HS 202 Fundamentals of Mycology PDF

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EnviousPurple

Uploaded by EnviousPurple

2024

Maria Christina Filomena R. Batac, MD

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fungi mycology pathogenic fungi biology

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This document, "Fundamentals of Mycology," provides an outline of the history, characteristics, classification, reproduction, and diagnostic procedures related to fungi. It also discusses the mode of nutrition for different types of fungi and briefly touches upon the impact of fungi on various ecosystems.

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hs 202: Biopsychosocial Dimension of Illness FUNDAMENTALS OF MYCOLOGY Maria Christina Filomena R. Batac, MD | September 24, 2024 OUTLINE we are and have paved the way for human c...

hs 202: Biopsychosocial Dimension of Illness FUNDAMENTALS OF MYCOLOGY Maria Christina Filomena R. Batac, MD | September 24, 2024 OUTLINE we are and have paved the way for human civilization, they I. Introduction V. Fungal Classification sometimes cause harm and can even kill humans.” A. History of Fungi A. Previous Classification “Fungi often hit when humans suffer, causing outbreaks B. Mycology Rules after tsunamis, hurricanes, and other natural disasters.” II. Characteristics of Fungi B. Current Classification A. HISTORY OF FUNGI A. Basic Characteristics Rules Fungi may have played a role in the extinction of dinosaurs and B. Mode of Nutrition C. Medically-Important the rise of mammals C. Pathogenic Fungi Phyla → Around 63 million years ago, dinosaurs and other reptiles were III.Basic Fungal Growth D. Renaming of Fungi dominating the Earth → an asteroid hit the Earth’s surface → A. Yeast VI.Fungal Diagnostic cause death of many living creatures B. Molds Procedures → Cloud of dust surrounded the Earth’s surface → prevented the C. Dimorphism A. Direct Microscopy Sun from reaching the planet IV. Fungal Reproduction B. Histopathology → Darkness & increase in organic matter from dead flora & fauna A. Asexual Reproduction C. Culture → caused a fungal bloom → infected the cold-blooded B. Sexual Reproduction D. Fungal Identification reptiles (e.g. dinosaurs) VII. How Fungi Cause Disease Fungi and the Rise of Mammals A. Host Risk Factors Mammals are warm-blooded with an average body temperature B. Determinants of of 36-40ºC Pathogenicity → But most environmental fungi are NOT thermotolerant & do VIII. References NOT grow between 20-50ºC → Thus, mammals are naturally resistant to fungi I. INTRODUCTION → Fungi can infect dinosaurs & other reptiles that are Debunking Statements from the “Last of Us” episode: cold-blooded → “Fungi seem harmless enough” No second age of reptiles due to fungal bloom → led to the rise ▪ Fact: fungal infections more commonly affect of mammals immunocompromised patients & those unable to mount → Fungi have made us who we are and have paved the way for an appropriate immune response against a pathogenic human civilization threat Review: Warm- vs Cold-blooded Animals → “Fungi cannot survive if its host temperature is over 94ºC, and currently there are no reasons for fungi to evolve to withstand Table 1. Examples of warm- and cold-blooded animals high temperatures. But what if…” Warm-blooded Cold-blooded ▪ Fact: TRUE; may be the reason why there are not a lot of (Homoiotherms) (Poikilotherms) fungal infections that happen in mammals, especially Birds Worms humans Mammals Insects → “There are no treatments, no preventatives, no cures…” Fish ▪ Fact: NOT entirely true; there are antifungals available in Amphibians the market but, Reptiles − May be referring to the limited antifungals available in the B. MYCOLOGY market Science that deals with the study of fungi o Limited armamentarium & are very difficult to treat From the Greek word “myke” meaning “mushroom”, and “logos” o Antifungal treatment requires dosing adjustments meaning “to study” − No available vaccines currently available for preventative Mushrooms measures → Are among the most popular members of fungi A quote from “When disaster strikes, fungi take control” → The first fungi known because they are large enough to be (2022): seen by the naked eye “During 1 billion years of evolution, fungi have not only become → The obvious structure of complex fungi with a massive masters of survival, but have actually leveraged disasters. 65 network of fungal cells found beneath the soil million years ago, when an asteroid strike wiped out 70% of all ▪ Like the tip of the iceberg life on Earth by sending dusty debris into the atmosphere, − Only a small part is seen on the surface fungi strived, taking advantage of the plants decaying due − Around 90% is hidden beneath to the lack of sunlight. Back then, fungi infected and killed The largest living organism in the world is a fungus reptiles in their masses, potentially contributing to the → A specific honey fungus measuring 2.4 miles (3.8 km) wide extinction of the dinosaurs.” found in the Blue Mountains in Oregon “By contrast, fungi were not able to survive at the high body ▪ An organism composed of genetically identical cells that temperatures of mammals and thereby contributed to the can communicate and have a common purpose or at least succession of mammals as the new dominant species on coordinate themselves Earth.” Study of fungi is divided into two groups: “Ever since then, fungi have maintained their integral role in → (1) Macroscopic the development of life. Although fungi have made us who ▪ E.g. Mushrooms Trans 04 TG9A: Ching, G., Ching, T., Chuatico, Clave, Collantes TH: Edeza 1 of 17 → (2) Microscopic Cell Wall ▪ Focus of Medical Mycology as it can cause disease in humans ▪ E.g. Molds and Yeast II. CHARACTERISTICS OF FUNGI A. BASIC CHARACTERISTICS Fungal cells have a lot of similarities with human cells → What can harm fungi can also harm humans → This makes antifungal development extra challenging → Molecules unique to fungi & not present in human cells should be the target of antifungal drugs → Most antifungals (e.g. azoles, allylamines) target ergosterol to inhibit cell wall synthesis Figure 2. Fungal Cell Wall and Cell Membrane Fungi are encased in rigid cell wall → In contrast to animals which have no cell walls Cell wall → Composed primarily of chitin and glucan → Whereas plant cell wall is composed of cellulose Cell membrane → Ergosterol is the main sterol component → Whereas animal cell membrane has cholesterol as its main sterol component B. MODE OF NUTRITION Fungi are heterotrophs → They embed themselves in organic substrates and obtain nourishment by: ▪ (1) secreting enzymes that cause external digestion, then Figure 1. Summary of basic characteristics of fungi ▪ (2) absorbing nutrients released from the substrate [2026 Trans] Aerobic Fungi cannot produce their own food, being either: Most fungi are aerobic → Saprotrophic ALL fungi known to be pathogenic to humans are aerobic ▪ Feed on remnants of dead plants or animals ALL fungi were known to be aerobic until the 1970s, ▪ Derive their nutrients from decaying matter → What was thought to be flagellated protozoan isolated from the → Parasitic digestive tract of ruminants turned out to be anaerobic fungi ▪ Require a living host; comprise only a few fungi Nucleated “There is no known organic material on earth that cannot be attacked by fungi” ALL fungi are nucleated; therefore, are eukaryotic → Can be seen on food, even refrigerated ones (e.g., → Nuclear material is arranged into chromosomes and strawberries, bread) enclosed by a nuclear membrane → Can also be seen on the wooden ceiling of houses → Unlike bacteria where the nuclear material is not arranged in Because of their heterotrophic features: chromosomes and is not enclosed by a nuclear membrane → They can be used in food processes such as making bread, Although bacteria and yeast cells look more alike, humans are cheese, beer, etc. actually more similar to yeasts at the cellular level than bacteria → They affect agriculture by infecting corn, wheat, and other Achlorophyllous grains, consequently affecting the economy Fungi are achlorophyllous → They can cause disease in humans → Lack chlorophyll and are mostly heterotrophic, NOT C. PATHOGENIC FUNGI photoautotrophic The healthy human immune system is well equipped in fighting → Do not produce their own food, and instead consume organic off invasive fungal infection. compounds for growth and development → However, fungi have emerged in the past few decades as Most are saprophytic and digest dead organic matter major causes of human disease, especially among those: Sexual and/or Asexual Reproduction ▪ Immunocompromised Many reproduce by sexually and asexual means ▪ Hospitalized, with serious underlying diseases (e.g., → Most fungi reproduce by forming spores that can survive transplant and cancer patients) extreme conditions such as cold and lack of water [2026 Trans] ▪ With COVID-19, and diabetes mellitus → Both sexual meiotic and asexual mitotic spores may be ▪ On prolonged steroids produced, depending on the species and conditions [2026 Trans] Many fungi serve as opportunistic pathogens, causing Simpler in structure than plants and animals considerable morbidity and mortality → Cells are NOT divided into tissues or organs → Overall incidence of specific invasive mycoses consistently increases with time → List of opportunistic fungal pathogens increases each year “There are no non-pathogenic fungi” Among the 50,000-250,000 species of fungi that have been named: → Fewer than 500 have been associated with human disease and are medically important HS 202 Fundamentals of Mycology 2 of 17 → Less than 100 are capable of causing infection in otherwise Eumycetoma / subcutaneous mycosis normal individuals → The remainder are ONLY able to produce disease in hosts that are debilitated or immunocompetent Hippocrates → Father of medicine → Recognized that persons with oral thrush (due to Candida albicans) were already debilitated by other diseases Prof. Graham S. Wilson → Director of the U.K’s Public Health Laboratory Service Figure 6. Eumycetoma affecting the right foot → Same sentiment as Hippocrates ▪ Even before the advent of AIDS or the association of Acquired from traumatic implantation esophageal candidiasis with AIDS was established → Usually from contaminated wood stepped on by people who → “Candida is a much better clinician than we are, in its ability to walk barefoot detect abnormalities earlier in the course of development of Suppurative and granulomatous subcutaneous fungal such abnormalities than we can with all our chemical tests” infection [2026 trans] Sample Diseases III. BASIC FUNGAL GROWTH 2 Basic Fungal Growth Forms: Tinea corporis (“Buni”) → Yeast: independent single cell → Hypha: filament-like cells Species grow as either filaments or yeasts only, but some can grow as either form (dimorphic) [2026 trans] A. YEAST Unicellular units Reproduce by budding or by fission → A progenitor (mother) cell pinches off a portion of itself to produce a progeny (buds/daughter cell) → Buds are termed blastoconidia Figure 3. Tinea corporis Relatively benign fungal cutaneous disease Erythematous annular plaques on the body [2026 trans] Caused by dermatophytes [2026 trans] Cryptococcal meningitis Figure 7. Structure of blastoconidia & pseudohyphae Blastoconidia may elongate to form pseudohyphae → Occurs when blastoconidia remain attached in a chain of round to elongate cells often resembling a string of pearls Yeast cells on culture media appear like bacterial colony → They are usually round, pasty or mucoid colonies on agar Figure 4. Cryptococcal Cells on Negative Stain with India Ink Cryptococcal cells surrounded by capsules appearing as halos around the cell Patients with AIDS are at risk of acquiring cryptococcal Figure 8. Yeast on agar meningitis and cutaneous cryptococcosis B. MOLDS Mucocutaneous candidiasis / oral thrush Multicellular Produce filamentous, hairy, or wooly colonies Figure 5. Mucocutaneous candidiasis Oral mucosa (tongue, mouth, inner cheeks) lined with a whitish film of Candida colonies Caused by proliferation of Candida albicans [2026 trans] → Affects immunocompromised patients Figure 9. Examples of mold HS 202 Fundamentals of Mycology 3 of 17 Features of Molds Hyphae Threadlike tubular structures that form molds Elongate at their tips by a process known as apical extension Spitzenkörper mediates apical extension → Appear as reddish marks at the apical extension hyphal tips → Complex, multicomponent structures that function to support directional growth of the hypha Figure 13. Septate Hyphae Mycelia Hyphae form together to produce a mat-like structure called mycelium Mycelia can be categorized according to location: → Vegetative mycelia: submerged in the substrate → Aerial mycelia: above the substrate and are visible ▪ Conidia: the round, black, pinpoint structures on top of the aerial mycelium (Fig. 15, Strawberry) − Easily be airborne by which the fungus gets disseminated Figure 10. Hyphae with visible Spitzenkörper Septa of Hyphae Cross-walls found in hyphae that divide the hyphae into cells or Figure 14. Types of conidia according to location cytoplasmic compartments Hyphae may or may not have septa → Septate: hyphae WITH septa ▪ Septa are perforated ▪ Allowing exchange of cytoplasmic material and sometimes even of nuclei → Coenocytic or Aseptate: hyphae WITHOUT septa ▪ Septa are sparse and when they occur, they are not usually perforated ▪ Allowing isolation of cellular structures Figure 15. Conidia seen on aerial mycelium on a strawberry Fungal Life Cycle Hyphae may also be classified according to function: → Vegetative Hyphae ▪ Both surface and submerged filaments that digest, absorb, and distribute nutrients from the substrate ▪ As this particular mold matures, it develops reproductive hyphae → Reproductive Hyphae ▪ Produce sporangia ▪ When sporangia ruptures, asexual sporangiospores are released into the air Figure 11. Difference between septate and coenocytic hyphae ▪ Sporangiospores settle on a substrate and send out germ tubes that elongate into hyphae which eventually becomes an extensive mycelium ▪ A single colony of mold easily contains 5,000 spore-bearing structures which can each release 2,000 spores Figure 12. Coenocytic Hyphae HS 202 Fundamentals of Mycology 4 of 17 Etiologic Agents of Endemic Mycoses Traditionally, there are 4 genera of fungi that are associated with endemic mycoses: → Histoplasma (histoplasmosis) → Blastomyces (blastomycosis) → Paracoccidioides (paracoccidioidomycosis) → Coccidioides (coccidioidomycosis) These four genera are considered primary pathogens since they cause disease even among immunocompetent individuals → Other fungi may cause endemic mycoses, but are opportunistic (e.g. Talaromyces marneffei endemic in Southeast Asia) Figure 16. Fungal life cycle Examples of Mold Organisms Rhizopus sp. Figure 17. (L) Rhizopus on an agar plate; (Mid) Ruptured Rhizopus sporangia releasing spores into the air; (R) Reproductive hyphae of Rhizopus → Colony usually white at beginning, becoming gray after sporulation → Some of the dark structures are the sporulating heads or sporangia (sac-like structures that contain sporangiospores) Aspergillus niger Figure 19. Mold (Left) & Yeast (Right) Forms of Dimorphic Fungi: (A) Histoplasma capsulatum, (B) Blastomyces dermatitidis, (C) Paracoccidioides brasiliensis, (D) Coccidioides immitis, (E) Talaromyces marneffei These fungi are soil-dwelling molds → Spores and hyphal fragments are inhaled → upon infection of tissues (usually the lungs), convert to yeast → Conversion is temperature-sensitive ▪ Incubation of dimorphic fungi needs to be at 35-37°C on an enriched medium, to observe their transformation in the laboratory setting Figure 18. (L) Macroscopic colony; (R) Microscopic picture of A. niger sporing head IV. FUNGAL REPRODUCTION → Whitish colony covered by black sporing heads Fungi have many complex and successful reproductive strategies → Have naked spores not contained in sac Modes of Reproduction: C. DIMORPHISM → Primary Fungal pathogens that change their growth form during the ▪ Production of various types of spores process of tissue invasion are called dimorphic ▪ Different fungi produce different types of spores and it is → Usually change from molds in the natural environment to yeast largely through these spores that fungi are identified in tissue → Simple lengthening A form of adaptation of the fungus to the host environment and ▪ Outward growth of existing hyphae improves its pathogenic ability ▪ Most fungi can propagate through this means Exists as mold in the environment and yeast in the host tissues → Fragmentation (37℃) ▪ A separated piece of mycelium can generate a whole new Dimorphic fungi cause endemic or systemic mycoses colony There are various ways of classifying spores, and the most basic MNEMONIC: one is by how they arise MOLD in the COLD (exist as mold form inside the environment) YEAST in the BEAST (exist as yeast form inside the host) HS 202 Fundamentals of Mycology 5 of 17 Table 2. Sexual vs Asexual Reproduction Asexual Sexual Products of mitotic division of a From fusion of two parental single parent cell nuclei followed by meiosis Anamorph: phase that Teleomorph: phase that produces asexual spores produces sexual spores Common sexual spore types: Two subtypes: zygospores, ascospores, sporangiospore and conidia basidiospores Classification of fungi is largely based on appearance or morphological characteristics → Rather than nutritional and biochemical differences used in bacterial classification → “Sexual spores and the structure in which they are produced form the main bases for fungal classification” In some fungi, anamorphic state is so successful as a means of rapid dispersal to new habitats, → Teleomorphic state has diminished or even disappeared Figure 20. Diagram of sporangiospores in a sporangium Many fungi bear 2 names: → (1) Sexual stage → (2) Asexual stage → E.g. Histoplasma capsulatum (anamorph) or Ajellomyces capsulatus (teleomorph) → Anamorphic and teleomorphic stages were described and named at different times by different scientists without the connection between them being recognized. → Both names are valid, but teleomorph phase name should take precedence ▪ In practice, it is more common and correct to refer to a fungus by its asexual (anamorph) designation − This is the stage that is usually obtained in culture Discovery of Malassezia globosa sexual state Fungus associated with dandruff and lives on the head Researchers sequenced the genes and found that its genome sequence has mating-type genes that indicate that they can reproduce sexually → It has never been observed in the past that Malassezia has a sexual state or a teleomorph state A. ASEXUAL REPRODUCTION Figure 21. Sporangiospores in the sporangium Sporangiospores Conidia Asexual spores produced in a containing structure or Asexual spores that are borne naked on specialized structures sporangium → Free spores: not enclosed by a spore-bearing sac → Sporangiophore: a supporting stalk (a special hyphal structure) E.g. Aspergillus spp., Penicillium spp., dermatophytes to which the sporangium is attached Formed by successive cleavages within the sporangium, which is attached to the sporangiophore Released when the sporangium ruptures → Usually produced by fungi from Order Mucorales ▪ Coenocytic types of fungi that are the etiologic agents of the “black fungus infection” ▪ E.g. Rhizopus sp., Mucor sp. Figure 22. Conidia HS 202 Fundamentals of Mycology 6 of 17 Table 3. Etymology of Some Types of Conidia Phialospore Conidia Etymology and Description → Conidium budded from the mouth of a vase-shaped spore-bearing cell called a phialide/sterigma, leaving a small collar From aspergillum: liturgical → From the Greek word, phialos, which means a vessel implement used to sprinkle holy water Aspergillus sp. From penicillium (paint brush): similar microscopic structure Penicillium sp. Types of Conidia Arthrospore → Rectangular spore formed when a septate hypha fragments at the cross walls → From the Greek word, arthron, which means joints Figure 26a. Phialospore Figure 23. Arthrospore Chlamydospore Figure 26b. (Bottom to Top) Different Types of Conidiophores (White), Rami (Dark Blue and Grey), Metulae (Light Blue), and Philades (Green), with → Spherical conidia formed by the thickening of a hyphal cell Phialospores → Released when the surrounding hypha fractures → Serves as a survival or resting cell → E.g. Penicillium in Fig. 26b (L→R) → From the Greek word, chlamys, which means cloak ▪ Monoverticillate: simple − No branching ▪ Biverticillate-symmetrical: one-stage branched − Has metulae ▪ Biverticillate-asymmetrical: two-stage branched − Has rami ▪ Quaterverticillate: three-stage branched − Has metulae and two branches of rami (blue and grey) Microconidium & Macroconidium Figure 24. Chlamydospore → Smaller and larger conidia formed by the same fungus under Blastospore varying conditions → Produced by budding from a parent cell (e.g. yeast, conidium) → I.e. “bud” Figure 27. Microconidia and macroconidia. Figure 25. Blastospore HS 202 Fundamentals of Mycology 7 of 17 B. SEXUAL REPRODUCTION Zygospores Diploid spores formed when hyphae of two opposite strains (+ and -) fuse → Create a diploid zygote that swells and becomes covered by strong, spiny walls Germinates when wall is disrupted and conditions are suitable → Forms a mycelium that gives rise to a sporangium → Diploid zygospore undergoes meiosis to form haploid nuclei that develop into sporangiospores Figure 31. Ascospore Formation Basidiospores Haploid sexual spores formed outside a club-shaped cell called a basidium Figure 28. Zygospores Formed similarly to ascospores, from two opposite strains of hyphae, forming dikaryons that differentiate into basidia → After meiosis, 4 haploid nuclei are formed and extruded through the top of the basidium, forming basidiospores Mushrooms are examples of basidiocarps → Structures designed to protect the basidiospores → Basidia are found along the gills (fleshy part/fruiting body/basidiocarp) Figure 32. Basidiospores Figure 29. Zygospore Formation and Germination Ascospores Created inside a special fungal sac or ascus Formed when two different strains or sexes join, producing an offspring of terminal cells called dikaryons (diploid nuclei) → Differentiate and enlarge to form asci, which undergo meiosis and mitosis to form 4-8 haploid nuclei which mature into ascospores Ascocarp: fruiting body that holds asci → Present in some species only Figure 33. Basidiospore Formation Figure 30. Ascospores. Figure 34. Basidiospores in the mushroom HS 202 Fundamentals of Mycology 8 of 17 V. FUNGAL CLASSIFICATION ▪ Eventually found to be genetically diverse using molecular methods A. PREVIOUS CLASSIFICATION RULES → Occur in many genera, many of which have now been Fungi were previously classified based on type of sexual spores renamed into revised identification and taxonomy each species produced ▪ E.g., Aspergillus, Fusarium, etc. Table 4. Classification based on known sexual state (teleomorph) Phylum Type of Sexual Spores Zygomycota Zygospores Ascomycota Ascospores Basidiomycota Basidiospores Deuteromycota Special classification for fungi whose sexual phase has not been observed Temporary classification created out of convenience Also referred to as “fungi imperfecti” → In contrast to sexual or teleomorph Figure 36. Aspergillus lentulus (R) as cryptic species of Aspergillus fumigatus (L) state, which were considered as the Molecular studies looking at broader relationships in fungi have “perfect state” found that some older morphological groupings are incorrect → Asexual or anamorph state was → E.g., Phylum Zygomycota referred to as the “imperfect state” ▪ NO longer considered a valid taxon because its members B. CURRENT CLASSIFICATION RULES are NOT monophyletic (cannot be traced to a single common ancestor) Classification Based on Morphological Features ▪ Phylum was removed Fungi are currently classified based on microscopic features and ▪ Previous members reclassified into different phyla reproductive structures observed, Names of clinically-relevant fungi have also been changed → Particularly the sexual structures of the fungi Mitosporic fungi Table 5. Old and new names of clinically-relevant fungi → NO sexual reproductive cycle is observed Old Name New Name → Classified using cladistic analysis Absidia corymbifera Lichtheimia corymbifera ▪ Genetic sequences are compared with other fungi with Penicillium marneffei Talaromyces marneffei previously-observed sexual phase Scedosporium prolificans Lomentospora prolificans Aspergillus fumigatus Aspergillus fumigatus species Heitman (2011) complex, Aspergillus lentulus General theme that has emerged from fungal genomics is that (cryptic species) there appear to be few, if any, truly asexual fungi Ramichloridium mackenzei Rhinocladiella mackenzei → Instead, each genome has revealed, even for anamorphic fungi with no known sexual cycle, that the machinery for C. MEDICALLY-IMPORTANT PHYLA both mating and meiosis, including the mating type locus, Medically-important phyla is conserved → Ascomycota We are drawn to the conclusion that the vast majority of → Basidiomycota fungi, perhaps even all, have a sexual nature that in many → Mucoromycota cases remains to be discovered under laboratory conditions → Glomeromycota → For pathogenic fungi, these sexual cycles are often rare → Basidiobolomycota and cryptic, leading to clonal populations punctuated by → Entomophtoromycota limited recombination, with broad implications for the → There may be a 7th fungal phyla evolution of eukaryotic microbial pathogens including fungi, Phylum Deuteromycota has been removed parasites, and oomycetes Members of Zygomycota have been reclassified to Mucoromycota, Glomeromycota, Basidiobolomycota, and Changes in Classification and Naming of Fungi Entomophthoromycota Molecular methods make use of internal transcribed spacer region 1 (ITS) and other genes to identify and classify fungi Phylum Ascomycota → Led to changes in classification and naming of fungi Contains the most number of fungi that are medically-relevant 3 subphyla, 3,200 genera, 32,000 species Examples → Pneumocystis → Candida → Aspergillus → Penicillium → Sporothrix → Dermatophytes → Endemic fungi Figure 35. Molecular-based methods to identify and classify fungi using ITS Phylum Basidiomycota Cryptic species Approximately 22,000 species → Fungi previously considered to be isolates of the same species Examples → Manifest low morphological disparity but considerable → Malassezia genetic disparity → Cryptococcus ▪ Difficult to distinguish morphologically → Trichosporon → Rhodotorula HS 202 Fundamentals of Mycology 9 of 17 → Hyalohyphomycosis Phylum Mucoromycota ▪ Infection caused by transparent hyaline (transparent) fungi Etiologic agents of mucormycosis or black fungus infection 1992: the International Society for Human and Animal Mycology → NOT actually “black fungi” proposed that instead of naming diseases according to the → Term comes from fact that these fungi cause necrotic lesions pathogen causing the disease, we should name fungal diseases which are dark or black in color accordingly: → Members of this phylum are not actually darkly-pigmented → “Pathology A due to Fungus B” ▪ Members are hyaline (transparent), but some structures may → Instead of “Aspergillosis”, we can say: have dark pigment ▪ Pneumonia due to Aspergillus Examples ▪ Rhinosinusitis due to Aspergillus → Mucor → This naming convention has NOT yet caught on → Rhizomucor → Rhizopus VI. FUNGAL DIAGNOSTIC PROCEDURES → Cunninghamella High index of suspicion is needed to diagnose fungal diseases, → Lichtheimia → Including the recognition of certain risk factors that lead to → Saksenaea them Phylum Enteromophthoromycota Common fungal diagnostic procedures: → Direct microscopy E.g., Conidiobolus causing conidiobolomycosis → Culture Phylum Basidiobolomycota → Histopathology E.g., Basidiobolus → Immunodiagnostic techniques D. RENAMING OF FUNGI → Antigen detection → Antibody detection Necessary due to the reclassification of fungi in light of more → Molecular diagnosis detailed investigation of its characteristics In the past, fungi were named according to morphological and A. DIRECT MICROSCOPY physical characteristics Direct examination methods are the simplest in terms of sample With the advent of genome sequencing, gene sequences of preparation for microscopic examination some fungi have been noted to be more similar with other fungi, Provides rapid diagnosis and information regarding possible requiring a change in genus name need for treatment in a patient → Eg., Reclassification of Pneumocystis from Kingdom Protista to → KOH smear can be done within an hour Kingdom Fungi ▪ Compared to a specimen culture, which can take days or ▪ Thought to be a protozoa due to morphology, lack of weeks in order to isolate and identify organisms from a ergosterol in cell membrane, and response to antiprotozoal specimen drugs Determines significance of an organism that may later be ▪ Genetic analysis showed that it is more akin to Fungi identified in a culture Fungi naming conventions also previously followed the → E.g., Fungus isolated in culture can be determined as International Code of Botanical Nomenclature pathogenic or simply part of the normal flora → Despite the fact that fungi are clearly NOT plants Directs the need for special culture media, as well as the need → Reason for having separate nomenclature for anamorph and for additional specimens from other sites and/or serological teleomorph states tests ▪ Given different names because fungi were named at Detects and identifies fungi that do not grow in vitro different times → E.g., Pneumocystis ▪ People did not realize that both nomenclatures were actually Common techniques done for direct microscopic examination in referring to the same organism medical mycology → If teleomorph state is known: → KOH smear ▪ Genus and species of teleomorph form takes priority over → India ink the anamorph name Table 6. Advantages and disadvantages of direct microscopy → Anamorph name is used in clinical and laboratory practice Advantages Disadvantages ▪ Asexual state is more commonly encountered in these Quick Insensitive settings Cheap Only small amounts of each One Fungus = One Name Initiative (2013) sample examined → Proposed revising nomenclature to prevent using two distinct Uses widely available Does NOT allow identification names when referring to the same organism equipment of the causative fungus without Renaming Fungal Diseases culture Previous nomenclature according to the generic name of the Can help with the interpretation Staff require training to become causal organism of the disease of culture results competent in recognizing → E.g., Aspergillosis, Candidiasis, Sporotrichosis specific structures → Becomes problematic when names and classifications of KOH Smear organisms change ▪ A change in the name of the phylum or species should Used to visualize fungal elements in specimens involving skin accordingly change the name of the disease scrapings, nail and hair clippings ▪ E.g., Zygomycosis caused by Phylum Zygomycota is now KOH is alkaline called Mucormycosis → Dissolves keratinous debris Previously, mycotic diseases of similar origin also had the same → Fungi possess chitin on their cell walls and are refractory to names alkaline treatment → Phaeohyphomycosis ▪ Allows fungal elements to stand out ▪ Infection caused by dematiaceous (darkly pigmented) fungi HS 202 Fundamentals of Mycology 10 of 17 KOH Smear Procedure Begins with sterilization of area of specimen collection → Sterile technique is important in specimen collection for fungal diagnostic procedures ▪ Especially applicable for skin surfaces, nails, and hair which may be contaminated by saprobic fungi and bacteria → Skin surface should be swabbed with 70% ethanol and allowed to air dry before sampling Sampling involves scraping the skin surface to remove skin scales or hair that contains the fungus Scales are placed on the slide, to which a drop or two of KOH is added to the specimen → Allowed to sit for an hour or so to allow KOH to digest keratinous debris Slide is visualized under the microscope Figure 37. Transparent fungal hyphae (refractile) using KOH smear VIDEO: The KOH Exam Applications of KOH Smear Method for microscopically preparing and examining a KOH smear can be requested to confirm clinical diagnosis specimen from the skin, hair, or nails for the presence of along with clinical presentation fungal elements Fast, simple, sensitive, and inexpensive test that can be Tinea corporis or “Buni” performed in the clinic setting Erythematous, annular, scaly plaques KOH causes separation and gradual destruction of stratum Cutaneous mycosis corneum cells, making it easier to visualize foreign materials → Involvement of keratinized layers of skin, hair, nails like hyphae and spores Similar to eczematous lesions (itchy, red lesions that have nothing Fungal elements take longer to be affected by the alkaline to do with fungi) solution Skin scrapings are obtained from edges/periphery of the skin → Will be degraded eventually so prepared specimens must lesions because it is where the active fungus is found be analyzed promptly. Pigmented hyphae are seen under the microscope Use when suspecting superficial fungal infections: Pityriasis versicolor or “An-an” → Red scaly rashes Multiple hypopigmented patches ▪ May or may not have central clearance Superficial mycosis → Rashes that do not respond to topical steroids → Fungus is present only on the most superficial areas of the → Tinea pedis/ Tinea cruris skin (stratum corneum) → Nail infections (onychomycosis) Scrape at the whole “white” or hypopigmented patch area → Hair loss with scaling No signs of inflammation Materials → Patients usually do not complain of itchiness → Rubbing alcohol wipes Skin scraping may be obtained via “Scotch-tape method” or → Scalpel (10- or 15-blade) scrape using blunt end of a scalpel → Toothbrush/ cotton swab “Spaghetti and meatballs” configuration are typical when viewed ▪ When swabbing the scalp of a child under the microscope → Nail clipper or small curette → Spaghetti: pseudohyphae → Nail infection → Meatballs: oval budding cells → Glass slides, cover slips → KOH solution ▪ 10% KOH – skin samples, 20% - nail specimens ▪ Stain (Chicago sky blue or chlorazol black) − Highlighting fungal walls makes them easier to see (helpful with nail specimens) → Fungal culture plate/slant/sterile specimen container → Microscope Make sure the patient is seated in a well-lit room and choose an appropriate site for specimen collection Figure 38. Tinea corporis (L) and Pityriasis versicolor (R) lesions of the skin Specimen Collection Skin Scales → Collected from border of the lesion, adjacent to normal appearing skin → After rubbing with alcohol, collect loosely adherent skin by scraping perpendicular to skin surface ▪ Do not scrape hard enough to draw blood → Collect scales on the microscope slides ▪ Hold the slide just underneath as you scrape to collect the scales ▪ If vesicles are present as in bullous tinea pedis, scrape Figure 39. Typical microscopic picture of Tinea corporis after KOH smear (L); and on the blister roof for fungal elements (R) classic “spaghetti and meatballs” configuration of Pityriasis versicolor after KOH smear ▪ If the scales adhere to the blade, gently wipe the blade at the edge of the slide to transfer the scales HS 202 Fundamentals of Mycology 11 of 17 → Using a plain glass coverslip, heap the fine skin scale → Adjusting the fine focus up and down, the hyphae scrapings into an area small enough to fit under a coverslip appearance also shift (from looking darker to lighter with → Place the cover slip over the piled up scales and proceed to dark edges) the microscope area Covers multiple squamous cells ▪ Putting the coverslip over the specimen even without the Uniform thickness and parallel walls liquid will keep the scales from flying off the slide May have septated walls ▪ Some providers prefer to apply the solution in an exam May be branching room and put the cover slip on to avoid losing specimen Always cross the cell membranes of the remnant ghost like in transit epithelial cells when the fine focus knob is adjusted Nails → Clipping part of the nail to be sent to the pathology lab to be evaluated with PAS or other fungal stains is the most sensitive method of diagnosing nail fungus but is also the most expensive ▪ KOH preparation in the clinic is the least expensive method that can be done immediately → Wipe clean with alcohol wipe ▪ Best specimens come from beneath the nail plate, sampling the subungual debris → Scrape out debris from the nail bed after cutting back a Figure 40. Hyphae/Dermatophytes portion of the affected nail ▪ In case the specimen requires PAS or other fungal stain False Positives analysis Mosaicism → Collect specimen on microscope slides → Bubbles around cell edges (lipid drops surrounding outside → Process it the same way as a skin specimen of stratum corneum cells) Scalp → Form a non-uniform mosaic pattern like a chicken wire → Performed when suspecting Tinea capitis due to a patch of fence or irregular polygons with lines that do not cross cell hair loss or scaling membranes → Wipe with rubbing alcohol Fibers → Remove few broken pieces of hair from scalp and place on → Larger than hyphae and often twisted, non-uniform slide diameter, non-parallel walls, lack of septate, do not cross → May use a cotton swab or toothbrush instead of scalpel cell membranes for children to scrape scales from scalp. Be sure to include broken pieces of hair on fungal agar → Fungal culture also recommended for tinea capitis in order to direct the therapy ▪ Rubbing a sterile cotton swab in a scant amount of fluid on top of a fungal plate/slant or with a small amount of water then rubbing it vigorously on the affected part → improve the yield of the fungal culture → KOH Specimen Processing Figure 41. Mosaicism (Left), Fibers (Right) Place cover slip Dimorphic Yeast (Tinea Versicolor) Apply several drops of KOH (with or without fungal stain) immediately adjacent to edge of cover slip Short, linear hyphae like structures and clusters of uniformly The solution will be drawn under the cover slip. sized round spores → By capillary action Spaghetti and meatball like appearance Tilt the slide (about 10o) and gently apply pressure to loosen Fungal stains (chlorazol black) makes them appear blue or the bubbles formed under the cover slip, OR place solution green over specimen, gently drop coverslip and blot the edge Heat is generally NOT necessary → However, general application of heat can help accelerate dissolving the keratinocytes ▪ 10 minutes is enough time to dissolve the keratinocytes, longer may be needed for nail specimens Fungal stains with KOH are better visualized Microscopy Use microscopy with low intensity light on 10x magnification (high power or 40x is rarely needed) Figure 42. Dimorphic Yeast Use coarse focus to look at a cluster of epithelial cells or hair Tinea Capitis (Spores) debris In the hair, one looks for presence of spores rather than Use fine focus to focus in and out as you move along hyphae Perform a systematic search of the area under the coverslip Can be found inside the hair shaft (endothrix) or outside the Hyphae / dermatophytes hair (ectothrix) Long, straight or curved, filamentous structures Refractile (change how they bend light) HS 202 Fundamentals of Mycology 12 of 17 Figure 43. Spores in Hair False Negatives Recent use of antifungals Inadequate specimen analyzed Lack of experience of the user → If there is lack of experience to diagnose, collect around ⅛ of a teaspoon of fine scale into a sterile specimen cup and properly label before submitting to the Microbiology Figure 45. Negative staining of cryptococcus using India Ink laboratory B. HISTOPATHOLOGY Calcofluor White Another form of microscopy Binds to chitin in the fungal cell wall Sample preparation steps: When illuminated in the correct wavelength of fluorescent light, → Fixed in formalin fungal structures will fluoresce and show up much more clearly → Thin sections of the tissue samples are cut in contrast to surrounding material → Various stains are applied to highlight features that may be present in the fungus or tissue reaction to it Allows us to determine that fungi are not merely colonizing the area but invading the tissue Samples used in Histopathology: → Cytologic preparations → Fine needle aspirates → Tissues → Body fluids → Exudates If fungal disease is expected, good to add: → Gomori Methenamine Silver (GMS), Mucicarmine, or Fonatana-Masson stains It is NOT possible to specifically identify the fungus under histopathology → Only helps suspect a certain fungus over another Stains Routine histological stains can detect but are NOT optimized for Figure 44. Fungal hyphae (fluoresce) using Calcofluor white fungal cells: → If only small amounts of fungi, can easily be missed India Ink (Negative Staining) Used to detect presence of encapsulated cryptococcal cells in Hematoxylin-Eosin (H&E) CSF → In suspected cases of cryptococcal meningitis Some dyes like lactophenol cotton blue stain the fungal cells contrasting with the background, allowing fungi to be seen In contrast, India ink darkens the background rather than the cell → Does NOT stain capsule of the cell, allowing it to be seen as a clear halo around the yeast cell ▪ This is why India Ink is referred to as a negative stain Figure 46. H&E stain of fungal elements (Aspergillus with acute-angle branching ▪ Halo around yeast cell: cryptococcal capsule of dichotomous hyphae) Gram stains may be used for bacteria, fungi and yeasts → Yeasts are Gram-positive Figure 47. H&E stain revealing non-septated wide open-angle branching hyphae infiltrating blood vessels (typical of agents of Mucormycosis) HS 202 Fundamentals of Mycology 13 of 17 Periodic Acid-Schiff (PAS) patients if they are prone to Detects glycogen and stains fungal cell walls pink bleeding CANNOT provide full identification of the etiologic agent C. CULTURE Uses, Advantages, and Disadvantages Identification of fungi for the purposes of: → Developing the treatment plan ▪ Knowledge of the pathogen influence the choice of Figure 48. PAS stain of a nail sample from a patient with Onychomycosis, with dark pink-staining fungal elements periodic acid schiff stain. Keratinous layer of nails diagnostic tests, medical and surgical procedures, as well with fungal elements. as antifungal therapy Gomori Methenamine Silver (GMS) → Investigating outbreaks ▪ To identify the fungal culprit when one is investigating Fungus-specific stain outbreaks Outlines fungal elements black against a blue green background ▪ Important to identify the source and find ways to prevent future infection ▪ The same fungus that was isolated from the patients should also be isolated from the suspected sources → Determining the susceptibility to antifungal agents ▪ Identifying the fungus will provide the appropriate antifungal medicines for the patient ▪ Some fungi are more responsive to certain antifungals → Estimating the significance of fungi generally considered to be opportunists or saprobes Figure 49. GMS stain of Aspergillus with acute-angle branching ▪ Possible to isolate fungi that are laboratory contaminants since they are ubiquitous like Aspergillus Mucicarmine − However, these fungi may be the cause of the patient’s Detects mucin disease if the patient is immunocompromised or has Stains inner layer of Cryptococcus capsule red some risk factors ▪ Identifying the fungi gives an idea of its significance especially if considered as an opportunist or saprobe Culture is a traditional method for diagnosis of fungal infections → Samples are placed on agar plate or liquid media → Incubated at an appropriate temperature and duration for the fungi to grow Media Used in Culture Sabouraud’s Dextrose Agar Figure 50. Mucicarmine stain of a lung of a patient with AIDS showing presence of Most common agar used in mycology Cryptococcus Selective for fungi because of its acidic pH and high sugar Fontana-Masson Stain concentration Stains melanin → 4% dextrose, 1% peptone, 2% agar, pH 5.5 Reacts with Cryptococcus and many fungi Will support growth of many fungi including Candida, Aspergillus, Cryptococcus and Fusarium → Candida and Aspergillus only need a few days to grow Cryptococcus and dimorphic fungi require long incubation period Allows the growth of fungal contaminants which can be fast growing and overgrow the actual fungal agent because some fungi grow slowly Mycobiotic Agar 2% glucose, 1% neopeptone, 2% agar + cycloheximide + chloramphenicol → Cycloheximide/Actidione: inhibits growth of saprobic fungi Figure 51. Cryptococcus stained with Fontana-Masson Stain ▪ Does NOT inhibit growth of dermatophytes and other Advantages vs Disadvantages of Histopathology endemic fungi Table 7. Advantages vs Disadvantages of Histopathology → Chloramphenicol: controls growth of bacterial Advantages Disadvantages contaminants[2025 Trans] More sensitive than direct field May take a long time to obtain Can be used for dermatophytes and other pathogens (EXCEPT microscopy as it can highlight results Cryptococcus, Aspergillus, and Candida) specific fungal structures → These three fungi are inhibited by cycloheximide One of the criteria for proven Requires specialist equipment → Dermatophytes are usually incubated for 2-4 weeks at 25-30°C fungal disease and staff to perform staining → Candida and Aspergillus only need a few days to grow and to interpret results → Cryptococcus and dimorphic fungi need long incubation Can be difficult to obtain periods tissues and biopsies in some HS 202 Fundamentals of Mycology 14 of 17 Specialized Media Table 9. Macroconidia and Microconidia of the Three Dermatophyte Genera Some fungi, like Malassezia, require specialized media Genus Macroconidia Microconidia → Lipophilic yeast requiring the addition of olive oil or other Trichophyton Rare, smooth, Numerous, long-chain fatty acids in the culture medium thin-walled spherical, teardrop Table 8. Advantages vs Disadvantages of Fungal Culture or peg shaped Advantages Disadvantages Microsporum Numerous, large, Rare Allows identification of fungus Slow thick, rough-walled Susceptibility testing of the Staff require experience to Epidermophyton Smooth-walled, Absent fungus can be performed become competent in borne in clusters of identifying fungi two or three Used for any clinical sample Insensitive Trichophyton rubrum Is one of the requirements for May isolate contaminants Microscopic Characteristics proven fungal disease Cannot always differentiate → Spores are small isolates from harmless → Numerous, small, piriform (pear shaped) microconidia commensals → Only some macroconidia Cannot be used for organisms Colony Characteristics such as Pneumocystis which → Downy white obverse cannot currently be cultured → Blood-red reverse → Anthropophilic (confined to humans) D. FUNGAL IDENTIFICATION → Common cause of tinea cruris, tinea pedis, and tinea corporis, Fungi growth on culture are classified based on morphologic tinea manuum, and tinea unguium characteristics → Macroscopic ▪ Colonial Form ▪ Shape and Texture ▪ Surface color and pigmentation → Microscopic ▪ Conidia type and color − Spore formed via asexual reproduction of the fungi at the tip of a specialized hypha[2025 Trans] ▪ Presence or absence of capsule Figure 52. Microscopic image of Trichophyton rubrum ▪ Presence or absence of cross walls in hyphae ▪ Pseudohyphae → Biochemical Tests ▪ Differentiate fungi from one another ▪ Yeasts (Fermentation of sugar) Application: Identification of Dermatophytes Derived from Greek; “dermatos” = skin and “phyte” = plant Some species grow in the soil; others grow in animals Keratinophilic fungi that cause cutaneous mycosis → Can infect the keratinous layer of skin, hair, and nails → Causes some forms of cutaneous mycosis, dermatophytosis, and tinea or “ring-worm infections” Dermatophyte infections are called: tinea + a name to denote the Figure 53. Colonies of Trichophyton rubrum. (Up) Bloody-Red Reverse; (Down) Downy White Obverse body site infected → Tinea capitis: scalp → Tinea corporis: body ▪ Specifically areas of glabrous or hairless skin → Tinea manuum: hands (palm) → Tinea cruris: intertriginous areas/groin ▪ Aka. “Jock itch” → Tinea pedis: feet ▪ Aka. “Athlete’s foot” Figure 54. (L) Nail infected with T. rubrum; (R) Arthrospores → Tinea unguium: toenails or fingernails ▪ Specifically caused by dermatophytes Trichophyton mentagrophytes Onychomycosis Microscopic Characteristics → General term for fungal infection in the nails caused by both → Numerous microconidia dermatophytes and non-dermatophytes → Some macroconidia 3 Genera that cause ring-worm infections: → Grape-like spores with spiral-shaped hyphae → Trichophyton ▪ Spiral hyphae similar to “grape-vines” where the → Microsporum microconidia/spores are the grapes and the spiral shaped → Epidermophyton hyphae are the vines The 3 genera of ring-worms can be differentiated based on their Colony Characteristics macroconidia and microconidia → White-cream colonies HS 202 Fundamentals of Mycology 15 of 17 Colony Characteristics: → Colonies are usually flat and velvety with a tan to olive green tinge[2025 Trans] Figure 55. Trichophyton mentagrophytes (L) Microscopic Image; (R) Colony Appearance Figure 59. Epidermophyton floccosum (L) Microscopic Image; (R) Colony Appearance Microsporum canis Trichophyton erinacei Microscopic Characteristics Microscopic Characteristics: → Numerous spindle-shaped macroconidia with knob-shaped → No macroconidia ends → Many microconidia → Some microconidia Colonial Characteristics Colony Characteristics → Yellow reverse with pigment similar to Microsporum canis → Canary yellow obverse → Thus, despite macroscopic similarities, the microscopic → Produces white cottony colonies with yellowish outer differences allow us to determine the proper species pigment[2025 Trans] Zoophilic dermatophyte usually present in hedgehogs → Causes tinea corporis[2025 Trans] → Humans get infected by handling hedgehogs that has been → Natural host are dogs and cats[2025 Trans] infected with Trichophyton erinacei Figure 60. Trichophyton erinacei (L) Microscopic Image; (R) Colony Figure 56. Microscopic Image of Microsporum canis Appearance New Additional Genera of Dermatophytes Nannizzia Lophyton Arthroderma Ctenomyces VII. HOW FUNGI CAUSE DISEASE Epidemiological triad is the traditional model for infectious diseases, consisting of: Figure 57. Colonies if Microsporum canis, showing Canary Yellow Obverse → Fungus/fungal agent → Host/susceptible host Microsporum gypseum → Opportunity (to bring host and fungus together) Microscopic Characteristics Fungal disease results from interaction between fungus and → Numerous elliptical macroconidia (compared with spindle susceptible host shape and knobbed ends of M. canis) → In an environment that supports transmission of the fungus → Very few microconidia from a source to the host E.g. Analysis of mucormycosis: → Fungus: Mucoromycota fungi → Host: Patients with recent recoveries from COVID-19 who are immunocompromised or with uncontrolled diabetes → Opportunity: Immunocompromised state provides the opportunity ▪ Fungus is ubiquitous in the environment and can come in Figure 58. Microsporun gypseum (L) Microscopic Image; (R) Colony contact with the host at any time Appearance Epidermophyton floccosum Microscopic Characteristics: → No microconidia → Many macroconidia → Smooth-walled, club-shaped macroconidia formed in small clusters[2025 Trans] HS 202 Fundamentals of Mycology 16 of 17 Cutaneous Infection of the keratinized layer of the skin, hair, and nails Examples: → Tinea/ringworm infections (tinea capitis, tinea pedis etc.) and other dermatophyte infections → Mucocutaneous candidiasis Subcutaneous Infection of the deeper layers of the skin including the cornea, muscle, and connective tissues Endemic mycoses Fungi usually found in localized and limited regions of the world Can affect any organ in the body but usually infect the lungs Figure 61. Epidemiological Triad primarily then spread to other tissues Caused by classic dimorphic fungal pathogens A. HOST RISK FACTORS → Histoplasma capsulatum Main risk factors → Blastomyces dermatitidis → Immunodeficiency Diseases (whether inborn or acquired) → Coccidioides immitis → Immunosuppressive therapies for cancer, organ transplant → Paracoccidioides brasiliensis maintenance, and autoimmune diseases → Talaromyces marneffei Additional risk factors → Extremes of age Systemic mycoses → Burns Refers to the mycosis caused by true pathogenic fungi among → Chronic respiratory disease which are endemic fungi → Debilitating illness dialysis Opportunistic mycoses → Endocrine disorders Infections attributed to fungi that normally live as commensals → Intensive care requiring parenteral nutriti

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