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INVENTION
Russian Federation Patent RU2289813
A method for detecting Trichomonas Forms
Name of the inventor: Garas'ko Ekaterina (RU); Moreva Jeanne Germanovna (RU)
The name of the patentee: State educational institution of higher education "Ivanovo State Medical Academy of the Russian Federation Ministry of Health" (RU)
Address for correspondence:. 153012, Ivanovo, pr Engels, 8, GOU VPO "Ivanovo State Medical Academy of the Russian Federation Ministry of Health"
Starting date of the patent: 2004.11.09
The invention relates to medicine, namely to medical microbiology. To identify forms Trichomonas produce blood culture in medium supplemented with Terrace inactivated for 5-10 minutes at + 56 ° C in human blood or horse serum and antibiotics in an amount of 500 U / ml. The culture was incubated for 2 days at + 37 ° C. Make subcultured to a similar medium but containing horse serum or human blood, inactivated for 15-20 minutes, and antibiotics in an amount of 250 U / ml. Re-incubated for 2 days at + 37 ° C. Make subcultured on a medium containing serum, inactivated for 30 minutes, without the addition of antibiotics, and the sample was incubated at + 37 ° C for 2 days. 1-2 reseeding is performed on a medium with fully inactivated human serum or horse without the addition of antibiotics. By going on the active budding or schizogony judge the living presence of atypical forms of trichomonads, and in the absence of reproduction of degenerative, decaying forms. Using the method makes it possible to identify atypical forms of trichomonads.
DESCRIPTION OF THE INVENTION
The invention relates to medicine, namely to the obstetrics and gynecology, parasitology, hematology, urology, venereology, Medical Microbiology.
According to the literature known methods for detecting Trichomonas vaginalis from clinical specimens. The most reliable method is a culture, which serves for detecting Trichomonas typical forms in the material on nutrient media. The greatest growth quality manifest in products of complex composition, such as Terrace or Johnson-Trussell containing as main components liver broth, Ringer's solution, peptone, maltose, agar-agar, L-cysteine added as an enriching factor serum of human or horse blood inactivated for 30 minutes at + 56 ° C. In order to suppress the accompanying microflora added to the medium the antibiotics penicillin, streptomycin, and nystatin in an amount 1500-2000 U / ml, previously dissolved in Ringer's solution. Establish medium pH 6,0-6,3.
The essence of the known methods in that, after the fence material with pathologic mucosal urinary organs, it is carried out seeding method the liquid inoculation, the nutrient medium, followed by incubation at + 37 ° C for 5-7 days (B.V.Klimenko, E.R.Avazov et al. Trichomoniasis men, women and children. Saint-Petersburg, OOO "Story", 2001, s.131-138). However, known dwelling Trichomonas not only on the mucous membranes of genito-urinary organs, but also in the peripheral blood, where Trichomonas, while in not their usual habitat, presents a significant number of atypical forms and method allowing culturally identify atypical forms Trichomonas blood, now not developed (PPSemenov, V.P.Semenov trichomonas defeat of the genito-urinary organs. L. Dep th, "Medicine", 1972, s.23-25).
The disadvantage of these methods is that they do not allow the identification of atypical forms Trichomonas, particularly in blood cultures who, showing low metabolic activity, hardly propagated in nutrient media.
The technical result of the proposed method is to identify atypical forms of trichomonads by adding to the culture medium of human serum or horse serum carried out by increasing the time of inactivation of from 5 to 30 minutes in three phases, which activates the proliferative activity of the elementary cells.
This result is achieved in that the nutrient medium is added stress factor - biologically active substance - human serum or horse just inactivated, which promotes active reproduction by budding or schizogony amoeboid, cyst-like, or various irregular, without nuclear and fixed forms of Trichomonas having low metabolic activity.
Proposed method is as follows
Blood taken from a vein of the patient, at least 2.0 mL were mixed with 15 U / ml heparin and poured into centrifuge tubes in an amount of not more than 1 ml in each. Samples were centrifuged for 20-30 minutes at 3000 rev / min. After the supernatant liquid portion - the plasma decanted and the residue in 0.5 ml is inoculated with a Pasteur pipette into 4 ml of culture medium. The first passage atypical forms produced in a culture medium supplemented with Terrace as a stress factor and enriching human serum or horse inactivated for 5-10 minutes at + 56 ° C. In order to suppress the accompanying microflora antibiotics added to the medium in an amount of 500 U / ml penicillin, streptomycin, and nystatin, which is an additional stress factor.
The culture was incubated for 2 days at + 37 ° C under a layer of paraffin oil to create anaerobic conditions. Then make reseeding a similar environment, but contains less stress factor: partially inactivated human serum or horse (for 15-20 minutes) and antibiotics in an amount of 250 U / ml. Samples were incubated at + 37 ° C. After 2 days, producing a third passage of culture on medium containing no stress factors, antibiotics and blood completely inactivated serum (30 min inactivation time at + 56 ° C). Samples were incubated at + 37 ° C for 2 days. Then perform another 1-2 reseeding culture medium and containing no stress factors. Such prolonged passaging allows isolation of a pure culture of Trichomonas which contains the highest number of different typical and atypical forms in the form of whitish bottom suspension.
The proposed method provides a diagnosis of atypical culture - amoeboid and rounded, and various changes and irregularly shaped Trichomonas: dumbbell, biskvitovidnyh and other different in size, even if they are present in clinical samples in a single quantity. This occurs under the influence of weak-inactivated human serum or horse, which is a stress factor that causes the active reproduction by budding or Trichomonas schizogony for the purpose of self-preservation.
This process is important in the diagnosis of generalized trichomoniasis when the infection is asymptomatic and latent, and the causative agent of an atypical form has a low metabolic activity, which is difficult to detect with conventional culture methods of diagnosis.
example 1
Patient Kiricheva R., age 64.
Diagnosis: cr colon. In the history of Trichomonas coleitis. For the treatment prescribed protistotsidnye drugs. After the first passage when blood cultures revealed Trichomonas 36 × 10 5 cells / ml. After 5 passages in culture medium increased the number of trichomonads to 280 × 10 5 cells / ml. Consequently, the patient revealed live typical and atypical forms of trichomonads.
example 2
Bezruchenkova Patient A., age 67 years.
Diagnosis: cr sigmae. In the history of Trichomonas coleitis. For the treatment prescribed protistotsidnye drugs. After the first passage when blood cultures revealed Trichomonas 18 × 10 4 cells / ml. After 5 passages were observed in a nutrient growth medium, and the number of Trichomonas decreased. Consequently, the patient revealed degenerative, decaying forms of Trichomonas.
CLAIM
A method for detecting Trichomonas forms comprising seeding a biological material in a nutrient medium containing inactivated human serum and antibiotics, or horse, characterized in that produce blood cultures in Terrace medium supplemented with human serum or horse inactivated for 5-10 minutes at temperature + 56 ° C, and antibiotics in an amount of 500 U / ml - penicillin, streptomycin, and nystatin; then the culture was incubated for 2 days at + 37 ° C, making reseeding a similar medium but containing human serum or horse inactivated for 15-20 min, and antibiotics in an amount of 250 U / ml, incubation was carried repeated for 2 days at + 37 ° C; then making reseeding with medium containing serum, inactivated for 30 minutes, without the addition of antibiotics, and the sample was incubated at + 37 ° C for 2 days, followed by subculturing 1-2 carried on the medium, with fully inactivated human serum or horse without the addition of antibiotics, and what is going on actively budding or schizogony judge the living presence of atypical forms of trichomonads, and in the absence of reproduction - a degenerative, decaying forms.
print version
Publication date 06.01.2007gg
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