Mycology and Virology PDF

| Topic # | [MLS 415] Mycology and Virology P1: INTRODUCTION TO MYCOLOGY Professor: Thynee Tago, RMT, MSMT Date: January 15, 2024 ETYMOLOGY MYCOLOGY Greek word: mukes → fungus Logia → study The study of fungus Seen in overripe fruits Grows in bread (beyond expiry date) FOOD FUNGAL CHARACTERI...

| Topic # | [MLS 415] Mycology and Virology P1: INTRODUCTION TO MYCOLOGY Professor: Thynee Tago, RMT, MSMT Date: January 15, 2024 ETYMOLOGY MYCOLOGY Greek word: mukes → fungus Logia → study The study of fungus Seen in overripe fruits Grows in bread (beyond expiry date) FOOD FUNGAL CHARACTERISTICS Eukaryotic ○ Believed to be true organisms ○ Eu - means true; true organisms or true cells ○ Because they have the genetic material encased in a membrane that is not found in bacteria Facultative anaerobic/strictly aerobic ○ Organisms that would acquire oxygen but some can survive without Gastronomic delights Penicillium Saccharomyces Chemotrophic; nutrition by absorption ○ Able to survive with different types of chemicals; and absorbs things within the environment - Non-photo-synthetic; achlorophyllos ○ May look like plants, but are nonphotosynthetic ○ Some have the ability to light up dark spaces ○ Nonphotosynthetic → does NOT have chlorophyll / chloroplast - Blue cheese → is not actually blue; and it only appears blue due to its veins; those are Fungal Mycelia (molds) These molds are necessary to achieve the palatable texture in cheese Yeast is needed for the growth of bread THERAPEUTICS/ANTIMICROBIALS pH 5-6 (Acidic) ○ In natural environments, survive in areas that are acidic High sugar concentration favors growth ○ They easily grow in people with high sugar levels ○ Many cases of diabetic people with fungal infections USES OF FUNGAL ORGANISMS SAPROPHYTES Pencillium notatum → Penicillin ○ P. notatum is a member under Penicillium chrysogenum In 2011, a DNA test was done on Penicillium chrysogenum and found out it was Penicillium rubens ○ The best source of Penicillin Tolypocladium inflatum → Cyclosporin ○ Bottom right picture ○ Cyclosporin is an antidepressant Widely distributed in nature Break down dead or decaying matter; saprophytic Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 1 NEGATIVE EFFECTS OF FUNGI PHYTOPATHOGEN Scarring after budding Every time a bud is stripped off from the mother cell, it will leave a scar. BUDDING YEAST Smuts ○ Pseudohyphae (Filamentous structure on the right image) The smuts get their name from a Germanic word for 'dirt' because of their dark, thick-walled, and dust-like teliospores Blights Rusts Dark spots Mycoses Scars are really evident ➔ ➔ ➔ ➔ Most common fungal infection: Ringworm infection (Dermatophytosis) (Topmost left) Includes athlete’s foot, jock’s itch Fingernails/toenails - Onychomycosis Oral Thrush - Candidiasis MORPHOLOGIC CLASSIFICATION OF FUNGI Yeast (unicellular) Mold (Multicellular) Dimorphic (1) YEAST Unicellular fungi MICROSCOPIC YEAST Oval to round Budding Bud (other terms) Blastospore Blastoconidia ➔ ➔ Process of Budding Buds arise as small cytoplasmic outgrowth from the parent cell Followed by the division of the nucleus into two One of the daughter nuclei migrates into the bud The bud enlarges and detaches from the parent cell by forming a constriction at the base ◆ scarring at the constriction point are evident on the surface of the parent cell ◆ number of scars indicate how many times the parent cell has undergone budding ◆ there are instances wherein the daughter cell does not separate from the parent cell even with a presence of constriction it simply extends forming a hyphal-like or filamentous structure known as the PSEUDOHYPHAE (False hyphae) The daughter cell matures into a new yeast cell ◆ generally smaller than the mother cell ◆ Can continue budding until it dies The parent cell remains intact; unlike in binary fission MACROSCOPIC YEAST Pasty, opaque, cream-colored colonies Much bigger compared to bacterial colony; sometimes it makes use of the entire media Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 2  MONOMORPHIC YEAST type of yeast that exists in a single form or morphology Candida albicans Cryptococcus neoformans Function: Specialized for reproduction, forming structures like mushrooms, sporangia, and producing spores for dispersal. MOLDS: Mycelia Surrounded by a very thick capsule Geotrichum candidum (2) MOLD Multicellular - made up of several cells Hypha [2-10 um] ○ These are the filaments that we see MICROSCOPIC (a) Hyphae/filaments ➔ ➔ When a spore lands in a conducive environment (soil) it can germinate into a hypha (looks like a “bean sprout) Eventually the hypha will extend and produce a network of hyphae called MYCELIUM MYCELIA Mycelia is divided based on where they are found A. Vegetative/Thallus Location: Underground in the substrate (soil, decaying matter). Function: Responsible for nutrient absorption, supporting fungal growth. B. Reproductive/Aerial Location: Aboveground, developing from the vegetative mycelium. Structure: Thread-like, tubular structures forming the body of the fungus. Appearance: Microscopically thin, elongated strands often branch out. Function: Constitutes the mycelium; involved in nutrient absorption and fungal growth. (b) Conidia Structure: Small, asexual reproductive structures. Appearance: Varied shapes (spherical, cylindrical, or irregular), often borne on specialized structures like conidiophores. Function: Serve as a means of asexual reproduction, capable of germinating into new fungal colonies. Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 3 ➔ SUBCUTANEOUS 1. Sporothrix schenckii SYSTEMIC 1. Histoplasma capsulatum MACROSCOPIC Cottony, wooly, velvety, granular, filamentous MONOMORPHIC MOLDS These organisms are considered TRUE MOLDS; maintain a consistent appearance regardless of their surroundings. Flower appearance; has buds Listed below are collectively known as DERMATOPHYTE ➔ Microsporum ➔ Epidermophyton ➔ Trichophyton 2. Blastomyces dermatitidis 3. Coccidioides immitis They are a group of fungi that infect and thrive on the skin, hair, or nails of humans and animals; common agent of Ringworm infections Belt appearance 4. DIMORPHIC FUNGI Paracoccidioides brasiliensis mariner’s wheel appearance OPPORTUNISTIC ➔ ➔ ➔ Group of fungal elements that are able to express themselves as yeast or molds depending on the temperature on where they are cultured ◆ Mold form → 25°C - 30°C ◆ Yeast form → 35°C - 37°C Therefore the most common form of fungal organism in our body is in YEAST FORM Thermal dimorphism (a group of pathogenic fungi) ◆ Able to cause fatal and deadly infections; common in dusty areas 1. - Penicillium marneffei Very common in patients with HIV Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 4 CLASSIFICATION OF HYPHAE A. EXISTENCE OF SEPTA/CROSSWALL ➔ ➔ PIGMENTATION Sometimes, the pigmentation on the surface is different from the reverse Observed from the reverse (A) SEPTATE HYPHAE Structure: Consists of distinct cells separated by septa (cross-walls). Appearance: Segmented appearance, with visible cross-walls dividing the hypha into compartments. Function: Septa regulate the flow of cytoplasm and nutrients between cells, contributing to the organization of the hypha. C. HYPHAL SHAPES (B) COENOCYTIC HYPHAE Structure: Lacks septa, forming a continuous, multinucleate structure. Appearance: Appears as a continuous, undivided tube. Function: Cytoplasm and nuclei can move freely throughout the hypha, allowing for rapid nutrient transport and growth. B. HYPHAL PIGMENTATION Usually appear in these colors: Brown, Brown-black or Black Dematiaceous hyphae Spiral hyphae Dark and pigmented hyphae Favic Chandelier (Antler Hyphae) Hyaline hyphae Non-pigmented hyphae Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 5 SUBCELLULAR STRUCTURE OF FUNGI (1) CAPSULE Structure ○ Polysaccharide Functions ○ Antiphagocytic factor e.g. Cryptococcus neoformans (encapsulated yeast) Can break the blood brain barrier and cause meningitis Pectinate Body Nodular Organ Racquet Hyphae (2) CELL WALL Provides shape, rigidity and strength, protection from osmotic shock and mediates attachment of the organism to the host cell Antigenic ○ Triggers immune response ○ Activate complement fixation ○ Provoke inflammatory reactions ○ Induce hypersensitivity Multilayered ○ polysaccharides poorly degraded by host cells ○ proteins and glycoproteins Major Polysaccharides of Fungal Cell Wall sparsely septated DEMATIACEOUS FUNGI dark and pigmented septate hyphae HYALINE MOLDS Septate, non-pigmented hyphae Monomer Chitin N-acetyl glucosamine (NAG) D-Glucosamine D-Glucose D-Glucose D-Glucose D-Mannose Chitosan Cellulose α-Glucan β-Glucan Mannan 3 TYPES OF HYPHAE IN MEDICALLY IMPORTANT FUNGI COENOCYTIC Polymer NOTE: (NAG) is also a main component of the peptidoglycan layer of the cell wall of a bacteria; The type & amount of polysaccharide vary from one fungal species to another (3) CELLULAR MEMBRANE A. Protects cytoplasm, regulates intake and secretion of solute, facilitate capsule and cell wall synthesis a. Undergoes respiration; also responsible for the entry and exit of nutrients B. Bilayered Structure: a. Phospholipids i. phosphatidylcholine ii. phosphatidylethanolamine b. Sterols i. ergosterol - very common target of our antifungal drugs ii. zymosterol Main sterol found in humans → Cholesterol (4) CYTOPLASM Nucleus - Brain of the cell Nuclear membrane - Outer covering of nucleus Nucleolus - Main producer of ribosomes Ribosomes - Protein synthesis sER - Production of lipids rER - Site for protein synthesis Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 6  Mitochondria - Powerhouse of the cell Vacuoles - Temporary storage and transport of nutrients FUNGAL REPRODUCTION Fungal elements that are clinically significant are known to produce both sexually and asexually. A. SEXUAL REPRODUCTION The most significant manner of reproduction; those who are able to reproduce sexually are referred as PERFECT FUNGI SEXUAL SPORES ○ Zygospore ○ Ascospore ○ Basidiospore ZYGOSPORE Actual image of a Rhizopus (bottom right) Enclosed in a thick wall Commonly produced by Rhizopus (Bread mold) and Mucor Parents of different genetic composition fused (top illustration) producing the gamete, eventually becoming a zygote. ASCOSPORE Process of Asexual Reproduction Produced in ascus (sac-like container) ○ Container-like structure of the spore ○ 4-8 spores per ascus Ascocarp (actual fungal organism) ○ The ascus is bound in a containing structure called the ascocarp ○ It is the actual body of the fungi e.g. Histoplasma capsulatum - A dimorphic fungi Note: The designation of fungi is “+” and “-” In summary, ascospores are the reproductive cells, ascus is the structure that enclose and release ascospores, and ascocarps are the larger fruiting bodies that house the asci. Occurs when two genetically different of fungal organisms mate; it involves 3 processes (highlighted BASIDIOSPORE in blue) ➔ ➔ ➔ (1) PLASMOGAMY - mating of 2 genetically different strains ◆ It produces the dikaryon (a cell with 2 nuclei with different genetic composition) (2) KARYOGAMY - mating of the 2 nucleus inside the dikaryon ◆ a fusion of two nuclei to form a diploid nucleus (Only 1 nucleus but has 2 different genetic material in it; Half from both father and mother) Which then undergoes (3) MEIOSIS produces 4 haploid spores Commonly produced by mushrooms Formed externally on a base pedestal (Basidium) ○ e.g. mushroom Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 7 Macroscopic view of the gills ➔ These are referred to as gills; each of those gill is lined with our basidium ➔ Microscopic view a gill ➔ ➔ Blue stained structures near the top are the Basidium and the pink stained oval shaped structures are the Basidiospore Every time the mushroom is agitated the basidiospores are released. SPORE GERMINATION Asexual Spore ◆ found on the fruiting body which is a principal structure. B. ASEXUAL REPRODUCTION For this process to happen it only needs: 1. Spore 2. Conducive growing ground Once the spores are released from their fruiting body and land on a suitable growing ground, the spores will undergo MITOSIS and germinate to form the hyphae ➔ ➔ ➔ ➔ Process of Asexual Reproduction At the tip of the conidiophore/sporangiophore are the SPORANGIA (With outer covering) which houses the spores or the CONIDIA (Without outer covering) When it is agitated the spores will be released into the environment Then the spore will produce a hyphae and it will elongate, stretch and branch out producing the mycelium a specialized hyphae will be produced from the mycelium which will grow erectly and out into the surface known as CONIDIOPHORE or SPORANGIOPHORE ➔ ➔ SPORE DISPERSAL Spore can be dispersed when they are ripe for dispersal or when they are physically disturbed (e.g. wind, rain) CONIDIOSPORE/CONIDIA (Asexual spore) Unicellular/multicellular → Produced in a chain at the end of conidiophore. Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 8 ➔ ➔ CONIDIOGENESIS Is the process of asexual spore (conidia) formation in fungi. It involves the initiation, differentiation, and formation of specialized structures called conidiophores, which produce and release conidia. Conidia play a crucial role in the rapid reproduction and dispersal of fungi, allowing them to adapt to different environments. TYPES OF CONIDIOGENESIS A. THALLIC CONIDIA A SEPTUM FIRST APPEARS before a cell develops into a spore ○ Starts of with a one long filament ○ Spores develops by septation or fragmentation of a hypha (at the tip or in between segments) ○ All layer of the hyphal wall of the parent cell are involved in spore formation ARTHROCONIDIA B. BLASTIC CONIDIA PARENT CELL HAS ALREADY EXTENDED first before the septum has been created ○ Spore is already evident before it separates from the conidia hyphae ○ Budding is an example of spores that are produced blastically BLASTOSPORES (Holoblastic) Formed by budding of a hypha or yeast cell ALL WALL LAYERS ARE INVOLVED The spore may remain attached and bud further blastospores (branched chain of spores) POROSPORES (Enteroblastic) Fragmented septate hyphae ○ Each fragment is rounded off an liberated in succession ○ The separation would be due to the breakdown of the middle region of each septum single, slightly thickened cells → e.g. Coccidioides, Geotrichum CHLAMYDOSPORE Also known as “Tretic” or “Poroconidia” Spore emerges through a distinct 'pore' in the hyphal wall The new spore develops its own inner wall layer With a scar at the point of detachment ANELLOSPORES (Enteroblastic) Thick walled formed along the periphery or tip of the hyphae → e.g. Candida Forms on scars or on the septum A new spore DOES develop at the scar A chain of spores may develop; cars will stack on top of each other The conidiophore gets a little longer with each spore produced due to annellations (ring-like scars) Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 9  Phialide is where the conidia is released; found at the tip of conidiophore Aspergillus (Left) → the tip of the conidiophore ends in a global structure called VESICLE where phialide grow Penicillium (Right) → the tip of the conidiophore branches out to form the metula where phialide grow PHIALOSPORES (Enteroblastic) Spores that are formed on top of a phialide Forms in succession Each spore is pushed up from the end of the conidiophore (PHIALIDE) The first spore has a cap ○ The oldest spore (the first to form) carries the tip of the phialide ○ The one nearest to the phialide is the youngest spore. B. SPORANGIOPHORE POROSPORES Produced on PORES ANELLOSPORES Produced on top of SCARS PHIALOSPORES Produced on top of PHIALIDES SPECIAL STRUCTURES FOR REPRODUCTION A. CONIDIOPHORE Has a sporangia which is a sac-like structure that encases the conidia it breaks open when it is ripe and mature to release the conidia TAXONOMIC CLASSIFICATION Depends primarily on the type of sexual spore ➔ Phylum “-mycota” ➔ Class “-mycetes” ➔ Order “-ales” ➔ Family “-ceae” For example: The stem that holds the spores; specially if the spore has no outer covering Specialized hyphae that grows erect; on top of it are the spores Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 10 Sexual Spore Zygospore Basidiospore A. ZYGOMYCOTA Class Zygomycetes Basidiomycetes Ascospore Ascomycetes Unknown Deuteromycetes (Fungi imperfecti; mitosporic fungi) ➔ Taxonomically, there are 4 classes of fungal elements and they are based on the types of spores that they produce sexually. Sporangium fungi (common molds) ○ Nakabalot ang kanilang spores Molds & blights Rhizopus stolonifer (bread mold) ○ Coenocytic (No septa) PARTS OF A ZYGOMYCOTA (1) Rhizoids Description: Root-like structures that extend from the lower surface Function: Anchor the fungus and aid in the absorption of nutrients from the surrounding environment. (2) Stolon Description: a horizontal, above-ground stem or runner that grows along the surface of the substrate. Function: They enable the plant or fungus to produce new individuals by growing horizontally, forming roots and shoots at nodes along their length. LIFE CYCLE OF A ZYGOMYCETE Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 11 B. ASCOMYCOTA SAC FUNGI Reproduction through: ○ Conidiospores (asexual) ○ Ascospores (sexual) ○ Buds Ascus (sac-like) Ascocarp LIFE CYCLE OF AN ASCOMYCETE C. BASIDIOMYCOTA CLUB FUNGI Some are used as food Others cause crop damage Seldom reproduce asexually ○ e.g. mushroom PARTS OF A MUSHROOM Basidiocarp ○ largest part ○ protects the gills Gills (with basidia that connects the basidiospores) ○ 1 basidia holds 4 basidiospores Stipe (stem) Annulus (ring) ○ may or may not be present Volva ○ protects the immature mushroom Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 12 LIFE CYCLE OF A BASIDIOMYCETE D. DEUTEROMYCOTA Heterogenous group of unrelated species in which SEXUAL REPRODUCTION HAS NEVER BEEN OBSERVED Asexual spores: conidia of various types Septate hyphae E.g. Penicillium SUMMARY 2 Forms of Fungi Yeast and Mold Crx of Yeast unicellular; budding; pasty, moist colonies Crx of Molds multicellular; hyphal extension & fragmentation; fluffy, cottony colonies Classif of Hyphae shape, septum, pigment Ex. of asexual spores Conidia: arthro-, blasto-, chlamydo-, tretic, annellospores,phialospores Fungal classes of medical significance: Zygomycetes, Ascomyctes, Basidiomycetes, Deuteromycetes Zygomycetes sporangium fungi; breadmold Ascomycetes sac fungi; morel, truffle Basidiomycetes club fungi; mushroom Deuteromycetes imperfect fungi; Penicillium Astronomo, Budo, Kintanar / Proofreading: Palco, Aguinaldo | 13 | Topic #2 | [MLS 415] Mycology and Virology P2: Laboratory Diagnosis, Specimen Collection & Serologic Test Professor: Thynee Tago, RMT, MSMT Date: January 24, 2024 ➔ ➔ ➔ ➔ ➔ ➔ SPECIMEN COLLECTION Aseptic technique Collect specimen from actual infection site Adequate quantity Accurate label Prompt delivery CLINICAL SPECIMENS 1. BLOOD Important specimen if the infection is a disseminated type ◆ Provide accurate measure of determining the etiology of the disseminated fungal infection 30 deg C up to 21 days Broth media (5mL to BHI) Biphasic broth-agar system ◆ Contains both solid and liquid media Organisms would be able to grown and solid and liquid environment ◆ If bottle is inverted, specimen will be inoculated on the solid media Lysis centrifugation technique (Isolator) ◆ Optimal for isolation of H. capsulatum & filamentous fungi ◆ Cells that contain the organism will be lysed by the machine centrifuge → gather concentrates ◆ Centrifugation of concentrates is best before culture ◆ As fast as 4 days of incubation, fungal elements can be already be detected on the plated media for pathogenic fungal elements, it is best incubated for as long as 10-15 days (e.g. Histoplasma capsulatum) Automated blood culture systems: ◆ BACTEC ◆ BacT/ALERT ◆ ESP ➔ 2. RESPIRATORY SECRETIONS Such as: Sputum, Bronchoalveolar lavage (BAL), Bronchial washings, Tracheal aspirate ◆ Common ◆ Use culture media with antibiotics ◆ Cycloheximide ◆ At least 0.5 mL of inoculate/ media NO 24 hr. Collections; Sterile NSS for washings ➔ 3. CSF Such as: Lumbar tap ◆ Trained physicians; Collection is not done by medical technologists too invasive; requires a lot of training. ◆ Filtered through a 0.45 um membrane pore filter Can filter big viruses; e.g. Ebola, Pox viruses ◆ Filter will be placed on plated media without antibiotics Part of the filter facing the barrel comes in contact with the culture media Position of the filtered paper is changed everyday to ensure that all the surface area of the media is inoculated Without antibiotics ⇒ to gather organisms present on the filtered CSF > 2 mL: Centrifuge, smear, culture (supernatant can be discarded) < 1mL: centrifuge & use 1 drop aliquots (supernatant is kept) scattered on the media source. Keep at room temperature or at 37 deg C Must not be refrigerated as it might kill any fungal elements present in the specimen 4. ➔ ➔ (left) Isolator machine; (right) Biphasic broth-agar system BONE MARROW AND STERILE BODY FLUIDS Bone marrow ◆ Placed directly on surface of plated media ◆ specifically collected for isolation of Histoplasma Sterile body fluid ◆ Almost all are abundant except for synovial fluid ◆ Centrifuge then culture ◆ at least 1 mL inoculum/plate @mlstranses | 1 ➔ ➔ ➔ ➔ ➔ Both may be processed as blood culture ◆ Biphasic broth-agar system can be utilized 5. URINE If ambulatory: Midstream, clean-catch, Early morning, Catheterized ◆ Centrifuge → smear → culture (sediments) Process in 2hrs (at RT) or refrigerate ◆ Do not let it stay for > 6 hrs Culture on media with antibiotics; because it has many bacteria ➔ ➔ ➔ Collection procedure: 1. Apply alcohol to decontaminate 2. Let it dry before collection HAIR May contain contaminants Pluck hair by roots (forceps or tweezers) ○ Check for fluorescent, broken, scaly hair ○ Be very careful in plucking because of the hair is infected with fungal elements, the hair gets fragile Woods lamp (UV lamp) allows the visualization of fluorescence which could aid in collecting the correct hair specimen Placed on petri dishes or specimen bags Yeast in urine sample Mucus threads are normally seen in the urine sample 6. VAGINAL AND CERVICAL DISCHARGES NAIL 70% alcohol → scrape discolored areas → collect inner infected nail ○ scrape underneath the discolored nails Nail clippings: cut into small pieces Microscopy with KOH ➔ ➔ ➔ ➔ ➔ Cervix (+): few to moderate Candida colonies ○ Candida is a normal flora of this area 2 swabs: for KOH (microscopic) & culture 7. SUBCUTANEOUS Such as: Lesions, abscesses, tissues ◆ Needle aspiration or biopsy ◆ Check for granules (microscopic) ◆ If the tissue collected is larger than normal Macerate or mash the tissue to release fungal elements that may be present machine: Stomacher ○ contains paddles that can press the tissue 8. MUCO-CUTANEOUS Such as: Throat & tongue (+): few yeast & contaminants ◆ Candida is also a normal flora of this area Use 2 sterile swabs ◆ One for culture; one for smear Scrape plaque w/ tongue depressor (Candida) 9. CUTANEOUS Most Common Dermatophytes are the suspected organisms for this type of specimen CANNOT be REFRIGERATED SKIN (+): few Candida & contaminants Scrape infected area (using blunt scalpel) ○ After scraping use swab to collect all debris from the scrapings Microscopy with KOH Mycosel agar: perfect for recovery of dermatophytes ○ Incubation at 30 deg C (21 days) CULTURE MEDIA Media must include: ➔ (1) Amino acids or urea (as a source of Nitrogen), (2) Glucose (as a source of Carbohydrate), etc. ➔ Antimicrobials: (for non-sterile specimens) ◆ Cycloheximide (perfect for fungal organisms) + Chloramphenicol ◆ Gentamicin + Chloramphenicol (better isolation) ◆ Ciprofloxacin - inhibits contaminating molds & bacteria Aerobic environment ; ↑ humidity & moisture ➔ ➔ ➔ TYPES OF CULTURE MEDIA Plated and Tubed Primary or Non-selective ◆ permit the growth of virtually all clinically relevant fungi Selective ◆ tailored to isolate specific pathogenic fungi of interest @mlstranses | 2 (1) AGAR PLATES ADV DADV Better aeration → more oxygen enter due to its wide opening Large surface area for better isolation Ease of handling Easily dehydrated (use 40 mL agar) → Due to wide opening Require BSCs Hazardous to handle → Use biosafety cabinets → Greater chances for aerial mycelium to release spores SABOURAUD HEART INFUSION (SABHI Agar) (2) SCREW-CAPPED TUBE ADV DADV Easy storage Less space for incubation More easily handled Less hazardous Lower dehydration rates Poor colony isolation Reduced surface area Promote anaerobiosis → open every now and then; slightly opened caps → if not dimorphic, they just use cotton plugs ➔ ➔ ➔ Primary recovery/ isolation of (1) saprophytic and (2) dimorphic fungi, particularly fastidious strains. SDA + BHI (Sabouraud Dextrose Agar + Brain Heart Infusion) Contains antibiotics → chloramphenicol and cycloheximide INHIBITORY MOLD AGAR A. PRIMARY CULTURE MEDIA SABOURAUD DEXTROSE AGAR ➔ ➔ ➔ ➔ ➔ ➔ Recovery of (1) saprobic & (2) pathogenic fungi Sufficient for recovery of dermatophytes ◆ but not recommended as a primary isolation medium or for dimorphic fungi because it lacks nutrients needed to recover fastidious pathogens. Initially without antibiotics to isolate C. neoformans, candida & aspergillus ◆ they are sensitive to Cycloheximide & chloramphenicol Emmons modification (contains 2% dextrose) ◆ enhances typical sporulation ◆ provides more characteristic colony morphology BRAIN-HEART INFUSION AGAR (BHI Agar) For the primary recovery of (1) saprophytic and (2) dimorphic fungi ➔ BHI with sheep blood + antibiotics = for tissue specimen w/ dimorphic fungi ➔ ✓BHI broth → widely used as an INITIAL CULTURE media from collection Usually used for research Primary recovery of dimorphic pathogenic fungi ◆ Saprophytic fungi and dermatophytes will NOT BE RECOVERED POTATO DEXTROSE AGAR ➔ ➔ ➔ ➔ ➔ Contains: Dextrose & Potato infusion ◆ Dextrose serves as growth stimulant and carbohydrate source ◆ Potato infusion provides a nutrient base for luxuriant growth of fungi General purpose BASAL MEDIUM for the cultivation of yeasts & molds ◆ It aint too expensive Stimulates sporulation and pigmentation Aids in cultivating and differentiating pathogenic and non-pathogenic fungi Commonly used in finding out fungal organisms from food and beverages @mlstranses | 3 ➔ To control the growth of the contaminating bacterial agent ◆ 10% sterile tartaric acid may be added to lower the pH to about pH 3.5 POTATO FLAKE AGAR Highly selective for dermatophytes Contains antibiotics: (1) cycloheximide & (2) chloramphenicol For isolation of pathogenic fungi from non-sterile materials ○ For the recovery of dermatophytes DERMATOPHYTE TEST MEDIUM ➔ ➔ ➔ Primary recovery of (1) saprophytic and (2) dimorphic fungi, particularly fastidious and slow-growing strains Cultivation, isolation, & induction of sporulation (Spore formation) of fungi from clinical specimens Accidentally discovered ◆ Advantageous for molds because diagnostically useful sexual reproductive structures develop more readily on PFA ➔ Recommended for selective isolation of pathogenic fungi (dermatophytes) from cutaneous sources CORN MEAL AGAR w/ TWEEN 80 V-8 AGAR ➔ ➔ ➔ Recommended for the cultivation of yeasts and molds Contains: Yeast extract ○ provides essential growth nutrients L-asparagine ○ Source of amino acids Glucose ○ Source of energy (carbohydrates) V-8 juice ○ vegetable juice that contains extract of 8 different vegetables ○ supplies the trace ingredients needed to stimulate the growth of fungal elements that are found in the specimen. ➔ ➔ For use in the cultivation of fungi and for the inducement of chlamydospore formation by Candida albicans Tween 80 ◆ Allows the growth of the organism vertically so it would lessen the surface tension on the agar; very helpful for candida so that from the base of the agar they will go out on the surface. ◆ mixture of lake esters which can stimulate the production of chlamydospores Chlamydospore formation is best seen when the organisms are incubated 25 deg C All Candida spp. are able to grow chlamydospore and pseudohyphae in CMA (except C. glabrata) CZAPEK AGAR B. SELECTIVE CULTURE MEDIA MYCOSEL/MYCOBIOTIC AGAR ➔ ➔ For the routine cultivation of fungi, especially: ◆ Aspergillus ◆ Penicillium ◆ non-sporulating molds ROUTINE CULTURE MEDIA for fungi but is perfect for aspergillus Candida albicans @mlstranses | 4 NIGER SEED/ BIRD SEED AGAR ➔ Identification of C. neoformans ◆ C. neoformans are able to release the enzyme phenoloxidase Contains: Caffeic acid ○ serves as a substrate for the detection of phenoloxidase Creatinine ○ enhances melanization of some strains of C. neoformans Glucose - source of energy Chloramphenicol - antibiotic The action of phenoloxidase to caffeic acid results in the production of MELANIN which is absorbed by the yeast cell wall forming tan to reddish brown pigmentation CHROMAGAR CANDIDA (Blue) C. tropicalis; (Green) C. albicans; (Big and fluffy colonies) C. krusei; (smooth pink colonies) C. glabrata ➔ For isolation and differentiation of major clinically significant Candida species ➔ Chromogenic agar that allows each Candida spp. to produce its characteristic colonies and pigmentation. ➔ Really expensive D. DIFFERENTIAL CULTURE MEDIA ➔ ➔ TRICHOPHYTON AGARS Differential media used in the presumptive identification of Trichophyton species based on nutritional requirements ◆ contains the nutrients that allow trichophyton to grow The succeeding three agars contain different additional enrichment material to show the growth of a particular group of Trichophyton COTTONSEED CONVERSION AGAR (1) Vitamin 1; (2) Inositol; (3) Inositol + Thiamine; (4) Thiamine UREA AGAR ➔ Conversion of Blastomyces dermatitidis from mold to yeast form ◆ Used for dimorphic fungi so that we could see their other form C. SELECTIVE/ DIFFERENTIAL CULTURE MEDIA RICE EXTRACT AGAR ➔ ➔ ➔ Detection of C. neoformans ◆ able to produce urease Differentiates T. mentagrophytes vs T. rubrum Detection of Trichosporon spp. SUMMARY ➔ ➔ For differentiation of yeasts by means of their typical chlamydospores and on basis of micromorphological criteria Useful in growing Candida spp. Spx. collection guidelines Aseptic tchnq Correct site and amt. Prompt delivery Types of spx. Blood, Respiratory spx, CSF, Hair, Skin, Nails, Urine, Body fluids, Bone Marrow Culture media Plated/tubed Primary, Selective or Differential @mlstranses | 5  ➔ ➔ ➔ Specimen Rejection Criteria Hemolysis Improper transport temperature Incorrect patient identifiers Incorrect specimen Insufficient volume No requisition/test order Unlabeled Unacceptable specimen age Unacceptable containers Adhesive is used to collect specimen on culture plate DOUBLE LAYER TAPE PREP 1 drop LPCB + aerial mycelia attached to the adhesive tape + another 1 drop LPCB + coverslip ✓No streaking for mold colonies C. INDIA INK METHODS OF IDENTIFICATION DIRECT MICROSCOPIC METHOD This method allows the medical technologists to obtain information regarding the morphology of the organism ○ From the specimen directly to the slide Samples used are from plated cultures except for Wet Mount A. WET MOUNT KOH (10%) ◆ destroy keratin layer particularly the hydroxide component (working component) ◆ clears debris; if not removed it obscures microscopy ◆ makes fungi prominent Drop of 10% KOH in a clean glass slide with the specimen then cover with cover slip Directly gather sample from the infected sample site ◆ useful for cutaneous specimens ➔ B. LACTOPHENOL COTTON BLUE (LPCB) ➔ ➔ ➔ ➔ ➔ Negative staining method ◆ → the background is stained while the target element remains colorless Perfect for encapsulated organisms ◆ e.g. capsule of C. neoformans Capsules appear as clear halo against dark background D. CALCOFLUOR WHITE + KOH Can be used by itself, but it is more effective with KOH Fluorescent staining method for rapid detection of yeast, fungal elements and parasites Uses a non-specific fluorochrome that binds to CHITIN and CELLULOSE in cell wall of organisms Result: apple-green/bluish-white: fungi and parasites reddish-orange: other materials Not a direct method; sample used is the one that is used in the culture ➔ a.k.a. AMAN’S MEDIUM Contains: Lactic acid ○ serve as a preservative Phenol ○ antimicrobial agent; can kill any live organisms in the specimen Cotton blue ○ a stain with affinity to CHITIN and fungal cell walls Method: Basic Tease Mount ➔ ➔ ➔ View under Wood’s light or UV light Perfect for dermatophytes Addition of 10% KOH helps dissolve keratinized particles for hair and nail specimens and emulsify solid and viscous material that may mask fungal elements Care must be taken when choosing the swabs when collecting the sample. Use dacron/rayon swabs instead of cotton swabs Cotton fibers may intensely fluorescent in Calcofluor white (interference) BASIC TEASE MOUNT rapid method 1 drop LPCB + sample Purpose of teasing, is to separate the individual growth of the organism CELLOPHANE FLAG PREP 1 drop LPCB + aerial mycelia attached to the adhesive tape @mlstranses | 6 STAIN PREPARATION ➔ TISSUE SECTION Stain Use Gomori’s/ Grocott Methenamine Silver → Simply coat the surface of the organism ➔ Periodic Acid Schiff (PAS) → Affinity to carbohydrates Gridley’s Black Cell Wall Stains hyphae of molds & some yeast (red) Mycelia (deep blue) Conidia (deep rose-purple) PIGMENTED TISSUE Stain ➔ ➔ Dermatophyte cultures should be incubated 6 weeks at room temperature before reporting it as negative Use ➔ Fontana-Masson → A follow-up after H&E staining → has affinity to melanin Dematiaceous hyphae (brown) ➔ MALIGNANT CELLS Stain ➔ Use Papanicolau ➔ Better demo of B. dermatitidis than wet mounts BONE MARROW Stain Detects intracellular H. capsulatum in blood smears. Small, oval yeast cells (light or dark blue or purple) GENERAL TYPE Stain LABORATORY SAFETY BSC (Level 2) ◆ Inoculations & manipulation of mold colonies ◆ Separate BSC for isolation of molds Clean surfaces of BSCs and incubators regularly ◆ use 70% ethanol or bleach; but bleach tends to corrode metal in the BSC Dimorphic fungi ◆ Risk Group 3 (may cause lethal diseases and treatment may not be available) ◆ BSL3 / reference labs − RITM MACROSCOPIC EXAMINATION OF FUNGAL GROWTH Use Giemsa Wright’s Concentration is necessary for body fluids over 5 mL ◆ Purpose of conc. is to make sure that the sediments/ solid particles are pooled together therefore easier to isolate Mucolytic agents may be added to liquefy highly viscous samples & facilitate plating on agar media Tissue samples are ground (Stomacher) with NSS before being inoculated onto media A battery of agar media should be used. Cultures are incubated at 30°C (yeast) or at 22-25°C (molds) for 21-30 days A relative humidity of 40-50% is desired to prevent the agar from drying out A. RATE OF GROWTH Rapid or slow grower ○ yeast: can grow after 24 hours to 4 days ○ molds: can grow after 4 days to several weeks Specify days or weeks ○ molds tend to grow downward first creating a mycelium and will grow upwards creating the aerial mycelia Use B. TOPOGRAPHY Kinyoun’s AFS Hucker’s Gram Stain H&E ➔ Fungi are weakly G (+) but poorly stained (pale red - pink) Useful for Candida CULTURE METHOD Allows the medical technologists to see actual colonies growing in the culture media and take note the microscopic details that would help in identifying the organism ○ gather enough information to name the genus of the organism. Flat, heaped or folded Rugose (wrinkled) Umbonate CULTIVATION OF FUNGI If delayed, the specimen can be refrigerated for a short time except blood, CSF, skin, hair, nails. @mlstranses | 7 C. TEXTURE Cottony or wooly (e.g. mold*) Velvety or silky* Powdery or granular* Moist, creamy, pasty (e.g. yeast) D. PIGMENTATION On surface and reverse side Color description is very specific. MICROSCOPIC EXAMINATION OF FUNGAL GROWTH SPORES/ CONIDIA & HYPHAE Hyphae can easily be observed microscopically because we only need to check for the presence of (1) septum or (2) cross walls, (3) hyphal shapes and (4) pigmentations. Pigmentation: ○ pigmented = dematiaceous ○ non-pigmented = hyaline Spores has different sizes and shapes ○ smooth, reticulate, spiny, pitted, etc. All can be visible by performing: ○ Basic Tease Mount ○ Cellophane Tape Mount ○ Double Layer Tape Prep SLIDE CULTURE/ MICROCULTURE METHOD Most accurate method to preserve & observe fungi Fungi are preserved in original state (as they are living) Requires more skill & time than tease mount NOT FOR DIMORPHIC FUNGI because it is too open and because the MOT for this is respiratory except for Sporothrix (skin puncture) ○ Histoplasma ○ Coccidioides ○ Blastomyces ○ Sporothrix ○ Paracoccidioides Materials: ➔ 7-10 day culture ➔ SDA Plate ➔ U-shaped glass tube placed in the petri dish in which a sterile filter paper (9cm) is placed ➔ glass slides and slips ➔ scalpel and inoculating loop ➔ 95% LPCB ➔ sterile distilled water Procedure: 1. Aseptically, place a sheet of sterile filter in a petri dish 2. Place a sterile U-shaped glass tube on a filter paper 3. Pour sterile distilled water on filter paper 4. Flame the scalpel and cut a 5mm2 square block of the medium from the SDA plate 5. Pick up the block of agar by inserting the scalpel and carefully transferring the block aseptically to the glass slide then place a cover slip 6. Flame the inoculating loop @mlstranses | 8 7. Take the plate containing fungal culture to be examined and remove a small amount of fungal culture 8. Inoculate four sides of the glass square with suppose mycelial fragments of the fungus to be examined 9. Cover the petri dish and incubate at RT for 48 hour 10. Observe micellar growth and spore protection is observed through the four corners of the agar cube spreading over to the cover slip 11. Procedure for Application fo Stain: a. place a drop of LPCB on a clean glass slide b. remove the cover glass from the slide culture and discard the block of agar c. place the cover glass with mold-grown side down on the drop of LPCB stain d. Examine slide in LPO and HPO ➔ ➔ 25 deg C for 10 to 14 days, examine microscopically (+) Conical perforations on the hair shaft BIOCHEMICAL TEST ➔ CHO Assimilation Test For Yeast ◆ (+) growth around disk ◆ CHO is utilized by the organism REDUCTION OF NITRATE TO NITRITE GERM TUBE TEST ➔ ➔ ➔ ➔ ➔ ➔ ➔ Used to differentiate between fungal or bacterial organisms Its procedure is similar to COAGULASE TEST ◆ We can use 0.5 mL serum/plasma + colony/inoculum To differentiate Candida albicans from other Candida spp. Similar to pseudohyphae without constrictions 0.5 mL rabbit serum + colony Incubate for 2 ½ - 3 hours at 37°C (+) Germ tube formation ◆ How do we differentiate it from a pseudohyphae? Pseudohyphae is a clingy daughter so the crosswall now is evident. In the germ tube the cell has no crosswall, it's just that the cell wall was just extended. HAIR BAITING/ PERFORATION ➔ ➔ RAPID UREASE TEST ➔ ➔ ➔ ➔ T. mentagrophytes Filter paper + water + sterile hair on a culture plate ◆ Can also utilize soil C. neoformans Rgnt: Alpha naphthyl & Sulphanilic acid ◆ change in color (red) = positive ◆ If negative add Zinc dust; the nitrite was further reduced into gas form No change in color = positive For PRELIMINARY ID of C. neoformans → (+) color change: peach to magenta SEROLOGICAL TEST Due to the increase in invasive fungal infections the need for serological test were raised ○ Provides enough information to name the species of the fungal organism Provide key elements that needed to improve management of invasive fungal infections ○ Rapid diagnosis ○ Early initiation of antibiotic fungal therapy @mlstranses | 9  Culture methods ○ Time consuming; mold cultures grow after several weeks ○ It has low sensitivity due to the low concentration of the organism in the samples infested. Non-culture methods (now opted) ○ Detection of specific host immune responses to fungal antigens using immunological reagents ○ Detection and quantitation of specific fungal metabolite byproducts Can be used as a prognostic marker, and back up positive cultures to rule out laboratory contamination. ○ Specially if monitoring treatment DISADVANTAGES: Test kits are very EXPENSIVE The detection will not tell us whether the presence of antibodies is because of colonization somewhere in the body or if it's really a deep infection. Abs are not detected in the early stage of the disease or the individual is immunocompromised. Presence of cross-reactive reactions; due to shared antigenicity Targets for serological tests: Antigen Antibody Metabolites Types: (1) Agglutination, (2) Immunodiffusion, (3) Complement Fixation Test, (4) Enzyme Linked Immunosorbent Assay, (5) Lateral Flow Assay ➔ ➔ ➔ LATEX AGGLUTINATION Good for Cryptococcosis Detect polysaccharide antigen (glucuronoxylomannan) Qualitative & semi-quantitative ➔ ➔ Invasive Candidiasis ◆ Detection of Heat labile glycoprotein, Mannan antigen and anti–Mannan antibody mannan is a component of cell wall C. Paracoccidioidomycosis ◆ Caused by: Paracoccidioides brasiliensis Sensitivity: 84% ; Specificity: 81%. DISADVANTAGES: False positives due to cross-reactivity Eliminated by: ○ Pretreatment of crude exoantigens: exoantigens are components of hyphal cell wall that can be released as they expand ○ Treat it with: Sodium metaperiodate ○ Pretreating serum with 2β mercaptoethanol IMMUNODIFFUSION Look for antibodies of specific antigens Aspergillosis ○ Diagnosis of Allergic Bronchopulmonary Aspergillosis, Aspergilloma (Detects IgE antibodies) Candidiasis → Detects Anti-Mannan abs Blastomycosis → Qualitative assay; Detects precipitating antibodies (+) precipitation band; SENSITIVITY: 80-90% & SPECIFICITY : >90% DADV: Costly Difficult to standardize Long turnaround time ○ About 3 days Histoplasmosis ○ Ags: M protein & H protein (reagent) ○ Abs: Anti-M Ab & Anti-H Ab (+) Precipitation Band Sensitivity – 90.9% ; Specificity – 95% LAT GRADING Grade Interpretation +1 Small clumping, cloudy background +2 Small to moderately sized clumps, slightly cloudy background +3 Moderate to large clumps, clear background +4 Large sized clumps, clear background Negative (+) M and H (+) M only Active infection Early/Chronic Infection Coccidioidomycosis ○ C. immitis/C. posadasii ○ We are looking for: IgM: which reacts w/ tube precipitin (TP) Ag (part of the set-up) IgG: which reacts w/ complement fixation (CF) Ag (part of the set-up) No agglutination @mlstranses | 10 DADV: False negatives ○ Immunocompromised ○ Early stage of infection ➔ ➔ ➔ ➔ ➔ ➔ ➔ ➔ COMPLEMENT FIXATION TEST Aspergillosis (Allergic) ◆ Detection of IgE antibodies Histoplasmosis ◆ Ab detected against M & H Ags ◆ high sensitivity, low specificity Coccidioidomycosis ◆ Detects IgG Ab to Coccidioides culture filtrate ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA) Cryptococcosis/ Blastomycosis/ Histoplasmosis/Coccidioidomycosis ◆ Qualitative or Screening assay Aspergillosis ◆ Allergic = detection of IgE Ab ◆ Invasive = Screening test; detects galactomannan ➔ DADV: Sensitivity decreases following Itraconazole prophylaxis Circulating galactomannan (GM) is rapidly eliminated by formation of immune complexes ○ That is why, collect samples not too early and not too late ➔ Candidiasis ◆ Presumptive diagnosis of Invasive candidiasis by Mannan Ag & Anti-Mannan Ab Assay Detection of metabolites ➔ D-Arabinitol ➔ Secreted Aspartyl Proteinase (metabolites of Candida) 1,3 β D-Glucan Assay (Fungitell) Adjunct in diagnosis of invasive fungal infections Pan fungal marker ◆ can be paired with any serologic tests Exo antigen: component in majority of fungal cell walls High levels of 1,3 β-D-glucan (BG) seen in: ◆ Candida sp. ◆ Trichosporon sp. ◆ Sporothrix sp. ◆ Fusarium sp. ◆ Aspergillus sp. ◆ Penicillium sp. ◆ Saccharomyces MOLECULAR IDENTIFICATION Uses PCR ○ Multiplex PCR makes use of several different target genes to identify an organism ○ Nested PCR makes use of two sets of primers Molecular Amplifications; most fungal elements amplified using target genes are: ○ Candida (5S gene, actin, etc.) 28S gene can also be used ○ Pneumocystis (dihydrofolate reductase, mitochondrial rRNA) ○ Aspergillus (28S gene, IGS regions) Part of the rDNA complex of the target gene SUSCEPTIBILITY TESTING E-TEST Agar MIC Method (Minimum Inhibitory Concentration) ○ Allows the physician to choose the right antifungal agent ○ not really required ○ Elipse: clear zone DADV: Mannan is rapidly eliminated by immune complexes & endocytosis by Kuppfer cells ➔ ➔ LATERAL FLOW ASSAY Cryptococcosis ◆ Screening test & determination of endpoint titres ◆ Caused by: C. neoformans & C. gattii Aspergillosis ◆ detects IgE Ab (allergic) ◆ detects GM (invasive) The point at which the edge of the ellipse meet a particular measurement that is considered as the MIC Sensititre Yeast One ○ Widely used for Candida albicans ○ Microbroth dilution MIC test ○ Tests several species of Candida against different antifungal agents ○ Reliable sensitivity test @mlstranses | 11 SUMMARY Microscopic KOH, LPCB, CFW, Special stains Culture (30C/ 22-25C for 21-30 days) Macroscopic & Microscopic details Serological LAT, ID, CFT, ELISA, LF Molecular PCR, Genetic amplification Sensitivity tests E-Test, Sensititere Aftercare Autoclave, Disinfect FungiTest ○ Microbroth dilution breakpoint test ○ Allows us to identify which concentration of drugs is susceptible ○ Pink (+) (Resistant); Blue (-) (Sensitive) FOR CANDIDA KRUSEI Drugs Culture 5Fc (Flucytosine) I 4-32 ug/mL AmB (Amphotericin) S