Detection method of microfungi coccidioides posadasii 36 s and coccidioides immitis c-5

FIELD: biotechnologies.

SUBSTANCE: detection method of microfungi Coccidioides posadasii 36 S and Coccidioides immitis C-5 in vitro involves pre-growth of culture in mycelial phase, preparation of a suspension corresponding to 5 units of activity of a standard opacity sample, possibility of spherules formation and detection of spherules filled with endospores. Culture in mycelial phase is grown during 3 days. Possibility of spherules formation is provided by infection of the one-day culture of cells of murine splenocytes, which is obtained on RPMI-1640, and further cultivation during 5 days at the temperature of 37°C, with content of CO2 in atmosphere of 5%. In order to detect spherules in the form of round double-outline formations filled with endospores, a sample is taken, deactivated with formalin and investigated by means of a light microscopy method.

EFFECT: invention allows simplifying the method and reducing the investigation period.

1 ex


The invention relates to medicine, for laboratory diagnosis of causative agents of coccidioidomycosis.

AIDS (disease Wernicke-Posada) - non-communicable systemic mycosis, which is manifested as a primary pulmonary infection, or as a progressive granulomatous lesions of the skin, joints, bones, internal organs and brain membranes. The causative agents of coccidioidomycosis, Coccidioides spp. - until recently, were represented by single species .immitis. In-depth study of the genome of fungi is possible to divide the strains into two groups - California and ncalifornia. In accordance with these data, since 2004, fungi of the genus Coccidioides are divided into two types: .immitis and .posadasii. However, to date the morphological differences among the cultures of different species is not installed.

The problem of identification of phytopathogenic fungi (microscopic fungi of importance in the development of pathological conditions, highly relevant, as it determines the further tactics of treatment and selection of effective drugs. In mycological practice, the most frequently used methods, aimed at the study of macro - and micro-morphology of the cultures. This concept of diagnosis was named morphological or phenotypic, when the determination unit type is defined is the morphological characteristics of the studied strain and comparative analysis by establishing differences with closely related species. This methodological approach is implemented by microscopic fungi cultivation under conditions that allow to obtain a culture of micromycetes with a characteristic morphology.

The causative agents of coccidioidomycosis refer to dimorphic fungi, characterized by the ability to exist in two different physiological phases and respectively in two morphological forms. Saprophyte phase represented by the filamentous form in which the causative agents of coccidioidomycosis live in the environment, and grow on nutrient media used for culturing, microscopic fungi. Strains of Coccidioides spp. within 3-5 days of incubation at 28°C on the surface of the dense nutrient agar form a grayish mold colonies. Microscopic study of the cultures found in filamentous form, revealed hyphae, consisting of a barrel-shaped of arthropod, alternating with smaller sterile dividing cells. Along with the development and maturation of the mycelium is its disintegration into smaller fragments and detached arthroconidia (4-6 µm in length) with the "scraps" cell separators in the form of antennae located at the corners. However, the formation of arthroconidia characterized not only by Coccidioides spp., but some yeast-like fungi and dermatophytes, which has a similar structure: Auxarthron spp., Oidiodendron spp. Gymnoascus spp., followed the flax morphology of filamentous forms of Coccidioides spp, is not strictly specific.

Inhalation of arthroconidia Coccidioides spp. in the tissues of the lung is the development of tissue or parasitic phase. It is represented by spherule - rounded formations from 10 to 200 microns in diameter, filled with endospore (from single to several hundred). Endospores are oval or irregular in shape. Double wall Sterol up to 2 microns in diameter. The final diagnosis of coccidioidomycosis is the identification of the morphological elements of microscopic fungus, characteristic of the parasitic phase, namely Sterol. Note that the immature spherule, i.e. undifferentiated education may not be taken into account in the absence of typical signs, such as a double shell, and the presence of endospore.

Cultivation of fungi in tissue (parasitic) phase in nutrient medium, i.e. iv vitro is difficult. There is a method of cultivation 3-4 monthly cultures of Coccidioides spp. in the medium RPMI 1640 containing 10% inactivated serum, 2,835 mg Na2PO4×7H2O per liter, of 0.8 mg N-Tamol, 40 units of penicillin and 40 mg of streptomycin per milliliter, in an atmosphere of 5% CO2at 35°C. After 48 h after initial inoculation of fungal suspension is filtered and re-seeded in fresh portion of the nutrient medium of the same composition. Full spherule formed within 8 days it is mandatory re-seeding culture in a fresh portion of the nutrient medium every 48 h of cultivation (Alan F. Petkus, Linda L. Baum, Robert B. Ellis, Malvin Stern and David L. Danley "Pure Spherules of Coccidioides immitis in continuous culture", Journal of Clinical Microbaiology, Aug., 1985, p.165-167). The presented method is time-consuming, costly and very time-consuming, making impractical their utilization for detection of microscopic fungi of the genus Coccidioides spp.

The closest analogue is the way to detect the causative agents of coccidioidomycosis in vivo. It provides for the infection of white mice particles 2-week cultures of Coccidioides spp., and observing them within 7 days. After that, kill mice, extract the liver from three animals and add 10 ml of 10%KOH solution, heated in a boiling water bath for 15 min and after dissolution bioprobes material sterilized with formalin. Then decontaminated suspension in a 1.5 ml centrifuged at 5000 rpm, the supernatant removed, and the precipitate prepare smears for light microscopy without staining and detection of Sterol judge the presence of the causative agent of coccidioidomycosis. (T.A. Grishkin, B.C. forest, E.V. Timofeeva, V.A. Spiridonov "Method for detection of the causative agent of coccidioidomycosis". RF patent №2265844, C1 G01N 33/48, 2004). A significant disadvantage of this method is that its implementation required odimo work with infected bioprobe animals as well as a longer research time for comparison with the proposed method.

The task of the invention is to devise simple and effective way of detecting microscopic fungi of the genus Coccidioides spp. in vitro.

The technical result is a reduction in terms of research and simplification of the method, due to the fact that the study does not require multiple transfers, performed in vitro without the use of biotronik animals.

The technical result is achieved by conducting a preliminary crop in the mycelial phase, the preparation of the suspension corresponding to 5 units at a time standard sample turbidity conversion in tissue culture phase and detection Sterol in the form of a rounded double-loop formations filled with endospores. The method differs in that the culture is grown for 3 days and cooked it in suspension infect the daily cell culture murine splenocytes, cultured for 5 days at 37°C, with content in the atmosphere of 5% CO2. then select a sample volume of 0.1 ml, sterilized with formalin and examined by light microscopy in drug "crushed" drop at magnification ×600.

Example 1 (optimal):

Strains of Coccidioides posadasii 36 S and Coccidioides immitis C-5 grown on the environment Saburo at 28°C for 3 days. Then the cult of the s wash 0.15 M solution of sodium chloride, pH 6.8, prepare a suspension, corresponding to the 5 UNITS of the industry standard sample turbidity gisk named after. Lytvyn (CCA) and 0.5 ml of suspension make tablets with cell cultures of splenocytes.

Cell culture murine splenocytes in the form of a monolayer prepare for the day before infection with fungal cultures. As a donor splenocytes using inbred BALB/c mice. Allocate the spleen in compliance with the rules of asepsis, homogenize it in a serum-free medium RPMI-1640 with glass cage, then twice washed cell suspension by centrifugation 1000 rpm for 10 min, resuspended sediment in 5-10 ml of the same medium. Counting splenocytes hold the camera Goryaeva, evaluate their concentration and viability using a 0.4% solution Trypanosoma blue. Seeding of splenocytes to produce tablets based 5,0-8,0×104CL/cm2. Cultivation of murine splenocytes carried out in medium RPMI-1640, pH 7.2 in the presence of 2 mM sodium bicarbonate, 20 mM Hepes, 10% ETS (Biolot"), without the addition of antibiotics, in CO2incubator (Hereus) at 37 atmosphere containing 5% CO2within 24 hours, and then infect obtained in the form of a monolayer culture of murine splenocytes by suspensions of the fungi and additionally cultivated under the same conditions for 5 days. Samples decontaminated by adding formalin at 10% of otoyama samples within 24 h, prepare native drugs "crushed" drop and examined by light microscopy (magnification ×600). The result is that in all fields of view observed spherule different degrees of maturity, the young form having a size of 5-10 microns, to Mature, the size of 60-80 microns, with a pronounced two-shell and formed by endospore.

The detection of microscopic fungi of the genus Coccidioides posadasii 36 S and Coccidioides immitis C-5 in vitro, including preliminary cultivation of rice in the mycelial phase, the preparation of the suspension corresponding to 5 units at a time standard sample turbidity, providing the possibility of formation of Sterol and detection Sterol in the form of a rounded double-loop formations filled with endospore, characterized in that the culture in the mycelial phase is grown within 3 days, moreover, providing the possibility of formation of Sterol carried out by infecting the daily cell culture murine splenocytes obtained in the medium RPMI-1640, and subsequent culturing for 5 days at 37°C content in the atmosphere of 5% CO2and to detect Sterol in the form of a rounded double-loop formations filled with endospore selected sample, sterilized with formalin and examined the sample by light microscopy.


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