TRACING THE CRIMINAL Part ten: Criminals – fungi Institute for microbiology shows L 67 Muchomurka_cervena_i Instead of an introduction bdadafoto.webzdarma.cz/rostliny_houby.htm [USEMAP] Survey of topics Characteristics of fungi Examples of mycotic diseases Morphology and physiology of fungi Diagnostics of mycoses Pictures of fungi Characteristics of fungi Common characteristics of fungi lFungi are eukaryotic organisms, unlike procaryotic bacteria lTheir cell wall is formed by polysacharids, it has a different composition than that of G+ bacteria. Neverthreless, it stains violet („gram-positive“) lFungi use to have a slower cell cycle than bacteria à infections use to be prolonged lMajority of antibacterial agents is not effective, so we have to use special drugs – antimycotics, that are not effective againts bacterial infections Fungi and health lIn the practical we are going to speak about microscopical fungi. But we shoud not forget about fungi with macroscopical fruits lFruits of macroscopical fungi (Amanita phalloides, Inocybe patouillardi, Amanita panterina, Entoloma eulividum, halucinogenous fungi) cause diseases and death of tens of persons every year (especially in countries like Czechia, where fungi picking is a very common hobby). In case of Amanita phalloides, the result is often death. Some toxical mushrooms 1 Amanita phaloides 2 Inocybe Patouillardi 3 Amanita panterina 4 Entoloma eulividum Do you know? 68 430px-Amanita_phalloides cs.wikipedia.org/wiki/Otrava_houbami 72 Amanita_pantherina_001 http://www.micologia.net/g3/Amanita-panterina/Amanita_pantherina_001 70 art_inocybeerubescens vláknice http://www.houbar.cz/default.aspx?show=3&text=3 71 450px-Entoloma_Sinuatum_1 cs.wikipedia.org/wiki/Z%C3%A1vojenka_olovov%C3%A 1 2 3 4 Medically important fungi lMicroscopical fungi may cause in human body lMycoses – fungal inflammations lMycotoxicoses – toxical action lMykoalergoses – allergy to fungi lMycetisms – fungus present in the body, acting only in form of pressure to surronding tissues lThe most imporant are mycoses, that may be subclassified into lsurface (skin and mucosal) mycoses and lorgan and systemic mycoses [USEMAP] Examples of mycotic diseases Story one nMrs. Udder came to dpt. of professional medicine with „fungus“ on her hand. nShe wanted her disease to be accepted as proffessional disease, as she works with cattle on a farm. Good luck 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; nevertheless, actual guidlines say that mere professional exposition is sufficient for considering such case professional, not regarding the species diagnostics Dermatophytes nSo named dermatophytes belong among the most common agents of skin mycoses (including mycoses of skin adnexa, i. e. hair, hairs and nails) nAmong dermatophytes there are genera Trichophyton, Epidermophyton and Microsporum nAccording to the most common ways of transmission, they are anthropophilic (person-to-person), zoophilic (from animals) and geophilic (from environment) nThe disease have various names in relation with their localisation (tinea manus, pedis, barbae etc.). nTreatment is usually local (ointments, shampoo). The mostly used drugs are nystatin, clotrimazol, ketokonazol etc. Dermatomycoses of various parts of body 04 23-1_tinea_capitis_Ttonsurans 06 23-3_Epidermophyton_or_Trichophyton 08 23-5_Trichophyton_hand 09 23-6_Trichophyton_foot 10 23-7_onychomycosis_EorT www.mycolog.com/chapter23.htm Tinea barbae 30 1110tinea_barbe1 www.emedicine.com Tinea pedis 31 tinea pedis www.itg.be Onychomycosis www.itg.be 33 onychomykóza A severe infection of Epidermophyton floccosum before and after treatment 05 23-2_Epidermophyton_floccosum_BA www.mycolog.com/chapter23.htm Story two nMr. 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. asnig3 Causative agent was nAspergillus niger nAspergilli usually attack diseased people, nevertheless, they are able to attact a heathy one, too. nAspergillosis is often a professional disease of persons working in wet, dusty places, full of mold spores nAspergillus disease is an example of organ or systemic mycoses www.medmicro.info 24 aspergilóza rtg aapredbook.aappublications.org 29 ct mozku aspergilus www.nature.com Aspergillus infections 1 Aspergillus infections 2 25 aspergilosis brain9 www.njmoldinspection.com 23 invazivní aspergilóza www.nlm.nih.gov A real case: aspergillosis as an influenza complication in a 38 year old woman nA 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 26th 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 lThey attack more organs, often the whole body lUsually they follow after a primary disease: lDiabetes mellitus lImmunity defects, WBC tumors etc. lTransplanted pacients lCaused by: Candida, Penicillium, Aspergillus, Histoplasma, Pneumocystis and other lTreatment: strong, broad-spectered and highly effective antimycotics are used (amphotericin B, voriconazole, itraconazole, flucytosin) l Zygomycets nZygomycets – true molds form non-septed hyphae. They produce a strong growth, they are even able to pull the lid of the Petri dish up. nInfections 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. nAnother typical feature is quick growth from nasal cavity to brain, even during a few hours nThe most important genera: Rhizopus & Mucor Dimorph fungi nCause mycoses in immunodeficient patients nCoccidioides 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. nHistoplasma capsulatum is seen mostly in the USA, but also in Africa. nMore genera: Blastomyces, Paracoccidiodioides, Sporothrix and other Blastomycosis 18 23-20_NAmerBlastomycosis www.mycolog.com/chapter23.htm Coccidioides immitis 36 Coccidioides immitis 35 coccidiomykóza http://www.mycology.adelaide.edu.au/gallery Coccidioides immitis: „strange fungus“ 17 lifecycleedit-NEW Coccidioides www.vfce.arizona.edu Histoplasma capsulatum 16 23-18_Histoplasma www.mycolog.com/chapter23.htm 48 histioplasmosa http://www.mycology.adelaide.edu.au/gallery Story three nEllen was scared. She loved her boyfried very much, but the intime moments were complicated by vaginal pruritus. nWell, she allready visited her gynecologist, and she got vaginal suppositoria. They helped always for a moment only. nEllen was really angry. She changed her gynecologist. The new gynecologist understood, that local treatment will not be sufficient in this specific case. Systemic treatment was able to destroy the causative agent both in vagina and intestinal reservoir. So her problems finished. Causative agent was nCandida albicans, the most common among medically important yeasts. Vaginal mycoses (mostly candidoses) are very unpleasant and difficult. nThe vaginal candidoses are multifactorial. Important are dietary influences (yeasts love sweet, so if does so their host, they would enjoy it), but also hormonal influences, pregnancy, diabetes and others. nThe reservoir of the infection is the intestine. Recidivating infections should not be treated only localy (suppositoria) but by combination of a local and systemic treatment. nVaginal mycosis of course should not be solved without thinking about the total status of the organism. Candida treatment nCandidosis may be both surface (skin, mucosal) and systemic. nAmong mucosal candidoses, beside vaginal mycoses also oral mycosis is seen (in sucklings and people with diseased immunity) nSkin candidoses are common, too (for example „diaper dermatitis“ in sucklings) nSystemic infections are present mostly in immunodefficient persons and persons treated by combination of broad-spectered antibiotics nThe most common is Candida albicans, also C. tropicalis, C. glabrata, C. krusei, C. parapsillosis etc. nIn some of them, we can see natural resistances (e. g. C. krusei for fluconazole) Genital candidosis 01 vulvovaginální kandidóza 25 candida www.vita.csc.pl/zakazenia-drozdzakowe.php www.telemedicine.org/common/common.htm. Oral candidosis 02 ústní kandidóza 03 ústní kandidóza chronická ww.asnanak.net/ar/article.php?sid=62. 12 23-9_intertrigo_Candida www.mycolog.com/chapter23.htm Intertrigo and diaper dermatitis 32 plenková dermatitida www.itg.be 64 candida_nappy_rash http://webs.wichita.edu/mschneegurt/biol103/lecture21/lecture21.html Intestinal candidosis 21 candida-gut http://george-eby-research.com/html/depression-anxiety.html Other yeasts and yeast-like organisms nVery dangerous is Cryptococcus neoformans, in immunodeficient persons it may cause pneumonia, meningitis, sepsis nPneumocystis jiroveci is a very strange fungus, some time ago it was supposed to be a protozoon (for example a stage of trypanosomas) nGenus 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. 26 moldcartoon [USEMAP] 65.254.85.56 Morphology and physiology of fungi Morphology of fungi (micromycetes) nA blastoconidia is an oval or round cell, characteristic for yeasts. Often we see budding blastoconidia (blastospores). nA hypha is a fiber. It may be branched, septed or not septed. A sample of hyphae is called mycelium, that may be nvegetative, anchoring the fungus in the substrate ngenerative or air mycelium, bearing generatory organs of the fungus Multiplication of fungi nMultiplication 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: nfor sexual multiplication particles use term spore (do not confuse with bacterial spores!) nfor asexual, vegetative reproductory particles to use term conidia Some morphological features in fungi 15 Fungi_Pic 14 pseudohyfy a hyfy gsbs.utmb.edu education.med.nyu.edu How individual parts of a fungus are called 18 části houby www.mc.uky.edu Life cycle of a fungus 19 cyklus houby /media.wiley.com How an arthroconidia are formed 16 vznik arthrokonidie gsbs.utmb.edu Physiology of micromycetes nFungi multiply usually more slowly than bacteria, but there are big differences. They grow easily even on poor media. nMajority 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 nBiochemical activity is rich especially in yeasts [USEMAP] Diagnostics of mycoses Sampling an diagnostics in surface mycoses nSampling: 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) nProper 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 nMain rules for sampling: ndo not send swab only, send several particles of skin (nail, hair, hairs etc.) nperform surface disinfection nsuperficial layer should be discarded, not used nin larger infections sample from margins (here the fungus is active) Diagnostics of systemic mycoses nNot 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. lDiagnostics: lfor direct diagnostics any relevant material: blood for blood culture, punctates, excisions etc. lmodern methods enable e. g. direct detection of antigens in blood (mannans, glucans) lindirect detection – serum antibodies (aspergilli) Sampling in candidosis lIn skin and mucosal form we use swabs mostly in transport medium FungiQuick or (in genital swabs only) C. A. T. lIn systemic form swabs, too, or blood, punctate etc. C. A. T. Foto O. Z. P1010010 Diagnostics of candidoses lThe basic is culture. For identification of candida we use chromogenous media and biochemical methods (mutual differences in metabolism between candida) lMicroscopically in a wet mount (C. A. T.), in Gram and Giemsa stain we can see oval cells, often budding, sometimes even so called pseudomyelia lIt is also possible to test in vitro susceptibility, but tests are less reliable than in bacteria lA modern method is the direct detection of mannan antigens in blood Fungi on bacteriological media nAlthough 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. nTo differenciate colonies of Candida from colonies of staphylococci is often difficult. Smell may help (bread, yeast); when nothing other helps, smear is useful. A selective medium for fungi nThe typical medium for yeasts, Sabouraud agar, is not selective itself, and many bacteria could grow on it nFor culture of mycoorganisms we use Sabouraud agar with antibiotics, that nearly excludes growth of bacteria. (In practice, nevertheless, we often meet very resistant strains of Pseudomonas, that grow where they want J) Chromogenic media – principle (Review from spring term) nCHROMOGENIC media contain a stuff that is originally colourless (a chromogene) nOnly in presence of a specific reaction they become coloured (splitting of a substrate) nThe medium may contain more chromogenes with bound substrates for warious bacteria or fungi nFLUOROGENIC media are principially simillar, but with a fluorescent stain Chromogenic medium at diagnostics of Candida nWe use various chromogenic media. Some differenciate Candida albicans from other media only, some other differenciate mutually several species of Candida. nOn the medium used in our Task 2c, C. albicans is green, C. tropicalis blue, C. glabrata smooth pink and C. krusei rough pink. nIf a strain is not determined using this medium, we have to use another test (e. g. biochemical test) S27 cakru caal cagla catro C. albicans C. glabrata C. tropicalis C. krusei Biochemical identification of yeasts nLike 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.) nOne 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 nWith some exceptions it is valid, that antibacterial agents are useless in mycotic diseases. nSimilarly, antimycotics do not act to majority of bacterial agents nFungi cannot be cultured on MH, they need Sabouraud agar To reading of antimycotic tests nIn amphotericin B a strain is considered to be susceptible even in small zone, but there should be no colonies inside the zone nIn 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 nAlso microscopy is different than that of yeasts. It is more important here. We can observe various types of spores and conidiae. nWe observe without immersion, objective multiplying 4× or 10×, eventually 40 × Microscopy of filamentous fungi nResults of culture in filamentous fungi are different from yeast, both on Sabouraud agar and eventually blood agar. nSome of them, especially dermatophytes, grow very slowly. This is because of them, why Sabouraud agar is poured into test tubes. nBiochemical differenciation is usually not performed here, unlike the situation in yeasts. Culture of filamentous fungi Example of indirect diagnostics of fungi: microprecipitation in agar nFrom the middle hole, antigen diffunds (marked red) nFrom the positive hole with serum No. 2 the antibody diffunds (blue) nFrom negative holes (sera No. 1, 3, 4) of course nothing diffunds. nIn place of meeting of antigen and antibody, precipitation line is formed (green) MPA ouchterlony nThe test is a repeating from J 07. Precipitation line is formed between the hole with antigen and the hole with antibody Holes with patient‘s sera 1–4 Hole with antigen positive Precipitation line – reason of positivity Example of indirect diagnostics of fungi: microprecipitation in agar [USEMAP] Pictures of fungi trimen1 Dermatophytes epifloc trirub 1 Epidermophyton floccosum 2 Trichophyton rubrum 3.Trichophyton mentagrophytes 1 3 2 3× www.medmicro.info Penicillium marneffei 20 Penicilliummarnaffei www.pasteur.fr asnig16 09 aspergillus_niger Aspergillus niger asnig1 www.medmicro.info www.medmicro.info http://fungifest.com 12 Aspergillus%20niger%20nakres www.sci.muni.cz Aspergillus sp. 11 Aspergillus%20fumigatus%20nakres www.sci.muni.cz 07 Asexual%20structures%20of%20Aspergillus%20niger 129.215.156.68 13 4_Aspergillus_herbariorum www.mycolog.com Aspergillus sp. 10 Aspergillus education.med.nyu.edu 28 aspergillus healthresources.caremark.com Mucor mucor14 www.medmicro.info Mucor sp. 50 mucor1 http://www.mycology.adelaide.edu.au/gallery 52 Mucor mucedo www.zsdukla.cz/nature/article86.php 03 candida Causative agents: Candida albicans 04 candida-alb-dk3 pathmicro.med.sc.edu 02 CAAL www.doctorfungus.org www.schoolwork.de 06 candida Candida albicans S27 www.medmicro.info 05 candida1 www.pferdemedizin.com www.medizin-forum.de 27 candida Candida http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/icu8/std/candidgram.html l17 Pneumocystis jiroveci www.medmicro.info Saccharomyces cerevisiae 51 Saccharomycees cerevisiae www.zsdukla.cz/nature/article86.php Geotrichum candidum geotrichum www.medmicro.info rhoru6 Rhodotorula rubra www.medmicro.info Cryptococcus neoformans 37 kryktokokóza http://www.higiene.edu.uy/ciclipa/parasito/Cryptococcus.jpg 73 Cryptococcus http://www.mycology.adelaide.edu.au/gallery The End http://www.jiricisar.com/blog/photo/20050824_kremenac.jpg 03 20050824_kremenac [USEMAP]