Method for identifying forms of urogenital trichomonads

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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The invention relates to medicine, namely venereology, urology, obstetrics and gynecology, Parasitology, medical Microbiology.

The known method of identification of typical forms of Trichomonas, which after the receipt of pathological material from mucous membranes of the urogenital organs carry out sowing his method of inoculation in liquid nutrient medium, followed by incubation at +37°C for 3-17 days (Appendix №2 to the order of the Ministry of health of the USSR from July 12, 1985, No. 936, "guidelines on application of standardized clinical diagnostic and bacteriological studies in the diagnosis of gonorrhea and trichomoniasis", p.2.6).

The disadvantage of this method is that it is not possible to identify atypical forms of Trichomonas that, showing low metabolic activity, hardly propagated in nutrient media. Study long, not giving the possibility of rapid diagnosis.

A method for detecting Trichomonas forms, including blood cultures in a nutrient medium Terrace with the addition of inactivated for 5-10 minutes at a temperature of 56°C human serum or horse and antibiotics in the amount of 500 IU/ml Carried out the incubation of the culture for 2 days at 37°C. Then twice repeat these cycles with various�CNAME nutrient environments and exposure modes, then identify forms of trichomonads (Patent RF 2289813, IPC G01N 33/48, publ. 2006).

The disadvantages of this method are its complexity, length of performance, there is a risk for questionable or negative results due to the study of pathological material - blood, which is an unusual habitat for Trichomonas. Mode centrifugation 3000 rpm for 20-30 minutes is a gross stress factor leading to the destruction of the protozoa. With rounded and amoeboid forms of Trichomonas, and also other forms are probably not atypical cells, and variants simplest adaptation to changing environmental conditions.

The closest is a method for detecting Trichomonas forms (Patent RF 2262104, IPC G01N 33/48, publ. 2005), including the capture of biological material, the addition of biologically active substances, exposure of the mixture in a thermostat at 37°C, followed by incubation for 24-48 h, the conducting microscopic examination for the detection of Trichomonas forms. As a result of repeated treatment every 12 hours a new dose of a biologically active substance (enzyme) mixture is the destruction of red blood cells, microscopically manifested in the form of fragments of cellular elements, among which a notable modified and atypical forms of Trichomonas.

The object of the invention is to eliminate these drawbacks, increasing the reliability of detection in pathological material Trichomonas forms, reducing the time needed to obtain result, detection of the modified typical and atypical forms of Trichomonas by adding nitrosoguanidine to the precipitate overnight culture of protozoa with subsequent laundering and the addition of nutrient medium, which causes changes in the nucleus (polymorphism: rounded, elongated - 2/3 of the length of a simple, curved, reduced in size of the nucleus); a supporting unit (axostyle) - shortening, thickening, curved axostyle, no spinules; the loss of the organs of movement - flagella; changing the metabolism of the simplest - damage hydrogenosome (analogues of mitochondria), activation Pino - and phagocytosis, the formation of giant vacuoles.

To do this, in the method for detecting Trichomonas forms, including z�their biological tissue, introduction of biologically active substances, exposure of the mixture in a thermostat at 37°C, followed by incubation, conducting microscopic examination for the detection of atypical forms of Trichomonas proposed as a biological material to use the scrapings from the mucous membranes of the urogenital organs. This biological tissue was incubated in a day in a nutrient medium, separating the resulting precipitate and add to it as biologically active substances nitrosoguanidine in the amount of 500 μg/ml, incubated for 15 min at +37°C, add preheated to a temperature of +37°C phosphate buffer with a pH of 5.6 followed by a double by centrifugation at 1000 rpm for 10 min, then placed in a nutrient medium for the cultivation of Trichomonas incubated for at least a day and microscopy reveal atypical besthomemovies and typical rounded and amoeboid forms of trichomonads.

Fig.1 shows basiguttata form Trichomonas (example 1); Fig.2 - rounded Trichomonas (example 2); Fig.3 - amoeboid form of Trichomonas (example 3).

The proposed method provides culture diagnosis typical modified and atypical forms of Trichomonas. This occurs under the action of nitrosoguanidine, which is a chemical mutagen (stress factor) that causes the activation of Pinot and Fugazi�oz, active fixation simplest to the surface of epithelial cells and to each other, the formation of colonies of protozoa for the purpose of self-preservation.

The method is as follows.

Material taken from the vaults of the vagina (in women) or urethra (men) sterile cotton micronova swab was immersed in 5 ml of nutrient medium for the cultivation of Trichomonas, the swab was rinsed in the environment. Were incubated for 24 h at +37°C. When grown on liquid nutrient media urogenital Trichomonas give demersal growth in the form of dense whitish precipitate.

For the preparation of nutrient media used "the Basis of the nutrient medium for the isolation of vaginal Trichomonas (dry) (production FSUE "NPO "Microgen" MOH", cat. No. C01). The composition of the basis 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 preparation is stirred in 1 l of distilled water, boiled for 1-2 minutes and poured into vials. The medium was sterilized by autoclaving at t +112°C for 20 min To a cooled to t +45-50°C culture medium was added 20% bovine serum, thoroughly stirred and poured into 5 ml sterile tubes. The surface of the medium in test tubes filled with sterile paraffin oil layer height of 3-5 mm. Tubes with nutrients such a�filled, the medium was incubated at t +36±1.0°C for 22-24 h to control the sterility of the environment.

To obtain atypical forms of urogenital Trichomonas precipitate overnight culture was adjusted with physiological saline to a volume of 1.0 ml and was added nitrosoguanidine in the amount of 500 μg/ml. According to the literature on the effects of the mutagen on the simplest indicates of their 50% death in culture (Pimenov M. N., Grechushkina N. N., Azov L. G., Netrusov A. I. a Guide to practical exercises in Microbiology/ edited by Egorova N. With. - 3rd ed.; revised and enlarged extra - M.: Publishing house of IPA, 1995. - Pp. 173-179).

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

After washing the culture of protozoa was added 5.0 ml of the nutrient medium for the cultivation of Trichomonas and were incubated for 24 h at +37°C. the cultivation mutated culture Trichomonas vaginalis allows to identify various typical modified and atypical (besthomemovies) shape in the form of near-bottom dense whitish precipitate.

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

Example 1

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

Investigated the pathological material from the posterior and lateral vaginal fornix. Conducted researched�e of the proposed method.

After being exposed nitrosoguanidine to precipitate overnight culture of Trichomonas detected besthomemovies forms of trichomonads.

The diagnosis was confirmed - urogenital trichomoniasis.

Treatment: metronidazole well-known scheme. After treatment is complete recovery.

Example 2

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

Investigated the pathological material from the posterior and lateral vaginal fornix. After being exposed nitrosoguanidine to precipitate overnight culture of Trichomonas detected rounded Trichomonas.

The diagnosis of urogenital trichomoniasis.

The patient was treated with metronidazole to cure.

Example 3

Patient F., 30 years.

Investigated the pathological material from the posterior and lateral vaginal fornix.

By the above procedure is carried out the impact nitrosoguanidine to precipitate overnight culture of trichomonads found amoeboid forms of trichomonads.

The diagnosis of urogenital trichomoniasis.

The patient was treated with metronidazole.

The proposed method was applied in 2245 patients, the identification of the forms allowed to pick up adequate drug therapy and to achieve a complete etiological cure patients.

This method is of great importance for the diagnosis of urogenital trichomoniasis when infection occurs asymp�UMNO and latent, while the pathogen has amastigote (basiguttata) form, which is difficult to detect with conventional culture methods of diagnosis. With the help of this technique confirmed the existence of atypical (besthomemovi) forms of protozoa identified the phenomenon of dropping flagella and transformation of Trichomonas in besthomemovies form (amastigote).

A method of detecting Trichomonas forms, including the taking of biological tissue, the introduction of biologically active substances, exposure of the mixture in a thermostat at 37°C, followed by incubation, conducting microscopic examination for the detection of Trichomonas forms, characterized in that as a biological tissue carry out the extraction of the material from the mucous membranes of the urogenital organs, which were incubated in a day in a nutrient medium, separating the resulting precipitate and add to it as biologically active substances nitrosoguanidine in the amount of 500 μg/ml, incubated for 15 min at +37°C, add preheated to a temperature of +37°C phosphate buffer with a pH of 5.6 followed by a double by centrifugation at 1000 rpm for 10 min, then placed in a nutrient medium for the cultivation of Trichomonas incubated for at least a day and microscopy reveal atypical besthomemovies and typical rounded and amoeboid forms of trichomonads.



 

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