RU2713911C1 - Method for assessing the activity of an inflammatory process in chronic abacterial prostatitis/chronic pelvic pain syndrome - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to urology, and enables assessing the activity of a local inflammatory process in the prostate gland in chronic abacterial prostatitis/chronic pelvic pain syndrome (CAP/CPPS). That is ensured by collecting the biomaterial after clinical examination of the patients and prostate massage followed by placing the biomaterial in test tubes with a stabilizer. Further, the obtained biomaterial samples are analyzed by solid-phase enzyme-linked immunosorbent assay to determine interleukin-8 concentration. Biomaterial is presented by post massage portion of urine wherein the method of solid-phase immunoassay enables additionally determining the concentrations of interleukin-6 and monocytic chemoattractant protein. Obtained concentration of cytokines shows activity of a local inflammatory process. If one of the concentrations of said cytokines increases: more than 14 pg/ml for interleukin-6; 48 pg/ml for interleukin-8 and 362 pg/ml for monocytic chemoattractant protein shows the presence of an inflammatory process in the prostate gland.

EFFECT: invention enables assessing the activity of the inflammatory process in the prostate gland in CAP/CPPS in all patients with high accuracy and sensitivity.

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Description

The invention relates to medicine, namely to urology, and can be used to study the activity of a local inflammatory process in the prostate gland in order to select an adequate method for the treatment of chronic abacterial prostatitis / chronic pelvic pain syndrome (CAP / SCTB), as well as to monitor the effectiveness of therapy.

Chronic prostatitis (CP) is one of the most common diseases in modern urological practice and has a significant negative impact on the quality of life of men. According to the European Association of Urology (EAU), symptoms of CP occur in 1-14.2% of men [D. Engeler et al. Chronic pelvic pain. EAU Guidelines. 2018]. Moreover, according to the modern classification [G. Bonkat, R. Pickard et al. Urological infections. EAU Guidelines. 2018] distinguish 4 categories of CP: I-acute bacterial prostatitis, II- chronic bacterial prostatitis; III - chronic abacterial prostatitis or chronic pelvic pain syndrome (A-inflammatory, B-non-inflammatory) and IV - asymptomatic prostatitis (established histologically). However, the bacterial origin of inflammation is confirmed in no more than 10% of patients; other men are diagnosed with CAP / CFS [G. Bonkat, R. Pickard et al. Urological infections. EAU Guidelines. 2018]. Guidelines EAU HAP / SCTD is determined by the presence of constant or periodic pain in the prostate gland, at least 3 of the last 6 months, which are reproduced upon palpation, in the absence of data for infectious or any (from other organs and systems) genesis of pathology [ D. Engeler et al. Chronic pelvic pain. EAU Guidelines. 2018]. Categories III A (inflammatory) and III B (non-inflammatory) differ from each other by the presence of leukocytes in the secretion of the prostate / semen / post-massage portion of urine. Two categories of CAP / SCTB are currently presented as varieties of one disease with a complex, multifaceted and pathogenesis that differs in a number of aspects [V. Bozhedomov. Chronic prostatitis: a new treatment paradigm. Urology. 2016; No. 3. Appendix 3. S. 1-12]. In this regard, in the medical science and clinical practice, a multimodal approach to the treatment of CAP / CFS is currently prevailing [Magri V. et al. Multidisciplinary approach to prostatitis. Arch Ital Urol Androl. 2019 Jan 18; 90 (4): 227-248. doi: 10.4081 / aiua.2018.4.227], and the treatment tactics are determined by the diagnostic criteria obtained on the basis of patient complaints, physical and laboratory-instrumental examination data. One of the modern approaches to the diagnosis and treatment of patients with CAP / CFS is phenotyping, in particular, using the UPOINT system [Nickel J.C, Shoskes D. Phenotypic approach to the management of chronic prostatitis / chronic pelvic pain syndrome. Curr Urol Rep. 2009 Jul; 10 (4): 307-12]. Nevertheless, most experts state that the problem of CAP / CFS is currently far from being resolved due to the low rates of its treatment effectiveness [Magri V. et al. Multidisciplinary approach to prostatitis. Arch Ital Urol Androl. 2019 Jan 18; 90 (4): 227-248. doi: 10.4081 / aiua.2018.4.227]. Significant is the fact that CP and, in particular, HAP / SKhTB affects mainly men of working age and can lead to the formation of chronic pain, impaired sexual function, impaired reproductive potential of men and, accordingly, demographic indicators of the population. Thus, the search for new methods for the diagnosis and treatment of CAP / APS is one of the priority and socially important tasks of modern urology.

A known method for diagnosing CP is known, which consists in conducting a test proposed by Meares and Stamey in 1968 [Meares E.M, Stamey TA. Bacteriologic localization patterns in bacterial prostatitis and urethritis. Invest Urol. 1968 Mar; 5 (5): 492-518], the essence of which is a microscopic examination of the first (VB1), medium (VB2) portions of urine, prostate secretion (EPS) after massage and the last portion of urine (VB3). This method is used to topically diagnose the inflammatory process in the lower urinary tract. Also, for the differential diagnosis of various forms of prostatitis, a two-glass test is performed according to Nickel [Nickel J.C. The Pre and Post Massage Test (PPMT): a simple screen for prostatitis. Tech Urol. 1997 Spring; 3 (1): 38-43], which consists in conducting microscopic and microbiological studies of pre- (VB2) and post-massage (VB3) portions of urine.

Significant disadvantages of the above methods are:

• the absence of guaranteed secretion of the prostate gland during massage, which may be due to both the features of the manipulation technique and the obstruction of the excretory ducts of the gland;

• the possibility of contamination of prostate secretion and post-massage portion of urine by microbiota of a different location;

• dependence of the results of a microscopic examination on the qualifications of a specialist and equipment (a standardized approach and an objective assessment of the study are difficult);

• long periods of time and the large number of bacteriological studies, the need for appropriate laboratory equipment;

• lack of information on the pathogenetic mechanisms of CAP / APS.

Other methods for the diagnosis of CP and, in particular, HAP / SKTB are also known.

In the patent of the Russian Federation No. 2210779 bull. No. 23, 2003. IPC G01N 33/92 describes “A method for diagnosing the activity of the course of chronic prostatitis,” based on a study of the secretion of the prostate gland, in which the concentration of diene conjugates and Schiff bases is determined, the coefficient of inflammation activity is calculated, and with a decrease in this indicator, the activity of the course chronic prostatitis is increasing.

Patent RU (11) 2,450,270 IPC G01N 33/483 describes a "Method for the differential diagnosis of chronic prostatitis" based on microscopy of prostate secretion and microbiological analysis of sperm before and after three sessions of low-intensity infrared laser therapy, followed by the final determination of the CP category, depending on the results research.

In RF patent No. 2103691, Bull. No. 3, 1998. IPC G01N 33/53, 33/48 describes a "Method for diagnosing the activity of the course of chronic prostatitis", which consists in determining the level of soluble leukocyte antigen - 2 (RAL - 2) in the ejaculate and in the diagnosis of chronic prostatitis with its content of 20 μg / ml and above.

Patent RU 11 2269783 IPC G01 N 33/573 presents a "Method for the diagnosis of chronic abacterial prostatitis", the essence of which is to determine the activity of a 1-proteinase inhibitor in prostate secretion. With its value from 2.0 to 3.0 IE / ml, chronic abacterial prostatitis (HAP) of a non-inflammatory form is diagnosed, and at 3.0 IE / ml and higher, an inflammatory HAP is diagnosed.

Patent RU 11 2344426 IPC G01 N33 / 84 describes a "Method for the differential diagnosis of forms of chronic abacterial prostatitis", characterized in that the semen determines the content of nitric oxide released by macrophage NO synthase and by reducing the amount of NO produced by sperm cells from 38 to 26 [n / M 2 · 105 cells] · 10-3 diagnosed with chronic abacterial prostatitis (CAP) of the inflammatory form, and with a content of 23 [n / M 2 · 105 cells] · 10-3 and below - CAP non-inflammatory form.

The disadvantages of the above methods are the inaccuracy of the diagnosis of the inflammatory form of CAP / CFS, related, including when studying the secretion of the prostate gland, with the difficulty of obtaining a sufficient amount of this type of material in some patients with suspected CAP / CTC; and in the study of sperm - with the presence of a sufficiently high probability of other reasons for increasing the above indicators (for example, inflammation of a different localization).

Also known are methods of differential diagnosis of CP forms based on the determination of kallikrein activity in blood serum (Patent No. RU (11) 2272294); the activity of superoxide dismutase and catalase (Patent No. RU (11) 2315318), the activity of nitric oxide released by macrophage NO synthase (Patent No. RU (11) 2359273). The use of these methods requires the need for a highly qualified laboratory and specific equipment, which can lead to difficulties in reproducing these methods. In addition, the concentration of these substances in the peripheral blood gives little information about the state of local immunity in the prostate gland.

 In the patent RU 11 2263915 IPC G 01 N 33/493 authors Eseneev S.M. and Boryak V.P. describe the "Method for the diagnosis of chronic prostatitis", which determines the number of leukocytes in 1 ml of pre-massage and post-massage portions of morning urine, and based on the quantitative increase in the concentration of leukocytes, the category of chronic prostatitis is established. The disadvantages of this method are the inaccuracy in the diagnosis of the inflammatory form of CAP / SCTB, subjectivity in determining and counting white blood cells.

There is also a diagnostic method described in the article by Naresh V. Desireddi, Phillip L. Campbell, Jeffrey A. Stern, Rudina Sobkoviak et.al. “Monocytic chemoattractant protein-1 (MCP) and macrophage inflammatory protein-1α (MIP) as possible biomarkers of chronic pelvic pain syndrome” [The J Urol. 2008 May; 179 (5): 1857–1862. doi: 10.1016 / j.juro.2008.01.028]. This method involves the study of the cytokines MIP and MCP-1 in the secretion of the prostate gland of patients with CAP / APS.

The closest analogue to the claimed invention in technical essence is the diagnostic method described in the article by Huang T.R., Li W., Peng B. "Correlation of inflammatory mediators in prostate secretion in patients with chronic prostatitis and chronic pelvic pain syndrome" [Andrologia. 2018 Mar; 50 (2). doi: 10.1111 / and.12860. Epub 2017 Aug 1.] This method is based on the determination of cytokines (including interleukin-8) in the secretion of the prostate gland of patients with CP and CAP / SCT.

The main disadvantage of this diagnostic method is its complexity, which consists in obtaining this type of biomaterial for research (for many patients, obtaining prostate secretion is difficult) and, accordingly, standardizing the stages of the study and interpreting the results.

The objective of the invention is the development of a method for assessing the activity of the inflammatory process in the prostate gland with CAP / SCTB, available for all patients, with high accuracy and sensitivity, as well as standardized at all stages of the process.

The essence of the claimed invention is that in the method for assessing the activity of a local inflammatory process in the prostate gland in case of chronic abacterial prostatitis / chronic pelvic pain syndrome, HAP / SCTD, which involves collecting the biomaterial after a clinical examination of the patients and carrying out massage of the prostate gland, followed by its placement in test tubes with stabilizer, the study of the obtained biomaterial samples by the method of enzyme-linked immunosorbent assay with the determination of the concentration of interleukin-8 (IL-8), as a biomaterial, use a post-massage portion of urine, in which the concentration of interleukin-6 (IL-6) and monocytic chemoattractant protein (MCP-1) is additionally determined by the method of enzyme-linked immunosorbent assay, according to the obtained concentration of cytokines about the activity of the local inflammatory process, with an increase in one of the concentrations of these cytokines: above 14 pg / ml for interleukin-6; 48 pg / ml for interleukin-8 and 362 pg / ml for monocytic chemoattractant protein indicate the presence of an inflammatory process in the prostate gland.

The technical result of the claimed invention.

Such a technique as the use of a post-massage portion of urine as a biomaterial under study allows an assessment of the activity of the inflammatory process in all patients in connection with the availability of biomaterial, which was absent in the closest analogues. At the same time, this type of biomaterial is stable when storing all the ingredients necessary for carrying out ELISA (up to a year). This method has high accuracy and sensitivity in connection with an additional study of the concentrations of IL-6 and MCP-1. The use of post-massage portion of urine as a biomaterial also allows standardizing all stages of the reaction, simplifying the process and improving the accuracy of the results.

A method for assessing the activity of a local inflammatory process in the prostate gland with CAP / SCTB, based on determining the levels of IL-6, IL-8 and MCP-1 cytokines in a post-massage portion of urine, allows:

• Based on the data obtained, evaluate the presence of local inflammation in the prostate gland;

• on the basis of the results obtained, carry out timely adequate pathogenetic treatment;

• based on the results obtained, monitor the ongoing therapy of the disease.

The invention is illustrated using Fig., Which depicts a system for collecting urine. In FIG. positions 1-5 are indicated:

1 - sterile container with a built-in holder of a vacuum tube for urine culture;

2 - sterile vacuum tubes for urine analysis with various stabilizers;

3 - holder for the safe selection of urine into a test tube from containers of various types;

4 - sterile container for urine;

5 - a sterile container for urine with a cross-shaped valve integrated in the lid.

A method for assessing the activity of the inflammatory process in chronic abacterial prostatitis / chronic pelvic pain syndrome is used as follows.

Initially, a clinical examination of the patient is carried out using standard methods, that is, with the assessment of complaints and somatic status, a detailed medical history with the identification of factors affecting the immune status, a urological examination with a digital examination of the prostate gland is transrectal. Also, the patient is additionally examined for sexually transmitted infections by polymerase chain reaction.

After a clinical examination of the patient, biomaterial is collected - a post-massage portion of urine. The collection of biomaterial is as follows: before conducting prostate massage, the patient urinates. Then, prostate massage is performed according to a standard technique. After massage of the prostate gland for up to 5 minutes, the patient urinates and the first 10-30 ml of post-massage portion of urine is collected in a container with a valve cover. Immediately after collecting urine, the container is tightly closed with a screw cap (Fig.). A urine sample is delivered to the laboratory within 2 hours after collection at + 4 ° C; the sample must not be frozen.

In the laboratory, for sample preparation of samples of a post-massage portion of urine and subsequent determination of the profile of cytokines, it is transferred using the holder (Fig.) Into urine tubes with a stabilizer and numbered. In test tubes, urine can be stored for analysis no more than 2-3 hours. Using solid-phase ELISA test freshly prepared samples. If this is not possible, then to exclude bacterial growth, the sample can be transferred to Eppendorf tubes and stored frozen at T -20-40 ° C if later analysis is necessary, and even one-time thawing of the samples is not allowed.

Determination of the level of cytokines in the urine is carried out by the method of enzyme-linked immunosorbent assay using reagent kits.

Clinical example 1.

Patient "F", 47 years old. He sought medical help with complaints of pain in the inguinal region, radiating to the perineum, testicles, intensifying after intercourse, episodes of nocturia up to 2 times per night, periodically-frequent urge to urinate. These complaints bothered the patient for several years, several times received courses of antibacterial, anti-inflammatory therapy, but did not pass a control examination. The current deterioration during the week when, after hypothermia, he again noted the appearance of the above symptoms. The survey was conducted according to the standards:

1) Sample by Meares-Stamey:

VB1 - white blood cells 0-1 in the field of view, other indicators within normal limits

VB2 - leukocytes 0-1 in the field of view, other indicators within normal limits, microflora growth during bacteriological examination was not found

EPS-leukocytes 35-40 in the field of view, epithelium +, lecithin grains in a small amount.

VB3-leukocytes 25-30 in the field of view, microflora growth during bacteriological examination was not detected.

Prostate TRUS: volume 28 cm3, diffuse heterogeneous structure of the prostate gland, with multiple sites of inflammatory infiltration in the periurethral zone.

2) In addition to the standard survey conducted

• PCR testing for STIs: without pathology.

• The indicators of the cytokine profile in the post-massage portion of urine were determined:

IL-6 - 25.553 pg / ml, IL-8 - 384.18 pg / ml, MCP-1 - 603.175 pg / ml.

 Based on standard criteria and the level of cytokines, the diagnosis was established: Chronic abacterial prostatitis / chronic pelvic pain syndrome, category III A. A course of standard antibacterial, anti-inflammatory therapy was prescribed. Follow-up examination after 1 month:

 There are no clinical complaints.

1) Sample by Meares-Stamey:

VB1 - white blood cells 0-1 in the field of view, other indicators within normal limits

VB2 - leukocytes 0-1 in the field of view, other indicators within normal limits, microflora growth during bacteriological examination was not found

EPS-leukocytes 5-8 in the field of view, lecithin grains significantly.

VB3-leukocytes 0-1 in the field of view, microflora growth during bacteriological examination was not detected.

2) TRACES of the prostate: volume 23 cm3, areas of inflammatory infiltration not determined.

3) The indicators of the cytokine profile in the post-massage portion of urine:

IL-6 - 3.802 pg / ml, IL-8 - 36.333 pg / ml, MCP-1 - 122.352 pg / ml.

Thus, the effectiveness of the treatment based on a comprehensive examination (both standard methods and dynamics of the level of cytokines) was ascertained.

Clinical example 2.

Patient C, 34 years old. He sought medical help with complaints of periodic pain in the perineum, frequent urination, deterioration in the quality of an erection. These complaints bothered the patient for 4 months, he took the drugs on his own (it is difficult to indicate). The survey was conducted according to the standards:

1) Sample by Meares-Stamey:

VB1 - white blood cells 0-1 in the field of view, other indicators within normal limits

VB2 - leukocytes 1-2 in the field of view, other indicators within normal limits, microflora growth during bacteriological examination was not found

EPS-leukocytes 25-30 in the field of view, epithelium ++, lecithin grains in a small amount.

VB3-leukocytes 10-15 in the field of view, microflora growth during bacteriological examination was not detected

1. Prostate TRUS: volume 26 cm3, diffuse-heterogeneous structure of the prostate gland, with isolated sites of inflammatory infiltration.

In addition to the standard survey conducted

• PCR testing for STIs: without pathology.

• The indicators of the cytokine profile in the post-massage portion of urine were determined:

IL-6 - 20.424 pg / ml, IL - 8 - 294.368 pg / ml, MCP-1 - 826.856 pg / ml.

The diagnosis was established: Chronic abacterial prostatitis / chronic pelvic pain syndrome, category III A. A course of standard antibacterial, anti-inflammatory therapy was prescribed. Follow-up examination after 1 month:

Clinically: against the background of treatment, positive dynamics, however, complaints remain about periodic aching pain in the perineum, worsening erection.

1) Sample by Meares-Stamey:

VB1 - leukocytes 0-1 in the field of view, other indicators within normal limits.

VB2 - leukocytes 1-2 in the field of view, other indicators within normal limits, microflora growth during bacteriological examination was not found

EPS-leukocytes 5-8 in the field of view, lecithin grains moderately.

VB3-leukocytes 0-1 in the field of view, microflora growth during bacteriological examination was not detected.

2) TRACES of the prostate: volume 24 cm3, sites of inflammatory infiltration not determined.

3) The indicators of the cytokine profile in the post-massage portion of urine:

IL-6 - 13.047 pg / ml, IL-8 - 204.344 pg / ml, MCP-1 - 654.324 pg / ml.

Thus, on the basis of clinical data and the levels of IL-8 and MCP-1, insufficient treatment efficiency and a persistent high activity of the inflammatory process were ascertained, although standard indicators (white blood cell count in EPS and VB3) reached normal values. An additional course of drug treatment in combination with physiotherapy (pneumovibromassage of the prostate) was prescribed.

Follow-up examination after 1 month:

Clinically: against the background of treatment, positive dynamics, no complaints.

1) Sample by Meares-Stamey:

VB1 - white blood cells 0-1 in the field of view, other indicators within normal limits

VB2 - leukocytes 0-1 in the field of view, other indicators within normal limits, microflora growth during bacteriological examination was not found

EPS-leukocytes 0-5 in the field of view, lecithin grains significantly.

VB3-leukocytes 0-1 in the field of view, microflora growth during bacteriological examination was not detected.

2) The indicators of the cytokine profile in the post-massage portion of urine:

IL-6 - 6.025 pg / ml, IL-8 - 40.538 pg / ml, MCP-1 -74.78 pg / ml.

Remission was ascertained.

Clinical example 3.

Patient "A", 22 years old. He sought medical help with complaints of pain in the suprapubic region, as well as dysuric phenomena - frequent urination, in small portions, mainly in the daytime. These complaints bothered the patient for a year, did not seek medical help. Sexual life is irregular. The survey was conducted according to the standards:

1) Sample by Meares-Stamey:

VB1 - white blood cells 0-1 in the field of view, other indicators within normal limits

VB2 - leukocytes 0-1 in the field of view, other indicators within normal limits, microflora growth during bacteriological examination was not found

EPS- was not obtained after prostate massage.

VB3-leukocytes 10-12 in the field of view, the growth of microflora during bacteriological examination was not detected

2) Prostate TRUS: the patient refused to perform the procedure due to psychological discomfort.

In addition to the standard survey conducted

• PCR testing for STIs: without pathology.

• The indicators of the cytokine profile in the post-massage portion of urine were determined:

IL-6 - 18.492 pg / ml, IL-8 - 271.101 pg / ml, MCP-1 - 529.671 pg / ml.

 The diagnosis was established: Chronic abacterial prostatitis / chronic pelvic pain syndrome, category III A. A course of standard antibacterial, anti-inflammatory therapy, physiotherapy treatment was prescribed. Follow-up examination after 1 month:

 There are no clinical complaints.

1) Sample by Meares-Stamey:

VB1 - white blood cells 0-1 in the field of view, other indicators within normal limits

VB2 - leukocytes 0-1 in the field of view, other indicators within normal limits, microflora growth during bacteriological examination was not found

EPS-leukocytes 5-8 in the field of view, lecithin grains significantly.

VB3-leukocytes 0-1 in the field of view, microflora growth during bacteriological examination was not detected.

4) The indicators of the cytokine profile in the post-massage portion of urine:

IL-6 - 5.92 pg / ml, IL-8 - 19.728 pg / ml, MCP-1 - 114.855 pg / ml.

Thus, the effectiveness of the treatment based on a comprehensive examination was ascertained. In this case, the study of cytokine concentrations provided important additional information about the presence of CAP / SCTB IIIA, which could not be obtained by other methods (due to the impossibility of obtaining prostate secretion and the patient's refusal to perform ultrasound examination) and made it possible to verify the positive dynamics as a result of the therapy.

The method was tested in the urology clinic of the Saratov State Medical University, the Research Institute for Fundamental and Clinical Uronephrology of the Saratov State Medical University and the Central Scientific Research Laboratory of the Northern State Medical University for 112 patients and showed high information content.

In order to assess the reliability of the application of the proposed method, the number of leukocytes in the post-massage portion of urine was simultaneously counted. Microscopy of this type of biomaterial was carried out with the calculation of the number of leukocytes. For the norm, the number of leukocytes is up to 10 cells in the field of view, an increase in the number of leukocytes indicates the presence of an inflammatory process (CAP / SCTB category IIIA). These data coincided with the conclusions made by the claimed method - the presence of an inflammatory process in the tissue of the prostate.

The advantage of the proposed method compared to the closest analogue is that it allows you to get biomaterial in all patients (the study of a post-massage portion of urine is possible in all cases, unlike microscopy of the secretion of the prostate gland), to conduct an objective standardized diagnosis of CAP / CPS and to evaluate the presence of the inflammatory process , and can also be used to monitor the effectiveness of treatment using common criteria.

Claims (1)

A method for evaluating the activity of a local inflammatory process in the prostate gland in case of chronic abacterial prostatitis / chronic pelvic pain syndrome HAP / SCTB, which includes collecting the biomaterial after a clinical examination of the patients and performing massage of the prostate gland, then placing it in test tubes with a stabilizer, studying the obtained biomaterial samples using the solid-phase method enzyme-linked immunosorbent assay with determination of the concentration of interleukin-8, characterized in that as of the material, a post-massage portion of urine is used, in which the concentration of interleukin-6 and the monocytic chemoattractant protein are additionally determined by the enzyme-linked immunosorbent assay, the activity of the local inflammatory process is judged by the obtained concentration of cytokines, with an increase in one of the concentrations of these cytokines: above 14 pg / ml for interleukin- 6; 48 pg / ml for interleukin-8 and 362 pg / ml for monocytic chemoattractant protein indicate the presence of an inflammatory process in the prostate gland.

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