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MYCOLOGY AND VIROLOGY (LECTURE) Quitaleg, Jenny Rose Maghuyop LEC PRELIM 1 INTRODUCTION TO MYCOLOGY OUTLINE...

MYCOLOGY AND VIROLOGY (LECTURE) Quitaleg, Jenny Rose Maghuyop LEC PRELIM 1 INTRODUCTION TO MYCOLOGY OUTLINE I. CHARACTERISTICS OF FUNGI I. Characteristics of Fungi II. Two Forms of Fungi III. Reproduction of Fungi FUNGI, BACTERIA AND PLANTS IV. Portal of Entry V. Modes of Transmission VI. Fungal Culture Process FUNGI BACTERIA PLANTS VII. Pretreatment of Clinical Specimens eukaryotic prokaryotic contains nucleus bound by a do not contain these membrane, endoplasmic structures reticulum and mitochondria morphologically same with has plants but has no chlorophyll chlorophyll dependent upon enzymes systems to derive energy from organic substrates can be: 1. Saprophytes – live on dead organic matter 2. Parasites – live on living organisms has cell wall – composed of chitin food storage – in the form of lipids and glycogen stain gram positive stain gram positive and gram negative all require water and oxygen – no obligate anaerobes – grows best at neutral pH – needs moisture for growth spore & conidia spore – dry condition – survival – reproductive structure structure yeast and molds – two forms of fungi Page 1 of 8  CLASSIFICATION OF HYPHAE BASED ON THE II. TWO FORMS OF FUNGI APPEARANCE OF ITS CROSS WALLS YEAST AND MOLDS 1. SEPTATE o has numerous cross – walls 2. COENOCYTIC OR SPARSELY SEPTATE o was called aseptate o has few cross - walls CLASSIFICATION OF HYPHAE BASED ON THE PIGMENTS  YEASTS – smooth ,creamy pasty or mucoid form of fungal growth – reproduce through budding resulted to blastoconidium (daughter cell) – bacterial colony without aerial hyphae  MOLDS – exhibit filamentous type of growth due to mycelia MYCELIUM  HYALINE o non – pigmented, clear, transparent  mass of branching intertwined hyphae o colorless without stain o will be blue with stain (for visualization of morphology) AERIAL HYPHAE / AERIAL VEGETATIVE HYPHAE / MYCELIUM VEGETATIVE MYCELIUM  DEMATIACEOUS o filamentous part o responsible for the o pigmented o holds the conidia absorption of nutrients o black or brown pigment without stain from culture media or agar o roots VEGETATIVE TYPES OF HYPHAE III. DIMORPHISM AND POLYMORPHISM 1. antlers hyphae - like an antler’s horn 2. nodular hyphae - knot - like 3. racquet hyphae - resembles racquet shape 4. spiral hyphae - curled hyphae 5. rhizoids hyphae - root – like structure of hyphae Page 2 of 8  DIMORPHISM ASEXUAL REPRODUCTION  exhibit different forms in different temperature CONIDIA grows at 25 oC (RT) 37 oC form mold form yeast form  reproductive structure of fungi  aka conidium or spore  form vary from different fungi POLYMORPHISM  can be classify based on structure  have both yeast and mould forms in the same culture grows at 25 oC (RT) 37 oC form mold form mold form yeast form yeast form DIMORPHIC FUNGI AND POLYMORPHIC FUNGI  CONIDIOPHORE DIMORPHIC FUNGI POLYMORPHIC FUNGI  branch of hyphae that holds the conidia  Blastomyces  Exophiala spp. dermatitidis  Coccidioides immitis  Histoplasma capsulatum CLASSIFICATION OF CONIDIA  Paracoccidioides brasiliensis ARTHROCONIDIA III. REPRODUCTION OF FUNGI  depends on the environment of fungi ASEXUAL SEXUAL REPRODUCTION REPRODUCTION  everything is stable in  environment is  results from fragmentation of the two fertile hyphae the environment  harsh  rectangular or parallel shape with thick wall conidia  nutrients  changing  temperature  undergo meiosis  moisture  from one hyphae to  just going to maintain another genetic MACROCONIDIA the characteristics of characteristic  mate fungi that will thrive in  new daughter cell this environment Page 3 of 8  large  Pseudohyphae  round and elliptical – chain of elongated yeast cells  multi septate = numerous cross - walls – like a hyphae (elongated)  multicelled – elongates because of high protein content – has constriction between each section MICROCONIDIA CHLAMYDOCONIDIA  small  unicellular  round, elliptical, pyriform and pear shape PHIALOCONIDIA  chlamydospores o round and thick wall conidia  appears in 3 types: 1. terminal – conidia appears at the tip of hyphae 2. intercalary – in between of the hyphae 3. sessile – on the side of the hyphae  the conidia is produced from phialide o vase - like structure that holds the conidia o basic petal – youngest conidia – base ANNELLOCONIDIA – oldest conidia – top BLASTOCONIDIA  has mother cell (larger, flattened edge) and daughter cell (smaller, blastoconidia)  fungal cell that is produced through budding Page 4 of 8  annelide ASCOSPORES o has transverse bond o length increases at the tip o becomes narrower at the bottom  annelophore o holds the annelide SPORANGIOSPORES  ascus o the one that holds the ascospores  ascocarp BASIDIOSPORES  has sporangium o sac - like vessel  has sporangiophore o the one that holds the sporangium o like a container o produce endogenous asexual spore by cytoplasmic cleavage, once becomes mature it will break morphology is important for the identification of fungi and if it’s a new specie or just a new form of fungi  physically same but genetically different mate  genetically different daughter cell SEXUAL REPRODUCTION ZYGOSPORES  “plus and minus” mating types o physically and morphologically same, genetically different  hyphae of different mating types fuse and give rise to a specialized structure that produces spores (diploid) o fuse to produced spores  most fungi are haploid throughout most of their life cycle environmental when unfavorable conditions stress the conditions are organism favorable asexual sexual reproduction occurs and the reproduction offspring have an increased likehood that occurs rapidly they will be better suited for the environment  has mature hyphae environment is not conducive for growth  genetically the same (plus and minus)  mate  genetic wanted a genetic variation of the organism – characteristic that will thrive exchange = mature zygosporangium in this condition Page 5 of 8 Teleomorph  fungus that produce sexually Anamorph  occasionaly teleomorph reproduce COLONIZATION asexually  asexual form of teleomorph Synanamorph  more than one anamorph is present for the same teleomorph  Multiplication of an organism at a given site without harm to the host ANAMORPH TELEOMORPH HOLOMORPH asexual sexual stage of whole fungus stage of a a fungus including INFECTION fungus anamorph and teleomorph SEXUAL OR  Invasion and multiplication of organisms in body tissue ASEXUAL Asexual Sexual Both resulting in local cellular injury STAGE  Destroys system and function of cells CELL Mitosis Meiosis Both mitosis DIVISION and meiosis SPORE V. MODES OF TRANSMISSION Only asexual Both sexual and Both sexual PRODUCTION spores asexual spores and asexual spores 1) Inhalation 2) Inoculation - through skin trauma / mucous membrane 3) Ingestion VI. FUNGAL CULTURE PROCESS  Specimen collection and transportation  Direct examination of specimen anamorph and telomorph  Selection and inoculation of media  Evaluation of fungal growth  Serological testing  Antifungal susceptibility testing IV. PORTAL OF ENTRY SPECIMEN COLLECTION  skin  hair  nails  respiratory tract  Specimen types  gastrointestinal tract  Collect from area most likely infected  urinary tract  Use sterile technique (especially in site with normal flora)  Keep specimen moist (fungi with die in humid environment)  Label container properly  Transport right away  Process right away  Only with appropriate handling of specimens can the recovery of fungal org be associated with disease process.  The best specimen for determining the etiologic agent is at active infection site.  The collection details for fungal cultures are similar with bacterial cultures. Page 6 of 8  DIRECT EXAMINATION  with sterile forceps  placed in collect 10-12 hairs dry HAIR with shaft intact container or  Provides preliminary report envelope  ≤15 min  Guides MD in treatment of patient RT  Observe yeast phase of dimorphic  ≤24hr RT  Gives clues to id causative agent Inoculate special media  May require more than one direct examination method  disinfect with 70%  placed in alcohol before the dry surface is scraped container NAILS  can be nail or SPECIMEN COLLECTION AND TRANSPORT scraping or envelope cuttings; complete  ≤72 hr RT nail  deeper scraping: KOH preparation, SPECIMEN COLLECTION TRANSPORT inoculate media  collect as bacterial  ≤ 2hr RT  bladder emptied  ≤15 min cultures; disinfect  ≤24hr RT PROSTATIC BLOOD with tincture of followed by RT FLUID prostatic massage  ≤24hr RT iodine  direct inoculation  use maximum onto media amount of blood recommended  1st morning  ≤2 hr RT  collect as bacterial  ≤15 min specimen, deep  ≤24hr RT cultures; at least RT cough 3mL in a sterile  ≤24hr RT RESPIRATORY  if patient cannot SPECIMEN obtain sputum- container, but as  never CSF much CSF as refrigerate nebulizer can be possible is preferred used  should be  oropharyngeal concentrated by – direct smear & centrifugation culture before inoculation  nasal sinus  if >5mL filtration is – plated directly recommended  disinfect with 70%  placed in SKIN alcohol before the dry, clean  collect as bacterial  ≤15 min cultures; should be surface is scraped container RT STERILE FLUIDS  scrape surface at  ≤72 hr RT concentrated by  ≤24hr 4oC centrifugation the margin of before inoculation lesions  use sediments for  placed in inoculation clean  prepare site for  ≤15 min TISSUE BIOPSIES  surgical collection container; BONE MARROW surgical incision add few RT drops of  heparinized bone  ≤24hr 4oC sterile marrow should be saline to plated directly onto keep moist media at bedside  ≤15 min  doctor will collect RT  collect as bacterial  ≤15 min  ≤24 hr RT culture RT  early-morning  wide-  5cm distal tip of  ≤24hr 4oC CATHETER specimen mouth catheter into sterile tubes with few drops URINE  midstream container of sterile water or collection technique  ≤15 min saline RT  ≤15 min  ≤24hr 4oC EAR, EXTERNAL  firmly rotate swab in RT  swab outer ear canal  ≤24hr 4oC VAGINA  as bacterial culture transport  ≤ 2hr RT  inoculate  ≤15 min  ≤24hr RT CORNEAL scrapings directly RT SCRAPING onto media and  ≤24hr RT EYE slides for staining  needle aspiration VITREOUS then direct FLUID inoculation  doctor will aspirate Page 7 of 8  VII. PRETREATMENT OF CLINICAL SPECIMENS SPECIMEN PRETREATMENT Blood Lysis and centrifugation Body fluids Filtration/ centrifugation Exudates Washing, centrifugation, crushing of granules Medical devices Gentle scraping of device with sterile scalpel to remove biofilm Nails Cutting into pieces Respiratory Liquefaction with mucolytic agents specimens Tissue Mincing, grinding Urine Centrifugation at 2000xg for 10min Page 8 of 8 MYCOLOGY AND VIROLOGY (LECTURE) Quitaleg, Jenny Rose Maghuyop LEC PRELIM 2 DERMATOPHYTES OUTLINE I. MYCOSES I. Mycoses II. (Classification) Site Of Infection III. Detection Of Clinical Materials  fungal infection IV. Culture Of Clinical Materials  seen most in immunocompromised patients  requires longer treatment as immune system becomes weaker overtime infection generally chronic (long-lasting) fungi grow slowly growth in culture media 7 days – 1 month II. (CLASSIFICATION) SITE OF INFECTION 1. Superficial o without tissue invasion o very rare o on the skin 2. Cutaneous / o with tissue invasion Dermatomycoses o cannot reach system 3. Subcutaneous o under the skin 4. Systemic / Deep o involves many deep mycoses organs o from superficial internal organs  (RES) Reticuloendothelial syste  (CVS) Chorionic villus sampling  (CNS) Central nervous system Opportunistic fungi o affects low immunity o people with other infectious disease (bacterial and viral infections) acquires secondary infection (fungal infection) Page 1 of 2  CUTANEOUS MYCOSES / DERMATOMYCOSES III. DETECTION OF CLINICAL MATERIALS  Keratin Loving (degrades keratin)  Infect only the epidermis , hair and nail SKIN SCRAPING  hyphae FROM BORDER o threadlike lines running  Do not invade underlying tissue OF ACTIVE through skin or nail cells LESION MODE OF TRANSMISSION  differentiation from artifacts o cell walls, branches or septate o chlamydospores of older  human to human hyphae  animal to animal HAIR, ECTOTHRIX  Microsporum mosaic pattern around hair  human to animal  Trichophyton parallel cell USUAL HABITAT arrangement around hair HAIR, ENDOTHRIX  Trichophyton arthroconidia in rows  geophilic soil  T. favus hyphae  zoophilic animals schoenleinii  anthropophilic human MANIFESTATION IV. CULTURE OF CLINICAL MATERIALS  ectothrix o infection in the hair: inside  Mycosel  DTM – dermatophyte test medium  endothrix o infection in the hair: – specific for dermatophytes inside fungi  wood’s lamp o fluorescent lamp  Cornmeal  scutula o infection of the skin/  Slide culture – to clearly see the colony cutaneous morphology o crusty lesion  Colony characteristic – surface pigment – reverse pigment ECTOTHRIX AND ENDOTHRIX See last page. ECTOTHRIX ENDOTHRIX Fungal parasite Trichophyton FEATURES OF TRICHOPHYTON RUBRUM AND TRICHOPHYTON MENTAGROPHYTES dermatophytes Trichophyton violaceum Trichophyton tonsurans sheath of outside of a hair as well invade the interior of the spores as growing within the hair hair shaft with mycelia See last page. shaft sometimes THREE GENERA – Trichophyton hair, skin and endothrix or nails ectothrix – Microsporum hair and skin ectothrix – Epidermophyton skin and nails Page 2 of 2  HABITAT INFECTION MOT MICROSPORUM CANIS zoophilic – tinea capitis in humans animals to humans (ectothrix) – simple ringworm in pets MICROSPORUM geophilic – hair infection (ectothrix) GYPSEUM MICROSPORUM anthrophilic – non-inflammatory infections infected hairs on AUDOUINII of scalp and skin especially in – hats children – caps – upholstery combs – barbers clippers EPIDERMOPHYTON anthropophilic invades skin and nails but never may become epidemic among personnel FLOCCOSUM hair using common shower or gym facilities such – Tinea cruris (groin area) as athletic teams, troops, ship crews and – Tinea pedis (ringworm on inmates of institutions feet) – Onychomycosis (infection in nails) – Tinea corporis (mammary gland) TRICHOPHYTON anthropophilic – common cause of tinea RUBRUM ungium (nail) – skin, nail , rarely scalp infection – hair infection (endothrix or ectothrix) TRICHOPHYTON zoophilic – common cause of Tinea laboratory / domesticated animal hosts MENTAGROPHYTES worldwide pedis (athletes foot) including distribution – inflammatory skin or scalp – mice lesions in humans – guinea-pigs – hair infection (ectothrix) – kangaroos – cats – horses – sheep – rabbits TRICHOPHYTON common cause of Tinea capitis TONSURANS - “Black dot” (endothrix) TRICHOPHYTON zoophilic – ringworm in cattle VERRUCOSUM – infections in humans – direct contact with cattle or infected fomites – Tinea barbae – beard – hair infection (ectothrix) TRICHOPHYTON anthropophilic favus in humans SCHOENLEINII TRICHOPHYTON “black dot” tinea capitis VIOLACEUM (endothrix) ZOOPHILIC GEOPHILIC ANTHROPOPHILIC – Microsporum canis – Microsporum gypseum – Epidermophyton floccosum – Trichophyton mentagrophytes – Trichophyton rubrum – Trichophyton verrucosum – Trichophyton schoenleinii COMMON CAUSE OF TINEA UNGIUM (NAIL) Trichophyton rubrum COMMON CAUSE OF TINEA PEDIS (ATHLETES FOOT) Trichophyton mentagrophytes COMMON CAUSE OF TINEA CAPITIS Trichophyton tonsurans  HAIR INFECTION (ECTOTHRIX) HAIR INFECTION (ENDOTHRIX) HAIR INFECTION (ENDOTHRIX OR ECTOTHRIX) – Microsporum gypseum – Trichophyton tonsurans – Trichophyton rubrum – Trichophyton mentagrophytes – Trichophyton violaceum – Trichophyton verrucosum SURFACE PIGMENT REVERSE PIGMENT MICROSPORUM CANIS – white – golden-yellow to brownish yellow MICROSPORUM GYPSEUM – white – yellow brown – reddish-brown may be present in some strains MICROSPORUM AUDOUINII – white – reddish-brown – salmon or peach –pink – some strain has no reverse pigment EPIDERMOPHYTON FLOCCOSUM – greenish brown or khaki – deep yellowish brown TRICHOPHYTON RUBRUM – white – reddish rose purple – deep wine-red – no reverse pigment when grown on 1% peptone agar TRICHOPHYTON – white to cream – yellow-brown to pinkish brown MENTAGROPHYTES TRICHOPHYTON TONSURANS – white – yellow, the so-called “sulfureum” form to dark- brown – yellow-brown to reddish-brown to deep mahogany TRICHOPHYTON VERRUCOSUM – white – yellow TRICHOPHYTON SCHOENLEINII – cream colored to yellow – orange brown TRICHOPHYTON VIOLACEUM – deep violet some strain has no reverse pigment Microsporum audouinii no reverse pigment when grown on 1% peptone agar Trichophyton mentagrophytes  WOOD’S LAMP RICE AGAR TEST / IN VITRO HAIR NUTRITIONAL UREASE TEST HAIR PERFORATION PENETRATION TEST TEST MICROSPORUM CANIS bright greenish - POSITIVE yellow MICROSPORUM GYPSEUM MICROSPORUM apple green or NEGATIVE T1 POSITIVE AUDOUINII bright greenish - very poor or absent T4 POSITIVE yellow brown discoloration EPIDERMOPHYTON FLOCCOSUM TRICHOPHYTON do not fluoresce 28 days or 4 weeks NEGATIVE >5 days NONE, RUBRUM NEGATIVE WEAK OR DELAYED 7 days POSITIVE TRICHOPHYTON do not fluoresce 14 days POSITIVE 7 days MENTAGROPHYTES POSITIVE POSITIVE commonly 3 - 5 days ALREADY POSITIVE TRICHOPHYTON T1 NEGATIVE TONSURANS T4 POSITIVE TRICHOPHYTON fluorescence in T1 NEGATIVE VERRUCOSUM cattle but not in T4 POSITIVE humans TRICHOPHYTON pale greenish SCHOENLEINII yellow TRICHOPHYTON do not fluoresce T1 NEGATIVE VIOLACEUM T4 POSITIVE MANY STRAINS REQUIRE INOSITOL - Growth is enhanced at TRICHOPHYTON VERRUCOSUM 37OC ALL STRAINS OF T. VERRUCOSUM PRODUCE TYPICAL BRAIN HEART INFUSION BROTH WITH PARA-AMINO CHAINS OF CHLAMYDOCONIDIA WHEN GROWN AT THIS BENZOIC ACID (PAB) AND AGAR AT 37OC 10% KOH AND PARKER INK SOLUTION shows typical large spored T. violaceum REQUIRED THIAMINE FOR GROWTH – MICROSPORUM AUDOUINII – TRICHOPHYTON TONSURANS – TRICHOPHYTON VERRUCOSUM – TRICHOPHYTON VIOLACEUM SABOURAUD AGAR MICROSPORUM GYPSEUM – powdery – cinnamon brown color – some with white aerial mycelium at first and become matted SABOURAUD DEXTROSE AGAR MICROSPORUM AUDOUINII – malted – velvety – light tan to brown  MACROCONIDIA MICROCONIDIA MICROSPORUM CANIS – 3-15 celled – few pyriform to clavate – large – multi celled – spindle shaped – rough thick walled – terminal end: sometimes curved with a terminal knob MICROSPORUM GYPSEUM – symmetrical – singly or in clusters – ellipsoidal – numerous clavate shaped – thin – moderately thick walled – 4-6 celled – terminal or distal ends are thin slightly rounded and filamentous MICROSPORUM AUDOUINII – rarely produce – absent or bizarre, if present atypical vegetative hyphae with terminal chlamydospore, racquet hyphae and favic chandeliers EPIDERMOPHYTON – monotypic genus (cluster or single) FLOCCOSUM – often in clusters – 1-5 celled not formed – thin-walled – club shaped – greenish yellow TRICHOPHYTON RUBRUM – pencil-shaped – teardrop shaped occurring singly and in – slender or pyriform, pear shape clusters along hyphae – not usually present – slender clavate to pyriform – if present, thin wall, slender associated with cigar shape microconidia – slender or pyriform, pear shape TRICHOPHYTON – thin-walled – grape-like clusters - spherical and sub MENTAGROPHYTES – smooth spherical – cigar shaped – subspherical on terminal branches – multiseptate – spherical often forming in dense cluster – pear shape TRICHOPHYTON – various size and shapes, flattened base TONSURANS – aged microconidia: balloon forms absent or rare, distorted – long-clavate to broad-pyriform at right angles to the hypha – hyphae are relatively broad, irregular and much branched with numerous septa – older cultures: swollen giant forms TRICHOPHYTON – rare, if present have a characteristic of – large clavate, lateral VERRUCOSUM rat tail or string bean shape – clavate to pyriform / pear shape – 3-5 cells – thin-walled TRICHOPHYTON absent SCHOENLEINII TRICHOPHYTON absent VIOLACEUM  COLONY SURFACE COLONY MICROSPORUM CANIS MICROSPORUM GYPSEUM – central white downy umbo (dome) or a fluffy white tuft of mycelium – some also show a narrow , white periphery – like rays of sun MICROSPORUM AUDOUINII – thick-walled intercalary – flat, spreading, downy white surface chlamydoconidium EPIDERMOPHYTON FLOCCOSUM – yellow to yellow tan – greenish-brown or khaki coloured with – flat and feathery edges a suede-like surface – small diameter – thin in older culture TRICHOPHYTON RUBRUM – cottony and white – later become velvety TRICHOPHYTON MENTAGROPHYTES TRICHOPHYTON TONSURANS – slow growing – sunken center – wrinkled – may not grow w/o thiamine TRICHOPHYTON VERRUCOSUM – dry crusty layer TRICHOPHYTON SCHOENLEINII – very slow growers (30 days or – dry and waxy more) – small wrinkled waxy – favic chandeliers and chlamydospore formation TRICHOPHYTON VIOLACEUM – very slow growing – glabrous or waxy – heaped and folded – deep violet in colour TEST RICE AGAR TEST MATERIALS – rice grain – hair sample – agar PROCEDURE – cut an agar in square shape – place the hair sample on the agar – place the agar with the hair sample at the center of the rice grain EXAMINATION – remove hair every 4 days of 1 week – examine microscopically RESULT – POSITIVE – good surface growth – sporulation COLLECTED AS BACTERIAL CULTURE blood CSF sterile fluids catheter vagina 1. blood 2. bone marrow 3. catheter 4. CSF 5. ear, external 6. eye 7. hair 8. nails 9. prostatic fluids 10. respiratory specimen 11. skin 12. sterile fluids 13. tissue biopsies 14. urine 15. vagina