TRACING THE CRIMINAL Part ten: Criminals – fungi Institute for microbiology shows Instead of an introduction Survey of topics Characteristics of fungi Common characteristics of fungi Fungi and health Some toxical mushrooms Medically important fungi Examples of mycotic diseases Story one n Mrs. Udder came to dpt. of professional medicine with „fungus“ on her hand. n She wanted her disease to be accepted as proffessional disease, as she works with cattle on a farm. Unfortunatelly for mrs. Udder, the causative agent of the disease was found to be Epidermophyton floccosum, that is supposed to by rather antropofilic species of so named dempatophyta. So it is transmitted rather person-to-person than from animals. Dermatofytes n So named dermatophytes belong among the most common agents of skin mycoses (including mycoses of skin adnesa, i. e. hair, hairs and nails) n Among dermatophytes there are genera Trichophyton, Epidermophyton and Microsporum n According to the most common ways of transmission, they are anthropophilic (person-to-person), zoofilic (from animals) and geophilic (from environment) n The disease have various names in relation with their localisation (tinea manus, pedis, barbae etc.). n Treatment is usually local (ointments, shampoo). The mostly used drugs are nystatin, clotrimazol, ketokonazol etc. Dermatomycoses of various parts of body Tinea barbae Tinea pedis Onychomycosis A severe infection of Epidermophyton floccosum before and after treatment Story two n Mr. Leopold worked for an archive. All days he spent in the wet and dusty archive. Step by step he started to cough. For a moment, he was affraid of TB, but if was not TB. After assessment of the true reason and after the proper treatment Leopold problems started to disapear – again, slowly, step by step. Causative agent was n Aspergillus niger n Aspergilli usually attack diseased people, nevertheless, they are able to attact a heathy one, too. n Aspergillosis is often a professional disease of persons working in wet, dusty places, full of mold spores n Aspergillus disease is an example of organ or systemic mycoses Aspergillus infections 1 Aspergillus infections 2 A real case: aspergillosis as an influenza complication in a 38 year old woman n A female, born 1970, Brno-city, primary infection of upper and lower respiratory ways, as a causative agent proven influenza type B and Staphylococcus aureus. Death as a result of a heavy mycotic – Aspergillus pneumonia and septicemia, with lung and tracheobronchial lymphonodes anthracosis, exitus 26^th Mar 2008. No risk factors in anamnesis, only about 15 years of smoking 15–20 cigarettes daily. (From regional public health office of South Moravia) Systemic mycoses Zygomycets n Zygomycets – true molds form non-septed hyphae. They produce a strong growth, they are even able to pull the lid of the Petri dish up. n Infections are rare, but they occur more and more e. g. in diabetics. Normally they live as saprophytes, e. g. on fruits. They are able to grow quickly, e. g. through wall of large vessels. They may cause even so called living trombus with a quick death of such a patient. n Another typical feature is quick growth from nasal cavity to brain, even during a few hours n The most important genera: Rhizopus & Mucor Dimorph fungi n Cause mycoses in immunodeficient patients n Coccidioides immitis grows more quickly than the others. In patients with small immune defficiencies the infection is asymptomatical or with small symptomas only. It is worse in persons with a developped AIDS, where you can see primary lung infections etc. n Histoplasma capsulatum is seen mostly in the USA, but also in Africa. n More genera: Blastomyces, Paracoccidiodioides, Sporothrix and other Blastomycosis Coccidioides immitis Coccidioides immitis: „strange fungus“ Histoplasma capsulatum Story three n Ellen was scared. Her boy-friend was unpleased that she does not want to make love with him. In fact, the problem was not the boy friend, but allways when they made love, she started to have pruritus „there down“. n Well, she allready visited her gynecologist, and she got vaginal suppositoria. They helped always for a moment only. n Ellen was really angry. She changed her gynecologist. The new gynecologist understood, that local treatment will not be sufficient. Systemic treatment was able to destroy the causative agent both in vagina and intestinal reservoir. So her problems finished. Causative agent was Candida treatment n Candidosis may be both surface (skin, mucosal) and systemic. n Among mucosal candidoses, beside vaginal mycoses also oral mycosis is seen (in sucklings and people with diseased immunity) n Skin candidoses are common, too (for example „diaper dermatitis“ in sucklings) n Systemic infections are present mostly in immunodefficient persons and persons treated by combination of broad-spectered antibiotics n The most common is Candida albicans, also C. tropicalis, C. glabrata, C. krusei, C. parapsillosis etc. n In some of them, we can see natural resistances (e. g. C. krusei for fluconazol) Genital candidosis Oral candidosis Intertrigo and diaper dermatitis Intestinal candidosis Other yeasts and yeast-like organisms n Very dangerous is Cryptococcus neoformans, in immunodeficient persons it may cause pneumonia, meningitis, sepsis n Pneumocystis jiroveci is a very strange fungus, some time ago it was supposed to be a protozoon (for example a stage of trypanosomas) n Genus Saccharomyces contains wine and bear yeasts. It was supposed to be non-pathogenous, but some studies say that 8 % of vaginal mycoses may be caused by this agent. Morphology and physiology of fungi Morphology of fungi (micromycetes) n A blastoconidia is an oval or round cell, characteristic for yeasts. Often we see budding blastoconidia (blastospores). n A hypha is a fiber. It may be branched, septed or not septed. A sample of hyphae is called mycelium, that may be n vegetative, anchoring the fungus in the substrate n generative or air mycelium, bearing generatory organs of the fungus Multiplication of fungi n Multiplication of fungi may be sexual or asexual. It is simillar like in plants, here, too, we have both asexual and sexual multiplication methods. It is recomended to use terminology like that: n for sexual multiplication particles use term spore (do not confuse with bacterial spores!) n for asexual, vegetative reproductory particles to use term conidia Some morphological features in fungi How individual parts of a fungus are called Life cycle of a fungus How an arthroconidia are formed Physiology of micromycetes n Fungi multiply usually more slowly than bacteria, but there are big differences. They grow easily even on poor media. n Majority of medically important media grow well even at lower temperature. We culture ther at 30 °C rather than at 37 °C. Another way is a parallel culture at 22.°C and 37 °C, suitable for dimorph fungi n Biochemical activity is rich especially in yeasts Diagnostics of mycoses Sampling an diagnostics in surface mycoses n Sampling: particles of skin, parts of nails, hairs etc; always the specimen should contain the site where the inflammation is active, and not to catch contamination; even surface disinfection is recommended (to destroy contaminants from skin surface) n Proper diagnostics: microscopical (files in tissue) and culture. Microscopy is more important – even contamination may be cultured, but hyphae growing through an epitelium are a clear sign of an infection Sampling for dermatomycoses n Main rules for sampling: n do not send swab only, send several particles of skin (nail, hair, hairs etc.) n perform surface disinfection n superficial layer should be discarded, not used n in larger infections sample from margins (here the fungus is active) Diagnostics of systemic mycoses Not only the proper mycosis diagnostic is to be performed. It is also always necessary to find what is primary cause of the disease (if we do not know): immunodefficienty, diabetes, tumor etc. l Diagnostics: l for direct diagnostics any relevant material: blood for blood culture, punctates, excisions etc. l modern methods enable e. g. direct detection of antigens in blood (mannans, glucans) l indirect detection – serum antibodies (aspergilli) Sampling in candidosis l In skin and mucosal form we use swabs mostly in transport medium FungiQuick or (in genital swabs only) C. A. T. l In systemic form swabs, too, or blood, punctate etc. Diagnostics of candidoses l The basic is culture. For identification of candida we use chromogenous media and biochemical methods (mutual differences in metabolism between candida) l Microscopically in a wet mount (C. A. T.), in Gram and Giemsa stain we can see oval cells, often budding, sometimes even so called pseudomyelia l It is also possible to test in vitro susceptibility, but tests are less reliable than in bacteria l A modern method is the direct detection of mannan antigens in blood Fungi on bacteriological media n Although we use special media for fungi, many fungi grow on bacteriological media, too. And not only this: some of them, mostly Candida, have often feature very simillar to bacterial colonies. n To differenciate colonies of Candida from colonies of staphylococci is often difficult. Smell may help (bread, yeast); when nothing other helps, smear is useful. Chromogenic media – principle (Review from spring term) n CHROMOGENIC media contain a stuff that is originally colourless (a chromogene) n Only in presence of a specific reaction they become coloured (splitting of a substrate) n The medium may contain more chromogenes with bound substrates for warious bacteria or fungi n FLUOROGENIC media are principially simillar, but with a fluorescent stain Chromogenic medium at diagnostics of Candida n We use various chromogenic media. Some differenciate Candida albicans from other media only, some other differenciate mutually several species of Candida. n On the medium used in our Task 2c, C. albicans is green, C. tropicalis blue, C. glabrata smooth pink and C. krusei rough pink. n If a strain is not determined using this medium, we have to use another test (e. g. biochemical test) Biochemical identification of yeasts n Like bacteria, fungi, too (but not filamentous fungi) may be identified biochemically. (Also use of a chromogenic medium is based on selective splitting of various substrates.) n One of commonly used test is Auxacolor, that replaced ancient sets of „auxanograms“ (testing use of sugars) and „zymograms“ (testing breakdown of sugars) Diffusion disc test of susceptibility to antimicromial agents n With some exceptions it is valid, that antibacterial agents are useless in mycotic diseases. n Similarly, antimycotics do not act to majority of bacterial agents n Fungi cannot be cultured on MH, they need Sabouraud agar To reading of antimycotic tests n In amphotericin B a strain is considered to be susceptible even in small zone, but there should be no colonies inside the zone n In azolic antimycotics (the names ending „-conazol“) the zone should be large enough, but „something“ may be present inside the zone, if this „sometning“ is not more than 20 % of intensity of growth inside the zone Microscopy of filamentous fungi n Also microscopy is different than that of yeasts. It is more important here. We can observe various types of spores and conidiae. n We observe without immersion, objective multiplying 4× or 10×, eventually 40 × Culture of filamentous fungi n Results of culture in filamentous fungi are different from yeast, both on Sabouraud agar and eventually blood agar. n Some of them, especially dermatophytes, grow very slowly. This is because of them, why Sabouraud agar is poured into test tubes. n Biochemical differenciation is usually not performed here, unlike the situation in yeasts. Example of indirect diagnostics of fungi: microprecipitation in agar n From the middle hole, antigen diffunds (marked red) n From the positive hole with serum No. 2 the antibody diffunds (blue) n From negative holes (sera No. 1, 3, 4) of course nothing diffunds. n In place of meeting of antigen and antibody, precipitation line is formed (green) Example of indirect diagnostics of fungi: microprecipitation in agar The test is a repeating from J 07. Precipitation line is formed between the hole with antigen and the hole with antibody Pictures of fungi Causative agents: Candida albicans Candida albicans Candida Mucor Aspergillus niger Aspergillus sp. Aspergillus sp. Mucor sp. Pneumocystis jiroveci Dermatophytes Saccharomyces cerevisiae Geotrichum candidum Rhodotorula rubra Penicillium marneffei Cryptococcus neoformans The End