Symptoms of mycosis of the feet. Laboratory diagnosis of fungal skin diseases Not used in the diagnosis of superficial mycoses
The scales or hair are placed on a glass slide (the glass must be degreased) and filled with one, two or three drops of a 30% solution of caustic potassium or caustic soda and covered with a coverslip. For 2-3 minutes, with slight heating on the flame of an alcohol burner, lightly press the preparation with a cover glass until
gray cloud when examining scales. The hair should not be destroyed; it only swells with such heating. Microscopic examination (magnification 100-200 times) with a darkened diaphragm reveals fungal elements - spores or mycelium threads. A necessary condition for the success of microscopic examination is the thoroughness of obtaining scales and hair, which must be taken from the affected areas with special tweezers (eyelash tweezers). It is necessary to pay attention not only to clearly visible hair, crusts and scales, but also to barely noticeable hair remnants (so-called stumps) and blackheads, which are removed with a scalpel or histological needle. You should never be content with pointing the patient to the affected areas; the doctor himself must carefully examine the entire scalp.
In hair affected by trichophytosis, fungal spores are arranged in a chain. With microsporia, the spores are much smaller than with ringworm, do not form chains and are located on the outside of the hair. There are cords of septate mycelium running inside the hair. The hair is as if dressed in a cover and dispute. With scab, polymorphic spores, coarse, varied threads and usually air bubbles are observed (Fig. 48).
In this case, fungi do not completely affect the entire hair, but unaffected areas remain.
To obtain material from smooth skin, scales are scraped off. peripheral areas of the lesion with a sharp spoon or scalpel. It is more convenient to scrape nails with a sharp medical knife (scalpel), removing small but deep sections from the inner surface of the nail plate or scraping off the pityriasis powder from the horny plates of the nail.
The starting material is boiled in a test tube with alkali and then left to stand in this test tube for 12-20 hours.
After this, the contents are centrifuged and the sediment is examined under a microscope (N.A. Chernogubov’s method).
Fungi grow well on artificial nutrient media containing carbohydrates and protein substances. Especially common are the so-called original Sabouraud medium, beer wort, agar and vegetables.
2. Laboratory examination of pathological material
2.1. Microscopic studies
To detect the morphological elements of the fungus - yeast cells, pseudomycelium, mycelium, conidiophores, conidia, tissue forms of deep mycoses - pathological material is examined in native and stained preparations.
Liquid pathological material is examined in an unstained state in the following clearing liquids: a mixture of alcohol with glycerin (ethyl alcohol 1 part, glycerin 2 parts, distilled water 2 parts), Lugol's solution (1 g crystalline iodine, 2 g potassium iodide, 150 ml water), as well as in water or saline. To prepare native preparations, a drop of material is applied to a glass slide using a loop or pipette, then 1-2 drops of clearing liquid are applied, covered with a cover glass and microscoped at a low magnification of 1:80 (10x eyepiece and 8x objective), at which you can see clusters of yeast cells and pseudomycelium , mycelium and other elements of mushrooms. At high magnification of 1:400, individual cells can be characterized.
Dense pathological material (skin, nail scales) is placed in a drop of 10-20% KOH solution, slightly heated over a burner flame (for better maceration) until alkali crystals appear around the periphery of the drop. Then the drop is covered with a cover slip, lightly pressed on it and examined microscopically, first under low magnification to find the scale, then at high magnification.
In hair material, the mantle of spores surrounding the hair (ectothrix) or within the hair (endothrix) fungal elements are usually very clearly visible. Hair lesions, as well as the size of the spores, are specific to different types of dermatophytes. Differential diagnosis between fungal structures and artifacts is necessary. Possible sources of false-positive results include lipid droplets, air bubbles, textile fibers and the so-called “mosaic mushroom”. Lipid droplets may resemble yeast cells, a finding most common in poorly cleared material. Textile fibers usually lie separate from the material of the epidermis, hair or nail. They are larger than fungal hyphae, have uneven thickness and do not contain septa. “Mosaic mushroom” is an artifact obtained during the crystallization of KOH due to excessive heating of preparations. Unlike fungi, there are no clear divisions into cells.
An important step in diagnosing fungal infections is the preparation of colored preparations. Any pathological material (smear preparations, organ prints, centrifugates and, of course, histological sections) must undergo three main types of processing: 1) staining using the PAS method to identify true fungi - eumycetes; 2) staining according to the Gram method or in modifications according to Gram-Weigert, according to Bogolepov, according to Brown-Brenna - to identify accompanying bacterial microbiota, to identify actinomycetes and nocardia; 3) staining according to the Ziehl-Nielson method or modification according to the Kinyon method - to identify acid-resistant microorganisms, primarily for indication and differentiation with Mycobacterium tuberculosis, to detect causative agents of leprosy, nocardia and spore-forming yeasts.
The PAS method (Piodic Acid Schiff) involves identifying neutral polysaccharides in the walls of microorganisms. In the walls of most eumycetes there is a glucan-mannan complex in varying concentrations, due to which coloring occurs.
Prokaryotes (bacteria) turn out to be PAS-negative, including the actinomycetes and nocardia of interest to the mycologist. However, during the formation of actinomycotic drusen, a so-called “cement” is formed, gluing the vegetative actinomycotic mycelium into a granule, which also gives a PAS-positive stain. In this connection, this method is also applicable in the diagnosis of actinomycosis.
The PAS reaction (and its modification) is the most important method for diagnosing tissue forms of fungal infection. The method is based on the oxidation of glycolic groups of carbohydrates with periodic or chromic acid. Periodic acid oxidizes 1,2 and 1,4 glycols to aldehydes and breaks the bonds between carbon atoms bearing hydroxyl groups. Aldehyde groups can also be detected using the aldehyde Schiff reagent. In situ, in the walls of the fungus, the heteropolysaccharide complex becomes intensely stained purple-red (methodology - see appendices).
To suppress the color of surrounding tissues, treatment (“countercolor”) with light green, methanil yellow, etc. is used. In this case, only fungal cells are detected, which is very useful at the stage of indicating the pathogen in tissues or in smear preparations. At the same time, it is not possible to judge the body’s response and tissue alteration to such drugs. It is always necessary to have parallel preparations stained using the PAS method with additional staining with hematoxylin.
In practice, not only the PAS method in the classical version is used, but also its various modifications: oxidation with chromic acid (instead of iodic acid) - this is the Bauer reaction, Gridley staining, impregnation with methenamine silver according to the Gomory-Grocott method. All of them are successfully used to detect tissue forms of fungi, and they are based on the same principle (technique - see Appendix). Table 3.
Table 3
Tinctorial properties of tissue forms of mushrooms
(in pathological material, in histological sections)
|
Opportunistic mycoses |
Detection methods |
| Candidiasis | PAS or Gridley staining |
| Aspergillosis | Hematoxylin and eosin, Gomori-Grocott impregnation |
| Zygomycosis | Hematoxylin and eosin |
| Cryptococcosis | Alcian blue (according to the Mowry method) + PAS reaction + hematoxylin |
| Pneumocystis | Staining according to the Bauer method, impregnation according to Gomory-Grocott, staining with thionin |
| Fusarium | Staining according to the Romanovsky-Giemsa method, according to the Wright method |
| Scedosporiasis | |
| Trichosporosis | Hematoxylin and eosin, Romanowsky-Giemsa staining |
| Pheohyphomycosis and chromomycosis | Hematoxylin and eosin |
| Primary pathogenic mycoses: | Detection methods |
| Coccidioidosis | PAS reaction + hematoxylin |
| Histoplasmosis | impregnation according to Gomory-Grocott, staining using the Bauer method, staining using the Romanovsky-Giemsa method |
| North American blastomycosis | |
| Paracoccidiosis | PAS reaction, impregnation according to Gomory-Grocott |
| Adiaspiromycosis | Hematoxylin and eosin, RAS reaction |
| Pseudomycoses: | Detection methods |
| Actinomycosis | Hematoxylin and eosin, Gram-Weigert stain |
| Nocardiosis | staining according to the Ziehl-Neelsen method, according to the Kinyon method, according to the Gram method. |
If a diagnosis of cryptococcosis is assumed, it is advisable to use methods for the specific detection of capsular material Cr.neoformans containing glycosaminoglycans (acidic polysaccharides). For this purpose, staining with alcian blue (according to the Mowry method), basic brown (according to Shubich method), and mucicarmine staining is used. Triple staining is very useful: PAS reaction, then treatment with Alcian blue, then hematoxylin. In this case, differentiation between cryptococci and tissue forms of other fungi with similar morphology becomes possible.
Description of pathological material (histological preparations) includes data on the morphology and size of tissue forms of fungi, their tinctorial characteristics, relationship to host tissues, the presence of phagocytosis, and determination of the accompanying microbiota.
2.2. Sowing of pathological material and quantitative counting of cells in mycoses caused by yeast fungi
Obtaining cultures of fungi is necessary for their identification and determination of sensitivity to antifungal drugs.
Before examination, the sputum is homogenized for 5-10 minutes by shaking with sterile beads. If the sputum contains a lot of mucus and is poorly homogenized, 1-2 ml of sterile saline solution can be added to it. Sputum is examined microscopically in native or stained preparations. If microscopy of sputum with high magnification shows fungal elements in the field of view, then it should be sown in dilutions of 1:10, 1:100, 1:1000; if fungal elements are not detected, the sputum is sown undiluted. Dilutions are prepared in a liquid nutrient medium (wort, Sabouraud, 1% peptone water) or in sterile saline. From each dilution, 0.1 ml is inoculated using a spatula onto 2 cups of wort agar, Sabouraud agar or MPA with the addition of antibacterial antibiotics - penicillin and streptomycin, biomycin (100-200 units/ml of medium). The nutrient medium is pre-dried in a thermostat at +37 C, because in the presence of condensate, the growth of colonies may be confluent. The crops are incubated in a thermostat at + 37 o C for 48 hours. Then, if there is growth of the same type of yeast colonies, they are quantitatively counted. Calculation of the number of yeast cells (n) in 1 ml or 1 g of the test material is carried out according to the formula n = авс, where a is the average number of colonies on one Petri dish, b = 10 with a volume of inoculum of 0.1 ml, c is the degree of dilution of excreta (10,100,1000).
Calculation example: on dishes with a dilution of 1:1000, an average of 60 colonies grew, while 1 ml of the excreta under study contains 60 x 10 x 1000 = 600,000 yeast cells.
BAL, WASHING fluid of the bronchi, maxillary cavities, bile (portions A, B, C), gastric juice, duodenal contents, urine are transferred into centrifuge tubes and centrifuged for 10-15 minutes, after which the supernatant liquid is quickly drained. Native preparations for microscopy are prepared from the sediment. If microscopy reveals yeast cells in each field of view, then sowing is carried out in dilutions of 1:100 and 1:1000, 0.1 ml each, onto solid nutrient media and incubated at 37 C for 48 hours. If microscopy of the sediment does not reveal yeast cells, the sediment sown without dilution. The amount of yeast flora is calculated per 1 ml of pathological material.
FECES are taken with a measuring spoon in the amount of 0.2 g, placed in 1.8 ml of liquid wort and stirred thoroughly with a glass rod. The resulting dilution (1:10) is left to stand for 5-10 minutes, dilutions of 1:100, 1:1000 are prepared and inoculated in a volume of 0.1 ml onto 2 Petri dishes with agar medium. The amount of yeast flora is recorded per 1 g of feces.
CEREBROSpinal fluid. Characterize its appearance (transparent or cloudy, colorless or colored, the presence of blood, sediment). Microscopy smears from the cerebrospinal fluid sediment after centrifugation. Three preparations are prepared: native - in a drop of sterile saline; native - in a drop of ink and a smear for staining using the PAS method, Gram and Alcian blue according to Mowry.
Microscopy cerebrospinal fluid in native and colored preparations, it allows us to determine the presence of budding yeast cells and fragments of fungal mycelium, or bacteria that cause purulent meningitis (meningococcus, pneumococcus, Haemophilus influenzae, etc.). A capsular fungus may be detected in the ink preparation. Cryptococcus neoformans. At the same time, budding yeast cells are visible on the gray background of the ink, surrounded by a light halo of the capsule. Ink preparations may also contain yeast forms Candida albicans that do not have a light halo of the capsule around the cell.
Cr. neoformans can also be detected using Alcian blue staining (Mowry). Coloring occurs due to the selective detection of capsular heteropolysaccharide characteristic of Cr.neoformans. If bacterial flora is detected in a Gram-stained preparation, further examination of the cerebrospinal fluid is carried out using appropriate bacteriological methods. After microscopy of the sediment, the liquid is inoculated onto 3 cups of freshly prepared, dried wort agar or Sabouraud agar, as well as onto liquid wort or liquid Sabouraud medium. Apply 2-3 drops of liquid sediment to the surface of the nutrient agar of cups 1 and 2 and rub it thoroughly with a spatula, and inoculate 1 drop at three points on the surface of the agar of cup 3 to detect filamentous fungi. The remaining liquid is transferred to 5 ml of liquid wort or Sabouraud agar. Crops are incubated at two temperature conditions: cup 1 - at +37 o C, and cups 2 and 3 and sowing in liquid medium - at +28 o C - +30 o C. Crops at a temperature of +37 o C are viewed at 2 and 5 day, and at 28 - 30 o C - on the 4th, 7th, 10th day of growth. If on the 5th day at a temperature of +37 o C and on the 10th day at a temperature of 28-30 o C there is no growth, sow from a liquid nutrient medium onto plates with wort or Sabouraud agar. If there is no growth of yeast flora, the result of inoculating the liquid is recorded as negative.
DISCHARGE of fistulas. The study begins with microscopy of the material in native or stained preparations. Then the material is inoculated onto an agar medium (wort or Sabouraud), for which 2-3 drops of the discharge are applied to a cup and spread over the surface of the agar with a spatula. The crops are incubated for 48 hours at +37 o C.
DISCHARGE of mucous membranes.
1. The tampon is placed in a test tube with 2 ml of liquid medium (wort, Sabouraud's medium or MPB) and shaken for 5-7 minutes without soaking the stopper. Prepare dilutions of 1:10, 1:100 and inoculate 0.1 ml of each dilution onto two cups of wort agar, Sabouraud agar or MPA. Inoculations on solid media and a test tube with a liquid medium with a swab (for enrichment) are incubated at a temperature of +37 C. After 48 hours, the number of colonies is counted and the number of yeast cells taken with a swab is approximately determined. To do this, the number of grown yeast colonies is multiplied by 20 and the dilution. If there is no growth of colonies on the plates, the taken dilutions are re-seeded from the enrichment medium onto one plate with wort agar.
2. Sowing can be done by moving a swab with rotation over the surface of the nutrient medium. In this case, the number of yeast colonies is not taken into account, but only the presence and intensity of growth are noted: single colonies, significant or continuous growth, lack of microbiota growth.
BLOOD. Venous blood samples for blood culture must be diluted with enrichment medium at least 1:5 so that the bactericidal properties of the blood do not inhibit fungal growth. Inoculate 5-10 ml of freshly drawn blood, respectively, into 50-100 ml of nutrient medium (liquid Sabouraud with 2% glucose or Kitt-Tarozzi medium after its regeneration). For culture, you can take blood with an anticoagulant (1:10 5% sodium citrate solution). Crops are grown for 10 days at +37 o C with control sowing on days 5 and 10. Using a sterile pipette, take the sediment, 3 drops of which are scattered with a bacteriological loop over the surface of the wort agar into Petri dishes. The seeded cups are placed in a thermostat with a temperature of +37 o C for 2-5 days. In case of growth of yeast flora, a preliminary answer is given about the presence of the fungus in the blood, and the culture is determined to the genus and species.
Another method of blood culture for mycoflora is described (H. Rieth). In this case, 5-10 ml of blood is inoculated in drops. Apply 40-50 drops of blood to the surface of a dense nutrient medium in a Petri dish with a sterile pipette, at a distance of 0.5 cm between drops. Inoculation is carried out on two Petri dishes, incubating in one dish at a temperature of +37 o C, and in the other at room temperature for 2-5 days.
PIECES of organ tissue. An imprint is made with the test piece of tissue on the surface of a dense nutrient medium in a Petri dish, then sieving is carried out with a loop. The same piece of tissue is placed in 50 ml of liquid nutrient medium (wort, Sabouraud medium). The crops are incubated in a thermostat at a temperature of +37 o C for 5 days.
SKIN and nail scales. Sowing of scales is carried out regardless of the microscopy results. To do this, using a sterile mycological spatula moistened in the condensate of the medium, the scales are transferred into a test tube onto slanted wort agar at 2-3 points, pressing them to the surface of the medium. Depending on the amount of material received for research, inoculation is done in 2-3 test tubes. The crops are incubated in a thermostat at a temperature of 28-37 o C for up to 5 days.
Rapid identification methods are widely used C. albicans. This species is capable of forming germ tubes and short filaments of pseudomycelium within several hours (2-4 hours at 37 o C) on blood serum, egg white, Eagle's medium, 199 medium, etc. In practice, laboratories use human serum (residues from serological reactions), where germ tubes are formed at 37 o C, and after 24 hours tangles of pseudomycelium are formed. For the sake of appearance C. albicans this phenomenon is typical in 90% of cases. Sprouts form less frequently C. tropicalis.
2.3. Microscopic examination and culture of pathological material for suspected mycoses caused by molds
Pathological material can be examined in native and stained preparations. Slides and cover glasses intended for the preparation of microslides should be stored in a mixture of alcohol and ether (1:1) to avoid contamination of the air with mycobiota. Before use, slides and coverslips are sterilized over a burner flame.
Microscopy of material
MICROSCOPY of sputum. To prepare native preparations, sputum is transferred to a sterile Petri dish and examined against a black background to detect small particles (lumps). Lumps can be purulent, purulent-mucous, purulent-bloody. The size of the lumps varies in size within 0.3-3 mm in diameter, their color can be gray, yellowish, greenish. To prepare native microslides, individual lumps are transferred with dissecting needles or a bacteriological loop into a drop of alcohol with glycerin, or into a drop of 10% KOH solution. Cover with a cover glass, apply slight pressure with a dissecting needle and microscope at low (1:80, 10x eyepiece and 8x objective) and high (1:400, 10x eyepiece and 40x objective) microscope magnifications.
Preparations from rinsing waters, exudate, as well as bile, urine, gastric juice, and liquor are prepared from native sediment or from sediment obtained by centrifugation (at 1500 rpm for 5 minutes). The sediment is transferred with a loop or Pasteur pipette into a drop of 10% KOH solution onto a glass slide, covered with a coverslip and examined at low and high magnifications of the microscope.
To prepare stained preparations, the lumps or drop of sediment to be examined are evenly distributed with dissecting needles or a smaller slide over the surface of a sterile slide until a thin smear is obtained. The resulting smear is dried in air, fixed with methyl alcohol or Nikiforov’s mixture (equal parts of 96% ethyl alcohol and ether) for 3-5 minutes, or by flaming it three times over a burner flame. The fixed smear is stained using the Gram method, the PAS method, and calcofluor white. The stained specimen is microscoped using an immersion microscope system (1:900, 10x eyepiece, 90x objective).
Sowing material
When examining any pathological material for filamentous fungi, it is inoculated on solid Sabouraud medium or wort with the addition of penicillin and streptomycin (100-200 units/ml medium). Sowing is carried out in two repetitions, taking into account different temperature conditions for growing filamentous fungi (+37 o C and +28 o C), always at 3 points in the center of the cup. Incubation time is 4-5 days.
Sputum (selected lumps) is transferred with a bacteriological loop or Pasteur pipette to the surface of Sabouraud's medium or wort. The seeding site is marked with a pencil on the back of the bottom of the Petri dish. The seeded Petri dishes are placed in a thermostat with the lid facing up.
After a certain period of incubation, the seeded dishes are examined and if sporulation is detected, the fungal culture is determined. If there is no sporulation, the fungus is subcultured on Czapek’s differential medium for further identification.
The sediment of the washing water of the bronchi, maxillary cavities, exudate, urine, gastric juice (native or after centrifugation) is taken with a pipette and inoculated in a volume of 0.1 ml. Feces are diluted 1:10 (1 g of feces and 9 ml of liquid) in liquid Sabouraud's medium or liquid sterile isotonic sodium chloride solution, emulsified, allowed to settle for 10 minutes to sediment large particles, and the supernatant is inoculated in a volume of 0.1 ml. The discharge from the external auditory canal and pharynx, taken with a swab, is inoculated by carefully passing each side of the swab over the surface of the nutrient medium. Swabs from swabs can be inoculated. To do this, tampons are placed in 10 ml of liquid Sabouraud medium or liquid wort with glass beads and emulsified for 10 minutes, inoculated with 0.1 ml of swab washout with a lawn (according to Leshchenko V.M., 1973), or at three points.
Skin and nail scales are placed on the surface of the nutrient medium, carefully pressing them.
The cerebrospinal fluid sediment (centrifuge at 1500 rpm for 5 minutes) is inoculated into two 0.1 ml cups of medium, and the remainder is inoculated into an enrichment medium (Sabouraud liquid medium or liquid wort), poured into 5 ml tubes. The inoculated dishes are incubated as usual, and the tubes inoculated on the enrichment medium are incubated at + 28 C for 10 days.
If there is growth of a filamentous fungus on solid media, its culture is determined from this inoculation; if there is no growth on Sabouraud agar or wort agar, the fungus is studied from the enrichment medium. To do this, the fungal culture is subcultured again on Czapek’s differential dense medium and subsequently identified.
From a piece of organ tissue (biopsy, autopsy), an imprint is made on the surface of a solid medium with the cut side of the piece being examined at three points. At the same time, pieces of tissue are placed in 50 ml of liquid nutrient medium (Saburo, wort).
If fungemia is suspected, blood is examined in two to three repetitions. Inoculate 5 or 10 ml of blood, respectively, in 50 or 100 ml of Sabouraud liquid medium with 2% glucose. Crops are grown at +37 C and +28 C for 10 days. The first inspection of crops is carried out after 5 days, the second - after 10 days. On the fifth day, you can observe the growth of the filamentous fungus in the form of a felt lump at the bottom and a surface film. The mycelium is subcultured on Czapek's differential medium to determine the genus and type of fungus. If no fungal growth is observed on day 5, the crops are kept for up to 10 days and if there is no growth, the test results are recorded as negative.
When sowing at three points, the growth of the fungus at two and three points is defined as diagnostically significant, at one point - random. If necessary, re-seeding is possible.
Identification of isolated mold cultures
After isolating the culture, filamentous fungi are subcultured on Czapek’s differential medium for generic and, if possible, species determination.
In the practice of diagnostic laboratories, as a rule, they use cultural and morphological identification criteria: they evaluate the growth pattern of a fungal culture on agar media (cultural diagnostics, macromorphology) and the micromorphology of the fungus.
In difficult cases, additional diagnostic methods are used (study of enzymatic activity, temperature characteristics of the growth of some molds).
The concept of macromorphology (cultural characteristics) includes the structure of the colony (fluffy, felt, velvety, cobwebby, woolly, ragged, mealy, etc.), surface (flat, folded, bumpy, dome-shaped, core-shaped, zonal, etc.), pigmentation of the fungal colony and substrate (various shades of green, blue, purple, black, gray, etc.), the presence of exudate on the surface of the colony.
The micromorphology of a fungus from a culture is studied using preparations, which, depending on the genus of the fungus, are prepared as follows: a drop of liquid for preparing preparations (equal parts of alcohol, glycerin and water) is applied to a glass slide; a piece of mycelium is placed in it, cut with a mycological spatula from the colony in the form of a triangle, capturing the central and peripheral parts, and the cut piece is straightened with two dissecting needles with care to avoid the formation of air bubbles. In some cases (mucor and rhizopus), when preparing the drug, the mycelium is spread out on a dry glass slide, a drop of liquid is applied to it and covered with a coverslip. The preparations are viewed under a microscope at low and high magnifications. Substrate and aerial mycelium are studied, the presence or absence of septa (septa) is noted, and attention is paid to the nature of sporulation: conidiophores with conidia and sporangia with sporangiespores.
Conidiophores vary in structure: from simple single spore-bearing hyphae to branched tree-like formations. Conidiophores are located singly or in groups; they differ markedly from the vegetative hyphae of the mycelium, colorless or colored, ascending, erect, cascading, creeping. They can consist of one cell and of a large number of cells of different shapes and sizes, each of which has its own name. For example, in the genus Aspergillus, the conidiophore consists of the following cells: stalk, vesicular swelling, sterigmata, chains of conidia. In the genus Penicillium, the conidiophore has the form of simple or complex brushes, also consisting of various cells: ramie branches, metulae, phialids, chains of conidia.
The mold fungi Mucor and Rhizopus sporulate in the form of sporangia with endosporangiospores. The sporangium is located at the end of the sporangiophore. Sporangia are spherical or pear-shaped, most with a special column, which is a continuation of the sporangiophore inside the sporangium. Sporangiospores are round, colorless or colored.
Conidia (spores) of mold fungi are polymorphic (cylindrical, spherical, oval, ellipsoidal, ovoid, pear-shaped, club-shaped), single- and multicellular, vary in size and color, single, in chains or collected in heads and arranged in clusters. The surface of conidia can be smooth, rough, spinous, warty, bristly, etc.
Microscopic examination and culture of pathological material for mycoses caused by dimorphic fungi
With these mycoses, it is necessary to take into account the dimorphism of pathogens.
Pathological material for primary pathogenic mycotic infections (coccidioidosis, histoplasmosis, paracoccidioidosis, North American blastomycosis), as well as chromomycosis, sporotrichosis, and mycetomas can be pus, sputum, scrapings from ulcerative lesions on the skin and mucous membranes, cerebrospinal fluid, blood, biopsied pieces from lesions defeats.
The difficulty in identifying the causative agents of these mycoses is that due to their dimorphism, microscopy of the pathological material will reveal tissue forms of the fungus, most often yeast cells of various morphologies or spherules with endospores, completely different from the elements of the same fungus extracted from its culture grown at a temperature of +28 - +30 o C on regular Sabouraud agave, with a pH value closer to acidic. However, it must be borne in mind that in some dimorphic fungi, it is possible to obtain in culture a yeast phase of fungal growth, often reminiscent of its tissue form, but when grown in media rich in proteins with a slightly alkaline reaction (pH = 7.6 - 7.8 ), at a temperature of 37 o C.
The above applies to mushrooms: Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Coccidioides immitis And Sporothrix schenckii.
Laboratory diagnosis of mycoses caused by the fungi mentioned here is based on the detection of tissue forms in pathological material by microscopy, isolation of the pathogen culture and its identification by cultural and morphological characteristics.
Microscopic examination is carried out in unstained preparations on a glass slide in a drop of 10% solution of caustic alkali or a mixture of alcohol and glycerin in equal volumes or in stained smears (Table 3).
Inoculation of pathological material is carried out on the above-mentioned nutrient media in Petri dishes using a bacteriological spatula according to the generally accepted method.
2.5. Microscopic examination and culture of pathological material for dermatomycosis (keratomycosis and dermatophytosis).
The object of the study is superficial mycoses, affecting only the keratin layer of the epidermis, and dermatophytes, affecting the skin and its appendages (hair, nails), the causative agents of which are fungi related to the genus Trichophyton, Microsporum and Epidermophyton.
Laboratory mycological research includes the same steps as for other mycoses: microscopy of the material and obtaining a pure culture when sowing it. Proper collection of material greatly influences the success of microscopy and culture acquisition.
Pathological material is examined as soon as possible after collection. It is first divided into three parts: for microscopy, cultivation and re-examination. Microscopy of pathological material is the simplest and fastest method for establishing the presence of a fungus in tissues. The crushed material is placed in the middle of a glass slide in a drop of 10-20% KOH solution and slightly heated over the flame of an alcohol lamp until a whitish rim is obtained along the edge of the drop, covered with a cover glass and left for 5-10 minutes (hair, skin scales) and 30 - 40 minutes. (nail) for maceration and clarification; the material can be processed without heating; for this, the preparation is left in a 20% KOH solution for 30-60 minutes.
Microscopy first under low, then high magnification microscope.
Culture is necessary to isolate and identify the pathogen. The inoculated material is crushed as much as possible and inoculated in minimal quantities onto slanted agar in test tubes at 2-3 points at a distance of 1-2 cm. At least 2-3 test tubes (hair) and 4-5 test tubes (skin and nail scales) are inoculated with the material of one sample. . For the primary isolation of dermatophytes, it is best to use standard Sabouraud agar medium with 2-4% glucose or wort agar containing antibacterial antibiotics (penicillin 50 μg/ml + streptomycin 50 μg/ml or biomycin 200 units/ml) and anti-mold antibiotic actidione ( cycloheximide) 0.1 - 0.5 mg/ml. Actidione does not affect the growth of dermatophytes and inhibits many molds, as well as Candida and Cryptococcus species.
Crops are incubated at 22-30 o C (best 28 o C). The appearance of dermatophyte growth is observed from the 4th to the 12th day of incubation at the sowing points along the edges of the introduced material. If there is no growth within 30 days, the culture results are considered negative. Under optimal conditions, primary cultures of many dermatophytes can be identified 7-10 days after sowing, but crops should be monitored for 20-30 days. Primary cultures grow relatively slowly and when using media without antibiotics, dermatophytes may be suppressed by faster growing bacteria or molds. When growth appears in the primary seeding, it is necessary to sift from the edge of the colony onto a fresh differential medium to obtain a pure culture, which will serve as material for identifying the isolated dermatophyte.
2.6. Serological methods for diagnosing mycoses
The significance of serological methods consists mainly in the following: identification of patients with probable invasive mycoses; confirmation of the mycotic nature of allergic diseases; screening examination of groups at risk of developing mycoses.
False positive results of serological tests are possible with mycocarriage and in healthy people sensitized with fungal antigens; negative tests can occur in immunodeficiency even against the background of ongoing invasive mycosis.
Routine generally accepted methods for serological diagnosis of mycoses
described in sufficient detail / P.N. Kashkin, V.V. Lisin. "Practical
guide to medical mycology", Medicine, 1983/. However, in recent times
decades there have been noticeable shifts in methodological approaches /Elinov N.P.,
2001/. Original procedures for detecting antigens and antibodies were proposed
some metabolites of fungal cells; special diagnostic tests have been created
kits (“whales”), for example Pastorex ® Candida, for determination in
"latex agglutination" reactions of repeating oligomannose epitopes of antigenic
structures/expressed on a large number of macromolecules
mushroom; for the determination of Candida mannan antigen, e.g.
serum of a patient with candidemia, you can use the Platelia ® Candida kit.
Using the first set, the threshold for determining antigenic structures is 2.5 ng/ml, using the second in conjunction with the _____________ method, the threshold for determining -
0.5 ng/ml.
The causative agents of mycoses most often identified during laboratory testing of various clinical materials
Blood
- Candida
- Cryptococcus
- Mycelial pathogens are rarely detected in blood tests except Fusarium
Cerebrospinal fluid
- Candida
- Cryptococcus
Pus discharge from abscesses, ulcers, etc.
- Candida
- Cryptococcus
- Fusarium
- Aspergillus
- Sporotrix
Respiratory secretions (sputum, BAL fluid, bronchial brush biopsy, transtracheal aspirate)
- Aspergillus
- Candida
- Cryptococcus
- Mucor
- Scedosporium
- Rhizopus
- Sporotrix
Discharge, biopsy material from wounds
- Aspergillus
- Candida
- Fusarium
- Rhizopus
Other biosubstrates
- Candida
- Cryptococcus
Material from the chest and abdominal cavity; synovial fluid
- Aspergillus
- Candida
- Fusarium
Vitreous body
- Candida
- Aspergillus
IV. Criteria for the diagnosis of systemic mycoses: clinical and laboratory parameters of the final diagnosis
Esophagitis
- presence of characteristic changes during esophagoscopy
- identification of the fungus during cultivation of biopsy material
- presence of pseudomycelium in stained smears or signs of invasive fungal growth in biopsy material
Pneumonia
Pneumonia due to Candida spp.
- acute infiltrative changes on a chest X-ray, coinciding with clinical manifestations of fungal pneumonia
- identification Candida spp. When culturing material from the lower respiratory tract obtained from transthoracic biopsy, transbronchial biopsy, lung biopsy findings, or thoracoscopically guided biopsy
- identification of pseudomycelium in adequately stained biopsy material
Pneumonia due to Aspergillus spp., Fusarium spp., Scedosporium apiospermum
- identification of fungal elements in tissues and positive culture
- persistent or progressive infiltrates in the lungs, resistant to antibiotic therapy
- detection of one of these pathogens by sputum or BAL culture
- clinical signs of pneumonia (cough, shortness of breath, “pleural” pain, wheezing, pleural friction noise)
- characteristic changes on X-ray or CT scan of the lungs:
Subpleural infiltrative, nodular, acute-angled or cavernous changes
Symptom of “halo” on CT scan of the lungs
Progression of infiltrative changes with the formation of cavities and the appearance of the “sickle” symptom
- absence of other pathogens when culturing BAL fluid that could cause the presented changes in the lungs
Sinusitis
- clinical and radiological signs of acute sinusitis
- microscopic and cultural signs of mycosis when examining aspirate or biopsy material
Urinary tract infection
- detection of >1x10 CFU/ml with repeated cultures of properly collected urine
Fungemia
- Single detection of fungi during blood culture during a period of rising body temperature > 38 o C
Acute disseminated mycosis
- Fungemia in combination with cultural or histological signs of damage to deep tissues (including subcutaneous tissue) or identification of the pathogen from two normally sterile biosubstrates
Endophthalmitis
- ophthalmological signs of endophthalmitis
- identification of the pathogen from the eye, blood or other foci of dissemination
Abscess or osteomyelitis
- radiographic/CT/MRI signs of osteomyelitis
- identification of the pathogen in aspirate or biopsy material
Meningitis
- determination of CSF changes confirming the presence of inflammation, and detection of fungi by CSF microscopy
- detection of fungi by culture of CSF or determination of antigens Cr.neoformans, Candida and Aspergillus in the CSF
Chronic disseminated (hepatolienal) candidiasis
Possible
- persistent or intermittent fever after the end of a period of neutropenia in combination with characteristic signs of liver, spleen or kidney damage
Proven
- the above in combination with seeding Candida spp. from the blood before the appearance of signs of damage to the liver, spleen or kidneys or with cultural, histological signs of candidiasis in biopsy material from the lesions
V. CAUSES OF FUNGAL INFECTIONS
There are different principles for classifying fungal infections and their causative agents. It seems to us that it is practically the simplest and most expedient to divide pathogenic fungi into 5 main groups:
1. Pathogens of superficial mycoses;
2. Pathogens of dermatomycosis;
3. Pathogens of subcutaneous mycoses;
4. Causative agents of deep mycoses (primary pathogenic micromycetes, causative agents of opportunistic infections).
5. Pathogens of pseudomycosis.
* It should be emphasized that pathogens of the first three groups in immunosuppressed patients can cause fungemia and multiple organ lesions.
In the following sections, we give a description of the causative agents of mycoses according to a single scheme: its name according to the accepted nomenclature, a list of synonyms, macroscopic characteristics of colonies on nutrient media and microscopic characteristics of fungal elements visible under a microscope in native preparations and from colonies. Data are also provided on the distribution of fungi in nature and the diseases they cause are listed.
Laboratory diagnosis of fungal diseases is based on detecting the fungus and determining its genus and type. It consists of two main stages: microscopic and cultural studies.
Microscopic examination
Microscopic examination is the first and important link in confirming the preliminary diagnosis.
The success of microscopic examination largely depends on the correct collection of pathological material. For microscopic examination, it is necessary to select hair that has visible signs of fungal damage (dull, broken, thickened). Hair that has changed in appearance is removed with epilation tweezers. To detect single affected hairs with microsporia, you can use a fluorescent lamp with a Wood filter (greenish-blue glow).
When selecting affected hair, a number of additional characteristics can be used. Hair affected by microsporums has a gray sheath of externally located spores at the base. In case of chronic trichophytosis, short gray affected hairs, curved in the form of “commas” and “question marks,” as well as “black dots” (thickened black affected hairs broken off at the mouth of the follicle) are found in the thickness of the scales. In case of infiltrative-suppurative trichophytosis, for microscopic examination, in addition to the affected hair, you can use pus and crusts from the lesion.
From skin lesions with microsporia, trichophytosis and mycosis of inguinal folds, scales must be scraped off from the peripheral zone of the lesion, where the fungus is found in larger quantities. The vellus hair is scraped off along with the skin flakes.
When examining affected hair with microsporia and trichophytosis, attention is paid to the location of the spores (inside or outside the hair) and their size. These data make it possible in some cases to clarify the diagnosis, clinical form of mycosis and epidemiology.
In the interdigital form of mycosis of the feet, skin scales and scraps of the macerated stratum corneum are used for microscopic examination. The area of the nail that needs to be taken for microscopic examination depends on the form of onychomycosis. With a superficial form, it is necessary to scrape the surface of the nail plate.
In the most common distal-lateral form, a scraping is used from the nail bed, from under the plate (subungual hyperkeratosis) with a part of the cut off altered nail plate. For the proximal subungual form, special methods are used to collect material (drilling windows using a drill, nail biopsy).
In the squamous-hyperkeratotic form of mycosis of the feet, the scales are scraped off from the plantar surface. In the dyshidrotic form of mycosis of the feet, the covers of the blisters are cut off for examination.
Technique for preparing material for microscopic examination of hair . A small drop of 30% KOH is applied to a glass slide and the affected hair is placed into it with a dissecting needle. The drop with the hair is slightly heated over the flame of an alcohol lamp until vapor appears above the surface of the liquid or a rim of crystals falls out along the edge of the drop of alkali. After covering with a coverslip, excess alkali is removed with filter paper. The drug is examined first under low and then under high (x 400) microscope magnification.
Skin and nail scales . Thin nail scales for microscopic examination are placed on a glass slide in a drop of 30% KOH and heated, adding alkali as it evaporates. The cooled, unstained specimen is covered with a coverslip and examined under a microscope.
Thick skin and nail scales are placed in a centrifuge tube and filled with a few drops of 30% KOH. The test tube is heated to a boil and left for 20-30 minutes. Part of the softened material is transferred to a glass slide with a glass rod, pressing with a match until a “cloud” appears, after which it is examined under a microscope.
Pus . A drop of pus is mixed with a drop of alcohol and half-and-half glycerin and examined in a native preparation.
Cultural diagnostics
Cultural diagnostics are carried out to definitively clarify the diagnosis and clarify the epidemiology. It involves obtaining a culture of the fungus followed by microscopic examination.
Affected hair, scales (skin and nail), blisters or pus are inoculated on an artificial nutrient medium. By the appearance of giant colonies on Petri dishes, one can get an idea of the genus of the pathogen (Microsporum, Trichophyton, Epidermophyton), its type (L. canis or ferrugineum, T. violaceum, verrucosum or gypseum). Final clarification of the genus and species of the fungus is possible only on the basis of a microscopic examination of the resulting culture.
Laboratory diagnosis of superficial candidiasis
For laboratory testing for yeast-like fungi, fresh material is required. For microscopic examination, depending on the clinical manifestations and localization of the lesions, skin flakes, scrapings from the nails, a drop of pus from under the nail fold, whitish deposits from the affected areas of the oral mucosa and external genitalia, vaginal walls, scrapings from the mucous membrane can be taken. membranes of the urethra, as well as washings from the red border of the lips, affected areas of the skin of large and small folds.
Depending on the location of the lesion and the nature of the clinical manifestations, the material for research is taken with a cotton swab, scalpel, loop, etc. Skin and nail scales, scraps of the epidermis and scrapings of the mucous membrane are pre-treated with 30% KOH. Pathological material is examined in unstained or stained preparations.
In the first case, the material is mixed with an equal amount of alcohol and glycerin. When stained by Gram, yeast cells and pseudomycelium appear dark purple, by Ziehl-Neelsen - blue, and by Romanovsky-Giemsa - pink-violet. In this case, a distinctive feature of a yeast cell is budding - the discovery of an “hourglass” figure. Taking material from the mucous membrane of the oral cavity, genitals, from the skin of the red border of the lips, from the corners of the mouth, from the skin of large and small folds is carried out with a sterile swab. After taking the material, the swab is placed in another sterile tube with liquid wort. The test tube with the swab is sent to the microbiology laboratory. Isolation of a pure culture of yeast-like fungi of the genus Candida is carried out according to generally accepted microbiological methods.
According to scientists, 70% of the world's population has symptoms of mycosis of the feet. This disease affects the interdigital folds and the skin on the soles. The cause of the disease is a fungus that was initially found only in limited areas of southeast Asia and Africa. The First World War, causing mass migration of people and deteriorating sanitary conditions, led to the spread of the disease throughout the world.
What causes mycosis of the feet
The main causative agent of the disease is Trichophyton rubrum. Infection can be caused by T. mentagrophytes and Epidermophyton floccosum. Fungi of the genus Candida and mold microorganisms can become pathogenic microbes much less frequently.
The most significant risk factors for the disease:
- diabetes;
- immunodeficiency state (AIDS);
- flat feet;
- atherosclerosis of peripheral arteries;
- varicose veins of the lower extremities.
External conditions conducive to the development of infection:
- closed non-absorbent shoes;
- foot injuries (calluses, abrasions);
- playing sports.
Symptoms of athlete's foot most often occur in adult men. Children rarely get sick.
Symptoms of mycosis of the feet
As the disease develops, peeling and dry skin, itching and burning appear, especially in the spaces between the fingers, and the appearance of painful cracks under the fingers. Sometimes the first symptoms of mycosis of the foot are blisters that burst with the formation of erosions. Often the disease occurs in an erased form, manifested only by slight peeling, reminiscent of flour, in the folds between the fingers.
There are 4 clinical forms of the disease.
The interdigital, or intertriginous, variant is the most common. The skin between the fingers turns red, cracks, the surface layer becomes wet and peels. These signs extend to the sole and are accompanied by severe itching and burning. Bacterial inflammation is often associated.
The squamous-hyperkeratotic variant is associated with severe thickening and cracking of the skin. The sole turns red and peels. Deep, painful cracks appear in the heel area; itching is usually uncharacteristic. This is often a bilateral lesion and is also called “moccasin foot.”
The dyshidrotic variant is accompanied by the appearance of multiple small itchy, painful blisters. They merge with each other, forming large bubbles. The covers of the blisters burst, revealing a shiny, vulnerable, painful surface - erosion. External manifestations resemble eczema.
Microbial inflammation is often associated with enlarged inguinal lymph nodes, fever, pain in the leg, nausea, headache and other signs of intoxication. With the dyshidrotic form, an allergy to fungi often occurs - mycotic eczema. It is accompanied by rashes on areas of the body that are not infected with the fungus, for example, on the hands.
The erased version usually goes unnoticed. It is accompanied by slight peeling of the skin between the big and index and/or ring and little fingers on the foot. There is no itching.
Signs of mycosis of the feet
Different types of mycosis of the feet can be independent diseases or occur as part of a general fungal infection of the body. Sometimes the sign “two feet - one hand” occurs with the involvement of these organs. Onychomycosis, a fungal destruction of the nail, may occur. Sometimes the inguinal folds are affected at the same time.
The main symptoms and treatment of mycosis of the foot are presented in the photo:
Peeling skin
Dry and cracked skin
Bubbles and erosions
Diagnostics
An experienced dermatologist can recognize the different types of mycosis of the feet during the first examination. However, microscopic examination is necessary to confirm the diagnosis. For it, scales from the lesion are used, scraped off with a spatula and treated with an alkali solution. The resulting material is examined under a microscope and pathogens are detected.
Direct microscopy is fast, cheap and easy to perform, but cannot determine which type of fungus is causing the disease. Therefore, the material is inoculated on a nutrient medium, followed by a cultural examination of the resulting material. However, it is possible to obtain a culture of the fungus after detecting it under a microscope only in 20–6% of cases.
Types of treatment for mycosis of the feet
Drugs for the treatment of fungal diseases should be prescribed by a dermatologist. Usually, treatment of mycosis of the foot is carried out using external means.
One of the effective drugs for this disease is clotrimazole. In our store you can buy it at a low cost. A medicine in the form of a lotion, Clotrimazole for nails and skin, suppresses the proliferation of fungi in the thickness of the stratum corneum of the epithelium. If the interdigital folds are affected, the lotion is applied daily to clean, dry skin of the feet for a week, longer if necessary.
In case of severe keratinization and cracking of the skin, it is first necessary to remove dead skin deposits. This requires the use of exfoliating medications. For example, salicylic ointment, creams with lactic acid or urea are prescribed. After removing horny deposits, the lotion is used 1 – 2 times a day.
In the dyshidrotic variant, the first step is to reduce weeping. For this, lotions with tannin or boric acid are used. In severe cases, glucocorticoids are added to treatment. Then apply Clotrimazole lotion according to the usual regimen.
If the foot is worn out, treat it with lotion once a day for 7-10 days, but the duration of the course is individual and determined by the doctor.
Systemic therapy
Long-term or recurrent athlete's foot may require oral antifungal medications. They pass from the gastrointestinal tract into the bloodstream and then into the skin, where they destroy fungi. Three main drugs are used:
- fluconazole;
- itraconazole;
- terbinafine
The duration of taking these drugs is at least a month. Their price is quite high. Therefore, preventing mycosis of the foot is always easier and more profitable than curing it.
Systemic drugs are especially often prescribed if the fungus has affected not only the skin, but also the nails. In this case, the drugs accumulate in the growing part of the nail plate, and a healthy nail gradually grows back. To improve the effect, the nail can be completely removed surgically, after which it is restored without fungus.
A combination of nail removal and systemic and local antifungal therapy is often necessary in elderly patients. In this group of patients, nails often grow slowly, blood circulation in the feet is impaired, so a large dose of drugs and a long course of treatment are required to achieve an effect.
Treatment with folk remedies
Using only traditional medicine recipes will not help get rid of the fungus. However, such an addition to conventional therapy shortens the course of treatment and speeds up recovery.
It is useful to take warm foot baths every evening for 10 minutes, then pat your feet thoroughly with a towel, especially between the toes, and apply Clotrimazole medicinal lotion for nails and skin. Useful bath ingredients that relieve inflammation and reduce itching:
- herb celandine and St. John's wort;
- burdock roots;
- wormwood grass;
- eucalyptus leaves;
- fir needles;
- fresh grounds from brewed ground coffee;
- salt;
- a mixture of grated laundry soap, baking soda, potassium permanganate and mustard powder.
The affected areas can be lubricated with birch tar or a self-prepared ointment made from 100 grams of butter mixed with a crushed head of garlic. Propolis, which can be bandaged to sore nails, is also useful.
It is useful to make compresses from natural remedies. First, they are left for 1 to 2 hours, and if tolerated, overnight. The following ingredients are used:
- pumpkin pulp;
- crushed black radish seeds;
- peppermint, ground with salt;
- burdock or rowan leaves, slightly softened with a rolling pin.
It is effective to lubricate the affected areas with the juices of some plants and other natural remedies:
- alcohol solution of propolis;
- onion or garlic juice;
- celandine juice;
- tea tree oil.
Disease prevention
In order to avoid mycosis or prevent its relapse, simple but constant prevention is needed:
- in summer, wear breathable shoes made of natural material;
- when visiting swimming pools, baths, public showers, wear individual rubber slippers;
- do not wear someone else’s shoes, for example, when visiting;
- use only your own hygiene equipment - scissors, pumice stone, nail file.
To avoid re-infection, the insoles and inner surfaces of shoes should be regularly treated with disinfectants. A well-known folk recipe is a solution of vinegar essence, but it has a sharp, unpleasant odor.
Doctors recommend using the drug Mikospray, which has not only an antifungal but also an antibacterial effect. Mycospray is great not only for treating shoes, but also for applying to feet before visiting public places to protect feet.
Residents of Moscow and the regions can purchase drugs for the treatment of mycosis of the foot and for its prevention in our online store. They have proven effectiveness and safety. Their use is recommended for all people who do not want to become infected with foot fungus or quickly get rid of it.
Precision diagnosis of invasive mycoses not easy. This is explained not only by difficulties in obtaining a culture of fungi, but also in interpreting research results, since fungi, both yeast and filamentous, can colonize mucous membranes and contaminate the studied samples. In this regard, the diagnosis of invasive mycoses is based on an integrated approach, including not only the results of mycological (cultural) and serological (determination of fungal antigen) studies, but also clinical symptoms of fungal infection, data from auxiliary research methods (computer or magnetic resonance imaging, ultrasound ).
European-American Cooperation Group for the study of invasive mycoses Criteria for diagnosing invasive mycoses have been developed in immunocompromised patients. They were presented in 2001 at the International Conference on Antimicrobials and Chemotherapy (ICAAC, Chicago), and in 2002 in print. Criteria for proven, probable and possible invasive mycosis have been defined, which are recommended for use in clinical and epidemiological studies
Proven invasive mycosis caused by filamentous fungi: detection of fungal mycelium in biopsies or aspirates during histological or cytological examination or isolation of a culture from samples obtained under aseptic conditions from a normally sterile lesion, which, according to the results of clinical and radiological studies, is associated with infection, with the exception of studies of urine and mucous membranes.
Proven invasive mycosis caused by yeast fungi: detection of yeast cells (fungi of the genus Candida can form pseudomycelium or true mycelium) in biopsies or aspirates, with the exception of samples from mucous membranes, or isolation of culture from samples obtained under aseptic conditions from a normally sterile lesion, which according to the results of clinical and radiological examination associated with infection, with the exception of urine, samples from the sinuses and mucous membranes, or the detection by microscopy and specific staining (in a drop of India ink, mucicarmine stain) of yeast cells or a positive antigen of Cryptococcus spp. in the cerebrospinal fluid.
Fungemia caused by filamentous fungi: isolation of blood culture of fungi, with the exception of Aspergillus spp. and Penicillium spp., including Penicillium marneffei, in combination with clinical symptoms of an infectious process compatible with the isolated pathogen.
Fungemia caused by yeast fungi: blood culture isolation of Candida or other yeast fungi from patients with clinical signs of infection associated with this pathogen.
Complex of diagnostic studies for invasive mycoses
| Biomaterial under study | Indications, media used, meaning |
| Blood |
Indications:
persistent fever (4-5 days or more) during therapy with broad-spectrum antibiotics; second “wave” of fever during antibiotic therapy Collecting blood from a vein into vials for aerobic bacteria* or into a selective medium for fungi, repeated (2-3 times a day with an interval of 1 hour) Diagnostic significance: isolation of yeast fungi, careful interpretation when isolating filamentous fungi, with the exception of Fusarium spp. |
| Venous catheter |
Indications:
isolation of yeast fungi from the blood The central or peripheral venous catheter is removed in all cases of yeast isolation from the blood For mycological research, an aseptically removed distal section of the catheter 5-6 cm long is used. The study is carried out semi-quantitatively (Maki method) or quantitative method on Sabouraud's medium Diagnostic significance:
|
| Upper respiratory tract discharge, sputum, washings from the trachea, bronchi, bronchoalveolar lavage fluid |
Indications:
suspicion of mycoses caused by filamentous fungi or Cryptococcus neoformans; prolonged fever during broad-spectrum antibiotic therapy and neutropenia Microscopy of samples with calcofluor white (detection of mycelium or pseudomycelium); sowing on Sabouraud's medium; determination of Aspergillus antigen in bronchoalveolar lavage fluid in the presence of lesions in the lungs characteristic of invasive aspergillosis Diagnostic significance: isolation of filamentous fungi or Cryptococcus neoformans |
| Cerebrospinal fluid |
Indications:
symptoms of meningitis; detection of a lesion(s) in the brain using computed tomography or magnetic resonance imaging; "brain" symptoms due to fever and neutropenia Microscopy with calcofluor white, in a drop of ink; determination of Aspergillus, Cryptococcus antigen; sowing on Sabouraud Wednesday Diagnostic significance:
|
| Biopsies, aspirates, peritoneal fluid, pleural fluid |
Indications:
clinical and/or radiological signs of invasive mycosis; fever during therapy with broad-spectrum antibiotics. Microscopy with calcofluor white, culture on Sabouraud's medium Diagnostic significance:
|
Possible invasive mycosis diagnosed based on the combination of the following criteria:
one sign from the category of microbiological criteria;
one sign from the category of “significant” or two from the group of “less significant” clinical symptoms of the infectious process.
Possible invasive mycosis diagnosed based on a combination of the following criteria:
the presence of at least one risk factor inducing the development of invasive mycosis;
one sign from the category of microbiological criteria or one sign from the category of “significant” (two from the group of “less significant”) clinical symptoms of the infectious process.
The concept " possible invasive mycosis» It is not recommended for use in clinical trials studying the effectiveness of antifungal drugs. You can use this term when analyzing empirical antifungal therapy, epidemiological studies, and studying pharmacoeconomics.
At mycological research sterile aspirates or biopsies take into account not only the isolation of fungal cultures, but also the detection of mycelium or pseudomycelium by microscopy. In histological preparations, Aspergillus is difficult to differentiate from Fusarium spp., Sceclosporium apiospermum and some other filamentous fungi. For differential diagnosis, an immunohistochemical study with antibodies to Aspergillus should be performed.
Isolation of yeast fungi from the blood in at least one study belongs to the category of “proven” invasive mycosis and is an absolute indication for the prescription of systemic antimycotics in patients with neutropenia. The frequency of detection of yeast fungi from the blood is low, even with disseminated candidiasis it is 35-50%.
Carrying out repeated blood cultures increases the likelihood of obtaining positive results.
Other interpretation results in case of detection of filamentous fungi in the blood. A high frequency of isolation of filamentous fungi is characteristic of Fusarium spp. and amounts to 40-60%. Aspergillus is detected extremely rarely, in most cases it is considered as contamination, with the exception of Aspergillus terreus.
Selection Aspergillus terreus from the blood of patients with hemoblastoses may indicate true aspergillemia, and in the presence of clinical symptoms of infection, it is the basis for prescribing antimycotics.
Criteria for invasive mycosis
| Index | Criteria |
| Factors inducing the occurrence of invasive mycosis (macroorganism) | Neutropenia (< 0,5*109/л в течение 10 дней)
Persistent fever for more than 96 hours during broad-spectrum antibiotic therapy Body temperature above 38 °C or below 36 °C and any of the following predisposing signs: prolonged neutropenia (more than 10 days) during the previous 60 days, intensive immunosuppressive therapy within the last 30 days, proven or probable invasive mycosis in the previous period neutropenia or AIDS Presence of symptoms of GVHD, primarily cases of severe course (II degree) or extensive course of chronic disease Long-term (more than 3 weeks) use of glucocorticoids within the last 60 days |
| Microbiological signs | Culture isolation of filamentous fungi (including Aspergillus spp., Fusaruim spp., Sceclosporium spp. and zygomycetes) and Cryptococcus neqformans from sputum or bronchoalveolar lavage fluid Positive results of cultural or cytological examination (direct microscopy) for the detection of filamentous fungi from aspirates of the paranasal sinuses Detection of filamentous fungi or Cryptococcus neoformans by cytology/direct microscopy from sputum or bronchoalveolar lavage fluid Positive Aspergillus antigen in bronchoalveolar lavage fluid, cerebrospinal fluid and blood samples (at least two) Positive cryptococcal antigen in blood samples Detection of fungal elements by cytological examination or direct microscopy in samples of normally sterile fluids (for example, Cryptococcus spp. in cerebrospinal fluid) Two positive results of studies on the detection of yeast cultures in urine in the absence of a urinary catheter Candida crystals in urine without a urinary catheter Isolation of Candida spp. from blood cultures |
|
Clinical signs
Lower respiratory tract |
Must be associated with the locus from which samples are taken for microbiological research Any of the following types of new pulmonary infiltrates according to CT: halo sign, crescent sign, cavity with areas of consolidation* Symptoms of lower respiratory tract infection (cough, chest pain, hemoptysis, dyspnea), pleural friction rub, any new infiltration not included in the signs of high significance; pleural effusion |
| Upper respiratory tract Signs of high significance Signs of lesser significance |
Radiological signs of invasive infection in the nasal sinuses (wall erosion or spread of infection to adjacent structures, extensive destruction of the skull bones) Runny nose, nasal congestion, nasal ulceration, epistaxis, periorbital edema, pain in the upper jaw, black necrotic ulceration or perforation of the hard palate |
| central nervous system Signs of high significance Signs of lesser significance |
Radiological signs of suspected CNS infection (mastoiditis or other parameningeal focus, extradural empyema, multiple lesions in the substance of the brain or spinal cord) Focal neurological symptoms and signs, including focal seizures, hemiparesis; disorders of consciousness, meningeal symptoms, disturbances in the biochemical composition of the cerebrospinal fluid and its cellular composition (in the absence of other pathogens, according to culture and microscopy, in the absence of tumor cells) |
At detection in blood or other sterile biosubstrates of yeast fungi, it is necessary to carry out identification to the species and determine sensitivity to antifungal drugs; when isolating filamentous (mold) fungi, only identification to the species, sensitivity is not determined.
In clinical practice The sensitivity of filamentous fungi is not studied due to imperfect standards for determining the sensitivity of such fungi to antimycotics. Moreover, only one study demonstrated a correlation between the susceptibility of Aspergillus spp. and the results of treatment of invasive aspergillosis in patients with hematological malignancies. None of the subsequent studies found similar results.
Recently, isolated reports have begun to appear about the formation of acquired resistance of A. fumigatus fungi to itraconazole and voriconazole.
Identification of mushrooms to species, especially those obtained from sterile loci, is necessary primarily for choosing an antimycotic and conducting adequate antifungal therapy. Thus, Candida krusei is resistant to fluconazole and less sensitive than other yeast species to amphotericin B; Aspergillus terreus, Scedosporium apiospermum (Pseudallescheria boydii), Trichosporon beigelii, Scopulariopsis spp. resistant to amphotericin B; Mucorales are resistant to itraconazole, voriconazole, Candida glabrata exhibits dose-dependent sensitivity to fluconazole, and when this type of fungus is isolated, even sensitive strains, the dose of fluconazole should be increased (adults are prescribed 800 mg instead of 400 mg); Candida lusitaniae is resistant to amphotericin B.
Identification of mushrooms to species is also important for conducting epidemiological analysis in a hospital - identifying the causative agents of outbreaks of infection and, if possible, the source of infection. Outbreaks of infection caused by such rare fungi as C. lusitaniae, C. krusei, C. lipolytica have been described.
Based mushroom species identification Invasive mycosis or fungal colonization of the mucous membranes can be assumed. For example, Aspergillus niger is significantly less likely than Aspergillus fumigatus to cause invasive aspergillosis in patients with acute leukemia. Isolation of Aspergillus niger from bronchoalveolar lavage fluid is most often regarded as colonization of the respiratory tract, and from sputum as contamination from the air and requires additional research when confirming the diagnosis of invasive aspergillosis.
Based secretions of filamentous fungi from sputum, bronchoalveolar fluid, and aspirate of the paranasal sinuses, one can only assume invasive mycosis, without including it in the “proven” category. However, the detection of Aspergillus in sputum, especially Aspergillus fumigatus or Aspergillus flavus, in neutropenic patients receiving allogeneic bone marrow should always be taken into account. This requires repeat mycological examination and computed tomography of the lungs. Thus, with neutropenia, the likelihood of detecting invasive aspergillosis in the case of a positive culture of Aspergillus spp. in sputum is 80%.
Selection Cryptococcus neoformans in immunocompromised patients from the respiratory tract (washes, lavage) is diagnostically significant. If the identification of yeast fungi from fluids obtained from the respiratory tract (tracheal, bronchial lavages, bronchoalveolar lavage) of immunocompromised patients is not required research, then screening to identify Cryptococcus neoformans from these samples is necessary.
Detection of candida in urine in patients with neutropenia and fever, it is usually considered a manifestation of disseminated candidal infection.
In a timely manner diagnostics invasive successfully use a commercial test to detect the circulation of a specific antigen of Aspergillus spp. galactomann (polysaccharide water-soluble component of the cell wall of the fungus).
Galactomann can be determined by two methods: the latex agglutination method (Pastorex Aspergillus, BioRAD) and the enzyme immunoassay method (Platelia Aspergillus, BioRAD).
Advantage enzyme immunoassay method is a lower sensitivity threshold for determining the level of galactomann in the blood - 1 ng/ml or less, and using latex agglutination - 15 ng/ml. The determination of galactomann in the blood (at least 2 samples), cerebrospinal fluid, and bronchoalveolar lavage is of diagnostic value. The sensitivity of the enzyme immunoassay method is about 90%, the specificity is 90-99%, in recipients of allogeneic bone marrow these indicators are lower and are equal to 60-70% and 80-90%, respectively, due to the prophylactic use of antifungal drugs (antimycotics reduce the threshold level of galactomann).
In 40% of cases, detection galactomann in the blood is ahead of the manifestations of invasive aspergillosis, determined by computer examination of the lungs, and in 70% it is ahead of the clinical symptoms of infection.
Diagnostic value of antigen detection test Aspergillus is the case if the study is carried out repeatedly. Determination of Aspergillus antigen in the blood should be carried out during fever during treatment with broad-spectrum antibiotics in patients with neutropenia 2 times a week; for pneumonia that occurs or persists during antibacterial therapy; when lesions are detected in the lung tissue (computed tomography).
