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Laboratory diagnostics of mycosis

General Characteristics of Fungi   Microbiologists use the term fungus [pl., fungi; Latin fungus, mushroom] to include eukaryotic, spore-bearing organisms with absorptive nutrition, no chlorophyll, and that reproduce sexually and asexually. Scientists who study fungi are mycologists [Greek mykes, mushroom, and logos, discourse], and the scientific discipline dealing with fungi is called mycology. The study of fungal toxins and their effects is called mycotoxicology, and the diseases caused by fungi in animals are known as mycoses [s., mycosis]. The five-kingdom system places the fungi in the kingdom Fungi. Fungi are eukaryotic microorganisms (domain eucarya) that occur ubiquitously in nature. Only about 200 of the thousands of species have been identified as human pathogens, and among these known pathogenic species fewer than a dozen are responsible for more than 90% of all human fungal infections. The basic morphological element of filamentous fungi is the hypha and a web of intertwined hyphae is called a mycelium. The basic form of a unicellular fungus is the yeast cell. Dimorphic fungi usually assume the form of yeasts in the parasitic stage and the form of mycelia in the saprophytic stage. The cell walls of fungi consist of nearly 90% carbohydrate (chitin, glucans, mannans) and fungal membranes are rich in sterol types not found in other biological membranes (e.g., ergosterol). Filamentous fungi reproduce either asexually (mitosis), by hyphal growth and tip extension, or with the help of asexual spores. Yeasts reproduce by a process of budding. Sexual reproduction (meiosis) on the other hand, produces sexual spores. Fungi imperfecti or deuteromycetes are the designation for a type of fungi in which the fructification forms are either unknown or missing entirely.
Mycosis A fungal infection in or on a part of the body.
Morphology of Fungi     Two morphological forms of fungi are observed: - Hypha: this is the basic element of filamentous fungi with a branched, tubular structure, 2–10 µm in width. Mycelium: this is the web or matlike structure of hyphae. Substrate mycelia (specialized for nutrition) penetrate into the nutrient substrate, whereas aerial mycelia (for asexual propagation) develop above the nutrient medium. Fungal thallus: this is the entirety of the mycelia and is also called the fungal body or colony. - Yeast is the basic element of the unicellular fungi. It is round to oval and 3 – 10 µm in diameter. Several elongated yeast cells chained together and resembling true hyphae are called pseudohyphae. Some fungal species can develop either the yeast or the mycelium form depending on the environmental conditions. This property called dimorphism. Dimorphic pathogenic fungi take the form of yeast cells in the parasitic stage and appear as mycelia in the saprophytic stage.
Structure of Fungi   The body or vegetative structure of a fungus is called a thallus [pl., thalli]. It varies in complexity and size, ranging from the single-cell microscopic yeasts to multicellular molds, macroscopic puffballs, and mushrooms. The fungal cell usually is encased in a cell wall of chitin. Chitin is a strong but flexible nitrogen-containing polysaccharide consisting of N-acetylglucosamine residues. Yeast is a unicellular fungus that has a single nucleus and reproduces either asexually by budding and transverse division or sexually through spore formation. Each bud that separates can grow into new yeast, and some group together to form colonies. Generally, yeast cells are larger than bacteria, vary considerably in size, and are commonly spherical to egg shaped. They have no flagella but do possess most of the other eukaryotic organelles.
Reproduction in Fungi   Asexual reproduction includes the vegetative propagation of hyphae and yeasts as well as vegetative fructification, i.e., formation of asexual spores. Asexual reproduction is accomplished in several ways: 1. A parent cell can divide into two daughter cells by central constriction and formation of a new cell. 2. Somatic vegetative cells may bud to produce new organisms. This is very common in the yeasts. 3. The most common method of asexual reproduction is spore production. Asexual spore formation occurs in an individual fungus through mitosis and subsequent cell division. There are several types of asexual spores: a. A hyphae can fragment (by the separation of hyphae through splitting of the cell wall or septum) to form cells that behave as spores. These cells are called arthroconidia or arthrospores. b. If the cells are surrounded by a thick wall before separation, they are called chlamydospores. c. If the spores develop within a sac [sporangium; pl., sporangia] at a hyphal tip, they are called sporangiospores. d. If the spores are not enclosed in a sac but produced at the tips or sides of the hypha, they are termed conidiospores. e. Spores produced from a vegetative mother cell by budding are called blastospores. Sexual reproduction in fungi perfecti (eumycetes) follows essentially the same patterns as in the higher eukaryotes. The nuclei of two haploid partners fuse to form a diploid zygote. The diploid nucleus then undergoes meiosis to form the haploid nuclei, finally resulting in the haploid sexual spores: zygospores, ascospores, and basidiospores. Sexual spores are only rarely produced in the types of fungi that parasitize human tissues. Sexual reproduction structures are either unknown or not present in many species of pathogenic fungi, known as fungi imperfecti (deuteromycetes).
General Aspects of Fungal Disease Fungal diseases fall into four clinical patterns: superficial infections on surface epithelial structures (skin, hair, nails), systemic infections of deep tissues, and subcutaneous and opportunistic infections, mycotoxicoses (aflatoxicosis).
Aspect of Laboratory Diagnosis   The laboratory diagnosis of a fungal infection depends on the direct microscopic detection of fungal structures in clinical samples and/or the recovery in culture and subsequent identification of the fungus. Fungi may be isolated from a variety of clinical specimens representing the focus of infection (sputum, spinal fluid, tissue, pus aspirated from lymph nodes or other lesion, bone marrow aspirates, skin scrapings). All specimens of sufficient quantity submitted for fungal culture should be examined microscopically for fungi. When there is not sufficient specimen to allow both a culture and direct microscopic examination, the culture has priority over the smear because culture is more sensitive than microscopic examination. However, observing a fungus in a clinical specimen is often valuable in establishing the significance of the fungus (i.e., ruling out contamination) and in providing early information that may be crucial for determining appropriate patient therapy. In general, serological tests have limited application for the diagnosis of most fungal infections. Exceptions to this rule include certain dimorphic fungal diseases, such as histoplasmosis and coccidioidomycosis. The purpose of this laboratory exercise is to acquaint the student with some direct microscopic and cultural methods that are available for establishing the laboratory diagnosis of a human mycosis. Histopathology. The visualization of fungal structures (hyphae, conidia, etc.) in tissue obtained by biopsy or at autopsy. Specialized tissue stains such as Giemsa, methenamine silver or mucicarmine may be used to facilitate the detection of the fungus in tissue. The particular fungal structures that are seen in tissue can sometimes confirm the identity of the fungus (e.g., spherules [the yeast form] of Coccidioides immitis or cysts of neumocystis carinii) or suggest the presence of a particular fungal group. In this latter case, culture is used to confirm the presence and identity of the fungal pathogen. Direct smears of patient material other than tissue are often made to detect the resence of fungal elements microscopically. Several types of stains or reagents are used to facilitate the detection of certain fungi. 1. 10% KOH are used to examine a variety of clinical samples including hair, nails, skin scrapings, fluids, or exudates. The potassium hydroxide solution serves to clear away tissue cells and debris, making the fungi more prominent. Slides must be examined with reduced illumination to allow fungal structures to be seen. 2. Calcofluor white is used with most specimen types to detect the presence of fungi by fluorescence microscopy. The cell walls of the fungi bind the stain and fluoresce blue-white or apple green, depending on the filter combination used with the microscope. This stain is useful for examining skin scrapings for the presence of dermatophytes and tissues and body fluids for yeast and filamentous fungi. 3. India ink is usually ordered to screen for the presence of Cryptococcus neoformans in spinal fluid samples. This yeast is encapsulated, and the capsule can be visualized readily against the black background of the India ink as a clear halo surrounding the yeast cell. The India ink test is very insensitive (detecting only 40% of cases of cryptococcal meningitis) and therefore has been superseded by other tests, such as the cryptococcal antigen latex agglutination test, which detects more than 90% of cases of cryptococcal meningitis. The India ink test is rarely performed in clinical microbiology laboratories. 4. Wright, Giemsa, or Diff-Quik stains. These specialized stains are often used on blood and bone marrow smears to look for intracellular yeast forms of Histoplasma capsulatum. 5. Most fungi are not stained well by the Gram-stain procedure, and therefore, it is of limited use when examining specimens for fungal forms. It is generally reliable only for detecting the presence of Candida species, Sporothrix schenkii, and perhaps a few other fungi in clinical material. In Gram-stained spinal fluid specimens, Cryptococcus neoformans may appear as irregularly staining gram-positive yeast cells surrounded by an orange capsule. Culturing is possible on universal and selective mediums. Sabouraud dextrose agar can contain selective agents (e.g., chloramphenicol and cycloheximide), this medium has an acid pH of 5.6. The main identifying structures are morphological, in particular the asexual and, if present, sexual reproductive structures. Biochemical tests are used mainly to identify yeasts and are generally not as important in mycology as they are in bacteriology. Serology is using for identification of antibodies to special fungal antigens in patient’s serum. The Interpretation of serological findings is quite difficult in fungal infections. Antigen detection is using for finding of specific antigens in the diagnostic material by direct means using known antibodies, possible in some fungal infections (e.g., cryptococcosis). Cutaneous (allergy) tests with specific fungal antigens can be useful in diagnosing a number of fungal infections. Nucleic acid detection. Combined with amplification, such tests are useful for rapid detection of mycotic diseases in immunocompromised patients.
Histoplasmosis Histoplasmosis is caused by Histoplasma capsulatum var. capsulatum, a facultative parasitic fungus that grows intracellularly. It appears as a small budding yeast in humans and on culture media at 37°C. At 25°C it grows as a mold, producing small microconidia (1 to 5 µm in diameter) that are borne singly at the tips of short conidiophores. Large macroconidia or chlamydospores (8 to 16 µm in diameter) are also formed on conidiophores. In humans the yeastlike form grows within phagocytic cells. H. capsulatum var. capsulatum is found as the mycelial form in soils throughout the world and is localized in areas that have been contaminated with bird or bat excrement.
Pathogenesis and clinical picture of Histoplasmosis The natural habitat of H. capsulatum is the soil. Spores (conidia) are inhaled into the respiratory tract, are taken up by alveolar macrophages, and become yeast cells that reproduce by budding. Small granulomatous inflammatory foci develop. The pathogens can disseminate hematogenously from these primary infection foci. The reticuloendothelial system (RES) is hit particularly hard. Lymphadenopathies develop and the spleen and liver are affected. Over 90% of infections remain clinically silent. The clinical picture depends heavily on any predisposing host factors and the infective dose. A histoplasmosis can also run its course as a respiratory infection only. Disseminated histoplasmoses are also observed in AIDS patients. Histoplasmosis is a disease of the monocyte-macrophage system; thus, many organs of the body can be infected. More than 95% of “histo” cases have either no symptoms or mild symptoms such as coughing, fever, and joint pain. Lesions may appear in the lungs and show calcification; most infections resolve on their own. Only rarely does the disease disseminate.
Diagnosis of Histoplasmosis Suitable material for diagnostic analysis is bronchial secretion, urine, or scrapings from infection foci. For microscopic examination, Giemsa or Wright staining is applied and yeast cells are looked for inside the macrophages and polymorphonuclear leukocytes. Cultures on blood or Sabouraud agar must be incubated for severalweeks. Laboratory diagnosis is accomplished by complement fixation tests and and agar gel precipitation. Most individuals with this disease exhibit a hypersensitive state that can be demonstrated by the histoplasmin skin test.
Epidemiology, therapy and prevention Histoplasmosis Histoplasmosis is endemic to the midwestern USA, Central and South America, Indonesia, and Africa. Prevention and control involve wearing protective clothing and masks before entering or working in infested habitats. Soil decontamination with 3 to 5% formalin is effective where economically and physically feasible. The most effective treatment is with amphotericin B, ketoconazole, or itraconazole.
Coccidioidomycosis   Coccidioidomycosis, also known as valley fever, San Joaquin fever, or desert rheumatism because of the geographical distribution of the fungus, is caused by Coccidioides immitis. C. immitis exists in the dry, highly alkaline soils of North, Central, and South America.
Morphology and culture C.immitis C. immitis is an atypical dimorphic fungus. In cultures, this fungus always grows in the mycelial form; in body tissues, however, it neither buds nor producesmycelia. What is found in vivo are spherical structures (spherules) with thick walls and a diameter of 15–60 µm, each filled with up to 100 spherical-to-oval endospores. C. immitis is readily cultivated on the usual fungus nutrient mediums. After five days of incubation, a white, wooly (fuzzy) mycelial colony is observed. One of the morphological characteristics of the mycelium is the asexual arthrospores seen as separate entities among the hyphae.
Pathogenesis and clinical picture of Coccidioidomycosis   The infection results from inhalation of dust containing arthrospores. Primary coccidioidomycosis is always localized in the lungs, whereby the level of manifestation varies from silent infections to severe pneumonia. Five percent of those infected develop a chronic cavernous lung condition. In fewer than 1%, hematogenous dissemination produces granulomatous lesions in skin, bones, joints, and meninges. Most cases of coccidioidomycosis are asymptomatic or indistinguishable from ordinary upper respiratory infections. Almost all cases resolve themselves in a few weeks, and a lasting immunity results. A few infections result in a progressive chronic pulmonary disease. The fungus also can spread throughout the body, involving almost any organ or site.
Diagnosis of Coccidioidomycosis The available tools are pathogen detection in sputum, pus, cerebrospinal fluid or biopsies, aspirates and antibody identification. The spherules can be seen under the microscope in fresh material. Culturing clinical samples in the presence of penicillin and streptomycin on Sabouraud agar also is diagnostic. The fungus can be readily cultured on Sabouraud agar at 25°C. The resulting arthrospores are highly infectious and must be handled very carefully. Newer methods of rapid confirmation include the testing of supernatants of liquid media cultures for antigens, serology (the complement fixation test, gel precipitation or latex agglutination), and skin testing.
Therapy and Prevention of Coccidioidomycosis Amphotericin B can be used to treat the disseminated forms. An oral azole derivative will serve as an alternative, or for use, in clinically less severe forms: miconazole (Lotrimin), itraconazole, ketoconazole, and amphotericin B are the drugs of choice for treatment. Prevention involves reducing exposure to dust (soil) in endemic areas.
Cryptococcosis   Cryptococcosis is a systemic mycosis caused by Cryptococcus neoformans. This fungus always grows as large budding yeast. In the environment C. neoformans is a aprophyte with a worldwide distribution. Aged, dried pigeon droppings are an apparent source of infection. Cryptococcosis is found in approximately 15% of AIDS patients. The fungus enters the body by the respiratory tract, causing a minor pulmonary infection that is usually transitory. Some pulmonary infections spread to the skin, bones, viscera, and central nervous system. Once the nervous system is involved, cryptococcal meningitis usually results. Diagnosis is accomplished by detection of the thick-walled spherical yeast cells in pus, sputum, or exudate smears using India ink to define the organism. The fungus can be easily cultured on Sabouraud dextrose agar. Identification of the fungus in body fluids is made by immunologic procedures. Treatment includes amphotericin B or itraconazole. There are no preventive or control measures.
Opportunistic Mycoses - Candidiasis   Candidiasis is the mycosis caused by the dimorphic fungus Candida albicans, C. parapsilosis, C. tropicalis, C. guillermondii, C. kruzei, and a few other rare Candida species. In contrast to the other pathogenic fungi, C. albicans is a member of the normal microbiota within the gastrointestinal tract, respiratory tract, vaginal area, and mouth.
Morphology and culture of Candida spp. Gram staining of primary preparations reveals Candida spp. to be a Gram-positive, budding, oval yeast with a diameter of approximately 5 µm. Gram-positive pseudohyphae are observed frequently and septate mycelia occasionally. Candida spp. can be grown on the usual culture mediums. After 48 hours of incubation on agar mediums, round, whitish, somewhat rough-surfaced colonies form. They are differentiated from other yeasts based on morphological and biochemical characteristics.
Pathogenesis and clinical pictures of Candidiasis Candida is a normal inhabitant of human and animal mucosa (commensal). Candida infections must therefore be considered endogenous. Candidiasis usually develop in persons whose immunity is compromised, most frequently in the presence of disturbed cellular immunity. The mucosa is affected most often, less frequently the outer skin and inner organs (deep candidiasis). Oral candidiasis or thrush is a common disease in newborns. It is seen as many small white flecks that cover the tongue and mouth. At birth, newborns do not have a normal microbiota in the oropharyngeal area. If the mother vaginal area is heavily infected with C. albicans, the upper respiratory tract of the newborn becomes colonized during passage through the birth canal. Thrush occurs because growth of C. albicans can not be inhibited by the other microbiota. Once the newborn has developed its own normal oropharyngeal microbiota, thrush becomes uncommon. Paronychia and onychomycosis are associated with Candida infections of the subcutaneous tissues of the digits and nails, respectively. These infections usually result from continued immersion of the appendages in water. Intertriginous candidiasis involves those areas of the body, usually opposed skin surfaces, that are warm and moist: axillae, groin, skin folds. Napkin (diaper) candidiasis is typically found in infants whose diapers are not changed frequently and therefore are not kept dry. Candidal vaginitis can result from diabetes, antibiotic therapy, oral contraceptives, pregnancy, or any other factor that compromises the female host. Normally the omnipresent lactobacilli (Döderlein’s bacilli) can control Candida in this area by the low pH they create. However, if their numbers are decreased by any of the aforementioned factors, Candida may proliferate, causing a curdlike, yellow-white discharge from the vaginal area. Candida can be transmitted to males during intercourse and lead to balanitis; thus it also can be considered a sexually transmitted disease. Balanitis is a Candida infection of the male glans penis and occurs primarily in uncircumcised males. The disease begins as vesicles on the penis that develop into patches and are accompanied by severe itching and burning.
Diagnosis of Candidiasis Diagnosis of candidiasis is difficult because (1) this fungus is a frequent secondary invader in diseased hosts, (2) a mixed microbiota is most often found in the diseased tissue, and (3) no completely specific immunologic procedures for the identification of Candida currently exist.
Criteria of diagnostics of Candidiasis - An exposure of exciter is in sterile liquids (blood, spinal fluid), punctuate of the closed cavities (pleura cavity). - A discovery in pathological material of the mycelium or pseudomicelium is. - The repeated excretions of the same type of fungus are in great numbers from the hearths of defeat (mucous membranes, skin and its appendages; abscesses). - A discovery in urine Candida in high concentrations (≥104 cell in 1 ml) is. - A discovery in the serum of antigens of fungi is.
Therapy of Candidiasis There is no satisfactory treatment for candidiasis. Cutaneous lesions can be treated with topical agents such as sodium caprylate, sodium propionate, gentian violet, nystatin, miconazole, and trichomycin. Ketoconazole, amphotericin B, fluconazole, itraconazole, and flucytosine also can be used for systemic candidiasis.
Pneumocystosis   The disease that this protist causes, pneumocystis pneumonia or Pneumocystis carinii pneumonia (PCP), occurs almost exclusively in immunocompromised hosts. Extensive use of immunosuppressive drugs and irradiation for the treatment of cancers and following organ transplants accounts for the formidable prevalence rates noted recently. This pneumonia also occurs in more than 80% of AIDS patients. Both the organism and the disease remain localized in the lungs—even in fatal cases. Within the lungs, Pneumocystis causes the alveoli to fill with a frothy exudate.
Morphology and developmental cycle of the P. carinii. Three developmental stages are known for P. carinii. The trophozoites are elliptical cells with a diameter of 1.5–5 µm. Presumably, the trophic form reproduces by means of binary transverse fission, i.e., asexually. Sexual reproduction does not begin until two haploid trophozoites fuse to make one diploid sporozoite (or precyst), which are considered to be an intermediate stage in sexual reproduction. After further nuclear divisions, the sporozoites possess eight nuclei at the end of their development. The nuclei then compartmentalize to form eight spores with a diameter of 1–2 lm each, resulting in the third stage of development, the cyst. The cysts then release the spores, which in turn develop into trophozoites.
Diagnosis of pneumocystis pneumonia Laboratory diagnosis of pneumocystis pneumonia can be made definitively only by microscopically demonstrating the presence of the microorganisms in infected lung material or by a PCR analysis.

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Date: 2016-01-14; view: 597

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