with a cone is 1 ml and the needles have a diameter of 1.2 mm. Use of the method of claim 1 for detecting joint diseases. A joint disease detection device according to any one of claims 1, 2, 3 and or 4, characterized in that it further comprises parts and / or parts of the device consisting of at least: a digital agitator, rotator and / or shaker, centrifuge or microcentrifuge; applicator, separator, column, UV detector or microscope, which are used to: mixing the biological sample of the joint exudate in the stabilizing agent with acetonitrile; - mixing the sample by means of a centrifuge, - centrifuging the sample, - separating the joint exudate sample on the HPLC device using a linear gradient from 5% acetonitrile to 70% acetonitrile at a mobile phase flow rate of at least 1.0 ml / min .; acidifying the mobile phases with trifluoroacetic acid; - detection of the substance under ultraviolet radiation; - injecting the centrifuged sample. 6. A substance for the determination and monitoring of a joint disease consisting of alpha-defensin obtained from joint exudate and detected by HPLC analysis according to claim 2, characterized in that: - for infection of joint replacements, the cut-off concentration of alpha-defensin is 38 mg / l of the sample; - for reactive arthritis, the cut-off concentration of alpha-defensin is 8 mg / l of the sample; - for arthritis of rheumatic origin, the cut-off concentration of alpha-defensin is 16.5 mg / l of the sample; - 7 2017 - 590 A3 - for purulent arthritis, the cut-off concentration of alpha-defensin is 98 mg / l of sample; Use of the device according to claims 2, 3, 4 and or 5 for detecting joint disease and / or determining a suitable treatment for the joint apparatus. ío Use of a substance including a device according to claims 2 and 3, 4a or 5 for determining the cause of a joint disease and monitoring it.