4402 0
Unfortunately, even now it can be stated that the diagnosis of fungal diseases is often untimely (thinning hair zones, peeling are often mistaken for "dandruff", "dryness"). At the same time, subjective sensations (itching, pain, etc.) often do not occur in the lesions, and patients, because of this, do not go to a specialist for a long time.
Changes in nails (ugly, crumbling, thinned) are regarded as "onychodystrophy" after injury, frostbite, etc. At the same time (even isolated lesions, including nails) can lead to the formation of allergic restructuring of the body, affect the blood and lymphatic vessels, etc. Patients with this indefinitely remain a source of spread of fungal infection. In connection with the foregoing, timely laboratory diagnostics of mycoses, as well as possible early treatment, are invariably relevant.
The variety of clinical variants of fungal diseases, predominantly found in humans, as well as the features of microbiology, morphostructure, immunological and other parameters of various types (and genera) of fungi have led to the presence of a significant number of methods for diagnosing mycoses; It should be noted that the available methods are constantly being improved and (relatively recently) have a peculiar bias towards immunological and molecular genetic tests.
On the other hand, cultural studies are still "in the category of standards close to gold" and are used to confirm the dubious results of other studies; There is an opinion that the combination of cultural and molecular genetic methods for diagnostically "dubious" forms of mycoses (especially disseminated, against the background of immunosuppression of any genesis, with damage to internal organs, etc.) is one of the most reliable ways to register the mycotic nature of the process.
However, one should not neglect the "old" techniques, in particular bacterioscopy (especially at the initial examination), especially since in everyday practice microscopic "verification" of mycosis in many dermatological institutions is used most widely in comparison with other tests.
Given the significant increase in the number of allergic manifestations on the skin (and sometimes visceral) - with a prolonged, chronic, periodically exacerbating course of mycosis (skin, nails, etc.), it is advisable to conduct allergological tests to identify the degree of sensitization of the body, including combined fungal and bacterial infection; this fact can affect the specificity of treatment in a particular patient - for example, determine the rational appointment of antimycotics and desensitizing agents, etc.
Traditionally, a presumptive diagnosis of a fungal disease is made on the basis of clinical manifestations and confirmed by laboratory tests.
In this section of the book, we briefly present the basic methods for registering mycoses (regardless of their location), taking into account the "type of research and the material taken." It should be noted that the polymorphism of the clinical manifestations of mycoses (including those with an allergic component) determines the diversity of the pathological material to be studied. In this case, the success of the search for elements of the fungus depends on the correct taking of it.
So, the peripheral zone of erythemato-squamous, often curly eruptions is richer in mycelium, fungal spores; on the hairy areas of the lesion, twisted, whitish, discolored, dull hair or their fragments - "hemp" are taken (it is advisable to control the hair intake using a Wood lamp). Certain skills are required by the sampling of material (using a needle) from the so-called. "Black dots" - dark horny cones at the mouths of the follicles.
In everyday practice, skin flakes are usually examined (collected by scraping, smear, using adhesive tape), scraping of altered nails, a zone of subungual hyperkeratosis, as well as mucous membranes. According to indications, sputum, lavage fluid, urine are examined (in patients with uncatheterized bladder); urine from urine bags, bed vessels cannot be taken for examination.
Blood is also of diagnostic value (for culture research, as well as ELISA, PCR), cerebrospinal fluid and other biofluids of the body (pleural, intra-articular, intraperitoneal - including those collected by aspiration or drainage); in some cases (depending on the topical diagnosis), bile, feces, punctates of subcutaneous abscesses, fistula discharge (especially with deep mycoses) are important. Even simple diagnostic tests require strict adherence to a number of conditions.
Methods are currently used to diagnose fungal diseases:
- microscopy; based on the detection of the pathogen in the test material, incl. in human tissues;
- cultural research followed by microscopic examination of the culture of the fungus;
- histological examination (G.O. with deep mycoses);
- immune and molecular methods.
Microscopic diagnostics
Microscopic diagnostics - became possible from the period of the appearance of special optical, and later electronic microscopic technology, which made it possible to study in detail the ultrastructure of fungi. At the same time, the detection of fungal elements - thin branching filaments that form the mycelium (mycelium), rounded bodies (spores; are the reproductive "organ" of fungi) is of diagnostic value.Microscopic diagnostics involves the study of unstained (native) and stained preparations. This method, as noted, is most common in dermatological practice due to its comparative simplicity and low cost, but, on the other hand, it is not sensitive enough, requires repeated analyzes in some cases, confirmation by other methods.
So, when studying unstained preparations, in addition to fungal elements, one can find epithelial cells, blood cells, various contaminants from the external environment, which makes it difficult to find the causative agent of mycosis, requires additional "preparation" of the material - the so-called. Its "enlightenment" (maceration), concentration, dilution, etc.
Nevertheless, direct microscopy of native preparations allows you to quickly diagnose mycosis, to determine on which nutrient media (if necessary) the material should be inoculated, there is an opinion that its positive result may remain the only laboratory confirmation of mycosis with a negative response in culture (A.Yu. Sergeev, Yu.V. Sergeev, 2003).
Among the various options for "enlightenment" of drugs, the most common is the addition of KOH or NaOH to the test material (it is more often used to detect fungi in skin scales, hair, as well as a number of causative agents of deep mycoses in sputum, biopsies).
For maceration of crushed and placed on a glass slide, 10-20% sodium hydroxide (or potassium) solution is carried out - 1-3 drops, for 10-20 minutes; the preparation is lightly pressed with a cover glass, for faster maceration - heated on a flame until vapors appear. Viewing is carried out first under low magnification, then under high magnification (dry system).
A properly prepared preparation, not subjected to rough mechanical, thermal and chemical influences, presents a picture of a homogeneous mass consisting of epithelial cells, products of cellular decay of fungal elements - mycelium filaments and spores.
The modified hair is placed on a glass slide with 10-30% alkali and treated in the same way, but for a longer period - from 20 minutes to 3-4 hours. With an abundant pigment content, it is recommended to pre-bleach the hair with a 5% hydrogen peroxide solution. They attach importance to the detection of the elements of the fungus, as well as their location in relation to the hair.
The preparation of material from pathologically altered nails (preferably a fine powder obtained by scraping from the depth of the focus) is carried out in the same way, but using 30% caustic soda, mandatory careful heating on a flame to light vapors and exposure for about 1 hour (sometimes up to several hours). Alkali-treated preparations should not be stored for more than 1.5-2 hours due to their "deterioration" (crystallization of the reagent, decrease in the diagnostic reliability determined by the microscopic picture).
Instead of KOH or NOOH, you can use: a) a solution of a mixture of KOH with 15% DMSO; b) a mixture of phenol (2 parts) with chloral hydrate (2 parts) and lactic acid (1 part); c) calcofluoric white, which has an affinity for higi and cellulose; for research, a fluorescent microscope is required (a blue or green glow is observed, depending on the filter used). In the study of cerebrospinal fluid (with suspicion of cryptococcosis), ink staining was often used.
Kulaga V.V., Romanenko I.M., Afonin S.L., Kulaga S.M.
LABORATORY DIAGNOSTICS OF MYCOSIS
Taking material for laboratory research for fungus.
1.OST 42-21-2-854: order No. 222/80 dated 27.06.00
2. Equipment: tweezers, microscope slides, scissors, folkman spoon.
4. Indications: fungal diseases.
5. Complications: no.
Preparation of a treatment room:
Change of solutions.
Prepare:
-1% solution of chloramine for rags
-3% chloramine solution - for disinfection of dressings and tweezers
- washing solution (156 ml. hydrogen peroxide + 5 g. detergent powder + 839 ml. distilled water) - for processing tweezers
- 6% solution of hydrogen peroxide - for the treatment of gloves.
Algorithm for performing manipulation.
You need to take tweezers, a glass slide, a Folkman spoon, scissors;
- invite the patient to the dressing room;
- the patient sits on a chair or couch;
- m / s stands.
Execution technique:
Take skin scales and hair from the lesion with tweezers;
- put the taken material on a glass slide and cover with another glass slide.
- wash your hands with soap and water;
- send the taken material to the laboratory.
COLLECTION OF MATERIAL
Take scissors and glass slides;
- cut off a piece from the free edge of the nail with scissors;
- cover the taken material with another glass slide;
Soak scissors and tweezers in 3% formalin solution.
The correct collection of material from the affected nails is the key to a successful microbiological study. When picking up material, they do not always capture areas of the nail containing viable fungi. Naturally, non-viable mushrooms in culture will not grow, and their species cannot be established.
The area of the nail to be taken is determined by the form of onychomycosis.
So, with a superficial form of onychomycosis, scrapings should be made from the surface of the nail plate.
In the most common distal subungual form, the most viable fungi are located under the nail plate. The material that is sent for research should include not only a trimming of the nail plate, but also scraping from the nail bed, from under the plate.
In addition, the areas of the unchanged nail should be captured, since the most active fungi are located on the border between them and the affected areas of the nail.
In the proximal subungual form, material is difficult to pick up. In these cases, sometimes, especially if they are going to conduct a histological examination or differential diagnosis, they take a biopsy of the nail, and occasionally use a drill.
With paronychia, scrapings are made from and from the proximal roller.
In all cases, to avoid bacterial contamination, the nail should be treated with ethyl alcohol before taking the sample.
MICROSCOPIC STUDY
Microscopic examination of pathological material for fungi is performed in native and colored preparations.
For the preparation of unstained preparations, the resulting material is crushed with a scalpel or dissecting needle and placed in the middle of a slide. For a clearer identification of the elements of the fungus, the material is enlightened (macerated). For this purpose, they resort to the help of various substances, most often caustic alkali (KOH, NaOH), which dissolve epidermal scales, mucus, pus, clarify the hair pigment and thereby make mushrooms available for research.
On the softened scales of the skin or nail, which are placed in the middle of the slide, apply 1-3 drops of 20-30% KOH (NaOH) solution. The test material in alkali drops is carefully heated over the flame of an alcohol lamp until a delicate white rim of alkali crystals appears along the periphery of the drop. Do not preheat to a boil. After heating, the drop is covered with a cover glass, avoiding the ingress of air bubbles.
RA Arabiyskiy and GI Gorshkova (1995) recommend leaving the skin scales and hair preparations covered with a cover glass for 5 - 10 minutes, and the nail plates - for 30 - 40 minutes before microscopy.
Enlightenment of preparations can be carried out without heating, for this they are left in a 20% KOH solution for 30 - 60 minutes or other methods of enlightenment of pathological material are used: chlorallactophenol according to Aman; lactophenol; a solution containing 15% dimethyl sulfoxide and KOH in water. Good results are obtained after the enlightenment of the nail plates, placed in a 5% KOH solution for 24 hours, no heating is required in this case.
Microscopic examination is performed on a conventional laboratory microscope without immersion.
The microscope condenser must be lowered, the diaphragm narrowed. At the beginning, the drug is found on glass at low magnification (40x), the subsequent study is performed at higher magnification (100x);
the preparation is studied in detail at a magnification of 400x. It is necessary to investigate several drugs in order to increase the reliability of the test and to avoid false positive results.
Errors in the microscopic diagnosis of fungi can occur due to both defects in the preparation of the drug and the lack of experience of the laboratory assistant.
Manufacturing defects are primarily related to:
with overheating of the drug, which can lead to the loss of alkali crystals, the destruction of the hair and the appearance of fine-grained disintegration in the pathological material.
The linear arrangement of elongated, even alkali crystals very much resembles the filaments of septic mycelium even on clean glass without pathological material.
Differential diagnostic features are the exceptional uniformity of crystals, their glassy transparency, versatility of edges and the absence of an inextricable connection between one element and another. In doubtful cases, it is recommended to add droplets of slightly warmed distilled water to the preparation, which quickly dissolve alkali crystals.
The following can be mistaken for the elements of the mushroom:
- droplets of fat,
- air bubbles,
- cotton threads of clothing
- and the so-called "mosaic fungus".
Lipids of the skin, fatty breakdown of cells and keratohyalin grains, especially those with the correct shape, can resemble individual spores of the fungus. But the variety of shapes and, most importantly, sizes, the absence of the internal structure of formations (vacuoles, shells) speaks against the fungal nature of these elements. Lipids can also get into the drug when taking pathological material from an insufficiently purified lesion.
Air bubbles may resemble the spores of yeast-like cells, but unlike the latter, they are surrounded by a dense dark membrane, and even the smallest air bubbles are always larger than the cells of the fungus.
Threads from the fabric of socks, clothing, etc. usually lie separately from the pathological material, they are always larger than hyphae, coarser and not septic.
"Mosaic fungus" is an artifact that occurs during crystallization (possibly due to the breakdown of cholesterol). It has the form of a mesh or loops, the outlines of which correspond to the boundaries of the horny scales, unlike the filaments of the mycelium, it never crosses the walls of the cells of the epidermis.
In some laboratories, the clarification of preparations for microscopic examination is carried out with 15 - 30% KOH solution, to which 5 - 10% of Parker's commercial dark blue ink (Parker's Superchrome Blue-Black Ink) is added.
With this color, the hyphae and spores turn blue.
Microscopy reveals filamentous fungal hyphae or budding cells (Fig. 1).
Thus, microscopy gives a conclusion only about the fungal nature of the infection, but not about the type of fungus-causative agent.
Of course, the effectiveness of microscopic examination depends on the qualifications of the laboratory employee.
Rice. 1. Microscopy of scrapings from nails affected by T. rubrum. The hyphae of the fungus are visible.
CULTURAL RESEARCH
The material is inoculated on a standard Sabouraud medium, often with antibiotics. In the diagnosis of dermatophyte infections, it is customary to add cycloheximide to Sabouraud's medium, which suppresses the growth of contaminating fungi that come from the air. There are commercial off-the-shelf media with antibiotic and cycloheximide additives. It should be remembered that many non-dermatophytic molds and some Candida species do not grow on medium with cycloheximide, therefore it is recommended to inoculate on Sabouraud medium with cycloheximide and on medium without it. Species identification is usually carried out by microscopic examination of the grown culture or by reseeding on selective media (Fig. 2-15).
Rice. 2. Culture of T. rubrum fungus isolated from affected nails. Obtained on Sabouraud's medium (left) and corn agar (right).
Rice. 3. Culture of the fungus T. mentagrophytes var. interdigitale isolated from affected nails. Obtained on the environment of Saburo.
Rice. 4. Culture of the fungus Candida albicans. Obtained on the environment of Saburo.
Rice. 5. Culture of the fungus Torulopsis glabrata isolated from the affected nails. Obtained on the environment of Saburo.
Rice. 6. Culture of the fungus Ulocladium sp., Isolated from the affected nails.
Rice. 7. Micromorphology of Acremonium sp. Isolated from affected nails.
Rice. 8. Micromorphology of Fusarium sp. Isolated from affected nails.
Rice. 9. Micromorphology of Scopulariopsis sp. Isolated from affected nails.
Rice. 10. Micromorphology of Candida albicans isolated from affected nails.
Rice. 11. Micromorphology of Altemaria sp. Isolated from affected nails.
Rice. 12. Micromorphology of Aspergillus sp. Isolated from affected nails.
Rice. 13. Micromorphology of Ulocladium sp isolated from affected nails.
Rice. 14. Micromorphology of Chaetomium sp. Isolated from affected nails.
Fig 15. Panel of nutrient fed for the identification of dermatophytes (left - culture T rubrum, right - T mentagrophytes var. Mterdigitale).
Left to right: Sabouraud's medium, Baxter's medium, Christensen's medium, corn agar
It should be noted that some mold fungi, including dermatophytes, grow slowly in culture, in 2-3 weeks.
Even if all the rules for collecting material are observed, with good laboratory equipment and high qualifications of its personnel, the number of positive results of a culture study is very small.
According to foreign literature, the percentage of positive studies does not exceed 50.
The percentage of positive results in the best domestic laboratories barely reaches 30.
Thus, in 2 out of every 3 cases of onychomycosis, its etiology cannot be established.
LUMINESCENT RESEARCH
In 1925, Margaret and Deveze found that hairs affected by certain dermatophytes gave off a characteristic glow when exposed to ultraviolet rays passed through a Byda filter. Byda glass consists of barium sulfate, contains about 9% nickel oxide; it transmits rays with a length of 365 nm. Various devices can be used as a source of ultraviolet rays. The nature of the glow has not been precisely established. The hair continues to glow after the death of the fungus and after attempts to extract the fluorescent material with hot water or cold sodium bromide solution. The intensity and nature of the glow depend on the pH of the solution. It is believed that the fluorescent substance appears in the process of interaction between the fungus and the growing hair.
Glow in ultraviolet rays, passed through the Wood filter, is typical only for hair affected by fungi of the genus Microsporum (M. canis, M. audouinii, M. ferrugineum, M. distortium, occasionally M. gypseum and M. nanum), as well as Trichophyton schonleinii ... Hair affected by microsporums, especially M. canis and M. audouinii, give the brightest glow; hairs affected by T. schonleinii have a dull greenish fluorescence.
The glow is observed only in hair completely affected by the fungus. It may not be in fresh lesions. In these cases, the hair should be epilated from the most active edge zone, and the glow can be found in the root part of the hair.
The luminescent method can be used both for diagnostics and monitoring the effectiveness of treatment in individual patients and in epidemiological foci. Compact mobile units are convenient for examining contact people in schools, kindergartens, etc.
Luminescent examination must be performed in a darkened room, lesions must be pre-cleaned of crusts, ointment residues, etc. The luminescent method can be used to diagnose pityriasis lichen, especially when lesions are localized on the scalp. The lesions in this disease have a reddish-yellow or brown glow. This glow, however, is not strictly specific, as it can be observed in the presence of dandruff on the scalp and even in healthy people in the area of the mouths of the hair follicles on the face and upper body. Affected hairs identified using the luminescent method must be subjected to microscopic examination.
IMMUNOLOGICAL AND BIOLOGICAL RESEARCH
Immunological research methods are used to identify specific restructuring of the body and serological diagnosis of fungal diseases. To detect specific antibodies in the serum of the sample, the following serological reactions are carried out: agglutination, precipitation, complement binding, immunofluorescence with the corresponding antigens.
An allergic condition of the patient's body is detected using allergic skin tests. Allergens are applied to scarified skin according to Pirque or by rubbing into the skin according to Moro, intradermally according to Mantoux, and also by injection into the skin. With the help of these tests, allergic reactions of both immediate and delayed type are detected, which makes it possible to assess the state of humoral and cellular immunity.
To detect specific sensitization of lymphocytes, basophil degranulation, agglomeration and alteration reactions, blast transformation test, inhibition of macrophage migration, etc. are used.
Comparison of the results of serological and allergic reactions is useful for both diagnosis and prognosis of the course of mycoses.
Biological method. It is used for laboratory diagnostics of deep and especially dangerous mycoses. Based on the infection of animals with pathological material from a patient or culture of the studied fungus. Carried out in special laboratories.
HISTOLOGICAL EXAMINATION
Histology of skin mycoses caused by dermatophytes
Pathomorphological changes in the lesions are caused by the introduction of fungi into the stratum corneum of the epidermis, hair and nails and an inflammatory response of the skin, which can be acute, subacute or chronic. The diagnosis can be considered established only if fungal elements are found in histological preparations. For this, various histological stains are used, the most informative is the periodic acid reaction (PAS), which makes it possible to identify the polysaccharides present in the cellulose and chitin of the cell wall of most dermatophytes (Shifu stain and its modifications). You can also use the reaction of sulfation and the impregnation of histological sections with silver [Khmelnitsky OK, 1973; Lewer W. F. and Schaumburg-Lewerl., 1983].
Fungi in the stratum corneum of the epidermis, even when using special stains, are detected in small quantities in the form of mycelium filaments and spores. In rare cases, when there are many fungi in the lesions, they can be found in sections stained with hemotoxylin-eosin, in the form of delicate basophilic structures in the stratum corneum.
Inflammatory changes in the epidermis can be different: from minor intra- and extracellular edema of spiny cells to severe spongiosis. Spongiosis usually develops with dyshidrotic variants of mycoses of the feet and hands, clinically in these cases, bubbles are noted. This reaction is usually caused by T. mentagrophytes var. interdigitale. Sometimes in the epidermis there is a pronounced hyperkeratosis, which is most often observed in mycosis caused by T. rubrum.
Histological changes in the dermis are nonspecific and correspond to acute, subacute and chronic inflammation.
In mycosis of smooth skin caused by T. rubrum, fungi are sometimes found in vellus hair and hair follicles. An inflammatory reaction develops around the follicles, which, due to the entry of fungi into the dermis, can acquire a granulomatous character. The central part of the infiltrate in these cases can undergo suppuration and necrosis, and the peripheral part consists of lymphocytes, histocytes, epithelioid and multinucleated giant cells, inside which fungal spores are sometimes found. The spore sizes here reach 6 microns in diameter, in the hair they usually do not exceed 2 microns.
With the infiltrative-suppurative form of mycoses of the scalp and the growth area of the beard and mustache, fungal elements are found in the hair follicle, inside and around the hair. In the hair, they are determined just above the zone of the beginning of keratinization (approximately at the level of 30 microns). In the dermis, an inflammatory reaction of varying intensity is noted, most pronounced with kerion Celsii. In case of an acute purulent reaction, a large number of neutrophilic leukocytes are noted in the composition of the infiltrate, the elements of the fungi in this case can completely disappear. In the chronic course of the process, the infiltrate can acquire a granulomatous character, multinucleated giant cells appear in it. To confirm the diagnosis in the absence of fungi in the infiltrate, immunofluorescent staining methods can be used. For these purposes, fluorescein-labeled antiserum to T. mentagrophytes is used, which makes it possible to detect fungal antigens in the hair and in the perifollicular infiltrate.
The formation of an infiltrative-suppurative skin reaction in mycosis of the scalp (kerion Celsii) and the growth area of the beard and mustache caused by the fungi M. canis, T. tonsurans and T. verrucosum is a manifestation of an immunological reaction. This is evidenced by:
1. The tendency of lesions to spontaneous resolution.
2. Absence of fungal elements with a very pronounced inflammatory reaction on the part of the skin with mycosis caused by T. verrucosum (faviforme) and T. tonsurans.
3. A constant positive reaction in response to the intradermal administration of trichophytin in infiltrative-suppurative forms of mycosis caused by zoophilic trichophytins (for example, T. tonsurans), and negative - in superficial mycoses caused by the same T. tonsurans.
With favus, a large number of mycelium filaments and single spores of the fungus are found in the stratum corneum of the epidermis. The scutula is represented by exudate, parakeratotic cells of the epidermis, cells of the inflammatory infiltrate, as well as mycelium filaments and fungal spores, which are located mainly in the peripheral zone of the scutula. In the active stage of the disease, a pronounced inflammatory infiltrate containing multinucleated giant and plasma cells is noted in the dermis around the degenerative hair follicles. In old lesions, hair and sebaceous glands are absent, there are phenomena of fibrosis.
Histology of mycoses of the skin and mucous membranes caused by yeast-like fungi
With candidiasis of the skin and mucous membranes, fungi of the genus Candida are found in the stratum corneum of the epidermis or in the superficial layers of the epithelium of the mucous membrane. Fungus elements are usually few, they are well stained by PAS-reaction or Gram; are presented in the form of septate branching mycelium filaments, 2-4 microns in diameter, or ovoid spores, 3-5 microns in diameter. Detection of the mycelial form of the fungus is of diagnostic value.
In a histological study of chronic granulomatous candidiasis of the skin and mucous membranes, fungal elements are also predominantly found in the stratum corneum of the epidermis or in the uppermost parts of the epithelium of the mucous membrane, but sometimes in the prickly layer, inside the hair and in the dermis. There is also marked hyperkeratosis and papillomatosis; in the dermis - a dense inflammatory infiltrate consisting of lymphoid cells, neutrophils, plasma and multinucleated giant cells. The infiltrate can spread into the subcutaneous fatty tissue.
With pityriasis versicolor, a large number of fungal elements are found in the stratum corneum of the epidermis in the form of delicate basophilic structures, which are clearly visible even when the preparations are stained with hemotoxylin-eosin. Mushrooms are represented by both filaments and spores.
With the follicular form of pityriasis lichen, there is an accumulation of horny masses and cells of an inflammatory infiltrate in the dilated mouths of the hair follicles. Inflammatory infiltration is also noted around the follicles. In the PAS reaction, spherical or oval spores of the fungus, 2-4 microns in diameter, are found inside the mouth of the hair follicles, and sometimes in the perifollicular infiltrate. The mycelium is never detected.
Impaired skin pigmentation in patients with pityriasis versicolor is due to the ability of the Pityrosporum fungus to produce a substance that inhibits the process of pigmentation in the epidermis. Electron microscopic examination of skin biopsies from hypopigmented areas showed that very small melanosomes are formed in melanocytes, which are not able to penetrate into keratinocytes. On the other hand, in hyperpigmented areas of the skin, melanosomes are large and contain a large amount of melanin.
The basis for the diagnosis of fungal diseases is microscopic examination of preparations prepared from the affected areas of the skin and nails. However, the microscopic picture in different types of mycoses is similar: in the skin scales and nails, fungal spores and branched septate mycelium with a diameter of 4-7 microns are visible. Therefore, the genus and species of the fungus in most cases cannot be determined by the microscopic picture in the skin scales or in the scraping from the nail. To identify the pathogen, inoculations are carried out on nutrient media, most often on Sabouraud's medium.
Epidermophytosis. The stratum corneum of the skin, most often the feet, and the nails of the lower extremities are affected. Hair is never affected. Yellow spots or stripes appear on the nails, then hyperkeratosis (thickening of the nails) develops, their deformation and destruction. Lamellar peeling of the soles and interdigital folds, redness appears on the skin of the feet. Sometimes bubbles, diaper rash, cracks form. The disease is accompanied by itching, burning, soreness.
Microscopic examination of scrapings, scales, bladder caps reveals low-septic filaments of mycelium with a diameter of 3-5 mm, some of the filaments break down into round and rectangular spores.
Trichophytosis. It is a fungal disease of the skin and its appendages with a particular tendency to affect the hair [lat. trichos hair + phyton fungus]. Numerous lesions with a diameter of about 1.5 cm appear on the scalp. The skin on them is edematous, hyperemic, covered with scales. Hair in the lesions breaks off at the level of 2-3mm above the surface of the skin, hence the name "ringworm".
Microscopic examination reveals a characteristic feature of Trichophyton fungi - the arrangement of their spores in chains. Depending on the properties of mushrooms, there are:
Endothrix (causative agent of superficial trichophytosis). Mushrooms grow inside the hair, dramatically changing its structure. The entire hair is filled (stuffed) with parallel rows of chains, consisting of large rounded or square spores;
Ectothrix (causative agent of deep trichophytosis), in which the hair is enveloped in a sheath of small or large spores arranged along the axis in chains.
Favus is scab. Hair and skin are affected, less often nails. Hair becomes thin, dull, “powdered”, like old wigs, but does not break off. Skin lesions are characterized by the appearance of yellow-gray crusts (scutes) with raised edges, like those of saucers.
Microscopic examination reveals air bubbles inside the affected hair.
Microsporia. Skin and hair are affected. In lesions, hair breaks off at a level of 6-8 mm above the skin surface. Whitish sheaths are visible around the remaining stumps.
Microscopic examination of the affected hair reveals spores located around and inside the hair in a mosaic (irregularly) pattern. They are very small (1-3mm), hence the name of the disease. The characteristic fluorescent glow of the hair helps diagnose microsporia.
Microscopic examination reveals rounded budding cells, often in the form of a bunch of grapes.
Deep (mold) mycoses. They are more common as occupational diseases in antibiotic factories, in agricultural workers in contact with moldy grain, hay, compost, etc. The causative agent of penicilliosis (brush fungus) has a rough, wide, septic mycelium ending in a brush. The causative agent of mucorosis has a wide non-septic mycelium ending in a bag of spores. The causative agent of aspergillosis (leukemia mold) is often found on moldy fruits and bread. It has a coarse septate mycelium, ending in an expansion, from which threads with spores, resembling a watering can with streams of water, branch off.
Actinomycosis. Caused by different types of radiant fungus. It is characterized by the formation of dense infiltrates on the skin, mucous membranes and in the internal organs, prone to suppuration and the appearance of fistulas. On the eye in the discharge of the fistula, small yellow grains with a characteristic radiant edge are visible - actinomycete druses. Preparations for microscopy are prepared from the discharge of fistulas and sputum.
At low magnification, the druses of the radiant fungus have the appearance of yellowish, rounded formations with a light amorphous middle and a darker color at the edges. At high magnification, mycelium filaments are determined in the center of the drusen, and flask-shaped swellings along the periphery. When staining according to Gram, the filaments of the mycelium are G +, and the cones are G-.
Precise diagnosis of invasive mycoses not easy. This is explained not only by difficulties in obtaining a culture of fungi, but also in the interpretation of research results, since fungi, both yeast and mycelial, may colonize mucous membranes and contaminate the samples under study. In this regard, the diagnosis of invasive mycoses is based on an integrated approach, which includes 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 (computed or magnetic resonance imaging, ultrasound ).
European-American Cooperative Group for the study of invasive mycoses in immunocompromised patients, criteria for the diagnosis of invasive mycoses have been developed. They were presented in 2001 at the International Conference on Antimicrobials and Chemotherapy (ICAAC, Chicago), and in 2002 in print. The criteria for proven, probable and possible invasive mycosis, which are recommended for use in clinical and epidemiological studies, have been determined.
Proven invasive mycosis due to filamentous fungi: detection of fungal mycelium in biopsies or aspirates during histological or cytological examination or isolation of culture from samples obtained under aseptic conditions from a sterile focus, which, according to the results of clinical and radiological studies, is associated with infection, with the exception of urine and mucous membrane studies.
Proven invasive mycosis due to yeast: 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 focus, which, according to the results of clinical and radiological studies associated with infection, with the exception of urine, sinus and mucous membrane specimens, or detection by microscopy and specific staining (in a drop of ink, staining with mucicarmine) yeast cells or positive antigen Cryptococcus spp. in the cerebrospinal fluid.
Fungaemia due to filamentous fungi: isolation of blood cultures of fungi, except for Aspergillus spp. and Penicillium spp., including Penicillium marneffei, in combination with clinical symptoms of an infectious process consistent with the isolated pathogen.
Fungaemia due to yeast fungi: Isolation of blood cultures of Candida or other yeast fungi in patients with clinical signs of infection associated with this pathogen.
Complex of diagnostic studies for invasive mycoses
| Investigated biomaterial | Indication, media used, value |
| Blood |
Indications:
persistent fever (4-5 days or more) during therapy with broad-spectrum antibiotics; second "wave" of fever on the background of antibiotic therapy Blood collection from a vein into vials for aerobic bacteria * or in a selective medium for mushrooms, repeated (2-3 times during the day with an interval of 1 hour) Diagnostic relevance: isolation of yeast, careful interpretation when isolating filamentous fungi, except for Fusarium spp. |
| Venous catheter |
Indications:
isolation of yeast from the blood The central or peripheral venous catheter is removed in all cases of yeast secretion from the blood For mycological examination, an aseptically removed distal segment of the catheter 5-6 cm long is used. The study is carried out by a semi-quantitative (Maki method) or quantitative method on Sabouraud Diagnostic relevance:
|
| Discharge of the upper respiratory tract, sputum, lavage from the trachea, bronchi, bronchoalveolar lavage fluid |
Indications:
suspicion of mycoses caused by filamentous fungi or Cryptococcus neoformans; prolonged fever during therapy with broad-spectrum antibiotics and neutropenia Microscopy of samples with white calcofluor (detection of mycelium or pseudomycelium); sowing on Saburo's Wednesday; determination of Aspergillus antigen in bronchoalveolar lavage fluid in the presence of foci in the lungs characteristic of invasive aspergillosis Diagnostic relevance: Isolation of filamentous fungi or Cryptococcus neoformans |
| Cerebrospinal fluid |
Indications:
symptoms of meningitis; detection of a focus (foci) in the brain with computed or magnetic resonance imaging; "Cerebral" symptoms associated with fever and neutropenia Microscopy with white calcofluor, in a drop of ink; determination of the antigen of Aspergillus, Cryptococcus; sowing Wednesday Saburo Diagnostic relevance:
|
| Biopsies, aspirates, peritoneal fluid, pleural fluid |
Indications:
clinical and / or radiological signs of invasive mycosis; fever during therapy with broad-spectrum antibiotics. Microscopy with white calcofluor, plating on Sabouraud medium Diagnostic relevance:
|
Probable invasive mycosis diagnosed with a combination of the following criteria:
one trait 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 that induces 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»Not recommended for use in clinical trials investigating the effectiveness of antifungal drugs. You can use this term in the analysis of empirical antifungal therapy, epidemiological studies, the study of pharmacoeconomics.
At mycological research Sterile aspirates or biopsies take into account not only the isolation of the fungal culture, 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 blood at least in one study belongs to the category of "proven" invasive mycosis and is an absolute indication for the appointment of systemic antimycotics in patients with neutropenia. The frequency of detecting yeast fungi from the blood is low, even with disseminated candidiasis, it is 35-50%.
Carrying out repeated blood cultures increases the likelihood of getting positive results.
Other interpretation results in the case of detection of filamentous fungi in the blood. The high frequency of isolation of filamentous fungi is characteristic of Fusarium spp. and is 40-60%. Aspergillus is rarely found and is considered contamination in most cases, with the exception of Aspergillus terreus.
Highlighting Aspergillus terreus from the blood of patients with hemoblastosis may indicate true aspergilloma, and in the presence of clinical symptoms of infection, it is the basis for the appointment of antimycotics.
Criteria for invasive mycosis
| Index | Criteria |
| Factors inducing the onset of invasive mycosis (macroorganism) | Neutropenia (< 0,5*109/л в течение 10 дней)
Persistent fever for more than 96 hours with 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) in the previous 60 days, intensive immunosuppressive therapy within the last 30 days, proven or probable invasive mycosis in the previous period neutropenia or AIDS Symptoms of GVHD, primarily cases of severe course (II degree) or extensive course of chronic illness Long-term (more than 3 weeks) use of glucocorticoids within the last 60 days |
| Microbiological signs | Isolation of a culture of filamentous fungi (including Aspergillus spp., Fusaruim spp., Sceclosporium spp. And zygomycetes) and Cryptococcus neqformans from sputum or bronchoalveolar lavage fluid Positive results of culture 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 Aspergillus antigen positive in bronchoalveolar lavage fluid, cerebrospinal fluid, and blood samples (at least two) Positive antigen of cryptococcus in blood samples Detection by cytological examination or direct microscopy of fungal elements in normally sterile fluid samples (for example, Cryptococcus spp. In cerebrospinal fluid) Two positive results of studies on the detection of a culture of yeast fungi in urine in the absence of a urinary catheter Candida crystals in urine in the absence of 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 examination Any of the following types of new lung infiltrates on CT: halo symptom, crescent symptom, cavity with areas of consolidation * Symptoms of a lower respiratory tract infection (cough, chest pain, hemoptysis, dyspnea), pleural friction murmur, any new infiltrate not included in the signs of high severity; pleural effusion |
| Upper respiratory tract Signs of high significance Signs of lesser importance |
Radiological signs of invasive sinus infection (wall erosion or spread of infection to adjacent structures, extensive destruction of the skull bones) Runny nose, nasal congestion, ulceration of the nasal mucosa, epistaxis, periorbital edema, upper jaw pain, black necrotic ulceration, or perforation of the hard palate |
| central nervous system Signs of high significance Signs of lesser importance |
X-ray signs of suspected CNS infection (mastoiditis or other parameningeal focus, extradural empyema, multiple lesions in the brain or spinal cord) Focal neurologic symptoms and signs, including focal seizures, hemiparesis; disorders of consciousness, meningeal symptoms, violations of the biochemical composition of 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 imperative to identify to the species and determine the 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 investigated due to imperfect standards for determining the sensitivity of such fungi to antimycotics. Moreover, only one study has demonstrated a correlation between the sensitivity of Aspergillus spp. and the results of treatment of invasive aspergillosis in patients with hemoblastosis. None of the studies that have been carried out since then have obtained similar results.
Recently, isolated reports began to appear on the formation of acquired resistance of A. fumigatus fungi to itraconazole, voriconazole.
Identification of fungi by species, especially those obtained from sterile loci, is necessary, first of all, for the selection of an antimycotic and for an adequate antifungal therapy. Thus, Candida krusei are 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 isolating this type of fungus, even sensitive strains, the dose of fluconazole should be increased (adults are prescribed 800 mg instead of 400 mg); Candida lusitaniae are resistant to amphotericin B.
Identification of fungi by species It is also important for conducting epidemiological analysis in a hospital - determining the causative agents of outbreaks and, if possible, the source of infection. Outbreaks of infection caused by such rare fungi as C. lusitaniae, C. krusei, C. lipolytica are described.
Based species identification of fungi 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 to confirm the diagnosis of invasive aspergillosis.
Based excretion of filamentous fungi from sputum, bronchoalveolar fluid, aspirate of the paranasal sinuses, one can only assume invasive mycosis, not including it in the category of "proven". However, the detection of Aspergillus in sputum, especially Aspergillus fumigatus or Aspergillus flavus, in neutropenic patients, recipients of allogeneic bone marrow, should always be considered. This requires repeated mycological examination and computed tomography of the lungs. So, with neutropenia, the likelihood of detecting invasive aspergillosis in the case of a positive culture of Aspergillus spp. in sputum is 80%.
Highlighting Cryptococcus neoformans in immunocompromised patients from the respiratory tract (washings, lavage) is diagnostically significant. If identification of yeast fungi from fluids obtained from the respiratory tract (lavage from the trachea, bronchi, bronchoalveolar lavage) of immunocompromised patients is not mandatory, then screening for the detection of 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 diagnostics invasive successfully used a commercial test to detect the circulation of the specific antigen of fungi Aspergillus spp. galactomanna (polysaccharide water-soluble component of the fungal cell wall).
Galactomann can be determined by two methods: the latex agglutination method (Pastorex Aspergillus, BioRAD) and the enzyme-linked immunosorbent assay (Platelia Aspergillus, BioRAD).
The advantage enzyme immunoassay is the lower threshold of sensitivity for determining the level of galactomann in the blood - 1 ng / ml or less, and with the help of latex agglutination - 15 ng / ml. Determination of galactomancy in blood (in at least 2 samples), cerebrospinal fluid, bronchoalveolar lavage is of diagnostic value. The sensitivity of the enzyme-linked immunosorbent assay is about 90%, the specificity is 90-99%; in allogeneic bone marrow recipients, these indicators are lower and 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 galactomancy in blood, it is ahead of the manifestations of invasive aspergillosis, determined by a computer study of the lungs, and in 70% it is ahead of the clinical symptoms of infection.
Diagnostic value of antigen detection test Aspergillus has in the event that the study is carried out repeatedly. Determination of Aspergillus antigen in the blood should be carried out in case of fever during treatment with broad-spectrum antibiotics in patients with neutropenia 2 times a week; with pneumonia that occurs or persists against the background of antibiotic therapy; upon detection of foci in the lung tissue (computed tomography).
The main methods in the etiological laboratory diagnosis of mycoses to this day continue to be classical methods, including microscopy of the material, isolation of a pure culture of the pathogen with its subsequent identification. Immunological diagnostic methods are of secondary importance. They are used to identify the most significant pathogens: pathogenic dimorphic fungi, as well as the causative agents of cryptococcosis, candidiasis and aspergillosis. Genoindication methods (polymerase chain reaction) also have limited application due to their lack of specificity, but are mainly used in the diagnosis of deep and opportunistic mycoses.
Material for research in laboratory diagnosis of mycoses, depending on the form of the disease, can be: skin and its appendages (hair, nails), discharge of wounds and fistulas, sputum, blood, cerebrospinal fluid and urine, tissue biopsies. The accuracy of taking pathological material is largely determined by the effectiveness of further laboratory research. In case of skin lesions, the material is selected most often by scraping or using adhesive tape (mainly for superficial forms) before treatment from fresh, but fully developed foci along the periphery, where the most viable pathogens are located.
When detecting dermatophyte fungi, hair is an extremely informative material, since the nature of the hair lesion makes it possible to identify the pathogen. In some cases, the use of a Wood lamp helps to determine the lesion focus. The affected hair, together with scales, is removed with epilation tweezers. In chronic black-point trichophytosis, the hair is removed from the skin with a dissecting needle.
The nails are taken along all the layers, cutting with a sharp scalpel or scissors. Selection using a dental drill is more efficient. In case of candidiasis lesions, the material is selected by scraping from the nail roller. The selected material is delivered to the laboratory in dark paper bags to avoid drying out and contamination with extraneous microflora. The material containing pus is delivered in sterile bacterial prints or Petri dishes. Sputum is collected in a sterile container. Sputum examination should be carried out no later than 2 hours after collection. When the study period is extended, the samples should be stored in a refrigerator at + 4 ° C, otherwise diagnostic errors associated with the appearance of pseudo mycelial forms of yeast cells may occur.
Blood and cerebrospinal fluid are collected aseptically. Sowing blood is carried out in the liquid medium of Sabouraud. To exclude contamination of blood samples, they are re-examined. The cerebrospinal fluid is centrifuged, and the sediment is used for microscopy and inoculation.
Urine is collected in the morning in a sterile container, while it is necessary to avoid contamination of the material by the microflora of the skin of the perineum. Urine culture is performed using quantitative culture methods.
In the diagnosis of mycoses, preparations are used both from native material and stained. When examining unstained preparations, the dense material is preliminarily clarified (macerated) with a 10-30% KOH solution (solutions of dimethyl sulfoxide can be used), and then microscoped using the “crushed drop” method. To give contrast, the preparations are stained with an aqueous solution of methylene blue.
Staining of fixed smears is carried out in various ways: according to Gram, methylene blue, according to Romanovsky - Giemsa. The latter method allows you to see small yeast cells and stages of phagocytosis. For cryptococci, use ink stain, Southgate mucicarmine stain. Melanin-containing mushrooms (dark colored), for example Cladophialophora bandana, stained by Mass - Fontana (color of melanin, which is part of the cell wall). Fluorescent sera are used in the diagnosis of pathogens of deep mycoses, pathogens of dermatophytosis, fungi of the genus Candida.
Isolation of a pure culture of the pathogen makes it possible to establish the generic and species affiliation of the fungus, to study its properties and sensitivity to antimicrobial drugs. For the isolation and identification of fungi, dense and liquid culture media of Sabouraud, wort-agar are used. These environments provide the growth of most pathogenic and opportunistic fungi. In addition, the Sabouraud environment (Fig. 8.2) stimulates the processes of pigmentation in fungi, which is important for identification.
To suppress the growth of contaminating bacteria, antibiotics are added to the media: chloramphenicol, streptomycin, penicillin, tetracycline. For the isolation of whimsical pathogenic fungi, media enriched with blood and cerebral heart extract are used. Special media are used for the cultivation and identification of fungi: Czapek's medium - for the formation of conidia when identifying molds, potato (potato-carrot) agar, rice agar - for identifying growth types and obtaining chlamydospores when identifying fungi of the genus Candida, carrot (vegetable) agar - for obtaining typical fungal culture colonies Trichophyton schonleini, Kashkin's medium - for the isolation of pigment-forming fungi. Differential diagnostic environments are also used, for example, to identify C. albicans - chromogenic media or media that allows you to determine the phospholipase activity of the fungus.
The incubation time of mushrooms ranges from several days to 1 month or more. If pathogenic dimorphic fungi are suspected, cultivation is carried out for 8 weeks to give a negative response.
Inoculations are carried out on media poured into test tubes, vials and Petri dishes. Inoculation of the material on dermatophyte fungi is carried out simultaneously on several test tubes with the medium. Materials suspected of having molds (sputum) are inoculated at 3 points and incubated at 28 and 37 ° C. To resolve the issue of the absence of contamination during the sampling of material, control air cultures are carried out for the content of mold fungi in the laboratory premises and wards where patients are located.
When cultivating fungi in a mycological laboratory, safety measures are observed, similar to those in bacteriological laboratories. In order to avoid allergization, the personnel carry out work with molds and mycotoxins in boxed rooms in gauze bandages or in table boxes with a hood. Work with cultures belonging to the II group of pathogenicity (pathogenic dimorphic fungi) is possible only in specialized laboratories.
Identification of fungal species is carried out on the basis of a complex of cultural, morphological and other characteristics. The first include the morphology of the colony, its color and size when cultivated on special media, the structure of the edge and center, the nature of the surface, the presence and nature of the reproductive organs, the second - the features of the microscopic structure of the mycelium, the structure, shape and size of the reproductive organs - conidiophores, conidia, chlamydospore, arthrospore, etc.
Fungi form various types of colonies on solid nutrient media. Scientists from the G. Venter Institute (USA), taking into account the ability of fungi to release pigment substances into the medium, created a "Christmas tree" in the nutrient medium (Fig. 8.3).
Rice. 8.3. "Christmas tree" of mushrooms (top: Talaromyces stipitatus; wood Aspergillus nidulans; decorations: Penicillium marneffei; stump: Aspergillus terreus)
The enzymatic and assimilative abilities of cultures are of great importance in the identification of yeast (yeast-like fungi). Currently, commercial test systems are used to identify common fungi - causative agents of human and animal diseases: BBL Mycotube, API 20C bio Merieux, etc.
Loading ...