Fungi (fungi) are widely found in nature. Although approximately 400,000 species have been described to date, only 100-150 of them can cause infection in humans. Diseases caused by fungi are called mycosis. The branch of science that studies fungi and fungal diseases is called mycology.
Fungi are microorganisms with a eukaryotic structure. Since they do not contain chlorophyll, they cannot perform photosynthesis and differ from plants with this feature. They are facultative anaerobes or obligate aerobes. Fungi are heterophilic microorganisms, meaning they use organic compounds as carbon and energy sources. They digest foods by releasing hydrolytic enzymes into the external environment and take the digested nutrients into the cells through absorption.
Some mushrooms have capsules and have a polysaccharide structure. Capsule polysaccharide consists of at least three different polymers: glucuronoxylomannan, galactoxylomannan and mannoprotein. In fungi, 90% of the cell wall consists of polysaccharide polymers such as chitin, glucan, mannan and chitosan, and 10% consists of proteins, glycoproteins and lipids. The type and amount of polysaccharides varies depending on the mushroom species. The cell wall gives the cell its shape, protects it from osmotic shock, is antigenic and has physiological activity because it contains some enzymes. The cell membrane is a bilayer structure composed of phospholipids, proteins and sterols, similar to mammalian membranes. Unlike the mammalian membrane, it contains ergosterol and zymosterol instead of cholesterol. The functions of the cell membrane include protecting the cytoplasm, regulating substance exchange, and helping capsule and wall synthesis. The target site of most of the antifungal group drugs used today for therapeutic purposes is ergosterol, located in the membrane.
Fungal spores are responsible for reproduction. Reproduction occurs through sexual spores (zygospore, ascospore, basidiospore), asexual spores (blastospore, arthrospore, chlamydospore, macroconidium, microconidium, sporangiospore) or parasexually. The scientific classification of fungi is based on sexual spores. According to their morphological appearance; It is divided into two: yeasts (Candida, Saccharomyces, etc.) and molds (Ascomycetes, Zygomycetes, Deuteromycetes, etc.).
Diagnostic Tests
Isolation and identification of a wide variety of fungi are performed by applying culture-based and non-culture-based (serological, molecular) methods. Before starting antifungal treatment, clinical samples should be collected under aseptic conditions, not placed in formalin, and delivered to the laboratory in sterile containers with the carrier medium most suitable for the material.
Due to the long time for culture results to be obtained, direct microscopic evaluation is of great importance. Clinical samples sent to the laboratory are first evaluated macroscopically, and initial information about the presence of yeast or mold is obtained in the microscopic evaluation. In direct microscopic examination, KOH is used at a rate of 10-20-30%, generally 30%. Skin and nail scrapings, feathers, hairs, wool, pieces of skin, etc. KOH is added to the collected materials to dissolve the chitin layer and reveal the fungal hyphae. For stained microscopic examination, dyes such as gram stain, lactophenol cotton blue, calcofluor white, India ink, Grocott-Gomori matamine silvering, pyrodic acid-Schiff (PAS), Giemsa, Wright and Mason-Fontana are used. Wood's light can also be used to search for fungi on infected surfaces. It is ultraviolet A ray passing through a nickel oxide filter. Under this light, some fungi can be identified by giving colors such as pale yellow, green, yellow fluorescence, and coral red fluorescence.
Culture is a more specific diagnostic method than direct microscopy. It is applied when a definitive diagnosis cannot be made or to determine the type of fungi. In culture evaluation, the morphology of the colonies and the resulting pigment color are the priority. The media used vary depending on the clinical sample taken. Culture media are divided into primary isolation purposes and specific purposes.
Primary isolation purposes;
- Sabauraud-dextrose-agar (SDA) and Brain heart infusion agar. (BHI); It is used for primary isolation of saprophytic and pathogenic fungi. For samples taken from non-sterile environments, SDA and BHI with antibiotics are used. They reproduce in 1-4 weeks at room temperature and 37°C and form colonies that are generally off-white or cream colored, have a soft consistency and are typically yeasty and smelly.
-Inhibitor mold agar and Yeast extract phosph. horse agar; Primary isolation of pathogenic fungi.
Specific purposes;
-Corn flour jelly with Tween 80 and trypan blue (MUJ); Chlamydospore diagnosis of Candida albicans,
-Cotton seed transformation agar; Transformation of Blastomyces dermatitidis from mold phase to yeast phase,
-Czapek's agar; Aspergillus isolation,
Bird seed agar (Staib agar); Cryptococcus neoformas isolation,
-CHROMagar Candida; Isolation of Candida species based on enzymatic reaction formation,
-Yeast fermentation broth; Fermentation properties of yeasts,
-Yeast nitrogen agar; Carbohydrate assimilation properties of yeasts.
In addition, BACTEC and BacT/Alert methods accelerate the detection of fungi in blood cultures.
Germ -Tube Test; In this test, germ tube formation from cells is sought. A light suspension of yeast-like microorganisms is made in 0.5-1.0 ml of sterile serum and incubated at 37°C for 2-4 hours. A drop of the mixture is placed on the slide and covered with a coverslip. It is investigated under the microscope whether the germ tube has formed from the cells. This situation is characteristic of C. albicans.
Tests to detect fungal antigens or metabolites in serum or body fluids are more valuable for the serological diagnosis of systemic fungal infections. Antibodies can be detected by latex agglutination (LA), ELISA, Enzyme Immuno Assay (EIA), Radioimmuno Assay (RIA), immunoelectrophoresis (IE) and immunodiffusion (ID) methods. With these methods, antigens in the cell structure or metabolites; mannan, D-arabinitol, (1-3)-β-glucan, polysaccharide glucoroxylomannan, galactomannan and enolase can be detected.
Although molecular diagnostic methods are increasingly popular, their application as a routine method in every clinical laboratory is due to economic reasons and It is not common due to lack of standardization. Signal amplification methods using nucleic acid amplification and hybridization probes are used to detect and identify infectious fungi. Nucleic acid amplification technologies use polymerase chain reaction (PCR) or similar methods. The fungal load is very low and culture and serological diagnosis are not yet available. It can be said that PCR-based diagnostic methods will be much more effective in the insufficient phase. Reverse transcriptase (RT) PCR aims to amplify the target RNA.
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