RU2265656C1 - Nutrient medium for detecting ureaplasma urealyticum and method for diagnosis of urogenital ureaplasmosis - Google Patents

Abstract

FIELD: medicinal microbiology.

SUBSTANCE: invention proposes a nutrient medium for detecting Ureaplasma urealyticum that comprises nutrient base, growth stimulating agents, cysteine, urea, phenol red, equine serum, selective additives and distilled water. Medium comprises heart-brain extract and tryptose as nutrient base; yeast extract and medium 199 as growth stimulating agents; antibiotics as selective additives. Proposed method for diagnosis of infections caused by Ureaplasma urealyticum involves addition of nutrient medium of abovementioned qualitative and quantitative composition promoting to detection of disease pathogens into wells of cultural plate, analyzed material and substance providing anaerobic conditions and analysis of color change of the nutrient medium. Diagnosis method is simple in carrying out, it doesn't require special equipment and allows carrying out both detection and identification of pathogens, to assay degree of insemination and sensitivity spectrum to different antibacterial preparations and to estimate effectiveness of treatment carried out. Using the nutrient medium allows simplifying the composition, to increase rate and precision of method in diagnosis of ureaplasma infection due to enhancing sensitivity of medium. Invention can be used in culturing Ureaplasma urealyticum and diagnosis of urogenital ureaplasmosis by isolation of ureaplasma from clinical material.

EFFECT: valuable properties of nutrient medium.

4 cl, 4 tbl, 3 ex

Description

The invention relates to medical microbiology and can be used to cultivate Ureaplasma urealyticum and diagnose urogenital ureaplasoses by isolating ureaplasis from clinical material.

The etiological structure of urogenital infections is constantly changing. Recently, the frequency of chlamydial, viral, mycoplasma, ureaplasma and mixed infections has sharply increased, the fight against which presents significant difficulties in connection with developing resistance to antibiotics and the characteristics of the body's response.

Of particular interest are mycoplasma and ureaplasma infections. Human diseases caused by ureaplasmas are combined into a group of human ureaplasmoses. Man is the natural host of eleven species of mycoplasmas. Five of them, including Ureaplasma urealyticum, are pathogenic in full accordance with Koch's postulates.

There is convincing evidence of the etiological role of ureaplasmas in habitual abortions and premature births. Ureaplasma infection of the upper genital tract leads to inflammatory processes that violate the reproductive function of the body. Population of the endometrial ureaplasma in 50% of cases leads to spontaneous abortion. Colonization of the fallopian tubes with ureaplasma can cause salpingitis, leading to a narrowing of the lumen or even to complete obstruction. There is evidence of serologically confirmed cases of intrauterine respiratory infection of the fetus caused by ureaplasmas. Thus, the need for a special examination of all pregnant women to identify cases of urogenital ureaplasmosis and subsequent rehabilitation is obvious.

The methods of detecting pathogens and their antigens are of the greatest diagnostic value. This is determined by a number of features of urogenital infections and their pathogens: their wide distribution and the duration of circulation of antibodies after infection (especially IgG antibodies detected in enzyme-linked immunosorbent assay), the presence of chronic and latent forms, frequent mixed infections, high serological heterogeneity and antigenic variability, a wide range of cross-serological reactions, low titers of specific immunoglobulins in local forms of infections, etc. The simplest and most The cultural method for diagnosing urogenital infections is important.

From the patent literature known "Nutrient medium for the isolation of Ureaplasma urealyticum" To prepare the medium in 1 liter of distilled water contribute, g / l: nutrient broth 20-27, yeast extract 3-8; cystine 0.1-0.2; urea 0.01-0.05; thiamine bromide 0.003-0.007; bromocresol purple 0.01-0.03 and potassium phosphate 6-10. The mixture is boiled and sterilized. After sterilization, horse serum 150-250 g and amphotericin B 30-70 units are added to the cooled medium. The medium has a pH of 5.4-5.8, a sensitivity of 10 ^-5 CI / ml. (See RF patent No. 1495381, 1989)

Also known as "Culture medium for the isolation of ureaplasmas." Prepare the broth from fresh placenta, washed with tap water and freed from the shells. The placenta is cut into small pieces, weighed, placed in a pan, poured with a double amount of tap water and boiled for 30 minutes. The resulting broth is clarified and sterilized by autoclaving. Take placental broth 700-800 g / l, fodder yeast extract 3.5-4.5 g / l hydrolysis of casein 6-10 g / l, urea 0.5-10.0 g / l, horse serum 40-100 g / l, phenol red 0020-0025, add distilled water to 1 l. The pH is adjusted to 6.0-6.2, poured into vials and freeze-dried. (See RF patent No. 1723128, C 12 Q 1/20, 1992).

Closest to the claimed method is the "Method of counting, detection and identification of mycoplasmas in general and urogenital mycoplasmas in particular, and a biological environment specially adapted for this purpose." The method is characterized by enzymatic reactions that occur under anaerobic conditions in a liquid medium for growing mycoplasmas containing broth for culturing mycoplasmas, horse serum, ampicillin, bactrim, sodium thioglycolate reducing agent, yeast extract, cysteine and arginine. Phenol red is used as a color indicator. To create anaerobic conditions, paraffin oil is used. Analyzing the color change of the indicator within one to two days, conclusions are made about the presence of mycoplasma infection. (See US patent No. 5091307, 1992).

The cultural method for the diagnosis of urogenital infections, for the detection of Ureaplasma urealyticum in a selective liquid nutrient medium, is the most simple and reliable. Using this method allows the identification and identification of Ureaplasma urealyticum, as well as to assess the degree of contamination of the studied material in color-forming units (COE / ml). The latter is especially important for the clinical evaluation of the etiology of inflammatory processes. Of etiological significance is the concentration of ureaplasmas in the test material not less than 10,000 CFU / ml. Determination of the sensitivity of the cultures to antibiotics and the targeted use of drugs significantly increase the effectiveness of the therapy, which distinguishes the cultural method from other methods for diagnosing urogenital ureaplasmosis (polymerase chain reaction (PCR) and immunofluorescence (RIF)). The cultural method for the diagnosis of urogenital ureaplasmosis is more reliable in comparison with RIF, whose lower sensitivity and specificity is determined by the small linear dimensions of these microorganisms, expressed by their heterogeneity and variability, often encountered by a low concentration of ureaplasmas in the primary material and, accordingly, the need for their accumulation to identify, the difficulty of taking a quality scraping for this reaction.

A technology has been developed for obtaining a selective nutrient medium, which makes it possible to identify the selected cultures of ureaplasmas and evaluate their amount (titer) in the samples. During the development, optimal ratios of the components of the selective liquid nutrient medium for the detection of Ureaplasma urealyticum were obtained, which led to an increase in its sensitivity.

The medium provides the growth of Ureaplasma urealyticum, accompanied by a change in the color of the medium due to the manifestation of enzymatic activity, and inhibits the growth of concomitant gram-positive and gram-negative bacteria and fungi.

Nutrient medium for detecting Ureaplasma urealyticum, contains a nutrient base, growth stimulants, cysteine, urea, phenol red, horse serum, selective additives and distilled water. It differs in that, as a nutrient base, it contains cardiopulmonary extract and tryptose, as growth stimulants - yeast extract and medium 199, as selective additives - antibiotics; in the following ratio of ingredients, g / l:

- a cerebral extract ("Becton Dickinson microbiology systems "USA, Cat. No. 237500, 2002)  18-22 - tryptose ("Becton Dickinson microbiology systems", USA, cat. No. 211709.2002)  8-12 - yeast extract ("Becton Dickinson microbiology systems ", USA, cat. No. 211929, 2002)  3-4 - medium 199 dry ("Sigma", cat. No. M5017, 2002)  4.7-5.2 - L-cysteine hydrochloride ("Sigma", USA, cat. No. 8277, 2002)  0.10-0.15 urea ("Sigma", USA, cat. No. U 4128, 2002)  0.40-0.60 - indicator - phenol red ("Sigma", USA, cat. No. P 5530, 2002)  0.04-0.07 - horse serum  0.25-0.30 - selective additives - antibiotics  0.63-1.25 - distilled water  rest

The nutrient medium contains selective additives - antibiotics in the following ratio of ingredients, g / l:

- ampicillin sodium salt  0.50-1.00 - vancomycin hydrochloride  0.10-0.20 - polymyxin B sulfate  0.01-0.02 - amphotericin B  0.01-0.02 - lincomycin hydrochloride  0.02-0.03

The proposed composition of the nutrient medium presents the optimal quantitative ratio of the components, the concentration of antibiotics and indicator dye. The proposed method for the diagnosis of infections caused by Ureaplasma urealyticum involves the introduction of a nutrient medium that facilitates the detection of pathogens into the wells of the culture plate, the test material and the substance that creates anaerobic conditions and analysis of the color change of the nutrient medium. As a nutrient medium use:

a nutrient medium containing a nutrient base, growth stimulants, cysteine, urea, phenol red, horse serum, selective additives and distilled water, as a nutrient base - a cerebral extract and tryptose, as growth stimulants - yeast extract and medium 199, as selective additives - antibiotics. The diagnostic method is simple to set up, does not require special equipment, allows both the identification and identification of pathogens, determine the degree of contamination and sensitivity spectrum to various antibacterial drugs, evaluate the effectiveness of the treatment.

The use of the claimed composition of the nutrient medium allows to simplify the composition, increase the speed and accuracy of the method for the diagnosis of ureaplasma infection by increasing the sensitivity of the environment.

To prepare the basics of the medium, a cerebral extract (Becton Dickinson microbiology systems ", USA, Cat. No. 237500, 2002) is dissolved in 650 ml of distilled water, 18-24 g, tryptose (" Becton Dickinson microbiology systems ", USA, Cat. No. 211709, 2002) - 8-12 g, yeast extract ("Becton Dickinson microbiology systems", USA, Cat. No. 211929, 2002) - 3-4 g. The resulting medium base is sterilized by autoclaving.

Separately, dry 199 medium is dissolved in 100 ml of distilled water (Sigma, Cat. No. M5017, 2002) - 4.7-5.2 g, L-cysteine hydrochloride (Sigma, USA, Cat. No. 8277, 2002) - 0.1-0.15 g, urea ("Sigma", USA, Cat. No. U 4128, 2002) - 0.4-0.6 g, phenol red ("Sigma", USA , Cat. No. P 5530, 2002) - 0.04-0.07 g; the resulting solution of additives is sterilized by filtration.

To prepare the medium, 650 ml of the base of the medium, 100 ml of a solution of additives and 250-300 ml of horse serum are mixed. The mixture is thoroughly mixed, then add ampicillin sodium salt - 0.5-1.0 g, vancomycin hydrochloride - 0.1-0.2 g, polymyxin B sulfate - 0.01-0.02 g, amphotericin B - 0.01 -0.02 g, lincomycin hydrochloride 0.02-0.03 g. The pH is adjusted to 6.1-6.3.

Example 1

To prepare the basics of the medium in a 650 ml distilled water, dissolve a cardiac extract ("Becton Dickinson microbiology systems", USA, cat. No. 237500, 2002) - 18 g, tryptose ("Becton Dickinson microbiology systems", USA, cat. No. 211709, 2002) - 8 g, yeast extract ("Becton Dickinson microbiology systems", USA, Cat. No. 211929, 2002) - 3 g. The resulting medium base is sterilized by autoclaving.

Separately, dry 199 medium is dissolved in 100 ml of distilled water (Sigma, Cat. No. M5017, 2002) - 4.7 g, L-cysteine hydrochloride (Sigma, USA, Cat. No. 8277, 2002). - 0.1 g, urea ("Sigma", USA, cat. No. U 4128, 2002) - 0.4 g, phenol red ("Sigma", USA, cat. No. P 5530, 2002) - 0.04 g; the resulting solution of additives is sterilized by filtration.

To prepare the medium, 650 ml of the base of the medium, 100 ml of a solution of additives and 250 ml of horse serum are mixed. The mixture is thoroughly mixed, then add ampicillin sodium salt - 0.5 g, vancomycin hydrochloride - 0.1 g, polymyxin B sulfate - 0.01-0.02 g, amphotericin B - 0.01 g, lincomycin hydrochloride - 0.02 d. The pH is adjusted to 6.1-6.3.

Example 2 (optimal).

To prepare the basics of the medium in a 650 ml distilled water, dissolve a cardiac extract ("Becton Dickinson microbiology systems", USA, Cat. No. 237500, 2002) - 20 g, tryptose ("Becton Dickinson microbiology systems", USA, Cat. No. 211709, 2002) - 10 g, yeast extract (Becton Dickinson microbiology systems, USA, Cat. No. 211929, 2002) - 3 g. The resulting medium base is sterilized by autoclaving.

Separately, dry 199 medium is dissolved in 100 ml of distilled water (Sigma, Cat. No. M5017, 2002) - 4.95 g, L-cysteine hydrochloride (Sigma, USA, Cat. No. 8277, 2002). - 0.125 g, urea ("Sigma", USA, cat. No. U 4128, 2002) - 0.5 g, phenol red ("Sigma", USA, cat. No. P 5530, 2002) - 0, 05 g; the resulting solution of additives is sterilized by filtration.

To prepare the medium, 650 ml of the base of the medium, 100 ml of a solution of additives and 250 ml of horse serum are mixed. The mixture is thoroughly mixed, then ampicillin is added sodium salt of 0.75 g, vancomycin hydrochloride is 0.15 g, polymyxin B sulfate is 0.01-0.02 g, amphotericin B is 0.015 g, lincomycin hydrochloride is 0.025 g. The pH is adjusted. 6.1-6.3.

Example 3

To prepare the basics of the medium, a cardiopulmonary extract (Becton Dickinson microbiology systems, USA, Cat. No. 237500, 2002) was dissolved 24 g, tryptose (Becton Dickinson microbiology systems, USA, Cat. No. 211709, 2002) - 12 g, yeast extract ("Becton Dickinson microbiology systems", USA, cat. No. 211929, 2002) - 4 g. The resulting medium base is sterilized by autoclaving.

Separately, dry 199 medium is dissolved in 100 ml of distilled water (Sigma, Cat. No. M5017, 2002) - 5.2 g, L-cysteine hydrochloride (Sigma, USA, Cat. No. 8277, 2002). - 0.15 g, urea ("Sigma". USA, cat. No. U 4128, 2002) - 0.6 g, phenol red ("Sigma", USA, cat. No. P 5530, 2002) - 0.07 g; the resulting solution of additives is sterilized by filtration.

To prepare the medium, 650 ml of the base of the medium, 100 ml of a solution of additives and 300 ml of horse serum are mixed. The mixture is thoroughly mixed, then add ampicillin sodium salt - 1 g, vancomycin hydrochloride - 0.2 g, polymyxin B sulfate - 0.01-0.02 g, amphotericin B - 0.02 g, lincomycin hydrochloride - 0.03 g. The pH is adjusted to 6.1-6.3.

A selective nutrient medium should ensure the growth of Ureaplasma urealyticum, accompanied by a change in the color of the medium from yellow to red due to the manifestation of urease activity, and inhibit the growth of related microorganisms (gram-positive and gram-negative bacteria, fungi). A method for the diagnosis of urogenital microplasmoses.

1. Thaw the nutrient medium at room temperature or in a water bath at a temperature of 40 ° C-45 ° C.

2. Pipette 0.225 ml of culture medium into 4 consecutive wells of a culture 96-well plate (or 4 microtubes per 1.5 ml or 0.5 ml), mark the wells of the tablet (microtubes): first. - 3, the second - 4, the third - 5, the fourth - 6.

3. Suspend the test sample on the vortex.

4. Make a series of 10-fold dilutions of the test sample in a nutrient medium: add 0.25 ml of the test sample to the well (microtube) 3, pipet thoroughly, transfer 0.25 ml from the dilution to the next well (microtube) and repeat the dilution procedure.

5. In 4 consecutive wells of the culture plate (microtubes) marked "K-" add 0.200 ml of culture medium and carry out a 10-fold dilution procedure as described in step 4, using a transport medium instead of the test sample.

6. Add 0.70 ml of sterile mineral oil to each well of the plate (microtube) to create anaerobic conditions.

7. The tablet (microtubes) is placed in a thermostat and incubated at a temperature of (37 ± 1) ° С.

8. The results are recorded on the third day of incubation in a thermostat with daily visual viewing of crops and the degree of contamination of the material is judged by the color change of the nutrient medium.

As a transport medium using the medium in the following ratio of ingredients: g / l:

- a cerebral extract ("Becton Dickinson microbiology systems "USA, Cat. No. 237500, 2002)  18-22 - tryptose ("Becton Dickinson microbiology systems", USA, cat. No. 211709.2002)  8-12 - yeast extract ("Becton Dickinson microbiology systems ", USA, cat. No. 211929, 2002)  3-4

- medium 199 dry ("Sigma", cat. No. M5017, 2002) 4.7-5.2

- L-cysteine hydrochloride ("Sigma", USA, cat. No. 8277, 2002)  0.10-0.15 - indicator - phenol red ("Sigma", USA, cat. No. P 5530, 2002)  0.04-0.07 - horse serum  0.20-0.30 - ampicillin sodium salt  0.50-1.00 - vancomycin hydrochloride  0.10-0.20 - polymyxin B sulfate  0.01-0.02 - amphotericin B  0.01-0.02 - distilled water  rest

For the diagnosis, the following clinical material is used:

- scraping cells of the mucous membrane of the urethra, cervical canal, vagina;

- the first portion of morning urine (sediment obtained by centrifugation of 10 ml of the first portion of morning urine);

- secretion of the prostate gland (V - 0.50-0.10 ml), ejaculate (V - 0.50-0.10 ml).

The key point in the diagnosis of pathogens of urogenital mycoplasmosis is the capture of the test material and its storage conditions. It is necessary:

1) take the material before antibiotic therapy;

2) the procedure for taking material should be standard;

3) it is important to obtain a sufficient number of cells, since ureaplasma is a microorganism that colonizes the cell surface;

4) do not use local antiseptics when taking material;

5) thorough removal of mucus from the cervical canal;

6) taking urethral samples should be carried out no earlier than 2-3 hours after urination;

7) obtaining the secretion of the prostate gland and ejaculate is carried out immediately after urination;

8) the test material is placed in a special transport medium that best ensures the preservation of viability of ureaplasmas.

Table 1
Storage conditions for samples. Storage conditions Max. storage time, hour. At room temperature from 18 ° C to 24 ° C 5 In the refrigerator (on the bottom shelf of the refrigerator) at a temperature of 2 ° C to 6 ° C 96

Table 2
Interpretation of the results. results
+ - there is a growth of Ureaplasma urealyticum
- - lack of growth of Ureaplasma urealyticum Interpretation Well (test tube) Identification of Ureaplasma urealyticum Seed of test sample TSOE (color-forming units) / ml sample 3 4 5 6 - - - - He revealed + - - - Detected 10 ³ + + - - Detected 10 ⁴ + + + - Detected w ⁵ + + + + Detected 10 ⁶ - + + + Detected 10 ⁶

Accounting for results.

Analysis of the results is carried out by visual viewing of seeded tablets (test tubes). Ureaplasma urealyticum growth is observed within 48-72 hours. The growth of Ureaplasma urealyticum is accompanied by a change in the color of the nutrient medium from yellow to red, without clouding, due to the manifestation of urease activity, the formation of ammonia and a shift in pH. Growth is noted on the second day of incubation. In case of low contamination of the material, the color change of the medium occurs on the third day.

Table 3
Ureaplasma urealyticum detection medium culture medium number of crops number of selected crops absolute value % famous 197 76 38.6 proposed 197 87 44,2 Table 4
The growth of Ureaplasma urealyticum when sowing the test material in different dilutions dilution of the test material (inoculum dose) growth on a known medium growth on the proposed environment 10 ^-3 76 87 10 ^-4 76 87 10 ^-5 69 87 10 ^-6 fifty 72

Claims (4)

1. Nutrient medium for the detection of Ureaplasma urealyticum, containing a nutrient base, growth stimulants, cysteine, urea, phenol red, horse serum, selective additives and distilled water, characterized in that it contains a cardiac extract and tryptosis as a nutrient base, as growth stimulants - yeast extract and medium 199, as selective additives - antibiotics in the following ratio of ingredients, g / l: Cardiac Hood ("Becton Dickinson microbiology systems ", USA, Cat. No. 237500, 2002) 18-22 Tryptose ("Becton Dickinson microbiology systems ", USA, cat. No. 211709, 2002) 8-12 Yeast Extract ("Becton Dickinson microbiology systems ", USA, cat. No. 211929, 2002) 3-4 Dry medium 199 (Sigma, Cat. No. M5017, 2002) 4.70-5.20 L-Cysteine Hydrochloride ("Sigma", USA, Cat. No. 8277, 2002) 0.10-0.15 Urea (Sigma, USA, Cat. U 4128, 2002) 0.40-0.60 Indicator-phenol red ("Sigma", USA, Cat. No. P 5530, 2002) 0.04-0.07 Horse serum 0.25-0.30 Selective Supplements - Antibiotics 0.63-1.25 Distilled water Rest 2. The nutrient medium according to claim 1, characterized in that it contains selective additives - antibiotics in the following ratio of ingredients, g / l: Ampicillin sodium salt 0.50-1.00 Vancomycin hydrochloride 0.10-0.20 Polymyxin B Sulfate 0.01-0.02 Amphotericin B 0.01-0.02 Lincomycin hydrochloride 0.02-0.03 3. A method for the diagnosis of infections caused by Ureaplasma urealyticum, including the introduction of a nutrient medium that facilitates the detection of pathogens into the wells of the culture plate, the test material and the substance that creates anaerobic conditions, and analysis of the color change of the nutrient medium, characterized in that the nutrient medium is used nutrient-based medium, growth stimulants, cysteine, urea, phenol red, horse serum, selective additives and distilled water, as pi atelnoy bases - heart brain extract and tryptose as growth stimulants - yeast extract and medium 199, as selective additives - antibiotics with the following ratio of ingredients in g / l: Cardiac extract ("Becton Dickinson microbiology systems", USA, cat. No. 237500, 2002) 18-22 Tryptose ("Becton Dickinson microbiology systems ", USA, cat. No. 211709, 2002) 8-12 Yeast extract ("Becton Dickinson microbiology systems", USA, cat. No. 211929, 2002) 3-4 Wednesday 199 dry ("Sigma", cat. No. M5017, 2002) 4.70-5.20 L-Cysteine Hydrochloride ("Sigma", USA, Cat. No. 8277, 2002) 0.10-0.15 Urea (Sigma, USA, Cat. U 4128, 2002) 0.40-0.60 Indicator-phenol red ("Sigma", USA, Cat. No. P 5530, 2002) 0.04-0.07 Horse serum 0.25-0.30 Ampicillin sodium salt 0.50-1.00 Vancomycin hydrochloride 0.10-0.20 Polymyxin B Sulfate 0.01-0.2 Amphotericin B 0.01-0.02 Lincomycin hydrochloride 0.02-0.03 Distilled water Rest bring the transport medium into the wells of the culture plate, mark the wells of the tablet; suspend the test material sample on a vortex, make a series of 10-fold dilutions of the test sample in a nutrient medium and repeat the dilution procedure; nutrient medium is introduced into designated control wells of the culture plate and a dilution procedure is carried out using a transport medium; sterile mineral oil is added to each well of the plate, creating anaerobic conditions; the tablet is placed in a thermostat and incubated at a temperature of (37 ± 1) ° C; registration of the results is carried out on the third day of incubation in a thermostat with daily visual viewing of crops. 4. The method according to claim 3, characterized in that as a transport medium using a nutrient medium in the following ratio of ingredients, g / l: Cardiac extract ("Becton Dickinson microbiology systems" USA, cat. No. 237500, 2002) 18-22 Tryptose ("Becton Dickinson microbiology systems ", USA, cat. No. 211709, 2002) 8-12 Yeast Extract ("Becton Dickinson microbiology systems ", USA, cat. No. 211929, 2002) 3-4 Dry medium 199 (Sigma, Cat. No. M5017, 2002) 4.70-5.20 L-Cysteine Hydrochloride ("Sigma", USA, Cat. No. 8277, 2002) 0.10-0.15 Indicator-phenol red ("Sigma", USA, Cat. No. P 5530, 2002) 0.04-0.07 Horse serum 0.25-0.30 Ampicillin sodium salt 0.50-1.00 Vancomycin hydrochloride 0.10-0.20 Polymyxin B Sulfate 0.01-0.02 Amphotericin B 0.01-0.02 Distilled water Rest