RU2553307C1 - Method for identifying forms of urogenital trichomonads - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves sampling a biological tissue, introducing a biologically active substance therein, keeping the prepared mixture in a thermostat at 37°C and incubating, performing microscopic examination for identifying the forms of trichomonads. The sampled biological tissue represents the urogenital mucosa that is incubated for 24 h in a nutrient medium. The prepared sediment is separated and added with the biologically active substance nitrosoguanidine in an amount of 500 mcg/ml. That is followed by incubating for 15 min at +37°C; a phosphate buffer with pH 5.6 heated to +37°C is added and centrifuged twice at 1000 rpm for 10 min. The material is placed into the nutrient medium for trichomonad growing, incubated for at least 24 hours, and microscopic examination is used to identify atypical nonflagellate and typical round and amoeboid forms of trichomonads.

EFFECT: using the declared invention enables the effective, fast and reliable identification of typical changed and atypical forms of trichomonads.

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Description

The invention relates to medicine, namely venereology, urology, obstetrics and gynecology, parasitology, medical microbiology.

A known method for identifying typical forms of trichomonads, in which, after receiving pathological material from the mucous membranes of the genitourinary organs, it is inoculated by inoculation into a liquid nutrient medium, followed by incubation at a temperature of + 37 ° C for 3-17 days (Appendix No. 2 to the order of the Ministry USSR Healthcare dated July 12, 1985 No. 936 "Methodological guidelines for the use of unified clinical diagnostic and bacteriological studies in the diagnosis of gonorrhea and trichomoniasis", clause 2.6).

The disadvantage of this method is that it is not possible to identify atypical forms of Trichomonas, which, exhibiting low metabolic activity, are difficult to reproduce on nutrient media. The study is long, not giving the possibility of express diagnostics.

A known method for detecting forms of Trichomonas, including sowing blood in a Teras culture medium with the addition of human or horse serum and antibiotics in the amount of 500 IU / ml inactivated for 5-10 minutes at a temperature of 56 ° C. The culture is incubated for 2 days at a temperature of 37 ° C. Then these cycles are repeated twice with various nutrient media and exposure modes, after which the forms of trichomonads are revealed (RF Patent 2289813, IPC G01N 33/48, publ. 2006).

The disadvantages of this method are its complexity, duration, high risk of obtaining doubtful or negative results due to the study of pathological material - blood, which is an unusual habitat for Trichomonas. The centrifugation mode of 3000 rpm for 20-30 minutes is a gross stress factor, leading to the destruction of protozoa. Moreover, the rounded and amoeboid forms of trichomonads, as well as possible other forms, are more likely not atypical cells, but options for adaptation of the simplest to changing environmental conditions.

The closest is a method for identifying forms of trichomonads (RF Patent 2262104, IPC G01N 33/48, publ. 2005), which includes taking biological material, adding biologically active substances to it, holding the mixture in an incubator at 37 ° C, followed by incubation for 24 -48 h, microscopic examination to identify forms of Trichomonas. As a result of repeated treatment every 12 hours with a new dose of a biologically active substance (enzyme) of the mixture, blood cells are destroyed, microscopically manifested in the form of fragments of cellular elements, among which visible and altered atypical forms of trichomonads.

The disadvantages of this method are its complexity, the duration of the run, the high risk of obtaining doubtful and negative results due to the study of pathological material (blood), which is not the usual habitat for Trichomonas, and prolonged repeated exposure of the mixture to a proteolytic enzyme, as well as inadequate centrifugation mode (3000 rpm / min, 20-30 min), which contributes to the destruction and death of protozoa cells.

The objective of the invention is to eliminate these drawbacks, increase the reliability of identifying all forms of trichomonads present in the pathological material, reduce the time required to obtain a result, identify altered typical and atypical forms of trichomonads by adding nitrosoguanidine to the precipitate of a protozoa daily culture, followed by washing and adding a nutrient medium , which causes changes in the nucleus (polymorphism: round, elongated - 2/3 of the length of the simplest, curved, reduced by a factor of Leray nucleus); supporting apparatus (axostyle) - shortening, thickening, curved axostyle, lack of spine; loss of organs of movement - flagella; a change in the metabolism of the simplest - damage to hydrogenosomes (analogues of mitochondria), activation of pinotag and phagocytosis, the formation of giant vacuoles.

To do this, in a method for identifying forms of trichomonads, including taking biological tissue, introducing biologically active substances into it, holding the resulting mixture in an thermostat at 37 ° C followed by incubation, conducting a microscopic examination to identify atypical forms of trichomonads, it is proposed to use scraping with biological material genitourinary mucous membranes. This biological tissue is incubated for 24 hours in a nutrient medium, the resulting precipitate is separated and 500 μg / ml nitrosoguanidine is added to it as a biologically active substance, incubated for 15 min at a temperature of + 37 ° C, phosphate heated to a temperature of + 37 ° C is added a buffer with a pH of 5.6 followed by double centrifugation at 1000 rpm for 10 minutes, then placed in a nutrient medium for growing Trichomonas, incubated for at least 24 hours and microscopic examination revealed atypical flagellates and typical round and amoebiform ormy trichomonas.

Figure 1 presents the flagellate form of Trichomonas (example 1); figure 2 - the rounded shape of Trichomonas (example 2); figure 3 - amoebic form of Trichomonas (example 3).

The proposed method provides a cultural diagnosis of typical altered and atypical forms of Trichomonas. This occurs under the influence of nitrosoguanidine, which is a chemical mutagen (stress factor), which causes the activation of pino- and phagocytosis, the active fixation of protozoa to the surface of epithelial cells and to each other, the formation of protozoa colonies for self-preservation.

The method is as follows.

Material taken from the vaults of the vagina (in women) or the urethra (in men) with a sterile cotton dacron swab was immersed in 5 ml of culture medium for growing Trichomonas, the rinse was rinsed in the medium. Incubated for 24 hours at a temperature of + 37 ° C. When grown on liquid nutrient media, urogenital trichomonads give a bottom growth in the form of a dense whitish sediment.

For the preparation of the nutrient medium, “The basis of the nutrient medium for the isolation of vaginal Trichomonas (dry)” was used (produced by FSUE NPO Microgen of the Ministry of Health of the Russian Federation, cat. No. C01). The composition of the base in g / l: extract of fodder yeast, D-maltose, sodium chloride, potassium chloride, sodium carbonate (pH 6.3 ± 0.3). Method of preparation: 28.5 g of the drug was stirred in 1 liter of distilled water, boiled for 1-2 minutes and poured into vials. The medium was autoclaved at t + 112 ° C for 20 minutes. 20% cattle serum was added to the nutrient medium cooled to t + 45-50 ° C, mixed thoroughly and poured into 5 ml sterile tubes. The surface of the medium in the test tubes was poured with sterile liquid paraffin oil with a layer height of 3-5 mm. Tubes with a nutrient medium were kept in a thermostat at t + 36 ± 1.0 ° C for 22-24 hours to control the sterility of the medium.

To obtain atypical forms of urogenital trichomonads, the daily culture precipitate was adjusted with saline to a volume of 1.0 ml and nitrosoguanidine was added in an amount of 500 μg / ml. According to the literature, the effects of mutagen on protozoa are indicated by their 50% death in culture (Pimenova M.N., Grechushkina N.N., Azova L.G., Netrusov A.I. Guide to practical studies in microbiology / Ed. Egorova N.S. - 3rd ed.; Revised and additional - M: Publishing house of the ISU, 1995. - S. 173-179).

After the exposure time (15 min) at a temperature of + 37 ° C, add 3 ml of phosphate buffer heated to a temperature of + 37 ° C with a pH of 5.6, followed by double centrifugation at 1000 rpm for 10 minutes.

After washing, 5.0 ml of culture medium for growing Trichomonas were added to the protozoa culture and incubated for 24 hours at + 37 ° C. Such a cultivation of a mutated culture of vaginal trichomonas allows us to identify various typical, altered and atypical (flagellate) forms in the form of a bottom dense whitish sediment.

The presence of the above forms of trichomonas allows you to confirm the diagnosis of trichomoniasis, and in their absence to exclude this diagnosis.

Example 1

Patient M., 45 years old, was admitted with a diagnosis of trichomoniasis.

Pathological material from the posterior and lateral vaginal arches was examined. A study on the proposed method.

After exposure to nitrosoguanidine on the precipitate of a daily culture of trichomonads, flagellate forms of trichomonads were found.

The diagnosis is confirmed - urogenital trichomoniasis.

The treatment: metronidazole according to the known scheme. After the end of treatment - a complete recovery.

Example 2

Patient O., 27 years old, was admitted with a diagnosis of trichomoniasis.

Pathological material from the posterior and lateral vaginal arches was examined. After exposure to nitrosoguanidine on the precipitate of a daily culture of Trichomonas, rounded forms of Trichomonas were found.

Diagnosis: urogenital trichomoniasis.

Metronidazole was treated until complete cure.

Example 3

Patient F., 30 years old.

Pathological material from the posterior and lateral vaginal arches was examined.

According to the method described above, nitrosoguanidine was exposed to the precipitate of a daily Trichomonas culture, and amoeboid forms of Trichomonas were found.

Diagnosis: urogenital trichomoniasis.

Metronidazole was treated.

The proposed method was used in 2245 patients, the identification of forms made it possible to select adequate drug therapy and achieve complete etiological cure of patients.

This method is of great importance for the diagnosis of urogenital trichomoniasis, when the infection is asymptomatic and latent, and the pathogen has an amastigotic (non-flagellate) form, which is difficult to detect with conventional culture diagnostic methods. Using this technique, the existence of atypical (flagellate) forms of protozoa was confirmed, the phenomenon of dropping flagella and the transformation of Trichomonas into flagellate forms (amastigotes) was revealed.

Claims (1)

A method for identifying forms of trichomonads, including taking biological tissue, introducing biologically active substances into it, holding the resulting mixture in an thermostat at 37 ° C followed by incubation, conducting a microscopic examination to identify the forms of trichomonads, characterized in that the material is sampled as biological tissue the mucous membranes of the genitourinary organs, which are incubated for 24 hours in a nutrient medium, the resulting precipitate is separated and added to it as a biologically active substance nitrosoguanidine in an amount of 500 μg / ml, incubated for 15 min at a temperature of + 37 ° C, add phosphate buffer at a temperature of + 37 ° C with a pH of 5.6, followed by double centrifugation at 1000 rpm for 10 minutes, then placed in a nutrient medium for the cultivation of Trichomonas, incubated for at least a day and microscopy reveals atypical flagellates and typical round and amoebiform forms of Trichomonas.

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