RU2739758C1 - Method for identifying candida spp_ and other yeast-like fungi from positive blood culture by matrix laser desorption ionization time-of-flight mass spectrometry (maldi-tof ms) in patients with blood flow infection - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, particularly to microbiology. Disclosed is a method for identifying yeast-like fungi of the genus Candida and genus Rhodotorula, comprising sample preparation of positive blood culture and identification of yeast-like fungi by matrix-activated laser desorption ionization time-of-flight mass spectrometry. At that, sample preparation of positive blood culture is performed by means of its transfer from flask with liquid nutrient medium to a test tube with separating gel and then centrifuged at 1000 rpm for 2 minutes. Obtained supernatant is exposed to multiple sequential centrifugation and addition of deionised water. Supernatant is then removed, deionised water and 96 % ethyl alcohol are added to the sediment and mixed once more with a vortex, followed by centrifugation at 14000 rpm for 2 minutes, then supernatant is removed. Then, microcentrifuge test tube with open cover is left to complete evaporation of alcohol, followed by extraction of protein extract with formic acid and acetonitrile, the obtained supernatant is applied on a target of a mass spectrometer, after drying is coated with a matrix and identification of yeast-like fungi of the genus Candida and genus Rhodotorula is carried out until the generic identity by the value of the identification coefficient (score) from 1.1 to 1.5 and to species identity - from 1.6 and higher.

EFFECT: invention provides a wider range of technical means for accelerated identification of microorganisms from blood containing yeast-like fungi with high reliability.

1 cl, 3 tbl, 4 ex

Description

The invention relates to medicine, in particular to microbiology with the possibility of using patients in hematology, and can be used to identify Candida spp. and other yeast-like fungi from positive blood culture by matrix laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) in patients with bloodstream infection.

Currently, there is a clear trend towards an increase in the number of fungal infections and, accordingly, it is urgent to improve the level of their diagnosis. Various factors play a role in the development of invasive fungal infections, including not only the patient's condition, but also the environment. Fungi are among the main pathogens of opportunistic infections in immunocompromised patients.

Over the past 10-15 years, there has been a clear trend towards an increase in the number of mycotic infections. Thus, yeast-like fungi are among the ten most frequently detected nosocomial pathogens; in intensive care units, they are ranked fifth, reaching 17%; among bloodstream infections Candida spp. take 4th place, accounting for 7.6%; about 7% of cases of fevers of unknown etiology in patients undergoing treatment in a hospital are caused by fungi. Moreover, these microorganisms are problematic not only in hematological cancer patients. Thus, when analyzing 837 cases of candidemia, it was shown that they most often occurred in patients with malignant tumors (26%) and after surgical treatment (18.5%). At the same time, among patients with a surgical profile, the highest frequency was registered in abdominal surgery, amounting to 13.5% (number of patients 113). The incidence of candidemia in cardiothoracic surgery was 4.3%, in general surgery departments - 1.7%. Fungal infections are common complications in patients with burns and after trauma. Mortality in patients with invasive candidiasis remains high and reaches 30-40%. According to a multicenter prospective study conducted in Russia from 2005 to 2013, among 55 patients with candidemia, the need to transfer patients to intensive care had a negative impact on the results of treatment of patients with candidemia, reducing the cure rate by 50 times.

Among infections caused by yeast-like fungi, the main share (up to 90%) are fungi of the genus Candida.

The risk of developing a fungal infection is determined by many factors that can act as synergists. In patients with malignant tumors, additional risk factors are prolonged granulocytopenia after polychemotherapy, impaired cellular immunity, colonization of the mucous membranes of Candida spp., Severe mucositis with cytostatic exposure.

One of the most serious complications is candidemia - the circulation of Candida in the bloodstream. Invasive candidiasis caused by fungi of the genus Candida is a frequent and severe complication, which is characterized by a higher mortality rate that requires long-term treatment. Among yeast-like fungi, C. albicans predominate, however, in recent years, their decrease and an increase in such fungi as C. parapsilosis, C. tropicalis, C. krusei, C. glabrata have been observed.

In some cases (5-7%) infections can be caused by other yeast-like fungi, such as Saprochaete capitata, Saccharomyces cerevisiae, Rhodotorula spp., And some of them require the appointment of special antimycotics, since they exhibit natural resistance to some antifungal drugs.

One of the leading causes of high mortality in patients with candidemia is the untimely and inadequate prescription of antimycotics. In this regard, it is extremely important to present the results of identification of infectious agents as soon as possible.

To date, it is known to use a polymerase chain reaction (PCR) to identify the causative agent of a fungal infection, with the help of which material obtained by bronchoalveolar lavage or puncture is examined [Bretagne, Costa, Marmorat-Khuong. Detection of Aspergillus spesies DNA in bronchoalveolar lavage samples by competitive PCR // J. Clin. Mcrobiol. 1995 Vol. 33, No. 5.-P. 1164-1168]. It is also possible to determine specific DNA using PCR in blood [Einsele, Hebart, Roller. Detection and identification of fungal pathogens in blood by using molecular probes // J. Clin. Mcrobiol - 1997. - Vol. 35, No. 6.-P. 1353-1360]. Microbiological research very rarely reveals the presence of a pathogen in a blood culture; also routine serological tests are not effective enough.

There is also known a method for identifying microorganisms, including yeast-like fungi, from a test sample of hemoculture. The method provides for obtaining a test sample, selective lysis and dissolution of cells that are not microorganisms of the test sample, layering the resulting lysate onto a density buffer in a sealed container and further centrifugation. The density buffer has a uniform density from about 1.025 g / ml to 1.120 g / ml. In this case, microorganisms, passing through the specified buffer, form a sediment at the bottom of the container. The sediment is examined using Raman spectroscopy, which allows the identification of the microorganism at the genus or species level. The invention makes it possible to identify yeast-like fungi from clinical samples in less than 120 minutes (RU 2541775, 20.02.2015).

The closest technical solution to the claimed invention is a method for the express identification of yeast-like fungi of the genus Candida from a positive blood culture using matrix-activated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), while from vials containing polymer granules as a sorbent, to 1 ml of its contents, placed in a microtube, add 200 μl of a 5% aqueous solution of saponin for the lysis of erythrocytes. The mixture is incubated for 5 min at room temperature, the lysate is centrifuged (12000 rpm, 1 min), after which the supernatant is removed. Then the precipitate is washed with 1 ml of phosphate-buffered saline, then re-centrifuged (12000 rpm, 1 min) and the supernatant is removed. First, 300 μl of bidistilled water is added to the sediment, stirred, then 900 μl of ethanol, centrifuged (12000 rpm, 2 min), the supernatant is removed, the sediment is dried at room temperature for several minutes, after which 20 μl of formic acid is added successively to it. acid, then an equal amount of acetonitrile. The resulting suspension is stirred, then centrifuged (12000 rpm, 2 min), 1 μl of the protein extract is applied to the target of the mass spectrometer in duplicate, dried, and then a matrix solution (a-cyano-4-hydroxycinnamic acid) is added in the ratio 1: 1 and left to dry completely. Mass spectra are recorded automatically in the range of 2-20 kDa. The identification of microorganisms is carried out by automatically comparing the obtained mass spectra with a reference database containing information on more than 950 clinically significant types of microorganisms (Popov D.A., Ovseenko S.T., Vostrikova T.Yu. Express identification of positive blood cultures using method of direct MALDI-TOF-mass spectrometry. Anesthesiology and resuscitation, 2015, No. 5, pp. 71-75). The disadvantages of these methods are the small number of investigated fungal isolates (only 2) and, accordingly, a small number of positive results (only 1 out of 2).

The technical result of the claimed method consists in expanding the arsenal of technical means intended for identification of microorganisms from the bloodstream containing not only Candida spp., But also other yeast-like fungi with high reliability.

The technical result is achieved in that the identification of yeast-like fungi of the genus Candida and the genus Rhodotorula is carried out by sample preparation of positive blood culture and identification of yeast-like fungi by the method of matrix-activated laser desorption ionization time-of-flight mass spectrometry, while sample preparation of positive blood culture is carried out by means of its liquid transfer from a culture medium with , intended for the cultivation of microorganisms, into a tube with a separating gel and then centrifuged at 1000 rpm for 2 minutes, the resulting supernatant is mixed, transferred to a microcentrifuge tube and centrifuged at 14000 rpm for 10 minutes, after which the supernatant is removed, and the sediment add deionized water and mix on a vortex, then centrifuge at 14000 rpm for 2 minutes, remove the supernatant again, and add deionized water to the sediment again and mix on vortex, then centrifuged at 14000 rpm for 2 minutes, remove the supernatant, then add 0.1% sodium dodecyl sulfate to the sediment, vortex, then leave the microcentrifuge tube for 10 minutes to completely dissolve the sediment, then centrifuge at 14000 rpm in for 2 minutes, after which the supernatant is removed, and deionized water is added to the sediment and vortexed, then centrifuged at 14000 rpm for 2 minutes, the supernatant is removed, deionized water is added to the sediment again and vortexed, after which it is centrifuged at 14000 rpm for 2 minutes, remove the supernatant, then add deionized water and 96% ethyl alcohol to the sediment and vortex again, then centrifuge at 14000 rpm for 2 minutes, then remove the supernatant, then leave a microcentrifuge tube with open the lid until the alcohol completely evaporates (when approximately for 5-10 minutes), after which the protein extract is extracted with formic acid and acetonitrile, the resulting supernatant is applied to the target of the mass spectrometer, after drying, it is covered with a matrix and the yeast-like fungi Candida spp. and Rhodotorula spp. to genus according to the value of the coefficient of identification (score) from 1.1 to 1.5 and to species - from 1.6 and higher.

The method is carried out as follows.

Blood for microbiological examination is taken from patients at a temperature of 38 ° C and higher from a peripheral vein and / or from a central venous catheter. Blood samples are introduced into commercial vials with a liquid nutrient medium intended for the cultivation of microorganisms (VASTEC Plus Aerobic / F, VASTEC Plus Anaerobic / F, VASTEC Mycosis-1C / F). The vials of blood are incubated in an automatic blood culture analyzer (BD VASTEC FX, Washington Dickinson). After the signal from the device about the presence of a positive blood culture (that is, the growth of a microorganism in a vial with a nutrient medium), the proposed sample preparation method is used to identify yeast-like fungi by the method of matrix laser desorption ionization time-of-flight mass spectrometry. To do this, 5-6 ml of blood is collected from a bottle with a liquid nutrient medium into a test tube (Monovetta 7.5 ml) containing serum-gel (5 min) and centrifuged at 1000 rpm (2 min). The resulting supernatant fluid is gently mixed with a sterile disposable pipette (15 sec), 1 ml of the supernatant fluid (15 sec) is transferred into a microcentrifuge tube (1.5 ml) and centrifuged at 14000 rpm (10 min). The supernatant is removed (30 sec), and 1 ml of deionized water is added to the sediment and stirred on a vortex (30 sec). Then centrifuged at 14000 rpm (2 min). The supernatant is removed again (30 sec), 1 ml of deionized water is added to the sediment, vortexed (30 sec), centrifuged at 14000 rpm (2 min) and after removing the resulting supernatant, 1 ml of 0.1% dodecyl sulfate solution is added to the sediment sodium, mix the mixture on a vortex (30 sec). Defend for 10 minutes and centrifuge at 14000 rpm (2 min). Then the supernatant liquid is removed (30 sec), 1 ml of deionized water is added to the sediment and vortexed (30 sec), centrifuged at 14000 rpm for 2 min, the supernatant is removed (30 sec), deionized water is added to the sediment again and vortex, centrifuge at 14000 rpm for 2 min, then remove the supernatant, and add 300 μl of deionized water and 900 μl of 96% ethanol to the sediment, vortex (30 sec) and centrifuge at 14000 rpm for 2 min ... The resulting supernatant is removed (30 sec) and the centrifuge tube is left with the lid open for 5 min to completely evaporate the alcohol. Then add 30 μl of formic acid and 30 μl of acetonitrile. The supernatant is applied to the target, after drying (5 min) of which it is coated with a matrix (α-cyano-4-hydroxycinnamic acid) (5 min).

Next, microorganisms are identified by matrix-activated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) on a Microflex LT analyzer (Bruker Daltonics, Germany) (5 min). As a criterion for reliable generic identification of yeast-like fungi, an identification coefficient (Score) from 1.1 to 1.5 is used, and that of species identification is from 1.6 and higher.

In the period from June 2016 to November 2019, 15 positive blood cultures obtained from patients were studied. Blood sampling from patients was carried out at a temperature of 38 ° and more from a peripheral vein and / or from a central venous catheter. Blood samples were added to microbial culture vials and placed in an automatic blood culture analyzer. After receiving a signal on an automatic analyzer about the presence of microorganism growth in the vial, identification of Candida spp. and other yeast-like fungi by the proposed method using matrix-activated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and simultaneously studied by the classical method on solid nutrient media in order to obtain a culture of microorganisms and carry out their identification.

Of all Bactec positive vials, microorganisms were isolated in monoculture, of which 14 were Candida spp. and 1 Rhodotorula mucilaginosa. The spectrum of microorganisms obtained from vials for the cultivation of microorganisms is presented in table 1.

When using the proposed method, successful identification of microorganisms to genus was obtained in 80% (in 12 out of 15) cases, to a species in 66.7% (in 10 out of 15). The results of identification to the genus and species of microorganisms obtained by the proposed method completely coincided with the results of identification after cultivation of microorganisms by the classical method (table 2).

The median duration of incubation of vials with hemoculture until a positive signal was obtained was 35 h 14 min (range from 5 h 38 min to 91 h 08 min), and the median duration of incubation of different types of yeast-like fungi differed and was minimal for Candida tropicalis - 15 h 31 min ( spread from 5 h 38 min to 18 h 34 min), and the maximum for Candida krusei is 91 h 08 min. The median time from the start of incubation of vials with hemoculture to obtaining the results of identification of microorganisms of all yeast-like fungi (n = 12) by the proposed method was statistically significantly less and amounted to 36 h 20 min (range 6 h 37 min to 92 h 05 min) versus 55 h 31 min (range from 16 h 21 min to 112 h 38 min) when identifying microorganisms by the classical method (p = 0.028). The identification of fungi to the genus Candida (n = 11) by the proposed method was 38 h 05 min (range 6 h 37 min to 92 h 05 min) versus 60 h 54 min (range from 16 h 21 min to 112 h 38 min) by the classical method ( p = 0.039), table 3.

The time spent on identification of microorganisms by the proposed method, including sample preparation and identification on a mass spectrometer, ranged from 55 minutes to 59 minutes, while identification using the classical method was obtained only after 18-48 hours. The median time difference between the identification of yeast-like fungi by the proposed and the classical method was 19 h 20 min (range from 9 h 44 min to 26 h 00 min), Table 3.

Examples of specific implementation of the proposed method for identifying microorganisms.

Example 1

Patient Y., 36 years old, was admitted to the hospital on May 22, 2018 with a diagnosis of lymphogranulomatosis for 2 courses of chemotherapy according to the Dexa Beam program. Against the background of myelotoxic agranulocytosis on May 30, 2018, the patient had diarrhea and an increase in body temperature up to 38.5 ° C. A microbiological study of blood from a peripheral vein and a central venous catheter was performed, and cefoperazone / sulbactam was prescribed. Against the background of the use of cefoperazone / sulbactam, an increase in temperature to 39.8 ° C was observed, septic screenings appeared on the skin, and on June 1, 2018, an escalation of antibiotic therapy was made, including the cancellation of cefoperazone / sulbactam and the appointment of imipenem, amikacin and vancomycin. Repeatedly on 01.06.2018, a microbiological blood test was carried out. A repeated study revealed Klebsiella pneumoniae from a blood culture (06/04/2019). The patient's condition worsened, and further modification of the antimicrobial treatment was carried out, including the appointment of meropenem and kolistan and the withdrawal of previous antibiotics. After a repeated microbiological study of blood from 05.06.2018 from a peripheral vein 37 hours 07 minutes after the start of incubation (06.06.2018), the growth of microorganisms was recorded. We used the proposed method for identifying microorganisms from positive blood cultures by matrix laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) from a vial for microorganism cultivation, and in parallel, blood cultures were sieved onto solid nutrient media. Using the proposed method, in 59 minutes after the signal of the automatic analyzer about a positive blood culture, the identification of microorganisms - Candida albicans was obtained. The identification coefficient (Score) was 1.6. Thus, the identification of Candida albicans was carried out 38 hours from the moment of placing the vial with blood in the automatic analyzer for blood cultures. On the same day (06.06.2018), the patient's condition worsened in the form of an increase in temperature to 40 ° C with chills. Immediately after receiving the results of preliminary identification of microorganisms, a modification of antibacterial therapy was carried out and mycamine, an antifungal drug from the echinocandin group, was prescribed. The result of routine identification of microorganisms from the culture was obtained only the next day after the positive signal of the automatic analyzer for blood cultures; at this time, with the addition of an antifungal agent, according to the results of preliminary identification, clinical improvement was achieved in the form of a decrease in temperature from 40 ° C to 37.5 ° C. As a result of treatment, normalization of temperature and regression of foci of screenings on the skin were noted. Adequate antifungal therapy was started 38 hours after blood collection. Repeated microbiological blood tests from the patient within 3 days (according to the recommendations) were negative. The duration of treatment was 14 days (according to the recommendations, at least 14 days).

Example 2

Patient G., 40 years old, diagnosed with acute myeloid leukemia, was admitted to the hospital for the first induction course "7 + 3" with constant administration of cytarabine according to the AML-17 protocol. The patient's condition on admission was serious, he had a temperature up to 37.8 ° C, sinusitis, and the course of chemotherapy was started on 02.02.2018 against the background of antimicrobial therapy with cefoperazone / sulbactam, clarithromycin and amikacin. Microbiological studies of blood from a peripheral vein and a central venous catheter were performed daily from 02.02.2018. On 02/09/2018, a turn of fever with an increase in temperature to 39.6 ° C was noted, in connection with which an escalation of antibiotic therapy was carried out, including the cancellation of previously used antibiotics and the appointment of imipenem. After 5 hours 38 minutes after the start of incubation of blood in vials for the cultivation of microorganisms (from 10.02.2018), growth was recorded. The identification of microorganisms was carried out by the proposed method, and in parallel, blood culture was sieved onto solid nutrient media. In 59 minutes after the positive signal of the automatic analyzer for blood cultures Bactec was obtained the identification of microorganisms - Candida tropicalis (identification coefficient of 1.7). After obtaining the results of the identification of microorganisms by the proposed method (the time from the receipt of the vial with blood in the laboratory to identification - 19 hours 30 minutes), the antifungal drug caspofungin was added to the therapy. The result of the classical identification of microorganisms on solid nutrient media was obtained only the next day (February 11, 2018) after a positive signal from the automatic analyzer for blood cultures (the time from the receipt of a vial of blood to the laboratory to the identification of microorganisms by the proposed method was 39 hours 04 minutes). At this time (02/10/2018), against the background of the addition of an antifungal drug, clinical improvement was achieved in the form of a decrease in temperature from 39.6 ° C to 37 ° C. Repeated microbiological blood tests from the patient within 3 days (according to the recommendations) were negative. Caspofungin was canceled 15 days later.

Example 3

Patient S., 55 years old, with a diagnosis of acute myeloblastic leukemia, was admitted to the hospital on February 10, 2017 for the first induction course of polychemotherapy (AzaAralda) with the inclusion of a hypomethylating drug azacitidine. On admission, the patient's condition was extremely serious, due to the onset of acute leukemia, bilateral pneumonia, occurring with hydrothorax and respiratory failure, enteropathy, syndrome of massive tumor disintegration. Taking into account an episode of fever with an increase in temperature to 39 ° C, microbiological studies of blood from a peripheral vein and a central venous catheter were performed daily from 10.02.2017. On February 14, 2017, 91 hours 08 minutes after the start of blood incubation in vials with a liquid nutrient medium, the growth of microorganisms was recorded. The identification of microorganisms by the proposed method was carried out by the method of matrix laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in parallel the sieving of blood cultures on solid nutrient media (by the classical method). After 57 minutes (02/14/2017) after the signal of the automatic analyzer about a positive blood culture, the identification of microorganisms to the genus Candida spp. Was obtained. (identification coefficient 1.1). After obtaining the results of the identification of microorganisms by the proposed method (02/14/2017), the antifungal drug Micafungin was added to the therapy. The result of classical identification of microorganisms isolated on solid nutrient media was obtained only the next day (15.02.2017) after the signal of the automatic analyzer about a positive blood culture. Isolates of Candida spp. have been identified as C. krusei. At the same time, against the background of antifungal therapy, a slight decrease in temperature was achieved from 39.0 ° C to 37.1 ° C, but with a subsequent increase to 39.4 ° C and on February 17, 2017, a modification of the antifungal therapy was carried out, including the abolition of micafungin and the appointment of amphotericin-B. During the treatment, clinical improvement was achieved in the form of a decrease in temperature from 39.4 ° C to 37.0 ° C. Repeated microbiological blood tests from the patient within 3 days (according to the recommendations) were negative. Amphotericin-B was canceled 17 days later.

Example 4

Patient I., 48 years old, was admitted to the hospital on January 10, 2017 with a diagnosis of acute myeloid leukemia for a course of chemotherapy according to the program "7 + 3 with idarubicin" due to the progression of the underlying disease. The course of polychemotherapy was started on January 11, 2017. The appearance of febrile fever was noted on January 20, 2017 in the patient. Blood microbiology from a peripheral vein and central venous catheter was performed, and piperacillin / tazobactam was prescribed. Against the background of the use of piperacillin / tazobactam, a decrease in body temperature to subfebrile values was observed. In order to induce a graft-versus-leukemia reaction, on January 24, 2017, a donor lymphocyte transfusion was performed, followed by the administration of interleukin-2. Considering an episode of fever with an increase in temperature to 39.0 ° C, the patient daily from 01.02.2017 was performed microbiological studies of blood from a peripheral vein and a central venous catheter, and amikacin was added to the treatment, however, against this background, fever persisted with a spontaneous decrease. to subfebrile and normal values during the day. 02/03/2017 42 hours 09 minutes after the start of incubation of blood in vials for an automatic analyzer with a liquid nutrient medium, the growth of microorganisms was recorded. Were carried out the identification of microorganisms by the proposed method and in parallel sieving blood cultures on solid nutrient media. In 59 minutes after the signal of the analyzer about a positive blood culture, the proposed method identified microorganisms up to the genus - Candida spp. with an identification factor of 1.5. After receiving the results of the identification of microorganisms by the proposed method, an antimycotic (echinocandin) - micafungin was added to the therapy on 03.02.2017. The result of the classical identification of microorganisms isolated on solid culture media was obtained only the next day (02/04/2017) after the signal of an automatic analyzer for blood cultures, and microorganisms from blood cultures were identified as C. parapsilosis. Repeated microbiological blood tests from the patient within 3 days (according to the recommendations) were negative. Antifungal therapy was not changed. Against the background of the use of micafungin on 02/03/2017, clinical improvement was achieved in the form of a decrease in temperature from 39.0 ° C to 37.1 ° C. Given the stable normalization of body temperature, according to the recommendations, micafungin was canceled on the 17th day.

Thus, the study showed a high reliability of the proposed method for identifying microorganisms in patients with candidemia, as well as a reduction in the time to identification of yeast-like fungi in a positive blood culture, which is fundamental for ensuring the maximum effectiveness of treatment, preventing the development of life-threatening conditions such as septic shock and multiple organ failure. ... It has been proven that in candidemia, a delay in etiotropic therapy for every 12 hours increases the mortality rate by 2 times, therefore, early identification of the pathogen is extremely important. Based on the results obtained, the proposed method for identifying yeast-like fungi from positive blood cultures using matrix-activated laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be recommended for use in routine practice in microbiology laboratories in order to reduce the time of results presentation.

Claims (1)

A method for identifying yeast-like fungi of the genus Candida and the genus Rhodotorula by the method of matrix laser desorption ionization time-of-flight mass spectrometry, including the preparation of positive blood culture and identification of yeast-like fungi by the method of matrix-activated laser desorption ionization time-of-flight mass spectrometry, characterized in that it is positive by transfer from a bottle with a liquid nutrient medium, intended for the cultivation of microorganisms, into a test tube with a separating gel and centrifuged at 1000 rpm for 2 minutes, the resulting supernatant is mixed, transferred to a microcentrifuge tube and centrifuged at 14000 rpm for 10 minutes, after which the supernatant liquid removed, and deionized water is added to the sediment and vortexed, then centrifuged at 14000 rpm for 2 minutes, the supernatant is removed, the sediment is again d add deionized water and vortex, then centrifuge at 14000 rpm for 2 minutes, remove the supernatant, then add 0.1% sodium dodecyl sulfate to the sediment, vortex the microcentrifuge tube, leave the microcentrifuge tube for 10 minutes to completely dissolve the sediment, then centrifuged at 14000 rpm for 2 minutes, after which the supernatant liquid is removed, and deionized water is added to the sediment and mixed on a vortex mixer, then centrifuged at 14000 rpm for 2 minutes, the supernatant liquid is removed, deionized water is added to the sediment again and mixed on a vortex mixer , after which it is centrifuged at 14000 rpm for 2 minutes, the supernatant is removed, then deionized water and 96% ethyl alcohol are added to the sediment and vortexed again, after which it is centrifuged at 14000 rpm for 2 minutes, then the supernatant is removed, then leave the microcentrifuge tube with an open lid until the alcohol has completely evaporated, after which the protein extract is extracted with formic acid and acetonitrile, the resulting supernatant is applied to the target of the mass spectrometer, after drying, it is covered with a matrix and the yeast-like fungi of the genus Candida and the genus Rhodotorula are identified to the generic belonging by the value of the identification coefficient ( score) from 1.1 to 1.5 and to species - from 1.6 and higher.