Mycology Midterm PDF

General properties, virulence, pathogenesis and classification of pathogenic fungi Chapter One  Fungal Structure, Metabolism & Reproduction Fungi are eukaryotes with a higher level of biologic complexity than bacteria. They are spore-b...

General properties, virulence, pathogenesis and classification of pathogenic fungi Chapter One  Fungal Structure, Metabolism & Reproduction Fungi are eukaryotes with a higher level of biologic complexity than bacteria. They are spore-bearing, reproducing both sexually and asexually. Fungi may be unicellular or may differentiate and become multicellular by the development of long, branching filaments.  The fungal cell has many typical eukaryotic features, including a nucleus with a nucleolus, nuclear membrane, and linear chromosomes. The cytoplasm contains a cytoskeleton with actin microfilaments and tubulin-containing microtubules. Ribosomes and organelles, such as mitochondria, endoplasmic reticulum, and the Golgi apparatus, are also present. The fungal cell wall consists primarily of chitin (not peptidoglycan as in bacteria); thus fungi are insensitive to certain antibiotics, such as penicillins and cephalosporins, that inhibit peptidoglycan synthesis. Chitin is a polysaccharide composed of long chains of N-acetylglucosamine. The fungal cell wall contains other polysaccharides as well, the most important of which is β-glucan, a long polymer of d-glucose. The medical importance of β-glucan is that it is the site of action of the antifungal drug. The fungal cell membrane contains ergosterol, in contrast to the human cell membrane, which contains cholesterol. The selective action of amphotericin B and azole drugs, such as fluconazole and ketoconazole, on fungi is based on this difference in membrane sterols  There are two types of fungi: yeasts and molds. Yeasts grow as single cells that reproduce by asexual budding. Molds grow as long filaments (hyphae) and form a mat (mycelium). Some hyphae form transverse walls (septate hyphae), whereas others do not (nonseptate hyphae). Nonseptate hyphae are multinucleated (coenocytic). The growth of hyphae occurs by extension of the tip of the hypha, not by cell division all along the filament. Several medically important fungi are thermally dimorphic (i.e., they form different structures at different temperatures). They exist as molds in the environment at ambient temperature and as yeasts in human tissues at body temperature. These morphological transitions are often very important for the pathogenesis of human infections.  Many fungi, including the most common human fungal pathogen Candida albicans, display a striking ability to modify their cellular shape and structure in order to adapt to new environments. A distinct group of human pathogens are the thermally dimorphic fungi, shifting from yeast-like to hyphal growth based on temperature. These fungal species tend to grow in the mold form in their environmental reservoir as well as when incubated in culture at ambient temperatures. However, they convert to a yeast-like growth form in the mammalian host or when incubated in culture at 37°C. Dimorphism in fungi is reversible The importance of the dimorphism in fungal virulence has been demonstrated in several fungi, including C albicans and H capsulatum.  Most fungi are obligate aerobes; some are facultative anaerobes; but none are obligate anaerobes. All fungi require a preformed organic source of carbon—hence their frequent association with decaying matter. The natural habitat of most fungi is, therefore, the environment. An important exception is Candida albicans, which is part of the normal human flora. Some fungi reproduce sexually by mating and forming sexual spores (e.g., zygospores, ascospores, and basidiospores). Zygospores are single large spores with thick walls; ascospores are formed in a sac called ascus; and basidiospores are formed externally on the tip of a pedestal called a basidium. The classification of these fungi is based on their sexual spores. Most fungi of medical interest propagate asexually by forming conidia (asexual spores) from the sides or ends of specialized structures. The shape, color, and arrangement of conidia aid in the identification of fungi Some important conidia are: (1) arthrospores which arise by fragmentation of the ends of hyphae and are the mode of transmission (2) chlamydospores, which are rounded and thick-walled (3) blastospores, which are formed by the budding process by which yeasts reproduce asexually (some yeasts can form multiple buds that do not detach, thus producing sausage-like chains called pseudohyphae, which can be used for identification) (4) sporangiospores, which are formed within a sac (sporangium) on a stalk by molds  Virulence, pathogenesis and classification Approximately 80,000 species of fungi have been described, but only about 400 are medically important, and less than 50 are responsible for more than 90% of the fungal infections of humans and other animals. Rather, most species of fungi are beneficial to humankind. They reside in nature and are essential in breaking down and recycling organic matter. Some fungi greatly enhance our quality of life by contributing to the production of food, cheese and bread. Other fungi have served medicine by providing useful bioactive secondary metabolites, such as antibiotics (eg, penicillin) and immunosuppressive drugs (eg, cyclosporine).  The diseases caused by fungi are called mycoses. These infections vary greatly in their manifestations but tend to present with subacute or chronic features, often relapsing over time. Acute disease, such as that produced by many viruses and bacteria, is uncommon with fungal infections. Most pathogenic fungi are exogenous, their natural habitats being water, soil, and organic debris. The mycoses with the highest incidence— candidiasis and dermatophytosis—are caused by fungi that are frequent components of the normal human microbiota and highly adapted to survival on the human host.  Most fungi are opportunists, causing serious disease only in individuals with impaired host defense systems, or with altered normal floral community due to excessive broad-spectrum antibiotic intake. Fungal infections are acquired from the environment either through inhalation of infectious conidia generated from molds growing in the environment or direct contact with injured skin. The infection also could be endogenous in the few instances where they are members of the resident flora. Fungal diseases are generally not spread from person to person.  Compared with bacterial, viral, and parasitic disease, less is known about the pathogenic mechanisms and virulence factors involved in fungal infections. Most fungi are opportunists, causing serious disease only in individuals with impaired host defence systems, or with altered normal floral community due to excessive broad-spectrum antibiotic intake. Only a few fungi are able to cause disease in immunocompetent persons. Virulence factors have been known for fungi, including: The organism’s size (with inhalation, the organism must be small enough to reach the alveoli) The organism’s ability to grow at 37°C at a neutral pH Conversion of the dimorphic fungi from the mold form into the corresponding yeast form in the host Toxin production Fungal pathogenesis is similar to bacteria in terms of infection steps, including adherence to mucosal surfaces, invasiveness, extracellular products, interaction with phagocytes and the ability to cause local and invasive infections.  Fungal adherence Several fungal species, particularly the yeasts, are able to colonize the mucosal surfaces of the gastrointestinal and female genital tracts. The ability of fungal adherence to host cell surfaces is associated with colonization and virulence. Adherence usually requires a surface adhesin on the fungus and a receptor on the epithelial cell. A few binding mediators have been identified for other fungi, usually a surface mannoprotein.  Invasion Passing an initial surface barrier—skin, mucous membrane, or respiratory epithelium is an important step for most successful pathogens. Some fungi are introduced through mechanical breaks. Fungi that initially infect the lung must produce conidia small enough to be inhaled and minimize the upper airway defences. Dimorphic fungi from the environment completely change their morphology and growth to a more invasive form. Extracellular enzymes (eg, proteases, elastases) might be associated with the invasive forms of many of the dimorphic and other pathogenic fungi, and they may contribute to some aspect of invasion or spread.  Injury Several fungi do produce exotoxins, called mycotoxins, in the environment but not in vivo. The structural components of the cell do not cause effects similar to those of the endotoxin of Gram-negative bacteria The injury caused by fungal infections seems to be due primarily to the destructive aspects inflammatory and immunologic responses. only the most immunocompromised patients appear to have extensive injury due to direct fungal destruction of the surrounding tissue, such as neutropenic patients with invasive mold infections. In addition to mycotic infections, there are two other kinds of fungal disease: (1) mycotoxicoses, caused by ingested toxins, and (2) allergies to fungal spores. The best-known mycotoxicosis occurs after eating Amanita mushrooms. These fungi produce five toxins, two of which—amanitin and phalloidin—are among the most potent hepatotoxins. The toxicity of amanitin is based on its ability to inhibit cellular RNA polymerase, which prevents mRNA synthesis. Other ingested toxins, aflatoxins, are coumarin derivatives produced by Aspergillus flavus that cause liver damage and tumors in animals and are suspected of causing hepatic carcinoma in humans. Aflatoxins are ingested with spoiled grains and peanuts and are metabolized by the liver to the epoxide, a potent carcinogen. Aflatoxin B1 induces a mutation in the p53 tumor suppressor gene, leading to a loss of p53 protein and a consequent loss of growth control in the hepatocyte. Allergies to fungal spores, particularly those of Aspergillus, are manifested primarily by an asthmatic reaction (rapid bronchoconstriction mediated by IgE), eosinophilia, and a “wheal and flare” skin test reaction. These clinical findings are caused by an immediate hypersensitivity response to the fungal spores  Host Defense and Fungal Resistance Innate and adaptive immunity Healthy persons have effective innate immunity to most fungal infections, especially the opportunistic molds. This resistance is mediated by the professional phagocytes (neutrophils, macrophages, and dendritic cells), the complement system, and pattern recognition receptors. Important receptors recognizing fungal elements include a lectin- like structure on phagocytes (dectin-1) that binds glucan, and toll-like receptors (TLR2, TLR4). In most instances, neutrophils and alveolar macrophages are able to kill the conidia of fungi if they reach the tissues. The small number of species that are able to cause clinically apparent infection are usually cleared from the host, most often through a combination of the innate activity of neutrophils and through the development of an adaptive, TH1-mediated immune response. Life-threatening fungal infections are commonly associated with depressed or absent cellular immune responses. Humoral and Cellular Immunity Antifungal antibodies can be detected at some time during the course of almost all fungal infections, but the appearance of antibodies does not necessarily correlate with resistance. In some mycotic infections, high titers of specific fungal antibodies don’t act against the infection. In contrast, antibodies directed against other infections may actually contribute to the cell-mediated clearance of the infection from the site of infection. Antibody may also play a role in control some mycotic infections by enhancing fungus–phagocyte interactions. Cellular Immunity Considerable clinical and experimental evidence points toward the importance of cellular immunity in the resolution of fungal infections. Most patients with invasive mycoses have neutropenia, defects in neutrophil function, or depressed TH1 immune responses.  Dimorphic fungi resist killing by phagocytes possibly because of a change in their size surface structures. Dimorphic fungi are able to bind complement components in a way that interferes with phagocytosis. Other pathogenic fungi contain a component in the wall of its conidia that is antiphagocytic. Some fungi produce substances such as melanin, which interfere with oxidative killing by phagocytes. The tissue yeast form of certain fungi multiplies within macrophages by interfering with lysosomal killing mechanisms in a manner similar to that of some bacteria. Finally, some fungi possess polysaccharide capsule that has antiphagocytic properties similar to those of encapsulated bacterial pathogens. Laboratory Diagnosis of fungal Infections Chapter 2  Collection and transportation of clinical specimens The diagnosis of fungal infections depends entirely on the selection and collection of an appropriate clinical specimen for microscopic analysis and culture. Many fungal infections are similar clinically to mycobacterial infections, and often the same specimen is cultured for both fungi and mycobacteria. Many infections have a primary focus in the lungs; respiratory tract secretions are almost always included among the specimens selected for culture. Proper collection of specimens and rapid transport to the clinical laboratory are crucial to the recovery of fungi. Specimens often contain not only the etiologic agent, but also contaminating bacteria or fungi that rapidly overgrow some of the slower-growing pathogenic fungi.  Specimens of hair, skin scrapings or biopsies, and nail clippings are usually submitted for dermatophyte culture and are contaminated with bacteria or rapidly growing fungi or both. Samples collected from lesions may be obtained by scraping the skin or nails with a scalpel blade or microscope slide; infected hairs are removed by plucking them with forceps. Only the leading edge of skin lesions should be sampled, because the centers often contain nonviable organisms. These specimens should be placed in a sterile container; they should not be refrigerated. Blood cultures for fungi is performed via automated blood culture systems for the recovery of yeasts  Vaginal samples should be transported to the laboratory within 24 hours of collection using culture transport swabs. Swabs should be kept moist in sterile tubes. This method of collection provides a specimen suitable for a wet preparation. Both selective and inhibitory agars should be plated. Vaginal cultures should be screened for yeasts and incubated at 30°C for 7 days. Urine samples collected for fungal culture should be processed as soon after collection as possible. The 24-hour urine sample is unacceptable for culture. The usefulness of quantitative cultures is undetermined. All urine samples should be centrifuged and the sediment cultured using a loop to provide adequate isolation of colonies. Routinely, yeasts are diagnosed in urine and vaginal samples accidently no special physician order is requested  Laboratory Diagnosis & Management Laboratory safety precautions followed in mycology laboratories are similar to those applied in any microbiology sections, with paying more attention toward the ability fungal infective spores spreading in the surround and being inhaled by the technicians. Without exception, mold cultures and clinical specimens must be handled in a class II biosafety cabinet.  In general, mycology laboratory workup is much related to bacteriology than virology laboratories; moreover, conventional staining and culturing on agar media are routinely performed with minimal load of sero and molecular diagnostics. There are four approaches to the laboratory diagnosis of fungal diseases: (1) Direct microscopic examination (2) Culture of fungi (3) DNA probe tests (4) Serologic tests.  Direct microscopic examination Fungi often demonstrate distinctive morphologic features on direct microscopic examination of infected pus, fluids, or tissues owing to their large size. Direct microscopic examination of clinical specimens such as sputum, lung biopsy material, and skin scrapings depends on finding characteristic spores, hyphae, or yeasts in the light microscope. Traditionally the potassium hydroxide (10% KOH) preparation has been the recommended method for direct microscopic examination of specimens. However, the calcofluor white stain now is believed to be superior. Slides prepared by this method may be observed using fluorescent or bright-field microscopy, as is used for the potassium hydroxide preparation.  10% KOH examination To visualize the fungal element in the clinical specimens and to examine skin scrapings or flakes, nail, tissues and hair for the presence of hyphae and arthroconidia in suspected dermatophyte infections. Potassium hydroxide (KOH) can be used on clinical specimens to clear cellular material and for better visualization of fungal elements. KOH preparation is a commonly used method for the diagnosis of superficial fungal infections and for the rapid detection of fungal elements in a clinical specimen. KOH is a strong alkali.  KOH separates the fungal elements from intact cells as it digests the protein debris and dissolves cement substances that hold the keratinized cells together surrounding the fungi so that the hyphae and conidia (spores) of fungi can be seen under the microscope. The specimen is placed in a few drops of 10% to 20% KOH and incubated for 5 to 10 minutes where gentle heating can clear samples more quickly. A coverslip is placed over the KOH-digested sample, and the slide is examined microscopically without staining. Different fungal elements like hyphae, pseudohyphae, yeast cells, spores, spherules, and sclerotic bodies can be seen clearly in a KOH wet mount. Usually, the results are reported as positive or negative. Slide method Place the specimens like epidermal scales, nail, hair, skin scraping or tissue on a clean glass slide. Pour a drop of 10% KOH on the specimen and place a coverslip over it. Heat the slide gently over the flame. Leave the slide for 5-10 minutes or place the slide in a petri dish, or other containers with a lid, together with a damp piece of filter paper or cotton wool to prevent the preparation from drying out. Examine the slide under the microscope using 10X and 40X objectives. Tube test Place the homogenized tissue material in a test tube and add 10% KOH. Incubate the tube overnight at 37°C. Following incubation, place a drop of suspension in the clean slide and cover with a coverslip. Examine the slide under the microscope in 10X and 40X objectives.  Tube method: This procedure can also be used for nail clippings and skin biopsies which dissolve with difficulty and the concentration of KOH may be increased.  Limitations Potassium hydroxide is a highly corrosive deliquescent chemical; therefore it should be handled with great care. Experience required since background artifacts are often confusing. Clearing of some specimens like biopsy material, nail clippings may require extended time.  Culture of fungi The most commonly used medium for cultivating fungi is Sabouraud’s dextrose agar, which contains glucose and peptones as nutrients. Its pH is 5.6, which is optimal for growth of fungi and inhibit bacteria. Blood agar or another enriched bacteriologic agar medium is used when pure cultures would be expected. However, selective agar is used for non-sterile specimens Plates of fungal cultures that are suspected of being pathogens should be placed in the incubator on their but with the cover up, this will keep fungal spores in the plate, otherwise they will be dropped on the cover entire and being risk for contamination. In addition, agar tends to dehydrate during the extended incubation period required for fungal recovery, but this problem can be minimized by using culture dishes containing at least 40 mL of agar and placing them in a humidified incubator. Also fungal plates should be sealed with tape or parafilm to prevent laboratory contamination and to keep the culture wet and should be autoclaved as soon as the definitive identification is made.  In contrast to most pathogenic bacteria, many fungi grow best at 25°C to 30°C, and temperatures in this range are used for primary isolation. Paired cultures incubated at 30°C and 35°C may be used to demonstrate dimorphism. Once a fungus is isolated, identification procedures depend on whether it is a yeast or a mold. Culture for fungi are usually incubated for 7 to 14 days. Yeasts are identified by biochemical tests analogous to those used for bacteria. The ability to form pseudohyphae is also taxonomically useful among the yeasts. Molds are most often identified by the morphology of their conidia and conidiophores. Other features such as the size, texture, and color of the colonies help characterize molds.  Microscopic fungal morphology is usually demonstrated by methods that allow in situ microscopic observation of conidia and their shape and arrangement. Lactophenol cotton blue and methylene blue are dyes that stain the hyphae, conidia, and spores. The significance of fungal growth should be decided before complete the identification process. Rapid-growing fungi, environmental fungi and unexpected fungi all must be excluded and must be considered as contamination. The knowledge about expected fungi-sample type would be helpful for correct diagnosis. After decision is made, stained slide and growth morphology of fungi must be correlated together using Mycology atlas. Usually the result is reported at Genus level.  Molecular Assays Tests involving DNA probes can identify colonies growing in culture at an earlier stage of growth than can tests based on visual detection of the colonies. As a result, the diagnosis can be made more rapidly. At present, DNA probe tests are available for Coccidioides, Histoplasma, Blastomyces, and Cryptococcus.  Immunoassays Tests for the presence of antibodies in the patient’s serum or spinal fluid are useful in diagnosing systemic mycoses but less so in diagnosing other fungal infections. A significant rise in the antibody titer must be observed to confirm a diagnosis. The complement fixation test is most frequently used in suspected cases of certain fungal infections. In addition, the presence of the polysaccharide capsular antigens can be detected by the latex agglutination test.