RU2332671C2 - Method of chronic urogenital gonococcal infection course forecast - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medical microbiology. The method of chronic urogenital gonococcal infection course forecast involves seeding of accompanying fungi of Candida genus in case of gonococcus detection, and persistence factors of accompanying microorganisms is evaluated. If titration of fungi of Candida genus gives not less than 10² colony-forming cells per millilitre and antilysozyme activity evolves simultaneously in the quantity not less than 1.3 mcg/ml per optical density unit, and anticomplementary activity not less than 1.5·10⁶ antilytic complement units, then chronic character of urogenital infection is confirmed diagnosed.

EFFECT: increased accuracy of chronic urogenital gonococcal infection course forecast.

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Description

The invention relates to medicine, in particular to medical microbiology, and in particular to methods for predicting the course of infectious and inflammatory diseases of the urogenital tract.

In particular, the method is designed to predict the protracted course of urogenital infection, an etiological agent that is gonococcus (Nesseria gonorrhoeae), and is also aimed at assessing the likelihood of infectious and inflammatory complications in urogenital gonorrhea.

Gonococcal infection remains one of the main sexually transmitted infections [1, 7]. An increase in the incidence of gonorrhea is facilitated by an increase in sluggish chronic forms that are difficult to identify and lead to an even greater spread of infection, as well as the development of serious complications - inflammatory diseases of the pelvic organs, often leading to male and female infertility [2].

Given the above, the need for an effective method for predicting the chronic course of gonococcal infection and possible complications in order to prevent their timely optimal therapy becomes apparent.

A known method for predicting the adverse course of a purulent-inflammatory disease of microbial etiology, where a pure culture of the pathogen is isolated from a sample of material, which is then identified and determined by its ability to inactivate factors of natural anti-infection resistance, namely, complement activity is determined by 50% hemolysis on agar plates in the supernatant of the culture fluid of the pathogen in an uninhabited nutrient medium and when the ability of the pathogen to degrade ement host predict chronic infection [3]. The specified method was developed mainly for surgical infections, the etiological agent of which was conditionally pathogenic microorganisms - staphylococci or E. coli, and is not intended for neiserial infections.

A known method for predicting the course of urogenital gonorrhea, based on the ability of the pathogen to inactivate factors of local immunity, which leads to the long-term preservation of the microorganism in the host cells [4]. The method consists in assessing the ratio of anti-complementary activity of gonococcus, determined by the percentage of suppression of complement-dependent hemolysis of red blood cells in the presence of a reference amount of serum complement, causing 100% hemolysis, as well as the activity of the local complement system of the genitourinary tract, determined by the percentage of hemolysis of sensitized red blood cells (calculated by the formula anticomplementary activity) / (complement activity), and with a value of this ratio less than 1 expect non a short and / or uncomplicated course of the disease, with a ratio greater than or equal to 1, a prolonged course of the disease is predicted with the likelihood of infectious and inflammatory complications. In terms of features, this method is the closest to the claimed. This method is taken as a prototype.

In this method, a comprehensive approach to the analysis of the relationship "pathogen - macroorganism" is carried out, which allows us to predict the nature of the course of infection.

However, it is possible to predict the course of gonococcal infection using this method only provided that the diagnosis of gonorrhea is made by the bacteriological (cultural) method. The disadvantage of this method is the need for a cultural diagnosis of gonorrhea, which is associated with difficulties caused by the high demand of the pathogen for cultivation conditions - rich nutrient media, microaerophilic conditions, the absence of competitive microflora in the test material, the presence of uncultivated forms, susceptibility of colonies to autolysis [4]. Thus, in some patients with gonococcal infection, it is not possible to isolate a pure pathogen culture, or if it is isolated once, it is lost during reseeding and, therefore, it becomes impossible to determine the properties of gonococcus.

A second attempt to isolate a pure culture is impractical due to the appointment of antibiotic therapy. Therefore, despite the fact that the diagnosis of gonorrhea is confirmed (which is possible both by cultural and microscopic methods - according to the order of M3 of the Russian Federation No. 415 of 08/20/2003) [5], a forecast cannot be made. The consequence is that with this method, some patients with a probable chronic course of gonococcal infection are not detected.

The main objective of the proposed method is to increase the accuracy of predicting the course of urogenital gonococcal infection.

To solve this problem, in the proposed method for predicting the chronic course of urogenital gonococcal infection, which includes identifying the presence of the pathogen and determining the persistence factors of microorganisms, determine the number of Candida fungi associated with the pathogen, as well as the presence of persistence factors - antilysocyme and anticomplementary activities and with a titer of Candida fungi at least February ¹⁰ colony forming cells per milliliter, and the manifestation of both anti-lysozyme activity is not less than 1.3 micro grammes per milliliter per unit of optical density and anticomplementary activity not less than 1.5 × 10 ⁶ units antiliticheskih complement diagnosed chronic urogenital infections.

The proposed method is not known from the prior art, that is, is new.

The novelty of the proposed method is that when gonococcus is detected, concomitant fungi of the genus Candida are simultaneously sown, and concomitant microorganisms evaluate persistence factors, which are informative criteria for predicting the course of urogenital gonorrhea.

A significant difference of the proposed method is that it is not the properties of the pathogen that are evaluated, but the characteristics of the concomitant microflora, namely, seeded Candida fungi and at a titer of fungi of at least 10 ² colony forming cells in a milliliter and at the same time, at least 1.3 micrograms per milliliter of antilysimic activity per unit of optical density and anti-complementary activity of at least 1.5 · 10 ⁶ antilithic complement units, a chronic course of urogenital infection is diagnosed.

The technical result achieved by the method implementation consists in the fact that the new forecasting method allows, excluding the determination of pathogen persistence factors (gonococcus), which is laborious and unstable due to its whimsicality, to predict the chronic course of urogenital gonococcal infection by the persistence factors of concomitant symbiotic microflora - fungi of the genus Candida, the determination of which is much simpler, more reliable, since it has better reproducibility, independent of the ant biotikoterapii. The inventive method does not require special equipment and is available for any bacteriological laboratory.

Thus, the forecast accuracy of the proposed method is increased by identifying those patients for whom it was impossible to predict due to the prototype method due to the non-isolation of the final pure pathogen culture, since gonococcus is very demanding on cultivation conditions, can be weakened due to the patient’s the moment of diagnosis of antibiotic therapy and even be in an uncultured form.

New features of the proposed method are:

- determine the number of fungi of the genus Candida associated with the pathogen (their boundary titer is set - at least 10 ² colony forming cells per milliliter);

- determine the presence of persistence factors in fungi of the genus Candida - antilysocyme and anticomplementary activities (the necessary simultaneous presence of antilysocyme activity at a level of at least 1.3 micrograms per milliliter per unit of optical density and anticomplementary activity at a level of at least 1.5 · 10 ⁶ antilithic units was established complement) in which a chronic course of urogenital infection is diagnosed.

The presence of the first new sign in the inventive method allows to identify a new, more informative sign of predicting the chronic course of urogenital gonococcal infection, which ultimately allows to increase the accuracy of prediction. The practice of treatment has shown that in patients with chronic gonococcal infection, the body's immune properties are significantly reduced, which entails an additional increased colonization of the urogenital tract by symbiotic microorganisms, primarily Candida fungi, to a level of at least 10 ² colony forming cells per milliliter.

The second new feature allows us to clarify the first new feature by establishing the simultaneous presence of two boundary values: the antilysocyme activity of fungi of the genus Candida and their anti-complementary activity. Each of these factors indirectly reflects the degree of decrease in the immune properties of the patient’s body, and together these factors ultimately allow for a simple implementation to increase the accuracy of the forecast.

The proposed method is based on new knowledge obtained as a result of experimental studies of fungal-bacterial interactions in the biocenosis of the urogenital tract in case of gonococcal infection. It was found that extracellular factors of Candida fungi in a significant percentage of cases increase the persistent properties of Neisseriae gonorhoea, namely antilysocyme and anticomplementary activities (Table 1).

Table 1. Change in gonococcus properties compared to baseline. Influencing factor Gonococcus Properties ALA AKA increase lowering lack of increase lowering lack of Candida mushroom supernatant 84.5% 2.82% 12.7% 45.1% 4.2% 50.1%

Also, new knowledge was revealed features of the microbiocenosis of the reproductive tract of patients with chronic gonococcal infection compared with fresh. In patients with chronic gonorrhea, at the same time with gonococcus, fungi were isolated. Candida, while both the pathogen and symbionts had high persistent properties - ALA and AKA. Considering the well-known fact that the high ability of the gonococcus to deplete the factors of the body's natural resistance leads to the chronicization of the infectious process [6], as well as the first information obtained on the presence and properties of symbionts, it was suggested that the fungi of the river. Candida, increasing the persistent properties of the neysseries and depleting the defenses of the macroorganism, have a synergistic effect with the pathogen on the colonization resistance of the mucosa of the urogenital tract, contributing to the chronization of the process.

This suggests that the detection, together with gonococcus, of fungi of the genus Candida with high persistent properties in fresh gonorrhea can serve as an informative prognostic criterion for the chronization of the process. Further clinical and laboratory studies confirmed this assumption and revealed threshold values of the proposed parameters (Table 2)

Table 2. Correspondence of the levels of the studied parameters to the nature of the course of the disease. Characterization of Candida fungi

Number of mushrooms ALA AKA <10 ² CFU / ml <1.3 mcg / ml <1.5 · 10 ⁶ AntiLEC

≥10 ² CFU / ml <1.3 mcg / ml <1.5 · 10 ⁶ AntiLEC

≥10 ² CFU / ml <1.3 mcg / ml ≥1.5 · 10 ⁶ AntiLEC

≥10 ² CFU / ml ≥1.3 μg / ml <1.5 · 10 ⁶ AntiLEC

<10 ² CFU / ml ≥1.3 μg / ml <1.5 · 10 ⁶ AntiLEC

<10 ² CFU / ml <1.3 mcg / ml ≥1.5 · 10 ⁶ AntiLEC

<10 ² CFU / ml ≥1.3 μg / ml ≥1.5 · 10 ⁶ AntiLEC

≥10 ² CFU / ml ≥1.3 μg / ml ≥1.5 · 10 ⁶ AntiLEC

Note - * Accepted abbreviations: SGI - fresh gonococcal infection; CGI is a chronic gonococcal infection.

The proposed method is as follows. To predict the course of urogenital gonorrhea, pathological material (discharge of the urethra, cervical canal) is sown on elective media. By the conventional method, a pure culture of the pathogen is isolated, which is identified by morphological, tinctorial, biochemical properties to a species as N. gonorrhoeae; 0.1 ml of pathological material is diluted to 10 ^-1 , 10 ^-2 , 10 ^-3 and sterile spatulas make inoculation of 0.1 ml of the suspension obtained in different Petri dishes with Candida agar. Crops are incubated at 30 ° C for 48 hours. The isolated culture of fungi is identified by the combination of cultural, morphological and saccharolytic properties as fungi of the genus Candida. Colonies grown on a Petri dish are counted and, multiplying by 100 and by the degree of dilution of the pathological material (10, 100, 1000, respectively), calculate the number (titer) of mushrooms in one milliliter of material. To predict the course of the disease, the known methods also determine the antilysocyme and anticomplementary activities [8].

The prognosis of the chronic course of urogenital infection is made if the yttrium of Candida mushrooms is at least 10 ² colony forming cells per milliliter, and if antilysocyme activity is manifested at least 1.3 micrograms per milliliter per unit optical density and anticomplementary activity is at least 1.5 · 10 ⁶ antilithic units of complement.

Example 1. Patient M., 35 years old (outpatient card No. 1121 for 2004) with a diagnosis of “Fresh gonorrhea; subacute anterior urethritis ”on the sixth day of the disease, upon initial treatment, 0.1 ml of urethra discharge was taken into a test tube with 0.9 ml of 0.9% sodium chloride solution. From this tube, a suspension in which a dilution of 10 ^-1 is transferred, 0.1 ml of the suspension was transferred to another tube together with 0.9 ml of a 0.9% sodium chloride solution. Thus, a dilution of 10 ^-2 was obtained. Dilutions of 10 ^-3 and 10 ^-4 were similarly obtained. 0.1 ml from each tube was applied to Petri dishes with Candida agar using sterile spatulas. Typical whitish-yellow colonies with a domed elevation formed on plates after 48-hour incubation at a temperature of 30 ° С, they were isolated into a pure culture and identified by the combination of cultural, morphological and saccharolytic properties as fungi of the genus Candida.

In order to clarify the prognosis of the course of the disease, the number of colonies was calculated in those plates where Candida growth was observed at maximum dilution. The selected culture was determined antilysocyme and anticomplementary activity. For this, a daily culture of mushrooms was prepared, which was sown with a standard bacteriological loop in 3 ml of liquid nutrient medium — nutrient broth (PB) (Nutrient broth for culturing dry microorganisms; NPO Nutrient mediums, Makhachkala). Cultivated at 37 ° C for 24 hours. On an SF-46 spectrophotometer (wavelength 540 nm), the optical density of the broth culture was measured against nutrient broth (Y). The supernatant was separated from the bacterial cells by centrifugation for 15 min at 3000 rpm. Micrococcus lysodeikticus NCTC 2665 daily agar culture was used as a test strain to determine the antilysocyme activity of microorganisms. The grown bacterial cells of the test strain were killed with chloroform for 60 minutes, washed, filtered through a large-pore filter, washed twice with 0.2 M trilon B phosphate buffer and once with 0.2 M phosphate buffer (pH = 6.2), then the optical density of the micrococcus suspension was adjusted to 0.300.

A solution of lysozyme with a concentration of 20 μg / ml was prepared on 0.2 M phosphate buffer (pH = 6.2).

The supernatant of the studied cultures of microorganisms with a volume of 0.9 ml was mixed with 0.1 ml of the prepared lysozyme solution and incubated for 40-60 minutes at a temperature of 37 ° C. Then, 0.5 ml of a mixture of supernatant and lysozyme was added to 2.0 ml of a suspension of the Microccocus lysodeikticus test strain and the absorbance of the mixture was measured after 30 and 150 s using an SF-46 spectrophotometer (wavelength 540 nm) against 0.2 M phosphate buffer .

As a control, a 9: 1 mixture of nutrient broth with lysozyme was used.

According to the degree of lysis of the suspension of the test culture, the antilysocyme activity of the studied culture was calculated by the formula

where A is the antilysocyme activity, ng of inactivated lysozyme / (ml of supernatant ^· optical density of the broth culture);

V ₁ - the volume (0.1 ml) of the lysozyme solution of the initial concentration (20,000 ng / ml);

V ₂ - volume (0.9 ml) of the supernatant of the broth culture of the studied strain;

C is the initial concentration of lysozyme (20,000 ng / ml);

Y is the optical density of the broth culture of the studied strain, units optical density;

ΔD ₀ - change in the optical density of the suspension of the test culture in the experiment between 30 and 150 s;

ΔD _K is the change in optical density of the suspension of the test culture in the control between 30 and 150 s.

The anti-complementary activity of microorganisms was determined as follows. The complement source was a preparation for CSC. Veronal buffer and sensitized sheep erythrocytes (SEB) were prepared as standard [Cabot E., Meyer M., 1968]. To determine the maximum lysis rate, an SF-46 spectrophotometer was used, into the processor of which an experimentally obtained calibration coefficient of 167.9 was introduced, which allows in mode A (3) with a measurement period of 10 s to obtain values of the lysis rate in million SEB / ml (10 ⁶ LEC) per second, where the LEC is a lytic complement unit. The reactions were carried out at 37 ° C in a 1 cm cuvette, the sample volume was 3 ml, and the wavelength was 800 nm. 50 μl of the supernatant of the daily broth culture of the studied microorganisms was mixed with an equal volume of serum, the source of complement, and thermostated at 37 ° C for 30 min, then 2.7 ml of chilled (at 4 ° C) veronal buffer was added, the tube was heated in a water bath in for 3 min at 37 ° С, 200 μl of a suspension of sensitized erythrocytes at a concentration of 1.5 × 10 ⁸ SEB / ml was added, mixed, the contents were transferred to a cuvette, and the measurement of erythrocyte lysis rate to its maximum value was immediately started. The result was compared with the control (instead of the supernatant - broth), the difference was counted per unit optical density of the culture (540 nm). Anticomplementary activity was expressed in antilithic units of complement.

In this example, the titer of fungi in the patient’s material did not exceed 10 colony forming cells per milliliter (growth only in the first Petri dish), the antilysimic and anticomplementary activities of microorganisms were 1.25 micrograms per milliliter per unit optical density and 1.5 · 10 ⁶ antilithic complement units , which did not allow to predict the long course of the inflammatory process. The accuracy of the prognosis was confirmed by the further course of the disease. Clinical and laboratory tests (negative bacterioscopy and inoculation of the urogenital discharge) studies after a single course of antibiotic therapy, as well as repeated two clinical and laboratory examinations with an interval of one month allowed to remove the patient from the register.

Example 2. Patient Sh., 28 years old, turned to the OKVD (outpatient card No. 2371 for 2005) with complaints of scanty discharge from the urethra, frequent urges to urinate. Sick for five weeks. I was not treated on my own. The disease binds to sexual contact. Gonococcus is isolated from the urethra. Diagnosed with fresh gonococcal infection; torpid anterior urethritis "the patient was prescribed a course of antibacterial therapy. In order to clarify the prognosis of the disease, the sore urethra was sown on Candida agar using the serial dilution method. The titer of the grown colonies was 10 ³ colony forming cells per milliliter; the culture was identified as the genus Candida and its antilysocyme and anticomplementary activity was determined, for which actions similar to those described in the methods of the previous example were performed. As a result, antilysocyme activity was 1.8 micrograms per milliliter per unit of optical density, and anticomplementary activity was 1.5 · 10 ⁶ antilithic complement units.

Thus, both the titer of the mushrooms and the value of their persistent properties exceeded the experimentally and statistically identified threshold values of the process chronicity indicators. Using the proposed method, a forecast was made for the development of chronic gonorrhea, which was confirmed by clinical and laboratory observations. When a laboratory control is repeated after treatment, gonococci are again detected in the discharge of the urethra. With a diagnosis of Chronic gonorrhea; torpid total urethritis; chronic prostatitis "the patient was prescribed an additional course of antibiotic therapy using drugs that affect the nonspecific reactivity of the body. Clinical and laboratory observations of this patient, therefore, lasted up to 4.5 months, when double laboratory tests yielded a negative result.

Analyzing the clinical and laboratory data of managing 65 patients, the authors concluded that the prognostic criteria of the proposed method for predicting the chronic course of urogenital gonococcal infection are highly accurate (reliable) compared to the prototype method (Table 3). The method is simple, does not require sophisticated equipment and is available for all bacteriological laboratories.

Table 3. A comparative analysis of the accuracy of the proposed method and the prototype method in an unfavorable course of urogenital gonorrhea. Name of characteristic Prototype The proposed method The number of subjects, people 65 Highlighted pure gonococcus cultures 65 The number of gonococcus cultures for which properties are defined 53 * Number of predictions made 16 twenty The number of confirmed forecasts 21 Forecast accuracy 76.2% 95.2% Note - * 12 strains did not grow when reseeding.

BIBLIOGRAPHY

1. Borisov S.D., Deryabin D.G., Minakov A.A., Mikhailenko S.V., Okshin A.M., Lapina I.A. Mixed urogenital infection in the context of reproductive health problems. B: V.M. Boev (eds.) Habitat and public health. Materials of the All-Russian Scientific and Practical Conference. Orenburg, 2001, p. 73-76.

2. Bukharin OV, Ivanov Yu.V., Kuzmin M.D., Mikhailenko S.V. Characteristics of changes in microbiocenosis in patients with chronic nonspecific urethritis. Zhurn. microbiol., 2001, 4: 86-90.

3. (19) RU (11) 2111493 (13) C1, (51) 6 G01N 33/543, 33/558. A method for predicting an adverse course of purulent-inflammatory disease of microbial etiology.

4. Prototype (19) RU (11) 2161312 (13) C2, (51) 7 G01N 33/571. A method for predicting the course of urogenital gonorrhea.

5. Order M3 of the Russian Federation No. 415 “Gonococcal infection” of 09.20.03, Moscow.

6. Voronina L.G. Improving the diagnosis, prognosis and treatment of gonorrhea under the control of the antilysocyme sign of gonococci. // Diss .... doct. medical sciences. - Orenburg, 1998 .-- 222.

7. Fetners K. Is bacterial vaginosis a sexually transmitted infection. J. Sex. Transm. Infect. 2001, 77 (5): 390.

8. Bukharin OV The persistence of pathogenic bacteria. - M.: Medicine. - Yekaterinburg: Ural Branch of the Russian Academy of Sciences. - 1999. ISB No. 5. - 7691-0689-4.

Claims (1)

A method for predicting the chronic course of urogenital gonococcal infection by detecting the presence of a pathogen and determining the persistence factors of microorganisms, characterized in that the number of Candida fungi associated with the pathogen is determined, as well as the presence of persistence factors - antilysocyme and anticomplementary activities and with a titer of Candida fungi of at least 10 ² colony forming cells per milliliter, and the manifestation of both anti-lysozyme activity is not less than 1.3 micrograms per milliliter per unit pticheskoy density and anticomplementary activity not less than 1.5 × 10 ⁶ units antiliticheskih complement diagnosed chronic urogenital infections.