Introduction to Mycology Lecture Notes PDF

MICROBIOLOGY-PARASITOLOGY INTRODUCTION TO MYCOLOGY Lecture 23.2 (11/19/2024) Dr. Tapia Finally, cytokinesis occurs forming a new daughter cell....

MICROBIOLOGY-PARASITOLOGY INTRODUCTION TO MYCOLOGY Lecture 23.2 (11/19/2024) Dr. Tapia Finally, cytokinesis occurs forming a new daughter cell. Buds may be solitary or in chains. WHAT IS FUNGI? Some yeasts multiply by fission. Events in the yeast cell are eukaryotic organisms that do not contain chlorophyll, cycle are finely orchestrated. but have cell walls, filamentous structures, and produce AKA Blastoconidia spores. These organisms grow as saprophytes and decompose dead organic matter. FISSION VS BUDDING About 300 species are presently known to be pathogenic for man. 4 TYPES OF MYCOTIC DISEASE HYPERSENSITIVITY ○ an allergic reaction to molds and spores MYCOTOXICOSIS ○ poisoning of man and animals by feeds and food products contaminated by fungi which produce toxins from the grain substrate MYCETISMUS ○ the ingestion of preformed toxin (mushroom poisoning) CANDIDA ALBICANS INFECTION BY A FUNGUS a yeast-like fungus that grows in a variety of forms: yeast, ○ we shall be concerned only with the last type pseudohyphae (a transitional form), and hyphae. (Infection by a fungus) Pseudohyphae can give rise to yeast cells by apical or FUNGI ARE SIMPLE EUKARYOTES WITH lateral budding. CHITIN-CONTAINING RIGID CELL WALLS Yeast can also convert to a hyphal form. All three forms are found in tissue invaded by the fungus. Medical mycology is mostly concerned with microfungi, specifically zoopathogenic fungi. They grow in two forms: ○ MOLD ○ YEAST WHERE DO FUNGI GROW? In contrast to plants and algae, fungi are “heterotrophs”: they cannot make their own food and instead obtain it by MOLD uptake of organic matter. A non-motile thallus constructed of apically elongating Plant pathogenic fungi cause damage to food crops, walled filaments (hyphae). trees, and other plants. A web of filaments constitutes a mycelium Some fungi are “commensals” living on the mucous membranes and skin of mammalian hosts. Various estimates of the number of fungal species range upwards of 1 million (Heitman, 2011). About 300 species are known human pathogens but any fungus capable of growth at 37 degrees C is potentially pathogenic in a suitably compromised host. DIFFERENCES BETWEEN PLANT CELL & FUNGAL CELL YEAST A unicellular fungus that reproduces by budding. Small, round projections from the ellipsoid shaped parent cell are produced during mitosis followed by migration of the nucleus and cytoplasm into the bud.  LECTURE 23.2: INTRODUCTION TO MYCOLOGY REPRODUCTION Bacteria reproduce by binary fission to two identical daughter cells. Fungi reproduce in various ways: ○ Budding ○ linear extension of the growing tips of hyphae ○ by the production of various types of spores, which in fungi are called conidia Size of Representative Bacteria, Viruses, Yeasts, and Note: This is an additional picture lifted from the net where doc Eukaryotic Cells got the first pic CELLULAR ORGANELLES Capsule ○ Polysaccharide capsules of bacterial pathogens are virulence factors. ○ A major fungal pathogen, Cryptococcus neoformans (also C. gattii) has such a capsule. Cell walls ○ Bacterial cell walls contain peptidoglycan, lipopolysaccharide, and teichoic acid. ○ Fungal walls contain glucan, mannan, and chitin. Cytoplasmic membranes ○ Membranes of fungi contain ergosterol, not present in From Doc: bacteria. Fungi are larger than viruses and bacteria. ○ The synthesis of and binding to ergosterol are potent antifungal drug targets. SIZE Episomes and plasmids The volume of a typical bacterium, ○ Bacterial resistance to antibiotics is mediated by E. coli , is 1 µm3, diameter 1 µm, extrachromosomal DNA and length ~2 µm. no such mechanisms are known to exist in fungi. In contrast, A budding yeast cell has Nucleus a V = 42 µm3 (haploid strain) and V= ~82 µm3 (diploid strain), diameter ○ As eukaryotes, fungal genes are organized into 3-6 µm. chromosomes, enclosed in a nuclear membrane. These differences in cellular ○ Baker’s yeast, Saccharomyces, has 16 chromosomes. organization help explain why ○ Bacteria, as prokaryotes, have a single chromosome, antibiotics active against bacteria not enclosed by a membrane, but packed into part of are, with exceptions, inactive the cytoplasm, the nucleoid, occupying ~1/3 of the against fungi. On the other hand, similarities between the organization cell volume. and metabolism of fungal cells and human cells Ribosomes complicate the development of antifungal agents with ○ Bacteria = 30s + 50s form 70s ribosomes selective toxicity for fungi. ○ fungi = 40s + 60s form 80s ribosomes. CLASSIFICATION OF FUNGI BASED ON THE PRIMARY SITE Dimorphism OF PATHOLOGY ○ Some fungi undergo morphogenesis into two forms, such as yeast and mold forms. ○ This feature is absent in bacteria. METABOLISM Bacteria are aerobic or anaerobic; fungi during tissue invasion of humans are aerobic, but metabolism by fungi under anaerobic conditions is known, e.g., fermentation by Saccharomyces beer yeast occurs at low oxygen concentrations. Energy transduction in bacteria occurs at the cell membrane; in fungi, mitochondria perform this function. Transcribed by Batch Dextera MICRO-PARA Lecture 23|2  LECTURE 23.2: INTRODUCTION TO MYCOLOGY SUPERFICIAL MYCOSES Rhizopus typified by Pityriasis versicolor, Mucormycosis is caused by various Mucorales species caused by Malassezia species. especially Rhizopus oryzae (syn: R. arrhizus) including: This yeast grows on the non-living ○ Rhinocerebral mucormycosis occurring in diabetic keratinized outer layer of the skin of ketoacidosis humans and dogs, also includes dandruff and other forms of seborrheic dermatitis, rarely the cause of invasive disease. CUTANEOUS MYCOSES Dermatophytosis, also known as ringworm, is caused by Trichophyton and Microsporum species. Fusarium They are restricted to grow on the Fusarium species mycosis includes: non-living keratinized outer layer of ○ Sino-pulmonary-disseminated disease skin of humans, dogs, and cats. ○ Keratitis Medical terminology assigns a Occurring in immune normal persons name to the diseases according to due to penetrating injury or contaminated contact the body site affected: lenses ○ Tinea capitis is scalp ringworm, ○ Tinea cruris is jock itch ○ Tinea unguium fungal nail infections, etc. A completely different category is the cutaneous site of disseminated mycoses. Skin is a frequent site for disseminated blastomycosis. OPPORTUNISTIC Opportunistic Yeasts and Pneumocystis Scedosporium Candidiasis includes mucocutaneous and deep seated Scedosporium species cause: disease caused by C. albicans and non-albicans Candida ○ Pulmonary disseminated disease species yeasts. ○ Eumycetoma ○ Their ecologic niche is the skin and mucosae of Occuring in immune-normal persons warm-blooded animals and humans. most often resulting from injury during barefoot Cryptococcal meningo-encephalitis is caused by the labor environmental yeasts Cryptococcus neoformans and C. gattii. Pneumocystis pneumonia (“PCP”) is caused by Pneumocystis jirovecii, an obligate endogenous commensal of the human lung. Opportunistic Mold Disease Disease is encountered in debilitated or immunocompromised hosts. The causative agents are non-pigmented molds SUBCUTANEOUS MYCOSES OF IMPLANTATION ○ ubiquitous in the environment ○ cause disease when their conidia are inhaled by a Confined to the subcutaneous susceptible host or when the conidia alight on skin of tissue and systemic spread is burn patients or on wounds. rare. Following a penetrating injury Aspergillus fumigatus with thorns and splinters, these Invasive pulmonary aspergillosis is caused by Aspergillus agents can develop into deep, fumigatus and related species. ulcerated skin lesions, subcutaneous cysts, or slowly enlarging warty masses. Transcribed by Batch Dextera MICRO-PARA Lecture 23|3  LECTURE 23.2: INTRODUCTION TO MYCOLOGY Subcutaneous Mycoses Sporotrichosis is the most common human subcutaneous mycosis, worldwide in its distribution, also affecting cats. It is caused by the dimorphic fungus Sporothrix schenckii. Melanized fungi (formerly referred to as “dematiaceous”) From Doc: cause a spectrum of disease separated into three Dematiaceous- usually dark in appearance. categories When you rotate in Sotto, do not be surprised if you Three categories: encounter this type of fungi. These cases are real. In ○ Chromoblastomycosis Sotto, it is not as rare as you think it might be like described as warty, slow growing tumor-like Madura foot which can be seen in Sotto. That is why cutaneous-subcutaneous masses caused by when you become senior clerks, any case that you see is Fonsecaea pedrosoi, among other species, with really gold. the characteristic dimorphic tissue form of round Tumefaction is like a swelling of a body part. copper-colored muriform cells. DIMORPHIC FUNGI Some fungi have two growth forms such as certain soil-dwelling molds that are primary respiratory pathogens. ○ Their conidia (or spores) become airborne and, when inhaled, can survive and undergo morphogenesis to the pathogenic yeast form at 37 degrees C. Specimens, such as sputum, when plated on mycologic medium and incubated at 30 degrees C, grow as molds. ○ Phaeohyphomycosis As mold at room temperature in the laboratory and as a Caused by Exophiala dermatitidis budding yeast or as spherules either in tissue or at 37°C. a term derived from the histopathologic appearance of the fungi in From Doc: cutaneous-subcutaneous cysts: In other words, the yeast forms prefer the temperature dark yeast-like, pseudohyphae-like, or closer to us humans. variously shaped hyphae or a combination of 30’C near room temperature, so in the laboratory they forms. appear as molds. But inside the human body they appear primarily as yeast form. This category includes the commensal yeast, Candida albicans which in tissue invasion may assume ○ Eumycetoma conformations of yeast, pseudohyphae and true hyphae. The hallmark of this mycosis is a triad of: Dimorphism in fungal pathogens includes Coccidioides Tumefaction species, filamentous in the environment, converting to Swelling endosporulating spherules in the human or animal host. sinus tracts draining “grains” (masses of fungal hyphae) ○ occurring mostly on the extremities ○ i.e., “Madura foot” Causative agents are many including Scedosporium and Madurella species. Exposure follows puncture wounds during barefoot labor in the endemic tropical and From Doc: subtropical areas of the world. Dimorphic fungi= the transformers Transcribed by Batch Dextera MICRO-PARA Lecture 23|4  LECTURE 23.2: INTRODUCTION TO MYCOLOGY ENDEMIC MYCOSES CAUSED BY DIMORPHIC HOST FACTORS AFFECTING PATHOGENICITY ENVIRONMENTAL MOLDS Patients receiving immunosuppressive therapy for maintenance of a transplanted organ or stem cell Several soil inhabiting saprobic fungi have gained the transplant, cancer chemotherapy, autoimmune disease, capacity to parasitize mammals causing systemic and in persons living with HIV/AIDS. infection in immune-normal individuals. Prolonged ICU stay, on mechanical ventilation. Disease occurs in defined geographic areas, following Very young (< 1 mo.) or aged (>65 y) patients inhalation of conidia, beginning asymptomatically in the Travel to or residence in an endemic area where this fungi lungs and progressing to an influenza-like illness or might be prevalent pneumonia. Invasive diagnostic and surgical procedures: abdominal Fungi belonging to dimorphic environmental molds surgery, prosthetic implants, indwelling catheters, renal ○ Coccidioidomycosis dialysis. ○ Histoplasmosis Inborn or acquired deficits: chronic granulomatous ○ Paracoccidioidomycosis disease, cystic fibrosis, diabetes. ○ Sporotrichosis Occupational or recreational exposure: ○ Talaromycosis ○ Barefoot labor Once inhaled, these agents convert from mycelial (since ○ Gardeners exposed to thorny plants mosulod na siya sa lawas) to yeast or spherule form in the ○ Workers in demolition of old buildings host. ○ If the inhaled dose of conidia is high and the host From Doc: immune response is insufficient extrapulmonary dissemination can ensue. Common in VSMMC. Persistence, dormancy, and reactivation may also occur. Grab the opportunity to make it as a case report, if you’ll (Partly because they can transform. That’s why, it is also encounter any of this case/disease. hard to kill these microorganisms, them being dimorphic.) The agents are Dialysis - one of the very challenging cases. Patients may ○ Blastomyces dermatitidis have bacterial, viral or fungi infection. ○ Histoplasma capsulatum ○ Paracoccidioides brasiliensis In the history, take note of their occupation. ○ P. lutzii ○ Lacazia loboi MICROBIAL FACTORS ○ Coccidioides immitis ○ C. posadasii, A fungi itself may produce adhesins. ○ Talaromyces (formerly Penicillium) marneffei ○ Adherence to endothelial cells is a prime requisite for Each genus has its own predilection for various organs tissue invasion. which will be described in discussing the individual ○ Examples of fungal adhesins are: diseases. ALS of C. albicans Each disease is prominent in some areas. BAD1 of Blastomyces dermatitidis ○ Talaromyces (was Penicillium) marneffei is endemic in Biofilm formation on biomaterials. SE Asia, especially in Thailand, also in Southern China ○ You will find in this biofilms fungi with a capsule. and Hong Kong. Capsule ○ Alone among these endemic mycoses, T. marneffei “Shape shifters” rarely infects immune normal humans. ○ Fungi with several forms. Thermotolerance ○ Pathogenic fungi can grow at 37oC Melanin ○ Melanin in fungal cell walls makes them resistant to phagocytosis and killing. ○ That’s why we have a separate category for these diseases of fungi that have melanin ○ Ex. Cryptococcus neoformans, Paracoccidioides brasiliensis, Sporothrix schenckii Resistance to the oxidative burst of polymorphonuclear From Doc: neutrophilic granulocytes ○ Primary respiratory pathogens So in studying fungi, as in other organisms causing disease, ○ e.g.: Blastomyces, Histoplasma, Paracoccidioides, you have to remember the mode of transmission as well as in and Sporothrix, but not opportunistic fungi, e.g.: which parts of the world they are located because there are Candida species, can resist the effects of the active some fungi that can be found in certain countries/states. It oxygen radicals released during the respiratory burst. will help you in the diagnosis and in identifying the culprit. From Doc: A clue among fungi is that it is common among immunocompromised. Oxidative burst are strong defense, yet, some fungi are resistant to it. Transcribed by Batch Dextera MICRO-PARA Lecture 23|5  LECTURE 23.2: INTRODUCTION TO MYCOLOGY FISH. Fluorescent in situ hybridization has found an DIAGNOSIS application in smears of blood cultures. ○ Fluorescent labeled peptide-nucleic acid probes are SPECIMEN PROCESSING hybridized to fungal RNA. Very important! ○ The AdvanDX® Peptide Nucleic Acid in Situ Within 2 hours of collection, especially to urine specimens, Hybridization Yeast Traffic Light system is available which also may be stored at ≤ 24 h at 4 CO for microscopic detection of Candida species directly Exceptions are that hair, nails, and skin scrapings may be from blood culture bottles. stored up to 72 hours at room temperature before culture, From Doc: and they may be shipped by mail. CSF specimens are stored at 30oC and never refrigerated In our setting, it’s more of the special stains, not this high-end because CSF is a good culture medium and fungi will method like FISH. But, be familiar with this lang, enough to continue to replicate at room temperature or 30oC. know that it exists. Microscopic detection is facilitated when wet mounts of certain specimens are digested with 10% KOH ± CULTURE Calcofluor: ○ skin scrapings, hair, nails, corneal scrapings, and Definitive diagnosis requires culture and identification. wound exudates. Emmons modification of Sabouraud dextrose agar Visibility of fungal elements is increased when wet mounts (SDA-Emmons) are combined with fluorescent brightener, like Calcofluor. ○ extensively used for primary isolation of pathogenic Sputum specimens may require digestion with Mucolyse® fungi but Mucorales, black molds, dermatophytes, and (which contains dithiothreitol in phosphate buffer) to yeast have higher percentage recoveries when plated reduce viscosity. on inhibitory mold agar (Scognamiglio et al., 2010). Tissue obtained by biopsy or surgery is prepared for ○ Cycloheximide may be added to suppress saprobic culture by grinding or homogenization if Histoplasma is fungi but then a medium without cycloheximide suspected should be paired. ○ but for other fungi and Mucorales species, mincing ○ Chloramphenicol is often included to inhibit bacterial and/or finely slicing are preferred so as to not disrupt growth. hyphal elements. ○ If Histoplasma or Blastomyces are suspected an enriched medium such as brain-heart-infusion agar is HISTOPATHOLOGY indicated, bearing in mind that media supplemented Formalin fixed, paraffin embedded (FFPE) tissues are with cycloheximide will inhibit the yeast form of these treated with special fungal stains, or subject to fluorescent dimorphic fungi. antibodies, to reveal fungal elements. There are many other formulations of mycologic media. The H&E stain does not always tint the organism, but it Cultures of primary systemic dimorphic fungi, (e.g.: will stain inflammatory cells. Blastomyces, Histoplasma) are identified in the laboratory The Gomori methenamine silver (GMS) stain is used to by reveal fungi which stain black against a green background. ○ slide culture and/or tease mount methods revealing ○ A combined H&E-GMS stain variation will characteristic microscopic morphology and demonstrate both fungi and the inflammatory ○ importantly by DNA probe tests of their mold forms response. grown at 25-30 degrees C (AccuProbe, Hologic Inc., Asci (cysts) of Pneumocystis jirovecii in lung tissue, San Diego, CA) stained with methenamine silver and hematoxylin and ○ Recently MALDI-TOF has become an important, rapid, eosin (H&E). The walls of the cysts are stained black. and reliable tool for identification Other Special Fungal Stains for FFPE Tissue Sections Periodic Acid Schiff is a good general stain for fungi, and can be counterstained with hematoxylin to demonstrate tissue reactions. Mucicarmine will stain the capsule of Cryptococcus species. Fontana Masson stains fungal cell wall melanin. Fluorescent antibodies may be used for microscopy on fixed tissue sections but commercial reagents are scarce. ○ One good example is the Merifluor® Pneumocystis kit (Meridian Bioscience, Inc.) Transcribed by Batch Dextera MICRO-PARA Lecture 23|6  LECTURE 23.2: INTRODUCTION TO MYCOLOGY Fungi grow best at 30 degrees C but if that temp is not ○ Double immunodiffusion and complement fixation available 25 degrees C will suffice. usually detect IgG antibodies. Some EIA tests detect It is unnecessary to incubate an additional culture at 37 o both IgG and IgM antibodies. C unless there is reasonable suspicion of a thermally Ag Test (Antigen Test) dimorphic fungus. Cultures are not considered negative for growth until after Cryptococcal antigen test in CSF, plasma, or serum 4 weeks incubation. (CrAG® LFA, IMMY Corp, Norman, OK) Once a pure culture is obtained identification is made by ○ Uses a lateral flow “dipstick” test and is rapid, observing colonies and microscopic morphology for sensitive and, with sample dilutions, can indicate a molds. titer. Galactomannan antigenemia is detected in invasive IDENTIFICATIONS BASED ON DNA SEQUENCE AND aspergillosis (Platelia Aspergillus EIA, Bio-Rad.com) PROTEOMICS ANALYSIS Histoplasma polysaccharide Matrix Assisted Laser Desorption/Ionization- ○ Antigen test (HPA) is a good indicator of active Time-of-Flight-Mass Spectrometry (MALDI- TOF) invasive or disseminated histoplasmosis ○ With minimal sample preparation from small isolated MiraVista Diagnostics, Indianapolis IN colonies, MALDI-TOF MS produces a spectrum of Screening serum test for fungi in general: protein fragments of known molecular mass. ○ (1→3)-ß- D-glucan detection (Fungitell® Assay, ○ The resulting peptide mass fingerprint leads to faster, Associates of Cape Cod Inc.) even same day, clinical decisions. Exceptions are that the Fungitell assay does not ○ When an incubated sample becomes a visible colony detect this polysaccharide in fungi in the genus (~ 105 cfu) it can be subject to laser analysis, taking Cryptococcus, or in the order Mucorales. less than 10 min. TREATMENT An organism ID can then be made with little or no reduction in reliability. Pharmacology An end-to-end system including a database is the Medicine Mechanism BioTyper® (Bruker Daltonics). Structure Biochemistry and metabolism ————————————nothing follows———————————— Commercial Multiplex PCR Assays for rapid detection of fungal infections MicroSeq® Rapid Microbial Identification System (Life Technologies Inc.) Luminex® xMAP Fungal Assay FilmArray® (BioFire Diagnostics, A bioMérieux Company) T2 Candida (FDA approved) (T2 Biosystems) IRIDICA assay (IBIS/Abbot Molecular) (not available in the USA). SEROLOGY Ab Test (Antibody Test) Serology may be helpful when applied to a specific fungal disease. The efficacy of serology varies with different mycoses. The most common serologic tests for fungi are based on double immunodiffusion, complement fixation and enzyme immunoassays (EIA). Transcribed by Batch Dextera MICRO-PARA Lecture 23|7